Research ArticleTranscriptome of the Deep-Sea Black ScabbardfishAphanopus carbo (Perciformes Trichiuridae) Tissue-SpecificExpression Patterns and Candidate Genes Associatedto Depth Adaptation
Sergio Stefanni12 Raul Bettencourt2 Miguel Pinheiro3 Gianluca De Moro4
Lucia Bongiorni5 and Alberto Pallavicini4
1 ISSIA-CNR Via de Marini 6 16149 Genova Italy2 LARSyS Associated Laboratory amp Centre of IMAR of the University of the Azores Department of Oceanography and FisheriesRua Prof Frederico Machado 4 9901-862 Horta Azores Portugal
3 School of Medicine University of St Andrews North Haugh St Andrews KY16 9TF UK4Department of Life Sciences University of Trieste Piazzale Valmaura 9 34148 Trieste Italy5 Institute of Marine Sciences National Research Council (ISMAR-CNR) Castello 2437F 30122 Venezia Italy
Correspondence should be addressed to Sergio Stefanni sergiostefannigeissiacnrit
Received 25 May 2014 Accepted 19 July 2014 Published 17 September 2014
Academic Editor Elena Pasyukova
Copyright copy 2014 Sergio Stefanni et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Deep-sea fishes provide a unique opportunity to study the physiology and evolutionary adaptation to extreme environments Wecarried out a high throughput sequencing analysis on a 454 GS-FLX titanium plate using unnormalized cDNA libraries from sixtissues ofA carbo Assemblage and annotationswere performed byNewbler and InterProPfam analyses respectivelyThe assemblyof 544491 high quality reads provided 8319 contigs 556 of which retrieved blast hits against the NCBI nonredundant database orwere annotated with ESTscan Comparison of functional genes at both the protein sequences and protein stability levels associatedwith adaptations to depth revealed similarities between A carbo and other bathypelagic fishes A selection of putative genes wasstandardized to evaluate the correlation between number of contigs and their normalized expression as determined by qPCRamplificationThe screening of the libraries contributed to the identification of new EST simple-sequence repeats (SSRs) and to thedesign of primer pairs suitable for population genetic studies as well as for tagging and mapping of genes The characterization ofthe deep-sea fish A carbo first transcriptome is expected to provide abundant resources for genetic evolutionary and ecologicalstudies of this species and the basis for further investigation of depth-related adaptation processes in fishes
1 Introduction
The deep-sea (gt1000m depth) covers about 70 of theEarthrsquos surface representing one of the last large unexploredareas on the planet Only within the last few decades thetechnology has advanced sufficiently to reach the deep-sea effectively revealing unexpected high levels of biodi-versity and extremely diverse habitats (canyons cold seepshydrothermal vents deep-water coral reefs mud volcanoesseamounts and trenches) of significant conservation interestand potential high economic values Deep-sea environmentsare characterized by extremely high hydrostatic pressures
(1MPa every 100m) lack of light and low temperatures(down to 1-2∘C)Therefore fish as well as any other organismliving in the deep-sea had to adapt to tolerate conditions ofthis extreme habitat [1]
First studies on adaptation to high pressure and lowtemperatures are dated back in the lsquo70s and they report com-parison of common proteins present in shallow and deep-water fishes [2 3] Key enzymes in muscle tissues that exhibitadaptive differences among species at different depths arethe lactate dehydrogenase (LDH) and malate dehydrogenase(MDH) presenting differences in structural stability (reviewsin [4ndash6]) More recent studies on evolutionary adaptation
Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2014 Article ID 267482 21 pageshttpdxdoiorg1011552014267482
2 International Journal of Genomics
of functional genes to high pressure report unique aminoacid substitutions in 120572-skeletal actin and myosin heavy chain(MyHC) proteins in deep-sea fishes [7ndash10] For deep-seaspecies inhabiting hydrothermal vents and cold seeps envi-ronments characterized by high pressure chronic hypoxiaand high concentrations of toxic compounds molecular andfunctional adaptation of hemoglobins (Hbs) are reviewedin Hourdez and Weber [11] Despite these studies ourknowledge on wide scale gene expression patterns in deep-sea fish remains elusive
The black scabbardfish Aphanopus carbo (Lowe 1839) isa bathypelagic species belonging to the Trichiuridae familyand is distributed in temperate-cold Atlantic waters at depthsbetween 200 and 1800m [12 13] A carbo represents acommercially valuable species for several regions of theIberic peninsula especially in Madeira where catches havereached up to 1000 tons per year [14] amounting to ca55 of the total landings Recently this species has becomeincreasingly targeted by Portuguese French and Irish fishingfleets ([15] and literature therein) and fishery data haveshown a constant decline in population [16]The informationavailable on the biology maturity spawning and growth ofthis species [17 18] is scattered Recent studies are reporting apanmictic distribution of this species in the NE Atlantic withmultiple breeding sites at low latitudes [19] It is also worthmentioning that in southern locations this species lives insympatry with A intermedius a close related species withvery similarmorphology [20] therefore attracting interest forevolutionary studies
High-throughput sequencing approaches applied to tran-scriptomics now provide a global perspective on taxonomicand functional profiling of genes expectedly expressed underthe influence of environmental conditions in which theseorganisms live Also known as next-generation sequencingthese techniques allow for a massive characterization ofexpressed sequence tags (ESTs) providing an overview ofthose genes expressed in a given tissue at any given time[21] In silico analyses of massive gene libraries may serveseveral interests among others For instances from discoveryand identification of new genes characterization of geneexpression to development of novel genetic markers forquantitative trait locus (QTL) and population of genomicanalysesThe breadth of next-generation sequencing applica-tions extends over a variety of biological questions includingthose addressing pertinent questions regarding a speciesrsquoecology life history and evolution [22 23]
Previous studies regarding transcriptome sequencing andgene expression studies in deep-water species were mostlylimited to hydrothermal vents invertebrates [24] microbialcommunities in hydrothermal plumes [25] deep-sediments[26] and in the water column [27] leaving vertebrates speciesvirtually under-represented The present work represents apioneer study for deep-sea fishes providing new insights intothe role of differential gene expression on the environmentaladaptation of deep-sea black scabbardfish
Here we describe the assembly and annotation of thetranscriptome of A carbo obtained by sequencing mRNAlibraries of six tissues (spleen brain heart gonads liverand muscle) and explore functional genes whose sequence
might be associated to depth adaptation Additionally wetested the correlation of selected candidate genes comparingthe number of contigs against the gene expression normalizedto a relative value of 10 as determined by qPCR amplifi-cation Furthermore the screening of the libraries allowedthe identification of newEST-simple-sequence repeats (SSRs)and the design of primer pairs suitable for population geneticstudies as well as tagging and mapping of genes
2 Methods
21 Fish and Tissue Samples Specimens of Aphanopus carbo(two males and two females) were collected in 2009 onboardof the RV ldquoArquipelagordquo A carbo were fished at depth range1100ndash1250m using deep-water long-lines in proximity of theCondor Seamount located approximately at 15 nm SW ofthe island of Fayal (Azores Portugal) The four specimensused in this study were caught on the same longline set andonce onboard the freshly caught animals were dissected andportions (or complete organs) of spleen brain heart gonadsliver and muscle tissues were preserved in formamide solu-tion and kept at minus20∘C until RNA extraction was performed
To validate the correct identification of the species a smallportion ofmuscle tissuewas also preserved in 95 ethanol formolecular screening following Stefanni et al [28] protocols
22 RNA Extraction and Sequencing Total RNA wasextracted from 20 to 40mg of each of the six preservedtissues of a pool of four A carbo individualsusing theRiboPure kit (Ambion Applied Biosystems) Quantity andpurity of the RNAwas determined on a 14 agarose-MOPS-formaldehyde denaturing gels and by assessing the 119860
260280
and 119860260230
ratios using the NanoVue spectrophotometer(GE Healthcare) Poly-A RNA was extracted from 15 120583gof each total RNA sample using the Poly(A)Purist mRNAPurification kit according to manufacturerrsquos instructions(Ambion Applied Biosystems) mRNAs were transcribedinto cDNA utilizing Mint-2 cDNA synthesis kit (Evrogenhttpwwwevrogencom) according to manufacturerrsquosinstructions for NGS platforms Six cDNA libraries wereconstructed from mRNA of individual pools of tissues andsequenced in a single 454 GS FLX Titanium run Each of thecDNA libraries was characterised by unique sequence tags(MIDs) that allowed to trace back the sequences generatedfrom single tissues after assembly
cDNAs were sheared by nebulization to yield randomfragments approximately 500ndash800 bp in length by applying30 psi (21 bar) of nitrogen for 1 minute on 4 120583g of eachlibrary The distribution of fragments was verified on aBioAnalyser DNA 7500 LabChip (Agilent Technologies)The fragmented cDNA sample was end-repaired with T4DNA polymerase and T4 polynucleotide kinase and adaptorsequences ligated according to the manufacturerrsquos instruc-tions [29] The fragments were immobilized onto strepta-vidin beads and nick-repaired with Bst polymerase ThecDNA fragments were denaturated with alkali to yield singlestranded cDNA (sscDNA) library Quality of the library wasassayed on a BioAnalyser RNA 6000 Pico LabChip (AgilentTechnologies) and quantity measured by spectrofluorimetry
International Journal of Genomics 3
with the Quant-iT RiboGreen RNA Assay kit (Invitrogen) Atitrationwas set up at 1 2 4 and 8 copies per bead (cpb) in theclonal amplification by emulsion PCR to optimize yield andsequence quality The percent enrichment of beads carryingthe sscDNA was determined and the amount of library inputcalculated to 18 A large scale emulsion PCR was set-up based on the previous value and sequenced at Biocant(Cantanhede Portugal) using the 454 GS FLX Titaniumpyrosequencing on a full 70X75 PicoTiterPlate according tomanufacturerrsquos instructions (Roche)
23 Bioinformatic Analyses High quality reads were assem-bled using Newbler ver 26 (Roche 454) sequence analysissoftware All reads were identified and grouped by theirunique MIDs to the tissue of origin Trimming and maskingthe polyAs was a common procedure for the assembling tool
The assemblage is characterized by read overlaps andmultiple alignments made in nucleotide space Consensusbase-calling and quality value determination for contigsare performed in flow space The use of flow space indetermining the properties of the consensus sequenceresults in an improved accuracy for the final base-calls Theimplementation of this software was performed using defaultparameters Assembled contigs were annotated throughsequence similarity searches against the National Centrefor Biotechnology Information (NCBI) nonredundant (nr)protein database using the BLASTx [30] with a cut-offcriterion of an expect-value (119890-value) lt 10minus6 The contigsthat did not find a hit were further processed with ESTScan(httpwwwchembnetorgsoftwareESTScan2html) Thetwo assemblages of amino acid sequences resulting from theBLASTx searches at high level of stringency and the ESTScanwere processed by InterProScan for functional annotationof transcripts applying the function for the mapping of geneontology (GO) termsThe GOmethod classifies genes withina hierarchy using a systematic nomenclature of attributesthat can be assigned to all gene products independentlyfrom the organism of origin To reduce the redundancyin the consensus sequences which correspond to the samegene we used BLASTClust to detect similar assemblieswith 95 identity and 90 coverage All the results fromboth assemblage methods were loaded into a SQL databasedeveloped for this purpose
To validate the accuracy of the assembly the result-ing contigs were compared to previously sequenced tran-scriptomes of 6 teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis using tBLASTn [30] tofind protein homologs at two levels of stringency (119890-value lt10minus3 and 119890-value lt 10minus10)To identify protein conserved domain specific for each
tissue analysed a new annotation was performed withHmmer against the Pfamdatabase (ver 250) Protein domainrepresentativeness for each tissue was obtained comparingprotein domain abundance in a particular tissue versus all thetissues compiled together using a hypergeometric test
24 cDNA Synthesis and qPCR Validation Tests Fresh cDNAwas synthesised from the six mRNAs that were used for
pyrosequencing cDNA synthesis was performed usingprimers with oligo(dT) and the ThermoScript RT-PCR Sys-tem (Invitrogen) following the manufacturerrsquos instructions
A set of 28 genes were selected including candidates thatwere tissue-specific and genes that were encountered in thetissue expressed at similar as well as at different amountsin all the six libraries with the aim of covering most of thepossible expression scenarios within the dataset Frequenciesof contigs for all candidates genes in themRNA libraries wereobtained by detecting orthologous gene sequences using theBLAST tool included in the A carbo database
For the design of all qPCR primers (Table 1) we usedthe web interfaceNCBI Primer-BLAST (httpblastncbinlmnihgov) Alignments of the sequences provided by theoutput from the internal blast search were used to select allprimer sets
Gene expression calculated as relative expression wasdetermined by means of real-time PCR using the CFX96(Bio-Rad) Primer concentrations and sample dilutions wereoptimized to meet highest efficiency in the PCR reactionin a total reaction volume of 20 120583L Fluorescent signal wasdetermined by the addition of SsoFast EvaGreen Supermix(Bio-Rad) which was included in the cocktail accordinglyto manufacturerrsquos instructions Baseline and threshold cyclewere always set to automatic in the sequence detectionsoftware CFXManager (Bio-Rad) All plates contained a ldquonotemplate controlrdquo (NTC) and each sample was tested in dupli-cate Cycling conditions for gene amplifications were 95∘Cfor 3min followed by 35 cycles of 95∘C for 10 s 56∘C for 10 sand 68∘C for 15 s An additional protocol for melting curvesanalysis included a cycle at 95∘C followed by a progressivereading of fluorescence for every cycle from 65∘C to 95∘Cfor 5 s at intervals of 05∘C Gene expression normalized toa relative value of 10 for all the genes selected and for eachtissue was compared to the contigs frequencies generatedby the assembly platform to determine the significance ofcorrelation between qPCRs values and 454 sequencing readsfrom unnormalized cDNA libraries
25 Characterization of Depth-Related Functional Genes Thepredicted amino acid sequences of functional genes of Acarbo possibly related to depth adaptations were comparedto those deposited in NCBI database searching for homolo-gies We aligned and compared translated sequences oflactate dehydrogenase (LDH-A andLDH-B) cytosolicmalatedehydrogenase (MDHc) hemoglobins (Hb-A and Hb-B)actin (ACTA1) and myosin heavy chain (MyHC) Proteinand nucleotide sequences were aligned using Clustal X [31]while sequence analysis and phylogenetic inferences wereperformed using CLC Main Workbench (v 682 CLC Bio)The neighbor-joining (NJ) algorithm [32] was implementedto construct a phylogenetic tree using HKY substitutionmodel and attributing a gap penalty of 10 The support forinternal branches was assessed using the bootstrap [33] with1000 replicates
Nucleotide alignments and ML trees built implementingthe most appropriate substitution model under the AkaikeInformation Criterion (AIC) were used in the program
4 International Journal of Genomics
Table 1 List of targeted genes using qPCR primer sets specifically designed for this study size for each of the product and NCBI accessionnumber for all the EST sequences
Gene Primer name Primer Sequences (51015840-31015840) Size(bp) NCBI Accession
Elongation factor 1-beta EF-1B L GCTTGGACATGTCGGTCTCGTC 229 bp All gs454 000396EF-1B H GTGGCTGACACCACATCTGGC
Ras-related GTP-binding protein A Rab-1A L AGTAGCCGTTCCACCTTGTCGG 247 bp All gs454 000598Rab-1A H TGCCAAGAAACCGTACGTGGGA
Basic Transcription Factor 3-like 4 BTF3 L CCCAAAGTTCAGGCCTCCCTGT 273 bp All gs454 000873BTF3 H TCATGTGCGTCAGTTCGCTTCG
CuZn Superoxide Dismutase SOD-1 L AAACGTGACTGCAGGAGGGGAT 240 bp All gs454 000925SOD-1 H CAGTGCTCCTGCTCCATGTTCG
2-Cys Peroxiredoxin PRDX1 L CCGATAACCTCGCAGCCGATAC 243 bp All gs454 000558PRDX1 H ACAGTCATTTGCCACCAGCATCA
Heat Shock Protein 90 HSP90 L TGACGATGTCCCCACAGATGAGG 221 bp All gs454 000008HSP90 H GCAACACTGGTCCACCACACAAC
Ferritin heavy subunit Ferr L CCTGCAGCTTGAGAAGAGCGTC 203 bp All gs454 000681Ferr H CAAACAGGTACTCGGCCATGCC
1205722
Globin Hb-A L AAATTGTTGGCCATGCGGAGGA 208 bp All gs454 001919Hb-A H CTGAGGTTCAGCAGACCTGCCT
1205732
Globin Hb-B L TCGTCTACCCCTGGTGTCAGAG 245 bp All gs454 001018Hb-B H AACCACAATGGTCAGGCAGTCC
Ependymin-1 precursor EPD-1 L CAGGTGTGAGGCAGTGCAGT 230 bp All gs454 000469EPD-1 H ACCCCGATCTCCTCCTGGTG
Fatty acid-binding protein brain BLBP L CAACACTTCTTGGCCGGTTTGG 239 bp All gs454 001220BLBP H GAGAGGAGTTCGACGAAGCCAC
CD63-like protein Sm-TSP-2 TSPAN-8 L TCGCTGGCTGCTCTGAGAAAGA 200 bp All gs454 000381TSPAN-8 H GGTCACGCCGAGCTGTATTCTG
Tropomyosin 4 isoform 1 TRPM-1 L GTGGAGGAGGAGTTGGACCGAG 221 bp All gs454 000222TRPM1 H TTGCGAGCCACCTCCTCGTATT
C-Myc-binding protein MYCBP L CGCCAGTTTACCTGCGTTCCAA 182 bp All gs454 001640MYCBP H GGCCGTCAACAACACCACCTTT
Cathepsin S CTSS L AACAGCCTACCCCTACACAGCC 200 bp All gs454 000156CTSS H TGTACACACCGTGGCGGTAGAA
Transferrin STF-1 L AGCTGCACCAGCTTCACAGTTG 215 bp All gs454 000004STF-1 H AAGGATGGCACCAGACAACCCA
Warm Temperature Acclimationrelated-like 65 kDa protein
HPX L TGATACCGGGTGGAACCTGGTG 207 bp All gs454 000060HPX H GCTGCTGTGGAGTGTCCCAAAG
Betaine HomocysteineS-methyltransferase
BHTM L GGGGGTTCGCTGTTACCAAGTG 194 bp All gs454 000088BHTM H TGTGAGACAGCAGCCTCAGGAG
FUCL1 Fucolectin-1 FUCL1 L CGCAAACCCTTTGGCTGGTGTA 196 bp All gs454 000758FUCL1 H GGCTTTTCCTTGGACTGCCAGG
Aldolase B ALDB L GCCATTGGTCTTGGCCCTGATC 220 bp All gs454 000115ALDB H CGCTGTGCCTGGTATCTGCTTC
Type-4 ice-structuring protein LS-12precursor
ISP LS12 L AAGACCTGACAAACCAGGCCCA 198 bp All gs454 001277ISP LS12 H GGAGGATGGCCTCCATCTGCTT
International Journal of Genomics 5
Table 1 Continued
Gene Primer name Primer Sequences (51015840-31015840) Size(bp) NCBI Accession
Alcohol Dehydrogenase 8a ADH L GGCAAGAAGGTGCTGCAGTTCA 228 bp All gs454 000105-6ADH H CATGACTGCAGCCAAACCCACA
Glyceraldehyde-3-phosphateDehydrogenase
GAPDH L GTCAACCACTGACACGTTGGGG 229 bp All gs454 000148GAPDH H CGGCATCATTGAGGGCCTGATG
Lactate Dehydrogenase-A LDH-A L TCTTAACCTGGTGCAGCGCAAC 219 bp All gs454 000149LDH-A H TGGAGCTTCTCGCCCATGATGT
Phosphoglycerate Mutase 2-1 (muscle) PglyM L ACACCTCTGTGCTGAAACGTGC 212 bp All gs454 000309PglyM H CATGGGTGGAGGTGGGATGTCA
Heat Shock Protein 70 HSP70 L CGGTGTTGTGTGCTGGGTGAAA 207 bp All gs454 000005HSP70 H CCACATAGCTGGGTGTGGTCCT
Fructose-bisphosphate Aldolase A FBPA L GGAACCAACGGCGAGACAACAA 208 bp All gs454 002732FBPA H CAATGGGGACGATGCCATGCAT
Phosphoglucose Isomerase-2 PGI L CCACACTGGGCCAATTGTCTGG 217 bp All gs454 000011PGI H GGCCTCCTCTGTGGTCTTACCC
Table 2 Global statistics for each of the nonnormalized libraries using Newbler software
Tissue Spleen Brain Heart Gonad Liver Muscle TotalTotal EST 15034 33337 73263 157275 134523 92788 544491Total bases 3426510 8219500 17647600 37123700 31792600 23342900 129412000Contigs 567 651 1260 3875 1274 626 8319Average contig length 470 619 567 465 612 689 555Contigs 119890-value lt 10minus6 220 420 622 951 617 409 2440ESTscan 584 345 977 2838 634 269 2715No similarity 36 70 109 406 211 74 1128GO annotation 202 338 473 623 509 307 1728InterPro annotation 223 417 610 908 649 395 2395
ldquocodemlrdquo in PAML 4 [34] to assess selective pressure onthose genes for which the complete sequences was availablePositive (or negative) selected sites were defined by the ratiobetween nonsynonymous versus synonymous substitutions(dNdS or 120596) Two models were tested comparatively M1which groups codons in two classes (120596 lt 1 and 120596 = 1)clustering sites under negative or neutral selection and M2which groups codons in three classes (120596 lt 1 120596 = 1 and 120596 gt1) adding a cluster for sites under positive selection to theones defined in M1 Probabilistic measures of how well thesemodels fits the evolutionary relationship of individual geneswere calculated from the likelihood values of fitted modelsand the number of ldquofree parametersrdquo for all genes
Protein stability was estimated using a virtual quantifica-tion software [35] calculating Gibbs free energy in terms ofkinetic and thermodynamic quantities taking into accounteach amino acid contribution for the maintenance of thenative structure of the protein For a protein to maintain itsstability there is a need of sufficient hydrophobic residueswhich will utilize free energy to guide proper folding [35]
26 EST-SSR Resources for Population Genetics Among vari-ous molecular markers simple sequence repeats (SSRs) arehighly polymorphic easier to develop and very useful for
researches such as genetic diversity assessment ThereforeA carbo database was further used to detect such regionsin the transcriptome sequencing data and provide a list ofcombination of primer sets on flanking regions that could beused for population genetic studies To identify EST-SSRs allthe contigs were searched using MISA [36] and for primerdesign we used Primer3 [37]The algorithm of the SSR finderidentifies a good quality repeat when one locus is present withadjacent loci at an up or downstream distance higher than100 bp and parameters were set to locate a minimum of 20 bpsequence repeats di-mers (x12) 3-mers (x8) 4-mers (x5) 5-mers (x5) and 6-mers (x5) Primer design was performedsetting parameters of a minimum size of 20 bp and meltingtemperatures of 60∘C
3 Results and Discussion
31 Sequences Assemblage and Functional Annotations Aftersequence trimming a total of 544491 high quality readswere produced with an average length of 237 bp correspond-ing to 1295Mb A total of 8319 contigs were assembledwith Newbler (ver 26) as high quality consensus sequenceswithout the presence of singletons A summary of ESTdata for each of the six tissues is reported in Table 2 Atotal of 2440 assembled contigs were annotated against
6 International Journal of Genomics
the NCBI nonredundant protein database at the cut-off (119890-value) lt 10minus6 Additional 3843 contigs with no proteinmatches were further processed with ESTScan to find 2715homologous proteins All the contigs were then processedby InterProScan for functional annotation of transcriptsand mapping functional information to gene ontology (GO)terms Of the 5155 amino-acid sequences (out of the 6283totally sequenced) 1728 could be annotated within theGO hierarchy (Figure 1) while 2395 could be annotatedaccordingly to InterProScan The complete GO mappingof A carbo for individual tissues can be accessed on thededicated online database (httptranscriptomicsbiocantptAphanopusCarbo) The largest proportions of GO func-tional categories are of similar proportion to the unnormal-ized libraries of Tilapia [38] In the Cellular Components GOgroup (Figure 1(a)) the genes involved in cell and cell partfunctional categories corresponded to the largest percentageof the pie chart (2815 each) In the Molecular FunctionGO group (Figure 1(b)) almost half of the total genes wereinvolved in binding (4740) followed by those related tocatalytic activity (2753) Finally for the Biological ProcessesGO group (Figure 1(c)) the largest portion of the genes wereinvolved in metabolic processes (3345) tightly followed bythe category of genes linked to the Cellular Process (3193)
To further validate the accuracy of the A carbo assemblywe compared our dataset to the transcriptomes of six otherprior sequenced teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis Similarity searches wereperformed comparing our assembled contigs against each ofthe available transcriptomes using tBLASTn [30] The resultshighlighted strong similarity between the transcriptomes ofA carbo and other teleosts indicating that about 30 of thetotal contigs matched protein homologs (119890-values lt 10minus3ranging between 2268 and 2457) and that our assemblypresents the highest similarity (by little margin) with thetranscriptome of Gasterosteus aculeatus (119890-value lt 10minus10 =2 223) (Table 3)
A total of 1639 (20) transcripts were annotated by Pfamprotein domainsmatches and the set of protein domains char-acterizing each single tissue was identified by hypergeometrictest (Table 4)
32 qPCR Assays and Validation Tests Quantitative PCRassays were performed using cDNA samples at 1 400 dilution(up to 100 ng approximately in the qPCR master mix)conditions at which the PCR resulted to be more efficientWe tested seven series of dilutions ranging from 1 50 to1 1000 Based on optimized qPCR conditions all targetedcDNAs were tested across all tissues for the complete set ofgenes selection used in this study Normalized expressionto a relative value of 10 for 28 genes is shown in Figure 2(see Figure S1 in Supplementary Material available online athttpdxdoiorg1011552014267482)
Statistical tests were performed to identify the correlationbetween the mean value of normalized expression and arelative value of 10 as determined by qPCR against contigfrequencies using Pearsonrsquos 119903 coefficient and its significance
Table 3 Similarity search for unigenes comparing transcriptomesof other teleosts against Aphanopus carbo using two levels ofstringency
Species Sequencesavailable
A carbo119890-value lt 10minus3
A carbo119890-value lt 10minus10
Danio rerio 42787 2457 2199Gasterosteusaculeatus 27576 2445 2223
Oreochromisniloticus 26763 2436 2202
Oryziaslatipes 24674 2412 2173
Takifugurubripes 47841 2268 2062
Tetraodonnigroviridis 23118 2300 2073
(Figure 3) Most of the genes showed highly significantcorrelation however one case indicated a reduced signifi-cance (elongation factor) and a few other cases resulted tobe not significant (eg Ras-related GTP-binding proteinBasic Transcription Factor 3 CuZn Superoxide Dismutaseand 2-Cys Peroxiredoxin) Reduced or lack of significanceappeared to be related to a low number of contigs from the454 sequencing (Table 5) which might be regarded as anindication of failure to reach library saturation Low valuesin contig frequency should correspond to a reduced levelof expression as the libraries are nonnormalized thereforecaution should be used when exploring 454 outputs belowa certain threshold Our data derived from the reading of asingle plate were insufficient for systematical comparison ofall the genes although the majority of the genes show a highcorrelation between the 454 data and the qPCR Howeverit should be emphasized that this exercise was not meant tobe a surrogate to the qPCR approach for the study of geneexpression but more a proxy for a preliminary screening ofdifferentially expressed genes in multiple libraries
33 Candidate Genes Associated to Depth The predictedamino acid sequences of A carbo functional genes puta-tively related to depth adaptations were compared to thosedeposited in NCBI database for homologies searches Com-plete alignments of orthologous protein sequences from theset of functional genes that have been reported as responsiveto depth adaptations included representatives of Teleostsbelonging to several families (Table 6) Exploring the levels ofsimilarities expressed as percentage of amino acid identity ornumber of amino acid that differ among sequences betweenA carbo and other fish species (Table 7) brings evidencesupporting the fact that deep-living aswell as polar species arenot the most similar to the black scabbard fish We attemptedto reconstruct the phylogenies for those species using aminoacid sequences of functional genes (LDH-A LDH-B MDHc-A Hb-A and Hb-B) and corresponding mtCOI nucleotidesequences (Figure 4) The resulting trees showed similartopologies suggesting that the signals embedded in thesefunctional genes reflect evolutionary divergence among taxarather than any enzyme adaptations relationships
International Journal of Genomics 7
Macromolecular complex 1541Organelle part 580Extracellular region 367
Organelle 1616Synapse part 031Synapse 031
Membrane-enclosed lumen 084Extracellular region part 120Cell 2815Cell part 2815
(a) Cellular components
Structural molecule activity 1054Catalytic activity 2753Enzyme regulator activity 294Electron carrier activity 152
Transporter activity 635Transcription factor activity 052Metallochaperone activity 016
Binding 4740Molecular transducer activity 058Antioxidant activity 094
(b) Molecular function
Cellular process 3193Death 010Signaling 127Cellular component biogenesis 215Cell wall organization or biogenesis 010
Metabolic process 3345Biological regulation 446
Viral reproduction 005
Biological adhesion 093
Cellular component organization 289
Signaling process 098Developmental process 024Immune system process 118Establishment of localization 847
Multicellular organismal process 049
(c) Biological processes
Figure 1 Functional categorization of unigenes with gene ontology (GO) term for theAphanopus carboEST collectionThese unigenes resultswere functionally classified from the six tissues pooled together as percentages under three main functional categories with respective GOSlim terms Data refer to assemblage derived by implementation of Newbler Roche 454 sequence analysis software
The lactate dehydrogenase A (LDH-A isotig01401)was encountered abundantly in the muscle tissue (Table 5Figure 2) and its comparison with other orthologoussequences differs for a number of amino acids between 16and 44 One indel at position 75 had to be added to the two
polar species to obtain a complete alignment (Table 7) Thepercentage of identity was higher with the strictly marinespecies ranging between 952 and 937 Unfortunatelyno sequences from neither deep-sea nor abyssal fisheswere available for comparison On the other hand lactate
8 International Journal of Genomics
Table 4 List of Pfam conserved domains that were tissue specifically expressed inAphanopus carbo transcriptome Frequencies and 119875-valuesof tissue specificity analysis are indicated
(a)
Gonads Heart LiverPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF00100 Zona pellucida 17 315 10minus13 PF00011 HSP20 3 403 10minus4 PF00084 Sushi 14 144 10minus10
PF00125 Histone 8 606 10minus5 PF13405 EF hand 4 5 677 10minus4 PF00089 Trypsin 20 759 10minus9
PF01400 Astacin 3 322 10minus3 PF00412 LIM 3 152 10minus3 PF00079 Serpin 12 563 10minus6
PF00069 Pkinase 3 001 PF05556 Calsarcin 3 360 10minus3 PF07678 A2M comp 4 135 10minus4
PF00653 BIR 2 002 PF01576 Myosin tail 1 3 360 10minus3 PF01042 Ribonuc L-PSP 3 126 10minus3
PF13424 TPR 12 2 002 PF00056 Ldh 1 N 3 360 10minus3 PF00045 Hemopexin 3 126 10minus3
PF13695 zf-3CxxC 2 002 PF05300 DUF737 2 550 10minus3 PF00059 Lectin C 6 169 10minus3
PF09360 zf-CDGSH 2 002 PF00992 Troponin 4 640 10minus3 PF00701 DHDPS 4 464 10minus3
PF01712 dNK 2 002 PF00595 PDZ 3 682 10minus3 PF00386 C1q 8 663 10minus3
PF00538 Linker histone 2 002 PF00022 Actin 3 001 PF00021 UPAR LY6 5 001PF10178 DUF2372 2 002 PF02874 ATP-synt ab N 2 002 PF00754 F5 F8 type C 9 001PF04856 Securin 2 002 PF13895 Ig 2 3 002 PF08702 Fib alpha 2 001PF00250 Fork head 2 002 PF00191 Annexin 2 003 PF03982 DAGAT 2 001PF01498 HTH Tnp Tc3 2 3 003 PF00212 ANP 2 003 PF01048 PNP UDP 1 2 001PF00268 Ribonuc red sm 2 006 PF05347 Complex1 LYR 2 007 PF01014 Uricase 2 001
(b)
Muscle Brain SpleenPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF01410 Troponin 7 199 10minus5 PF01669 Myelin MBP 4 403 10minus5 PF00042 Globin 5 133 10minus6
PF02807 COLFI 3 967 10minus4 PF01453 B lectin 2 644 10minus3 PF00078 RVT 1 5 133 10minus6
PF00041 ATP-guaPtransN 3 359 10minus3 PF00612 IQ 2 644 10minus3 PF00993 MHC II alpha 4 502 10minus6
PF02453 fn3 3 359 10minus3 PF11414 Suppressor APC 2 644 10minus3 PF07686 V set 5 249 10minus6
PF13895 Reticulon 3 359 10minus3 PF05768 DUF836 2 644 10minus3 PF07654 C1 set 5 471 10minus4
PF00365 Ig 2 4 797 10minus3 PF05196 PTNMK N 2 644 10minus3 PF00089 Trypsin 5 201 10minus3
PF01216 PFK 2 984 10minus3 PF04300 FBA 2 644 10minus3 PF01391 Collagen 2 230 10minus3
PF01267 Calsequestrin 2 984 10minus3 PF00287 Na K-ATPase 2 644 10minus3 PF00240 ubiquitin 3 328 10minus3
PF00261 F-actin cap A 2 984 10minus3 PF11032 ApoM 3 853 10minus3 PF09307 MHC2-interact 2 667 10minus3
PF00856 Tropomyosin 2 984 10minus3 PF00061 Lipocalin 4 001 PF00643 Zf-B box 2 001PF01661 SET 2 984 10minus3 PF00007 Cys knot 2 002 PF13445 Zf-RING LisH 2 001PF01667 Macro 2 003 PF01275 Myelin PLP 2 002 PF01498 HTH Tnp Tc3 2 2 002PF00036 Ribosomal S27e 2 005 PF00300 His Phos 1 2 002 PF02301 HORMA 1 005PF01576 efhand 2 005 PF00230 MIP 2 002 PF14259 RRM 6 1 005PF01410 Myosin tail 1 2 008 PF00091 Tubulin 3 002 PF09004 DUF1891 1 005
dehydrogenase B (LDH-B isotig01423) inA carbowas highlyrepresented in the heart while very scarcely recorded in theother tissues (Table S1) The largest sequence divergence interms of amino acid substitutions ranges between 47 and 55when compared with deep-water macrourids and similarlyto LDH-A an indel had to be added in the sequences ofthe gadiform species at position 76 to obtain a completealignment (Table 7) Previous studies on benthopelagic fishreport a significant decline of LDH activities with increasingwater depth [39] However it is known that variation inenzyme activities of fish at a given depth is influenced byfeeding behaviour and locomotory modes [40 41]
Cytosolic malate dehygrogenase A (MDHc-Aisotig01010) was detected in most of the tissues withthe exception of the muscle (Table S1) An isoform of MDHcwas also detected but its sequence covered only the last 110amino acids (isotig01731) However the type of substitutionsand tissue expression pattern (Table S1) according to Merritand Quattro [42] lead us to believe that this cytosolicisoform is MDHc-B It has been reported that the two teleostMDHc isozymes are the products of a gene duplicationevent after the separation of teleosts and tetrapods (see [42]and references therein) although the exact timing of thisduplication has not been inferred
International Journal of Genomics 9
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Elongation factor 1-beta
Basic transcription factor 3-like 4
B G H L M S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
2-Cys peroxiredoxin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ferritin heavy subunit
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
1205732-Globin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ependymin-1 precursor10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
1205722-Globin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 90
12
14
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CuZn superoxide dismutase
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
Ras-related GTP-binding protein A12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Figure 2 Continued
10 International Journal of Genomics
Fatty acid-binding protein brain10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B GH LM S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CD63-like protein Sm-TSP-2
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Tropomyosin 4 isoform 1
10
12
08
06
04
02
00Re
lativ
e fol
d ex
pres
sion
B G H L M S
C-Myc-binding protein
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Cathepsin S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Transferrin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
GH L M S
Warm temperature acclimation related-like proteinlowast
10
12
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Betaine homocysteine S-methyltansferase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
FUCL1 fucolectin-110
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Aldolase B
Figure 2 Continued
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Nucleic AcidsJournal of
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Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
2 International Journal of Genomics
of functional genes to high pressure report unique aminoacid substitutions in 120572-skeletal actin and myosin heavy chain(MyHC) proteins in deep-sea fishes [7ndash10] For deep-seaspecies inhabiting hydrothermal vents and cold seeps envi-ronments characterized by high pressure chronic hypoxiaand high concentrations of toxic compounds molecular andfunctional adaptation of hemoglobins (Hbs) are reviewedin Hourdez and Weber [11] Despite these studies ourknowledge on wide scale gene expression patterns in deep-sea fish remains elusive
The black scabbardfish Aphanopus carbo (Lowe 1839) isa bathypelagic species belonging to the Trichiuridae familyand is distributed in temperate-cold Atlantic waters at depthsbetween 200 and 1800m [12 13] A carbo represents acommercially valuable species for several regions of theIberic peninsula especially in Madeira where catches havereached up to 1000 tons per year [14] amounting to ca55 of the total landings Recently this species has becomeincreasingly targeted by Portuguese French and Irish fishingfleets ([15] and literature therein) and fishery data haveshown a constant decline in population [16]The informationavailable on the biology maturity spawning and growth ofthis species [17 18] is scattered Recent studies are reporting apanmictic distribution of this species in the NE Atlantic withmultiple breeding sites at low latitudes [19] It is also worthmentioning that in southern locations this species lives insympatry with A intermedius a close related species withvery similarmorphology [20] therefore attracting interest forevolutionary studies
High-throughput sequencing approaches applied to tran-scriptomics now provide a global perspective on taxonomicand functional profiling of genes expectedly expressed underthe influence of environmental conditions in which theseorganisms live Also known as next-generation sequencingthese techniques allow for a massive characterization ofexpressed sequence tags (ESTs) providing an overview ofthose genes expressed in a given tissue at any given time[21] In silico analyses of massive gene libraries may serveseveral interests among others For instances from discoveryand identification of new genes characterization of geneexpression to development of novel genetic markers forquantitative trait locus (QTL) and population of genomicanalysesThe breadth of next-generation sequencing applica-tions extends over a variety of biological questions includingthose addressing pertinent questions regarding a speciesrsquoecology life history and evolution [22 23]
Previous studies regarding transcriptome sequencing andgene expression studies in deep-water species were mostlylimited to hydrothermal vents invertebrates [24] microbialcommunities in hydrothermal plumes [25] deep-sediments[26] and in the water column [27] leaving vertebrates speciesvirtually under-represented The present work represents apioneer study for deep-sea fishes providing new insights intothe role of differential gene expression on the environmentaladaptation of deep-sea black scabbardfish
Here we describe the assembly and annotation of thetranscriptome of A carbo obtained by sequencing mRNAlibraries of six tissues (spleen brain heart gonads liverand muscle) and explore functional genes whose sequence
might be associated to depth adaptation Additionally wetested the correlation of selected candidate genes comparingthe number of contigs against the gene expression normalizedto a relative value of 10 as determined by qPCR amplifi-cation Furthermore the screening of the libraries allowedthe identification of newEST-simple-sequence repeats (SSRs)and the design of primer pairs suitable for population geneticstudies as well as tagging and mapping of genes
2 Methods
21 Fish and Tissue Samples Specimens of Aphanopus carbo(two males and two females) were collected in 2009 onboardof the RV ldquoArquipelagordquo A carbo were fished at depth range1100ndash1250m using deep-water long-lines in proximity of theCondor Seamount located approximately at 15 nm SW ofthe island of Fayal (Azores Portugal) The four specimensused in this study were caught on the same longline set andonce onboard the freshly caught animals were dissected andportions (or complete organs) of spleen brain heart gonadsliver and muscle tissues were preserved in formamide solu-tion and kept at minus20∘C until RNA extraction was performed
To validate the correct identification of the species a smallportion ofmuscle tissuewas also preserved in 95 ethanol formolecular screening following Stefanni et al [28] protocols
22 RNA Extraction and Sequencing Total RNA wasextracted from 20 to 40mg of each of the six preservedtissues of a pool of four A carbo individualsusing theRiboPure kit (Ambion Applied Biosystems) Quantity andpurity of the RNAwas determined on a 14 agarose-MOPS-formaldehyde denaturing gels and by assessing the 119860
260280
and 119860260230
ratios using the NanoVue spectrophotometer(GE Healthcare) Poly-A RNA was extracted from 15 120583gof each total RNA sample using the Poly(A)Purist mRNAPurification kit according to manufacturerrsquos instructions(Ambion Applied Biosystems) mRNAs were transcribedinto cDNA utilizing Mint-2 cDNA synthesis kit (Evrogenhttpwwwevrogencom) according to manufacturerrsquosinstructions for NGS platforms Six cDNA libraries wereconstructed from mRNA of individual pools of tissues andsequenced in a single 454 GS FLX Titanium run Each of thecDNA libraries was characterised by unique sequence tags(MIDs) that allowed to trace back the sequences generatedfrom single tissues after assembly
cDNAs were sheared by nebulization to yield randomfragments approximately 500ndash800 bp in length by applying30 psi (21 bar) of nitrogen for 1 minute on 4 120583g of eachlibrary The distribution of fragments was verified on aBioAnalyser DNA 7500 LabChip (Agilent Technologies)The fragmented cDNA sample was end-repaired with T4DNA polymerase and T4 polynucleotide kinase and adaptorsequences ligated according to the manufacturerrsquos instruc-tions [29] The fragments were immobilized onto strepta-vidin beads and nick-repaired with Bst polymerase ThecDNA fragments were denaturated with alkali to yield singlestranded cDNA (sscDNA) library Quality of the library wasassayed on a BioAnalyser RNA 6000 Pico LabChip (AgilentTechnologies) and quantity measured by spectrofluorimetry
International Journal of Genomics 3
with the Quant-iT RiboGreen RNA Assay kit (Invitrogen) Atitrationwas set up at 1 2 4 and 8 copies per bead (cpb) in theclonal amplification by emulsion PCR to optimize yield andsequence quality The percent enrichment of beads carryingthe sscDNA was determined and the amount of library inputcalculated to 18 A large scale emulsion PCR was set-up based on the previous value and sequenced at Biocant(Cantanhede Portugal) using the 454 GS FLX Titaniumpyrosequencing on a full 70X75 PicoTiterPlate according tomanufacturerrsquos instructions (Roche)
23 Bioinformatic Analyses High quality reads were assem-bled using Newbler ver 26 (Roche 454) sequence analysissoftware All reads were identified and grouped by theirunique MIDs to the tissue of origin Trimming and maskingthe polyAs was a common procedure for the assembling tool
The assemblage is characterized by read overlaps andmultiple alignments made in nucleotide space Consensusbase-calling and quality value determination for contigsare performed in flow space The use of flow space indetermining the properties of the consensus sequenceresults in an improved accuracy for the final base-calls Theimplementation of this software was performed using defaultparameters Assembled contigs were annotated throughsequence similarity searches against the National Centrefor Biotechnology Information (NCBI) nonredundant (nr)protein database using the BLASTx [30] with a cut-offcriterion of an expect-value (119890-value) lt 10minus6 The contigsthat did not find a hit were further processed with ESTScan(httpwwwchembnetorgsoftwareESTScan2html) Thetwo assemblages of amino acid sequences resulting from theBLASTx searches at high level of stringency and the ESTScanwere processed by InterProScan for functional annotationof transcripts applying the function for the mapping of geneontology (GO) termsThe GOmethod classifies genes withina hierarchy using a systematic nomenclature of attributesthat can be assigned to all gene products independentlyfrom the organism of origin To reduce the redundancyin the consensus sequences which correspond to the samegene we used BLASTClust to detect similar assemblieswith 95 identity and 90 coverage All the results fromboth assemblage methods were loaded into a SQL databasedeveloped for this purpose
To validate the accuracy of the assembly the result-ing contigs were compared to previously sequenced tran-scriptomes of 6 teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis using tBLASTn [30] tofind protein homologs at two levels of stringency (119890-value lt10minus3 and 119890-value lt 10minus10)To identify protein conserved domain specific for each
tissue analysed a new annotation was performed withHmmer against the Pfamdatabase (ver 250) Protein domainrepresentativeness for each tissue was obtained comparingprotein domain abundance in a particular tissue versus all thetissues compiled together using a hypergeometric test
24 cDNA Synthesis and qPCR Validation Tests Fresh cDNAwas synthesised from the six mRNAs that were used for
pyrosequencing cDNA synthesis was performed usingprimers with oligo(dT) and the ThermoScript RT-PCR Sys-tem (Invitrogen) following the manufacturerrsquos instructions
A set of 28 genes were selected including candidates thatwere tissue-specific and genes that were encountered in thetissue expressed at similar as well as at different amountsin all the six libraries with the aim of covering most of thepossible expression scenarios within the dataset Frequenciesof contigs for all candidates genes in themRNA libraries wereobtained by detecting orthologous gene sequences using theBLAST tool included in the A carbo database
For the design of all qPCR primers (Table 1) we usedthe web interfaceNCBI Primer-BLAST (httpblastncbinlmnihgov) Alignments of the sequences provided by theoutput from the internal blast search were used to select allprimer sets
Gene expression calculated as relative expression wasdetermined by means of real-time PCR using the CFX96(Bio-Rad) Primer concentrations and sample dilutions wereoptimized to meet highest efficiency in the PCR reactionin a total reaction volume of 20 120583L Fluorescent signal wasdetermined by the addition of SsoFast EvaGreen Supermix(Bio-Rad) which was included in the cocktail accordinglyto manufacturerrsquos instructions Baseline and threshold cyclewere always set to automatic in the sequence detectionsoftware CFXManager (Bio-Rad) All plates contained a ldquonotemplate controlrdquo (NTC) and each sample was tested in dupli-cate Cycling conditions for gene amplifications were 95∘Cfor 3min followed by 35 cycles of 95∘C for 10 s 56∘C for 10 sand 68∘C for 15 s An additional protocol for melting curvesanalysis included a cycle at 95∘C followed by a progressivereading of fluorescence for every cycle from 65∘C to 95∘Cfor 5 s at intervals of 05∘C Gene expression normalized toa relative value of 10 for all the genes selected and for eachtissue was compared to the contigs frequencies generatedby the assembly platform to determine the significance ofcorrelation between qPCRs values and 454 sequencing readsfrom unnormalized cDNA libraries
25 Characterization of Depth-Related Functional Genes Thepredicted amino acid sequences of functional genes of Acarbo possibly related to depth adaptations were comparedto those deposited in NCBI database searching for homolo-gies We aligned and compared translated sequences oflactate dehydrogenase (LDH-A andLDH-B) cytosolicmalatedehydrogenase (MDHc) hemoglobins (Hb-A and Hb-B)actin (ACTA1) and myosin heavy chain (MyHC) Proteinand nucleotide sequences were aligned using Clustal X [31]while sequence analysis and phylogenetic inferences wereperformed using CLC Main Workbench (v 682 CLC Bio)The neighbor-joining (NJ) algorithm [32] was implementedto construct a phylogenetic tree using HKY substitutionmodel and attributing a gap penalty of 10 The support forinternal branches was assessed using the bootstrap [33] with1000 replicates
Nucleotide alignments and ML trees built implementingthe most appropriate substitution model under the AkaikeInformation Criterion (AIC) were used in the program
4 International Journal of Genomics
Table 1 List of targeted genes using qPCR primer sets specifically designed for this study size for each of the product and NCBI accessionnumber for all the EST sequences
Gene Primer name Primer Sequences (51015840-31015840) Size(bp) NCBI Accession
Elongation factor 1-beta EF-1B L GCTTGGACATGTCGGTCTCGTC 229 bp All gs454 000396EF-1B H GTGGCTGACACCACATCTGGC
Ras-related GTP-binding protein A Rab-1A L AGTAGCCGTTCCACCTTGTCGG 247 bp All gs454 000598Rab-1A H TGCCAAGAAACCGTACGTGGGA
Basic Transcription Factor 3-like 4 BTF3 L CCCAAAGTTCAGGCCTCCCTGT 273 bp All gs454 000873BTF3 H TCATGTGCGTCAGTTCGCTTCG
CuZn Superoxide Dismutase SOD-1 L AAACGTGACTGCAGGAGGGGAT 240 bp All gs454 000925SOD-1 H CAGTGCTCCTGCTCCATGTTCG
2-Cys Peroxiredoxin PRDX1 L CCGATAACCTCGCAGCCGATAC 243 bp All gs454 000558PRDX1 H ACAGTCATTTGCCACCAGCATCA
Heat Shock Protein 90 HSP90 L TGACGATGTCCCCACAGATGAGG 221 bp All gs454 000008HSP90 H GCAACACTGGTCCACCACACAAC
Ferritin heavy subunit Ferr L CCTGCAGCTTGAGAAGAGCGTC 203 bp All gs454 000681Ferr H CAAACAGGTACTCGGCCATGCC
1205722
Globin Hb-A L AAATTGTTGGCCATGCGGAGGA 208 bp All gs454 001919Hb-A H CTGAGGTTCAGCAGACCTGCCT
1205732
Globin Hb-B L TCGTCTACCCCTGGTGTCAGAG 245 bp All gs454 001018Hb-B H AACCACAATGGTCAGGCAGTCC
Ependymin-1 precursor EPD-1 L CAGGTGTGAGGCAGTGCAGT 230 bp All gs454 000469EPD-1 H ACCCCGATCTCCTCCTGGTG
Fatty acid-binding protein brain BLBP L CAACACTTCTTGGCCGGTTTGG 239 bp All gs454 001220BLBP H GAGAGGAGTTCGACGAAGCCAC
CD63-like protein Sm-TSP-2 TSPAN-8 L TCGCTGGCTGCTCTGAGAAAGA 200 bp All gs454 000381TSPAN-8 H GGTCACGCCGAGCTGTATTCTG
Tropomyosin 4 isoform 1 TRPM-1 L GTGGAGGAGGAGTTGGACCGAG 221 bp All gs454 000222TRPM1 H TTGCGAGCCACCTCCTCGTATT
C-Myc-binding protein MYCBP L CGCCAGTTTACCTGCGTTCCAA 182 bp All gs454 001640MYCBP H GGCCGTCAACAACACCACCTTT
Cathepsin S CTSS L AACAGCCTACCCCTACACAGCC 200 bp All gs454 000156CTSS H TGTACACACCGTGGCGGTAGAA
Transferrin STF-1 L AGCTGCACCAGCTTCACAGTTG 215 bp All gs454 000004STF-1 H AAGGATGGCACCAGACAACCCA
Warm Temperature Acclimationrelated-like 65 kDa protein
HPX L TGATACCGGGTGGAACCTGGTG 207 bp All gs454 000060HPX H GCTGCTGTGGAGTGTCCCAAAG
Betaine HomocysteineS-methyltransferase
BHTM L GGGGGTTCGCTGTTACCAAGTG 194 bp All gs454 000088BHTM H TGTGAGACAGCAGCCTCAGGAG
FUCL1 Fucolectin-1 FUCL1 L CGCAAACCCTTTGGCTGGTGTA 196 bp All gs454 000758FUCL1 H GGCTTTTCCTTGGACTGCCAGG
Aldolase B ALDB L GCCATTGGTCTTGGCCCTGATC 220 bp All gs454 000115ALDB H CGCTGTGCCTGGTATCTGCTTC
Type-4 ice-structuring protein LS-12precursor
ISP LS12 L AAGACCTGACAAACCAGGCCCA 198 bp All gs454 001277ISP LS12 H GGAGGATGGCCTCCATCTGCTT
International Journal of Genomics 5
Table 1 Continued
Gene Primer name Primer Sequences (51015840-31015840) Size(bp) NCBI Accession
Alcohol Dehydrogenase 8a ADH L GGCAAGAAGGTGCTGCAGTTCA 228 bp All gs454 000105-6ADH H CATGACTGCAGCCAAACCCACA
Glyceraldehyde-3-phosphateDehydrogenase
GAPDH L GTCAACCACTGACACGTTGGGG 229 bp All gs454 000148GAPDH H CGGCATCATTGAGGGCCTGATG
Lactate Dehydrogenase-A LDH-A L TCTTAACCTGGTGCAGCGCAAC 219 bp All gs454 000149LDH-A H TGGAGCTTCTCGCCCATGATGT
Phosphoglycerate Mutase 2-1 (muscle) PglyM L ACACCTCTGTGCTGAAACGTGC 212 bp All gs454 000309PglyM H CATGGGTGGAGGTGGGATGTCA
Heat Shock Protein 70 HSP70 L CGGTGTTGTGTGCTGGGTGAAA 207 bp All gs454 000005HSP70 H CCACATAGCTGGGTGTGGTCCT
Fructose-bisphosphate Aldolase A FBPA L GGAACCAACGGCGAGACAACAA 208 bp All gs454 002732FBPA H CAATGGGGACGATGCCATGCAT
Phosphoglucose Isomerase-2 PGI L CCACACTGGGCCAATTGTCTGG 217 bp All gs454 000011PGI H GGCCTCCTCTGTGGTCTTACCC
Table 2 Global statistics for each of the nonnormalized libraries using Newbler software
Tissue Spleen Brain Heart Gonad Liver Muscle TotalTotal EST 15034 33337 73263 157275 134523 92788 544491Total bases 3426510 8219500 17647600 37123700 31792600 23342900 129412000Contigs 567 651 1260 3875 1274 626 8319Average contig length 470 619 567 465 612 689 555Contigs 119890-value lt 10minus6 220 420 622 951 617 409 2440ESTscan 584 345 977 2838 634 269 2715No similarity 36 70 109 406 211 74 1128GO annotation 202 338 473 623 509 307 1728InterPro annotation 223 417 610 908 649 395 2395
ldquocodemlrdquo in PAML 4 [34] to assess selective pressure onthose genes for which the complete sequences was availablePositive (or negative) selected sites were defined by the ratiobetween nonsynonymous versus synonymous substitutions(dNdS or 120596) Two models were tested comparatively M1which groups codons in two classes (120596 lt 1 and 120596 = 1)clustering sites under negative or neutral selection and M2which groups codons in three classes (120596 lt 1 120596 = 1 and 120596 gt1) adding a cluster for sites under positive selection to theones defined in M1 Probabilistic measures of how well thesemodels fits the evolutionary relationship of individual geneswere calculated from the likelihood values of fitted modelsand the number of ldquofree parametersrdquo for all genes
Protein stability was estimated using a virtual quantifica-tion software [35] calculating Gibbs free energy in terms ofkinetic and thermodynamic quantities taking into accounteach amino acid contribution for the maintenance of thenative structure of the protein For a protein to maintain itsstability there is a need of sufficient hydrophobic residueswhich will utilize free energy to guide proper folding [35]
26 EST-SSR Resources for Population Genetics Among vari-ous molecular markers simple sequence repeats (SSRs) arehighly polymorphic easier to develop and very useful for
researches such as genetic diversity assessment ThereforeA carbo database was further used to detect such regionsin the transcriptome sequencing data and provide a list ofcombination of primer sets on flanking regions that could beused for population genetic studies To identify EST-SSRs allthe contigs were searched using MISA [36] and for primerdesign we used Primer3 [37]The algorithm of the SSR finderidentifies a good quality repeat when one locus is present withadjacent loci at an up or downstream distance higher than100 bp and parameters were set to locate a minimum of 20 bpsequence repeats di-mers (x12) 3-mers (x8) 4-mers (x5) 5-mers (x5) and 6-mers (x5) Primer design was performedsetting parameters of a minimum size of 20 bp and meltingtemperatures of 60∘C
3 Results and Discussion
31 Sequences Assemblage and Functional Annotations Aftersequence trimming a total of 544491 high quality readswere produced with an average length of 237 bp correspond-ing to 1295Mb A total of 8319 contigs were assembledwith Newbler (ver 26) as high quality consensus sequenceswithout the presence of singletons A summary of ESTdata for each of the six tissues is reported in Table 2 Atotal of 2440 assembled contigs were annotated against
6 International Journal of Genomics
the NCBI nonredundant protein database at the cut-off (119890-value) lt 10minus6 Additional 3843 contigs with no proteinmatches were further processed with ESTScan to find 2715homologous proteins All the contigs were then processedby InterProScan for functional annotation of transcriptsand mapping functional information to gene ontology (GO)terms Of the 5155 amino-acid sequences (out of the 6283totally sequenced) 1728 could be annotated within theGO hierarchy (Figure 1) while 2395 could be annotatedaccordingly to InterProScan The complete GO mappingof A carbo for individual tissues can be accessed on thededicated online database (httptranscriptomicsbiocantptAphanopusCarbo) The largest proportions of GO func-tional categories are of similar proportion to the unnormal-ized libraries of Tilapia [38] In the Cellular Components GOgroup (Figure 1(a)) the genes involved in cell and cell partfunctional categories corresponded to the largest percentageof the pie chart (2815 each) In the Molecular FunctionGO group (Figure 1(b)) almost half of the total genes wereinvolved in binding (4740) followed by those related tocatalytic activity (2753) Finally for the Biological ProcessesGO group (Figure 1(c)) the largest portion of the genes wereinvolved in metabolic processes (3345) tightly followed bythe category of genes linked to the Cellular Process (3193)
To further validate the accuracy of the A carbo assemblywe compared our dataset to the transcriptomes of six otherprior sequenced teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis Similarity searches wereperformed comparing our assembled contigs against each ofthe available transcriptomes using tBLASTn [30] The resultshighlighted strong similarity between the transcriptomes ofA carbo and other teleosts indicating that about 30 of thetotal contigs matched protein homologs (119890-values lt 10minus3ranging between 2268 and 2457) and that our assemblypresents the highest similarity (by little margin) with thetranscriptome of Gasterosteus aculeatus (119890-value lt 10minus10 =2 223) (Table 3)
A total of 1639 (20) transcripts were annotated by Pfamprotein domainsmatches and the set of protein domains char-acterizing each single tissue was identified by hypergeometrictest (Table 4)
32 qPCR Assays and Validation Tests Quantitative PCRassays were performed using cDNA samples at 1 400 dilution(up to 100 ng approximately in the qPCR master mix)conditions at which the PCR resulted to be more efficientWe tested seven series of dilutions ranging from 1 50 to1 1000 Based on optimized qPCR conditions all targetedcDNAs were tested across all tissues for the complete set ofgenes selection used in this study Normalized expressionto a relative value of 10 for 28 genes is shown in Figure 2(see Figure S1 in Supplementary Material available online athttpdxdoiorg1011552014267482)
Statistical tests were performed to identify the correlationbetween the mean value of normalized expression and arelative value of 10 as determined by qPCR against contigfrequencies using Pearsonrsquos 119903 coefficient and its significance
Table 3 Similarity search for unigenes comparing transcriptomesof other teleosts against Aphanopus carbo using two levels ofstringency
Species Sequencesavailable
A carbo119890-value lt 10minus3
A carbo119890-value lt 10minus10
Danio rerio 42787 2457 2199Gasterosteusaculeatus 27576 2445 2223
Oreochromisniloticus 26763 2436 2202
Oryziaslatipes 24674 2412 2173
Takifugurubripes 47841 2268 2062
Tetraodonnigroviridis 23118 2300 2073
(Figure 3) Most of the genes showed highly significantcorrelation however one case indicated a reduced signifi-cance (elongation factor) and a few other cases resulted tobe not significant (eg Ras-related GTP-binding proteinBasic Transcription Factor 3 CuZn Superoxide Dismutaseand 2-Cys Peroxiredoxin) Reduced or lack of significanceappeared to be related to a low number of contigs from the454 sequencing (Table 5) which might be regarded as anindication of failure to reach library saturation Low valuesin contig frequency should correspond to a reduced levelof expression as the libraries are nonnormalized thereforecaution should be used when exploring 454 outputs belowa certain threshold Our data derived from the reading of asingle plate were insufficient for systematical comparison ofall the genes although the majority of the genes show a highcorrelation between the 454 data and the qPCR Howeverit should be emphasized that this exercise was not meant tobe a surrogate to the qPCR approach for the study of geneexpression but more a proxy for a preliminary screening ofdifferentially expressed genes in multiple libraries
33 Candidate Genes Associated to Depth The predictedamino acid sequences of A carbo functional genes puta-tively related to depth adaptations were compared to thosedeposited in NCBI database for homologies searches Com-plete alignments of orthologous protein sequences from theset of functional genes that have been reported as responsiveto depth adaptations included representatives of Teleostsbelonging to several families (Table 6) Exploring the levels ofsimilarities expressed as percentage of amino acid identity ornumber of amino acid that differ among sequences betweenA carbo and other fish species (Table 7) brings evidencesupporting the fact that deep-living aswell as polar species arenot the most similar to the black scabbard fish We attemptedto reconstruct the phylogenies for those species using aminoacid sequences of functional genes (LDH-A LDH-B MDHc-A Hb-A and Hb-B) and corresponding mtCOI nucleotidesequences (Figure 4) The resulting trees showed similartopologies suggesting that the signals embedded in thesefunctional genes reflect evolutionary divergence among taxarather than any enzyme adaptations relationships
International Journal of Genomics 7
Macromolecular complex 1541Organelle part 580Extracellular region 367
Organelle 1616Synapse part 031Synapse 031
Membrane-enclosed lumen 084Extracellular region part 120Cell 2815Cell part 2815
(a) Cellular components
Structural molecule activity 1054Catalytic activity 2753Enzyme regulator activity 294Electron carrier activity 152
Transporter activity 635Transcription factor activity 052Metallochaperone activity 016
Binding 4740Molecular transducer activity 058Antioxidant activity 094
(b) Molecular function
Cellular process 3193Death 010Signaling 127Cellular component biogenesis 215Cell wall organization or biogenesis 010
Metabolic process 3345Biological regulation 446
Viral reproduction 005
Biological adhesion 093
Cellular component organization 289
Signaling process 098Developmental process 024Immune system process 118Establishment of localization 847
Multicellular organismal process 049
(c) Biological processes
Figure 1 Functional categorization of unigenes with gene ontology (GO) term for theAphanopus carboEST collectionThese unigenes resultswere functionally classified from the six tissues pooled together as percentages under three main functional categories with respective GOSlim terms Data refer to assemblage derived by implementation of Newbler Roche 454 sequence analysis software
The lactate dehydrogenase A (LDH-A isotig01401)was encountered abundantly in the muscle tissue (Table 5Figure 2) and its comparison with other orthologoussequences differs for a number of amino acids between 16and 44 One indel at position 75 had to be added to the two
polar species to obtain a complete alignment (Table 7) Thepercentage of identity was higher with the strictly marinespecies ranging between 952 and 937 Unfortunatelyno sequences from neither deep-sea nor abyssal fisheswere available for comparison On the other hand lactate
8 International Journal of Genomics
Table 4 List of Pfam conserved domains that were tissue specifically expressed inAphanopus carbo transcriptome Frequencies and 119875-valuesof tissue specificity analysis are indicated
(a)
Gonads Heart LiverPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF00100 Zona pellucida 17 315 10minus13 PF00011 HSP20 3 403 10minus4 PF00084 Sushi 14 144 10minus10
PF00125 Histone 8 606 10minus5 PF13405 EF hand 4 5 677 10minus4 PF00089 Trypsin 20 759 10minus9
PF01400 Astacin 3 322 10minus3 PF00412 LIM 3 152 10minus3 PF00079 Serpin 12 563 10minus6
PF00069 Pkinase 3 001 PF05556 Calsarcin 3 360 10minus3 PF07678 A2M comp 4 135 10minus4
PF00653 BIR 2 002 PF01576 Myosin tail 1 3 360 10minus3 PF01042 Ribonuc L-PSP 3 126 10minus3
PF13424 TPR 12 2 002 PF00056 Ldh 1 N 3 360 10minus3 PF00045 Hemopexin 3 126 10minus3
PF13695 zf-3CxxC 2 002 PF05300 DUF737 2 550 10minus3 PF00059 Lectin C 6 169 10minus3
PF09360 zf-CDGSH 2 002 PF00992 Troponin 4 640 10minus3 PF00701 DHDPS 4 464 10minus3
PF01712 dNK 2 002 PF00595 PDZ 3 682 10minus3 PF00386 C1q 8 663 10minus3
PF00538 Linker histone 2 002 PF00022 Actin 3 001 PF00021 UPAR LY6 5 001PF10178 DUF2372 2 002 PF02874 ATP-synt ab N 2 002 PF00754 F5 F8 type C 9 001PF04856 Securin 2 002 PF13895 Ig 2 3 002 PF08702 Fib alpha 2 001PF00250 Fork head 2 002 PF00191 Annexin 2 003 PF03982 DAGAT 2 001PF01498 HTH Tnp Tc3 2 3 003 PF00212 ANP 2 003 PF01048 PNP UDP 1 2 001PF00268 Ribonuc red sm 2 006 PF05347 Complex1 LYR 2 007 PF01014 Uricase 2 001
(b)
Muscle Brain SpleenPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF01410 Troponin 7 199 10minus5 PF01669 Myelin MBP 4 403 10minus5 PF00042 Globin 5 133 10minus6
PF02807 COLFI 3 967 10minus4 PF01453 B lectin 2 644 10minus3 PF00078 RVT 1 5 133 10minus6
PF00041 ATP-guaPtransN 3 359 10minus3 PF00612 IQ 2 644 10minus3 PF00993 MHC II alpha 4 502 10minus6
PF02453 fn3 3 359 10minus3 PF11414 Suppressor APC 2 644 10minus3 PF07686 V set 5 249 10minus6
PF13895 Reticulon 3 359 10minus3 PF05768 DUF836 2 644 10minus3 PF07654 C1 set 5 471 10minus4
PF00365 Ig 2 4 797 10minus3 PF05196 PTNMK N 2 644 10minus3 PF00089 Trypsin 5 201 10minus3
PF01216 PFK 2 984 10minus3 PF04300 FBA 2 644 10minus3 PF01391 Collagen 2 230 10minus3
PF01267 Calsequestrin 2 984 10minus3 PF00287 Na K-ATPase 2 644 10minus3 PF00240 ubiquitin 3 328 10minus3
PF00261 F-actin cap A 2 984 10minus3 PF11032 ApoM 3 853 10minus3 PF09307 MHC2-interact 2 667 10minus3
PF00856 Tropomyosin 2 984 10minus3 PF00061 Lipocalin 4 001 PF00643 Zf-B box 2 001PF01661 SET 2 984 10minus3 PF00007 Cys knot 2 002 PF13445 Zf-RING LisH 2 001PF01667 Macro 2 003 PF01275 Myelin PLP 2 002 PF01498 HTH Tnp Tc3 2 2 002PF00036 Ribosomal S27e 2 005 PF00300 His Phos 1 2 002 PF02301 HORMA 1 005PF01576 efhand 2 005 PF00230 MIP 2 002 PF14259 RRM 6 1 005PF01410 Myosin tail 1 2 008 PF00091 Tubulin 3 002 PF09004 DUF1891 1 005
dehydrogenase B (LDH-B isotig01423) inA carbowas highlyrepresented in the heart while very scarcely recorded in theother tissues (Table S1) The largest sequence divergence interms of amino acid substitutions ranges between 47 and 55when compared with deep-water macrourids and similarlyto LDH-A an indel had to be added in the sequences ofthe gadiform species at position 76 to obtain a completealignment (Table 7) Previous studies on benthopelagic fishreport a significant decline of LDH activities with increasingwater depth [39] However it is known that variation inenzyme activities of fish at a given depth is influenced byfeeding behaviour and locomotory modes [40 41]
Cytosolic malate dehygrogenase A (MDHc-Aisotig01010) was detected in most of the tissues withthe exception of the muscle (Table S1) An isoform of MDHcwas also detected but its sequence covered only the last 110amino acids (isotig01731) However the type of substitutionsand tissue expression pattern (Table S1) according to Merritand Quattro [42] lead us to believe that this cytosolicisoform is MDHc-B It has been reported that the two teleostMDHc isozymes are the products of a gene duplicationevent after the separation of teleosts and tetrapods (see [42]and references therein) although the exact timing of thisduplication has not been inferred
International Journal of Genomics 9
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Elongation factor 1-beta
Basic transcription factor 3-like 4
B G H L M S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
2-Cys peroxiredoxin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ferritin heavy subunit
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
1205732-Globin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ependymin-1 precursor10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
1205722-Globin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 90
12
14
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CuZn superoxide dismutase
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
Ras-related GTP-binding protein A12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Figure 2 Continued
10 International Journal of Genomics
Fatty acid-binding protein brain10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B GH LM S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CD63-like protein Sm-TSP-2
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Tropomyosin 4 isoform 1
10
12
08
06
04
02
00Re
lativ
e fol
d ex
pres
sion
B G H L M S
C-Myc-binding protein
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Cathepsin S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Transferrin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
GH L M S
Warm temperature acclimation related-like proteinlowast
10
12
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Betaine homocysteine S-methyltansferase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
FUCL1 fucolectin-110
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Aldolase B
Figure 2 Continued
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
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Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
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Stem CellsInternational
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Genomics 3
with the Quant-iT RiboGreen RNA Assay kit (Invitrogen) Atitrationwas set up at 1 2 4 and 8 copies per bead (cpb) in theclonal amplification by emulsion PCR to optimize yield andsequence quality The percent enrichment of beads carryingthe sscDNA was determined and the amount of library inputcalculated to 18 A large scale emulsion PCR was set-up based on the previous value and sequenced at Biocant(Cantanhede Portugal) using the 454 GS FLX Titaniumpyrosequencing on a full 70X75 PicoTiterPlate according tomanufacturerrsquos instructions (Roche)
23 Bioinformatic Analyses High quality reads were assem-bled using Newbler ver 26 (Roche 454) sequence analysissoftware All reads were identified and grouped by theirunique MIDs to the tissue of origin Trimming and maskingthe polyAs was a common procedure for the assembling tool
The assemblage is characterized by read overlaps andmultiple alignments made in nucleotide space Consensusbase-calling and quality value determination for contigsare performed in flow space The use of flow space indetermining the properties of the consensus sequenceresults in an improved accuracy for the final base-calls Theimplementation of this software was performed using defaultparameters Assembled contigs were annotated throughsequence similarity searches against the National Centrefor Biotechnology Information (NCBI) nonredundant (nr)protein database using the BLASTx [30] with a cut-offcriterion of an expect-value (119890-value) lt 10minus6 The contigsthat did not find a hit were further processed with ESTScan(httpwwwchembnetorgsoftwareESTScan2html) Thetwo assemblages of amino acid sequences resulting from theBLASTx searches at high level of stringency and the ESTScanwere processed by InterProScan for functional annotationof transcripts applying the function for the mapping of geneontology (GO) termsThe GOmethod classifies genes withina hierarchy using a systematic nomenclature of attributesthat can be assigned to all gene products independentlyfrom the organism of origin To reduce the redundancyin the consensus sequences which correspond to the samegene we used BLASTClust to detect similar assemblieswith 95 identity and 90 coverage All the results fromboth assemblage methods were loaded into a SQL databasedeveloped for this purpose
To validate the accuracy of the assembly the result-ing contigs were compared to previously sequenced tran-scriptomes of 6 teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis using tBLASTn [30] tofind protein homologs at two levels of stringency (119890-value lt10minus3 and 119890-value lt 10minus10)To identify protein conserved domain specific for each
tissue analysed a new annotation was performed withHmmer against the Pfamdatabase (ver 250) Protein domainrepresentativeness for each tissue was obtained comparingprotein domain abundance in a particular tissue versus all thetissues compiled together using a hypergeometric test
24 cDNA Synthesis and qPCR Validation Tests Fresh cDNAwas synthesised from the six mRNAs that were used for
pyrosequencing cDNA synthesis was performed usingprimers with oligo(dT) and the ThermoScript RT-PCR Sys-tem (Invitrogen) following the manufacturerrsquos instructions
A set of 28 genes were selected including candidates thatwere tissue-specific and genes that were encountered in thetissue expressed at similar as well as at different amountsin all the six libraries with the aim of covering most of thepossible expression scenarios within the dataset Frequenciesof contigs for all candidates genes in themRNA libraries wereobtained by detecting orthologous gene sequences using theBLAST tool included in the A carbo database
For the design of all qPCR primers (Table 1) we usedthe web interfaceNCBI Primer-BLAST (httpblastncbinlmnihgov) Alignments of the sequences provided by theoutput from the internal blast search were used to select allprimer sets
Gene expression calculated as relative expression wasdetermined by means of real-time PCR using the CFX96(Bio-Rad) Primer concentrations and sample dilutions wereoptimized to meet highest efficiency in the PCR reactionin a total reaction volume of 20 120583L Fluorescent signal wasdetermined by the addition of SsoFast EvaGreen Supermix(Bio-Rad) which was included in the cocktail accordinglyto manufacturerrsquos instructions Baseline and threshold cyclewere always set to automatic in the sequence detectionsoftware CFXManager (Bio-Rad) All plates contained a ldquonotemplate controlrdquo (NTC) and each sample was tested in dupli-cate Cycling conditions for gene amplifications were 95∘Cfor 3min followed by 35 cycles of 95∘C for 10 s 56∘C for 10 sand 68∘C for 15 s An additional protocol for melting curvesanalysis included a cycle at 95∘C followed by a progressivereading of fluorescence for every cycle from 65∘C to 95∘Cfor 5 s at intervals of 05∘C Gene expression normalized toa relative value of 10 for all the genes selected and for eachtissue was compared to the contigs frequencies generatedby the assembly platform to determine the significance ofcorrelation between qPCRs values and 454 sequencing readsfrom unnormalized cDNA libraries
25 Characterization of Depth-Related Functional Genes Thepredicted amino acid sequences of functional genes of Acarbo possibly related to depth adaptations were comparedto those deposited in NCBI database searching for homolo-gies We aligned and compared translated sequences oflactate dehydrogenase (LDH-A andLDH-B) cytosolicmalatedehydrogenase (MDHc) hemoglobins (Hb-A and Hb-B)actin (ACTA1) and myosin heavy chain (MyHC) Proteinand nucleotide sequences were aligned using Clustal X [31]while sequence analysis and phylogenetic inferences wereperformed using CLC Main Workbench (v 682 CLC Bio)The neighbor-joining (NJ) algorithm [32] was implementedto construct a phylogenetic tree using HKY substitutionmodel and attributing a gap penalty of 10 The support forinternal branches was assessed using the bootstrap [33] with1000 replicates
Nucleotide alignments and ML trees built implementingthe most appropriate substitution model under the AkaikeInformation Criterion (AIC) were used in the program
4 International Journal of Genomics
Table 1 List of targeted genes using qPCR primer sets specifically designed for this study size for each of the product and NCBI accessionnumber for all the EST sequences
Gene Primer name Primer Sequences (51015840-31015840) Size(bp) NCBI Accession
Elongation factor 1-beta EF-1B L GCTTGGACATGTCGGTCTCGTC 229 bp All gs454 000396EF-1B H GTGGCTGACACCACATCTGGC
Ras-related GTP-binding protein A Rab-1A L AGTAGCCGTTCCACCTTGTCGG 247 bp All gs454 000598Rab-1A H TGCCAAGAAACCGTACGTGGGA
Basic Transcription Factor 3-like 4 BTF3 L CCCAAAGTTCAGGCCTCCCTGT 273 bp All gs454 000873BTF3 H TCATGTGCGTCAGTTCGCTTCG
CuZn Superoxide Dismutase SOD-1 L AAACGTGACTGCAGGAGGGGAT 240 bp All gs454 000925SOD-1 H CAGTGCTCCTGCTCCATGTTCG
2-Cys Peroxiredoxin PRDX1 L CCGATAACCTCGCAGCCGATAC 243 bp All gs454 000558PRDX1 H ACAGTCATTTGCCACCAGCATCA
Heat Shock Protein 90 HSP90 L TGACGATGTCCCCACAGATGAGG 221 bp All gs454 000008HSP90 H GCAACACTGGTCCACCACACAAC
Ferritin heavy subunit Ferr L CCTGCAGCTTGAGAAGAGCGTC 203 bp All gs454 000681Ferr H CAAACAGGTACTCGGCCATGCC
1205722
Globin Hb-A L AAATTGTTGGCCATGCGGAGGA 208 bp All gs454 001919Hb-A H CTGAGGTTCAGCAGACCTGCCT
1205732
Globin Hb-B L TCGTCTACCCCTGGTGTCAGAG 245 bp All gs454 001018Hb-B H AACCACAATGGTCAGGCAGTCC
Ependymin-1 precursor EPD-1 L CAGGTGTGAGGCAGTGCAGT 230 bp All gs454 000469EPD-1 H ACCCCGATCTCCTCCTGGTG
Fatty acid-binding protein brain BLBP L CAACACTTCTTGGCCGGTTTGG 239 bp All gs454 001220BLBP H GAGAGGAGTTCGACGAAGCCAC
CD63-like protein Sm-TSP-2 TSPAN-8 L TCGCTGGCTGCTCTGAGAAAGA 200 bp All gs454 000381TSPAN-8 H GGTCACGCCGAGCTGTATTCTG
Tropomyosin 4 isoform 1 TRPM-1 L GTGGAGGAGGAGTTGGACCGAG 221 bp All gs454 000222TRPM1 H TTGCGAGCCACCTCCTCGTATT
C-Myc-binding protein MYCBP L CGCCAGTTTACCTGCGTTCCAA 182 bp All gs454 001640MYCBP H GGCCGTCAACAACACCACCTTT
Cathepsin S CTSS L AACAGCCTACCCCTACACAGCC 200 bp All gs454 000156CTSS H TGTACACACCGTGGCGGTAGAA
Transferrin STF-1 L AGCTGCACCAGCTTCACAGTTG 215 bp All gs454 000004STF-1 H AAGGATGGCACCAGACAACCCA
Warm Temperature Acclimationrelated-like 65 kDa protein
HPX L TGATACCGGGTGGAACCTGGTG 207 bp All gs454 000060HPX H GCTGCTGTGGAGTGTCCCAAAG
Betaine HomocysteineS-methyltransferase
BHTM L GGGGGTTCGCTGTTACCAAGTG 194 bp All gs454 000088BHTM H TGTGAGACAGCAGCCTCAGGAG
FUCL1 Fucolectin-1 FUCL1 L CGCAAACCCTTTGGCTGGTGTA 196 bp All gs454 000758FUCL1 H GGCTTTTCCTTGGACTGCCAGG
Aldolase B ALDB L GCCATTGGTCTTGGCCCTGATC 220 bp All gs454 000115ALDB H CGCTGTGCCTGGTATCTGCTTC
Type-4 ice-structuring protein LS-12precursor
ISP LS12 L AAGACCTGACAAACCAGGCCCA 198 bp All gs454 001277ISP LS12 H GGAGGATGGCCTCCATCTGCTT
International Journal of Genomics 5
Table 1 Continued
Gene Primer name Primer Sequences (51015840-31015840) Size(bp) NCBI Accession
Alcohol Dehydrogenase 8a ADH L GGCAAGAAGGTGCTGCAGTTCA 228 bp All gs454 000105-6ADH H CATGACTGCAGCCAAACCCACA
Glyceraldehyde-3-phosphateDehydrogenase
GAPDH L GTCAACCACTGACACGTTGGGG 229 bp All gs454 000148GAPDH H CGGCATCATTGAGGGCCTGATG
Lactate Dehydrogenase-A LDH-A L TCTTAACCTGGTGCAGCGCAAC 219 bp All gs454 000149LDH-A H TGGAGCTTCTCGCCCATGATGT
Phosphoglycerate Mutase 2-1 (muscle) PglyM L ACACCTCTGTGCTGAAACGTGC 212 bp All gs454 000309PglyM H CATGGGTGGAGGTGGGATGTCA
Heat Shock Protein 70 HSP70 L CGGTGTTGTGTGCTGGGTGAAA 207 bp All gs454 000005HSP70 H CCACATAGCTGGGTGTGGTCCT
Fructose-bisphosphate Aldolase A FBPA L GGAACCAACGGCGAGACAACAA 208 bp All gs454 002732FBPA H CAATGGGGACGATGCCATGCAT
Phosphoglucose Isomerase-2 PGI L CCACACTGGGCCAATTGTCTGG 217 bp All gs454 000011PGI H GGCCTCCTCTGTGGTCTTACCC
Table 2 Global statistics for each of the nonnormalized libraries using Newbler software
Tissue Spleen Brain Heart Gonad Liver Muscle TotalTotal EST 15034 33337 73263 157275 134523 92788 544491Total bases 3426510 8219500 17647600 37123700 31792600 23342900 129412000Contigs 567 651 1260 3875 1274 626 8319Average contig length 470 619 567 465 612 689 555Contigs 119890-value lt 10minus6 220 420 622 951 617 409 2440ESTscan 584 345 977 2838 634 269 2715No similarity 36 70 109 406 211 74 1128GO annotation 202 338 473 623 509 307 1728InterPro annotation 223 417 610 908 649 395 2395
ldquocodemlrdquo in PAML 4 [34] to assess selective pressure onthose genes for which the complete sequences was availablePositive (or negative) selected sites were defined by the ratiobetween nonsynonymous versus synonymous substitutions(dNdS or 120596) Two models were tested comparatively M1which groups codons in two classes (120596 lt 1 and 120596 = 1)clustering sites under negative or neutral selection and M2which groups codons in three classes (120596 lt 1 120596 = 1 and 120596 gt1) adding a cluster for sites under positive selection to theones defined in M1 Probabilistic measures of how well thesemodels fits the evolutionary relationship of individual geneswere calculated from the likelihood values of fitted modelsand the number of ldquofree parametersrdquo for all genes
Protein stability was estimated using a virtual quantifica-tion software [35] calculating Gibbs free energy in terms ofkinetic and thermodynamic quantities taking into accounteach amino acid contribution for the maintenance of thenative structure of the protein For a protein to maintain itsstability there is a need of sufficient hydrophobic residueswhich will utilize free energy to guide proper folding [35]
26 EST-SSR Resources for Population Genetics Among vari-ous molecular markers simple sequence repeats (SSRs) arehighly polymorphic easier to develop and very useful for
researches such as genetic diversity assessment ThereforeA carbo database was further used to detect such regionsin the transcriptome sequencing data and provide a list ofcombination of primer sets on flanking regions that could beused for population genetic studies To identify EST-SSRs allthe contigs were searched using MISA [36] and for primerdesign we used Primer3 [37]The algorithm of the SSR finderidentifies a good quality repeat when one locus is present withadjacent loci at an up or downstream distance higher than100 bp and parameters were set to locate a minimum of 20 bpsequence repeats di-mers (x12) 3-mers (x8) 4-mers (x5) 5-mers (x5) and 6-mers (x5) Primer design was performedsetting parameters of a minimum size of 20 bp and meltingtemperatures of 60∘C
3 Results and Discussion
31 Sequences Assemblage and Functional Annotations Aftersequence trimming a total of 544491 high quality readswere produced with an average length of 237 bp correspond-ing to 1295Mb A total of 8319 contigs were assembledwith Newbler (ver 26) as high quality consensus sequenceswithout the presence of singletons A summary of ESTdata for each of the six tissues is reported in Table 2 Atotal of 2440 assembled contigs were annotated against
6 International Journal of Genomics
the NCBI nonredundant protein database at the cut-off (119890-value) lt 10minus6 Additional 3843 contigs with no proteinmatches were further processed with ESTScan to find 2715homologous proteins All the contigs were then processedby InterProScan for functional annotation of transcriptsand mapping functional information to gene ontology (GO)terms Of the 5155 amino-acid sequences (out of the 6283totally sequenced) 1728 could be annotated within theGO hierarchy (Figure 1) while 2395 could be annotatedaccordingly to InterProScan The complete GO mappingof A carbo for individual tissues can be accessed on thededicated online database (httptranscriptomicsbiocantptAphanopusCarbo) The largest proportions of GO func-tional categories are of similar proportion to the unnormal-ized libraries of Tilapia [38] In the Cellular Components GOgroup (Figure 1(a)) the genes involved in cell and cell partfunctional categories corresponded to the largest percentageof the pie chart (2815 each) In the Molecular FunctionGO group (Figure 1(b)) almost half of the total genes wereinvolved in binding (4740) followed by those related tocatalytic activity (2753) Finally for the Biological ProcessesGO group (Figure 1(c)) the largest portion of the genes wereinvolved in metabolic processes (3345) tightly followed bythe category of genes linked to the Cellular Process (3193)
To further validate the accuracy of the A carbo assemblywe compared our dataset to the transcriptomes of six otherprior sequenced teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis Similarity searches wereperformed comparing our assembled contigs against each ofthe available transcriptomes using tBLASTn [30] The resultshighlighted strong similarity between the transcriptomes ofA carbo and other teleosts indicating that about 30 of thetotal contigs matched protein homologs (119890-values lt 10minus3ranging between 2268 and 2457) and that our assemblypresents the highest similarity (by little margin) with thetranscriptome of Gasterosteus aculeatus (119890-value lt 10minus10 =2 223) (Table 3)
A total of 1639 (20) transcripts were annotated by Pfamprotein domainsmatches and the set of protein domains char-acterizing each single tissue was identified by hypergeometrictest (Table 4)
32 qPCR Assays and Validation Tests Quantitative PCRassays were performed using cDNA samples at 1 400 dilution(up to 100 ng approximately in the qPCR master mix)conditions at which the PCR resulted to be more efficientWe tested seven series of dilutions ranging from 1 50 to1 1000 Based on optimized qPCR conditions all targetedcDNAs were tested across all tissues for the complete set ofgenes selection used in this study Normalized expressionto a relative value of 10 for 28 genes is shown in Figure 2(see Figure S1 in Supplementary Material available online athttpdxdoiorg1011552014267482)
Statistical tests were performed to identify the correlationbetween the mean value of normalized expression and arelative value of 10 as determined by qPCR against contigfrequencies using Pearsonrsquos 119903 coefficient and its significance
Table 3 Similarity search for unigenes comparing transcriptomesof other teleosts against Aphanopus carbo using two levels ofstringency
Species Sequencesavailable
A carbo119890-value lt 10minus3
A carbo119890-value lt 10minus10
Danio rerio 42787 2457 2199Gasterosteusaculeatus 27576 2445 2223
Oreochromisniloticus 26763 2436 2202
Oryziaslatipes 24674 2412 2173
Takifugurubripes 47841 2268 2062
Tetraodonnigroviridis 23118 2300 2073
(Figure 3) Most of the genes showed highly significantcorrelation however one case indicated a reduced signifi-cance (elongation factor) and a few other cases resulted tobe not significant (eg Ras-related GTP-binding proteinBasic Transcription Factor 3 CuZn Superoxide Dismutaseand 2-Cys Peroxiredoxin) Reduced or lack of significanceappeared to be related to a low number of contigs from the454 sequencing (Table 5) which might be regarded as anindication of failure to reach library saturation Low valuesin contig frequency should correspond to a reduced levelof expression as the libraries are nonnormalized thereforecaution should be used when exploring 454 outputs belowa certain threshold Our data derived from the reading of asingle plate were insufficient for systematical comparison ofall the genes although the majority of the genes show a highcorrelation between the 454 data and the qPCR Howeverit should be emphasized that this exercise was not meant tobe a surrogate to the qPCR approach for the study of geneexpression but more a proxy for a preliminary screening ofdifferentially expressed genes in multiple libraries
33 Candidate Genes Associated to Depth The predictedamino acid sequences of A carbo functional genes puta-tively related to depth adaptations were compared to thosedeposited in NCBI database for homologies searches Com-plete alignments of orthologous protein sequences from theset of functional genes that have been reported as responsiveto depth adaptations included representatives of Teleostsbelonging to several families (Table 6) Exploring the levels ofsimilarities expressed as percentage of amino acid identity ornumber of amino acid that differ among sequences betweenA carbo and other fish species (Table 7) brings evidencesupporting the fact that deep-living aswell as polar species arenot the most similar to the black scabbard fish We attemptedto reconstruct the phylogenies for those species using aminoacid sequences of functional genes (LDH-A LDH-B MDHc-A Hb-A and Hb-B) and corresponding mtCOI nucleotidesequences (Figure 4) The resulting trees showed similartopologies suggesting that the signals embedded in thesefunctional genes reflect evolutionary divergence among taxarather than any enzyme adaptations relationships
International Journal of Genomics 7
Macromolecular complex 1541Organelle part 580Extracellular region 367
Organelle 1616Synapse part 031Synapse 031
Membrane-enclosed lumen 084Extracellular region part 120Cell 2815Cell part 2815
(a) Cellular components
Structural molecule activity 1054Catalytic activity 2753Enzyme regulator activity 294Electron carrier activity 152
Transporter activity 635Transcription factor activity 052Metallochaperone activity 016
Binding 4740Molecular transducer activity 058Antioxidant activity 094
(b) Molecular function
Cellular process 3193Death 010Signaling 127Cellular component biogenesis 215Cell wall organization or biogenesis 010
Metabolic process 3345Biological regulation 446
Viral reproduction 005
Biological adhesion 093
Cellular component organization 289
Signaling process 098Developmental process 024Immune system process 118Establishment of localization 847
Multicellular organismal process 049
(c) Biological processes
Figure 1 Functional categorization of unigenes with gene ontology (GO) term for theAphanopus carboEST collectionThese unigenes resultswere functionally classified from the six tissues pooled together as percentages under three main functional categories with respective GOSlim terms Data refer to assemblage derived by implementation of Newbler Roche 454 sequence analysis software
The lactate dehydrogenase A (LDH-A isotig01401)was encountered abundantly in the muscle tissue (Table 5Figure 2) and its comparison with other orthologoussequences differs for a number of amino acids between 16and 44 One indel at position 75 had to be added to the two
polar species to obtain a complete alignment (Table 7) Thepercentage of identity was higher with the strictly marinespecies ranging between 952 and 937 Unfortunatelyno sequences from neither deep-sea nor abyssal fisheswere available for comparison On the other hand lactate
8 International Journal of Genomics
Table 4 List of Pfam conserved domains that were tissue specifically expressed inAphanopus carbo transcriptome Frequencies and 119875-valuesof tissue specificity analysis are indicated
(a)
Gonads Heart LiverPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF00100 Zona pellucida 17 315 10minus13 PF00011 HSP20 3 403 10minus4 PF00084 Sushi 14 144 10minus10
PF00125 Histone 8 606 10minus5 PF13405 EF hand 4 5 677 10minus4 PF00089 Trypsin 20 759 10minus9
PF01400 Astacin 3 322 10minus3 PF00412 LIM 3 152 10minus3 PF00079 Serpin 12 563 10minus6
PF00069 Pkinase 3 001 PF05556 Calsarcin 3 360 10minus3 PF07678 A2M comp 4 135 10minus4
PF00653 BIR 2 002 PF01576 Myosin tail 1 3 360 10minus3 PF01042 Ribonuc L-PSP 3 126 10minus3
PF13424 TPR 12 2 002 PF00056 Ldh 1 N 3 360 10minus3 PF00045 Hemopexin 3 126 10minus3
PF13695 zf-3CxxC 2 002 PF05300 DUF737 2 550 10minus3 PF00059 Lectin C 6 169 10minus3
PF09360 zf-CDGSH 2 002 PF00992 Troponin 4 640 10minus3 PF00701 DHDPS 4 464 10minus3
PF01712 dNK 2 002 PF00595 PDZ 3 682 10minus3 PF00386 C1q 8 663 10minus3
PF00538 Linker histone 2 002 PF00022 Actin 3 001 PF00021 UPAR LY6 5 001PF10178 DUF2372 2 002 PF02874 ATP-synt ab N 2 002 PF00754 F5 F8 type C 9 001PF04856 Securin 2 002 PF13895 Ig 2 3 002 PF08702 Fib alpha 2 001PF00250 Fork head 2 002 PF00191 Annexin 2 003 PF03982 DAGAT 2 001PF01498 HTH Tnp Tc3 2 3 003 PF00212 ANP 2 003 PF01048 PNP UDP 1 2 001PF00268 Ribonuc red sm 2 006 PF05347 Complex1 LYR 2 007 PF01014 Uricase 2 001
(b)
Muscle Brain SpleenPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF01410 Troponin 7 199 10minus5 PF01669 Myelin MBP 4 403 10minus5 PF00042 Globin 5 133 10minus6
PF02807 COLFI 3 967 10minus4 PF01453 B lectin 2 644 10minus3 PF00078 RVT 1 5 133 10minus6
PF00041 ATP-guaPtransN 3 359 10minus3 PF00612 IQ 2 644 10minus3 PF00993 MHC II alpha 4 502 10minus6
PF02453 fn3 3 359 10minus3 PF11414 Suppressor APC 2 644 10minus3 PF07686 V set 5 249 10minus6
PF13895 Reticulon 3 359 10minus3 PF05768 DUF836 2 644 10minus3 PF07654 C1 set 5 471 10minus4
PF00365 Ig 2 4 797 10minus3 PF05196 PTNMK N 2 644 10minus3 PF00089 Trypsin 5 201 10minus3
PF01216 PFK 2 984 10minus3 PF04300 FBA 2 644 10minus3 PF01391 Collagen 2 230 10minus3
PF01267 Calsequestrin 2 984 10minus3 PF00287 Na K-ATPase 2 644 10minus3 PF00240 ubiquitin 3 328 10minus3
PF00261 F-actin cap A 2 984 10minus3 PF11032 ApoM 3 853 10minus3 PF09307 MHC2-interact 2 667 10minus3
PF00856 Tropomyosin 2 984 10minus3 PF00061 Lipocalin 4 001 PF00643 Zf-B box 2 001PF01661 SET 2 984 10minus3 PF00007 Cys knot 2 002 PF13445 Zf-RING LisH 2 001PF01667 Macro 2 003 PF01275 Myelin PLP 2 002 PF01498 HTH Tnp Tc3 2 2 002PF00036 Ribosomal S27e 2 005 PF00300 His Phos 1 2 002 PF02301 HORMA 1 005PF01576 efhand 2 005 PF00230 MIP 2 002 PF14259 RRM 6 1 005PF01410 Myosin tail 1 2 008 PF00091 Tubulin 3 002 PF09004 DUF1891 1 005
dehydrogenase B (LDH-B isotig01423) inA carbowas highlyrepresented in the heart while very scarcely recorded in theother tissues (Table S1) The largest sequence divergence interms of amino acid substitutions ranges between 47 and 55when compared with deep-water macrourids and similarlyto LDH-A an indel had to be added in the sequences ofthe gadiform species at position 76 to obtain a completealignment (Table 7) Previous studies on benthopelagic fishreport a significant decline of LDH activities with increasingwater depth [39] However it is known that variation inenzyme activities of fish at a given depth is influenced byfeeding behaviour and locomotory modes [40 41]
Cytosolic malate dehygrogenase A (MDHc-Aisotig01010) was detected in most of the tissues withthe exception of the muscle (Table S1) An isoform of MDHcwas also detected but its sequence covered only the last 110amino acids (isotig01731) However the type of substitutionsand tissue expression pattern (Table S1) according to Merritand Quattro [42] lead us to believe that this cytosolicisoform is MDHc-B It has been reported that the two teleostMDHc isozymes are the products of a gene duplicationevent after the separation of teleosts and tetrapods (see [42]and references therein) although the exact timing of thisduplication has not been inferred
International Journal of Genomics 9
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Elongation factor 1-beta
Basic transcription factor 3-like 4
B G H L M S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
2-Cys peroxiredoxin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ferritin heavy subunit
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
1205732-Globin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ependymin-1 precursor10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
1205722-Globin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 90
12
14
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CuZn superoxide dismutase
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
Ras-related GTP-binding protein A12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Figure 2 Continued
10 International Journal of Genomics
Fatty acid-binding protein brain10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B GH LM S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CD63-like protein Sm-TSP-2
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Tropomyosin 4 isoform 1
10
12
08
06
04
02
00Re
lativ
e fol
d ex
pres
sion
B G H L M S
C-Myc-binding protein
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Cathepsin S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Transferrin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
GH L M S
Warm temperature acclimation related-like proteinlowast
10
12
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Betaine homocysteine S-methyltansferase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
FUCL1 fucolectin-110
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Aldolase B
Figure 2 Continued
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
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4 International Journal of Genomics
Table 1 List of targeted genes using qPCR primer sets specifically designed for this study size for each of the product and NCBI accessionnumber for all the EST sequences
Gene Primer name Primer Sequences (51015840-31015840) Size(bp) NCBI Accession
Elongation factor 1-beta EF-1B L GCTTGGACATGTCGGTCTCGTC 229 bp All gs454 000396EF-1B H GTGGCTGACACCACATCTGGC
Ras-related GTP-binding protein A Rab-1A L AGTAGCCGTTCCACCTTGTCGG 247 bp All gs454 000598Rab-1A H TGCCAAGAAACCGTACGTGGGA
Basic Transcription Factor 3-like 4 BTF3 L CCCAAAGTTCAGGCCTCCCTGT 273 bp All gs454 000873BTF3 H TCATGTGCGTCAGTTCGCTTCG
CuZn Superoxide Dismutase SOD-1 L AAACGTGACTGCAGGAGGGGAT 240 bp All gs454 000925SOD-1 H CAGTGCTCCTGCTCCATGTTCG
2-Cys Peroxiredoxin PRDX1 L CCGATAACCTCGCAGCCGATAC 243 bp All gs454 000558PRDX1 H ACAGTCATTTGCCACCAGCATCA
Heat Shock Protein 90 HSP90 L TGACGATGTCCCCACAGATGAGG 221 bp All gs454 000008HSP90 H GCAACACTGGTCCACCACACAAC
Ferritin heavy subunit Ferr L CCTGCAGCTTGAGAAGAGCGTC 203 bp All gs454 000681Ferr H CAAACAGGTACTCGGCCATGCC
1205722
Globin Hb-A L AAATTGTTGGCCATGCGGAGGA 208 bp All gs454 001919Hb-A H CTGAGGTTCAGCAGACCTGCCT
1205732
Globin Hb-B L TCGTCTACCCCTGGTGTCAGAG 245 bp All gs454 001018Hb-B H AACCACAATGGTCAGGCAGTCC
Ependymin-1 precursor EPD-1 L CAGGTGTGAGGCAGTGCAGT 230 bp All gs454 000469EPD-1 H ACCCCGATCTCCTCCTGGTG
Fatty acid-binding protein brain BLBP L CAACACTTCTTGGCCGGTTTGG 239 bp All gs454 001220BLBP H GAGAGGAGTTCGACGAAGCCAC
CD63-like protein Sm-TSP-2 TSPAN-8 L TCGCTGGCTGCTCTGAGAAAGA 200 bp All gs454 000381TSPAN-8 H GGTCACGCCGAGCTGTATTCTG
Tropomyosin 4 isoform 1 TRPM-1 L GTGGAGGAGGAGTTGGACCGAG 221 bp All gs454 000222TRPM1 H TTGCGAGCCACCTCCTCGTATT
C-Myc-binding protein MYCBP L CGCCAGTTTACCTGCGTTCCAA 182 bp All gs454 001640MYCBP H GGCCGTCAACAACACCACCTTT
Cathepsin S CTSS L AACAGCCTACCCCTACACAGCC 200 bp All gs454 000156CTSS H TGTACACACCGTGGCGGTAGAA
Transferrin STF-1 L AGCTGCACCAGCTTCACAGTTG 215 bp All gs454 000004STF-1 H AAGGATGGCACCAGACAACCCA
Warm Temperature Acclimationrelated-like 65 kDa protein
HPX L TGATACCGGGTGGAACCTGGTG 207 bp All gs454 000060HPX H GCTGCTGTGGAGTGTCCCAAAG
Betaine HomocysteineS-methyltransferase
BHTM L GGGGGTTCGCTGTTACCAAGTG 194 bp All gs454 000088BHTM H TGTGAGACAGCAGCCTCAGGAG
FUCL1 Fucolectin-1 FUCL1 L CGCAAACCCTTTGGCTGGTGTA 196 bp All gs454 000758FUCL1 H GGCTTTTCCTTGGACTGCCAGG
Aldolase B ALDB L GCCATTGGTCTTGGCCCTGATC 220 bp All gs454 000115ALDB H CGCTGTGCCTGGTATCTGCTTC
Type-4 ice-structuring protein LS-12precursor
ISP LS12 L AAGACCTGACAAACCAGGCCCA 198 bp All gs454 001277ISP LS12 H GGAGGATGGCCTCCATCTGCTT
International Journal of Genomics 5
Table 1 Continued
Gene Primer name Primer Sequences (51015840-31015840) Size(bp) NCBI Accession
Alcohol Dehydrogenase 8a ADH L GGCAAGAAGGTGCTGCAGTTCA 228 bp All gs454 000105-6ADH H CATGACTGCAGCCAAACCCACA
Glyceraldehyde-3-phosphateDehydrogenase
GAPDH L GTCAACCACTGACACGTTGGGG 229 bp All gs454 000148GAPDH H CGGCATCATTGAGGGCCTGATG
Lactate Dehydrogenase-A LDH-A L TCTTAACCTGGTGCAGCGCAAC 219 bp All gs454 000149LDH-A H TGGAGCTTCTCGCCCATGATGT
Phosphoglycerate Mutase 2-1 (muscle) PglyM L ACACCTCTGTGCTGAAACGTGC 212 bp All gs454 000309PglyM H CATGGGTGGAGGTGGGATGTCA
Heat Shock Protein 70 HSP70 L CGGTGTTGTGTGCTGGGTGAAA 207 bp All gs454 000005HSP70 H CCACATAGCTGGGTGTGGTCCT
Fructose-bisphosphate Aldolase A FBPA L GGAACCAACGGCGAGACAACAA 208 bp All gs454 002732FBPA H CAATGGGGACGATGCCATGCAT
Phosphoglucose Isomerase-2 PGI L CCACACTGGGCCAATTGTCTGG 217 bp All gs454 000011PGI H GGCCTCCTCTGTGGTCTTACCC
Table 2 Global statistics for each of the nonnormalized libraries using Newbler software
Tissue Spleen Brain Heart Gonad Liver Muscle TotalTotal EST 15034 33337 73263 157275 134523 92788 544491Total bases 3426510 8219500 17647600 37123700 31792600 23342900 129412000Contigs 567 651 1260 3875 1274 626 8319Average contig length 470 619 567 465 612 689 555Contigs 119890-value lt 10minus6 220 420 622 951 617 409 2440ESTscan 584 345 977 2838 634 269 2715No similarity 36 70 109 406 211 74 1128GO annotation 202 338 473 623 509 307 1728InterPro annotation 223 417 610 908 649 395 2395
ldquocodemlrdquo in PAML 4 [34] to assess selective pressure onthose genes for which the complete sequences was availablePositive (or negative) selected sites were defined by the ratiobetween nonsynonymous versus synonymous substitutions(dNdS or 120596) Two models were tested comparatively M1which groups codons in two classes (120596 lt 1 and 120596 = 1)clustering sites under negative or neutral selection and M2which groups codons in three classes (120596 lt 1 120596 = 1 and 120596 gt1) adding a cluster for sites under positive selection to theones defined in M1 Probabilistic measures of how well thesemodels fits the evolutionary relationship of individual geneswere calculated from the likelihood values of fitted modelsand the number of ldquofree parametersrdquo for all genes
Protein stability was estimated using a virtual quantifica-tion software [35] calculating Gibbs free energy in terms ofkinetic and thermodynamic quantities taking into accounteach amino acid contribution for the maintenance of thenative structure of the protein For a protein to maintain itsstability there is a need of sufficient hydrophobic residueswhich will utilize free energy to guide proper folding [35]
26 EST-SSR Resources for Population Genetics Among vari-ous molecular markers simple sequence repeats (SSRs) arehighly polymorphic easier to develop and very useful for
researches such as genetic diversity assessment ThereforeA carbo database was further used to detect such regionsin the transcriptome sequencing data and provide a list ofcombination of primer sets on flanking regions that could beused for population genetic studies To identify EST-SSRs allthe contigs were searched using MISA [36] and for primerdesign we used Primer3 [37]The algorithm of the SSR finderidentifies a good quality repeat when one locus is present withadjacent loci at an up or downstream distance higher than100 bp and parameters were set to locate a minimum of 20 bpsequence repeats di-mers (x12) 3-mers (x8) 4-mers (x5) 5-mers (x5) and 6-mers (x5) Primer design was performedsetting parameters of a minimum size of 20 bp and meltingtemperatures of 60∘C
3 Results and Discussion
31 Sequences Assemblage and Functional Annotations Aftersequence trimming a total of 544491 high quality readswere produced with an average length of 237 bp correspond-ing to 1295Mb A total of 8319 contigs were assembledwith Newbler (ver 26) as high quality consensus sequenceswithout the presence of singletons A summary of ESTdata for each of the six tissues is reported in Table 2 Atotal of 2440 assembled contigs were annotated against
6 International Journal of Genomics
the NCBI nonredundant protein database at the cut-off (119890-value) lt 10minus6 Additional 3843 contigs with no proteinmatches were further processed with ESTScan to find 2715homologous proteins All the contigs were then processedby InterProScan for functional annotation of transcriptsand mapping functional information to gene ontology (GO)terms Of the 5155 amino-acid sequences (out of the 6283totally sequenced) 1728 could be annotated within theGO hierarchy (Figure 1) while 2395 could be annotatedaccordingly to InterProScan The complete GO mappingof A carbo for individual tissues can be accessed on thededicated online database (httptranscriptomicsbiocantptAphanopusCarbo) The largest proportions of GO func-tional categories are of similar proportion to the unnormal-ized libraries of Tilapia [38] In the Cellular Components GOgroup (Figure 1(a)) the genes involved in cell and cell partfunctional categories corresponded to the largest percentageof the pie chart (2815 each) In the Molecular FunctionGO group (Figure 1(b)) almost half of the total genes wereinvolved in binding (4740) followed by those related tocatalytic activity (2753) Finally for the Biological ProcessesGO group (Figure 1(c)) the largest portion of the genes wereinvolved in metabolic processes (3345) tightly followed bythe category of genes linked to the Cellular Process (3193)
To further validate the accuracy of the A carbo assemblywe compared our dataset to the transcriptomes of six otherprior sequenced teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis Similarity searches wereperformed comparing our assembled contigs against each ofthe available transcriptomes using tBLASTn [30] The resultshighlighted strong similarity between the transcriptomes ofA carbo and other teleosts indicating that about 30 of thetotal contigs matched protein homologs (119890-values lt 10minus3ranging between 2268 and 2457) and that our assemblypresents the highest similarity (by little margin) with thetranscriptome of Gasterosteus aculeatus (119890-value lt 10minus10 =2 223) (Table 3)
A total of 1639 (20) transcripts were annotated by Pfamprotein domainsmatches and the set of protein domains char-acterizing each single tissue was identified by hypergeometrictest (Table 4)
32 qPCR Assays and Validation Tests Quantitative PCRassays were performed using cDNA samples at 1 400 dilution(up to 100 ng approximately in the qPCR master mix)conditions at which the PCR resulted to be more efficientWe tested seven series of dilutions ranging from 1 50 to1 1000 Based on optimized qPCR conditions all targetedcDNAs were tested across all tissues for the complete set ofgenes selection used in this study Normalized expressionto a relative value of 10 for 28 genes is shown in Figure 2(see Figure S1 in Supplementary Material available online athttpdxdoiorg1011552014267482)
Statistical tests were performed to identify the correlationbetween the mean value of normalized expression and arelative value of 10 as determined by qPCR against contigfrequencies using Pearsonrsquos 119903 coefficient and its significance
Table 3 Similarity search for unigenes comparing transcriptomesof other teleosts against Aphanopus carbo using two levels ofstringency
Species Sequencesavailable
A carbo119890-value lt 10minus3
A carbo119890-value lt 10minus10
Danio rerio 42787 2457 2199Gasterosteusaculeatus 27576 2445 2223
Oreochromisniloticus 26763 2436 2202
Oryziaslatipes 24674 2412 2173
Takifugurubripes 47841 2268 2062
Tetraodonnigroviridis 23118 2300 2073
(Figure 3) Most of the genes showed highly significantcorrelation however one case indicated a reduced signifi-cance (elongation factor) and a few other cases resulted tobe not significant (eg Ras-related GTP-binding proteinBasic Transcription Factor 3 CuZn Superoxide Dismutaseand 2-Cys Peroxiredoxin) Reduced or lack of significanceappeared to be related to a low number of contigs from the454 sequencing (Table 5) which might be regarded as anindication of failure to reach library saturation Low valuesin contig frequency should correspond to a reduced levelof expression as the libraries are nonnormalized thereforecaution should be used when exploring 454 outputs belowa certain threshold Our data derived from the reading of asingle plate were insufficient for systematical comparison ofall the genes although the majority of the genes show a highcorrelation between the 454 data and the qPCR Howeverit should be emphasized that this exercise was not meant tobe a surrogate to the qPCR approach for the study of geneexpression but more a proxy for a preliminary screening ofdifferentially expressed genes in multiple libraries
33 Candidate Genes Associated to Depth The predictedamino acid sequences of A carbo functional genes puta-tively related to depth adaptations were compared to thosedeposited in NCBI database for homologies searches Com-plete alignments of orthologous protein sequences from theset of functional genes that have been reported as responsiveto depth adaptations included representatives of Teleostsbelonging to several families (Table 6) Exploring the levels ofsimilarities expressed as percentage of amino acid identity ornumber of amino acid that differ among sequences betweenA carbo and other fish species (Table 7) brings evidencesupporting the fact that deep-living aswell as polar species arenot the most similar to the black scabbard fish We attemptedto reconstruct the phylogenies for those species using aminoacid sequences of functional genes (LDH-A LDH-B MDHc-A Hb-A and Hb-B) and corresponding mtCOI nucleotidesequences (Figure 4) The resulting trees showed similartopologies suggesting that the signals embedded in thesefunctional genes reflect evolutionary divergence among taxarather than any enzyme adaptations relationships
International Journal of Genomics 7
Macromolecular complex 1541Organelle part 580Extracellular region 367
Organelle 1616Synapse part 031Synapse 031
Membrane-enclosed lumen 084Extracellular region part 120Cell 2815Cell part 2815
(a) Cellular components
Structural molecule activity 1054Catalytic activity 2753Enzyme regulator activity 294Electron carrier activity 152
Transporter activity 635Transcription factor activity 052Metallochaperone activity 016
Binding 4740Molecular transducer activity 058Antioxidant activity 094
(b) Molecular function
Cellular process 3193Death 010Signaling 127Cellular component biogenesis 215Cell wall organization or biogenesis 010
Metabolic process 3345Biological regulation 446
Viral reproduction 005
Biological adhesion 093
Cellular component organization 289
Signaling process 098Developmental process 024Immune system process 118Establishment of localization 847
Multicellular organismal process 049
(c) Biological processes
Figure 1 Functional categorization of unigenes with gene ontology (GO) term for theAphanopus carboEST collectionThese unigenes resultswere functionally classified from the six tissues pooled together as percentages under three main functional categories with respective GOSlim terms Data refer to assemblage derived by implementation of Newbler Roche 454 sequence analysis software
The lactate dehydrogenase A (LDH-A isotig01401)was encountered abundantly in the muscle tissue (Table 5Figure 2) and its comparison with other orthologoussequences differs for a number of amino acids between 16and 44 One indel at position 75 had to be added to the two
polar species to obtain a complete alignment (Table 7) Thepercentage of identity was higher with the strictly marinespecies ranging between 952 and 937 Unfortunatelyno sequences from neither deep-sea nor abyssal fisheswere available for comparison On the other hand lactate
8 International Journal of Genomics
Table 4 List of Pfam conserved domains that were tissue specifically expressed inAphanopus carbo transcriptome Frequencies and 119875-valuesof tissue specificity analysis are indicated
(a)
Gonads Heart LiverPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF00100 Zona pellucida 17 315 10minus13 PF00011 HSP20 3 403 10minus4 PF00084 Sushi 14 144 10minus10
PF00125 Histone 8 606 10minus5 PF13405 EF hand 4 5 677 10minus4 PF00089 Trypsin 20 759 10minus9
PF01400 Astacin 3 322 10minus3 PF00412 LIM 3 152 10minus3 PF00079 Serpin 12 563 10minus6
PF00069 Pkinase 3 001 PF05556 Calsarcin 3 360 10minus3 PF07678 A2M comp 4 135 10minus4
PF00653 BIR 2 002 PF01576 Myosin tail 1 3 360 10minus3 PF01042 Ribonuc L-PSP 3 126 10minus3
PF13424 TPR 12 2 002 PF00056 Ldh 1 N 3 360 10minus3 PF00045 Hemopexin 3 126 10minus3
PF13695 zf-3CxxC 2 002 PF05300 DUF737 2 550 10minus3 PF00059 Lectin C 6 169 10minus3
PF09360 zf-CDGSH 2 002 PF00992 Troponin 4 640 10minus3 PF00701 DHDPS 4 464 10minus3
PF01712 dNK 2 002 PF00595 PDZ 3 682 10minus3 PF00386 C1q 8 663 10minus3
PF00538 Linker histone 2 002 PF00022 Actin 3 001 PF00021 UPAR LY6 5 001PF10178 DUF2372 2 002 PF02874 ATP-synt ab N 2 002 PF00754 F5 F8 type C 9 001PF04856 Securin 2 002 PF13895 Ig 2 3 002 PF08702 Fib alpha 2 001PF00250 Fork head 2 002 PF00191 Annexin 2 003 PF03982 DAGAT 2 001PF01498 HTH Tnp Tc3 2 3 003 PF00212 ANP 2 003 PF01048 PNP UDP 1 2 001PF00268 Ribonuc red sm 2 006 PF05347 Complex1 LYR 2 007 PF01014 Uricase 2 001
(b)
Muscle Brain SpleenPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF01410 Troponin 7 199 10minus5 PF01669 Myelin MBP 4 403 10minus5 PF00042 Globin 5 133 10minus6
PF02807 COLFI 3 967 10minus4 PF01453 B lectin 2 644 10minus3 PF00078 RVT 1 5 133 10minus6
PF00041 ATP-guaPtransN 3 359 10minus3 PF00612 IQ 2 644 10minus3 PF00993 MHC II alpha 4 502 10minus6
PF02453 fn3 3 359 10minus3 PF11414 Suppressor APC 2 644 10minus3 PF07686 V set 5 249 10minus6
PF13895 Reticulon 3 359 10minus3 PF05768 DUF836 2 644 10minus3 PF07654 C1 set 5 471 10minus4
PF00365 Ig 2 4 797 10minus3 PF05196 PTNMK N 2 644 10minus3 PF00089 Trypsin 5 201 10minus3
PF01216 PFK 2 984 10minus3 PF04300 FBA 2 644 10minus3 PF01391 Collagen 2 230 10minus3
PF01267 Calsequestrin 2 984 10minus3 PF00287 Na K-ATPase 2 644 10minus3 PF00240 ubiquitin 3 328 10minus3
PF00261 F-actin cap A 2 984 10minus3 PF11032 ApoM 3 853 10minus3 PF09307 MHC2-interact 2 667 10minus3
PF00856 Tropomyosin 2 984 10minus3 PF00061 Lipocalin 4 001 PF00643 Zf-B box 2 001PF01661 SET 2 984 10minus3 PF00007 Cys knot 2 002 PF13445 Zf-RING LisH 2 001PF01667 Macro 2 003 PF01275 Myelin PLP 2 002 PF01498 HTH Tnp Tc3 2 2 002PF00036 Ribosomal S27e 2 005 PF00300 His Phos 1 2 002 PF02301 HORMA 1 005PF01576 efhand 2 005 PF00230 MIP 2 002 PF14259 RRM 6 1 005PF01410 Myosin tail 1 2 008 PF00091 Tubulin 3 002 PF09004 DUF1891 1 005
dehydrogenase B (LDH-B isotig01423) inA carbowas highlyrepresented in the heart while very scarcely recorded in theother tissues (Table S1) The largest sequence divergence interms of amino acid substitutions ranges between 47 and 55when compared with deep-water macrourids and similarlyto LDH-A an indel had to be added in the sequences ofthe gadiform species at position 76 to obtain a completealignment (Table 7) Previous studies on benthopelagic fishreport a significant decline of LDH activities with increasingwater depth [39] However it is known that variation inenzyme activities of fish at a given depth is influenced byfeeding behaviour and locomotory modes [40 41]
Cytosolic malate dehygrogenase A (MDHc-Aisotig01010) was detected in most of the tissues withthe exception of the muscle (Table S1) An isoform of MDHcwas also detected but its sequence covered only the last 110amino acids (isotig01731) However the type of substitutionsand tissue expression pattern (Table S1) according to Merritand Quattro [42] lead us to believe that this cytosolicisoform is MDHc-B It has been reported that the two teleostMDHc isozymes are the products of a gene duplicationevent after the separation of teleosts and tetrapods (see [42]and references therein) although the exact timing of thisduplication has not been inferred
International Journal of Genomics 9
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Elongation factor 1-beta
Basic transcription factor 3-like 4
B G H L M S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
2-Cys peroxiredoxin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ferritin heavy subunit
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
1205732-Globin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ependymin-1 precursor10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
1205722-Globin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 90
12
14
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CuZn superoxide dismutase
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
Ras-related GTP-binding protein A12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Figure 2 Continued
10 International Journal of Genomics
Fatty acid-binding protein brain10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B GH LM S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CD63-like protein Sm-TSP-2
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Tropomyosin 4 isoform 1
10
12
08
06
04
02
00Re
lativ
e fol
d ex
pres
sion
B G H L M S
C-Myc-binding protein
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Cathepsin S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Transferrin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
GH L M S
Warm temperature acclimation related-like proteinlowast
10
12
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Betaine homocysteine S-methyltansferase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
FUCL1 fucolectin-110
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Aldolase B
Figure 2 Continued
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Genomics 5
Table 1 Continued
Gene Primer name Primer Sequences (51015840-31015840) Size(bp) NCBI Accession
Alcohol Dehydrogenase 8a ADH L GGCAAGAAGGTGCTGCAGTTCA 228 bp All gs454 000105-6ADH H CATGACTGCAGCCAAACCCACA
Glyceraldehyde-3-phosphateDehydrogenase
GAPDH L GTCAACCACTGACACGTTGGGG 229 bp All gs454 000148GAPDH H CGGCATCATTGAGGGCCTGATG
Lactate Dehydrogenase-A LDH-A L TCTTAACCTGGTGCAGCGCAAC 219 bp All gs454 000149LDH-A H TGGAGCTTCTCGCCCATGATGT
Phosphoglycerate Mutase 2-1 (muscle) PglyM L ACACCTCTGTGCTGAAACGTGC 212 bp All gs454 000309PglyM H CATGGGTGGAGGTGGGATGTCA
Heat Shock Protein 70 HSP70 L CGGTGTTGTGTGCTGGGTGAAA 207 bp All gs454 000005HSP70 H CCACATAGCTGGGTGTGGTCCT
Fructose-bisphosphate Aldolase A FBPA L GGAACCAACGGCGAGACAACAA 208 bp All gs454 002732FBPA H CAATGGGGACGATGCCATGCAT
Phosphoglucose Isomerase-2 PGI L CCACACTGGGCCAATTGTCTGG 217 bp All gs454 000011PGI H GGCCTCCTCTGTGGTCTTACCC
Table 2 Global statistics for each of the nonnormalized libraries using Newbler software
Tissue Spleen Brain Heart Gonad Liver Muscle TotalTotal EST 15034 33337 73263 157275 134523 92788 544491Total bases 3426510 8219500 17647600 37123700 31792600 23342900 129412000Contigs 567 651 1260 3875 1274 626 8319Average contig length 470 619 567 465 612 689 555Contigs 119890-value lt 10minus6 220 420 622 951 617 409 2440ESTscan 584 345 977 2838 634 269 2715No similarity 36 70 109 406 211 74 1128GO annotation 202 338 473 623 509 307 1728InterPro annotation 223 417 610 908 649 395 2395
ldquocodemlrdquo in PAML 4 [34] to assess selective pressure onthose genes for which the complete sequences was availablePositive (or negative) selected sites were defined by the ratiobetween nonsynonymous versus synonymous substitutions(dNdS or 120596) Two models were tested comparatively M1which groups codons in two classes (120596 lt 1 and 120596 = 1)clustering sites under negative or neutral selection and M2which groups codons in three classes (120596 lt 1 120596 = 1 and 120596 gt1) adding a cluster for sites under positive selection to theones defined in M1 Probabilistic measures of how well thesemodels fits the evolutionary relationship of individual geneswere calculated from the likelihood values of fitted modelsand the number of ldquofree parametersrdquo for all genes
Protein stability was estimated using a virtual quantifica-tion software [35] calculating Gibbs free energy in terms ofkinetic and thermodynamic quantities taking into accounteach amino acid contribution for the maintenance of thenative structure of the protein For a protein to maintain itsstability there is a need of sufficient hydrophobic residueswhich will utilize free energy to guide proper folding [35]
26 EST-SSR Resources for Population Genetics Among vari-ous molecular markers simple sequence repeats (SSRs) arehighly polymorphic easier to develop and very useful for
researches such as genetic diversity assessment ThereforeA carbo database was further used to detect such regionsin the transcriptome sequencing data and provide a list ofcombination of primer sets on flanking regions that could beused for population genetic studies To identify EST-SSRs allthe contigs were searched using MISA [36] and for primerdesign we used Primer3 [37]The algorithm of the SSR finderidentifies a good quality repeat when one locus is present withadjacent loci at an up or downstream distance higher than100 bp and parameters were set to locate a minimum of 20 bpsequence repeats di-mers (x12) 3-mers (x8) 4-mers (x5) 5-mers (x5) and 6-mers (x5) Primer design was performedsetting parameters of a minimum size of 20 bp and meltingtemperatures of 60∘C
3 Results and Discussion
31 Sequences Assemblage and Functional Annotations Aftersequence trimming a total of 544491 high quality readswere produced with an average length of 237 bp correspond-ing to 1295Mb A total of 8319 contigs were assembledwith Newbler (ver 26) as high quality consensus sequenceswithout the presence of singletons A summary of ESTdata for each of the six tissues is reported in Table 2 Atotal of 2440 assembled contigs were annotated against
6 International Journal of Genomics
the NCBI nonredundant protein database at the cut-off (119890-value) lt 10minus6 Additional 3843 contigs with no proteinmatches were further processed with ESTScan to find 2715homologous proteins All the contigs were then processedby InterProScan for functional annotation of transcriptsand mapping functional information to gene ontology (GO)terms Of the 5155 amino-acid sequences (out of the 6283totally sequenced) 1728 could be annotated within theGO hierarchy (Figure 1) while 2395 could be annotatedaccordingly to InterProScan The complete GO mappingof A carbo for individual tissues can be accessed on thededicated online database (httptranscriptomicsbiocantptAphanopusCarbo) The largest proportions of GO func-tional categories are of similar proportion to the unnormal-ized libraries of Tilapia [38] In the Cellular Components GOgroup (Figure 1(a)) the genes involved in cell and cell partfunctional categories corresponded to the largest percentageof the pie chart (2815 each) In the Molecular FunctionGO group (Figure 1(b)) almost half of the total genes wereinvolved in binding (4740) followed by those related tocatalytic activity (2753) Finally for the Biological ProcessesGO group (Figure 1(c)) the largest portion of the genes wereinvolved in metabolic processes (3345) tightly followed bythe category of genes linked to the Cellular Process (3193)
To further validate the accuracy of the A carbo assemblywe compared our dataset to the transcriptomes of six otherprior sequenced teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis Similarity searches wereperformed comparing our assembled contigs against each ofthe available transcriptomes using tBLASTn [30] The resultshighlighted strong similarity between the transcriptomes ofA carbo and other teleosts indicating that about 30 of thetotal contigs matched protein homologs (119890-values lt 10minus3ranging between 2268 and 2457) and that our assemblypresents the highest similarity (by little margin) with thetranscriptome of Gasterosteus aculeatus (119890-value lt 10minus10 =2 223) (Table 3)
A total of 1639 (20) transcripts were annotated by Pfamprotein domainsmatches and the set of protein domains char-acterizing each single tissue was identified by hypergeometrictest (Table 4)
32 qPCR Assays and Validation Tests Quantitative PCRassays were performed using cDNA samples at 1 400 dilution(up to 100 ng approximately in the qPCR master mix)conditions at which the PCR resulted to be more efficientWe tested seven series of dilutions ranging from 1 50 to1 1000 Based on optimized qPCR conditions all targetedcDNAs were tested across all tissues for the complete set ofgenes selection used in this study Normalized expressionto a relative value of 10 for 28 genes is shown in Figure 2(see Figure S1 in Supplementary Material available online athttpdxdoiorg1011552014267482)
Statistical tests were performed to identify the correlationbetween the mean value of normalized expression and arelative value of 10 as determined by qPCR against contigfrequencies using Pearsonrsquos 119903 coefficient and its significance
Table 3 Similarity search for unigenes comparing transcriptomesof other teleosts against Aphanopus carbo using two levels ofstringency
Species Sequencesavailable
A carbo119890-value lt 10minus3
A carbo119890-value lt 10minus10
Danio rerio 42787 2457 2199Gasterosteusaculeatus 27576 2445 2223
Oreochromisniloticus 26763 2436 2202
Oryziaslatipes 24674 2412 2173
Takifugurubripes 47841 2268 2062
Tetraodonnigroviridis 23118 2300 2073
(Figure 3) Most of the genes showed highly significantcorrelation however one case indicated a reduced signifi-cance (elongation factor) and a few other cases resulted tobe not significant (eg Ras-related GTP-binding proteinBasic Transcription Factor 3 CuZn Superoxide Dismutaseand 2-Cys Peroxiredoxin) Reduced or lack of significanceappeared to be related to a low number of contigs from the454 sequencing (Table 5) which might be regarded as anindication of failure to reach library saturation Low valuesin contig frequency should correspond to a reduced levelof expression as the libraries are nonnormalized thereforecaution should be used when exploring 454 outputs belowa certain threshold Our data derived from the reading of asingle plate were insufficient for systematical comparison ofall the genes although the majority of the genes show a highcorrelation between the 454 data and the qPCR Howeverit should be emphasized that this exercise was not meant tobe a surrogate to the qPCR approach for the study of geneexpression but more a proxy for a preliminary screening ofdifferentially expressed genes in multiple libraries
33 Candidate Genes Associated to Depth The predictedamino acid sequences of A carbo functional genes puta-tively related to depth adaptations were compared to thosedeposited in NCBI database for homologies searches Com-plete alignments of orthologous protein sequences from theset of functional genes that have been reported as responsiveto depth adaptations included representatives of Teleostsbelonging to several families (Table 6) Exploring the levels ofsimilarities expressed as percentage of amino acid identity ornumber of amino acid that differ among sequences betweenA carbo and other fish species (Table 7) brings evidencesupporting the fact that deep-living aswell as polar species arenot the most similar to the black scabbard fish We attemptedto reconstruct the phylogenies for those species using aminoacid sequences of functional genes (LDH-A LDH-B MDHc-A Hb-A and Hb-B) and corresponding mtCOI nucleotidesequences (Figure 4) The resulting trees showed similartopologies suggesting that the signals embedded in thesefunctional genes reflect evolutionary divergence among taxarather than any enzyme adaptations relationships
International Journal of Genomics 7
Macromolecular complex 1541Organelle part 580Extracellular region 367
Organelle 1616Synapse part 031Synapse 031
Membrane-enclosed lumen 084Extracellular region part 120Cell 2815Cell part 2815
(a) Cellular components
Structural molecule activity 1054Catalytic activity 2753Enzyme regulator activity 294Electron carrier activity 152
Transporter activity 635Transcription factor activity 052Metallochaperone activity 016
Binding 4740Molecular transducer activity 058Antioxidant activity 094
(b) Molecular function
Cellular process 3193Death 010Signaling 127Cellular component biogenesis 215Cell wall organization or biogenesis 010
Metabolic process 3345Biological regulation 446
Viral reproduction 005
Biological adhesion 093
Cellular component organization 289
Signaling process 098Developmental process 024Immune system process 118Establishment of localization 847
Multicellular organismal process 049
(c) Biological processes
Figure 1 Functional categorization of unigenes with gene ontology (GO) term for theAphanopus carboEST collectionThese unigenes resultswere functionally classified from the six tissues pooled together as percentages under three main functional categories with respective GOSlim terms Data refer to assemblage derived by implementation of Newbler Roche 454 sequence analysis software
The lactate dehydrogenase A (LDH-A isotig01401)was encountered abundantly in the muscle tissue (Table 5Figure 2) and its comparison with other orthologoussequences differs for a number of amino acids between 16and 44 One indel at position 75 had to be added to the two
polar species to obtain a complete alignment (Table 7) Thepercentage of identity was higher with the strictly marinespecies ranging between 952 and 937 Unfortunatelyno sequences from neither deep-sea nor abyssal fisheswere available for comparison On the other hand lactate
8 International Journal of Genomics
Table 4 List of Pfam conserved domains that were tissue specifically expressed inAphanopus carbo transcriptome Frequencies and 119875-valuesof tissue specificity analysis are indicated
(a)
Gonads Heart LiverPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF00100 Zona pellucida 17 315 10minus13 PF00011 HSP20 3 403 10minus4 PF00084 Sushi 14 144 10minus10
PF00125 Histone 8 606 10minus5 PF13405 EF hand 4 5 677 10minus4 PF00089 Trypsin 20 759 10minus9
PF01400 Astacin 3 322 10minus3 PF00412 LIM 3 152 10minus3 PF00079 Serpin 12 563 10minus6
PF00069 Pkinase 3 001 PF05556 Calsarcin 3 360 10minus3 PF07678 A2M comp 4 135 10minus4
PF00653 BIR 2 002 PF01576 Myosin tail 1 3 360 10minus3 PF01042 Ribonuc L-PSP 3 126 10minus3
PF13424 TPR 12 2 002 PF00056 Ldh 1 N 3 360 10minus3 PF00045 Hemopexin 3 126 10minus3
PF13695 zf-3CxxC 2 002 PF05300 DUF737 2 550 10minus3 PF00059 Lectin C 6 169 10minus3
PF09360 zf-CDGSH 2 002 PF00992 Troponin 4 640 10minus3 PF00701 DHDPS 4 464 10minus3
PF01712 dNK 2 002 PF00595 PDZ 3 682 10minus3 PF00386 C1q 8 663 10minus3
PF00538 Linker histone 2 002 PF00022 Actin 3 001 PF00021 UPAR LY6 5 001PF10178 DUF2372 2 002 PF02874 ATP-synt ab N 2 002 PF00754 F5 F8 type C 9 001PF04856 Securin 2 002 PF13895 Ig 2 3 002 PF08702 Fib alpha 2 001PF00250 Fork head 2 002 PF00191 Annexin 2 003 PF03982 DAGAT 2 001PF01498 HTH Tnp Tc3 2 3 003 PF00212 ANP 2 003 PF01048 PNP UDP 1 2 001PF00268 Ribonuc red sm 2 006 PF05347 Complex1 LYR 2 007 PF01014 Uricase 2 001
(b)
Muscle Brain SpleenPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF01410 Troponin 7 199 10minus5 PF01669 Myelin MBP 4 403 10minus5 PF00042 Globin 5 133 10minus6
PF02807 COLFI 3 967 10minus4 PF01453 B lectin 2 644 10minus3 PF00078 RVT 1 5 133 10minus6
PF00041 ATP-guaPtransN 3 359 10minus3 PF00612 IQ 2 644 10minus3 PF00993 MHC II alpha 4 502 10minus6
PF02453 fn3 3 359 10minus3 PF11414 Suppressor APC 2 644 10minus3 PF07686 V set 5 249 10minus6
PF13895 Reticulon 3 359 10minus3 PF05768 DUF836 2 644 10minus3 PF07654 C1 set 5 471 10minus4
PF00365 Ig 2 4 797 10minus3 PF05196 PTNMK N 2 644 10minus3 PF00089 Trypsin 5 201 10minus3
PF01216 PFK 2 984 10minus3 PF04300 FBA 2 644 10minus3 PF01391 Collagen 2 230 10minus3
PF01267 Calsequestrin 2 984 10minus3 PF00287 Na K-ATPase 2 644 10minus3 PF00240 ubiquitin 3 328 10minus3
PF00261 F-actin cap A 2 984 10minus3 PF11032 ApoM 3 853 10minus3 PF09307 MHC2-interact 2 667 10minus3
PF00856 Tropomyosin 2 984 10minus3 PF00061 Lipocalin 4 001 PF00643 Zf-B box 2 001PF01661 SET 2 984 10minus3 PF00007 Cys knot 2 002 PF13445 Zf-RING LisH 2 001PF01667 Macro 2 003 PF01275 Myelin PLP 2 002 PF01498 HTH Tnp Tc3 2 2 002PF00036 Ribosomal S27e 2 005 PF00300 His Phos 1 2 002 PF02301 HORMA 1 005PF01576 efhand 2 005 PF00230 MIP 2 002 PF14259 RRM 6 1 005PF01410 Myosin tail 1 2 008 PF00091 Tubulin 3 002 PF09004 DUF1891 1 005
dehydrogenase B (LDH-B isotig01423) inA carbowas highlyrepresented in the heart while very scarcely recorded in theother tissues (Table S1) The largest sequence divergence interms of amino acid substitutions ranges between 47 and 55when compared with deep-water macrourids and similarlyto LDH-A an indel had to be added in the sequences ofthe gadiform species at position 76 to obtain a completealignment (Table 7) Previous studies on benthopelagic fishreport a significant decline of LDH activities with increasingwater depth [39] However it is known that variation inenzyme activities of fish at a given depth is influenced byfeeding behaviour and locomotory modes [40 41]
Cytosolic malate dehygrogenase A (MDHc-Aisotig01010) was detected in most of the tissues withthe exception of the muscle (Table S1) An isoform of MDHcwas also detected but its sequence covered only the last 110amino acids (isotig01731) However the type of substitutionsand tissue expression pattern (Table S1) according to Merritand Quattro [42] lead us to believe that this cytosolicisoform is MDHc-B It has been reported that the two teleostMDHc isozymes are the products of a gene duplicationevent after the separation of teleosts and tetrapods (see [42]and references therein) although the exact timing of thisduplication has not been inferred
International Journal of Genomics 9
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Elongation factor 1-beta
Basic transcription factor 3-like 4
B G H L M S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
2-Cys peroxiredoxin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ferritin heavy subunit
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
1205732-Globin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ependymin-1 precursor10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
1205722-Globin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 90
12
14
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CuZn superoxide dismutase
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
Ras-related GTP-binding protein A12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Figure 2 Continued
10 International Journal of Genomics
Fatty acid-binding protein brain10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B GH LM S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CD63-like protein Sm-TSP-2
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Tropomyosin 4 isoform 1
10
12
08
06
04
02
00Re
lativ
e fol
d ex
pres
sion
B G H L M S
C-Myc-binding protein
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Cathepsin S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Transferrin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
GH L M S
Warm temperature acclimation related-like proteinlowast
10
12
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Betaine homocysteine S-methyltansferase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
FUCL1 fucolectin-110
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Aldolase B
Figure 2 Continued
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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PeptidesInternational Journal of
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Nucleic AcidsJournal of
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Stem CellsInternational
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 International Journal of Genomics
the NCBI nonredundant protein database at the cut-off (119890-value) lt 10minus6 Additional 3843 contigs with no proteinmatches were further processed with ESTScan to find 2715homologous proteins All the contigs were then processedby InterProScan for functional annotation of transcriptsand mapping functional information to gene ontology (GO)terms Of the 5155 amino-acid sequences (out of the 6283totally sequenced) 1728 could be annotated within theGO hierarchy (Figure 1) while 2395 could be annotatedaccordingly to InterProScan The complete GO mappingof A carbo for individual tissues can be accessed on thededicated online database (httptranscriptomicsbiocantptAphanopusCarbo) The largest proportions of GO func-tional categories are of similar proportion to the unnormal-ized libraries of Tilapia [38] In the Cellular Components GOgroup (Figure 1(a)) the genes involved in cell and cell partfunctional categories corresponded to the largest percentageof the pie chart (2815 each) In the Molecular FunctionGO group (Figure 1(b)) almost half of the total genes wereinvolved in binding (4740) followed by those related tocatalytic activity (2753) Finally for the Biological ProcessesGO group (Figure 1(c)) the largest portion of the genes wereinvolved in metabolic processes (3345) tightly followed bythe category of genes linked to the Cellular Process (3193)
To further validate the accuracy of the A carbo assemblywe compared our dataset to the transcriptomes of six otherprior sequenced teleosts including Danio rerio Gasterosteusaculeatus Oreochromis niloticus Oryzias latipes Takifugurubripes and Tetraodon nigroviridis Similarity searches wereperformed comparing our assembled contigs against each ofthe available transcriptomes using tBLASTn [30] The resultshighlighted strong similarity between the transcriptomes ofA carbo and other teleosts indicating that about 30 of thetotal contigs matched protein homologs (119890-values lt 10minus3ranging between 2268 and 2457) and that our assemblypresents the highest similarity (by little margin) with thetranscriptome of Gasterosteus aculeatus (119890-value lt 10minus10 =2 223) (Table 3)
A total of 1639 (20) transcripts were annotated by Pfamprotein domainsmatches and the set of protein domains char-acterizing each single tissue was identified by hypergeometrictest (Table 4)
32 qPCR Assays and Validation Tests Quantitative PCRassays were performed using cDNA samples at 1 400 dilution(up to 100 ng approximately in the qPCR master mix)conditions at which the PCR resulted to be more efficientWe tested seven series of dilutions ranging from 1 50 to1 1000 Based on optimized qPCR conditions all targetedcDNAs were tested across all tissues for the complete set ofgenes selection used in this study Normalized expressionto a relative value of 10 for 28 genes is shown in Figure 2(see Figure S1 in Supplementary Material available online athttpdxdoiorg1011552014267482)
Statistical tests were performed to identify the correlationbetween the mean value of normalized expression and arelative value of 10 as determined by qPCR against contigfrequencies using Pearsonrsquos 119903 coefficient and its significance
Table 3 Similarity search for unigenes comparing transcriptomesof other teleosts against Aphanopus carbo using two levels ofstringency
Species Sequencesavailable
A carbo119890-value lt 10minus3
A carbo119890-value lt 10minus10
Danio rerio 42787 2457 2199Gasterosteusaculeatus 27576 2445 2223
Oreochromisniloticus 26763 2436 2202
Oryziaslatipes 24674 2412 2173
Takifugurubripes 47841 2268 2062
Tetraodonnigroviridis 23118 2300 2073
(Figure 3) Most of the genes showed highly significantcorrelation however one case indicated a reduced signifi-cance (elongation factor) and a few other cases resulted tobe not significant (eg Ras-related GTP-binding proteinBasic Transcription Factor 3 CuZn Superoxide Dismutaseand 2-Cys Peroxiredoxin) Reduced or lack of significanceappeared to be related to a low number of contigs from the454 sequencing (Table 5) which might be regarded as anindication of failure to reach library saturation Low valuesin contig frequency should correspond to a reduced levelof expression as the libraries are nonnormalized thereforecaution should be used when exploring 454 outputs belowa certain threshold Our data derived from the reading of asingle plate were insufficient for systematical comparison ofall the genes although the majority of the genes show a highcorrelation between the 454 data and the qPCR Howeverit should be emphasized that this exercise was not meant tobe a surrogate to the qPCR approach for the study of geneexpression but more a proxy for a preliminary screening ofdifferentially expressed genes in multiple libraries
33 Candidate Genes Associated to Depth The predictedamino acid sequences of A carbo functional genes puta-tively related to depth adaptations were compared to thosedeposited in NCBI database for homologies searches Com-plete alignments of orthologous protein sequences from theset of functional genes that have been reported as responsiveto depth adaptations included representatives of Teleostsbelonging to several families (Table 6) Exploring the levels ofsimilarities expressed as percentage of amino acid identity ornumber of amino acid that differ among sequences betweenA carbo and other fish species (Table 7) brings evidencesupporting the fact that deep-living aswell as polar species arenot the most similar to the black scabbard fish We attemptedto reconstruct the phylogenies for those species using aminoacid sequences of functional genes (LDH-A LDH-B MDHc-A Hb-A and Hb-B) and corresponding mtCOI nucleotidesequences (Figure 4) The resulting trees showed similartopologies suggesting that the signals embedded in thesefunctional genes reflect evolutionary divergence among taxarather than any enzyme adaptations relationships
International Journal of Genomics 7
Macromolecular complex 1541Organelle part 580Extracellular region 367
Organelle 1616Synapse part 031Synapse 031
Membrane-enclosed lumen 084Extracellular region part 120Cell 2815Cell part 2815
(a) Cellular components
Structural molecule activity 1054Catalytic activity 2753Enzyme regulator activity 294Electron carrier activity 152
Transporter activity 635Transcription factor activity 052Metallochaperone activity 016
Binding 4740Molecular transducer activity 058Antioxidant activity 094
(b) Molecular function
Cellular process 3193Death 010Signaling 127Cellular component biogenesis 215Cell wall organization or biogenesis 010
Metabolic process 3345Biological regulation 446
Viral reproduction 005
Biological adhesion 093
Cellular component organization 289
Signaling process 098Developmental process 024Immune system process 118Establishment of localization 847
Multicellular organismal process 049
(c) Biological processes
Figure 1 Functional categorization of unigenes with gene ontology (GO) term for theAphanopus carboEST collectionThese unigenes resultswere functionally classified from the six tissues pooled together as percentages under three main functional categories with respective GOSlim terms Data refer to assemblage derived by implementation of Newbler Roche 454 sequence analysis software
The lactate dehydrogenase A (LDH-A isotig01401)was encountered abundantly in the muscle tissue (Table 5Figure 2) and its comparison with other orthologoussequences differs for a number of amino acids between 16and 44 One indel at position 75 had to be added to the two
polar species to obtain a complete alignment (Table 7) Thepercentage of identity was higher with the strictly marinespecies ranging between 952 and 937 Unfortunatelyno sequences from neither deep-sea nor abyssal fisheswere available for comparison On the other hand lactate
8 International Journal of Genomics
Table 4 List of Pfam conserved domains that were tissue specifically expressed inAphanopus carbo transcriptome Frequencies and 119875-valuesof tissue specificity analysis are indicated
(a)
Gonads Heart LiverPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF00100 Zona pellucida 17 315 10minus13 PF00011 HSP20 3 403 10minus4 PF00084 Sushi 14 144 10minus10
PF00125 Histone 8 606 10minus5 PF13405 EF hand 4 5 677 10minus4 PF00089 Trypsin 20 759 10minus9
PF01400 Astacin 3 322 10minus3 PF00412 LIM 3 152 10minus3 PF00079 Serpin 12 563 10minus6
PF00069 Pkinase 3 001 PF05556 Calsarcin 3 360 10minus3 PF07678 A2M comp 4 135 10minus4
PF00653 BIR 2 002 PF01576 Myosin tail 1 3 360 10minus3 PF01042 Ribonuc L-PSP 3 126 10minus3
PF13424 TPR 12 2 002 PF00056 Ldh 1 N 3 360 10minus3 PF00045 Hemopexin 3 126 10minus3
PF13695 zf-3CxxC 2 002 PF05300 DUF737 2 550 10minus3 PF00059 Lectin C 6 169 10minus3
PF09360 zf-CDGSH 2 002 PF00992 Troponin 4 640 10minus3 PF00701 DHDPS 4 464 10minus3
PF01712 dNK 2 002 PF00595 PDZ 3 682 10minus3 PF00386 C1q 8 663 10minus3
PF00538 Linker histone 2 002 PF00022 Actin 3 001 PF00021 UPAR LY6 5 001PF10178 DUF2372 2 002 PF02874 ATP-synt ab N 2 002 PF00754 F5 F8 type C 9 001PF04856 Securin 2 002 PF13895 Ig 2 3 002 PF08702 Fib alpha 2 001PF00250 Fork head 2 002 PF00191 Annexin 2 003 PF03982 DAGAT 2 001PF01498 HTH Tnp Tc3 2 3 003 PF00212 ANP 2 003 PF01048 PNP UDP 1 2 001PF00268 Ribonuc red sm 2 006 PF05347 Complex1 LYR 2 007 PF01014 Uricase 2 001
(b)
Muscle Brain SpleenPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF01410 Troponin 7 199 10minus5 PF01669 Myelin MBP 4 403 10minus5 PF00042 Globin 5 133 10minus6
PF02807 COLFI 3 967 10minus4 PF01453 B lectin 2 644 10minus3 PF00078 RVT 1 5 133 10minus6
PF00041 ATP-guaPtransN 3 359 10minus3 PF00612 IQ 2 644 10minus3 PF00993 MHC II alpha 4 502 10minus6
PF02453 fn3 3 359 10minus3 PF11414 Suppressor APC 2 644 10minus3 PF07686 V set 5 249 10minus6
PF13895 Reticulon 3 359 10minus3 PF05768 DUF836 2 644 10minus3 PF07654 C1 set 5 471 10minus4
PF00365 Ig 2 4 797 10minus3 PF05196 PTNMK N 2 644 10minus3 PF00089 Trypsin 5 201 10minus3
PF01216 PFK 2 984 10minus3 PF04300 FBA 2 644 10minus3 PF01391 Collagen 2 230 10minus3
PF01267 Calsequestrin 2 984 10minus3 PF00287 Na K-ATPase 2 644 10minus3 PF00240 ubiquitin 3 328 10minus3
PF00261 F-actin cap A 2 984 10minus3 PF11032 ApoM 3 853 10minus3 PF09307 MHC2-interact 2 667 10minus3
PF00856 Tropomyosin 2 984 10minus3 PF00061 Lipocalin 4 001 PF00643 Zf-B box 2 001PF01661 SET 2 984 10minus3 PF00007 Cys knot 2 002 PF13445 Zf-RING LisH 2 001PF01667 Macro 2 003 PF01275 Myelin PLP 2 002 PF01498 HTH Tnp Tc3 2 2 002PF00036 Ribosomal S27e 2 005 PF00300 His Phos 1 2 002 PF02301 HORMA 1 005PF01576 efhand 2 005 PF00230 MIP 2 002 PF14259 RRM 6 1 005PF01410 Myosin tail 1 2 008 PF00091 Tubulin 3 002 PF09004 DUF1891 1 005
dehydrogenase B (LDH-B isotig01423) inA carbowas highlyrepresented in the heart while very scarcely recorded in theother tissues (Table S1) The largest sequence divergence interms of amino acid substitutions ranges between 47 and 55when compared with deep-water macrourids and similarlyto LDH-A an indel had to be added in the sequences ofthe gadiform species at position 76 to obtain a completealignment (Table 7) Previous studies on benthopelagic fishreport a significant decline of LDH activities with increasingwater depth [39] However it is known that variation inenzyme activities of fish at a given depth is influenced byfeeding behaviour and locomotory modes [40 41]
Cytosolic malate dehygrogenase A (MDHc-Aisotig01010) was detected in most of the tissues withthe exception of the muscle (Table S1) An isoform of MDHcwas also detected but its sequence covered only the last 110amino acids (isotig01731) However the type of substitutionsand tissue expression pattern (Table S1) according to Merritand Quattro [42] lead us to believe that this cytosolicisoform is MDHc-B It has been reported that the two teleostMDHc isozymes are the products of a gene duplicationevent after the separation of teleosts and tetrapods (see [42]and references therein) although the exact timing of thisduplication has not been inferred
International Journal of Genomics 9
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Elongation factor 1-beta
Basic transcription factor 3-like 4
B G H L M S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
2-Cys peroxiredoxin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ferritin heavy subunit
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
1205732-Globin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ependymin-1 precursor10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
1205722-Globin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 90
12
14
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CuZn superoxide dismutase
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
Ras-related GTP-binding protein A12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Figure 2 Continued
10 International Journal of Genomics
Fatty acid-binding protein brain10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B GH LM S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CD63-like protein Sm-TSP-2
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Tropomyosin 4 isoform 1
10
12
08
06
04
02
00Re
lativ
e fol
d ex
pres
sion
B G H L M S
C-Myc-binding protein
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Cathepsin S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Transferrin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
GH L M S
Warm temperature acclimation related-like proteinlowast
10
12
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Betaine homocysteine S-methyltansferase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
FUCL1 fucolectin-110
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Aldolase B
Figure 2 Continued
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Genomics 7
Macromolecular complex 1541Organelle part 580Extracellular region 367
Organelle 1616Synapse part 031Synapse 031
Membrane-enclosed lumen 084Extracellular region part 120Cell 2815Cell part 2815
(a) Cellular components
Structural molecule activity 1054Catalytic activity 2753Enzyme regulator activity 294Electron carrier activity 152
Transporter activity 635Transcription factor activity 052Metallochaperone activity 016
Binding 4740Molecular transducer activity 058Antioxidant activity 094
(b) Molecular function
Cellular process 3193Death 010Signaling 127Cellular component biogenesis 215Cell wall organization or biogenesis 010
Metabolic process 3345Biological regulation 446
Viral reproduction 005
Biological adhesion 093
Cellular component organization 289
Signaling process 098Developmental process 024Immune system process 118Establishment of localization 847
Multicellular organismal process 049
(c) Biological processes
Figure 1 Functional categorization of unigenes with gene ontology (GO) term for theAphanopus carboEST collectionThese unigenes resultswere functionally classified from the six tissues pooled together as percentages under three main functional categories with respective GOSlim terms Data refer to assemblage derived by implementation of Newbler Roche 454 sequence analysis software
The lactate dehydrogenase A (LDH-A isotig01401)was encountered abundantly in the muscle tissue (Table 5Figure 2) and its comparison with other orthologoussequences differs for a number of amino acids between 16and 44 One indel at position 75 had to be added to the two
polar species to obtain a complete alignment (Table 7) Thepercentage of identity was higher with the strictly marinespecies ranging between 952 and 937 Unfortunatelyno sequences from neither deep-sea nor abyssal fisheswere available for comparison On the other hand lactate
8 International Journal of Genomics
Table 4 List of Pfam conserved domains that were tissue specifically expressed inAphanopus carbo transcriptome Frequencies and 119875-valuesof tissue specificity analysis are indicated
(a)
Gonads Heart LiverPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF00100 Zona pellucida 17 315 10minus13 PF00011 HSP20 3 403 10minus4 PF00084 Sushi 14 144 10minus10
PF00125 Histone 8 606 10minus5 PF13405 EF hand 4 5 677 10minus4 PF00089 Trypsin 20 759 10minus9
PF01400 Astacin 3 322 10minus3 PF00412 LIM 3 152 10minus3 PF00079 Serpin 12 563 10minus6
PF00069 Pkinase 3 001 PF05556 Calsarcin 3 360 10minus3 PF07678 A2M comp 4 135 10minus4
PF00653 BIR 2 002 PF01576 Myosin tail 1 3 360 10minus3 PF01042 Ribonuc L-PSP 3 126 10minus3
PF13424 TPR 12 2 002 PF00056 Ldh 1 N 3 360 10minus3 PF00045 Hemopexin 3 126 10minus3
PF13695 zf-3CxxC 2 002 PF05300 DUF737 2 550 10minus3 PF00059 Lectin C 6 169 10minus3
PF09360 zf-CDGSH 2 002 PF00992 Troponin 4 640 10minus3 PF00701 DHDPS 4 464 10minus3
PF01712 dNK 2 002 PF00595 PDZ 3 682 10minus3 PF00386 C1q 8 663 10minus3
PF00538 Linker histone 2 002 PF00022 Actin 3 001 PF00021 UPAR LY6 5 001PF10178 DUF2372 2 002 PF02874 ATP-synt ab N 2 002 PF00754 F5 F8 type C 9 001PF04856 Securin 2 002 PF13895 Ig 2 3 002 PF08702 Fib alpha 2 001PF00250 Fork head 2 002 PF00191 Annexin 2 003 PF03982 DAGAT 2 001PF01498 HTH Tnp Tc3 2 3 003 PF00212 ANP 2 003 PF01048 PNP UDP 1 2 001PF00268 Ribonuc red sm 2 006 PF05347 Complex1 LYR 2 007 PF01014 Uricase 2 001
(b)
Muscle Brain SpleenPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF01410 Troponin 7 199 10minus5 PF01669 Myelin MBP 4 403 10minus5 PF00042 Globin 5 133 10minus6
PF02807 COLFI 3 967 10minus4 PF01453 B lectin 2 644 10minus3 PF00078 RVT 1 5 133 10minus6
PF00041 ATP-guaPtransN 3 359 10minus3 PF00612 IQ 2 644 10minus3 PF00993 MHC II alpha 4 502 10minus6
PF02453 fn3 3 359 10minus3 PF11414 Suppressor APC 2 644 10minus3 PF07686 V set 5 249 10minus6
PF13895 Reticulon 3 359 10minus3 PF05768 DUF836 2 644 10minus3 PF07654 C1 set 5 471 10minus4
PF00365 Ig 2 4 797 10minus3 PF05196 PTNMK N 2 644 10minus3 PF00089 Trypsin 5 201 10minus3
PF01216 PFK 2 984 10minus3 PF04300 FBA 2 644 10minus3 PF01391 Collagen 2 230 10minus3
PF01267 Calsequestrin 2 984 10minus3 PF00287 Na K-ATPase 2 644 10minus3 PF00240 ubiquitin 3 328 10minus3
PF00261 F-actin cap A 2 984 10minus3 PF11032 ApoM 3 853 10minus3 PF09307 MHC2-interact 2 667 10minus3
PF00856 Tropomyosin 2 984 10minus3 PF00061 Lipocalin 4 001 PF00643 Zf-B box 2 001PF01661 SET 2 984 10minus3 PF00007 Cys knot 2 002 PF13445 Zf-RING LisH 2 001PF01667 Macro 2 003 PF01275 Myelin PLP 2 002 PF01498 HTH Tnp Tc3 2 2 002PF00036 Ribosomal S27e 2 005 PF00300 His Phos 1 2 002 PF02301 HORMA 1 005PF01576 efhand 2 005 PF00230 MIP 2 002 PF14259 RRM 6 1 005PF01410 Myosin tail 1 2 008 PF00091 Tubulin 3 002 PF09004 DUF1891 1 005
dehydrogenase B (LDH-B isotig01423) inA carbowas highlyrepresented in the heart while very scarcely recorded in theother tissues (Table S1) The largest sequence divergence interms of amino acid substitutions ranges between 47 and 55when compared with deep-water macrourids and similarlyto LDH-A an indel had to be added in the sequences ofthe gadiform species at position 76 to obtain a completealignment (Table 7) Previous studies on benthopelagic fishreport a significant decline of LDH activities with increasingwater depth [39] However it is known that variation inenzyme activities of fish at a given depth is influenced byfeeding behaviour and locomotory modes [40 41]
Cytosolic malate dehygrogenase A (MDHc-Aisotig01010) was detected in most of the tissues withthe exception of the muscle (Table S1) An isoform of MDHcwas also detected but its sequence covered only the last 110amino acids (isotig01731) However the type of substitutionsand tissue expression pattern (Table S1) according to Merritand Quattro [42] lead us to believe that this cytosolicisoform is MDHc-B It has been reported that the two teleostMDHc isozymes are the products of a gene duplicationevent after the separation of teleosts and tetrapods (see [42]and references therein) although the exact timing of thisduplication has not been inferred
International Journal of Genomics 9
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Elongation factor 1-beta
Basic transcription factor 3-like 4
B G H L M S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
2-Cys peroxiredoxin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ferritin heavy subunit
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
1205732-Globin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ependymin-1 precursor10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
1205722-Globin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 90
12
14
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CuZn superoxide dismutase
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
Ras-related GTP-binding protein A12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Figure 2 Continued
10 International Journal of Genomics
Fatty acid-binding protein brain10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B GH LM S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CD63-like protein Sm-TSP-2
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Tropomyosin 4 isoform 1
10
12
08
06
04
02
00Re
lativ
e fol
d ex
pres
sion
B G H L M S
C-Myc-binding protein
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Cathepsin S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Transferrin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
GH L M S
Warm temperature acclimation related-like proteinlowast
10
12
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Betaine homocysteine S-methyltansferase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
FUCL1 fucolectin-110
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Aldolase B
Figure 2 Continued
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
8 International Journal of Genomics
Table 4 List of Pfam conserved domains that were tissue specifically expressed inAphanopus carbo transcriptome Frequencies and 119875-valuesof tissue specificity analysis are indicated
(a)
Gonads Heart LiverPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF00100 Zona pellucida 17 315 10minus13 PF00011 HSP20 3 403 10minus4 PF00084 Sushi 14 144 10minus10
PF00125 Histone 8 606 10minus5 PF13405 EF hand 4 5 677 10minus4 PF00089 Trypsin 20 759 10minus9
PF01400 Astacin 3 322 10minus3 PF00412 LIM 3 152 10minus3 PF00079 Serpin 12 563 10minus6
PF00069 Pkinase 3 001 PF05556 Calsarcin 3 360 10minus3 PF07678 A2M comp 4 135 10minus4
PF00653 BIR 2 002 PF01576 Myosin tail 1 3 360 10minus3 PF01042 Ribonuc L-PSP 3 126 10minus3
PF13424 TPR 12 2 002 PF00056 Ldh 1 N 3 360 10minus3 PF00045 Hemopexin 3 126 10minus3
PF13695 zf-3CxxC 2 002 PF05300 DUF737 2 550 10minus3 PF00059 Lectin C 6 169 10minus3
PF09360 zf-CDGSH 2 002 PF00992 Troponin 4 640 10minus3 PF00701 DHDPS 4 464 10minus3
PF01712 dNK 2 002 PF00595 PDZ 3 682 10minus3 PF00386 C1q 8 663 10minus3
PF00538 Linker histone 2 002 PF00022 Actin 3 001 PF00021 UPAR LY6 5 001PF10178 DUF2372 2 002 PF02874 ATP-synt ab N 2 002 PF00754 F5 F8 type C 9 001PF04856 Securin 2 002 PF13895 Ig 2 3 002 PF08702 Fib alpha 2 001PF00250 Fork head 2 002 PF00191 Annexin 2 003 PF03982 DAGAT 2 001PF01498 HTH Tnp Tc3 2 3 003 PF00212 ANP 2 003 PF01048 PNP UDP 1 2 001PF00268 Ribonuc red sm 2 006 PF05347 Complex1 LYR 2 007 PF01014 Uricase 2 001
(b)
Muscle Brain SpleenPfam ID Domain Freq 119875 Pfam ID Domain Freq 119875 Pfam ID Domain Freq 119875
PF01410 Troponin 7 199 10minus5 PF01669 Myelin MBP 4 403 10minus5 PF00042 Globin 5 133 10minus6
PF02807 COLFI 3 967 10minus4 PF01453 B lectin 2 644 10minus3 PF00078 RVT 1 5 133 10minus6
PF00041 ATP-guaPtransN 3 359 10minus3 PF00612 IQ 2 644 10minus3 PF00993 MHC II alpha 4 502 10minus6
PF02453 fn3 3 359 10minus3 PF11414 Suppressor APC 2 644 10minus3 PF07686 V set 5 249 10minus6
PF13895 Reticulon 3 359 10minus3 PF05768 DUF836 2 644 10minus3 PF07654 C1 set 5 471 10minus4
PF00365 Ig 2 4 797 10minus3 PF05196 PTNMK N 2 644 10minus3 PF00089 Trypsin 5 201 10minus3
PF01216 PFK 2 984 10minus3 PF04300 FBA 2 644 10minus3 PF01391 Collagen 2 230 10minus3
PF01267 Calsequestrin 2 984 10minus3 PF00287 Na K-ATPase 2 644 10minus3 PF00240 ubiquitin 3 328 10minus3
PF00261 F-actin cap A 2 984 10minus3 PF11032 ApoM 3 853 10minus3 PF09307 MHC2-interact 2 667 10minus3
PF00856 Tropomyosin 2 984 10minus3 PF00061 Lipocalin 4 001 PF00643 Zf-B box 2 001PF01661 SET 2 984 10minus3 PF00007 Cys knot 2 002 PF13445 Zf-RING LisH 2 001PF01667 Macro 2 003 PF01275 Myelin PLP 2 002 PF01498 HTH Tnp Tc3 2 2 002PF00036 Ribosomal S27e 2 005 PF00300 His Phos 1 2 002 PF02301 HORMA 1 005PF01576 efhand 2 005 PF00230 MIP 2 002 PF14259 RRM 6 1 005PF01410 Myosin tail 1 2 008 PF00091 Tubulin 3 002 PF09004 DUF1891 1 005
dehydrogenase B (LDH-B isotig01423) inA carbowas highlyrepresented in the heart while very scarcely recorded in theother tissues (Table S1) The largest sequence divergence interms of amino acid substitutions ranges between 47 and 55when compared with deep-water macrourids and similarlyto LDH-A an indel had to be added in the sequences ofthe gadiform species at position 76 to obtain a completealignment (Table 7) Previous studies on benthopelagic fishreport a significant decline of LDH activities with increasingwater depth [39] However it is known that variation inenzyme activities of fish at a given depth is influenced byfeeding behaviour and locomotory modes [40 41]
Cytosolic malate dehygrogenase A (MDHc-Aisotig01010) was detected in most of the tissues withthe exception of the muscle (Table S1) An isoform of MDHcwas also detected but its sequence covered only the last 110amino acids (isotig01731) However the type of substitutionsand tissue expression pattern (Table S1) according to Merritand Quattro [42] lead us to believe that this cytosolicisoform is MDHc-B It has been reported that the two teleostMDHc isozymes are the products of a gene duplicationevent after the separation of teleosts and tetrapods (see [42]and references therein) although the exact timing of thisduplication has not been inferred
International Journal of Genomics 9
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Elongation factor 1-beta
Basic transcription factor 3-like 4
B G H L M S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
2-Cys peroxiredoxin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ferritin heavy subunit
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
1205732-Globin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ependymin-1 precursor10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
1205722-Globin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 90
12
14
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CuZn superoxide dismutase
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
Ras-related GTP-binding protein A12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Figure 2 Continued
10 International Journal of Genomics
Fatty acid-binding protein brain10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B GH LM S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CD63-like protein Sm-TSP-2
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Tropomyosin 4 isoform 1
10
12
08
06
04
02
00Re
lativ
e fol
d ex
pres
sion
B G H L M S
C-Myc-binding protein
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Cathepsin S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Transferrin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
GH L M S
Warm temperature acclimation related-like proteinlowast
10
12
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Betaine homocysteine S-methyltansferase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
FUCL1 fucolectin-110
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Aldolase B
Figure 2 Continued
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
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Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Genomics 9
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Elongation factor 1-beta
Basic transcription factor 3-like 4
B G H L M S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
2-Cys peroxiredoxin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ferritin heavy subunit
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
1205732-Globin
B G H L M S
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
Ependymin-1 precursor10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
1205722-Globin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 90
12
14
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CuZn superoxide dismutase
10
08
06
04
02
00Relat
ive f
old
expr
essio
n
B G H L M S
Ras-related GTP-binding protein A12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Figure 2 Continued
10 International Journal of Genomics
Fatty acid-binding protein brain10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B GH LM S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CD63-like protein Sm-TSP-2
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Tropomyosin 4 isoform 1
10
12
08
06
04
02
00Re
lativ
e fol
d ex
pres
sion
B G H L M S
C-Myc-binding protein
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Cathepsin S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Transferrin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
GH L M S
Warm temperature acclimation related-like proteinlowast
10
12
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Betaine homocysteine S-methyltansferase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
FUCL1 fucolectin-110
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Aldolase B
Figure 2 Continued
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
10 International Journal of Genomics
Fatty acid-binding protein brain10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B GH LM S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
CD63-like protein Sm-TSP-2
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H LM S
Tropomyosin 4 isoform 1
10
12
08
06
04
02
00Re
lativ
e fol
d ex
pres
sion
B G H L M S
C-Myc-binding protein
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Cathepsin S
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Transferrin
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
GH L M S
Warm temperature acclimation related-like proteinlowast
10
12
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
Betaine homocysteine S-methyltansferase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BG H L M S
FUCL1 fucolectin-110
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Aldolase B
Figure 2 Continued
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Evolutionary BiologyInternational Journal of
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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Genomics 11
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
BH L M S
Type-4
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Alcohol dehydrogenase 8a
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Glyceraldehyde-3-phosphate dehydrogenase
10
08
06
04
02
00
Relat
ive f
old
expr
essio
nB G H L M S
Lactate dehydrogenase-A
12
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B H L M S
Phosphoglycerate mutase 2-1 (muscle)lowastlowast
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Heat shock protein 70
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Fructose-bisphosphate aldolase A
10
08
06
04
02
00
Relat
ive f
old
expr
essio
n
B G H L M S
Phosphoglucose isomerase-2
ice-structuring protein LS-12 precursorlowastlowast
Figure 2 Gene expression normalized to a relative value of 10 of all genes selected for this study in different tissues from Aphanopus carboS = spleen B = brain H = heart G = gonad L = liver M = muscle lowast= expression in the brain was below detection and lowastlowast= expression in thegonads was below detection
Further comparison analyses were carried out only forMDHc-A with orthologous sequences obtained from NCBIdatabase where only shallow-water fish sequences were avail-able The complete alignment did not include any indel andsequences differed from A carbo between 15 and 35 aminoacid substitutions representing a percentage of identityranging from 955 to 895
Two 120572-skeletal actins were detected from the A carbodatabase the isoform 1 (isotig01459) expressed in shallow-water species and probably not functioning in abyssal fishes
and the isoform 2a (isotig01561) The mutations specific forA carbo in the actin 2a were the substitutions Asp3GluAla155Ser (a mutation also commonwith the isoform 2b [7])Ser234Val Val165Ile Leu261Val and finally Ala278Thr Thistype of isoform was reported in other deep-sea species asCoryphaenoides acrolepis C cinereus C yaquinae and Carmarus [7] The isoform 2b so far reported only in abyssalspecies was not detected in A carbo
Actins 1 and 2a were significantly expressed in muscletissuewith evidence of its presence also in the heart (Table S1)
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
12 International Journal of Genomics
EF-1Blowast
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SBHSP90
10
08
04
06
02
00
H G L M SB
PRDX1∘10
08
04
06
02
00
H G L M SB
SOD-1∘10
08
04
06
02
00
H G L M SB
Ferr10
08
04
06
02
00
H G L M SB
Hb-A10
08
04
06
02
00
H G L M SB
Hb-B10
08
04
06
02
00
H G L M SB
EPD-110
08
04
06
02
00
H G L M SB
BLBP10
08
04
06
02
00H G L M SB
TSPAN-810
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
BTF3∘
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
10
08
04
06
02
00
H G L M SB
MYCBP10
08
04
06
02
00
H G L M SB
CTSS
STF-1
ContigsqPCR
Rab-1A∘
TRPM-1
Figure 3 Comparative plots of relative expression as contig frequencies versusmean qPCR values for all genes selected for this study Codesfor each of the gene are listed in Table 1 S = spleen B = brain H = heart G = gonad L = liver and M = muscle the correlation coefficient(Pearsonrsquos 119903) resulted to be highly significant (119875 lt 00001) for most of the genes except for lowast119875 lt 005 and ∘not significant
International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of Genomics 13
Table 5 Summary table of contig frequency for each of the tissues for those genes used to test the correlation with qPCR assays S spleen Bbrain H heart G gonad L liver and M muscle Names of genes based on their coding used in this table are found in table
Gene Contig code Spleen Brain Gonads Heart Liver MuscleEF-1B isotig00991 9 24 33 19 38 54Rab-1A isotig02689 4 0 3 3 0 4BTF3 isotig02213 3 5 7 10 8 2SOD-1 isotig02222 4 16 32 16 25 14PRDX1 isotig01838 4 50 59 17 23 27HSP90 isotig01406 3 64 94 86 86 13Ferr isotig01665 33 121 10 210 264 47Hb-A isotig06973 406 17 2 24 108 1Hb-B isotig00163 2303 81 16 313 707 22EPD-1 isotig01567 0 1190 0 1 0 0BLBP isotig02632 0 171 0 0 0 0TSPAN-8 isotig01397 17 7 3 407 24 9TRPM-1 isotig01595 1 4 0 637 2 2MYCBP isotig01988 0 2 135 1 1 1CTSS isotig01659 0 0 135 0 0 0STF-1 isotig00767 0 1 30 0 3218 0HPX isotig01479 0 0 0 0 1234 0BHTM isotig01489 1 0 0 0 1024 0FUCL1 isotig00473 1 0 0 0 22 0ALDB isotig01524 0 1 30 0 369 0ISP LS12 isotig03004 0 0 0 0 211 1ADH isotig01491-546 1 2 16 7 292 6GAPDH isotig00609 0 8 51 539 134 1281LDH-A isotig01401 2 7 2 5 0 789PglyM isotig01642 0 0 0 36 0 241HSP70 isotig01398 0 0 0 0 2 142FBPA isotig00492 2 2 0 233 0 4471PGI isotig01410 0 0 0 26 0 151
Direct comparison of A carbo actin 1 with orthologoussequences reported for other marine and deep-water speciesdiffered by just 1 amino acid at position 3 (Table 7) Thenumber of substitutions increases to 2 when this sequence iscompared to the isoform 2a of Coryphaenoides species (sub-stitution at position 155) and to 5 amino acids replacement(Table 7) when compared to the isoform 2b two of which areunique (positions 116 and 137) [7] Actin has a function inpolymerization of G-actin to F-actin in neutral salts Whilethe volume is increased following polymerization the reac-tion is strongly affected by high pressure [43] It is reportedthat the substitutions of Val54Ala or Leu67Pro reduced thevolume change associated with actin polymerization [7]
The assemblage of the myosin heavy chain protein(MyHC isotig02568 and isotig1394) obtained in A carbowas incomplete (All gs454 000756 and All gs454 000001)containing a gap of 711 amino acids at the positions from171 to 882 of the 1933 AA of the complete sequence Unfor-tunately within this gap there are the two loop regionswith characteristic structures that are uniquely reported fordeep-sea fish loop-2 region is shorter and the loop-1 regionhas a proresidue [9] MyHC was almost uniquely found in
muscle tissue (Table S1) and for comparison analyses withorthologous genes from other fish we only used the last 305AA (All gs454 000184) of the complete sequenceThe lowestnumber of amino acid substitutions ranged between 53 and69 (corresponding to sequence identity between 9495 to9343) when the sequence from A carbo is compared to itsrelative shallow water marine and freshwater species whilethis value increases up to 92 amino acid substitutions (andcorresponding sequence identity of 9125) when comparedto its relatives from deep water or polar regions (Table 7)
Variation in globin sequences was analysed exploring the1205722- and120573
2-chains (Hb-A isotig00247 andHb-B isotig00163)
whose relative expressions were detected more abundantly inthe spleen followed by liver heart brain andmuscle and vir-tually absent in the gonads (Figure 2 Table 5) Comparisonanalyses with orthologous sequences of deep-sea gadiformsand a notothenoid indicate that the number of amino acidsubstitutions ranged between 39 and 53 (Hb-A) and between31 and 42 (Hb-B) The complete alignments included theadditional single indel for the 120572
2-chain of Notothenia angus-
tata at position 102 Notothenioids acquired a completelydifferent globin genotype with respect to other teleostean
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
14 International Journal of Genomics
Table6Specieslist
with
inform
ations
ontheirtypeo
fenviro
nment(M
=marineBR
=brackish
andFW
=fre
shwater)climatedepthrange(
inmeters)andthetypeo
fgenes
used
inthe
study
with
relativeG
enbank
accessionnu
mbers
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Percifo
rmes
Trichiuridae
Aphanopu
scarbo
Blackscabbardfish
MDeep-water
200ndash
1700
COI
EU8540
76
Beloniform
esAd
rianichthydae
Oryzias
latip
esJapanese
ricefi
shFW
+BR
Subtropical
shallow
ACTA
1MDHcMyH
CCO
I
NM
00110
4806
NM
00116
3134
XM00
4071618AB4
9806
6
Scorpaenifo
rmes
Hexagrammidae
Pleurogram
mus
azonus
Okh
otsk
atka
mackerel
MTemperate
0ndash240
ACTA
1AB0
73381
Percifo
rmes
Scom
bridae
Scom
berscombrus
Atlanticmackerl
MTemperate
0ndash200
(0ndash100
0)AC
TA1CO
IEF
607093K
C015895
Percifo
rmes
Percihcthyidae
Sinipercachuatsi
Mandarin
fish
FWTemperate
10AC
TA1MyH
CCO
IAY
395872A
Y454304
NC
015822
Percifo
rmes
Sparidae
Sparus
aurata
Gilthead
seabream
MTemperate
1ndash30
(1ndash150)
ACTA
1AF190473
Percifo
rmes
Sphyraenidae
Sphyraenaidiaste
sPelican
barracud
aM
Trop
ical
3ndash24
ACTA
1LD
H-AM
DHc
mMDH
AF503593SIU80001
AF390559AF390561
Tetraodo
ntifo
rmes
Tetraodo
ntidae
Takifugu
rubripes
Japanese
pufferfish
M+FW
+BR
Temperate
0ndash200
(0ndash100
0)Hb-Am
MDHC
OI
XM003964767
XM003965959HM102315
Scorpaenifo
rmes
Ano
plop
omatidae
Anoplopomafim
bria
Sablefish
MDeep-water
0ndash2740
Hb-B
COI
BT082849JQ353978
Percifo
rmes
Serranidae
Epinepheluscoioides
Orange-spottedgrou
per
M+BR
Subtropical
1ndash100
Hb-B
GU982530
Gasteroste
iform
esGasteroste
idae
Gasteroste
usaculeatusTh
ree-spined
stickleb
ack
M+FW
+BR
Temperate
0ndash100
Hb-B
NM
001267638
Percifo
rmes
Nototheniidae
Nototheniacoriiceps
Blackrockcod
MPo
lar
0ndash550
LDH-AM
yHC
COI
AF0
79822AJ
243767
EU326390
Percifo
rmes
Nototheniidae
Nototheniaangusta
taMaorichief
MTemperate
0ndash100
Hb-AH
b-B
P62363P
29628
Gadifo
rmes
Macrouridae
Coryphaenoides
armatus
Abyssalgrenadier
MDeep-water
282ndash5180
LDH-BM
yHC
COI
AJ60
9232A
B330140
FJ1644
97Gadifo
rmes
Gadidae
Gadus
morhu
aAtlanticcod
M+BR
Temperate
0ndash60
0LD
H-BC
OI
AJ60
9233K
C015385
Gadifo
rmes
Gadidae
Arctogadus
glacia
lisArctic
cod
MDeep-water
0ndash1000
Hb-AC
OI
Q1AGS4K
C015200
Percifo
rmes
Latid
aeLa
tescalcarifer
Barram
undi
M+FW
+BR
Trop
ical
10ndash4
0LD
H-BC
OI
FJ439507JQ431879
Gadifo
rmes
Gadidae
Merlangiusm
erlangus
Whitin
gM
Temperate
30ndash100
(10ndash
200)
LDH-BC
OI
AJ60
9234JQ623954
Cyprinod
ontiformes
Poeciliidae
Poeciliareticulata
Gup
pyFW
+BR
Trop
ical
Shallow
LDH-BC
OI
EF40
8825JX9
68696
Gadifo
rmes
Macrouridae
Trachyrin
cusm
urrayi
Roug
hnoseg
renadier
MDeep-water
0ndash1630
LDH-BC
OI
AJ60
9235A
P008990
Percifo
rmes
Channichthyidae
Chionodraco
rastrospinosus
Ocellatedicefish
MPo
lar
0ndash1000
LDH-AC
OI
AF0
79829EU
326337
Percifo
rmes
Pomacentridae
Chromiscaud
alis
Blue-axilchrom
isM
Trop
ical
15ndash55
LDH-A
AY289558
Percifo
rmes
Gob
iidae
Rhinogobiops
nicholsii
Blackeye
goby
MSubtropical
-106
LDH-A
AF0
79534
Cyprinifo
rmes
Cyprinidae
Cyprinus
carpio
Com
mon
carp
FW+BR
Subtropical
shallow
LDH-AM
yHC
COI
AF0
76528D89992
HQ960709
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Genomics 15
Table6Con
tinued
Order
Family
Species
Com
mon
name
Environm
ent
Clim
ate
Depth
range
Gene
NCB
IAccN
r
Cyprinod
ontiformes
Fund
ulidae
Fund
ulus
heteroclitus
Mum
micho
gM
+FW
+BR
Temperate
shallow
LDH-ALDH-BC
OI
L43525L23792EU
524629
Osm
erifo
rmes
Osm
eridae
Osm
erus
mordax
Rainbo
wsm
eltM
+FW
+BR
Temperate
0ndash425
MDHcmMDH
BT075651B
T07560
0
Salm
onifo
rmes
Salm
onidae
Salm
osalar
Atlanticsalm
onM
+FW
+BR
Temperate
10ndash23
(0ndash210)
MDHcmMDH
BT06
0183B
T048216
Gadifo
rmes
Macrouridae
Coryphaenoides
acrolep
isPacific
grenadier
MDeep-water
900ndash
1300
MyH
CCO
IAB3
30141JQ
3540
60
Gadifo
rmes
Macrouridae
Coryphaenoides
yaquinae
na
MDeep-water
3400ndash5800
MyH
CCO
IAB3
30139GU44
0291
Percifo
rmes
Cirrhitid
aePa
racir
rhitesforste
riBlacksideh
awkfi
shM
Trop
ical
5ndash35
MyH
CCO
IAJ
243770H
Q561521
Percifo
rmes
Carang
idae
Serio
ladu
merili
Greater
amberja
ckM
Subtropical
18ndash72
(1ndash360)
MyH
CCO
IAB0
32020KC
015917
Gadifo
rmes
Gadidae
Boreogadus
saida
Polarc
odM
+BR
Polar
0ndash40
0Hb-AH
b-B
COI
DQ125471Q
1AGS6
KC015250
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Signal TransductionJournal of
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Evolutionary BiologyInternational Journal of
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Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Nucleic AcidsJournal of
Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
16 International Journal of Genomics
Table 7 Comparison of protein sequences of depth related genesbetween Aphanopus carbo database (All gs454 xxx) and otherfishes Diff amino acid differences Id percentage of identityGaps number of gaps introduced in the complete alignment andAcc nr NCBI Accession number 1Partial sequence 2only AAsequence available
(a) LDH-A (332 AA)
All gs454 00149 vs Diff Id Gaps Acc nrSphyraena idiastes 16 9518 0 U80001Rhinogobiops nicholsii 17 9488 0 AF079534Chromis caudalis 21 9367 0 AY289558Chionodraco rastrospinosus 26 9217 1 AF079829Notothenia coriiceps 27 9187 1 AF079822Fundulus heteroclitus 27 9187 0 L43525Cyprinus carpio 44 8675 0 AF076528
(b) MDHc-A (333 AA)
All gs454 00146 vs Diff Id Gaps Acc nrOryzias latipes 15 9550 0 NM 1163134Sphyraena idiastes 20 9399 0 AF390559Osmerus mordax 30 9099 0 BT075651Salmo salar 35 8949 0 BT060183
(c) Actin-1 (375 AA)
All gs454 00104 vs Diff Id Gaps Acc nrScomber scombrus 1 0 100 0 EF607093Iniperca chuatsi 1 0 100 0 AY395872Oryzias latipes 1 1 9973 0 NM 1104806Pleurogrammus azonus 1 1 9973 0 AB073381Coryphaenoides acrolepis 1 1 9973 0 AB021649Coryphaenoides cinereus 1 1 9973 0 AB021651Coryphaenoides armatus 2a 2 9947 0 AB086240Coryphaenoides yaquinae 2a 2 9947 0 AB086242Coryphaenoides acrolepis 2a 2 9947 0 AB021650Coryphaenoides cinereus 2a 2 9947 0 AB021652Cyprinus carpio 1 2 9947 0 AY395870Sphyraena idiastes 1 3 9920 0 AF503593Coryphaenoides armatus 2b 5 9867 0 AB086241Coryphaenoides yaquinae 2b 5 9867 0 AB086243Aphanopus carbo 2a 6 9840 0 All gs454 00102Sparus aurata 1 9 9760 0 AF190473
(d) LDH-B (334 AA)
All gs454 00143 vs Diff Id Gaps Acc nrLates calcarifer 14 9581 0 FJ439507Fundulus heteroclitus 21 9371 0 L23792Trachyrincus murrayi 47 8593 0 AJ609235Coryphaenoides armatus 50 8503 0 AJ609232Merlangius merlangus 52 8443 1 AJ609234Gadus morhua 55 8353 1 AJ609233
(e) MyHC1 (305 AA)
All gs454 00001 vs Diff Id Gaps Acc nrParacirrhites forsteri 53 9495 0 AJ243770Seriola dumerilii 54 9486 0 AB032020Siniperca chuatsi 59 9438 0 AY454304Oryzias latipes 62 9410 2 XM 4071618Cyprinus carpio 69 9343 2 D89992Coryphaenoides acrolepis 86 9182 1 AB330141Notothenia coriiceps 86 9182 1 AJ243767Coryphaenoides yaquinae 89 9153 1 AB330139Coryphaenoides armatus 92 9125 1 AB330140
(f) Hb-A1 (143 AA)
All gs454 01074 vs Diff Id Gaps Acc nrNotothenia angustata 39 7273 1 P623632
Boreogadus saida 43 6993 0 DQ125471Arctogadus glacialis 44 6993 0 DQ125475Takifugu rubripes 46 6783 0 XM 3964767Gadus morhua 53 6294 0 O424252
(g) Hb-B1 (146 AA)
All gs454 01018 vs Diff Id Gaps Acc nrEpinephelus coioides 27 8163 0 GU982530Anoplopoma fimbria 31 7891 0 BT082849Gasterosteus aculeatus 31 7891 0 NM 1267638Boreogadus saida 40 7279 0 Q1AGS62
Notothenia angustata 42 7143 0 P296282
groups The Antarctic ichthyofauna (dominated by a singletaxonomically uniform group) lost its globin multiplicity incorrelation with temperature stability On the other handfor the Arctic ichthyofauna it may have been advantageousto maintain a multiple globin system helping to deal withenvironmental changes and metabolic demands [44]
Selective pressure in the site-by-site patterns amongspecies was evaluated for LDH-A -B MDHc and ACTA1(isoform 1) There was no evidence of positive selection atthe nucleotide site level in any of those genes whose global120596 value was very low in all cases (from 0 in ACTA1 and 0032in LDH-B) (Table S2) The proportion of sites supportingpositive selection the model M2 was null (LDH-A and -B)or extremely low (03 in MDHc and 16 in ACTA1) (TableS2) These results indicate that the evolution of these fourgenes in teleosts is constrained by very stringent selectivepressure (model probabilities for M1 versusM2 ranging from87 in MDHc and 88 in all others)
In terms of protein stability calculated as Gibbs freeenergy and taking into consideration kinetic and thermo-dynamic quantities we explored only the functional genesfor which the complete sequences were obtained (Figure 5)In this context protein stability is defined by the ability of
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Genomics 17
02
01
Ep_coiGa_acuAn_fim
Ap_carBo_sai
No_angNo_ang
Ap_carTa_rub
Bo_saiAr_glaGa_mor
52
5452
66
100
5359
67
100 100
COI
LDH-A
LDH-B
MDHc-A
Hb-B
Hb-A
La_calCy_carAn_fim
Ap_carSi_chu
Se_dumSc_sco
Ta_rubPa_for
Ch_rasNo_cor
Or_latGa_morBo_saiAr_glaMe_mer
Tr_murCo_armCo_yaq
Co_acrPo_ret
Fu_hetLa_cha
62100
73
87
100
100
005
100
99
No_corCh_rasFu_hetCh_cauRh_nicSp_idiAp_car
Cy_carLa_calAp_car
Fu_hetTr_murCo_armGa_morMe_mer
Or_latAp_car
Sp_idiSa_salOs_mor
100
6479
100
100
100
100
100100
9699
7990
8055
70
Figure 4 At the top molecular phylogenetic tree of COI gene estimated by the HKY nucleotide substitution model constructed by neighborjoining and rooted including the sequence of Latimeria chalumnae (acc nr NC 001804) in the middle NJ tree of LDH andMDH genes andat the bottom NJ tree of globin (Hb) gene Numbers in proximity of the nodes denote the bootstrap value (above 50) out of 1000 replicatesThe scale indicates the evolutionary distance of the base substitution per site Taxon coding includes the first two letters for the genus followedby three letters for the species name (see Table 6) A black square helps to locate Aphanopus carbo in the trees
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
18 International Journal of Genomics
Ac carAc carAc car
Tr murNo corOs mor
Co armCh rasSa sal
Me merSp idiSp idi
Ga mor
Or lat
MDHc-A
LDH-ALDH-B
450
400
350
300
250
200
150
100
50
00
ΔG
(cal
mol
)
(a)
ΔG
(cal
mol
)
Ac car Co acr Co cin Co arm Co yaq460
480
500
520
540
560
580
600
Actin 1
Actin 2bActin 2a
(b)
1000
800
600
400
200
00
minus200
ΔG
(cal
mol
)
Hb-AHb-B
No ang Ac car Bo sai Ar gla Ga morAn fim
(c)
Figure 5 Comparative plots of normalised difference in protein stability (Δ119866) for different functional genes among several representativespecies Taxon coding includes the first two letters for the genus followed by three letters for the species name (see Table 6)
a protein to retain its structural conformation or its activ-ity when subjected to physical or chemical manipulationstherefore the energy consumed by activation to promotedfolding has to be compensated by thermal stability providedby the energy of denaturation [35] Hence protein stability isquantitatively calculated by the standardGibbs energy change(Δ119866) allowing comparison of stabilities for different proteins[45]
The normalised difference in protein stability (Δ119866) oflactate dehydrogenase A (LDH-A) was lower in A carbocompared to fish from polar waters and tropical marine(Figure 5) This gradual variation was primarily dictatedby the lower contributions of leucine and lysine in thehydrophobic trend of kinetic calculation in the A carbosequence (Table S3) The substantial drop in Δ119866 of LDH-B in A carbo compared to the orthologous sequences ofpolar abyssal and shallower marine fishes (Figure 5) wasdue to a larger contribution of glutamic acid (Table S3)promoting the thermal denaturation of the protein Other
studies investigating kinetic physical properties and ability towithstand high pressure of LDH-B of two gadiformes supportour calculations showing that the enzyme from the deep-sea species has a significant increased tolerance to pressureand higher thermostability [6] To provide protection to Acarbo LDH-B from pressure and temperature denaturationosmolytes might play an essential role Experiments addingTrimethylamine-N-oxide (TMAO) to samples resulted insubstantial increment of activity of LDH-B under conditionsat which the enzyme was previously sensitive [6]
In the cytosolicmalate dehygrogenaseA (MDHc-A) therewas a sharp decrease in protein stability of marine fishesgoing from shallower to deeper waters (no sequences ofabyssal specieswere available) Lowest values are encounteredin the sequences of the two euryhaline species included inthe comparison (Figure 5) This progressive decrement instability was associated to the larger contribution of asparticacid (Table S3) also a promoter of thermal denaturation ofthe protein The responses to pressure and temperature of
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Genomics 19
soluble enzymes like LDH andMDHdiffer adaptively amongspecies found at different depths
The isoforms 1 and 2a of 120572-skeletal actin of A carboshowed very similar numerical values of Δ119866 compared totheir homologues of deep-water (actin 1) and deep andabyssal water (actin 2a) Protein stability drops substantiallywhen these two isoforms were compared with actin 2bonly present in abyssal species (Figure 5) Such reduction instability was due to a higher number of hydrophobic aminoacids contributing to the thermodynamic calculation (TableS3) Morita [7] reported that the substitutions of Gln137Lysand Ala155Ser generate a mechanism for stabilizing enzyme-substrate interactions under high pressure
Comparing the stability of globin 1205722-chain (Hb-A) in
shallow deep-water and polar fish the larger Δ119866 was foundin polar and deep-water cods followed by A carbo beforedropping to negative values for the shallower representativesof Antarctic and temperate environments (Figure 5) Stabilityof protein in A carbo and its deep-water relatives was linkedto larger contributions by leucine in the kinetic calculationspromoting folding and thermal stability (Table S3) Thestability as Δ119866 of globin sequences of the 120573
2-chains (Hb-
B) showed a very similar trend as in Hb-A (although therewere no negative values) and the two deep-living species hadvery similar protein stability value Variation in Δ119866 amongenvironments was linked to the balanced contribution ofhydrophobic amino acids promoting folding versus thoselowering thermal stability (Table S3)
Although for key enzymes it was found that adap-tive differences among species at different depths showedstructural changes as well as structural stability (reviewsin [4ndash6]) several studies remarked on the importance ofregulatory regions of the genome acting on gene regulation(see [46]) Promoter regions often work together with othercys-regulatory elements (eg transcription factors bindingsites enhancers silencers and insulators) to regulate thetranscription and expression of mRNA in a specific tissueat different times and places throughout the genome Inaddition in regards to adaptation to depth it is remarkedthe importance of osmolyte concentrations of methylamines(TMAO is the most relevant) These are protein stabilizersthat counteract inhibition of proteins by hydrostatic pressure[47ndash49]
Finally we should also take into account the stressimposed on this fishes being captured at a depth range of1100ndash1250m and brought to surface in at least 3ndash5 hours Bythe time specimens of black scabbardfish reach the surfacethey are already dead or dying therefore the expressionof some of the transcripts might be different if comparedto a transcriptome of a fish sampled at a thousand metersdepth Notwithstanding several experiments using modelspecies have been targeting specific genes linked to stressfactors [50] and this might prove useful in interpretingtranscriptomes of animals undergoing stressful conditionsbefore being sampled
On the bases of this preliminary but encouraging resultswe are planning to explore further deep water adap-tivity employing different NGS sequence technology andexperimental design for instance using individual RADseq
Table 8 Statistics of EST-SSRs identified in Aphanopus carbotranscriptome
Searching item NumbersTotal number of sequences examined 7920Total size of examined sequences (bp) 4235839Total number of identified SSRs 153Number of SSR containing sequences 142Number of sequences containing more than 1 SSR 9Number of SSRs present in compound formation 8Di-nucleotide 63Tri-nucleotide 52Tetra-nucleotide 35Penta-nucleotide 3
approach on a larger number of specimens caught at deepor shallow waters or by comparing sister species that live atdifferent depth levels
34 EST-SSR Resources for Population Genetics In this studya total of 153 EST-SSRs were detected in 142 contigs (387)with a frequency of one EST-SSR per 2769 kb sequence(Table 8) A further selection was made taking into accountthe type of repeats size of the fragment and quality of theprimer sets reducing the EST-SSR to 98 Some of thosemicrosatellite markers were found in one tissue but notin others (163) while others resolved to be found inmore than one tissue (837) Among the identified EST-SSRs tri-nucleotide repeats represented the largest por-tion (408) followed by di-nucleotide (286) and tetra-nucleotide (257) and only 2 EST-SSRs penta-nucleotidewere identified (Table S4) Primer pairs have sizes rangingbetween 19 and 25 bp and melting temperatures from 571∘Cto 612∘C while expected PCR products are 100 to 280 bp inlength None of these EST-derived primer pairs have beentested for neutrality therefore not allmay be suitable for basicpopulation genetic studies
4 Conclusions
The transcriptome analysis of A carbo revealed a com-prehensive set of genes expressed in six tissues producingover 8000 genes 556 of which annotated by similarity toknown proteins or nucleotide sequences from freely acces-sible database The transcriptome of black scabbardfish setsthe stage for expanding the genetic resources available whileproviding sets of genes that are likely tied with physiologicaladaptations to depth particularly to low temperature andhigh pressure factorsThis study represents the first transcrip-tome analysis for a deep-sea fish providing insights based oncomparison analyses of homologous depth-related functionalgenes from shallow deep-water and abyssal fish highlight-ing similarities for A carbo isozyme patterns and stabilityto other bathypelagic fishes Direct sequence comparisonsuggested that the signals embedded in these functionalgenes reflect evolutionary divergence among taxa rather thanany kind of enzyme adaptations Organisms are adapted to
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
20 International Journal of Genomics
deep-sea environment and their physiological tolerance mayvary among taxa as well as their enzymatic activities underextreme conditions Osmolyte concentrations as protein sta-bilizers that counteract inhibition of proteins by hydrostaticpressure [6 47ndash49] play a key role in deep-sea adaptationFurthermore contribution to adaptation is also provided bypromoter regions that together with cys- and trans-regulatoryelements work concertedly at the mRNA transcription levelto drive the expression of a gene in a specific tissue or at aspecific time [46]
The strong correlation detected between the values ofstandardized contigs frequency with the expression level ofthe gene by qPCR supports the use of our data to explore geneexpression patterns in A carbo
Finally considering the importance of this species forfishery management a further exploration of this databasehas provided the characterization of new EST-SSR markersas additional resource for basic population genetic studies aswell as tagging and mapping of genes
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Sergio Stefanni conceived the study and design collectedsamples participated in the data analysis and drafted thepaper Raul Bettencourt contributed to gene validation exper-iments and discussion Miguel Pinheiro and Gianluca DeMoro developed the pipe-line analysis for all functionalannotations and the SQL database Lucia Bongiorni andAlberto Pallavicini participated in the structure discussionand paper drafting
Acknowledgments
All molecular work was supported by the research ProjectsDEECON (European Science Foundation under the EURO-CORES programme proposal no 06-EuroDEEP-FP-008)and ReDEco (MarinERA programme funded by the EU FP6ERA-NET Scheme MARIN-ERAMAR00032008) Speci-mens of A carbo were provided by the CONDOR Project(EEA Grants Financial Mechanism Iceland Liechtensteinand Norway proposal no PT0040) The authors are thank-ful to captain crew and technicians onboard of the RVArquipelago for their excellent contribution while at seaSergio Stefanni is a research fellow supported by the MarieCurie grant cofunded by the EU under the FP7-People-2012-COFUND Cofunding of Regional National and Inter-national Programmes GA no 600407 and the BandieraProject RITMARE IMARDOP is funded through the pluri-annual and programmatic funding scheme as research unitnumber 531 and associate laboratory number 9 The authorsare also grateful to Conceicao Egas for the support providedat Biocant Last but not least the authors thank three
anonymous reviewers for their comments and suggestionsthat greatly improved the paper
References
[1] G N Somero ldquoAdaptations to high hydrostatic pressurerdquoAnnual Review of Physiology vol 54 pp 557ndash577 1992
[2] J J Childress andMHNygaard ldquoThe chemical composition ofmidwater fishes as a function of depth of occurence off southernCaliforniardquo Deep-Sea Research and Oceanographic Abstractsvol 20 no 12 pp 1093ndash1109 1973
[3] J J Childress and G N Somero ldquoDepth-related enzymicactivities in muscle brain and heart of deep-living pelagicmarine teleostsrdquo Marine Biology vol 52 no 3 pp 273ndash2831979
[4] G N Somero ldquoBiochemical ecology of deep-sea animalsrdquoExperientia vol 48 no 6 pp 537ndash543 1992
[5] P Sebert ldquoFish at high pressure a hundred year historyrdquoComparative Biochemistry and Physiology A vol 131 no 3 pp575ndash585 2002
[6] A A Brindley R W Pickersgill J C Partridge D J DunstanD M Hunt and M J Warren ldquoEnzyme sequence and itsrelationship to hyperbaric stability of artificial and natural fishlactate dehydrogenasesrdquo PLoS ONE vol 3 no 4 Article IDe2042 2008
[7] TMorita ldquoStructure-based analysis of high pressure adaptationof 120572-actinrdquo Journal of Biological Chemistry vol 278 no 30 pp28060ndash28066 2003
[8] T Morita ldquoStudies on molecular mechanisms underlying highpressure adaptation of 120572-actin from deep-sea fishrdquo Bulletin ofFisheries Research amp Development Agency vol 13 pp 35ndash772004
[9] T Morita ldquoComparative sequence analysis of myosin heavychain proteins from congeneric shallow- and deep-living rattailfish (genus Coryphaenoides)rdquo The Journal of ExperimentalBiology vol 211 no 9 pp 1362ndash1367 2008
[10] T Morita ldquoHigh-pressure adaptation of muscle proteins fromdeep-sea fishes Coryphaenoides yaquinae and C armatusrdquoAnnals of the New York Academy of Sciences vol 1189 pp 91ndash94 2010
[11] S Hourdez and R E Weber ldquoMolecular and functionaladaptations in deep-sea hemoglobinsrdquo Journal of InorganicBiochemistry vol 99 no 1 pp 130ndash141 2005
[12] D W Tucker ldquoStudies on Trichiuroid fishesmdash3 A preliminaryrevision of the family Trichiuridaerdquo Bulletin of the BritishMuseum (Natural History) Zoology vol 4 pp 73ndash131 1956
[13] M R Martins A M Leite and M L Nunes ldquoPeixe-espada-pretoAlgumas notas acerca da pescaria do peixe-espada-pretordquoInstituto Nacional de Investigacao das Pescas 1987
[14] FAO ldquoFishstatrdquo FAO Fisheries Department Fishery Informa-tion Data and Statistics Unit 2002
[15] M Machete T Morato and G Menezes ldquoExperimental fish-eries for black scabbardfish (Aphanopus carbo) in the AzoresNortheast Atlanticrdquo ICES Journal of Marine Science vol 68 no2 pp 302ndash308 2011
[16] ICES ldquoReport of theWorking Group on the Biology and Assss-ment of Deep-Sea Fisheries Resources (WGDEEP)rdquo Tech RepICES CM 2008ACOM 14 ICEC Headquarters CopenhagenDenmark 2008
[17] I Figueiredo P Bordalo-Machado S Reis et al ldquoObservationson the reproductive cycle of the black scabbardfish (Aphanopuscarbo Lowe 1839) in the NE Atlanticrdquo ICES Journal of MarineScience vol 60 no 4 pp 774ndash779 2003
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
International Journal of Genomics 21
[18] B Morales-Nin and D Sena-Carvalho ldquoAge and growth of theblack scabbard fish (Aphanopus carbo) off Madeirardquo FisheriesResearch vol 25 no 3-4 pp 239ndash251 1996
[19] C Longmore C N Trueman F Neat et al ldquoOcean-scaleconnectivity and life cycle reconstruction in a deep-sea fishrdquoCanadian Journal of Fisheries and Aquatic Sciences pp 1ndash122014
[20] S Stefanni andH Knutsen ldquoPhylogeography and demographichistory of the deep-sea fish Aphanopus carbo (Lowe 1839) inthe NE Atlantic vicariance followed by secondary contact orspeciationrdquoMolecular Phylogenetics and Evolution vol 42 no1 pp 38ndash46 2007
[21] S H Nagaraj R B Gasser and S Ranganathan ldquoA hitchhikersguide to expressed sequence tag (EST) analysisrdquo Briefings inBioinformatics vol 8 no 1 pp 6ndash21 2007
[22] M EHudson ldquoSequencing breakthroughs for genomic ecologyand evolutionary biologyrdquo Molecular Ecology Resources vol 8no 1 pp 3ndash17 2008
[23] H Ellegren ldquoSequencing goes 454 and takes large-scalegenomics into the wildrdquo Molecular Ecology vol 17 no 7 pp1629ndash1631 2008
[24] R Bettencourt M Pinheiro C Egas et al ldquoHigh-throughputsequencing and analysis of the gill tissue transcriptome from thedeep-sea hydrothermal vent mussel Bathymodiolus azoricusrdquoBMC Genomics vol 11 no 1 article 559 2010
[25] R A Lesniewski S Jain K Anantharaman P D Schloss andG J Dick ldquoThemetatranscriptome of a deep-sea hydrothermalplume is dominated by water column methanotrophs andlithotrophsrdquo The ISME Journal vol 6 no 12 pp 2257ndash22682012
[26] J WuW GaoW Zhang and D R Meldrum ldquoOptimization ofwhole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysisrdquo Journalof Microbiological Methods vol 84 no 1 pp 88ndash93 2011
[27] Y Shi J McCarren and E F Delong ldquoTranscriptionalresponses of surface water marine microbial assemblages todeep-sea water amendmentrdquo Environmental Microbiology vol14 no 1 pp 191ndash206 2012
[28] S Stefanni R Bettencourt H Knutsen and G MenezesldquoRapid polymerase chain reaction-restriction fragment lengthpolymorphism method for discrimination of the two Atlanticcryptic deep-sea species of scabbardfishrdquo Molecular EcologyResources vol 9 no 2 pp 528ndash530 2009
[29] M Margulies M Egholm and W E Altman ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 pp 376ndash380 2005
[30] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990
[31] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997
[32] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987
[33] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 pp 783ndash791 1985
[34] Z Yang ldquoPAML 4 a program package for phylogenetic analysisby maximum likelihoodrdquoMolecular Biology and Evolution vol24 no 8 pp 1586ndash1591 2007
[35] S Prashanth Kumar and M Meenatchi ldquoVirtual quantificationof protein stability using applied kinetic and thermodynamicparametersrdquo IIOAB Letters vol 1 pp 21ndash28 2011
[36] T Thiel W Michalek R K Varshney and A Graner ldquoExploit-ing EST databases for the development and characterizationof gene-derived SSR-markers in barley (Hordeum vulgare L)rdquoTheoretical and Applied Genetics vol 106 no 3 pp 411ndash4222003
[37] S Rozen and H Skaletsky ldquoPrimer3 on the WWW for generalusers and for biologist programmersrdquo Methods in MolecularBiology vol 132 pp 365ndash386 2000
[38] B-Y Lee A E Howe M A Conte et al ldquoAn EST resource fortilapia based on 17 normalized libraries and assembly of 116899sequence tagsrdquo BMC Genomics vol 11 article 278 2010
[39] J C Drazen and B A Seibel ldquoDepth-related trends inmetabolism of benthic and benthopelagic deep-sea fishesrdquoLimnology andOceanography vol 52 no 5 pp 2306ndash2316 2007
[40] K M Sullivan and G N Somero ldquoEnzyme activities of fishskeletal muscle and brain as influenced by depth of occurrenceand habits of feeding and locomotionrdquoMarine Biology vol 60no 2-3 pp 91ndash99 1980
[41] J F Siebenaller GN Somero andR LHaedrich ldquoBiochemicalcharacteristics of macrourid fishes differing in their depths ofdistributionrdquoTheBiological Bulletin vol 163 pp 240ndash249 1982
[42] T J S Merrit and J M Quattro ldquoEvolution of the vertebratecytosolic malate dehydrogenase gene family duplication anddivergence in actinopterygian fishrdquo Journal of Molecular Evo-lution vol 56 no 3 pp 265ndash276 2003
[43] T Ikkai and T Ooi ldquoThe effects of pressure on F-G transforma-tion of actinrdquo Biochemistry vol 5 no 5 pp 1551ndash1560 1966
[44] C Verde M Balestrieri D de Pascale D Pagnozzi G Lecoin-tre and G Di Prisco ldquoThe oxygen transport system in threespecies of the boreal fish family Gadidae molecular phylogenyof hemoglobinrdquoThe Journal of Biological Chemistry vol 281 no31 pp 22073ndash22084 2006
[45] H J Him C Steif T Vogl et al ldquoFundamentals of proteinstabilityrdquo Pure andApplied Chemistry vol 65 pp 947ndash952 1993
[46] H E Hoekstra and J A Coyne ldquoThe locus of evolution evodevo and the genetics of adaptationrdquo Evolution vol 61 no 5pp 995ndash1016 2007
[47] P H Yancey A L Fyfe-Johnson R H Kelly et al ldquoTrimethy-lamine oxide counteracts effects of hydrostatic pressure onproteins of deep-sea teleostsrdquo Journal of Experimental Zoologyvol 289 p 172 2001
[48] P H Yancey W R Blake and J Conley ldquoUnusual organicosmolytes in deep-sea animals adaptations to hydrostaticpressure and other perturbantsrdquo Comparative Biochemistry andPhysiology A Molecular amp Integrative Physiology vol 133 no 3pp 667ndash676 2002
[49] P H Yancey M D Rhea K M Kemp and D M BaileyldquoTrimethylamine oxide betaine and other osmolytes in deep-sea animals depth trends and effects on enzymes under hydro-static pressurerdquo Cellular and Molecular Biology vol 50 no 4pp 371ndash376 2004
[50] P Prunet M T Cairns S Winberg and T G PottingerldquoFunctional genomics of stress responses in fishrdquo Reviews inFisheries Science vol 16 no 1 pp 157ndash166 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology