Insights into molecular profiles and genomic evolution of an IRAK4homolog from rock bream (Oplegnathus fasciatus): Immunogen- andpathogen-induced transcriptional expression
Navaneethaiyer Umasuthan a, b, S.D.N.K. Bathige a, b, Ilson Whang b, Bong-Soo Lim b,Cheol Young Choi c, Jehee Lee a, b, *
a Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic ofKoreab Fish Vaccine Research Center, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Koreac Division of Marine Environment and Bioscience, Korea Maritime University, Busan 606-791, Republic of Korea
a r t i c l e i n f o
Article history:Received 7 August 2014Received in revised form4 December 2014Accepted 15 December 2014Available online 31 December 2014
As a pivotal signaling mediator of toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signalingcascades, the IL-1R-associated kinase 4 (IRAK4) is engaged in the activation of host immunity. This studyinvestigates the molecular and expressional profiles of an IRAK4-like homolog from Oplegnathus fasciatus(OfIRAK4). The OfIRAK4 gene (8.2 kb) was structured with eleven exons and ten introns. A putative codingsequence (1395 bp) was translated to the OfIRAK protein of 464 amino acids. The deduced OfIRAK4protein featured a bipartite domain structure composed of a death domain (DD) and a kinase domain(PKc). Teleost IRAK4 appears to be distinct and divergent from that of tetrapods in terms of its exon-intron structure and evolutionary relatedness. Analysis of the sequence upstream of translation initia-tion site revealed the presence of putative regulatory elements, including NF-kB-binding sites, which arepossibly involved in transcriptional control of OfIRAK4. Quantitative real-time PCR (qPCR) was employedto assess the transcriptional expression of OfIRAK4 in different juvenile tissues and post-injection ofdifferent immunogens and pathogens. Ubiquitous basal mRNA expression was widely detected withhighest level in liver. In vivo flagellin (FLA) challenge significantly intensified its mRNA levels in intestine,liver and head kidney indicating its role in FLA-induced signaling. Meanwhile, up-regulated expressionwas also determined in liver and head kidney of animals challenged with potent immunogens (LPS andpoly I:C) and pathogens (Edwardsiella tarda and Streptococcus iniae and rock bream iridovirus (RBIV)).Taken together, these data implicate that OfIRAK4 might be engaged in antibacterial and antiviral im-munity in rock bream.
The invading microorganisms are initially recognized by thehost's innate immune system through evolutionarily conservedpattern recognition receptors (PRRs) [1]. Toll-like receptors (TLRs)are PRRs which recognize pathogen associated molecular patterns(PAMPs) and trigger the innate immune responses [2]. The
s Lab, Department of MarineUniversity, 66 Jejudaehakno,4 754 3472; fax: þ82 64 756
interleukin (IL)-1 receptor-associated kinases (IRAKs) operatedownstream of TLRs and play a crucial role in TLR/IL-1R pathways[3] by acting as inter-mediators of the signaling between MyD88and (TNF) receptor-associated factor 6 (TRAF6). The term IRAK wasfirst referred to a serine/threonine-specific kinase activity, whichco-precipitated with the IL-1 receptor in an IL-1-concentrationdependent manner [4]. To date, four different members of IRAKfamily, namely IRAK1, 2, 3 (M) and 4, have been described. Whilethese members share certain common structural features, theyperform distinct and non-redundant functional roles at differentlevels, and mediate the downstream signal transduction. The IRAKs(1, 2, and 4), except IRAK3, positively regulate the TLR/IL-1Rsignaling [5].
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As a novel member of IRAK family, IRAK4 was first reported in2002 [6], which shares the domain structure that is characteristic tothe IRAK family. However, it lacked the C-terminal stretch [3],similar to a IRAK functional homolog in fruitfly Drosophila (Pelleprotein). The IRAK1 and IRAK4 are considered to be the only truekinases in this family. IRAK4 was demonstrated to be involved inMyD88-dependent TLR/IL-1R signaling cascade [7]. Upon TLR-stimulation, MyD88 recruits the IRAK4 by interacting via thedeath domain (DD). Subsequently, this MyD88eIRAK4 complexrecruits IRAK2 and/or IRAK1 to form the Myddosome complexesleading to their phosphorylation and activation [6,8]. StimulatedIRAK1 transduces the signal to TRAF6, and activation of severalother downstream molecules culminates in NF-kB and AP-1-dependent transcriptional responses. This would result in amodulated expression of a wide range of immune-relevant genes.
Using mammalian systems, many aspects of IRAK4 have beenintensively explored in past decades. The kinase domain-deficientmutant IRAK4 diminished IL-1/LPS-induced NF-kB activation sug-gested that the kinase activity is required for its function [6]. Thecrucial functional importance of the IRAK4 was highlighted basedon knock-out studies in mice, in which IRAK4�/� mice werecompletely resistant to pathogen challenge or mitogen-shock, dueto the impaired TLR/IL-1R-mediated responses, mainly the pro-duction of pro-inflammatory cytokines and chemokines [9,10]. Inaddition, its necessity in eliciting the adaptive immune responsesand its involvement in crosstalk between innate and adaptive armsof immunity has also been demonstrated [11]. These genetic evi-dences emphasized an indispensable role for IRAK4 in the immunesignaling.
Following the discovery of IRAK4 gene of mouse and human, itsorthologs have been reported from both invertebrates and verte-brates. A few invertebrate IRAK4 homologs have been identified,including those from Euprymna scolopes [12], Suberites domuncula[13], Mya arenaria [14], Haliotis diversicolor [15], and their mRNAexpression has been investigated in healthy tissues and post-pathogen infection. To the best of our knowledge, a few teleos-tean IRAK4 homologs have also been reported from Danio rerio [16],Cynoglossus semilaevis [17], Trachidermus fasciatus [18] and threesalmonids [19]. However, these studies have not provided aninsight into the exon-intron structure (except the recently pub-lished data in salmonids [19]), and genomic evolution of IRAK4 inteleost with respect to other lineages.
Rock bream and its mariculture production are of great interestin Eastern Asian region due to its commercial value and deli-ciousness [20]. However, severe spread of pathogen infections havebrought a great loss to the rock bream industry in recent years[21,22]. Consequently, improving the disease resistance of rockbream has become an important objective to ensure its stablefishery production. As an initial step in establishing the immunesignaling pathways, we explored the genes involved in rock breamimmunity by means of transcriptomic- and genomic BAC-libraries.In the present study, we describe (1) the identification of an IRAK4homolog from rock bream at cDNA and genomic levels, (2) thegenomic organization of IRAK4 in a comparative context, (3) itsputative promoter sequence, and (4) spatial-as well as thetemporal-mRNA expressional responses under pathologicalconditions.
2. Materials and methods
2.1. Identification and analysis of rock bream IRAK4 molecularprofile
Sequence data of a previously constructed normalized cDNAlibrary was searched for genes involved in TLR signaling [23], and a
cDNA of Oplegnathus fasciatus IRAK4-like gene was identified. Inaddition, two partial sequences of a putative IRAK1 gene and threeoverlapping partial sequences of a IRAK3 (IRAKM) gene were alsoidentified. These partial cDNA sequences were used in appropriatecontexts when a comparative analysis is conducted, concerning theentire IRAK-family. The primers designed for IRAK4-like gene(Supplementary Table 1; F1 and R1) were employed in PCR toamplify its coding sequence. Amplicon was cloned into T-VectorpMD20 (TaKaRa) and confirmed by sequencing (Macrogen). BLASTanalysis affirmed this IRAK4-like gene as a true homolog ofO. fasciatus IRAK4 and it was denoted as OfIRAK4.
Putative CDS of OfIRAK4 was determined and translated toderive the corresponding amino acid sequence using DNAssist (2.2)and these sequences were subjected to BLAST analysis. Homolo-gous genomic and amino acid sequences of other IRAKs wereretrieved from GenBank or Ensembl databases. Molecular profile ofOfIRAK4 gene and its primary sequence were examined by variousin silico tools available in ExPASy Resource Portal (http://www.expasy.org/). Domain topology of OfIRAK4 was determined usingSMART server (http://smart.embl-heidelberg.de/). The amino acidsequence similarity and identity scores were computed by theMatGAT software using the BLOSUM62matrix. Multiple alignmentsof orthologous IRAK4 sequences were conducted using ClustalWalgorithm. Conceptually translated amino acid sequences ofOfIRAK4 and partial translations of OfIRAK1 and OfIRAK3, as well asIRAK-family members from other lineages were used to deduce theoverall evolutionary relationship. The Neighbor-Joining (NJ) algo-rithm embedded in Mega 6.0 software was engaged in recon-structing the phylogenetic trees. The distance matrix wascalculated using Poisson correction method and statistical confi-dence of the inferred phylogenetic relationships was evaluated byperforming 5000 bootstrap replicates.
2.2. Determination and analysis of gene structure and putativepromoter of rock bream IRAK4 gene
To identify the complete genomic length of OfIRAK4, wescreened a custom constructed, random-shear, bacterial artificialchromosome (BAC) genomic DNA (gDNA) library of rock bream asdescribed earlier [24]. A two-step PCR-based screening strategywas employed in identification of a positive BAC clone bearing theOfIRAK4, and sequenced by GS-FLX pyro-sequencing approach(Macrogen) to retrieve the full-length sequence of OfIRAK4. Theprimers used for screening were designed based on the OfIRAK4cDNA sequence (Supplementary Table 1; F2 and R2). The exon-intron organization of OfIRAK4 was inferred by aligning the cDNAwith the gDNA sequence using the Spidey mRNA-to-genomicalignment program. In addition, BAC DNA from positive clone wasisolated and purified by using QIAGEN Plasmid Midi Kit, and wassubjected to sequencing by primer walking with gene specificprimers (R3 and R4) to obtain the 50-flanking sequence upstream ofthe untranslated region (UTR). This putative promoter-like regu-latory region of OfIRAK4was inspected for the presence of potentialbinding sites of the important transcription factors using predictiontools including Aibaba, TFsearch and JASPAR.
2.3. Animal husbandry and immune challenges
HealthyO. fasciatus fishwere obtained from the Jeju Special Self-Governing Province Ocean and Fisheries Research Institute (Jeju,Republic of Korea). Animals were acclimatized for one week undercontrol conditions (salinity: 34 ± 1‰, pH 7.6 ± 0.5 at 24 ± 1 �C) intanks filled with aerated and sand-filtered sea water. In order toexamine the immune response of OfIRAK4, we conducted threeimmune stimulant challenges [flagellin (FLA-ST), lipopolysach
N. Umasuthan et al. / Fish & Shellfish Immunology 43 (2015) 436e448438
charide (LPS) and poly I:C] and three pathogen challenges [rockbream iridovirus (RBIV), Edwardsiella tarda and Streptococcus iniae].For the flagellin-challenge, fish (~96 g) were maintained with adensity of 20 animals per tank (150 L) and experiment was con-ducted in Fish Vaccine Research Center, Jeju National University(Jeju, Korea). For all the other challenges, rock bream juveniles(~50 g) were maintained in a 400 L tank in Marine Molecular Ge-netics Laboratory, Jeju National University (Jeju, Korea), and thendivided into separate 40 L tanks at a density of 40 animals per tank.Detailed profile of challenges devised in this study are summarizedin Table 1 [25].
2.4. Collection of tissues, RNA extraction and cDNA preparation
Four replicate rock breams (n ¼ 4) were obtained from FLA-STchallenged group (No. 1) at 3, 6, 12, and 24 h post-injection (p.i.)and blood (~1 mL) was withdrawn to isolate the peripheral bloodcells (PBCs) by subsequent centrifugation (10 min at 3000� g at4 �C). Then, animals were decapitated on ice and different tissuespecimens including head kidney, spleen, intestine, liver, gills andkidney were dissected, from both control and experimental groups.Meanwhile, three animals (n ¼ 3) were randomly sampled from allthe other challenge- and corresponding control-groups to collectliver and head kidney tissues at 3, 6, 12, 24, and 48 h post-challenges. PBCs and tissues were snap-frozen in liquid nitrogenand stored at �80 �C until the RNA was extracted.
In order to investigate the tissue mRNA distribution of OfIRAKs,specimens of gills, liver, skin, spleen, head kidney, kidney, heart,muscle, brain, intestine and PBCs were collected from three healthyfish. As described in our previous report [25], the total RNA wasisolated from all the tissues obtained from both healthy and chal-lenged rock breams using Tri Reagent™ (Sigma). Subsequently,reverse transcription was carried out using PrimeScript™ first-strand cDNA Synthesis Kit (TaKaRa), and the synthesized cDNAswere diluted 40� prior to store at �20 �C until use in quantitativereal-time PCR (qPCR).
2.5. Spatial and temporal mRNA expressional analysis of OfIRAK4
The mRNA levels of OfIRAK4 (and other two IRAK-family mem-bers in rock bream, OfIRAK1 and OfIRAK3) in healthy juveniles andOfIRAK4 mRNA levels in experimentally injected fish weremeasured by qPCR technique using a Thermal Cycler Dice Real TimeSystem (TP800; TaKaRa), by following the necessary MIQE guide-lines [26]. Briefly, qPCR was performed in a 10 mL reaction volumecontaining 3 mL of diluted cDNA, 5 mL of 2� SYBR Greenmaster mix,0.4 mL of each primer (10 pmol/mL), and 1.2 mL of PCR-grade water,under the following thermal cycling conditions: 1 cycle of 95 �C for30 s, followed by 40 cycles of 95 �C for 5 s, 58 �C for 10 s and 72 �Cfor 20 s, and a final single cycle of 95 �C for 15 s, 60 �C for 30 s and95 �C for 15 s. The same qPCR cycle profile was implemented for
Table 1Description of experimental challenges conducted in the current study.
5 Edwardsiella tarda CNUb 5 � 105 CFU6 Streptococcus iniae CNUb 1 � 107 CFU7 Un-challenged e e
8 PBS (control) e e
a 10� dilution; TCID50, tissue culture infectious dose 50.b Obtained from Department of Aqualife Medicine at Chonnam National University (R
detection of the reference gene, rock bream b-actin (GenBankaccession no. FJ975146). The primers used in qPCR assay are listedin Supplementary Table 1. The baselinewas set automatically by theTaKaRa Thermal Cycler Dice TP800 Real Time System software. Theexpression level of OfIRAK4 relative to that of b-actin was deter-mined by the Livak method [27]. In spatial expression analysis, therelative expression level calculated in each tissue was comparedwith the respective expression level in muscle which was set asbaseline. In temporal expression analysis, the relative expressionlevel calculated was further normalized to the corresponding PBS-injected controls at each time point. In representation, relativemRNA level in un-injected control (0 h) was considered as baseline.Statistical analysis was performed by one-way analysis of variance(ANOVA) followed by an appropriate multiple comparison test as apost-hoc comparison using the SPSS 16.0 program (USA). Differ-ences were considered statistically significant at p < 0.05.
3. Results
3.1. Molecular characterization of rock bream IRAK4
BLAST analysis of a previously constructed rock bream tran-scriptome database yielded a complete cDNA (contig10006,1927 bp)representing a putative IRAK4 homolog. This genewas cloned and itsidentity was confirmed by sequencing, which is mentioned in thefollowing as OfIRAK4 (Fig. 1A). The 50- and 30-UTRs flanking the pre-dicted open reading frame (ORF) of OfIRAK4were 187 bp and 345 bp,respectively. A canonical polyadenylation signal site (AATAAA) wasobserved 12 bp upstream of the poly(A) tail. The ORF of OfIRAK4was1395 bp and encoded a protein of 464 amino acids with a theoreticalmolecular mass and pI of 52.1 kDa and 5.26, respectively.
In silico analysis of OfIRAK4 protein indicated that it sharesseveral features described for previously characterized mammalianand teleost orthologs (Fig. 1B). According to the SignalP server,OfIRAK4 contained no N-terminal signal peptide suggesting that itis localized in cytosolic region. In addition, four potential N-glyco-sylation sites and eight protein kinase C phosphorylation sites werepredicted as potential sites of post-translational modifications.Conserved domain prediction using SMART tool revealed thatOfIRAK4was composed of a proto-typical N-terminal death domain(DD; 8e104) and a centrally located protein kinase catalytic domain(PKc; 181e449). Three important motifs were identified within PKcdomain of OfIRAK4 including an ATP-binding region signature, ahinge region and an activation segment. In the hinge region, a Tyr(254) residue was predicted as the gatekeeper of OfIRAK4. Similarto other IRAK4s, OfIRAK4 also possessed an Asp (303) as its catalyticbase (Fig. 1).
Multiple sequence alignment of conceptually translated aminoacid sequences of IRAK4 orthologs revealed that the OfIRAK4 sharesdifferential degree of homology with other known IRAK4s (Fig. 2A).The functionally important domains, DD and PKc, appear to be
Stock concentration Route of administration Volume Re-suspension
2.4 � 10�2 mg/mL Intraperitoneal (i.p.) 100 mL In 1 � PBS1.25 mg/mL Intraperitoneal (i.p.) 100 mL In 1 � PBS1.5 mg/mL Intraperitoneal (i.p.) 100 mL In 1 � PBSe Intramuscular (i.m.) 100 mL In 1 � PBS5 � 103 CFU/mL Intraperitoneal (i.p.) 100 mL In 1 � PBS1 � 105 CFU/mL Intraperitoneal (i.p.) 100 mL In 1 � PBSe e e e
e Intraperitoneal (i.p.) 100 mL e
epublic of Korea).
Fig. 1. Sequences and domains of rock bream IRAK4 (OfIRAK4). (A) Nucleotide (black) and amino acid (dark-blue) sequences of rock bream IRAK4 (OfIRAK4). In DNA sequence:UTRs are in upper case, while CDS is in lowercase. The RNA instability motifs are underlined and italicized. The canonical polyadenylation signal site is bold italicized and poly(A) tailis underlined. In amino acid sequence: the N-terminal death domain (DD; blue) and central kinase domain (PKc; red) are shaded. Protein kinases ATP-binding region signature inPKc domain are boxed and shaded by dark red. The hinge region and activation segment are enclosed with double line box in green and underlined with yellow line, respectively.Gatekeeper (Y254) is boxed and black-shaded. Catalytic base (D303) is boxed and shaded in green. Four possible N-glycosylation sites and eight potential PKc phosphorylation sitesare underlined and boxed, respectively. (B) The comprehensive domain architecture of rock bream IRAK4 (OfIRAK4) protein. OfIRAK4 is composed of DD and PKc. In PKc, ATP-signature, hinge region and activation segments are found. All motifs are shown in colors corresponding to Fig. 1A. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)
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highly conserved in both fish and mammalian lineages. However, ahighly variable nature was noted in the linker region connectingthese two domains (Fig. 2A), which also contained a PEST-like motif(PAEET). The OfIRAK4 possessed conserved contact sites interacting
with MyD88 (Arg12, Arg20, Glu57, Thr76 and Asn78) and IRAK2(Phe25, Gln50, Arg54 and Ala95) as observed in human IRAK4 [8]. Theinvariant Asp residue, which is crucial for the kinase activity ofIRAK4, was also preserved in OfIRAK4 (Asp303). While hinge region
Fig. 2. Multiple amino acid alignment and homology analyses of rock bream IRAK4 (OfIRAK4) with other known teleost and mammalian IRAK4s. (A) The alignment isgenerated by ClustalW with default parameters. Residues conserved across the lineages are shaded with specific colors in BioEdit alignment editor. Completely (100%) and stronglyconserved residues are marked with asterisk (*) and colon (:), respectively. Weak conservation is marked by a full stop (.). The N-terminal death domain (DD) and central kinasedomain (PKc) are highlighted by blue and red shades, and boxed with same colors, respectively. In the DD, the MyD88 binding sites are indicated with a red arrow (Y) and IRAK2contacting sites are shown by blue dots (C). In the PKc, the active site Asp (D303) is indicated with a star (✴). The accession numbers and references are shown next to eachsequence. The hinge region (260e274) and activation segment (329e358) corresponding to human IRAK4 are shown by black flanking arrows. (B) Homology grid of IRAK4orthologs. Deduced amino acid sequences of whole IRAK4 protein, and individual DD and PKc were used to estimate the degree of homology. Matrix was generated by the MatGATprogram using BLOSUM62 scoring matrix maintaining first gap penalty and extending gap penalty levels at 12 and 1, respectively. (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.)
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and activation segment, including the DFG motif (DFGXXR), werefound to be conserved, the activation segment revealed a fish-specific conservation (Fig. 2A). Pairwise sequence comparison ofIRAK4 showed that whole amino acid sequence of OfIRAK4 shared astrong homology with other fish species (>79% similarity), exceptwith zebrafish (Fig. 2B). At the same time, individual homologyacross its DD and PKc domain of other fish and mammalian speciesrevealed a strong overall conservation in examined lineages.
To infer the phylogenic relationship among IRAK members invertebrate lineage, an un-rooted phylogenetic tree was constructedusing NJ algorithm by including 38 different IRAK subfamilymembers (Fig. 3). During the course of this work, two putativepartial sequences corresponding to O. fasciatus IRAK1 and IRAK3were also identified. We included these two sequences along withthe complete sequence of OfIRAK4 in this analysis. Accordingly,four branches originated from the tree and represented the distinctIRAK subfamilies. Two putative IRAKs (OfIRAK1 and OfIRAK3) andOfIRAK4 clustered with the other fish orthologs belong to the cor-responding subfamilies. The branching order of each subfamily wasconsistent with the accepted evolutionary order.
Fig. 3. Molecular phylogenetic tree of vertebrate IRAK family. Analysis was based on preevolutionary distances were computed using the Poisson correction method using MEGA6.values of the 5000 replicates. The accession numbers are as follow: X. tropicalis IRAK1, NP_0NP_001035645; D. rerio IRAK1, XP_005166760; C. idella IRAK1, AFM09716; C. carpio IRAK1, AHXP_003962967; X. laevis IRAK2, NP_001079489; M. musculus IRAK2, AAO24761; M. mulattaG. gallus IRAK2, NP_001025776; T. guttata IRAK2, XP_002187461; L. oculatus IRAK3-like, XP_B. taurus IRAK3, NP_001177228; H. sapiens IRAKM, AAD40879; O. mykiss IRAK3, NP_001268
We also reconstructed a phylogenetic tree using the aligned full-length amino acid sequences of selected vertebrate and fewinvertebrate IRAK4 orthologs (Supplementary Fig. 1). The phylo-genetic tree clearly separated the IRAK4 proteins into two mainclusters: one comprising the IRAK4s from vertebrate origin and theother one containing IRAKs from invertebrate origin. The vertebratebranch further divides into two subclusters of tetrapods and tele-osts, in which OfIRAK4 was placed with other perciformes. It wasnoteworthy that while teleost IRAK4 homologs comprise a separatecluster, all four tetrapod classes demonstrate a closer relationshipamong them than with the fish group. The topology of this phylo-genetic tree suggested that teleost IRAK4s are evolutionarilydistinct from the other counterparts (Supplementary Fig. 1).
3.2. Genomic structure of rock bream IRAK4 and comparativeanalysis
Screening of the rock bream genomic BAC library using OfIRAK4-specific primers located a positive clone and the gDNA sequence ofOfIRAK4 was obtained by NGS. Comparison of the cDNA and
dicted/reported amino acid sequences, generated by neighbor-joining method and thePhylogeny is un-rooted. The numbers at the nodes indicate the percentage bootstrap01006713; H. sapiens IRAK1, AAC41949; M. musculus IRAK1, EDL29847; B. taurus IRAK1,B53116; S. chuatsi IRAK1, ACN64942;M. zebra IRAK1, XP_004568259; T. rubripes IRAK1,IRAK2, XP_001090790; H. sapiens IRAK2, NP_001561; B. taurus IRAK2, NP_001069164;006633634; L. chalumnae IRAK3, XP_005989512; R. norvegicus IRAK3, NP_001101571;333; For accession numbers of IRAK4 members, refer to Fig. 4.
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genomic sequences of OfIRAK4 revealed that the complete genomiclength (8271 bp) of OfIRAK4 is fragmented into eleven exons by tenintervening introns of variable lengths (GenBank accession no:KP215283; Fig. 4A). A part of the terminal exons represented the 50
and 30 UTRs and all the introns were located within the ORFspanning region. In addition, all the intron/exon boundary se-quences in OfIRAK4 essentially complied with the consensus GT/AGrule. The complete composition of the OfIRAK4 structure is sche-matically illustrated in Fig. 4A, and subjecting this element tosplicing results in a transcript described in Section 3.1 (Fig. 1).
In order to establish a comparative understanding about thegenomic organization of IRAK4 gene across different taxa, repre-sentative orthologous structures were determined and aligned tocompare them. As shown in Fig. 4B and C, the coding sequence ofvertebrate IRAK4 seems to be generally distributed among elevenexons segmented by ten introns except in some salmonids. Thelengths of many exons in IRAK4 orthologs were essentially identicaland conserved among species, whilst the intron lengths werehighly variable (Table 2; A and B).
In contrast, comparison of representative invertebrate IRAK4s ofarthropod-, echinoderm-, mollusc- and porifera-origins clearlyrevealed a lack of correlation among them (Supplementary Fig. 2).Exon-intron composition largely varied not only in between theanimal groups, but also within a specific class. The relative introndensity of invertebrate IRAK4s (Supplementary Fig. 2) was lowerthan that of vertebrate IRAK4s (Table 2) suggesting that IRAK4 genemight have gained introns during the evolution.
3.3. Putative 50-flanking region of rock bream IRAK4
From genomic sequencing, only a small stretch was obtainedupstream of the 50-UTR. Hence, we subjected the BAC clone to two-rounds of reverse primer-walking and recovered an additionalgenomic sequence of ~1.5 kb (Fig. 5). This promoter-like regulatory
Fig. 4. Genomic organization of the rock bream IRAK4 (OfIRAK4) and schematic com(E1eE11) and untranslated sequences are indicated by black and empty boxes, respectively.table shows the exon composition and features of exon-intron boundaries. *, intron sequenbold-boxed. Structure of OfIRAK4 transcript resulting from splicing is also shown. (B and C) Cthe exons and intron annotations are indicated within the exon boxes and below intron linesletters. Accession numbers and sizes of exons/introns of each sequence are tabulated in Ta
region was analyzed to examine the presence of cis-elements andputative transcription factor binding sites. There was no obviousTATA box or GC box present. However, isolated OfIRAK4 promoter,especially the TIS-proximal region (~370 bp), harbored copies of afew important transcription regulatory elements including NF-kB,Sp1, CTCF, TF-AP2C and AP-2aA. In addition, presence of CREB1 andc-Jun binding sites were also noted. Presence of these transcriptionfactor binding sites (TFBS), mainly the NF-kB binding elements,implied that OfIRAK4 transcription is rigorously governed andregulated.
3.4. Detection of IRAK mRNAs in healthy juvenile tissues
The spatial mRNA expression levels of IRAK-family memberswere examined in different tissues including PBCs, gill, liver, spleen,head kidney, kidney, skin, muscle, heart, brain and intestine inhealthy rock bream juveniles by qPCR (Fig. 6). The transcript levelsof IRAKs in each tissue was normalized to that of b-actin and rep-resented as relative-fold compared with the expression of corre-sponding IRAK gene in muscle. Ubiquitous expression of OfIRAKswas detected in all tissues investigated, signifying their crucialrole(s) in different organs and tissues. Generated Ct values for eachOfIRAK from qPCR were in the range of 23e31 (data not shown),indicating that a moderate level of constitutive transcription ofOfIRAK occurs in healthy juveniles. Predominant OfIRAK4 expres-sion was detected in liver, followed by heart (p < 0.05) (Fig. 6;inset). In spleen, gills, kidney, head kidney and PBCs, OfIRAK4 wasmoderately transcribed. Intestine, brain, skin andmuscle containedsignificantly low levels of OfIRAK4 mRNA (p < 0.05).
The tissuemRNA distribution pattern of OfIRAKs shared a certaindegree of similarity. For instance, mRNA levels of all three OfIRAKswere significantly higher in liver and heart (Fig. 6). While PBCspossessed a relatively large quantity of OfIRAK1 and OfIRAK3 tran-scripts, the level of OfIRAK4 transcript was moderate in PBCs.
parison of genomic organization of vertebrate IRAK4 gene. (A) In OfIRAK4, exonsWhereas, introns are shown as black lines with the corresponding sizes (bp). The insetces are shown in lowercase and the acceptor and donor sites in intron sequences areomparative analysis includes IRAK4 gene from pisces (B) and tetrapods (C). The size of, respectively. Conserved sizes of exons are indicated and if not conserved, labeled withble 2. While shaded boxes show CDSs, UTRs are indicated with blank boxes.
Table 2Comparison of genomic arrangement and summary of exon-intron structure for IRAK4 gene from different teleost species (A) and different tetrapod classes (B).
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3.5. Detection of IRAK4 mRNAs in fish injected with flagellin
Flagellin stimulates the TLR5 and signal is transduced to down-stream cascade through IRAK4. Thus, we initiated the temporaltranscriptional analysis of OfIRAK4 in different tissues collected from
Fig. 5. Analysis of the putative promoter region of rock bream IRAK4 (OfIRAK4). The sequbent-arrow. No TATA or GC box was detected. Possible NF-kB, Sp1, CTCF, TF-AP2C, AP-2aAdirections whenever possible. The available transcribed sequence (50 UTR) up to translation
FLA-ST immunized animals using qPCR (Fig. 7). While the OfIRAK4expression almost remained unchanged in PBCs, it was significantlydown-regulated in gills (p < 0.05) (Supplementary Fig. 3). Mean-while, a modestly but significantly up-regulated response was eli-cited by spleen (1.9-fold) and kidney (1.6-fold) (p < 0.05) (Fig. 7 and
ence numbers are relative to the translation-initiation site (þ1) which is indicated by a, CREB1, c-Jun binding sites are differentially marked and indicated below, with thestart site (atg) is shown with gray shade.
Fig. 6. Constitutive mRNA expression of rock bream IRAKs (OfIRAKs). Relative transcript levels of three OfIRAKs were examined in 11 different tissues of healthy juveniles by SYBRgreen qPCR. Rock bream b-actinwas chosen as internal reference gene. The calculation was performed using Livak method and values were calibrated against mRNA level in muscle.The results are reported as mean ± standard deviation (SD) of triplicates. Ms, muscle; It, intestine; Br, brain; Sk, skin; Gl, gill; Lv, liver; Bl, blood (PBCs); Sp, spleen; Ht, heart; Kd,kidney; Hk, head kidney. Inset figure shows the OfIRAK4 mRNA distribution in rock bream tissues in declining order. Statistical analysis was performed by one-way analysis ofvariance (ANOVA) followed by Turkey's Multiple Range test using SPSS 16 program. Means with the different letters are significantly different at p < 0.05 level. (For interpretation ofthe references to color in this figure legend, the reader is referred to the web version of this article.)
N. Umasuthan et al. / Fish & Shellfish Immunology 43 (2015) 436e448444
Supplementary Fig. 3). In contrast, a strong induction of OfIRAK4transcription was detected in head kidney (2.4-fold), liver (3.7-fold)and intestine (4.8-fold) (p < 0.05; Fig. 7).
3.6. Detection of IRAK4 mRNAs in fish challenged with PAMPs orpathogens
We further characterized the kinetic OfIRAK4 mRNA expressionin liver and head kidney post-injection of different immune stimuli
Fig. 7. Detection of rock bream IRAK4 (OfIRAK4) mRNA expression after flagellin injectimRNA level in rock bream spleen, head kidney, liver and intestine was calculated by the compis presented as relative to mRNA level of PBS-injected group at each time point. The vertical bvariance (ANOVA) followed by Duncan's Multiple Range test using SPSS 16 program. Meaninterpretation of the references to color in this figure legend, the reader is referred to the
(Fig. 8). The qPCR data indicated that all immune challenges causeda similar pattern of significantly induced OfIRAK4 transcript levelsin liver (Fig. 8A). The Gram-negative bacterial- and double-stranded (ds) RNA viral-mimics, LPS and poly I:C, stimulated theOfIRAK4 transcription to highest level at 3 h p.i. with 7.8-fold and8.4-fold, respectively (p < 0.05). Interestingly, bacterial strains usedin the current study increased the OfIRAK4 expression at 12 h p.i.S. iniae and E. tarda demonstrated a 11-fold and 32.5-fold dramaticincrease in its transcript levels, respectively (p < 0.05). In contrast,
on. Quantification was conducted in selected tissues by SYBR green qPCR. The relativearative Ct method using b-actin as reference gene. The fold change in mRNA expressionars represent the S.D. (n ¼ 4). Statistical analysis was performed by one-way analysis ofs with the different letters in a tissue are significantly different at p < 0.05 level. (Forweb version of this article.)
Fig. 8. Detection of rock bream IRAK4 (OfIRAK4) mRNA expression after flagellin injection. Quantification was conducted in selected tissues by SYBR green qPCR. The relativemRNA level in rock bream liver (A) and head kidney (B) was calculated by the comparative Ct method using b-actin as reference gene. The fold change in mRNA expression ispresented as relative to mRNA level of PBS-injected group at each time point. The vertical bars represent the S.D. (n ¼ 3). Statistical analysis was performed by one-way analysis ofvariance (ANOVA) followed by Tukey's Multiple Range test using SPSS 16 program. Means with the different letters in a challenge are significantly different at p < 0.05 level. (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
N. Umasuthan et al. / Fish & Shellfish Immunology 43 (2015) 436e448 445
an initial increase at 3 h (5.3-fold) followed by a secondary increaseat 48 h (7-fold) p.i. was noticed in animals injected with RBIV.
Temporal transcriptional profile of OfIRAK4 in head kidney isshown in Fig. 8B. Poly I:C and RBIV induced a relatively moderateOfIRAK4 transcription with a peak at 12 h (2.2- and 2.5-fold,respectively). However, dramatic changes occurred upon in vivoinjection of LPS and bacterial strains. While LPS induced an im-mediate response at 3 h p.i. (5.6-fold), E. tarda (4.7-fold) and S. iniae(5.4-fold) revealed their maximal induction at 6 h p.i. (p < 0.05).
These evidences suggest that OfIRAK4 was transcriptionallyresponsive to both immunogens and pathogens.
4. Discussion
In this study, we describe a new teleost member that belongs tothe growing IRAK4 subfamily from a marine fish, rock bream. TheIRAK4plays a crucial role inTLR/IL-1Rmediated signaling byacting asa central mediator. Transduction of signals perceived by all the TLRs
N. Umasuthan et al. / Fish & Shellfish Immunology 43 (2015) 436e448446
(except TLR3) occurs through an ancient and conserved pathwaymediated by MyD88-IRAK4-TRAF6 axis. This pivotal pathway regu-lates the immune and inflammatory responses through the produc-tion of numerous effector molecules including cytokines.
The rock bream IRAK4 identified hereinwas encoded byOfIRAK4gene (1395 bp CDS) and translated to 464 amino acids. As amember of multi-domain IRAK family protein, OfIRAK4 demon-strated a conserved bipartite structure composed of an N-terminalDD and a central kinase domain, PKc, with characteristic signatureswhich were also found in reported IRAKs including human IRAK4[9]. The MyD88 interacts with IRAK4 through a homotypic-DDinteraction [28], and activate the IRAK4, which subsequentlytransduces the signal to the other downstream components.However, IRAK4 lacks a C-terminal domain part that interacts withthe TRAF6 [29], when compared to that of other IRAK familymembers.
Highest homology of OfIRAK4 was noted with its homolog ofroughskin scuplin fish. Alignment and homology matrix datafurther supported the tight conservation of DD and PKc domains ofIRAK4s in different species. In contrast, the linker region possesseda number of residues that had undergone substitutions due to theselective pressure and positive selection during the evolution,causing a high degree of variability among different species [30].The DD of OfIRAK4 had conserved contact residues, known fromhuman IRAK4, binding with MyD88 and IRAK2 [8]. A pivotal res-idue that controls access to a preexisting hydrophobic pocket toavoid the exploitation of ATP is known as gatekeeper [31]. Tyrgatekeeper, an exclusive feature of IRAK family, was also present inOfIRAK4. A site-directed mutagenesis study has shown that Asp311
to Asn in human IRAK4 demolished its phosphorylation suggestingits importance in kinase activity. Among four IRAK paralogs, IRAK1and IRAK4 possess kinase activity, due to this critical Asp residue.However, IRAK2 and IRAK3/M lacked catalytic kinase activity, sincethis Asp residue was replaced [32]. As expected, this functionalcatalytic site was conserved in all the IRAK4s of examined species,including rock bream. All these shared domain architecture andother structural composition of OfIRAK4 suggested that it could befunctionally active and manifest biological roles similar to itsmammalian counterparts.
Even though IRAK family members from different species shareconserved features, evidence suggested that they have undergonevarious divergence in terms of sequence, structure and function[30]. Evolutionary aspects of IRAK family were examined through aphylogenetic analysis using members of all four subfamilies ofIRAK, including an IRAK1-like, IRAK3-like and the IRAK4 sequencesidentified from rock bream by transcriptome analysis. Not all thevertebrate classes contain all four IRAKs in their genome exceptmammals [30]. While the genomes of bony fish lack IRAK2, avespossess neither IRAK1 nor IRAK3. Interestingly, IRAK3 is only pre-sent in mammals and fish. Thus, it is apparent that IRAK subfamilieshave evolved in a discontinuous manner. Molecular phylogenyanalysis further indicated that IRAK4 might be the ancestor-likegene from which the other IRAK paralogs diverged through agene-duplication event, which took place in the early metazoanlineage [30]. The IRAK4 homolog has been identified in in-vertebrates, including some ancient species of sponge (Amphime-don queenslandica); and however, a different entity is present inarthropods (Pelle and Tube) [33,34] and nematodes (PIK-1). Thissporadic evolutionary existence of IRAK4 gene might be resultedfrom its discrete characteristics compared to that of other IRAKparalogs. Additionally, the branching pattern of the phylogenetictrees in this study was essentially consistent with the evolutionaryrelationships among the examined species.
Despite of the studies reporting the identification of IRAK4 gene,information regarding the genomic arrangement is rather limited
[19]. In the current study, we determined that OfIRAK4 is structuredwith eleven exons and ten introns. The OfIRAK4 essentiallyresembled the other teleost counterparts and/or the primordialIRAK4 gene. Moreover, inter-species genomic comparison of IRAK4gene dissected the possible modifications it might have undergonethroughout the evolution.
The teleost IRAK4s and other tetrapod IRAK4s slightly variedwith respect to certain aspects. For instance, tetrapod IRAK4members possessed additional exon(s) representing the extended50 UTR (Fig. 5; Table 2). Out of eleven, seven exons in vertebrateIRAK4s were identical in size. In addition, certain exons were groupspecifically equal in length (E8/E9, 187/184 and E10/E11, 165/159 inteleost and tetrapod lineages, respectively). According to a recentreport, IRAK4 of some salmonid species, such as rainbow trout andwhitefish has been subjected to rearrangement through an exon-fusion event (Table 2A) [19].
Another intriguing finding was the differences between verte-brate and invertebrate IRAK4s. While the vertebrate IRAK4spossessed a relatively ‘stable’ gene structure with higher intron-content, selected invertebrate IRAK4 members revealed a diversi-fied structures mainly featured by less number of introns. It hasbeen proposed that early branching eukaryotes are relativelyintron-poor compared with the intron-rich late-evolved eukary-otes, mainly due to the continuous accumulation and/or retentionof introns [35,36]. In consistent with this hypothesis, our dataimplicate that IRAK4 gene could have accumulated intronsthroughout the evolution, which allowed the higher-order verte-brate IRAK4s to be alternatively spliced. For instance, a pseudogeneof trout termed IRAK4swas suspected to be the spliced copy of troutIRAK4 gene and it was suggested that they originated from a recentgene duplication event [19].
Among IRAK-family, plenty of evidence showed the transcrip-tional regulation of IRAK1 and IRAK2, that encode two importantdownstream role players of IRAK4 [37,38]. However, to the best ofour knowledge, isolation and analysis of IRAK4 promoter-likesequence was not reported so far. Herein, we obtained the puta-tive promoter sequence of OfIRAK4 by using primer-walking tech-nique to sequence the BAC clone. Immediately upstream ofavailable 50-UTR, the proximal promoter of OfIRAK4 was rich interms of TFBS. Principally, two NF-kB binding motifs were presentwhich were surrounded by other TFBS, such as Sp1, CTCF, TF-AP2Cand AP-2aA. Considered that IRAK-family members may be tran-scriptionally regulated, at least in part, by common transcriptionfactors, we noticed some of the above TFBS in OfIRAK4 promoter,which have been evidently shown to regulate the expression ofother IRAKs [37,38].
Using qPCR, we showed that OfIRAK4 was constitutively tran-scribed in various rock bream tissues examined, as reported inhuman [39] and teleosts [16e18]. Zebrafish IRAK4was differentiallydetected in various developmental stages and tissues of adult fishand consequently, it was implicated in development and immunity,respectively [16]. Consistently, the omnipresence of OfIRAK4 tran-scripts implied its involvement in a broad spectrum of physiologicalprocesses [10,40]. Earlier reports revealed the teleostean IRAK4 tobe dominantly expressed in head kidney and spleen [16,17,19].Nevertheless, similar to a contrasting pattern noticed in roughskinsculpin with highest IRAK4 transcripts in skin [18], OfIRAK4 mRNAlevel was prominently detected in liver, along with moderate levelsin head kidney and spleen. Dual blood supply to the liver fromgastro-intestinal tract and via the hepatic artery makes the liverhighly susceptible to pathogens. Therefore, liver is likely enrichedin complex repertoires of immune competent cells [41,42], whichmight express IRAK4 to effectively mediate the TLR signaling. Theseexpressional characteristics signify the hallmark function of IRAK4in immunity. However, these comparative results need to be
N. Umasuthan et al. / Fish & Shellfish Immunology 43 (2015) 436e448 447
interpreted with caution, since discrepancies in transcription levelsmight have resulted from species-specific variation, developmentalstage and genetic background.
We investigated the transcript levels of OfIRAK1 and OfIRAK3along with OfIRAK4 using qPCR. Surprisingly, liver, PBCs and heartrepresented the tissues of higher OfIRAK abundance in an interre-lated manner (Fig. 6), albeit at different levels. So far, no IRAK3homolog has been characterized in teleosts and hence our datamaycontribute to the overall understanding about the expression ofIRAK homologs in teleosts. Moreover, OfIRAK1 expression patterncorroborates with that of grass carp IRAK1 [43]. Although, previousfindings suggested redundant roles for IRAKs, emerging evidencesupport that each IRAK member is devoted for distinct physiolog-ical role [3]. Tissue-specific transcription of OfIRAKs complied withthis hypothesis; however, further research should investigate thepertinent mechanisms.
Bacterial flagellin is a potent immunogen recognized by hostcells through TLR5 and the systemic defense responses are elicited[44]. This process is MyD88-dependent, in which IRAK4 functionsas a crucial transducer passaging the signals to the downstream.Apart from Basu (2012), there is a general lack of research in teleostresponse against FLA-ST administration [45]. This study was un-dertaken to evaluate the mRNA expressional response of OfIRAK4upon injection of immunogens including FLA-ST and potential live-pathogens.
Upon FLA-ST challenge, OfIRAK4 was significantly up-regulatedin intestine, liver, head kidney and spleen. While it persisted atbasal level in PBCs, it was down-regulated in gills. Intriguingly, thetranscriptional pattern of TLR5 signaling genes in rock breamincluding TLR5M, MyD88, and TRAF6 were found to be identical inhead kidney with highest levels at 24 h p.i. of FLA-ST ([46] and/orUnpublished data). The gastrointestinal bacterial flora from food-or environmental-origin could be recognized by TLRs in intestinalcells, and activate the TLR signaling cascade [47]. The maximuminduction of OfIRAK4 detected in intestine suggested that TLR5M-mediated FLA-ST recognition, subsequently up-regulated theOfIRAK4 (and adaptor molecules; our unpublished data and [45,46])to trigger the downstream cascade. This data indicate that juvenilerock breams possess an ability to respond to the in vivo FLAencounter, and do so in part through the transcriptional modula-tion of OfIRAK4 in a tissue-specific manner. To the extent of ourknowledge, the current investigation is the first to report theresponse of an IRAK4 homolog in a wide range of fish tissues uponFLA-ST challenge.
Increasing evidence support that teleost liver and liver-residingcells play a vital role in immunity [42]. Teleost head kidney, themammalian bone marrow equivalent, is one of the importanthaemopoietic and immunological organ. Consequently, these or-gans are likely candidates for examining the modulated mRNAexpression of immune genes. The qPCR in current study detectedthat hepatic-OfIRAK4 expression was enhanced to 7-fold or aboveduring different experimental injections compared with the cor-responding controls. The E. tarda bacterium, an emerging pathogenin aquaculture, causes edwardsiellosis in a wide array of species[48]. This pathogen drastically induced the OfIRAK4mRNA levels upto 32-folds in liver. In head kidney also we noticed significantlyelevated OfIRAK4 mRNA expression upon different challenges.However, the level of inductionwas low, when compared with thatof liver, suggesting that liver OfIRAK4 expression is sensitivelymodulated upon encountering a pathogen-insult.
While Pelle, the IRAK4 homolog in Drosophila, operates againstfungi and Gram-positive bacteria [33,34], evidence indicate thatmammalian IRAK4 is involved in generating innate responsesagainst both Gram-positive and Gram-negative bacteria [9,49]. Inrecent years, different researchers have investigated the temporal
expression of IRAK4 in various animal models and reported its up-regulated expression under pathological conditions. ChallengewithVibrio and/or Staphylococcus bacteria resulted IRAK4-induction insmall abalone and shrimp [15,50]. Similarly, increased IRAK4 tran-scription has also been reported in fish species such as roughskinsculpin (by LPS), half-smooth tongue sole (Vibrio anguillarum) andzebrafish (E. tarda) [16e18]. However, a contrasting finding wasreported in trout that IRAK4 transcription was barely modulated inan infection trial with Aeromonas salmonicida [19].
It was apparent that bacteria and bacterial PAMP (LPS)demonstrated relatively strong impact on OfIRAK4 transcription inhead kidney, when compared to that of RBIV and viral PAMP (polyI:C). Unlike in head kidney, hepatic-OfIRAK4 induction by bacteriaand virus or corresponding PAMPs was almost similar. Whenzebrafish was exposed to snakehead rhabovirus (SHRV), IRAK4transcription mostly remained unchanged [16].
Studies with IRAK4-deficient macrophages resolved that TLR3signaling requires IRAK4 [9]. Meanwhile, it has already beenproven in human that TLR7-, TLR8- and TLR9-dependent induc-tion of IFN-a/b and -l is strictly IRAK4-dependent [51]. Poly I:C isan agonist of TLR3 and multiple viral derivative compounds(ssRNA, unmethylated DNA) could act as agonists for TLR7, TLR8and TLR9, whose antiviral response depends on IRAK4. These factsmight explain, why poly I:C and RBIV mediated the OfIRAK4expression in rock bream tissues. The data of current study alongwith the evidence presented thus far support the idea that OfIRAK4might play an important role in bacterial- and viral-induced TLRsignaling. An attempt to functionally integrate the trout IRAK4 intomammalian system showed that it blunted the signaling cascade[19]. Therefore, future research needs to examine the detailedunderlying mechanism to investigate the functional roles andtranscriptional regulation of IRAK4. Moreover, further efforts toobtain the complete sequences of IRAK1 and IRAK3 would helpcharacterizing them and to completely identify the IRAK-membersin rock bream.
5. Conclusion
In summary, this study describes the genomic and cDNA iden-tification and characterization of a teleost IRAK4 from rock bream.The OfIRAK4 revealed the classic structural characteristics of IRAKfamily and shared higher identity and evolutionary relatednesswith its teleost counterparts. Of note, phylogenetic and genomicanalyses implied that teleost IRAK4 is distinct and diverged fromthat of other tetrapods. Our mRNA expression data demonstratedits basal expression in a wide range of tissues. Whereas, it wassignificantly modulated by immunogens and pathogens suggestingits putative involvement in different TLR signaling cascades.
Acknowledgment
Authors are grateful to Dr. Hyung-Bok Jung, Dr. W.D.N. Wick-ramaarachchi and Yucheol Kim, who helped in FLA challengeexperiment. Special thanks go to Prof. Willem B. Van Muiswinkelfor critical review of this manuscript and providing helpful sug-gestions. This research was supported by the project titled ‘Devel-opment of Fish Vaccines and Human Resource Training’, funded bythe Ministry of Oceans and Fisheries, Korea.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.fsi.2014.12.010.
N. Umasuthan et al. / Fish & Shellfish Immunology 43 (2015) 436e448448
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