A new antimicrobial peptide SCY2 identified in Scylla Paramamosainexerting a potential role of reproductive immunity
Kun Qiao a, 1, Wan-Fang Xu a, 1, Hui-Yun Chen a, b, c, Hui Peng a, b, c, Ya-Qun Zhang a,Wen-Shu Huang a, Shu-Ping Wang a, Zhe An a, Zhong-Guo Shan a, Fang-Yi Chen a, b, c,Ke-Jian Wang a, b, c, *
a State Key Laboratory of Marine Environmental Science, College of Ocean & Earth Science, Xiamen University, Xiamen, Fujian, PR Chinab Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen, Fujian, PR Chinac Fujian Engineering Laboratory of Marine Bioproducts and Technology, Xiamen University, Xiamen, Fujian, PR China
a r t i c l e i n f o
Article history:Received 15 November 2015Received in revised form15 February 2016Accepted 17 February 2016Available online 18 February 2016
A new antimicrobial peptide named SCY2 with 65.08% identity in amino acid sequence to the knownscygonadin (SCY1) was first characterized in Scylla paramamosain based on its cloned full-length cDNAand genomic DNA sequences. The SCY2 gene was dominantly expressed in the ejaculatory duct of malecrabs and its mRNA transcripts were discerned mainly in the glandular epithelium of the inner wall andthe secretion inside the ejaculatory duct. Although the SCY2 gene could not be induced with the chal-lenge of the bacteria and fungi tested, its induction reached the highest level at the peak period of matingin mature male crabs either in June or November, suggesting its induction was likely related to seasonalreproduction changes. Moreover, it was interesting to note that, from analysis of its transcripts andprotein, SCY2 was significantly expressed only in the ejaculatory duct of pre-copulatory males beforemating, however it was clearly detected in the spermatheca of post-copulatory females after matingaccompanied by the decreased level of SCY2 expression in the ejaculatory duct. These results suggestedthat the SCY2 was probably transferred from the male during mating action with the female for thepurpose of protecting fertilization. The recombinant SCY2 was more active against the Gram-positivethan the Gram-negative bacteria tested. It was further observed that the SCY2 transcripts were signifi-cantly increased with addition of exogenous progesterone in tissue cultures whereas the several otherhormones tested had no any effect on SCY2 expression, indicating that there might be a relationshipbetween the SCY2 expression and the induction of hormones in vivo. In summary, this study demon-strated that one role of SCY2 was likely to be involved in crab reproduction and it exerted its reproductiveimmune function through the mating action and the maintenance of inner sterility in the spermatheca ofthe female, thus leading to successful fertilization of S. paramamosain.
Crustaceans, which lack an acquired immunity [1], are depen-dent mainly on the innate immune system to protect themselvesfrom infection. Antimicrobial peptides (AMPs) are a majorcomponent of the innate immune defense in marine invertebrates
including crustaceans and exert wide activities against bacteria,fungi, viruses or parasites when necessary. The mud crab Scyllaparamamosain lives in a complex aquatic environment and theirreproductive tracts, which are usually exposed to the externalenvironment, are subject to microbial invasion simply owing totheir continuity with the environment [2]. However, the fact thatcrabs can maintain successful fertilization and survival of thezygote under these complex conditions suggests that someimmune-associated components exist in the reproductive systemin order to protect the semen from infection during the insemi-nation process, and their immune response may be exceptionallyefficient in pathogen elimination as witnessed by the invertebrateevolutionary success [3]. In invertebrates, some AMPs have been
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isolated from the male or female reproductive tract: andropin isexpressed exclusively in the male reproductive tract and is char-acterized in Drosophila melanogaster [4]; ceratotoxin A and B arepresent in the female reproductive tract of Ceratitis capitata [5]; incrustaceans, a 20 kDa protein from the seminal plasma of Scyllaserrata has antibacterial activity [6]; and, in addition, the recentlydiscovered novel AMP scygonadin (SCY1) inmud crabs is associatedwith the reproductive immunity in S. paramamosain [7,8]. As pre-viously reported in mammals, two major families of cationic AMPsdefensins and cathelicidins, which are characterized in mammals,are specifically produced in the male genital tract [9e11] and bothare involved in innate immunity.
The AMPs found in the reproductive tracts are thought to share acommon function in protection of the reproductive system frombacterial invasion in males or females. Recent investigation into therelationship between post-mating processes and immune functionreveals that mating and immunity are also intimately linked [12].Correlation between reproduction and the immune responsestudied inDrosophila shows that sperm and accessory gland proteintransferred by the male during mating up-regulate the expressionof antimicrobial genes [13]. This further strengthens that there arepotential components in the reproductive tract responsible forresistance to invading bacteria or other pathogenic microorgan-isms. Analysis of the specific proteins or peptides existing in thereproductive tract will facilitate elucidating the innate immunemechanism of crabs against the microorganisms present inambient sea water, thus giving an explanation why the marinecrabs can maintain a sterile environment in the reproductive tractand reach successful fertilization.
In our previous studies, an anionic AMP SCY1 is characterizedfrom the seminal plasma of S. serrata [14,15]. Both the native andrecombinant products of SCY1 are potently active against Gram-positive and Gram-negative bacteria in vitro [14,16,17]. Its expres-sion is predominantly in the ejaculatory duct of males and issignificantly present at the mature stage and during the matingperiod of male and female crabs [7,8], suggesting that its function isassociated with the reproductive process [7,8]. In the process ofmolecular cloning of SCY1, a homologous cDNA sequence wasscreened and the predicted amino acid sequences were approxi-mately 65% identical. The cDNA sequence homologous to SCY1 wasprobably a new gene encoding a variant of SCY1. The putativepeptide hitherto described was named SCY2, after the genus Scylla.The purpose of the present study was to characterize the newlyscreened SCY2 gene and further differentiate it from the knownSCY1. The cDNA sequence and its genomic DNA organization wereelucidated. Its mRNA transcript abundance was investigated indifferent tissues of male and female S. paramamosain. The geneexpression patterns of SCY2 in adult males challengedwith bacteria
and during the mating season were elucidated comparatively. Inaddition, the antimicrobial activity of SCY2 was investigated usingthe recombinant product. Moreover, considering that SCY2 mighthave a role in the reproductive process as an insightful observationin the study of SCY1, a detailed study potentially associated withreproductive immunity was subsequently evaluated. Character-ization of this new AMP SCY2 will provide valuable information tofurther understand how the species S. paramamosain successfullymaintains reproduction in the complex marine environment.
2. Materials and methods
2.1. Cloning of the SCY2 cDNA sequence
The sample RNA was extracted from tissues of S. paramamosainas in our previous study of SCY1 [15]. The 30 end region of SCY2 wasamplified using PCR with a primer pair of Adaptor/Sh1. The PCRassay was performed generally as described previously [15]. The 50
end region of SCY2 was amplified using the SMART™ RACE cDNAAmplification Kit (BD Biosciences Clontech) in accordance with themanufacturer's instructions. The male gonad cDNA was synthe-sized using a modified lock-docking Oligo (dT) primer (termed the50-RACE CDS Primer (50-CDS)) and the SMART II A Oligonucleotide;and one gene-specific primer Ah1 (Table 1) was designed followingthemanufacturer's instructions. The PCR products were cloned intothe pMD-18T vector (TaKaRa) and sequenced (Invitrogen).
2.2. Determination of SCY2 genomic DNA organization
The procedure for amplification of the SCY2 genomic DNA wasas described previously [15]. The primers used (the forward primerSh2 and reverse complementary primer Ah2) are shown in Table 1.Inverse PCR was used to amplify the 50- and 30-flanking sequencesof SCY2 with the specific nest primers Shy1, Ahy1, Shy2 and Ahy2.The PCR product was purified and its sequence was analyzed asdescribed above. Intron positions and transcription factors wereidentified in comparison with the SCY2 cDNA sequence.
2.3. Sequence analysis
Sequence data were analyzed using DNAStar software. PCRprimers were designed using Primer Premier 5. A homology searchwas performed with BLASTN 2.1.3 and BLASTP 2.1.2 using the NCBINet WWW Server (http://www.ncbi.nlm.nih.gov/blast). The amino-terminal signal sequence was predicted using the SignalP 3.0 server(http://www.cbs.dtu.dk/services/signalP). Both mass and pI of theputative peptide were calculated separately using the ProtParamtool in ExPASy (http://www.expasy.org/tool/protparam). The ORF
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was predicted using ORF Finder (http://www.ncbi.nih.gov/gorf/gorf.html).
2.4. Specific amplification and differentiation of the SCY2 gene fromthe SCY1 gene using the corresponding specific primers and probes
The mature sequence of SCY2 cDNA was amplified from thereverse transcript reactant using a pair of primers, Sh2 and Ah2(Table 1), which was designed as a template for making a specificSCY2 cDNA probe (281 bp). The PCR product was purified,sequenced and labeled with digoxigenin using the Dig High PrimeDNA-labeling and Detection Starter Kit I (Roche Applied Science).Southern blot was carried out to confirm the PCR products. The PCRproducts obtained from the S. paramamosain gonad RNA weretransferred from the 2% agarose gel to the membrane using vacuumblotting. The procedure was performed as described above usingthe Dig High Prime DNA-labeling and Detection Starter Kit I (RocheApplied Science).
2.5. Analysis of SCY2 mRNA transcripts in tissues of normal adultmale and female S. paramamosain
To determinewhether SCY2mRNA transcripts exist in bothmaleand female crabs and to analyze the distribution of SCY2 mRNAtranscripts in different tissues, 10 normal mature crabs (five malesand five females) were carefully dissected. The tissues tested weresubcuticular epithelia (body wall), gills, heart, hepatopancreas,reproductive system, muscle, mid gut, thoracic ganglion, brain andhemocyte, and they were individually collected for RNA extraction.For male crabs, the reproductive tissues included testis, seminalvesicle, ejaculatory duct, the posterior ejaculatory duct and penis.For female crabs, the reproductive tissues included spermatheca,ovary and vagina. Total RNA for each tissue was extracted usingTrizol reagent as described above.
2.6. Quantitative real-time PCR (qPCR)
qPCR was performed within a reaction mixture of 20 mL con-taining 0.5 ng of total transcribed cDNA to detect the expression ofthe SCY2 gene. SCY2-S2 and SCY2-A2 (Table 1) were used as for-ward and reverse primers, and five pmol of each primer was used.The reaction was carried out using 10 mL Power SYBR Green PCRMaster Mix (Applied Biosystems, UK) in accordance with themanufacturer's specifications in a 7500 Real-Time PCR system (ABI)with the following procedure: 50 �C for 2 min, 95 �C for 10 min,followed by 40 cycles of 95 �C for 15 s, 60 �C for 1 min. Amplifi-cations were repeated in triplicate. No-template controls wereincluded on each plate. Melting curve analysis was carried out forspecificity. For relative qPCR, b-actin gene was used as the internalcontrol for normalization of the gene expression data.
The absolute standard curve qPCR method was performed as inour previous study of SCY1 [7]. The PCR products of SCY2 wereligated into the pMD-18T simple vector (TaKaRa) following themanufacturer's specifications. A standard curve was constructedusing a 10-fold serial dilution of PCR product inserted plasmid DNAstandard (1 � 102e1 � 108 copies/mL). The concentration of eachsamplewas determined by comparing its cycle threshold (CT) valueagainst the standard curve using 7500 system SDS software version1.3.1.21. The reaction mixture and the procedure were followed asdescribed in relative qPCR.
2.7. Expression analysis of the SCY2 mRNA transcripts in differentdevelopmental stages of male S. paramamosain
To analyze the SCY2mRNA transcripts expressed in the different
developmental stages, samples of embryos and crabs at the zoea I,zoea III, megalops and juvenile stages were individually collectedfrom a local aquaculture farm at Zhao An, Fujian Province, China.Considering that the gonad maturity of a normal crab is normallyrelated to its body weight, three different weights (100, 200, 300 g/crab, n ¼ 5) of male crabs were chosen to investigate SCY2 mRNAtranscripts. Dissection examination revealed one group of smallercrabs (100 g) whose gonads were not fully developed, and twogroups of mature crabs (200 g and 300 g), whose gonads were fullydeveloped. Gonads were individually collected for RNA extraction.
2.8. Seasonal investigation of the SCY2 gene expression pattern inthe ejaculatory duct of mature male S. paramamosain
Sexually mature male crabs (250 ± 20 g, n ¼ 5) were carefullycollected from January to December. Dissection examination wascarried out at each sample time to confirm that the crabs sampledhad developed gonads, before they were used for absolute quan-titative measurement of SCY2 mRNA transcripts. Samples of theejaculatory duct frommale crabs were separately collected for RNAanalysis. The procedure for sample treatment and analysis of SCY2mRNA transcripts were the same as described above.
2.9. In situ hybridization (ISH)
The samples were freshly frozen with liquid nitrogen,embedded in OCT at �20 �C and then sectioned at 4e6 mm on amicrotome (LEICA CM 1900). The sections were spread on poly-L-lysine-treated slides for immediate use or stored at �80 �C. TheSCY2 gene was cloned into a pMD-18T plasmid (TaKaRa) and usedas the DNA template for making SCY2 probes using PCR and a PCRDIG Probe Synthesis Kit following the manufacturer's protocol(Roche). The procedure was similar to that in our previous study[7]. Frozen sections of the ejaculatory duct of mature male crabswere tested with DIG labeled cDNA probes and detected with AP-conjugated anti-DIG antibodies. The control for ISH consisted ofnon-DIG-labeled SCY2 probes with PCR production.
2.10. Recombinant expression and antimicrobial activity of SCY2
Primers SF and SR (Table 1) were synthesized in order togenerate the sequence encoding themature SCY2 peptide, and thenthe target sequence was inserted into a pPIC9K pTrc-CKS vector.The recombinant protein was expressed and purified based on ourprevious study [17]. The antimicrobial activity of the recombinantSCY2 was determined against a panel of microorganisms includingGram-positive Micrococcus luteus, Corynebacterium glutamicum,Bacillus subtilis and Micrococcus lysodeikticus and Gram-negativeAeromonas hydrophila, Vibrio parahaemolyticus and Escherichiacoli. The minimal inhibitory concentration (MIC) was calculated aspreviously described [16e18].
For the in vivo experiment: the cultured V. parahaemolyticus inmid-logarithmic growth phase was harvested by centrifugationwith 5000 rpm for 5 min followed by washing twice with 1 � PBS,and then resuspended in 1 � PBS. The purified SCY2 protein(200 mg/mL) was incubated with V. parahaemolyticus (7 � 107 cfu/mL) at room temperature for 1 h, and then 100 mL injected into eachcrab. V. parahaemolyticus treated with PBS buffer only was used as acontrol.
2.11. Anti-SCY2 polyclonal serum production in mice
To obtain an antibody against SCY2, the recombinant proteinSCY2 expressed in Pichia pastoriswas purified andused to immunizeBALB/C mice. The mice were maintained for more than one month
Fig. 1. Nucleotide sequence and genomic DNA organization of SCY2. (A) Nucleotide sequence of the mud crab SCY2 cDNA and predicted amino acid sequence. The numbers on theleft of the sequence give the positions of the last nucleotide and amino acid on each line. Polyadenylation signal is underlined in the 30 UTR. The stop codon is indicated with anasterisk. Splicing sites for introns 1 and 2 are indicated with triangles. The GenBank accession number is EF555444. (B) Genomic DNA organization of SCY2. The predicted or-ganization of the peptide domains (signal peptide and mature peptide) is shown by gray boxes. The genomic DNA sequence has been deposited in the GenBank nucleotide sequencedatabase and given the accession number EF555445.
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until the level of serum antibody reached the requirements of theexperiment. The anti-SCY2 IgG was purified using NAb™ Spin Kits(Thermo). The concentration, titer, purity and specificity of thepurified anti-SCY2 antibody were analyzed using an ultravioletspectrophotometer, ELISA, SDS-PAGE andwestern blot, respectively.
2.12. Protein extraction and western blot
The total proteins were extracted from the crab ejaculatory ductusing RIPA lysis buffer. The concentration of the total protein wasdetermined using the BCA Protein Assay Kit (Pierce™). Tricine-SDS-PAGE assay was performed with 40 mg total protein. Transfer ofproteins to PVDF-membrane (Amersham) and immune detection ofGAPDH, b-actin and SCY2 with GAPDH, b-actin antibody (SantaCruz Biotechnology) and anti-SCY2 IgG, were carried out following
the standard procedures. Secondary horseradish peroxidase-conjugated antibodies were used, and detected with ECL WesternBlotting Substrate (Millipore).
2.13. Immunofluorescence
The method of immunofluorescence was similar to that in ourearlier studies [19]. However, we used Cy5-conjuncted anti-SCY2polyclonal antibodies (1:50 dilution in PBS) for 1.5 h at 37 �C in ahumidified chamber. After washing, sections were incubated withan appropriately diluted DAPI solution (0.25 mg/mL) at room tem-perature for 15 min. Finally, the sections were mounted with cov-erslips using anti-fademountingmedium. The color of the antibodystaining in the tissue sections was observed under a Leica fluores-cence microscopy imaging system.
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2.14. Hormone induction
For in vitro experiments, ejaculatory duct obtained frommaturemale crabs were cut into 4e6 mm3 pieces under sterile conditions[20,21], and cultured in L15 (without phenol red)-crab saline me-dium supplemented with epidermal growth factor (20 ng/mL) andglucose (1 mg/mL) in 24-well flat-bottom plates [22]. The steroidhormones 17b-estradiol and progesterone were purchased fromSigma-Aldrich; testosterone from the Ningbo A second hormonefactory (China); and mifepristone from Tocris Biosciences (Bristol,UK). The steroid hormones (17b-estradiol, testosterone, progester-one) and mifepristone (an inhibitor of progesterone) were firstdissolved in ethanol as stock solutions (100 mM), and then dilutedin the medium to approximately 10 mM. Plates were incubated at28 �C for 24 h, followed by RNA extraction.
3. Results
3.1. Determination of the full-length cDNA and genomic DNAsequences of SCY2
The full-length cDNA sequence of SCY2 was determined from
Fig. 2. SCY2 mRNA and protein distribution. (A) Distribution of SCY2 mRNA transcripts andqPCR and western blot. T: Testis; SV: Seminal vesicle; ED: Ejaculatory duct; PED: PosterioSubcuticular epithelia; Gi: Gill; Mu: Muscle; Hp: Hepatopancreas; Mg: Mid gut; Ht: Heart;female S. paramamosain analyzed using absolute qPCR and western blot. OA: Ovary; Vg: VagiS. paramamosain using ISH. A: positive; A’: negative; Bars ¼ 10 mm. (D) Detection of SCY2immunofluorescence.
the ejaculatory duct RNA of the normal mature S. paramamosain(Fig. 1A). This new gene (GenBank accession no. EF555444) was947 bp including a 50 UTR of 274 bp and a 30 UTR of 301 bp with theputative polyadenylation consensus signal (AATAAA) and 49 addi-tional nucleotides before the poly (A) tail. The complete cDNAconsisted of an ORF with 375 bases, a start codon (Met) and a stopcodon (TAA) and encoded a 124-amino acid protein. The predictedpropeptide was composed of two domains with a hydrophobicsignal peptide (24 amino acids) and a mature peptide (100 aminoacids). The deduced amino acid sequence began with a 24-residuesignal peptide rich in hydrophobic amino acids. The putativecleavage site for signal peptidase was probably located after theAla24 residue, which was predicted using SignalP 3.0. The deducedmature peptide consisted of 53 polar amino acids and 47 apolaramino acids. Because the theoretical pI of the mature peptide was4.84 (http://www.expasy.org/tools/protparam.html), it was sug-gested to be an anionic molecule. Searches in the SWISS-PROTprotein data-base revealed that the identity between the pre-dicted peptide of the new gene and SCY1 was 65.08%, indicatingthat they may belong to one family. In addition, there was no sig-nificant homology in the sequence to other reported AMPs.
The nucleotide sequence for the SCY2 gene and upstream region
protein in different tissues of normal male S. paramamosain analyzed using absoluter ejaculatory duct; P: Penis; Es: Eyestalk; Ne: Thoracic ganglion; Hc: Hemocyte; BW:(B) Distribution of SCY2 mRNA transcripts and protein in different tissues of normal
na; S: Spermatheca. (C) Detection of SCY2 mRNA in the ejaculatory duct of mature maleprotein in the reproductive system of normal male and female S. paramamosain using
Fig. 3. SCY2 gene expression in different developmental stages of S. paramamosain. (A)qPCR analysis of SCY2 gene expression in different developmental stages ofS. paramamosain. Samples were collected from crabs at different developmental stagesincluding embryo (Em), zoea I (Z1), zoea III (Z3), megalops (Me), juvenile males (J) andthe mature males with different weights (100, 200, 300 g) (for each weight n ¼ 5). Thedifference is extremely significant (P < 0.01). (B) Detection of SCY2 protein in theembryo of S. paramamosain using western blot. EM1-4: different embryo stages; ED:ejaculatory duct.
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was determined for S. paramamosain (GenBank accession no.EF555445, Fig. 1B). The gene consisted of two introns and threeexons (Fig. 1B), with the three exons of 405, 125 and 417 basesseparated by two introns. Intron 1 was longer and contained 997bases, while intron 2 was much shorter with only 154 bases. Exon 1encoded for 50 UTR, the signal peptide, and the partially maturepeptide. Exon 2 encoded part of the mature peptide and Exon 3encoded the remainder of the mature peptide and the 30 UTR. TheSCY2 gene also appeared to contain typical spliceosomal intronswith the donor and acceptor (GT and AG) dinucleotide sequencesfound in most vertebrate and invertebrate splice sites.
3.2. Demonstration of SCY2 mRNA transcripts in normalS. paramamosain
To make sure whether the expression of SCY2 was specific, thecross-reactivity between SCY2 and SCY1 genes was determinedusing Southern blot and western blot assays. Southern blot assay tohybridize the PCR product of each isoform gene was performedusing a mixture of two probes, of which one probe was designedspecific to the SCY2 gene and the other specific to the SCY1 gene.Results from this experiment showed that there was no cross-reactivity between the SCY2 and SCY1 genes (data not shown).The two isoform gene expression was clearly discriminated usingPCR assay with our designed specific primer pairs corresponding toeach isoform sequence in this study. Tricine-SDS-PAGE was used toseparate these two proteins from crab tissues. Specificity of anti-bodies was determined using western blot, and polyclonal anti-bodies distinguished differences between SCY1 and SCY2.
To determine the overall expression profile of SCY2 in vivo, theSCY2 gene expression in various tissues was investigated usingqPCR and western blot assay (Fig. 2A,B). The highest expression ofthis gene was present in the ejaculatory duct, and a relatively lowexpression was also observed in the post-ejaculatory duct, whilethe other tissues of the male and female crabs tested showed veryweak expression. SCY2 gene expression in the ejaculatory duct wassignificantly higher than that in other tissues (P < 0.05). The SCY2protein expression pattern was studied using western blot. Thehighest expression was in the ejaculatory duct, but it was alsoobserved in the post-ejaculatory duct and penis. This peptide wasnot detected in other tissues of male or female crabs.
ISH was performed to confirm the localization of SCY2 mRNAtranscripts using cDNA probes. Strong positive signals weredetected in a number of cells in the glandular epithelia of the innerwall and in the secretion inside the ejaculatory duct (Fig. 2C). Anaccumulation of blue dye precipitates was obvious in the cytoplasmof these cells. No signal was observed in the control, and the resultsindicated that the SCY2 gene was specifically expressed in theglandular epithelial cells.
To confirm the specificity of SCY2 protein in the tissues,immunofluorescence and IHC were performed on separate tissuesections of male and female crabs using anti-SCY2 IgG. The resultsdemonstrated that SCY2 protein was present in the ejaculatoryduct, posterior ejaculatory duct and penis of male crabs. Strongsignals were present in the secretion and glandular epithelia of theinner wall and in the connective tissue outside the ejaculatory duct(Fig. 2D), while the secretion inside the posterior ejaculatory ductand the penis still showed positive signals. In other male tissuesand in female crabs, no signal was observed (data not shown).
3.3. Expression analysis of SCY2 in different developmental stagesof male S. paramamosain
The mRNA expression of SCY2 in the different developmentalstages of male S. paramamosain was investigated using qPCR. As
shown in Fig. 3A, SCY2 was significantly expressed in mature malecrabs. Crabs of 300 g weight had complete gonad developmentwhich was recognized under dissection. A relatively small amountof SCY2 mRNA transcripts was also detected in adult (100 g and200 g) crabs but no signal was seen at the zoea I, zoea III or meg-alops stages, suggesting that SCY2 mRNA transcripts were presentonly after the juvenile stage.
To further check the tissue distribution of SCY2 protein duringdevelopment, IHC was performed on sections of embryo using anti-SCY2 IgG, and no positive signals were observed in the embryos.Western blot showed the same results (data not shown).
3.4. Seasonal SCY2 gene expression pattern
Since SCY2 was highly expressed in the male reproductive sys-tem of S. paramamosain, especially in mature male crabs, the SCY2genewas likely to be related to sexual maturity, and so the seasonalchanges of SCY2 mRNA expression in the sexually mature crabswere examined. It was interesting to note that a high level of mRNAexpression was present in January, June and November, while theprotein appeared in January, May, June and November (Fig 4A,B).The expression pattern of this gene was consistent with the sexualmaturity seasons of S. paramamosain and matched the mating pe-riods. These results suggested that this peptide was likely to beassociated with the reproductive action.
Fig. 4. SCY2 gene expression from January to December. (A) qPCR analysis of SCY2 gene expression (mean ± S.E. of replicate qPCRs) in the ejaculatory duct of S. paramamosain fromJanuary to December (n ¼ 5). (B) Detection of SCY2 protein in the ejaculatory duct of male S. paramamosain from January to December using western blot.
Table 2Antibacterial and bactericidal activities of the recombinant SCY2 expressed inP. pastoris.
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3.5. Expression analysis of SCY2 pre- and post-mating of male andfemale S. paramamosain
H&E staining was performed to examine the changes of SCY2transcripts and protein concentration in pre- and post-mating crabs.In comparison with the pre-mating crabs, there were obviousspermatophores in the ejaculatory duct, posterior ejaculatory ductand spermatheca of post-mating crabs (data not shown).
SCY2 mRNA transcripts in the ejaculatory duct of the pre- andpost-mating males, evaluated using qPCR, showed that the mRNAof SCY2 in the ejaculatory duct decreased after mating, butincreased in the post-ejaculatory duct and penis of males, and inthe spermatheca of females (Fig. 5A). The SCY2 protein in pre- andpost-mating crabs was detected using western blot and immuno-fluorescence. We quantified western blot with Quantity One anal-ysis software, and SCY2 protein in the ejaculatory duct showed thesame pattern as gene expression (Fig. 5B). In female crabs, positivesignals were detected in the spermatheca and vagina after mating(Fig 5C,D), but were not found in pre-mating females.
Microorganisms CGMCC No.b P. pastoris-derived SCY2
The values of MIC was expressed as the interval of concentration [a]-[b], where [a] isthe highest concentration tested at whichmicrobial growth can be observed, and [b]is the lowest concentration yielding no detectable microbial growth or that killedmore than 99.9% of the microorganisms (n ¼ 3).
a MIC: minimal inhibitory concentration.b CGMCC No.: China General Microbiological Culture Collection Center number.
3.6. Analysis of SCY2 expression in relationship to hormoneinduction
Comparison of the effect of hormone and LPS on SCY2 expres-sion showed that LPS induced TLR which was the receptor of LPS,but could not significantly induce the gene expression of SCY2(Fig. 6A,B).
When qPCRwas used to analyze whether the gene expression ofSCY2 was induced by other hormones, it was found that only pro-gesterone significantly induced the SCY2 mRNA expression andcorrespondingly the inhibition of progesterone suppressed theexpression of SCY2 (Fig. 6C,D).
3.7. Recombinant expression and antimicrobial activity of SCY2
When we analyzed the antimicrobial and bactericidal activitiesof the recombinant SCY2 using MIC (Table 2), the purified recom-binant SCY2 expressed in P. pastoris showed antimicrobial activitiesin Gram-positive bacteria. In particular, the recombinant SCY2 wasactive against the important aquatic pathogens B. subtilis(12.5e25 mM) and M. lysodeikticus (12.5e25 mM).
We further investigated the survival of S. paramamosain chal-lenged by V. parahaemolyticus, which is one of the major pathogensin the mud crab. As shown in Fig. 7, the survival rate of the crabchallenged by V. parahaemolyticus treated with SCY2 protein was23% after 16 h, while the control (crabs challenged byV. parahaemolyticus treated with PBS buffer) percentage was only
Fig. 5. SCY2 gene expression with mating action. (A) SCY2 mRNA expression pre- and post-mating in the reproductive system of male and female S. paramamosain. (B) Detection ofSCY2 protein in the ejaculatory duct of male S. paramamosain pre- and post-mating using western blot. (C) Detection of SCY2 protein in the reproductive system of femaleS. paramamosain pre- and post-mating using immunofluorescence. C: chitin; S: spermatophores; ML: muscle layer. (D) Detection of SCY2 protein in the vagina of femaleS. paramamosain post-mating using immunohistochemistry.
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6.7%. These data suggested that SCY2 protein performed an impor-tant function in the antibacterial immunity of S. paramamosain.
4. Discussion
In this study, a new cDNA sequence encoding an AMP withapproximately 65% identity to the reported SCY1 [15] was identi-fied and it was named SCY2. Some AMPs have more than onevariant which exists in crustacean species such as ALF in crabs[23,24]. The phenomenon is also found in other species of marineanimals such as hepcidin in fish [25e28]. Two or more variants ofthe innate immune associated molecules are identified more often
in marine animals than in mammals, as described previously[29,30]. The full length cDNA sequence of SCY2 and its corre-sponding genomic DNA sequence were determined. The putativesignal peptide sequence was 24 residues in length and wasconserved in both isoforms. The mature peptide of SCY2 comprised100 amino acids, two amino acid residues less than that of SCY1.The calculated molecular mass of the SCY2 mature peptide wasapproximately 10.925 kDa with an estimated pI of 4.84, thus it wasan anionic antibacterial peptide andwasmore powerful in its acidicnature than SCY1 (pI 6.09). Similar to the SCY1 gene [15], SCY2genomic DNA was composed of three exons and two introns. TheSCY2 gene was split by two introns but the two introns of SCY2
Fig. 6. Inducted SCY2 expression by steroid hormones. (A) In vitro induction of TLR and (B) SCY2 expression by progesterone and LPS on the ejaculatory duct of S. paramamosain. (C)In vitro induction of SCY2 expression by steroid hormones and (D) an inhibitor on the ejaculatory duct of S. paramamosain.
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were much different in length to those of SCY1, especially the firstintronwhich was 997 bp, much shorter than that of SCY1 (1569 bp).Intron 1 of the SCY1 gene contained insertion of a tandem repeat CTof 108 nucleotides, whereas the 30 UTR of the SCY2 gene containedan insertion of 21 repeats of AT microsatellite sequences. The po-sitions of introns inserted into ORF were identical for the two ho-mologous genes, in terms of separating the coding amino acids inthe first exon (44 aa) and the third exon (39 aa) for both genes.However, in the case of the second exon (41 aa), SCY2 missed twoamino acid residues in comparison with SCY1. The 30 UTR of SCY2and SCY1 were very different from each other, SCY2 containing along additional portion.
Our earlier study demonstrates that SCY1 is significantlyexpressed in the ejaculatory duct of male S. paramamosain and hasa potential role associated with reproductive immunity [7]. In thepresent study, it was observed that SCY2was dominantly expressedin the ejaculatory duct of male crabs and had more than thousandsof copy numbers of SCY2 mRNA transcripts than in femalesmeasured using the absolute qPCR. The immunofluorescencestaining result further indicated that the SCY2 proteinwas detectedonly in the posterior reproductive system of males (from ejacula-tory duct to penis) but no signal was detected in the other tissues ofmale and female crabs. The collected data suggested that the SCY2protein present in the posterior ejaculatory duct and penis wasprobably transferred from the ejaculatory duct since the copynumbers of SCY2mRNA transcripts in the ejaculatory duct were 104
times that in the posterior ejaculatory duct and penis. However, thepossibility could not be excluded that both the posterior ejaculatoryduct and penis might synthesize a small amount of SCY2 protein.This result was different from that of SCY1 protein, which is
detected in both the male and female tissues [7,8], although theconcentration of SCY1 is greatly more in male than in female.
Some AMPs that are predominantly expressed in the repro-ductive tracts from various species, such as andropin [4] anddefensins [10,11], possess functions not only simply in terms ofantimicrobial activity but also associated with reproduction. TheSCY2might have a similar functionwith its antimicrobial activity inrelation to crab reproduction. Through one year of seasonal inves-tigation of SCY2 mRNA expression in crabs it was observed that thehighest level of SCY2 gene expression was present in the matingpeak periods (June and November) when the adult male crabs werealready mature and prone to mate females. The highest level ofSCY2 expression at the mating peaks suggested a potential inter-action exhibited between SCY2 induction and the associated hor-mones induced during the mating time. Thus, it was reasonablydeduced that SCY2 might play a role associated with reproductiveimmunity in S. paramamosain as does SCY1.
In general, most AMPs tend to be up-regulated by pathogens aspart of the host defense response, such as MjCru1-1 and SpALF5 indifferent decapod species [31,32]. Some AMPs exceptionally do notrespond to the bacterial challenge whereas they are induced whilemating, for example the two AMPs andropin and ceratotoxin[33,34]. In our study, the bacterial and fungal challenges (data notshown) did not cause any change in the level of SCY2 expression;conversely, the mating action induced a high expression of SCY2,which suggested a direct relationship between SCY2 expressionand the mating action. Moreover, it was interesting to note thatSCY2 transcripts were detected in the spermatheca after matingalthough the transcript numbers were greatly lower than in theejaculatory duct. The result from immunofluorescence and IHC
Fig. 7. Cumulative percentage survival of S. paramamosain after co-treatment withSCY2 protein and V. parahaemolyticus. The purified SCY2 protein (200 mg/mL) wasincubated with V. parahaemolyticus (7Х107 cfu/mL) at room temperature for 1 h, and100 mL injected into each crab.
K. Qiao et al. / Fish & Shellfish Immunology 51 (2016) 251e262260
further confirmed that the SCY2 protein was obviously present inthe spermatheca and vagina but no signal of SCY2 was detectedbefore mating. The increase in numbers of the SCY2 transcripts andprotein after mating suggested the activation of SCY2 as a protec-tive component against invading microorganisms in fertilization(as shown in Fig. 8). Where were the SCY2 mRNA transcripts in thespermatheca after mating? One possibility was that the SCY2 in thespermatheca may have been transferred from the ejaculatory ductof the males, the evidence being that after mating the numbers ofSCY2 transcripts were decreased in the ejaculatory duct of males,which suggested the termination of the task of SCY2 action as aprotective component. The numbers of SCY2 transcripts down-
Fig. 8. SCY2 immune function associated with the reproductive process. In this study, thconversely, the progesterone or mating action induced a high expression of SCY2. The SCYnumbers were much lower than in the ejaculatory duct. SCY2 protein was obviously presenincrease in numbers of the SCY2 transcripts and protein after mating suggested the activati
regulated in the ejaculatory duct but up-regulated in the sperma-theca indicated that the SCY2 transcripts were probably transferredfrom male to female to protect the sperm, or the female from anypathogenic infection. In addition, another possibility could not beexcluded: that the SCY2 gene was expressed in the spermathecaduring the mating time. Similar evidence is reported in Drosophilawhere the sperm and accessory gland protein may be transferred tothe female during mating, which then triggers many physiologicaland behavioral changes in females, including the AMP [13],although the female reproductive tract constitutively expressessome AMP at mating. From our observation, SCY2 protein wasclearly detected in the female spermatheca shortly after mating(1e3 days), but did not exist in female crabs a long time aftermating. Sowe preferred to consider that SCY2 protected thematingprocess. However, the exact protective role of SCY2 needs to befurther investigated.
Our study revealed that the SCY2 expression level tended toincrease with the maturity of male crabs and was much morehighly expressed in the larger weight mature males (300 g) than inthe smaller weights (100, 200 g). Correspondingly, dissectionrevealed that the larger crabs (300 g) had more fully developedgonads than the smaller ones, indicating that SCY2 expression wasassociated with gonad maturity. This phenomenon is also demon-strated in a mammalian AMP Bin-1b study, which shows that Bin-1b expression throughout the life-span of rats is developmentallyregulated [35]. However, when the crabs grew older and wereapproximately 400 g or more the level of SCY2 expressiondecreased (data not shown). This result might lead to a presump-tion that for maintaining qualified breeding and better fertilization,crabs would be prone to carry out the mating action at theirenergized developmental stages when their gonads werecompletely developed and their associated hormones were at theirhighest generation. To guarantee that fertilization was successful,the protective components should be pre-prepared or generated inthe reproductive system, and thus SCY2 might be one of thosepotential immune associated endogenous components which wasinduced to reach a high level with the mating action. The peptide
e bacterial and fungal challenges did not cause any change to the SCY2 transcripts;2 transcripts were detected in the spermatheca after mating although the transcript
t in the spermatheca and vagina but no signal of SCY2 was detected before mating. Theon of SCY2 as a protective component in fertilization against invading microorganisms.
K. Qiao et al. / Fish & Shellfish Immunology 51 (2016) 251e262 261
might also be genetically selected to act as the reproductive im-mune defense in the face of the marine complex environment, butthis presumption needs further investigation.
AMPs show multi-functionality except for their direct antimi-crobial properties [36,37]. As reported, the expression of crustin inthe crab Portunus pelagica changes over the moult cycle, suggestingthat it may protect the crab from bacterial infection [38]. Theepididymis-specific Bin1b is involved with sperm maturationand prevents the microorganisms entering the adjacent testeswhere sperm is produced [35]. b-defensin 15 exhibits an androgen-dependent expression pattern and may regulate the spermmotilityand male fertility [10]. In our study, based on the observation thatthe highest level of SCY2 expressionwas during themating periods,we further investigated the potential relationship between SCY2expression and the associated hormone induction. It was mostinteresting to note that SCY2 mRNA expression was significantlyup-regulated by exogenous progesterone in cultures, but that noeffect on SCY2 expression was found with the addition of 17b-estradiol or testosterone in cultures, and, furthermore, that therewas no significant difference between the control and the 20-Hydroxyecdysone challenged group (data not shown). A previousstudy of AMP HBD3 and HBD4 reports that the hormone proges-terone can induce the expression in endometrial explants from theproliferative phase by 2.6 fold although this effect is not significant[39]. Human defensin-5mRNA level correlates with the stage of themenstrual cycle [40]. Another study also reports that progesteronecan activate the cation channel of human sperm during fertilization[41]. Considering the potential interaction between SCY2 inductionand hormone induction, we carried out a longer term investigationto detect the modulation of hormone level during a whole year'sdevelopment cycle (data not shown) and it was observed that themodulation of progesterone was largely consistent with the SCY2gene expression. Thus, it could be derived from these data that theprogesterone was generated with the sexual maturity ofS. paramamosain which subsequently regulated the mRNA expres-sion of SCY2.
To our knowledge, two homologous genes existing in theS. paramamosain ejaculatory duct have not yet been reported.Considering that both genes are expressed mainly in adult mudcrabs and at a similar site, it was likely that the two genes played anindividual or overlapping role in the reproductive immunity againstspecific pathogens. The deduced mature peptide of SCY2 had 100amino acids, containing 13% acidic amino acids and 10% basicamino acids. The predicted pI was 4.84, and thus it was an anionicAMP. Compared to cationic AMPs, anionic peptides are relativelysmall and include only a few reported groups of molecules that arefound in different vertebrate and invertebrate species. Severalanionic peptides such as dermcidin [42], PvHCt [43], maximin H5[44], theromyzin [45], SCY1 [15] and stylicin [46] are reported,including three small ones rich in glutamic and aspartic acids [47].The mode of action of the anionic peptides is still not clearly un-derstood. The first found acidic AMPs H-GDDDDDD-OH, H-DDDDDDD-OH and H-GADDDDD-OH are isolated from ovine pul-monary surfactant [47]. These peptides require zinc as a cofactor formaximal activity and are active against both Gram-negative andGram-positive bacteria [48]. The anionic amphibian peptideMaximin H5 is active only against Gram-positive bacteria (Staphy-lococcus aureus) [44] whereas the anionic annelid's (Theromyzontessulatum) peptide theromyzin shows limited bacteriostatic ac-tivity against a Gram-positive bacterium (M. luteus) [45]. In ourstudy, we observed that the recombinant SCY2 had antimicrobialactivity against several Gram-positive bacteria, including M. luteus,C. glutamicum, B. subtilis and M. lysodeikticus. In vivo studiesrevealed that the SCY2 protein significantly increased the survivalrate of S. paramamosain under the challenge of V. parahaemolyticus.
Increasing evidence indicates that some AMPs can confer protec-tion by an indirect mechanism and not simply because they can killmicrobes. Penaeidins do not have a direct effect against Gram-negative bacteria, but they can eliminate bacteria through phago-cytosis to protect tissues from infection or participate in woundhealing processes [49]. However, the antibacterial mechanism ofanionic AMPs remains unclear, and further work needs to be un-dertaken on the exact way in which SCY2 might exert its effects.
In summary, a new AMP SCY2 was characterized inS. paramamosain. After SCY1, this was the second most potent AMPwhich was identified in the reproductive system of crabs [14,15].This peptide was dominantly expressed in the ejaculatory duct ofS. paramamosain. The SCY2 gene did not respond to bacterial andfungal challenge but was significantly induced during mating ac-tion. Our study revealed that the potential function of SCY2 may beclosely associated with reproductive immunity. This conclusionwas based on the observation that SCY2 protein may be transferredfrom male to female during mating action in order to protect thefertilization process from exogenous invading microorganisms.Evidence was also derived from the fact that the change in level ofSCY2 expression matched the mating peaks of male and female. Inaddition, with the decrease in level of the SCY2 gene in the ejacu-latory duct of males after mating, the SCY2 gene was highlyexpressed in the female spermatheca and the increase in the levelafter mating meant the activation of SCY2 as a protective compo-nent in the female spermatheca in order to maintain a sterileenvironment for fertilization. The recombinant SCY2 had potentantimicrobial activity against Gram-positive bacteria, suggestingthat SCY2 could play a powerful role in the defense reactions toinvading microorganisms, during which a sterile inner environ-ment would be created thus leading to the successful fertilization ofS. paramamosain.
Acknowledgments
This work was supported by grants (41176116, U1205123) fromthe National Natural Science Foundation of China (NSFC) and grants(2014N2004, 2013N5010) from the Fujian Science and TechnologyDepartment. Professor John Hodgkiss of The University of HongKong is thanked for his assistance in polishing the English.
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