Fish & Shellfish Immunology 34 (2013) 1448e1454

Contents lists available at SciVerse ScienceDirect

Fish & Shellfish Immunology

journal homepage: www.elsevier.com/locate/fsi

Molecular characterization of two kazal-type proteinase inhibitor genes in the surf clam Mesodesma donacium exposed to Vibrio anguillarum

Waleska Maldonado-Aguayo, Gustavo Núñez-Acuña, Valentina Valenzuela-Muñoz, Jacqueline Chávez-Mardones, Cristian Gallardo-Escárate*

Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Sustainable Aquaculture Research (INCAR), University of Concepción, PO. Box 160-C Concepción, Chile article info abstract

Article history: This study reports two kazal-type serine inhibitors (KPI) identified in a cDNA library from the Received 29 November 2012 surf clam Mesodesma donacium, and characterized through Rapid Amplification of cDNA Ends (RACE). Received in revised form The KPIs, denoted as MdSPI-1 and MdSPI-2, presented full sequences of 1139 bp and 781 bp respectively. 20 February 2013 MdSPI-1 had a 50untranslated region (UTR) of 175 bp, a 30UTR of 283 bp and an open reading frame (ORF) Accepted 11 March 2013 of 681 pb that encodes for 227 amino acids. MdSPI-2 showed a 50UTR of 70 bp, a 30UTR of 279 bp and Available online 22 March 2013 an ORF of 432 bp that encodes for 144 amino acids. Both sequences presented two kazal-type tandem domains. Phylogenetic analysis of MdSPI-1 and MdSPI-2 shows a main clade composed by other bivalve Keywords: inhibitor species and closely related crustaceans. Real time PCR analysis showed that MdSPI-1 is mainly SPI up-regulated in mantle, foot, gills and muscle tissues, while MdSPI-2 is expressed principally in foot Mesodesma donacium tissue. Moreover, to evaluate the immune response of MdSPI-1 and MdSPI-2, infections with Vibrio Vibrio anguillarum anguillarum were performed. Herein, MdSPI-1 and MdSPI-2 transcription expression were significantly Gene expression up-regulated at 2 and 8 h post-challenge. Our results suggest that MdSPI-1 and MdSPI-2 are important humoral factors of innate immunity in M. donacium. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Serine protease inhibitors have been identified in mollusks as humoral factors present in haemolymph and related to compo- Serine protease inhibitors (SPI) are a super-family of nents of innate immune response. For instance, in species like the with physiological functions, such as blood coagulation [1],inflam- Pacific oyster Crassostrea gigas [10,11], soft-shell clam Mya arenaria mation [2], metamorphosis [3], complement system [4] and innate [12], bay scallop Argopecten irradians [13] and Zhikong scallop immune response. In all these process, SPIs are mainly responsible Chlamys farreri [14], have been characterized as kazal-type in- for maintaining cellular homeostasis, inhibiting undesirable pro- hibitors (KPI) capable of blocking the action of some such teolytic cascades [5] or inhibiting exogenous protease secreted by as subtilisin A and . Furthermore, KPI immune response has pathogenic microorganisms that use the protease to penetrate the been studied in the shrimp Penaeus monodon and the river crab host and invade new tissues [6]. Among the families of serine pro- Procambarus clarkii, evidencing the presence of kazal-type domains tease inhibitors there is the I1, which is composed by proteins con- with inhibitory activity against subtilisin, trypsin and chymotrypsin taining the kazal-type domain and therefore are called kazal-type [5,15]. Four serine protease inhibitors in Fenneropenaeus chinensis SPIs (KPI) [7]. These have a single or multi kazal domain, which can have the capacity to inhibit subtilisin and proteinase K and have inhibit different proteases [6]. The latter are characterized by a well been associated with the innate antibacterial and antifungal im- conserved amino acid sequence, with six cysteines responsible of the mune response system [6]. formation of disulfide bridges (C1x (3) C2X (7) PVC3X (3) The surf clam Mesodesma donacium, the model species for GX2YXNXC4X (6) C5X (12) C) [8]. The specificity of the inhibitor is our study, has socio-economic importance all along the Chilean determined by the P1 residue located in the second residue after the coast. There have been few studies of this species at the molecular second cysteine conserved from the kazal domain [9]. level, which has limited the development of DNA markers to assess this over-exploited population at genetic level [16].The objective of this study was to characterize two EST sequences * Corresponding author. fi E-mail addresses: [email protected], crisgallardo@oceanografia.udec.cl homologs to kazal-type SPI identi ed by 454 pryrosequencing from (C. Gallardo-Escárate). M. donacium transcriptome, and determine their transcription

1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.03.356 W. Maldonado-Aguayo et al. / Fish & Shellfish Immunology 34 (2013) 1448e1454 1449 patterns in different tissues using qPCR and their role in antibacterial Table 1 immune response. Sequence of the oligonucleotide primer used in the study. Primers Sequence (50e30) 2. Materials and methods MD_8873F(MdSPI1) ATCCCAGAACGCTAAAAGCA MD_8873R(MdSPI1) TCTGCGATTCCCAGGACAGT 2.1. Experimental design MD_3594F(MdSPI2) GAGTCCGTCGCTCCAATGTA MD_3594R(MdSPI2) CTTGGTTGATCGTCCATGC QMD_8873F CGAATGGCAACAAGAACAAA Forty specimens with the same stage of gonadal maturation were QMD_8873R CAGGAGGAACAAATGGGAGA 0 0 obtained from Coquimbo, Chile (31 55 S, 71 30 W). The specimens QMD_3594F TGGGTTTGAGATGCCACTTC were used to characterize the SPIs mRNA, and also in challenge trials QMD_3594R CATGCTGCCTCACATTCATT against Vibrio anguillarum. Tissues from the foot, mantle, gills and MD_atub_1F ATGTACCACGTGCCGTCTTT MD_atub_1R ACGGATTCTGTCGAGAACCA muscle were fixed in RNAlater RNA Stabilization Reagent (Ambion, 0 3594_5 R inner AAGCCTTGATTCTGCCGTCGCA USA) and stored at 80 C for subsequent RNA extraction. The 3594_50R outer TGATCGTCCATGCACTGTCGTGT remaining specimens were first acclimatized for a week in filtered 3594_30F outer GCTTGTGACGGACCTTGCCCAT 0 seawater at 17 C with constant airing. The specimens were then 3594_3 F inner GCATGGACGATCAACCAAGTA 0 ¼ ¼ 8873_5 inner CCAGCTCAAAAACCTGTTCC divided into a control group (N 18) and a challenge group (N 18). 0 m 8 8873_5 outer GCCCAGCACGCCGAAATGAT For the challenge trials,100 LofV. anguillarum (3.2 10 cell/ml) in 8873_30inner GTCTTACCCTCGCAAAGTGC Luria Bertani (LB) culture medium was injected into the adductor 8873_30outer TGCAAATTGTGCGGCGGAAGGA muscle of M. donacium specimens, while the control group was Adapter 50 CTAATACGACTCACTATAGGGCAAGCA injected with 100 ml of LB medium. Both groups were sampled at GTGGTATCAACGCAGAGT Adapter 30 CTAATACGACTCACTATAGGGCAAGCA different exposure times: 2, 4, 6, 8, 16 and 32 h post-injection. GTGGTATCAACGCAGAGT Around 300 mg of foot, mantle, gills and muscle samples were dissected and fixed in RNAlater RNA Stabilization Reagent (Ambion, USA), and stored at 80 C for subsequent RNA extraction. Escherichia coli JM109 in LB/amp/IPTG/Xgal plates overnight at 37 C. The positive clones were selected and purified to obtain Ò 2.2. Extraction of total ARN plasmids using the E.Z.N.A Plasmid DNA Mini kit II (Omega Bio- tek, Doraville, GA, USA). The plasmids obtained were sequenced Total RNA was extracted from 100 mg of the previously in both directions and their sequences were then assembled using Ò described tissues using TRIzol Reagent (Invitrogen , Life Technol- the software Geneious 5.1.3. ogies, USA) according to the manufacturer’s instructions. The con- Blastn and Blastx searches were conducted against the non- centration and purity of the extracted RNA was determined by redundant Genbank database (http://www.ncbi.nlm.nih.gov/)to Ò spectrophotometry in a Nanodrop ND-1000 (NanoDrop Technol- identify the conserved sites of the kazal domain. Subsequently, a ogies, Inc) and its integrity was visualized by electrophoresis in multiple alignment was made of the kazal domains of MdIsp1 and 2 denaturing agarose gels at 1.2% stained with ethidium bromide at and the kazal-type domains publicly available for the species: P. 0.001%. Subsequently, from 200 ng of RNA of each sample, cDNA clarkii (hcPcSPI, hpPcSPI and PcSPI), P. monodon (SPIPm2 and SPIPm), F. was synthesized using the RevertAidTMÔ H Minus First Strand chinensis (hpFcSPI1), Litopenaeus vannamei (hcLvSPI and hpLvSPI), C. cDNA Synthesis kit (Thermo Scientific, Maryland, USA), in accor- farreri (CfKZSPI), C. gigas (CvSI) and A. irradians (AISPI). Finally, to dance with the supplier’s instructions. determine clusters among the amino acid sequences of the kazal domains, a phylogenetic analysis was conducted using the 2.3. KPI identification and characterization Neighbor-Joining method adjusted to 1000 iterations, and including Hidra magnipapillata as outgroup. We identified two annotated contigs for KPIs from a cDNA li- brary for M. donacium obtained by 454 pyrosequencing of gill and 2.4. Expression profiles of MdSPI in M. donacium mussel tissue from adult individuals of M. donacium that are available for download at the Dryad Digital 162 depository (http:// Sustainable primers were designed for qPCR reactions for the datadryad.org/) under the access http://dx.doi.org/10.5061/dryad. sequences obtained from MdSPI-1 (QMD_8873F, QMD_8873R) and 8jd18. For both sequences annotated as KPIs, specific primers MdSPI-2 (QMD_3594F, QMD_3594R) (Table 1). As well, the genes a- were designed with Primer 3 in the software Geneious 5.1.3 (Bio- tubulin, b-tubilin and GAPDH were identified and assessed as matters, New Zealand) [17] (Table 1). The partial sequence of each endogenous controls for the relative expression qPCR assays. After contig was then amplified by PCR in a final volume of 12.5 mLof an assessment of efficiency, a-tubulin was selected as an endoge- reaction. The amplification conditions were: 94 C for 2 min and nous control. All the primers were designed to amplify w100 bp of 30 s (holding stage), 35 cycles of 94 C for 30 s (denaturing), 60 C PCR products. (T annealing) for 30 s, 72 C for 45 s and 72 C for 5 min. The PCR The qPCR runs were performed with StepOnePlusÔ (Applied product was visualized through electrophoresis in agarose gels at Biosystems, Life Technologies, USA) using the comparative DCt 1.2% and, then sent to Macrogen Inc. (Korea) for sequencing in an method [18]. This method requires efficiency values close to 100%, ABI 3730xl capillary sequencer (Applied Biosystems). Subsequently, therefore we analyzed the dynamic range to calculate the reaction the sequences were analyzed with Geneious 5.1.3 software, efficiency of every gene. To perform this, we conducted five cDNA resulting in two partial sequences termed MdSPI-1 and MdSPI-2. dilution series, beginning with 80 ng of template and a dilution factor Based on these sequences, specific new primers were designed of 1:5. Each reaction was conducted with a volume of 10 mL using Ò (Table 1) for amplification of the 30 and 50UTR ends, using the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, Ò Ò FirstChoise RLM RACE kit (Ambion , Life Technologies, USA) in USA). The amplification conditions were: 95 C for 10 min (holding accordance with the manufacturer’s instructions. The fragments stage), 40 cycles of 95 C for 30 s (denaturation), T (annealing) 30 s, obtained for the 50 and 30UTR ends were ligated in the cloning 72 C for 30 s (extension). To determine the presence of single vector TOPO TA Cloning kit (InvitrogenÔ, Life Technologies, Carls- amplicons, non-specific products, gDNA contamination and primer bad, CA, USA) and transformed into electrocompetent bacteria of dimers, a melting curve was carried out following the amplification, 1450 W. Maldonado-Aguayo et al. / Fish & Shellfish Immunology 34 (2013) 1448e1454 from 60 Cto95C and taking fluorescence data every 0.03 C. Once the reaction efficiency of all the genes (including the endogenous controls) was between 90 and 110%, we proceeded to evaluate the expression profile of MdSPIs in specimens after being challenged with V. anguillarum. The data obtained were analyzed with the KruskaleWallis test with Statistica software (Version 7.0, StatSoft, Inc.), given the non-parametric distribution of the data. Statistically significant differences were accepted with a p < 0.05.

3. Results

3.1. Characterization of MdSPIs mRNA

The full length obtained for MdSPI-1 cDNA was 1181 bp, which is composed by 217 bp of 50-end untranslated region (UTR), 283 bp of 30UTR and 681 bp of ORF that encodes for a sequence of 227 amino acids. Equally, a full length of 781 bp was obtained for MdSPI-2, containing a 50UTR of 70 bp, a 30UTR of 279 bp and an ORF of 432 bp that encoding for 144 amino acids. Both sequences presented the conserved polyadenylation signal of AATAA and a poly a tail (A). As well, both sequences presented the characteristic Kazal domain, with six conserved cysteines responsible for forming disulfide bridges (C1x (3) C2X (7) PVC3X (3) GX2YXNXC4X (6) C5X (12)). The translated sequence of MdSPI-1 contains two kazal-type domains separated by fifteen amino acid residues and the P1 amino acids, phenylalanine (Phe) and alanin (Ala) for each respective domain (Fig. 1). Similarly, MdSPI-2 presented two kazal-type domains separated by 37 amino acid residues, and leucine (Leu) and lysine (Lys) were observed in the P1 residue of each domain (Fig. 2). Nucleotide sequences of MdSPI-1 and MdSPI-2 were analyzed by Blastx algorithm against the EST database from GenBank. The recorded e-values were 3E-9 and 5E-19 for MdSPI-1 and MdSPI-2 respectively, with a high level of homology to sequences described as kazal-type serine protease inhibitors (PROSITE motif PS00282). Both sequences were homologous to the KPI of marine invertebrates such as C. gigas (EKC34738), Azumapecten farreri (ACD88987) and A. irradians (AAX38588). The amino acid sequences deduced from MdSPI-1 and MdSPI-2 were compared to the GenBank database using Blastp, resulting in 38% identity and an e-value of 7E-11 for MdSPI-1 and 50% identity and an e-value of 5E-12 for MdSPI-2 with respect to the KPI of C. gigas (EKC34738). The alignment of the amino acid sequences of both transcripts for M. donacium with those of other marine invertebrates evidenced the conserved sites for the cysteines characteristic of the kazal-type domain and the diversity of P1 residues among the different domains (Fig. 3). The phyloge- netic analysis showed a consensus topology with well-sustained nodes by high bootstrap values. It can be noted that the MdSPI sequences form a separate clade supported by a bootstrap value of 68%, which is closely related to the clade of the bivalves (C. gigas, A. irradians and Pinctada fucata) and in a second order with a clade composed of crustacean species (P. clarkii, F. chinensis and P. monodon)(Fig. 4). It should be noted that the bootstrap values in the basal nodes are higher than those of the distal nodes.

3.2. MdSPIs expression patterns in M. donacium Fig. 1. Nucleotide and deduced amino acid sequence of MdSPI-1 gene from Mesodesma donacium (GenBank accession no. KC160531). The two Kazal-type domain are shadowed in black boxes. The reading frame are shadowed in gray boxes. The classical The relative expression of MdSPIs in the different tissues of polyadenylation signal is underlined. The poly-A tail is labeled with a black rectangle. M. donacium not challenged was evaluated by RT-qPCR. We found The asterisk (*) indicates the stop codon. higher levels of expression of MdSPI-1 in the mantle than in the foot, gills and muscle, with significant differences supported by a than in the mantle (Fig. 5). The expression analysis after exposure (p < 0.05). MdSPI-1 was expressed 5 times more in tissue from the to V. anguillarum were carried using a cDNA of mantle tissue. The mantle than from the foot, 3 times more than in tissue from the gills relative expression of both MdSPIs gradually increased compared to and 4 times than muscle tissue (Fig. 5). The highest levels of the control groups (Fig. 6). MdSPI-1 expression peaked at 2 hpi expression of MdSPI-2 were found in the foot, being 10 times higher (Fig. 6A), while MdSPI-2 expression peaked at 8 hpi, with significant than in the gills, 9 times more than in the muscles and 5 times more differences from the control (p < 0.05) at all times evaluated. W. Maldonado-Aguayo et al. / Fish & Shellfish Immunology 34 (2013) 1448e1454 1451

Specifically, MdSPI-1 increased its mRNA two times in challenged group compared to control group at 2 hpi, and it reached up to four times in respect to controls at 8 hpi. Meanwhile, MdSPI-2 increased its mRNA levels in challenged group up to nine times more than control groups at 2 hpi, and then its levels decreased.

4. Discussion

4.1. Serine protease inhibitors and their role in the innate immunity of marine invertebrates

Serine protease inhibitors play an important role in the innate immunity of marine invertebrates, acting as modulators of pro- teases from endogenous and pathogenic microorganisms [8].In this study we identified two mRNA encoding for the serine protease inhibitor in M. donacium, termed MdSPI-1 and MdSPI-2. The com- plete mRNA was characterized and the amino acid sequences of both inhibitors were deduced. Two domains were identified in both mRNA sequences that are homologous to those described for kazal- type serine protease inhibitors. A typical or canonical Kazal domain is composed of 40e60 amino acid residues including some spacer amino acids [8]. This was found in the deduced amino acid se- quences MdSPI-1 where the first and second domain had 42 and 41 amino acid. MdSPI-2 also showing amino acid 43 and 49 for the first and the second domain respectively. Kazal domain similar lengths have been found in other marine invertebrates such as in the swimming crab Portunus trituberculatus [19], L. vannamei [14] and P. clarkii [13]. The inhibitory specificity of a kazal domain arises mainly from the P1 residue, which varies widely, with more one domain being possible in the same sequence [5]. Each domain acts independently, inhibiting different protease. Generally, inhibitors with the amino acids Arg or Lys in the P1 residue trend to inhibit Trypsin, while those with the amino acids Ala or Ser inhibit elas- tase, and those with Tyr, Pro, Met, Leu or Phe inhibit chymotrypsin [20]. For example, in Bombix mori, it was established by recombi- nant enzymatic characterization of a kazal-type serine protease inhibitor, that there is inhibitory action against subtilisin featured by Ala is present as a P1 residue [21]. Nine kazal-domains have been identified in F. chinensis, corresponding to five Asp, Thr, two Gl and Ala, the latter showing inhibitory activity against elastase [6]. Consequently, the role of MdSPIs could be inferred according to the amino acid in the P1 position of the kazal domain. The P1 residues of the MdSPI-1 sequence in its first domain presents an Ala residue, the latter suggesting inhibitory activity against subtilisin, acting in this way as a component of the defense against the protease of pathogenic bacteria as well as those secreted by of V. anguillarum [21,22]. A Phe was found in the P1 residue of the second Mdlsp1 domain, suggesting inhibitory activity against trypsin and chymo- trypsin [23]. In turn, MdSPI-2 presents the Leu residue in its first domain, providing specificity of the inhibitor for chymo- trypsin or subtilisin, according to what is described in Pacifactacus leniusculus [24] and P. clarkii [25]. The second MdSPI-2 domain presents a Lys residue with inhibitory activity against trypsin ac- cording to what has been observed in P. monodon [26,27], H. mag- nipapillata [28] and C. farreri [29]. Overall, the SPIs characterized in this work suggest that both identified enzymes are important hu- moral factors of innate immunity in M. donacium. A clade was observed in the phylogenetic analysis formed by the kazal-type domains of MdSPI-1 and MdSPI-2. The two sequences of

Fig. 2. Nucleotide and deduced amino acid sequence of MdSPI-2 gen from Mesodesma donacium (GenBank accession no. KC160532). The two Kazal-type domain are shadowed in black boxes. The reading frame are shadowed in gray boxes. The classical polyadenylation signal is underlined. The poly-A tail is labeled with a black rectangle. The asterisk (*) indicates the stop codon. 1452 W. Maldonado-Aguayo et al. / Fish & Shellfish Immunology 34 (2013) 1448e1454

Fig. 3. Multiple alignment of Kazal-type domain of MdSPI-1 and MdSPI-2, and other Kazal-type inhibitor: The inhibitor the Kazal-type of P. clarkii (hcPcSPI, hpPcSPI and PcSPI), P. monodon (SPIPm2 and SPIPm), F. chinensis (hpFcSPI1), L. vannamei (hcLvSPI and hpLvSPI), C. farreri (CfKZSPI), C. gigas (CvSI) and A. irradians (AISPI). Identical amino acids are in white letters with black background and gray, those with high similarity. Gaps are indicated by dashes to improve the alignment.

M. donacium have a closer phylogenetic relationship to each other shown in studies with different invertebrates like P. monodon, than either has with other bivalve species, and in turn this bivalve where SPIm2 was identified from a cDNA library of hemocytes, with clade has a relatively distant phylogenetic relationship with other a high level of gene expression [30]. Similar patterns have been kazal-type inhibitors of crustacean species. It is important to keep observed with P. clarkii [25], C. farreri [29], and L. vannamei [14], in mind that this analysis is not a taxonomic inference. Neverthe- while in F. chinensis SPI expression has only been observed in the less, it is possible to suggest that these inhibitors have specific hepatopancreas [6]. The relative expression levels of MdSPI-1 immune system functions, probably coded by different genes for showed higher levels of expression in the mantle than in other each species. Our results are similar to those obtained with P. clarkii, tissue, while MdSPI-2 was expressed mainly in the foot. It is where it was found that the crustacean clade was evolutively possible that high level of MdSPI-1 expression in the mantle is due distant from serine protease inhibitors identified in other bivalve to the direct contact of this tissue with the external medium and mollusks like C. farreri [15]. consequently with pathogenic microorganisms, thus requiring the expression of genes that act as barriers to contagious pathogens. On 4.2. Patterns of KPIs expression in marine invertebrates the other hand, our results suggest that the higher level of expression of MdSPI-2 in the foot than in the mantle, gills and The expression of serine protease inhibitors in marine in- muscle could be related to the residue P1s found in this sequence, vertebrates is generally associated with hemocytes, as have been which correspond to Lys and Leu, where it can be inferred that their objective protease are trypsin and chymotrypsin, both proteins involved in digestion [31]. The relative expression of the mRNA of MdSPIs after the bacterial challenge had a time-dependent response, with statistically

Fig. 4. Phylogenetic relationships of MdSPI-1 and MdSPI-1 with Kazal inhibitor domain in different organism. GenBank accession numbers for KPIs are: C. gigas (GenBank accession no. EKC34738), F. chinensis (EU375464) P. monodon KPI (AY267200), L. van- Fig. 5. Temporal expression of the MdSPI-1 y MdSPI-2 transcript from different tissues namei KPI (AY544986), P. fucata (GU361540), A. irradians (AAX38588), P. clarkii in individuals not challenge. (A) Relative expression of MdSPI-1 in different tissues (B) (AY351957). The tree was constructed by Neighbor-Joining method and confirmed by Relative expression of MdSPI-2 in different tissues. The expression levels were Boostrap (1000 replications). The support value at the nodes are shown. Hydra mag- normalized by the a-tub endogenous control. The bars are shown the standard nipapillata (FJ496658) was used as outgroup. deviation SD (n ¼ 3). Significant differences were considered with p < 0.05. W. Maldonado-Aguayo et al. / Fish & Shellfish Immunology 34 (2013) 1448e1454 1453

Fig. 6. The relative expression level of mRNA from MdSPI in M. donacium after challenge to Vibrio anguillarum in the tissues Mantle. Temporal expression of MdSPI-1 (A) and MdSPI-2 DD (B) transcripts. The mRNA expression of MdSPI was measured at 2e4e6e8e16e32 h post-injection. The relative MdSPI expression level as expressed by 2 ct was determined for each group. a-tub was used as an internal reference for qPCR. Vertical bars represented the mean SD (n ¼ 3). Significant differences were considered with p < 0.05. significant differences from the control at all the exposure times. Acknowledgments Both MdSPI-1 and MdSPI-2 transcripts were overexpressed after the injection with V. anguillarum. Similar results have been reported for This work has been supported by FONDECYT (1120397) and P. clarkii, where hcPcSI1 showed positive regulation in response to a FONDAP (15110027) from CONICYT e Chile. bacterial challenge and negative regulation in response to a viral challenge. The authors suggested that the SPI response is associated References with an innate defense system that is only antibacterial [32]. With respect to bivalve mollusks, the levels of mRNA of CfKZSPI in C. [1] Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, mainte- nance, and regulation. Biochemistry 1991;30:10363e70. farreri are over-expressed 3 h after infection with V. anguillarum, [2] Shigetomi H, Onogi A, Kajiwara H, Yoshida S, Furukawa N, Haruta S, et al. Anti- decaying by 6 h and increasing again at 24 h [29]. In the case of inflammatory actions of serine protease inhibitors containing the Kunitz A. irradians, it was determined that expression peaked at 16 h post- domain. Inflammation Research 2010;59:679e87. [3] Bayer CA, Kalm Lv, Fristrom JW. Relationships between isoforms infection with V. anguillarum [13]. Consequently, the differences in and genetic functions demonstrate functional redundancy at the broad- expression of MdSPI-1 and MdSPI-2 at the analyzed time points complex during Drosophila metamorphosis. Developmental Biology could indicate that different transcription factors or activation 1997;187:267e82. cascades are regulate by the two transcripts. As well, the qPCR [4] Gál P, Dobó J, Beinrohr L, Pál G, Závodszky P. Inhibition of the serine proteases of the complement system complement therapeutics. US: Springer; results suggest that in the V. anguillarum challenge, there is positive 201323e40. regulation of MdSPIs expression, suggesting its participation in [5] Jarasrassamee B, Supungul P, Panyim S, Klinbunga S, Rimphanichayakit V, fi the regulation of innate antibacterial system in M. donacium.In Tassanakajon A. Recombinant expression and characterization of ve-domain Kazal-type serine proteinase inhibitor of black tiger shrimp (Penaeus mono- conclusion, the present study contributes relevant information at don). Marine Biotechnology 2005;7:46e52. the molecular level about two genes associated with innate im- [6] Wang Z-H, Zhao X-F, Wang J-X. Characterization, kinetics, and possible munity in M. donacium derived from NGS transcriptome function of Kazal-type proteinase inhibitors of Chinese white shrimp, Fen- neropenaeus chinensis. Fish & Shellfish Immunology 2009;26:885e97. sequencing, and their relation with presence of marine pathogens [7] Rawlings ND, Morton FR, Barrett AJ. MEROPS: the peptidase database. Nucleic such as V. anguillarum. Acids Research 2006;34:D270e2. 1454 W. Maldonado-Aguayo et al. / Fish & Shellfish Immunology 34 (2013) 1448e1454

[8] Rimphanitchayakit V, Tassanakajon A. Structure and function of invertebrate silkworm pupae (Bombyx mori). Comparative Biochemistry and Physiology Kazal-type serine proteinase inhibitors. Developmental & Comparative Part B: Biochemistry and Molecular Biology 2007;146:234e40. Immunology 2010;34:377e86. [22] Kanost MR. Serine proteinase inhibitors in arthropod immunity. Develop- [9] Whisstock J, Skinner R, Lesk AM. An atlas of serpin conformations. Trends in mental & Comparative Immunology 1999;23:291e301. Biochemical Sciences 1998;23:63e7. [23] Potempa J, Korzus E, Travis J. The serpin superfamily of proteinase inhibitors: [10] Xue Q, Itoh N, Schey KL, Cooper RK, La Peyre JF. Evidence indicating the ex- structure, function, and regulation. Journal of Biological Chemistry 1994;269: istence of a novel family of serine protease inhibitors that may be involved in 15957e60. marine invertebrate immunity. Fish & Shellfish Immunology 2009;27:250e9. [24] Johansson MW, Keyser P, Söderhäll K. Purification and cDNA cloning of a four- [11] Faisal M, MacIntyre EA, Adham KG, Tall BD, Kothary MH, La Peyre JF. Evidence domain Kazal proteinase inhibitor from crayfish blood cells. European Journal for the presence of protease inhibitors in Eastern (Crassostrea virginica) and of Biochemistry 1994;223:389e94. Pacific(Crassostrea gigas) oysters. Comparative Biochemistry and Physiology [25] Zeng Y, Wang W-C. Molecular cloning and tissue-specific expression of a five- Part B: Biochemistry and Molecular Biology 1998;121:161e8. Kazal domain serine proteinase inhibitor from crayfish Procambarus clarkii [12] Elsayed EE, McLaughlin SM, Faisal M. Protease inhibitors in plasma of hemocytes. Aquaculture 2011;321:8e12. the softshell clam Mya arenaria: identification and effects of disseminated [26] Donpudsa S, Tassanakajon A, Rimphanitchayakit V. Domain inhibitory and sarcoma. Comparative Biochemistry and Physiology Part B: Biochemistry and bacteriostatic activities of the five-domain Kazal-type serine proteinase in- Molecular Biology 1999;123:427e35. hibitor from black tiger shrimp Penaeus monodon. Developmental & [13] Zhu L, Song L, Chang Y, Xu W, Wu L. Molecular cloning, characterization and Comparative Immunology 2009;33:481e8. expression of a novel serine proteinase inhibitor gene in bay scallops (Argo- [27] Visetnan S, Donpudsa S, Supungul P, Tassanakajon A, Rimphanitchayakit V. pecten irradians, Lamarck 1819). Fish & Shellfish Immunology 2006;20:320e31. Kazal-type serine proteinase inhibitors from the black tiger shrimp Penaeus [14] Jiménez-Vega F, Vargas-Albores F. A four-Kazal domain protein in Litopenaeus monodon and the inhibitory activities of SPIPm4 and 5. Fish & Shellfish vannamei hemocytes. Developmental & Comparative Immunology 2005;29: Immunology 2009;27:266e74. 385e91. [28] Augustin R, Siebert S, Bosch TCG. Identification of a Kazal-type serine pro- [15] Li X-C, Zhang R-R, Sun R-R, Lan J-F, Zhao X-F, Wang J-X. Three Kazal-type tease inhibitor with potent anti-staphylococcal activity as part of Hydra’s serine proteinase inhibitors from the red swamp crayfish Procambarus clar- innate immune system. Developmental & Comparative Immunology kii and the characterization, function analysis of hcPcSPI2. Fish & Shellfish 2009;33:830e7. Immunology 2010;28:942e51. [29] Wang B, Zhao J, Song L, Zhang H, Wang L, Li C, et al. Molecular cloning and [16] Aguilar-Espinoza A, Guzmán-Riffo B, Haye PA, Gallardo-Escárate C. Mining of expression of a novel Kazal-type serine proteinase inhibitor gene from EST-SSR from 454 pyrosequencing in the surf clam Mesodesma donacium Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant (Lamark, 1818). Conservation Genetics Resources 2012;4:829e32. domain. Fish & Shellfish Immunology 2008;24:629e37. [17] Drummond A. Geneious. v. 5.1. Available from: http://www.geneious.com; [30] Ponprateep S, Tassanakajon A, Rimphanitchayakit V. A Kazal type serine 2009. proteinase SPIPm2 from the black tiger shrimp Penaeus monodon is capable of [18] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real- neutralization and protection of hemocytes from the white spot syndrome time quantitative PCR and the 2DDCT method. Methods 2001;25:402e8. virus. Fish & Shellfish Immunology 2011;31:1179e85. [19] Wang S, Cui Z, Liu Y, Li Q, Song C. The first Kazal-type serine proteinase in- [31] Garcia-Esquivel Z, Felbeck H. Activity of digestive enzymes along the gut of hibitor in the swimming crab Portunus trituberculatus involved in immune juvenile red abalone, Haliotis rufescens, fed natural and balanced diets. response to bacteria and fungi. Aquaculture 2012;356e357:55e60. Aquaculture 2006;261:615e25. [20] Laskowski M, Kato I. Protein inhibitors of proteinases. Annual Review of [32] Li X-C, Wang X-W, Wang Z-H, Zhao X-F, Wang J-X. A three-domain Kazal-type Biochemistry 1980;49:593e626. serine proteinase inhibitor exhibiting domain inhibitory and bacteriostatic [21] Zheng Q-L, Chen J, Nie Z-M, Lv Z-B, Wang D, Zhang Y-Z. Expression, purifi- activities from freshwater crayfish Procambarus clarkii. Developmental & cation and characterization of a three-domain Kazal-type inhibitor from Comparative Immunology 2009;33:1229e38.