Histochemical distribution of four types of enzymes and mucous cells in the intestine of koi carp (Cyprinus carpio var. koi).

The main purpose of this study was to investigate the distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non-specific esterase (NSE), peroxidase (POD), and mucous cells in the intestine of the koi carp Cyprinus carpio var. koi. ACP activity was located in the striated border, enterocytes, and lamina propria of the anterior and middle intestines. The ACP activity in the anterior intestine was higher than that in the middle and posterior intestines. ALP existed in the striated border of enterocytes and lamina propria, serosa, muscular layer, and the junction between muscular layer and submucosa layer of the intestine. The ALP activity in the anterior intestine was higher than that in the middle and posterior intestines. NSE activity was localized in the cytoplasm of enterocytes in the whole intestine, and the middle intestine showed the lower NSE activity than the anterior and posterior intestines. POD activity was localized in the blood cells of the lamina propria and cytoplasm of enterocytes in all intestinal segments. The POD activity among the anterior, middle, and posterior intestines was non-significantly different. Alcian blue periodic acid-Schiff histochemical results revealed three types of mucous cells in the intestine. The total number of mucous cells and percentage of type I cells among the anterior, middle, and posterior intestines were non-significantly different. The percentage of the type II cells was the highest in the posterior intestine, while the lowest in the anterior intestine. The percentage of the type III cells was the highest in the anterior intestine, while the lowest in the posterior intestine.

Transcriptome profiling of immune-responsive genes in the intestine of Cynoglossus semilaevis Günther challenged with Shewanella algae.

To better understand gene expression in the intestine after Shewanella algae infection and provide insights into its immune roles in the tongue sole, Cynoglossus semilaevis, sequencing-based high-throughput RNA analysis (RNA-Seq) for the intestines between the control group and 12 h post-injection group was performed. After assembly, there was an average of 23,957,159 raw sequencing reads, and 23,943,491 clean reads were obtained after filtering out low-quality reads. Then, 383 differentially expressed genes (DEGs) in the intestines in response to S. algae infection were identified. Subsequently, gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs were conducted to further explore their functions. Among all of the pathways involved, sixteen pathways were related to the immune system, among which the complement and coagulation cascades pathway was the most prominent for immunity-related DEGs, followed by the leukocyte transendothelial migration pathway. Furthermore, the expression levels of twelve selected DEGs in the immune-related pathways were identified by quantitative real-time polymerase chain reaction, substantiating the reliability and reproducibility of the RNA-Seq results. In summary, this study represents an important genomic resource for understanding the potential immune role of the tongue sole intestine from the perspective of gene expression.

Effect of environmental factors on allelopathic inhibition of Microcystis aeruginosa by berberine.

To understand how environmental conditions affect the allelopathic inhibition of toxic Microcystis aeruginosa by berberine, the independent effects of some environmental factors, including temperature, light, and aeration, on the growth and extracellular microcystin (MC) content of M. aeruginosa (FACHB 905) treated with 0.000 and 0.001% (w/v) berberine were investigated. The results showed that higher temperature and light density, and aeration in daytime were beneficial for the growth of M. aeruginosa under the measured environmental conditions. The allelopathic effects of berberine on M. aeruginosa were closely associated with the environmental conditions. Berberine had the best inhibitory effects when temperature, light and aeration were more optimal for growth. In darkness, no changes in the density of M. aeruginosa were observed with the prolongation of culture time and berberine could hardly exhibit algicidal effects. Disturbance in the photosynthesis process might be one of the main reasons responsible for algicidal function. Berberine could increase extracellular MC contents significantly via killing and lyzing algal cells. Other treatments coupled with berberine needed to be carried out to degrade or remove MC released from berberine-killed algal cells.

Biochemical responses of the copepod Centropages tenuiremis to CO(2)-driven acidified seawater.

An ecophysiological experiment was conducted to examine the biochemical effects of acidified seawater containing elevated concentration of CO(2) (C(CO2) 0.08, 0.20, 0.50 and 1.00%) on the copepod Centropages tenuiremis. AchE, ATPase, SOD, GPx, GST, GSH level and GSH/GSSG ratio of the copepod were analyzed. The results showed that elevated C(CO2) and the duration of culture time significantly influenced several biochemical indices in C. tenuiremis (ATPase, GPx, GST, GSH and SOD). Furthermore, the principal component analysis results indicated that 72.32% of the overall variance was explained by the first three principal components (GPx, SOD and GSH). Changes in GPx and GSH levels may play a significant role in the antioxidant defense of copepods against seawater acidification. The long-term response of copepods to seawater acidification and the synergistic effects of acidification with other environmental factors, such as temperature, salinity and trace metal need further investigation.

Bipolar properties of red seabream (Pagrus major) transforming growth factor-beta in induction of the leucocytes migration.

TGF-beta is one of the pleiotropic cytokines and plays a pivotal role in immune regulation and orchestrating the subsequent healing response. Recombinant red seabream TGF-beta (rTGF-beta) mature peptide was expressed and purified under native conditions in vitro. Bio-assay showed that the rTGF-beta could significantly induce head kidney (HK) and peripheral blood (PB) leucocytes migration in a dose dependent manner, whereas the rTGF-beta suppressed HK and PB leucocyte migration when the leucocytes was activated by primed with lipopolysaccharide (LPS). Both enhancing and suppressing roles of rTGF-beta on the HKL and PBL chemotactic activity indicated that the fish TGF-beta shared the similar bipolar nature with mammalian TGF-beta. Furthermore, the results indicated that the activity of TGF-beta induction of leucocyte migration appears not to be an innate feature but function by regulation the chemokines activity. This is the first time we reported that fish TGF-beta has innate bipolar property in regulation of fish immune function.

A putative endocrine factor SIBD (single insulin binding domain protein) involved in immune response of Chinese mitten crab Eriocheir sinensis.

Insulin growth factor binding proteins (IGFBPs), characterized by the conserved insulin binding (IB) domains, are important components of endocrine system and play key roles in metabolism and growth. In the present study, the full-length cDNA of a single IB domain protein (designated EsSIBD) was identified from Chinese mitten crab Eriocheir sinensis based on expressed sequence tag (EST) sequence. The 1187 bp EsSIBD cDNA contained a 321 bp open reading frame (ORF) encoding a putative protein of 106 amino acids, a 5'-untranslated region (UTR) of 189 bp, and a 3'-UTR of 677 bp. Multiple sequence alignment presented ten conserved cysteine residues critical for the fundamental structure and function of IB domain. BLAST analysis revealed that EsSIBD shared high similarity with previously known IB domains of IGFBPs with the identities ranging from 40% to 46%. The sequence similarity and domain conservation indicated that EsSIBD was a potential member of the IGFBP family. Phylogenic analysis presented that EsSIBD was closer to IGFBP7 than to the other IB domain containing proteins, suggesting its functional similarity with the endocrine factor IFGBP7. The mRNA expression of EsSIBD in different tissues including hepatopancreas, gill, gonad, muscle, heart and haemocytes, and the temporal expression in haemocytes challenged by Listonella anguillarum were measured by real-time RT-PCR. EsSIBD mRNA transcripts could be detected in all examined tissues with the highest expression level in gill. The EsSIBD mRNA expression in haemocytes was sensitive to L. anguillarum stimulation and it was up-regulated from 3 to 24 h after challenge. The structure conservation and functional similarity to IFGBPs, and its sensitivity to L. anguillarum stimulation collectively implied that EsSIBD was probably involved in endocrine and immune systems of Chinese mitten crab, and provided insight into the cross-talk between the invertebrate endocrine and immune system.

Identification and analysis of a Cu/Zn superoxide dismutase from Haliotis diversicolor supertexta with abalone juvenile detached syndrome.

A partial cDNA sequence of a putative Cu/Zn superoxide dismutase (SOD) from Haliotis diversicolor supertexta with abalone juvenile detached syndrome (AJDS) was isolated by suppression subtractive hybridization library screening. The full 988 base pair (bp) abalone Cu/Zn SOD cDNA representing full cDNA coding sequence was obtained by rapid amplification of cDNA ends (RACE), and included a 462-bp open reading frame encoding 154 amino acids, plus 49 bp of 5'-, and 477 bp of 3'-untranslated region. The coding sequence shared high similarity and identity with known Cu/Zn SODs. The deduced amino acids contained two typical Cu/Zn SOD motifs and the conserved geometry active sites. Moreover, the cysteines involved in dimer formation, and the copper- and zinc-binding sites were conserved. Recombinant abalone Cu/Zn SOD (abSOD) was expressed in Escherichia coli, purified under denaturing conditions, and refolded by direct buffer exchange or gel gradient recovery. The activity of the purified protein was enhanced by 1 microM Cu(2+) or 1 microM Cu(2+) plus 1 microM Zn(2+). The transcription of abSOD was significantly increased in AJDS abalones compared to controls, while protein expression and SOD enzyme activity both decreased significantly, suggesting that SOD may play an important role in AJDS, and that the disease etiology may be related to environmental stress.

Inhibitory effects of golden thread (Coptis chinensis) and berberine on Microcystis aeruginosa.

The effects of 40 Chinese herbs on Microcystis aeruginosa growth were monitored spectrophotometrically. Golden thread (Coptis chinensis) exhibited the best inhibitory effects. Cell density of M. aeruginosa decreased with the increasing concentrations of golden thread and the prolongation of exposure time. Decreases in protein content, carbohydrate content, and chlorophyll a content were observed in a golden thread concentration-dependent manner after 96 h exposure. Changes in cell density, protein content, carbohydrate content, and chlorophyll a content of M. aeruginosa exposed to berberine, the main component of golden thread, were also investigated. It was observed that berberine exhibited the same inhibitory effects on M. aeruginosa. The results suggested that golden thread could inhibit M. aeruginosas growth effectively, and berberine might be the main allelochemical implementing the inhibitory effects of golden thread.

Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei.

In invertebrates, C-type lectins play crucial roles in innate immunity responses by mediating the recognition of host cells to pathogens and clearing microinvaders, which interact with carbohydrates and function as pattern recognition receptors (PRRs). A novel C-type lectin gene (LvLec) cDNA was cloned from hemocytes of Litopenaeus vannamei by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of LvLec was of 618 bp, consisting of a 5'-terminal untranslated region (UTR) of 60 bp and a 3'-UTR of 87 bp with a poly (A) tail. The deduced amino acid sequence of LvLec possessed all conserved features critical for the fundamental structure, such as the four cysteine residues (Cys(53), Cys(128), Cys(144), Cys(152)) involved in the formation of disulfides bridges and the potential Ca(2+)/carbohydrate-binding sites. The high similarity and the close phylogenetic relationship of LvLec shared with C-type lectins from vertebrates and invertebrates. The structural features of LvLec indicated that it was an invertebrate counterpart of the C-type lectin family. The cDNA fragment encoding the mature peptide of LvLec was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvLec) could agglutinate bacteria E. coli JM109 depending on Ca(2+), and the agglutination could be inhibited by mannose and EDTA. These results indicated that LvLec was a new member of C-type lectin family and involved in the immune defence response to Gram negative bacteria in Litopenaeus vannamei.

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis.

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8 h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174 bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G+) and Gram-negative (G-) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture.

A modified method for genomic DNA extraction from the fish intestinal microflora.

A modified genomic DNA extraction method named the combination of lysozyme and ultrasonic lysis (CLU) method was used to analyze the fish intestinal microflora. In this method, the physical disruption and chemical lysis steps were combined, and some parameters in the key steps were adjusted. In addition, the results obtained by this method were compared with the results obtained by the Zirmil-beating cell disruption method and the QIAamp Fast DNA Stool Mini Kit. The OD260/OD280 ratio and concentration of the DNA extracted using the CLU method were 2.02 and 282.8 µg/µL, respectively; when the incubation temperatures for lysozyme and RNase were adjusted to 37 °C, those values were 2.08 and 309.8 µg/µL, respectively. On the agarose gel, a major high-intensity, discrete band of more than 10 kb was found for the CLU method. However, the smearing intensity of degraded DNA was lower when the incubation temperatures were 60 °C for lysozyme and 30 °C for RNase than when incubation temperatures of 37 °C for lysozyme and 37 °C for RNase were used. The V3 variable region of the prokaryotic 16S rDNA was amplified, and an approximately 600-bp fragment was observed when the DNA extracted using the CLU method was used as a template. The CLU method is simple and cost effective, and it yields high-quality, unsheared, high-molecular-weight DNA, which is comparable to that obtained with a commercially available kit. The extracted DNA has potential for applications in critical molecular biology techniques.

Potential mechanisms of phthalate ester embryotoxicity in the abalone Haliotis diversicolor supertexta.

The effects and associated toxicological mechanisms of five phthalate esters (PAEs) on abalone embryonic development were investigated by exposing the embryos to a range of PAEs concentrations (0.05, 0.2, 2 and 10 µg/mL). The results showed that PAEs could significantly reduce embryo hatchability, increase developmental malformations, and suppress the metamorphosis of abalone larvae. The possible toxicological mechanisms of PAEs to abalone embryos included, affecting the Na+-K+-pump and Ca2+-Mg2+-pump activities, altering the peroxidase (POD) level and the malondialdehyde (MDA) production, damaging the extraembryonic membranes structure, as well as disrupting endocrine-related genes (gpx, cyp3a, and 17ß-hsd 12) expression properties. Taken together, this work showed that PAEs adversely affected the embryonic ontogeny of abalone. The abilities of PAEs affecting the osmoregulation, inducing oxidative stress, damaging embryo envelope structure, and causing physiological homeostasis disorder, are likely to be a part of the common mechanisms responsible for their embryonic toxicity.

Enhanced activity and enantioselectivity of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1 by acetone treatment.

To improve the activity and enantioselectivity of hyperthermophilic archaeon Aeropyrum pernix K1 esterase (APE1547) and its mutants, they were purified by acetone-treated method. It was found that the acetone treatment not only caused APE1547 and its mutants to display higher activity and enantioselectivity but also saved more than 90% of time spent in purifying them by Ni-chelating column. In hydrolysis of p-nitrophenyl caprylate, the acetone-treated APE1547 and mutant A containing the following substitutions R11G, L36P, V225A, I551L, and A564T showed 5.7- and 6.9-fold active increase, respectively. In the resolution of 2-octanol acetate, the acetone-treated mutant A had a 9-fold enantioselective increase relative to that purified by Ni-chelating column. In addition, the impact of pH, temperature, and chemical reagents on activity of APE1547 and mutant A was discussed in this paper.

Functional characterization of the ELR motif in piscine ELR+CXC-like chemokine.

To elucidate the functional role of piscine incomplete ELR motif, the recombinant CXC and its mutants (mELR and mLoop) were produced in Escherichia coli M15 based on the predicted mature peptide coding sequence of the black sea bream CXC (BS CXC) chemokine. Assays showed that the BS rCXC proteins displayed strong ability to induce fish blood neutrophils and head kidney (HK) macrophage migration in a dose-independent manner (10 to 200 ng), both in black sea bream and common carp. Although the ELR motif and the N-terminal loop of ELR(+)CXC chemokines are essential for chemotactic activity and receptor binding in mammals, the mELR and mLoop mutants showed no significant difference in their induction of chemotaxis of fish blood neutrophils compared with the full-length rCXC at the same dose. Human recombinant IL-8 (hrIL-8) can clearly induce piscine blood neutrophil migration and has no effect on macrophages, whereas the BS rCXC cannot induce chemotaxis in higher vertebrates, such as rat blood neutrophils or macrophages, even if the incomplete ELR motif in rCXC was mutated to ELR. The BS CXC and its mutants can promote the phagocytosis ability of piscine blood neutrophils and HK macrophages both in black sea bream and common carp, but have no effect on rat neutrophils or macrophages. Results showed that the piscine ELR(+)CXC-like chemokine represents an ancient version of a CXC chemokine; the ELR motif still does not show the higher specific polarization of function as found in mammalian.

Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain.

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3h post-challenge. After a decrease at 6h, the expression level increased again and reached 207.8-fold at 12h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCfKZSPI-12) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (Ki) of rCfKZSPI-12 to trypsin was 173 nmol L(-1). These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response.

cDNA cloning, characterization and mRNA expression of a pacifastin light chain gene from the Chinese mitten crab Eriocheir sinensis.

The pacifastin family, characterized by several conserved arrays of six cysteine residues, is a newly identified serine protease inhibitor (SPI) family discovered uniquely in arthropods and plays important roles in multiple biological processes. In the present study, the full-length cDNA of a pacifastin light chain (designated ESPLC) was cloned from the Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1036bp ESPLC cDNA contained an 831bp open reading frame (ORF) encoding a putative pacifastin-related peptide of 276 amino acids, a 5'-untranslated region (UTR) of 67bp, and a 3'-UTR of 138bp. Six putative conserved domains sharing a characteristic cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys) were identified in the deduced amino acid sequence of ESPLC. The conservation of these PLDs (pacifastin light chain domains) and the relative higher similarity of ESPLC to other pacifastin-related precursors suggested that ESPLC was a member of pacifastin family. The mRNA transcripts of ESPLC were found to be higher expressed in hepatopancreas, gill and haemolymph than in gonad, muscle and heart, with the highest expression level in hepatopancreas. The ESPLC mRNA expression in haemolymph of Chinese mitten crab was up-regulated at 2h and 12h after challenged with Listonella anguillarum. The tissue distribution and temporal characteristics of ESPLC mRNA expression, similar to that of prophenoloxidase gene in E. sinensis, suggested that ESPLC was potentially involved in the response against invading bacteria, with the possibility that it functioned in the prophenoloxidase system in E. sinensis.

A lectin (CfLec-2) aggregating Staphylococcus haemolyticus from scallop Chlamys farreri.

Lectins are a family of carbohydrate-recognition proteins which play crucial roles in innate immunity. In this study, a new lectin (CfLec-2) gene was cloned from Chlamys farreri by EST and RACE approaches. The full-length cDNA of CfLec-2 was composed of 708bp, encoding a typical long form carbohydrate-recognition domain of 130 residues. The deduced amino acid sequence showed high similarity to Brevican in Homo sapiens, C-type lectin-1 and lectin-2 in Anguilla japonica. The cDNA fragment encoding the mature peptide of CfLec-2 was recombined into plasmid pET-32a (+) and expressed in Escherichia coli Rosseta-Gami (DE3). The recombinant CfLec-2 (rCfLec-2) protein exhibited aggregative activity toward Staphylococcus haemolyticus, and the agglutination could be inhibited by d-mannose but not EDTA or d-galactose, indicating that CfLec-2 was a Ca2+ independent lectin. Moreover, rCfLec-2 could suppress the growth of E. coli TOP10F'. These results suggested that CfLec-2 was perhaps involved in the recognition and clearance of bacterial pathogens in scallop.

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress.

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651bp in length, having a 5' untranslated region (UTR) of 96bp, a 3' UTR of 575bp, and an open reading frame (ORF) of 1980bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8h and lasted to 16h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop.