Integrated transcriptome analysis of immunological responses in the pearl sac of the triangle sail mussel (Hyriopsis cumingii) after mantle implantation.

For pearl culture of bivalve Hyriopsis cumingii, implantation of the sabio may cause nucleus discharge and increased host death rates. We performed a transcriptome analysis of the pearl sac of H. cumingii for 30 days after mantle implantation; 293863 unigenes were obtained, and 27176 unigenes were identified using nr, nt, KO, Swiss-Prot, Pfam, GO, and KOG databases. We detected 4878 differentially expressed genes (DEGs) through pairwise comparisons. We speculated that the physical condition of the recipient mussels returned to normal in about one month; the period was divided into six vital phases (0, 2 h-6 h, 12 h-24 h, 48 h to 7 days, 14 days and 30 days) on the basis of the overall similarities in DEGs. We compared the DEGs between time points and identified key immune-related genes. Our findings provide information on the immunological reactions induced by implantation in pearl mussels.

Identification of Hc-ß-catenin in freshwater mussel Hyriopsis cumingii and its involvement in innate immunity and sex determination.

ß-catenin is a multifunctional protein that participates in a variety of physiological activities, including immune regulation, sex determination, nervous system development and, cell differentiation. However, the function of ß-catenin in freshwater mussel Hyriopsis cumingii remains unclear. Herein, the gene encoding ß-catenin from H. cumingii (Hc-ß-catenin) was cloned and characterised. The full-length 5544 bp gene includes an open reading frame (ORF) of 2463 bp encoding a putative protein of 820 amino acids residues containing 12 armadillo (ARM) repeats. After injecting H. cumingii with Aeromonas hydrophila or lipopolysaccharides, Hc-ß-catenin transcription was induced in hemocytes and gills, and the greatest responses occurred at 24 h after bacterial challenge, confirming an important role in immune responses. Quantitative real-time PCR analysis showed that Hc-ß-catenin mRNA was distributed in the gill, foot, liver, kidney, mantle, adductor muscle and gonad of male and female mussels. In gonad, Hc-ß-catenin expression was markedly higher in females than males. During the embryonic period, Hc-ß-catenin expression was highest at 3 day. In 1-, 2- and 3-year-old mature mussels, Hc-ß-catenin expression in female gonad tissue was notably higher than in males. In situ hybridisation revealed a significant hybridisation signal in female gonads, indicating that Hc-ß-catenin is a pro-ovarian, anti-testis gene. Our findings demonstrate that Hc-ß-catenin is important in immune regulation and sex determination in freshwater mussel.

Molecular cloning of complement component C3 gene from pearl mussel, Hyriopsis cumingii and analysis of the gene expression in response to tissue transplantation.

Complement component C3 is well recognized as the central mediator of complement system, whose activation is responsible for the immune surveillance and elimination of non-self-antigens. In this study, C3 gene (HcC3) from a pearl making mussel, Hyriopsis cumingii, was successfully identified. The putative HcC3 possessed the canonical domains and highly conserved functional residues of C3 family members. In phylogenetic analysis, HcC3 was also clustered into C3 subfamily and separated from α2 macroglobulin clade. HcC3 gene was constitutively expressed in a wide range of tissues of pearl mussels, among which the immune-related tissues like hemocytes got highest expression. After allograft surgery of mantle tissues for aquaculture pearl production, the gene expression of HcC3 exhibited a rapid upregulation on day 1, dropped back on day 3, peaked the value on day 7, and restored to the level similar to control samples on day 14 after mantle allograft. The biphasic expression within the two weeks post the surgery suggests the important roles for HcC3 in alloimmune responses and an intricate complement activation mechanism in mollusks during tissue allograft.

A superoxide dismutase (MnSOD) with identification and functional characterization from the freshwater mussel Cristaria plicata.

Manganese superoxide dismutase (MnSOD) is a sort of important metalloenzyme that can catalyze ROS in the organisms. In this study, MnSOD cDNA of C. plicata, designated as CpMnSOD (accession no. MK465057), was cloned from hemocytes. The full-length cDNA of MnSOD was 1096 bp with a 672 bp open reading frame encoding 223 amino acids. The deduced amino acid sequence contained a mitochondrial-targeting sequence (MTS) of 18 amino acids in the N-terminus, and four conserved amino acids for manganese binding (H49, H97, D182, H186). CpMnSOD showed a high level (65-73%) of sequence similarity to MnSODs from other species. The results of Real-time quantitative PCR revealed that CpMnSOD mRNA constitutively expressed in tissues. The highest expression level was in hepatopancreas, followed by muscle, mantle and gill, and the lowest expression level was in hemocytes. After microcystin challenge, the expression levels of CpMnSOD mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpMnSOD was cloned into the plasmid pColdI-ZZ, and the recombinant protein was expressed in Escherichia coli BL21 (DE3). The enzyme stability assay showed that the purified CpMnSOD protein maintained more than 80% enzyme activity at temperature up to 70 °C, at pH 2.0-10.0, and resistant to 8 mol/L urea or 8% SDS.

A Kunitz proteinase inhibitor (HcKuPI) participated in antimicrobial process during pearl sac formation and induced the overgrowth of calcium carbonate in Hyriopsis cumingii.

Proteinase inhibitors with the ability to inhibit specific proteinases are usually closely connected with the immune system. Interestingly, proteinase inhibitors are also a common ingredient in the organic matrix of mollusk shells. However, the molecular mechanism that underlies the role of proteinase inhibitors in immune system and shell mineralization is poorly known. In this study, a Kunitz serine proteinase inhibitor (HcKuPI) was isolated from the mussel Hyriopsis cumingii. HcKuPI was specifically expressed in dorsal epithelial cells of the mantle pallium and HcKuPI dsRNA injection caused an irregular surface and disordered deposition on the aragonite tablets of the nacreous layer. These results indicated that HcKuPI plays a vital role in shell nacreous layer biomineralization. Moreover, the expression pattern of HcKuPI during LPS challenge and pearl formation indicated its involvement in the antimicrobial process during pearl sac formation and nacre tablets accumulation during pearl formation. In the in vitro calcium carbonate crystallization assay, the addition of GST-HcKuPI increased the precipitation rate of calcium carbonate and induced the crystal overgrowth of calcium carbonate. Taken together, these results indicate that HcKuPI is involved in antimicrobial process during pearl formation, and participates in calcium carbonate deposition acceleration and morphological regulation of the crystals during nacreous layer formation. These findings extend our knowledge of the role of proteinase inhibitors in immune system and shell biomineralization.

Three newly identified galectin homologues from triangle sail mussel (Hyriopsis cumingii) function as potential pattern-recognition receptors.

Galactoside-binding lectins, also known as galectins, play crucial roles in innate immune response in invertebrates. In this study, three cDNA sequences from Hyriopsis cumingii were identified and collectively called HcGalec genes. Each of the three deduced HcGalec proteins contained a galactose-binding lectin domain or a GLECT domain. All the three HcGalec genes are mainly present in the hepatopancreas and gills, and their expression is induced at 24 h after bacterial challenge. Three recombinant HcGalec proteins can bind and agglutinate (Ca2+-dependent) various microorganisms, including Gram-positive and Gram-negative bacteria. These proteins can attach to mannan and peptidoglycan. Meanwhile, the expression of the three HcGalec genes in the gills were significantly down-regulated after dsRNA interference (HcGalec1-RNAi, HcGalec2-RNAi, and HcGalec3-RNAi) and Vibrio parahaemolyticus injection. The expression levels of some antimicrobial peptides, including lysozyme 1 and lysozyme 2, were also markedly decreased after dsRNA interference. Overall, these results suggested that these three HcGalec proteins may function as potential receptors participating in the innate immune responses of H. cumingii against bacterial infection.

Identification of tumor necrosis factor receptor-associated factor 6 in the pearl mussel Hyriopsis cumingii and its involvement in innate immunity and pearl sac formation.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) acts as a central intracellular signal adapter molecule that mediates the tumor necrosis factor receptor superfamily and the interleukin-1 receptor/Toll-like receptor family in vertebrates and invertebrates. In the present study, HcTRAF6, a molluscan homologue of TRAF6 from Hyriopsis cumingii, has been cloned and identified. The entire open reading frame of HcTRAF6 was found to comprise a 1965-bp region that encodes a predicted protein of 654 amino acids, which contains conserved characteristic domains including a RING domain, two TRAF-type zinc finger domains, a typical coiled coil and the MATH domain. Phylogenetic analysis revealed that HcTRAF6 was aggregated closely with CsTRAF6 from Cyclina sinensis in the invertebrate cluster of mollusks. Further, qRT-PCR analysis showed that HcTRAF6 mRNA was extensively distributed in mussel tissues with a high expression in gills. After immune stimulation with Aeromonas hydrophila and lipopolysaccharides, the transcription of HcTRAF6 was obviously induced in the gills and hemocytes. In addition, significant fluctuation in HcTRAF6 expression was observed in the pearl sac, gills and hemocytes after mantle implantation. These findings confirmed its role in the alloimmune response. Dual-luciferase reporter assay showed that over-expression of HcTRAF6 could enhance the activity of the NF-κB reporter in a dose-dependent manner. Further, the RNA interference showed that the up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in HcTRAF6-silenced mussels and that depletion of HcTRAF inhibited the elimination of A. hydrophila. All these findings together prove that HcTRAF6 functions as an efficient regulator in innate immune mechanisms against invading pathogens and the alloimmune mechanism after mantle implantation in H. cumingii.

HcToll3 was involved in anti-Vibrio defense in freshwater pearl mussel, Hyriopsis cumingii.

Toll-like receptors (TLRs) play an important role in the activation of innate immune response but their functions in bivalves remain largely unknown. In this study, we identified a TLR from the freshwater pearl mussel Hyriopsis cumingii (HcToll3) and investigated its functions in immunity. The full-length cDNA of HcToll3 is 3852 bp and includes an open reading frame (ORF) of 3228 bp that encodes a polypeptide of 1075 amino acids. The predicted HcToll3 protein shares similar structural characteristics with other known Toll family proteins. Quantitative real-time PCR analysis revealed that HcToll3 mRNA is broadly expressed in all of the examined tissues; its transcript level was significantly up-regulated by challenge with gram-negative bacteria Vibrio parahaemolyticus or lipopolysaccharide, but not gram-positive Staphylococcus aureus or peptidoglycan. RNA interference by siRNA results showed that HcToll3 regulated expression of whey acidic protein (HcWAP) and lysozymes (HcLyso1 and HcLyso2) in vivo and knockdown of HcToll3 suppressed the elimination of V. parahaemolyticus. These findings suggest that HcToll3 might be involved in anti-Vibrio defense in H. cumingii.

Molecular characterization of two distinct Smads gene and their roles in the response to bacteria change and wound healing from Hyriopsis cumingii.

The proteins of Smad family are critical components of the TGF-ß superfamily signal pathway. In this paper, we cloned two intracellular mediators of TGF-ß signaling, Smad3 and Smad5, from the pearl mussel Hyriopsis cumingii. The full length cDNA of HcSmad3 and HcSmad5 were 2052 bp and 1908 bp and encoded two polypeptides of 418 and 461amino acid residues, respectively. The deduced amino acid of HcSmad3 and HcSmad5 possessed two putative conserved domains, MH1 and MH2, a conserved phosphorylation motif SSXS at the carboxyl-terminal. The two Smad genes were detected muscle, mantle, hepatopancreas and gill, but with a very low level in heamocytes. The transcripts of Smad3 and Smad5 were up-regulated in hemocytes and hepatopancreas after A. hydrophila and PGN stimulation. However, the expression of Smad3 and Smad5 were only up-regulated in hepatopancreas after A. hydrophila stimulation. The transcripts of Smad3 and Smad5 had a slight change in hepatopancreas after PGN stimulation. The transcripts of HcSmad3 showed very little increase and HcSmad5 mRNA significantly up-regulated after wounding.

Three STATs are involved in the regulation of the expression of antimicrobial peptides in the triangle sail mussel, Hyriopsis cumingii.

Janus kinase (Jak) and signal transducers and activators of transcription (STAT) signaling pathway is associated in antiviral and antibacterial immune response. Previous studies primarily investigated the function of STATs in mammals. For most invertebrates, only one STAT was found in each species, such as STAT92E was found in Drosophila melanogaster. The studies, which focus on the functional difference between various STATs in the same species of invertebrate, are limited. In the present study, three STATs (HcSTAT1, HcSTAT2 and HcSTAT3) were identified in triangle shell pearl mussel, Hyriopsis cumingii. Phylogenetic analysis showed that HcSTAT1 and HcSTAT3 were clustered with Homo sapiens STAT5, and HcSTAT2 was clustered with Pinctada fucata STAT and Crassostea gigas STAT6. All three STATs could be detected in all tested tissues (hemocytes, hepatopancreas, gill, mantle and foot), and were induced expression when challenged with Staphylococcus aureus or Aeromonas hydrophilia in hemocytes and hepatopancreas. HcSTAT1 regulated the expression of HcDef, HcWAP, HcThe and HcTNF. The expression of HcWAP and HcTNF was down-regulated in HcSTAT2-RNAi mussel. And HcSTAT3 affected the expression of HcTNF. The study is the first report of different functions in antibacterial immune responses between STATs in mollusks.

Molecular cloning, expression and antioxidative activity of 2-cys-peroxiredoxin from freshwater mussel Cristaria plicata.

Peroxiredoxins (Prxs) play an important role against various oxidative stresses by catalyzing the reduction of hydrogen peroxide (H2O2) and organic hydroperoxides to less harmful form. A 2-cys peroxiredoxin, designated as CpPrx, was cloned from hemocytes of freshwater mussel Cristaria plicata. The full length cDNA of CpPrx is 1247 bp, which includes an open reading frame (ORF) of 591bp, encoding 196 amino acids. CpPrx possesses two conserved cysteine residues (Cys49, Cys170). The deduced amino acid sequence of CpPrx showed a high level (67-74%) of sequence similarity to 2-Cys Prxs from other species. The results of real-time quantitative PCR revealed that CpPrx mRNA was constitutively expressed in tissues, and the highest expression levels were in hepatopancreas and gills. After peptidoglycan (PGN) and Aeromonas hydrophila challenge, the expression levels of CpPrx mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpPrx was cloned into the plasmid pET-32, and the recombinant protein was expressed in Escherichia coli BL21(DE3). Comparison with DE3-pET-32 and DE3 strain, the cells of DE3-pET-32-CpPrx exhibited resistance to the concentration of 0.4, 0.8 and 1.2 mmoL/L H2O2 in vivo.

Identification and characterization of two LBP/BPI genes involved in innate immunity from Hyriopsis cumingii.

Lipopolysaccharide-binding protein and bactericidal permeability-increasing protein (LBP/BPI) play crucial role in modulating cellular signals in response to Gram-negative bacteria infection. In the present study, two isoforms of LBP/BPI genes, designated as HcLBP/BPI1 and HcLBP/BPI2, respectively, were cloned from the mussel Hyriopsis cumingii by RACE approach. The full-length cDNA sequences of HcLBP/BPI1 and HcLBP/BPI2 were 1887 and 2227 bp and encoded two secreted proteins of 501 and 518 amino acid residues, respectively. The deduced amino acid of HcLBP/BPI1 and HcLBP/BPI2 contained several conserved domains, such as signal peptide, two BPI/LBP and one central domain. Phylogentic analysis further supported that HcLBP/BPI1 and HcLBP/BPI2 belonged to new members of invertebrate LBP/BPI family. The mRNA transcripts of HcLBP/BPI1 and HcLBP/BPI2 were ubiquitously expressed in all examined tissues, and the expression level of HcLBP/BPI1 was higher than that of HcLBP/BPI2. The mRNA expression of HcLBP/BPI1 in hepatopancreas and hemocytes was significantly up-regulate after Aeromonas hydrophila and LPS challenge, and HcLBP/BPI2 in hepatopancreas was only up-regulated at 6 and 12 h after LPS challenge and at 12 h after A. hydrophila challenge. In addition, the recombinant HcLBP/BPIs displayed antibacterial activity against Gram-negative bacteria, and the antibacterial index of HcLBP/BPI1 was higher than that of HcLBP/BPI2. These results indicated that HcLBP/BPI1 and HcLBP/BPI2 probably played distinct roles in bacterial mediating immune response in Mollusca.

Four C1q domain-containing proteins involved in the innate immune response in Hyriopsis cumingii.

C1q is a key subcomponent of the complement C1 complex. This subcomponent contains a globular C1q (gC1q) domain with remarkable ligand binding properties. C1q domain-containing (C1qDC) proteins are composed of all proteins with a gC1q domain. C1qDC proteins exist in many invertebrates and recognize non-self-ligands. In our study, four C1qDC genes, namely, HcC1qDC1-HcC1qDC4, were identified from Hyriopsis cumingii. HcC1qDC1-HcC1qDC4 encode a protein of 224, 204, 305, and 332 amino acids, respectively. All C1qDC proteins consist of a gC1q domain at the C terminal. In addition to the gC1q domain, a coiled-coil region is found in HcC1qDC4. Multiple alignments and phylogenetic tree analysis revealed that the C1qDC proteins highly differ from one another. Tissue distribution analysis demonstrated that HcC1qDC1-HcC1qDC4 are widely distributed in hemocytes, hepatopancreas, gills, mantle, and foot. These C1qDC genes are regulated by bacteria to varying degrees. These recombinant HcC1qDC proteins exhibit a binding activity against different bacterial species. Our results may suggest the roles of HcC1qDC genes in anti-bacterial immune defense.

Identification and function of a novel C1q domain-containing (C1qDC) protein in triangle-shell pearl mussel (Hyriopsis cumingii).

C1q is the target recognition sequence of the classical complement pathway and a major link that connects innate and acquired immunity. In this study, a C1qDC homolog, HcC1qDC5, from the triangle-shell pearl mussel (Hyriopsis cumingii) was identified. The complete nucleotide sequence of HcC1qDC5 cDNA consists of a 5'-untranslated terminal region (UTR) of 123 bp, a 3'-UTR of 105 bp with a poly(A) tail, and an open reading frame (ORF) of 1344 bp, which encodes a polypeptide of 447 amino acids. HcC1qDC5 contains a signal peptide and three typical C1q domains. The HcC1qDC5 gene was expressed in all tested tissues, with the highest expression in the mantle. Staphylococcus aureus or Vibrio parahaemolyticus infection increased the mRNA transcript levels of HcC1qDC5 in the hepatopancreas and mantle. The recombinant HcC1qDC5 protein could bind to Gram-negative and Gram-positive bacteria as well as to different PAMPs (LPS and PGN). RNAi results showed that HcC1qDC5 was involved in V. parahaemolyticus-induced HcTNF and HcWAP expression. The combined results demonstrated that HcC1qDC5 participates in the innate immunity of H. cumingii.

Characterization of allograft inflammatory factor-1 in Hyriopsis cumingii and its expression in response to immune challenge and pearl sac formation.

The allograft inflammatory factor-1 (AIF-1) is one of the key factors associated with inflammatory response and immune defense. In the present study, we report the identification and characterization of AIF-1 from triangle sail mussel Hyriopsis cumingii (HcAIF-1). The full-length cDNA of HcAIF-1 consisted of a 5'-terminal untranslated region (UTR) of 80 bp, a 3'-UTR of 420 bp with a poly (A) tail, and an open reading frame of 444 bp encoding a polypeptide of 147 amino acids with two conserved EF-hand Ca2+-binding motifs. HcAIF-1 mRNA and protein were expressed in all examined tissues and showed higher mRNA expression levels were observed in immune tissues, especially hemocytes and mantle, and the highest protein expression level was in mantle. The expression level of HcAIF-1 mRNA was significantly upregulated in hemocytes 12-48 h after lipopolysaccharide challenge. After mantle tissue implantation, the expression level of this gene in pearl sac decreased significantly at 3-48 h (P < 0.01), and then was significantly upregulated at 96 h (P < 0.05) and recovered to the control level at 21-28 d. There was significant increase HcAIF-1 transcript abundance in hemocytes 96 h (P < 0.05) after mantle tissue implantation. The phagocytosis rate was significantly enhanced in hemocytes 3-24 h (P < 0.01) after the injection of recombinant HcAIF-1 protein. These findings suggest that HcAIF-1 is important in the underlying mechanism of the innate immune responses and pearl sac formation of H. cumingii.

A novel C-type lectin with four CRDs is involved in the regulation of antimicrobial peptide gene expression in Hyriopsis cumingii.

C-type lectins (CTLs) are found in a wide number of invertebrates, and have been reported to participate in immune responses, such as the activation of prophenoloxidase, cell adhesion, bacterial clearance and phagocytosis. Previous studies on CTLs focused on the function of their carbohydrate recognition domains (CRDs). Currently, studies on lectins with multi-CRDs are limited. In this study, a lectin with four CRDs was cloned from Hyriopsis cumingii, and called HcLec4. HcLec4 was widely distributed in several tissues and was significantly down-regulated at the early stage (2 h) of bacterial infection. We further analyzed the bacteria and carbohydrate binding activities of HcLec4. The results showed that HcLec4 could bind to several bacteria, lipopolysaccharide (LPS) and peptidoglycan (PGN). In HcLec4 knockdown mussels, the bacterial clearance rate was increased, and the expression level of antimicrobial peptides (AMPs) was up-regulated. This study reveals that HcLec4 exerts its antibacterial effect by regulating the expression of AMPs at the early stage of bacterial infection.

A galectin from Hyriopsis cumingii involved in the innate immune response against to pathogenic microorganism and its expression profiling during pearl sac formation.

Hyriopsis cumingii is the most important freshwater pearl mussel cultured in China. The operation for implantation is one necessary technical step for pearl culture. However, implantation-induced trauma results in a series of immune responses and can enable the invasion of pathogenic microbes. Lectin proteins are found widely in nature and play important roles in innate immunity. Galectins are members of the lectin superfamily and are characterized by one or several carbohydrate recognition domains (CRDs) that produce multiple sugar binding sites on the protein. Here we cloned and characterized the H. cumingii galectin gene HcGal1, which encodes a 312 amino acid galectin protein. The HcGal1 transcript was detected in all tested H. cumingii tissues and showed higher expression specifically in immune tissues. The significant upregulation of HcGal1 expression was observed after challenging the mussel with lipopolysaccharide or Gram-negative and Gram-positive bacteria. After implantation, significant downregulation of the HcGal1 transcript was noted in the mantle, hemocytes, and pearl sac in the acute-stress stage (0-24 h) and the stage of wound healing and pearl-sac formation (24 h-7 d). In addition, significant upregulation of HcGal1 expression was observed in the liver in the stage of wound healing and pearl-sac formation. In the pearl-secretion stage (7-35 d), the HcGal1 transcript levels returned to normal in all tested tissues. We also show that recombinantly expressed and purified HcGal1 can agglutinate some Gram-negative and Gram-positive bacteria. In addition, in vivo experiments showed that the recombinant protein HcGal1 could promote phagocytosis by hemocytes. Our data suggest that HcGal1 plays a role in innate immune responses involved in pathogen recognition and wound healing.

Cloning and analysis of gene expression of interleukin-17 homolog in triangle-shell pearl mussel, Hyriopsis cumingii, during pearl sac formation.

Successful allograft of mantle tissues in certain bivalve mollusks can form pearl sacs secreting nacre for pearl production. Little was known, however, about the immune consequences in response to the tissue transplantation. In the present study, interleukin (IL)-17, one of the key regulatory genes of alloimmunity, was cloned from the triangle-shell pearl mussel (HcIL-17) Hyriopsis cumingii by high-throughput sequencing of the mantle transcriptome. The sequence of HcIL-17 contains an open reading frame of 567 bp encoding a putative protein of 188 amino acid residues. Analysis of sequence characteristics, multiple sequence alignment and phylogenetic analysis indicated HcIL-17 was a novel member in the mollusk IL-17 family. Expression of the HcIL-17 gene in donor mantle tissues and in hemocytes of recipient mussel was up-regulated dramatically within 7 days in response to the mantle tissue allograft for pearl aquaculture, suggesting remarkable proinflammatory responses during pearl sac formation in triangle-shell pearl mussels. Analysis of the time-course expression of HcIL-17 gene revealed the induction of HcIL-17 was time-dependent, reflecting the different periods of alloimmune events in triangle-shell mussels. The results of this study provide essential background information for further investigation of mollusk alloimmunity.

Molecular cloning and functional characterization of a novel i-type lysozyme in the freshwater mussel Cristaria plicata.

The freshwater bivalve Cristaria plicata, which is widely distributed in Eastern Asia, is a key species in the pearl culture industry. In this study, a novel invertebrate-type lysozyme, designated as CpLYZ2, was cloned from hemocytes of C. plicata. This lysozyme shares high sequence identity and is homologous to a previously identified lysozyme CpLYZ1 isolated from C. plicata and with HcLyso3 isolated from Hyriopsis cumingii. The full-length cDNA of CpLYZ2 is 913 bp long, which includes an open reading frame (ORF) of 486 bp, a 3' untranslated region (UTR) of 389 bp and a 5' UTR of 38 bp. The ORF encodes a putative polypeptide of 161 amino acids with a predicted molecular mass of 18.2 kDa and a theoretical isoelectric point of 6.56. CpLYZ2 mRNA transcripts can be detected in hemocytes, hepatopancreas, muscle, gills and mantle tissues, the greatest expression being observed in the gills. CpLYZ2 expression in hemocytes, hepatopancreas and gills increased significantly after the mussel was challenged with Aeromonas hydrophila. Furthermore, the optimal pH and temperature for enzyme activity of the recombinant CpLYZ2 were 5.5 and 50°C, respectively. The recombinant lysozyme protein exhibited bacteriolytic activity against Escherichia coli, A. hydrophila, Staphylococcus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis. The findings of this study help to elucidate immune responses in molluscs and will thus expedite disease management of these key freshwater species, in turn boosting pearl culture in eastern Asia.

Molecular cloning, characterization and expression analysis of trypsin-like serine protease from triangle-shell pearl mussel (Hyriopsis cumingii).

Trypsin-like serine protease (TLS) is ubiquitous in animals and plays a number of diverse roles, including dietary protein digestion, hemolymph coagulation, antimicrobial activity and immune responses, among others. This study reports the isolation of a 1048 bp full-length cDNA sequence of TLS from triangle-shell pearl mussel (Hyriopsis cumingii), including a 12 bp 5' UTR (untranslated region), a 172 bp 3' UTR, and an open reading frame (ORF) of 864 bp by rapid amplification of cDNA ends (RACE). Bioinformatic analysis shows that the gene belongs to the trypsin-like serine protease superfamily, and contains a 15 residues N-terminal signal peptide and a conserved C-terminal domain. In comparison to other serine proteases, the catalytic triad were identified as His-98, Asp-149, and Ser-240. Quantitative real-time PCR (qPCR) showed a broad expression of the TLS gene in ten tested tissues. Time-course expression analysis demonstrated that the expression level of the TLS mRNA was significantly up-regulated in eight tested tissues (liver, intestine, gill, heart, axe foot, adductor muscle, kidney and gonad), but down-regulated in mantle and stomach after Aeromonas hydrophila injection. This is one of the results indicate that TLS may be involved in innate defense reactions against A. hydrophila in triangle-shell pearl mussel.