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As members of arrestins family, β-arrestins are widely expressed in monocytes, macrophages, neutrophils and other immune cells. They can regulate the immune response of bodies through various ways. In the present study, a β-arrestin homolog named Hcβ-arrestin was cloned and identified from Hyriopsis cumingii. Predicted Hcβ-arrestin protein contained a conserved arrestin domain, which could be further divided into arrestin-N (39-192aa) and arrestin-C (211-365aa). Amino acid sequence alignment showed that it had the highest identity with Mytilus galloprovincialis and Mytilus edulis counterpart, which was 89.02% and 87.68%, respectively. Furthermore, real-time quantitative PCR analysis showed that the Hcβ-arrestin gene was widely expressed in the detected tissues and with the highest expression in hepatopancreas. The transcription of Hcβ-arrestin in hepatopancreas and gill of mussels was significantly up-regulated after stimulation with peptidoglycan, lipopolysaccharide (LPS) and polyinosinic polycytidylic acid. Knockdown of Hcβ-arrestin gene significantly increased the expression of some antibacterial effector genes, such as lysozyme, LPS-binding protein/bactericidal permeability increasing protein and theromacin in hepatopancreas and gills of LPS stimulated mussels, but only had little effect on TLR pathway genes. In addition, GST pull-down assay confirmed that Hcβ-arrestin can bind to HcTRAF6 protein in vitro. Dual luciferase reporter assay showed that the co-expression of HcTRAF6 and Hcβ-arrestin inhibited the activation of NF-κB reporter by HcTRAF6. These findings indicated that Hcβ-arrestins could interact with HcTRAF6 to negatively regulate the NF-κB pathway in H. cumingii.

The Betta fish displays a remarkable variety of phenotypes selected during domestication. However, the genetic basis underlying these traits remains largely unexplored. Here, we report a high-quality genome assembly and resequencing of 727 individuals representing diverse morphotypes of the Betta fish. We show that current breeds have a complex domestication history with extensive introgression with wild species. Using a genome-wide association study, we identify the genetic basis of multiple traits, including coloration patterns, the "Dumbo" phenotype with pectoral fin outgrowth, extraordinary enlargement of body size that we map to a major locus on chromosome 8, the sex determination locus that we map to

MAPK/MAK/MRK Overlapping Kinase (MOK) belongs to MAP kinase superfamily, which plays an important role in regulating cell growth, division, and differentiation. Caspase-3, as the final executor of apoptosis, has an important position in the caspase-mediated apoptotic signaling pathway. The full-length cDNA of MOK and caspase-3 were cloned from Cristaria plicata (designated CpMOK and CpCaspase-3). The CpMOK gene was sequence with a full-length of 1413 bp, encoding a total of 470 amino acids, and containing an S_TKc structural domain. CpCaspase-3 has a sequence of 2425 bp, encoding 322 amino acids, containing a CASc domain. Real-time fluorescence quantitative PCR analysis showed that CpMOK and CpCaspase-3 distributed in various tissues of C. plicata, and the highest expression of CpMOK and CpCaspase-3 mRNA was in hepatopancreas. The expression of CpMOK was significantly changed in hepatopancreas, gills, and kidneys by the construction of wound model as well as stimulation of LPS, PGN, Poly I: C and Aeromonas hydrophila. Subcellular localization experiments confirmed that CpMOK was localized in the nucleus. Furthermore, the double-stranded RNA (dsRNA) of CpMOK was constructed for interference experiment, and the results showed that the mRNA expression of apoptotic gene signals caspase-1, caspase-3, caspase-7, caspase-8, and caspase-9 were increased. The expression of caspase-1, -3, -7, -9, cytochrome C (Cyt-c) and tumor necrosis factor-α (TNF-α) was detected by ELISA. Fluorescent staining of apoptotic cells using the Tunnel method revealed an increase in the number of apoptotic cells after interference. These results suggested that CpMOK knockdown could induce caspase-mediated apoptosis in C. plicata, and the phosphorylation of the kinase was disrupted during the process.

Avian musculoaponeurotic fibrosarcoma (Maf) proteins play an important role in Nrf2/Keap1 signaling pathway, which mainly resist the oxidant stress. The members of sMaf have a high homology basic leucine zipper (bZIP) and lack trans activation domain, and could interact with other transcriptional regulatory factors as a molecular chaperone. In this study, a full-length MafG-like gene was cloned from Procambarus Clarkii, designated as PcMafG-like, which consisted of an ORF length of 246 bp encoding 82 amino acids, a 5' untranslated region (UTR) of 483 bp, and a 3' UTR of 111 bp. The domain of PcMafG-like had a bZIP-Maf domain that binds to DNA. The cDNA sequence of PcMafG-like was 99 % similar to that of Penaeus vannamei. The mRNA of PcMafG-like was expressed in all tested tissues, and the highest expression was in muscle tissue. Under stimulation of Cu

The Betta fish displays a remarkable variety of phenotypes selected during domestication. However, the genetic basis underlying these traits remains largely unexplored. Here, we report a high-quality genome assembly and resequencing of 727 individuals representing diverse morphotypes of the Betta fish. We show that current breeds have a complex domestication history with extensive introgression with wild species. Using a genome-wide association study, we identify the genetic basis of multiple traits, including coloration patterns, the “Dumbo” phenotype with pectoral fin outgrowth, extraordinary enlargement of body size that we map to a major locus on chromosome 8, the sex determination locus that we map to dmrt1 , and the long-fin phenotype that maps to the locus containing kcnj15 . We also identify a polygenic signal related to aggression, involving multiple neural system-related genes such as esyt2 , apbb2 , and pank2 . Our study provides a resource for developing the Betta fish as a genetic model for morphological and behavioral research in vertebrates.

To elucidate the antibacterial role of peroxinectin (referred to as PXN) and its molecular mechanism in Chinese mitten crab Eriocheir sinensis, we analyzed the bacterial binding and removal of the peroxinectin recombinant protein in vitro and the interaction of peroxinectin with integrin and CuZn-SOD through GST-pulldown and bimolecular fluorescence complementation methods. Concurrently, the effect of peroxinectin interference on the expression of other immune-related genes was studied using RNA interference. The results showed that the recombinant peroxinectin protein could bind to Bacillus subtilis, Staphylococcus aureus, Aeromonas hydrophila, and Vibrio parahaemolyticus with different affinities in vitro and could eliminate Vibrio parahaemolyticus in vivo. The findings also indicated that peroxinectin could establish interactions with integrin and CuZn-SOD in vitro. Furthermore, 48 h after the injection of the peroxinectin gene siRNA in vivo, the expression of peroxinectin mRNA decreased significantly (P 0.05), integrin mRNA expression decreased by 16.8%, and CuZn-SOD mRNA expression decreased by 62.84% (P 0.01). The expression levels of Dorsal, GPx, GST, PPAF, and Relish (P 0.01), as well as that of lectin (P 0.001) were significantly decreased. When peroxinectin siRNA was injected in vivo for 48 h and Aeromonas hydrophila was injected into mitten crabs, the expression of immune-related genes significantly increased. All data indicate that the recombinant peroxinectin protein in Chinese mitten crabs can recognize and bind different bacteria and promote the elimination of Vibrio parahaemolyticus from the body. Furthermore, peroxinectin may establish interactions with integrin and CuZn-SOD to activate the expression of related immune genes to elicit responses to bacterial infections and achieve immune protection.

Metalloproteinase tissue inhibitors (TIMPs) have the activity of inhibiting matrix metalloproteinases (MMPs), which can promote cell growth, bind to the matrix, inhibit angiogenesis, and play a key role in extracellular matrix (ECM) metabolism regulation. In this study, TIMP-1, 2 from Hyriopsis cumingii (designated as HcTIMP-1, 2) were cloned and identified. Full-length cDNA of HcTIMP-1, 2 was 1160 bp and 729 bp, encoding 235 and 150 amino acid residues, respectively. The predicted molecular weight of HcTIMP-1 and 2 protein was 27.26 and 16.58 kDa, with isoelectric points of 8.89 and 8.72, respectively. HcTIMP-2 contained only one netrin (NTR) domain at the N-terminal but lacked a C-terminal domain. The mRNA of HcTIMP-1, 2 was expressed in hepatopancreas, gills, muscles, hemocytes, and mantles, which had the highest expression in hemocytes and muscles. The expression of HcTIMP-1, 2 had increased remarkably in hemocytes after bacterial challenge. After trauma, HcTIMP-1, 2 genes had the highest expression level in the first day. This indicated that HcTIMP-1 and 2 were involved in the immune response of H. cumingii. The soluble recombinant proteins HcTIMP-1, 2 were expressed efficiently in Escherichia coli BL21 (DE3) by constructing pET32a-TIMP1, 2 recombinant plasmids. The concentration of the recombinant was 0.14 and 0.31 mg/mL, respectively. The recombinant HcTIMP-1, 2 proteins were shown to inhibit human MMP2 activity and promoted the growth of NBL-7 and HUVE cells.

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 70-day feeding trial was conducted to investigate the effects of dietary supplementation with prebiotics β-glucan (BG group) and probiotics Bacillus subtilis (BS group) on growth performance, fillet quality, immune response, and antioxidant status of Pengze crucian carp (Carassius auratus var. Pengze). The results showed that supplementation of B. subtilis significantly improved the growth performance than other groups whereas feed efficiency was unaffected by dietary treatments. The textures of muscle in terms of hardness, springiness, cohesiveness, gumminess, chewiness, and resilience were higher in BG and BS groups than Control group. Supplementation of β-glucan and B. subtilis acted as an hypolipidemic in terms of decreasing the total cholesterol, high-density lipoprotein, and low-density lipoprotein whereas, increased the immune responses in serum measured by acid phosphatase, alkaline phosphatase, and catalase activities. Dietary supplementation of β-glucan and B. subtilis significantly improved the fold height and microvillus height in contrast to basal diet. Moreover, β-glucan could significantly increase digestive capacity observed in terms of an increase in amylase and trypsase activities, and B. subtilis significantly increased amylase and lipase activities in intestine. β-glucan and B. subtilis supplementation enhanced the immunity and antioxidant capacity via an increase in acid phosphatase, alkaline phosphatase, glutathione peroxidase, reduce glutathione, catalase, and total superoxide dismutase activities, accompanied by a significant reduction in malondialdehyde. Overall, the results indicated that B. subtilis improved the growth performance by increasing the digestive enzymes activities and improving the intestinal morphology. β-glucan and B. subtilis supplementation significantly improved the fillet quality, immune responses and antioxidant status of Pengze crucian carp.

This study was aimed to evaluate the effects of dietary supplementation of Bacillus cereus as probiotics on growth, fillet quality, serum biochemical parameters and intestinal histology of Pengze crucian carp (Carassius auratus var. Pengze). Fish were fed basic diet (Control group) and three diets such as CD7, CD9 and CD11 supplemented with B. cereus at dose of 10⁷, 10⁹ and 10¹¹ CFU/kg for 70 days. The results showed that supplementation of 10⁹ CFU/kg B. cereus significantly improved the growth performance compared to that in the Control group. The textures of muscle were improved by dietary supplementation of probiotics, and the hardness, gumminess, chewiness and the resilience of fish fillet increased with increasing dose of B. cereus. Total cholesterol including high‐density lipoprotein and low‐density lipoprotein level in serum decreased correspondingly with increasing level of probiotics whereas, acid phosphatase and catalase activities increased in serum. B. cereus supplementation enhanced the immunity and antioxidant capacity via an increase in acid phosphatase, alkaline phosphatase, glutathione peroxidase, reduce glutathione, and catalase, accompanied by a significant reduction in malondialdehyde. Higher intestinal fold height was observed in CD9 group than other groups. The lowest enterocyte height was exhibited in the Control group, HE values increased with increasing dose of probiotics. Moreover, dietary inclusion of B. cereus significantly affected the digestive enzymes activities in terms of higher lipase and trypase activities. In conclusion, dietary supplementation of B. cereus could promote the growth, elevate the immunity and antioxidant status of carp, and importantly improve the texture of fish fillet.

Intestinal microbiota plays crucial roles in nutrient uptake and contributes to obesity in aquatic animals, although the association between intestinal microbiota and obesity remains unclear. In this study, high-throughput sequencing was employed to determine the composition, interspecific interactions, and potential function of the microbial community in the intestine of rice field eel (Monopterus albus) fed with basal diet (Control group, 6.30% lipid) or high-fat diet (HF group, 12.11% lipid). The high-fat diet decreased species richness, accompanied by a significant reduction in the diversity of intestinal microbiota, and significantly increased the relative abundance of Fusobacteriia (classified as Cetobacterium), concurrent with a significant reduction in Clostridia (classified as Clostridium) in the eel. However, the eels in both groups shared the same dominant gut microbiota, comprising Clostridia, Fusobacteriia, and Gammaproteobacteria. Molecular ecological network analysis described the complex inter-species interactions within the intestinal bacterial community, which in both groups was predominated by positive interactions. The dominant intestinal microbiota was found to be the major component of the ecological network and could play vital roles, for instance, as connectors, which have a major contribution in maintaining the stability of the bacterial community. The high-fat diet disturbed the intestinal microbiota homeostasis through the deterioration of the ecological network by reducing the average connectivity, negative interactions, and the number of connectors. Analysis of predicted functions (PICRUSt) showed that the high-fat diet significantly increased some potential functions of intestinal microbiota involved in lipid, amino acid, carbohydrate, and energy metabolism, in accordance with metabolic changes observed in terms of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, cholesterol, and glucose in the serum. These results suggested that the change in intestinal microbiota may contribute to the development of diet-induced fat deposition in eels.

The NF-κB pathway activated by bacteria and viruses produces a series of antimicrobial peptides that participate in the innate immune response. In this study, two NF-κB subunits were cloned and identified from Hyriopsis cumingii (named Hcp65 and Hcp105) using RT-PCR and RACE. The predicted Hcp65 protein possessed a N-terminal Rel homology domain (RHD) and an Ig-like/plexins/transcription factors domain (IPT); the Hcp105 contained an RHD, an IPT domain, 6 ankyrin (ANK) domain and a death domain. Quantitative reverse transcription PCR (qRT-PCR) showed that Hcp65 and Hcp105 were constitutively expressed in the detected tissues, and were significantly up-regulated in hemocytes, hepatopancreas and gill of mussels challenged with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly I: C). The dsRNA-mediated silencing of Hcp65 and Hcp105 caused significant reduction of immune genes such as lysozyme (HcLyso), theromacin (Hcther), whey acid protein (HcWAP), LPS-binding protein/bactericidal permeability protein (HcLBP/BPI) 1 and 2. In addition, subcellular localization experiments showed that Hcp65 and Hcp105 proteins were expressed in both the nucleus and cytoplasm of HEK-293T cells, and Hcp50 proteins (mature peptide of Hcp105) were mainly localized in the nucleus. The recombinant Hcp65 and Hcp50 protein could form homodimer and heterodimer and bind κB site in vitro. These results provide useful information for understanding the role of NF-κB in mollusks.

Glutathione-S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione, and play an important role in protecting organisms against the toxicity of reactive oxygen species. In this study, two distinct sigma-class GST (CpGSTσ1 and 2) cDNA sequences were cloned from freshwater bivalve Cristaria plicata. The full length cDNA of CpGSTσ1 and 2 was 826 bp and 1609 bp, which encoded 213 and 248 amino acid residues, respectively. Their transcripts were expressed in all detected tissues and the highest expression level was in hepatopancreas from C. plicata. The expression level of CpGSTσ1 and 2 in hepatopancreas and hemocytes showed a significantly increased trend after bacterial challenge. The recombinant CpGSTσ1 was successfully expressed as a soluble form in Escherichia coli DE3. The specific activity of recombinase toward CDNB was 46.965 ± 0.082 μmol/min/mg, and its optimum temperature and pH was 37 °C and 9.0, respectively. The recombinant of CpGSTσ1 could bear 6 M urea and 8% SDS, when the concentration of urea was 8 M and its activity was only below 20%. The results might provide a better perspective on the mechanisms of resistance to bacterial infection in molluscs.

The giant roundworm Ascaris infects pigs and people worldwide and causes serious diseases. The taxonomic relationship between Ascaris suum and Ascaris lumbricoides is still unclear. The purpose of the present study was to investigate the genetic diversity and population genetic structure of 258 Ascaris specimens from humans and pigs from 6 sympatric regions in Ascaris-endemic regions of China using existing simple sequence repeat data. The microsatellite markers showed a high level of allelic richness and genetic diversity in the samples. Each of the populations demonstrated excess homozygosity (Ho0). According to a genetic differentiation index (Fst=0.0593), there was a high-level of gene flow in the Ascaris populations. A hierarchical analysis on molecular variance revealed remarkably high levels of variation within the populations. Moreover, a population structure analysis indicated that Ascaris populations fell into 3 main genetic clusters, interpreted as A. suum, A. lumbricoides, and a hybrid of the species. We speculated that humans can be infected with A. lumbricoides, A. suum, and the hybrid, but pigs were mainly infected with A. suum. This study provided new information on the genetic diversity and population structure of Ascaris from human and pigs in China, which can be used for designing Ascaris control strategies. It can also be beneficial to understand the introgression of host affiliation.

The proteins of Smad family are critical components of the TGF-β superfamily signal pathway. In this paper, we cloned two intracellular mediators of TGF-β 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.

Peroxiredoxins (Prxs) play an important role against various oxidative stresses by catalyzing the reduction of hydrogen peroxide (H 2 O 2 ) 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 H 2 O 2 in vivo.

As a specific pearl mussel in China, Hyriopsis cumingii has enormous economic value. However, the organism damage caused by pearl insertion is immeasurable. TGF-β/Smad signal transduction pathways are involved in all phases of wound healing. We have previously reported on two cytoplasmic signal transduction factors, Smad3 and Smad5 in mussel H. cumingii (named HcSmads), suggesting their involvements in wound healing. Here, Smad4 was cloned and described. The full length cDNA of HcSmad4 was 2543 bp encoded 515 amino acids. Deduced HcSmad4 protein possessed conserved MH1 and MH2 domains, nuclear location signals (NLS), nuclear exput signals (NES) and Smad activation domain (SAD). Transcripts of Smad3, 4 and 5 were constitutively expressed in all detected tissues, at highest levels in muscles. Furthermore, HcSmad4 mRNA levels were significantly increased at incision site post wounding, and expression of downstream target genes of Smad4, such as HcMMP1, HcMMP19, HcTIMP1 and HcTIMP2 were upregulated to a certain extent. Whatever knocked down HcSmad3/4 or treated by specific inhibitors of Smad 3 (SIS3), expression levels of these genes displayed a significantly downregulated tendency compared with the wound group. In addition, histological evaluation suggested that Smad3 knockdown or SIS3 treatment was accelerated wound healing, and then Smad4 knockdown delayed the process of wound healing in mussels. These data implicate that Smad3/4 play an important role in tissue repair in mollusks.

The Ganjiang River (length: 823 km; drainage area: 82,809 km2) is the largest river that flows into Poyang Lake and an important tributary of the Yangtze River. In this study, fish fauna were collected from 10 stations in the lower reaches of the river (YC: Yichun, XY: Xinyu, SG: Shanggao, GA: Ganan, ZS: Zhangshu, FC: Fengcheng, NC: Nanchang, QS: Qiaoshe, NX: Nanxin, CC: Chucha) from March 2017 to February 2018. The species composition and distribution as well as spatio-temporal variation in biodiversity and abundance were then examined. Overall, 12,680 samples comprising15 families and 84 species were collected, the majority of which belonged to the Order Cypriniformes (69.05% of the total species collected) and Cyprinidae (64.29%). Moreover, of these 84 species, 36 (42.86%) were endemic to China. Dominant species were Cyprinus carpio (index of relative importance (IRI): 17.19%), Pseudobrama simoni (IRI: 10.81%) and Xenocypris argentea (IRI: 10.20%). Subsequent cluster analysis divided the samples into three significantly different groups by sample site. Meanwhile, Margalef species richness and Shannon−Wiener diversity indices were both low, and along with analyses of abundance-biomass curves suggested moderate disturbance. Current threats to the conservation of fish biodiversity in the lower reaches were also reviewed and management solutions suggested. The results will help form the basis for reasonable exploitation and protection of freshwater fish in the lower reaches of the Ganjiang River.

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.

Microcystin (MC) is a cyclic heptapeptide toxin. Nuclear factor erythocyte 2-related factor 2 (Nrf2) can enhance cellular survival by mediating phase 2 detoxification and antioxidant genes. In this study, CpNrf2 cDNA sequences were cloned from freshwater bivalve Cristaria plicata. The full-length CpNrf2 cDNA sequence was 4259 bp, and its homology was the highest with Mizuhopecten yessoensis, reaching 46%. CpNrf2 transcription levels were examined in all tested tissues, and the highest level was in hepatopancreas from C. plicata. The recombinant protein pET32-CpNrf2 was purified with the content of 1.375 mg/mL. The expression levels of CpNrf2 mRNA were raised in hepatopancreas after MC stimulation. After CpNrf2 knockdown, CpNrf2 mRNA levels were significantly down-regulated after 24 h. Compared with control group, the expression levels of ARE-driven enzymes (CpMnSOD, CpCuZnSOD, CpTRX, CpPrx, CpSe-GPx and Cpsigma-GST) were significantly increased, and those enzyme activities were also significantly up-regulated in MC-stimulated group. However, in CpNrf2-iRNA group, they were significantly down-regulated. The results revealed that Nrf2/ARE pathway is very crucial to protect molluscs from MC.

To investigate the regulation of

The small Mafs, MafF, MafG and MafK play critical roles in morphogenesis and homeostasis through associating with Cap “n” Collar family of transcription factors. In this study, we tried to identify a small Maf protein in the freshwater mussel Cristaria plicata. The MafK cDNA of C. plicata, designated as CpMafK, was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends PCR. The full length cDNA of CpMafK is 2170 bp, which includes an open reading frame of 570 bp, encoding 189 amino acids. CpMafK possesses four conserved domains and shows a low level (54–63%) of sequence similarity to small Mafs from other species. The results of Real-time quantitative PCR revealed that CpMafK mRNA was constitutively expressed in tissues, and the highest expression level was in hepatopancreas. After microcystin challenge, the expression levels of CpMafK mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpMafK was cloned into the plasmid pET-32, and the recombinant protein was expressed in Escherichia coli BL21(DE3). CpMafK could combine to the promoters of CpGST1 and CpGST2 with high-affinity in vitro. Therefore, CpMafK could regulate the expression of detoxification.

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.

The water mite Unionicola penicillatus (Wen, Gao & Zhu) (Acari: Unionicolidae) was collected in freshwater bivalve Cristaria plicata (Unionidae). The population variation of U. penicillatus was investigated in 504 bivalves during the period from August 2013 to July 2014 in Poyang Lake, East China. The results indicated that the overall prevalence and abundance of U. penicillatus was 9.52% and 1.58±10.38. The highest prevalence was 58.70% in September. The highest abundance levels were observed when the length of bivalves was in 220–300 mm. The number of U. penicillatus in a host was not correlated with the sex of bivalves. The outer and inner gills of bivalves were the preferred sites of U. penicillatus.

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.