Analysis of the immune function of Caspase-3 in Cristaria plicata.

Caspase-3 is generally considered to be the most important terminal shear enzyme in the process of apoptosis, as well as an important part of cytotoxic T lymphocytes (CTL) killing mechanism, which is confirmed to play an important role in vertebrate cell apoptosis and immune system, and is poorly reported in invertebrates. In this paper, we used bioinformatics to perform amino acid multiple sequence alignment and protein structural domain analysis, and constructed a phylogenetic tree to identify the full-length cDNA of the cloned caspase-3 of Cristaria plicata (Named CpCaspase-3). The expression of caspase-1, caspase-7, caspase-8, and caspase-9 was found to be down-regulated by double-stranded RNA interference of CpCaspase-3 in C. plicata. Some degree of disruption of the caspase signaling pathway occurs. The expression of CpCaspase-3 was affected after injection of Lipopolysaccharide (LPS), Peptidoglycan (PGN), polyinosinic-polycytidylic acid (poly(I:C)), and Aeromonas hydrophila. These results were suggested that CpCaspase-3 was involved in the immune response of C. plicata. The wound recovery process of C. plicata was simulated and CpCaspase-3 was found to promote wound recovery. An autophagy inhibition and autophagy activation model of mussels was constructed, where apoptosis and autophagy undergo crosstalk, and inhibition of autophagy induces the onset of apoptosis, and similarly autophagy activation inhibits the process of apoptosis instead. In addition, a recombinant CpCaspase-3-pEGFP-C1 plasmid was constructed for subcellular localization experiments and found that CpCaspase-3 was distributed in both the nucleus and the cytoplasm. This paper aims to unveil the immune mechanism of C. plicata and provide a theoretical basis for the healthy culture of shellfish.

Nuclear factor E2-associated factor 2 and musculoaponeurotic fibrosarcoma K mediate regulation glutathione peroxidase of Cristaria plicata after microcystin-induced oxidative stress.

Nuclear factor E2-associated factor 2 (Nrf2)/Antioxidant Response Element (ARE) signaling pathway is an endogenous antioxidant pathway that protects cells from oxidative damage. This pathway is triggered when aquatic organisms are exposed to environmental toxicants. In this study, CpMafK (musculoaponeurotic fibrosarcoma K of Cristaria plicata) mRNA expression in hepatopancreas and gills were up regulated after Cristaria plicata (C. plicata) was exposed to microcystin (MC), which showed that CpMafK protected C. plicata from MC. After MC treatment and CpNrf2 (Nrf2 of Cristaria plicata) knockdown, the mRNA expression of CpMafK was down regulated. After MC treatment and CpMafK knockdown, the mRNA expression of CpNrf2 was down regulated. Indicating that the expression of CpNrf2 was positively correlated with CpMafK. CpGPx (GPx of Cristaria plicata) mRNA was also down regulated with the down regulation of CpMafK and CpNrf2. CpGPx promoter contains a variety of transcription factor binding sites, including Nrf2, ARE elements, etc. Gel blocking experiments showed that CpNrf2/CpMafK heterodimers were bound to CpGPx promoters in vitro. Dual luciferase reporter assay showed that CpNrf2/CpMafK heterodimer negatively regulated CpGPx promoter in cells. In conclusion, Nrf2 and MafK mediate regulation of GPx play a crucial role in protecting bivalves from MC.

GST-Mu of Cristaria plicata is regulated by Nrf2/Keap1 pathway in detoxification microcystin and has antioxidant function.

Glutathione S-transferase is a crucial phase II metabolic enzyme involved in detoxification and metabolism in aquatic organisms. This study aimed to investigate the regulation of Nrf2/Keap1 pathway on microcystin-induced CpGST-Mu expression and CpGST-Mu resistance to hydrogen peroxide. A mu class GST from Cristaria plicata (CpGST-Mu) was identified. The full-length cDNA was 1026 bp, with an open reading frame of 558 bp. Subcellular localization revealed that CpGST-Mu was localized in cytoplasm. The optimum pH and temperature for the catalytic activity of CpGST-Mu protein was pH 6 and 40 °C, respectively. The results of Real-time quantitative PCR showed that CpGST-Mu mRNA was constitutively expressed in tissues, with the highest expression level in hepatopancreas and the lowest expression level in gill. The mRNA level of CpGST-Mu was significantly increased under the stress of microcystins and hydrogen peroxide. CpGST-Mu had an antagonistic effect on hydrogen peroxide. In the knockdown experiments, the mRNA levels of CpGST-Mu exhibited corresponding changes while Nrf2 and Keap1 genes were individually knocked down. These findings indicated that GST-Mu exhibited antioxidant properties and its expression was regulated by Nrf2/Keap1 signaling pathway. The study provided new information on the function of GST-Mu and could contribute to future studies on how to excrete microcystins in molluscs.

Immunotoxicity and transcriptome analysis of zebrafish embryos exposure to Nitazoxanide.

Nitazoxanide (NTZ) is a broad-spectrum immunomodulatory drug, and little information is about the immunotoxicity of aquatic organisms induced by NTZ. In the present study, reduced body length and decreased yolk sac absorption in the NTZ-treated group were observed. Meanwhile, the number of innate immune cells and adaptive immune cells was substantially reduced upon NTZ exposure, and the migration and retention of macrophages and neutrophils in the injured area were inhibited. Following NTZ stimulation, oxidative stress levels in the zebrafish increased obviously. Mechanistically, RNA-seq, a high-throughput method, was performed to analyze the global expression of differentially expressed genes (DEGs) in zebrafish embryos treated with NTZ. 531 DEGs were identified by comparative transcriptome analysis, including 121 up-regulated and 420 down-regulated genes in zebrafish embryos after NTZ exposure. The transcriptome sequences were further subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) and analysis, showing phototransduction and metabolic pathway, respectively, and were most enriched. In addition, some immune-related genes were inhibited after NTZ exposure. RNA-seq results confirmed by qRT-PCR were used to verify the expression of the 6 selected genes. The other immune-related genes such as two pro-inflammatory cytokines (IL-1ß, tnfα) and two chemokines (CXCL8b.3, CXCL-c1c) were further confirmed and were differentially regulated after NTZ exposure. In summary, NTZ exposure could lead to immunotoxicity and increased ROS in zebrafish embryos, this study provides valuable information for future elucidating the molecular mechanism of exogenous stimuli-induced immunotoxicity in aquatic ecosystems.

JNK regulates the Nrf2/NQO1-ARE pathway against Microcystins-Induced oxidative stress in freshwater mussel Cristaria plicata.

In response to stress, cells can utilize several processes, such as the activation of the Nrf2/Keap1 pathway as a critical regulator of oxidative stress to protect against oxidative damage. C-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, is involved in regulating the NF-E2-related nuclear factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway. NAD(P)H quinone redox enzyme-1 (NQO1), a downstream target gene of the Nrf2 pathway, plays a vital role in removing peroxide and providing resistance to oxidative injury. We found that microcystins (MCs) stimulated CpNrf2 to express and increase anti-oxidative enzyme activities in a previous experiment. In our current study, the full-length cDNAs of JNK and NQO1 from Cristaria plicata (designated CpJNK and CpNQO1) were cloned. The relative levels of CpJNK and CpNQO1 were high in hepatopancreas. Upon MCs induction, the relative level of CpNQO1 was increased, whereas that of CpJNK was decreased significantly. In contrast, CpNrf2 knockdown upregulated the expression of CpJNK mRNA and phosphorylation of CpJNK protein (Cpp-JNK), but inhibited CpNQO1 expression. Additionally, we found that JNK inhibitor SP600125 stimulated expression of CpNQO1 and CpNrf2 upon exposure to MCs, and we further confirmed that CpNrf2 protein combined with the ARE element in CpNQO1 gene promoter in vitro, and increased CpNQO1-ARE-luciferase activity in a CpNrf2-dependent manner. These findings indicated C. plicata effectively alleviated MC-induced oxidative injury through JNK participated in regulating the Nrf2/NQO1-ARE pathway.

Detoxification and antioxidant functions and regulatory mechanisms of two Delta-class GSTs in paddy crayfish (Procambarus clarkii) after imidacloprid stress.

Glutathione S-transferases (GSTs) are phase II metabolic detoxification enzymes, which are widely found in organisms, and play an important role in helping organisms to resist toxic compounds. In this study, the two Delta-class GSTs cDNA sequences were cloned from Procambarus clarkii (designated as PcGSTD1 and PcGSTD2). Tissue specific expression profile showed that PcGST1,2 were expressed in all 6 tissues, with the highest expression in hepatopancreas. Subcellular localization assay showed that PcGSTD1, 2 were mainly expressed in the cytoplasm of HEK-293 T cells. Recombinant PcGSTD1, 2 showed the highest catalytic activity to the GST model substrate 1-chloro-2,4-dinitrobenzene (CDNB) at 20 and 30 °C, pH 8 and 7, respectively. The mRNA expression of PcGSTD1, 2 and the GSTs activity varied with the time of imidacloprid challenge. The BL21(DE3) expressing PcGSTD1, 2 proteins could more resistant to H2O2. The dsRNA experiments showed that PcKeap1b, PcNrf1, and PcMafK affected the transcription levels of PcGSTD1, 2. The GST-Pulldown results revealed that PcbZIP and PcMafK recombinant proteins could bind to each other in vitro. The gel mobility shift assay demonstrated that PcMafK recombinant protein had affinity with the promoter of PcGSTD2. The Dual luciferase assays analyzed the activity of the promoters after different truncations, the core region of PcGSTD1 promoter was at -440 bp to +54 bp, and that of PcGSTD2 promoter was between -1609∼-1125 bp. These results suggested that PcGSTD1, 2 respond positively to imidacloprid stress in P. clarkii, and the transcriptional expressions of PcGSTD1, 2 were influenced by the factors of PcKeap1b/PcNrf1/PcMafK.

The thioredoxin expression of Cristaria plicata is regulated by Nrf2/ARE pathway under microcystin stimulation.

Thioredoxin plays an important role in inhibiting apoptosis and protecting cells from oxidative stress. This study was aimed to clarify how the expression of Trx from Cristaria plicata is regulated by Nrf2/ARE pathway. The expression of CpTrx mRNA was significantly up-regulated in gill and kidney tissues under microcystin stress. The Nrf2 gene of Cristaria plicata was identified to possess an auto active domain bit. While CpNrf2 was knocked down by specific small RNA, CpTrx mRNA expression was significantly down-regulated. The promoter of CpTrx gene had high transcriptional activity, and this basic transcriptional activity persisted after ARE element mutation. The region of promoter -206 to +217 bp was a core promoter region and had forward regulatory elements. Gel shift Assay exhibited that the CpTrx promoter could bind to the purified proteins CpNrf2 and CpMafK in vitro. The binding phenomenon disappeared after the ARE element mutation in promoter region. Subcellular localization experiments displayed that fluorescence overlap between CpNrf2 and Trx promoter increased under microcystin toxin stress. These results suggested that Trx expression was regulated by Nrf2/ARE pathway under oxidative stress.

Dietary supplementation of ß-1, 3-glucan improves the intestinal health of white shrimp (Litopenaeus vannamei) by modulating intestinal microbiota and inhibiting inflammatory response.

The phenomenon of intestinal dysfunction is widely observed in white shrimp (Litopenaeus vannamei) culture, and ß-1,3-glucan has been confirmed to be beneficial in intestinal health with a lack understanding of its underlying mechanism. Proteobacteria, Firmicutes, and Actinobacteria served as the predominant phyla inhabiting the intestine of white shrimp, whilst a significant variation in their proportion was recorded in shrimp fed with basal and ß-1,3-glucan supplementation diets in this study. Dietary supplementation of ß-1,3-glucan could dramatically increase the microbial diversity and affect microbial composition, concurrent with a notable reduction in the ratio of opportunistic pathogen Aeromonas, gram-negative microbes, from Gammaproteobacteria compared to the basal diet group. The benefits for microbial diversity and composition by ß-1,3-glucan improved the homeostasis of intestinal microbiota through the increase of specialists' number and inhibition of microbial competition caused by Aeromonas in ecological networks; afterward, the inhibition of Aeromonas by ß-1,3-glucan diet dramatically suppressed microbial metabolism related to lipopolysaccharide biosynthesis, followed by a conspicuous decrease in the intestinal inflammatory response. The improvement of intestinal health referred to the elevation in intestinal immune and antioxidant capacity, ultimately contributing to the growth of shrimp fed ß-1,3-glucan. These results suggested that ß-1,3-glucan supplementation improved the intestinal health of white shrimp through the modulation of intestinal microbiota homeostasis, the suppression of intestinal inflammatory response, and the elevation of immune and antioxidant capacity, and subsequently promoted the growth of white shrimp.

Prx5 of Cristaria plicata has antioxidant function and is regulated by Nrf2/ARE signaling pathway.

Cristaria plicata is one of the more important freshwater pearl bivalves in China, which is susceptible to pathogen infection, and greatly impacts the ability of breeding pearls. Nrf2/ARE signaling pathway and its downstream target gene Prx5 have endogenous antioxidant functions to protect cells from oxidative damage. The full-length cDNA of Prx5 was cloned from C. Plicata, which was 1420 bp, encoding a total of 189 amino acids and had two conserved cysteine residues (Cys78 and Cys179). The amino acid sequence of CpPrx5 was highly similar to Prx5 of other species. Real-time fluorescence quantitative PCR showed that CpPrx5 was distributed in various tissues of mussels, and the highest expression was in hepatopancreas. The expression of CpPrx5 up-regulated in hepatopancreas and gills after LPS, PGN and Poly:I:C stimulation. The recombinant plasmid DE3-PGEX-4T-1-CpPrx5 was expressed in Escherichia coli BL21 and showed antioxidant activity. With the increase of CpPrx5 protein concentration, the superhelical form of DNA was protected. The expression of CpPrx5 was up-regulated after interference CpKeap1 and down-regulated after interference CpNrf2. Gel block assay showed that CpNrf2 and CpMafK proteins blocked CpPrx5 promoter. Subcellular localization showed that CpPrx5 was located in 293T nucleus and cytoplasm and CpMafK was located in 293T nucleus. GST-Pull down verified that CpMafK and CpPrx5 could bind in vitro. These results indicated that Prx5 had antioxidant function and could protects DNA from oxidative damage, and participated in transcriptional regulation by combining with the transcription factor MafK. In addition, MafK could combine with Nrf2 to regulate the downstream target gene Prx5.

Identification of Nrf2/Keap1 pathway and its transcriptional regulation of antioxidant genes after exposure to microcystins in freshwater mussel Cristaria plicata.

Microcystins (MC) are one of the most abundant and widely distributed cyanotoxins in aquatic systems. MC inhibits the functions of protein phosphatase 1 and 2A (PP1/2A), which can seriously affect ecosystem integrity. The NF-E2-related nuclear factor 2 (Nrf2)/Kelch-like epichlorohydrin-related protein-1 (Keap1) signaling pathway protects against oxidative damage by activating phase II detoxification/antioxidant enzymes. Our previous study revealed that MC upregulates the expression and enhances the activities of the antioxidant enzymes by stimulating the CpNrf2 signaling pathway. In the current study, to further clarify the regulatory role of Keap1 in response to MC-induced oxidative stress in shellfish, we cloned the full-length cDNA of Keap1a and Keap1b from Cristaria plicata (designated CpKeap1a and CpKeap1b), which are 2952 and 3710 bp peptides, respectively. The amino acid sequence of CpKeap1a and CpKeap1b contained Tram-track and Bric-a-brac (BTB), Intervening region (IVR), and Double glycine repeat (DGR) domain. Additionally, CpKeap1a contained two cysteine residues analogous to Cys-273 and -288 in zebrafish, but CpKeap1b did not. Moreover, CpKeap1a and -1b formed a homodimer and heterodimer, respectively, and also formed a heterodimer with CpNrf2. In the hepatopancreas, the expression levels of CpKeap1a and -1b were the highest, but MC treatment down-regulated the expression of these proteins. Moreover, the transcription of antioxidant enzymes with antioxidant response element (ARE-driven enzymes), including CpMnSOD, CpCu/ZnSOD, CpTRX, CpPrx, CpSe-GPx, and Cpsigma-GST was upregulated by CpNrf2 in the hepatopancreas. Compared with the MC-induced group, CpKeap1a-siRNA1117 injection significantly increased the transcription of mRNAs for ARE-driven enzymes and Nrf2. CpKeap1a-siRNA1117 also enhanced the activities of antioxidation enzymes. These findings demonstrated that Keap1a negatively regulated the expression of Nrf2 protein and MC-induced oxidative stress response in C. plicata. Therefore, we speculated that CpKeap1a promoted CpNrf2 by recognizing and binding MC. These events then protected molluscs from MC-induced oxidative damage.

P62/SQSTM1 upregulates NQO1 transcription via Nrf2/Keap1a signaling pathway to resist microcystins-induced oxidative stress in freshwater mussel Cristaria plicata.

Microcystins (MCs) are the most frequent and widely distributed type of cyanotoxin in aquatic systems, and they cause an imbalance of the body's oxidative system. In a previous experiment, we demonstrated that the mollusk Cristaria plicata can protect against MC-induced oxidative damage through the nuclear factor erythroid 2-related factor 2(Nrf2)/Kelch-like epichlorohydrin-related protein-1 (Keap1) pathway. Here, we evaluated whether selective autophagy affects the Nrf2/Keap1a anti-oxidative stress pathway in C. plicata. Full-length cDNA sequences of p62/SQSTM1 from C. plicata (Cpp62) were divided into 2484 bp fragments. From N-terminal to C-terminal, the amino acid sequence of Cpp62 contained PB1 (Phox and Bem1p domain), ZNF (zinc finger domain) chain, LIR (LC3 interacting region) and UBA (ubiquitin-associated domain) domains, but not the KIR (Keap1 interacting region) domain. We confirmed that Cpp62 did not bind to CpKeap1a in vitro, and the relative level of Cpp62 was the highest in the hepatopancreas. Moreover, MCs significantly upregulated the mRNA and protein levels of Cpp62 in the hepatopancreas after CpKeap1a knockdown, whereas Nrf2 upregulated the transcription levels of Cpp62, suggesting that MCs increased Cpp62 expression via the Nrf2/Keap1a signaling pathway. Moreover, Cpp62 and CpNrf2 proteins have a strong affinity for the NQO1 promoter, but MCs inhibited the ability of CpNrf2 and Cpp62 to upregulate luciferase activity. The results show that Nrf2 and the p62 protein induced p62 expression by binding to ARE (antioxidant response element) sequences in the p62 promoter of C. plicata, thereby promoting p62 to resist MC-induced oxidative stress. Therefore, we speculate that MCs induce p62-dependent autophagy in C. plicata, resulting in the inhibition of Nrf2 transcription and Cpp62 promoter activity. These findings help to reveal the mechanism by which the p62-Nrf2/Keap1 pathway mitigates MC-induced oxidative damage in mussels.

Prevalence and genetic diversity of Aeromonas veronii isolated from aquaculture systems in the Poyang Lake area, China.

The area around Poyang Lake is the main aquaculture area in Jiangxi Province, China, and an important base for the supply of freshwater aquatic products. Aquaculture in the Poyang Lake area is severely threatened by diseases caused by bacterial pathogens, and Aeromonas veronii has been the main pathogen in recent years. In this paper, ERIC-PCR genotyping, virulence gene and antimicrobial resistance gene detection, and drug susceptibility tests were carried out on 46 A. veronii isolates obtained from aquaculture systems in the Poyang Lake area from 2016 to 2020. The results showed that the A. veronii strains in the Poyang Lake area had high genetic diversity, and 46 strains produced 36 ERIC genotypes. There were no geographical and temporal differences in the cluster analysis results and no dominant clones. All 13 virulence genes tested were detected, and all isolates had harbored 2 or more virulence genes, with a maximum of 12 virulence genes detected. Among the 22 antimicrobial resistance genes selected, 15 were detected; 97.8% of the isolates contained 2 or more antimicrobial resistance genes, with a maximum of 9 antimicrobial resistance genes. Drug susceptibility tests showed that some strains were resistant to a variety of traditionally effective drugs for Aeromomas, such as enrofloxacin and florfenicol. This study provides a reference for exploring the impact of aquaculture in the Poyang Lake area on public health.

Response of Intestinal Microbiota to the Variation in Diets in Grass Carp (Ctenopharyngodon idella).

The intestinal microbiota is important for the nutrient metabolism of fish and is significantly influenced by the host's diet. The effect of ryegrass and commercial diets on the intestinal microbiota of grass carp was compared in this study. In comparison to ryegrass, artificial feed significantly reduced the microbial diversity in the intestine, which was measured by a decrease in the observed OTUs, ACE, Shannon, and the InvSimpson index. Although grass carp fed with ryegrass and artificial feed shared a dominant phyla Firmicutes and Proteobacteria, the microbial composition was clearly distinguishable between the two groups. In grass carp fed with ryegrass, Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria predominated, whereas Bacilli was significantly higher in the artificial feed group due to an increase in Weissella and an unassigned Bacillales bacteria, as well as a significant increase in a potential pathogen: Aeromonas australiensis. Grass carp fed with ryegrass exhibited a more complex ecological network performed by the intestinal bacterial community, which was dominated by cooperative interactions; this was also observed in grass carp fed with artificial feed. Despite this, the increase in A. australiensis increased the competitive interaction within this ecological network, which contributed to the vulnerable perturbation of the intestinal microbiota. The alteration of the microbial composition through diet can further affect microbial function. The intestinal microbial function in grass carp fed with ryegrass was rich in amino acids and exhibited an increased energy metabolism in order to compensate for a low-nutrient diet intake, while the artificial feed elevated the microbial lipid metabolism through the promotion of its synthesis in the primary and secondary bile acids, together with a notable enhancement of fatty acid biosynthesis. These results indicated that diet can affect the homeostasis of the intestinal microbiota by altering the microbial composition and the interspecific interactions, whilst microbial function can respond to a variation in diet.

MafG-like contribute to copper and cadmium induced antioxidant response by regulating antioxidant enzyme in Procambarus clarkii.

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 Cu2+ and Cd2+, PcMafG-like was significantly up-regulated in hepatopancreas and gill, and the same result was testified by situ hybridization. The representative antioxidant genes, CAT, GPx and CZ-SOD, were significantly induced by Cu2+; CAT and GPx was induced by Cd2+. PcMafG-dsRNA significantly inhibited the expression of these up-regulated genes, but also inhibited the expression of other detected genes CZ-SOD, GST-θ and GST-1like. The antioxidant effect of PcMafG-like was further verified by oxidative stress markers (T-SOD, CuZnSOD, GPx, CAT, GSH and MDA) kits. Cu2+ and Cd2+ could induce the contents of these oxidative stress markers (MDA, GSH, CZ-SOD, CAT in Cu2+/Cd2+ treated group, and GSH-Px in Cd2+ group), while interference of PcMafG-like significantly inhibited the up-regulation. Furthermore, hematoxylin-eosin staining experiments showed that the degree of pathological damage was dose-dependent and time-dependent, and the pathological damage was more serious after dsRNA interfered with PcMafG-like. In addition, subcellular localization showed that PcMafG-like gene existed in nucleus. The recombinant protein PcMafG-like was expressed and purified in prokaryotic expression. The affinity analysis of promoter by agarose gel electrophoresis suggested that PcMafG-like could bind with CAT promoter in vitro. This indicated that PcMafG-like could activate antioxidant genes.

ß-arrestin interacts with TRAF6 to negatively regulate the NF-κB pathway in triangle sail mussel Hyriopsis cumingii.

As members of arrestins family, ß-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 ß-arrestin homolog named Hcß-arrestin was cloned and identified from Hyriopsis cumingii. Predicted Hcß-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 Hcß-arrestin gene was widely expressed in the detected tissues and with the highest expression in hepatopancreas. The transcription of Hcß-arrestin in hepatopancreas and gill of mussels was significantly up-regulated after stimulation with peptidoglycan, lipopolysaccharide (LPS) and polyinosinic polycytidylic acid. Knockdown of Hcß-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 Hcß-arrestin can bind to HcTRAF6 protein in vitro. Dual luciferase reporter assay showed that the co-expression of HcTRAF6 and Hcß-arrestin inhibited the activation of NF-κB reporter by HcTRAF6. These findings indicated that Hcß-arrestins could interact with HcTRAF6 to negatively regulate the NF-κB pathway in H. cumingii.

MAPK/MAK/MRK overlapping kinase mediated apoptosis through caspase signaling pathway from Cristaria plicata.

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.

Two NF-κB subunits are associated with antimicrobial immunity in Hyriopsis cumingii.

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.

Construction of wound repair model and function of recombinant TIMP from Hyriopsis cumingii.

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.

Characterization of a novel toll-like receptor and activation NF-κB signal pathway in triangle sail mussel Hyriopsis cumingii.

Toll-like receptor (TLR) family plays an important role in innate immunity for detection of and defense against microbial pathogens. In this study, a novel toll-like receptor (HcTLRn) was characterized from freshwater pearl mussel H. cumingii. The complete sequence of HcTLRn was 3725 bp, and the open reading frame (ORF) encoded 718 amino acid residues. Predicted HcTLRn protein possessed seven atypical leucine-rich repeat (LRR) domains, two typical LRR subfamily domains, a C-terminal domain LRR, a transmembrane domain and an intracellular Toll/interleukin-1 (IL-1) receptor domain. Transcripts of HcTLRn were constitutive expressed in the tissues of healthy mussels and were markedly induced in hepatopancreas and gills after lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic polycytidylic acid (ploy I: C) stimulation. Knockdown of HcTLRn in vivo significantly decreased the mRNA levels of TLR pathway transcription factors p65 and p105 as well as antimicrobial peptides (AMPs) including lysozyme (HcLys), theromacin (HcTher), whey acidic protein (HcWAP), LPS-binding protein/bactericidal permeability increasing protein (HcLBP/BPI) 1 and 2 after mussels challenged by LPS. In situ hybridization results showed that HcTLRn mRNA was significantly increased in hemocytes after LPS, PGN and poly I:C stimulation. HcTLRn protein was mainly expressed in hepatopancreas and gills and was significantly increased after LPS stimulation. Moreover, recombinant extracellular domain of HcTLRn (HcTLRn-ECD) proteins could bind to a variety of bacterial and pathogen-associated molecular patterns such as LPS, PGN, and poly I:C in vitro. Subcellular localization results showed that HcTLRn was mainly distributed near the cell membrane and in cytoplasm. Over-expression of HcTLRn activated the NF-κB luciferase reporter in HEK293T cells. Collectively, these results suggested that HcTLRn was a TLR family member that might play an important role in activation of NF-κB signal pathway in Mollusca.

Effect of microcystin on the expression of Nrf2 and its downstream antioxidant genes from Cristaria plicata.

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.