Identification and Functional Analysis of MAPKAPK2 in Hyriopsis cumingii.

MAPKAPK2 (MK2) is an important regulator of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, which is involved in a plethora of cellular processes concluding the development of gamete cells in meiosis and resisting pathogenic bacterial infestation. Hyriopsis cumingii is a significant mussel resource in China and a good material for pearl breeding. To explore the role of MK2 in H. cumingii, MK2 was identified and cloned, whose full-length cDNA was 1568 bp, including 87 bp in 5' UTR, 398 bp in 3' UTR, and 1083 bp in the open reading frame (ORF) region, encoding 360 amino acids. The expression of MK2 was the highest in the gills. Meanwhile, there was a significant difference in the gonads. After Aeromonas hydrophila and Lipopolysaccharide (LPS) infestation, the transcript level of the MK2 was upregulated in the gills. It indicated that MK2 might be involved in the innate immune response of H. cumingii after a pathogenic attack. After quantifying H. cumingii of different ages, it was found that the expression of MK2 was highest at 1 year old. In situ hybridization (ISH) results showed that the blue-purple hybridization signal was very significant in the oocytes and egg membranes of the female gonads of H. cumingii. The expression of MK2 increased gradually at the age of 1 to 5 months and showed a downward trend at the age of 5 to 8 months. It was suggested that MK2 might play an important role in the formation of primitive germ cells in H. cumingii. To sum up, MK2 might not only be involved in the immune response against pathogenic bacterial infection but also might play an important role in the development of the gonads in H. cumingii.

Histopathology, antioxidant responses, transcriptome and gene expression analysis in triangle sail mussel Hyriopsis cumingii after bacterial infection.

Bacterial disease outbreaks in filter feeder bivalve Hyriopsis cumingii as water contamination become more frequent in the water ecosystem, especially in intensive aquaculture habitats. To characterize host-pathogen interactions between H. cumingii and bacterial infection, we investigated the effects of Stenotrophomonas maltophilia HOP3 and Aeromonas veronii GL1 on the antioxidant response, tissue invasion and transcriptome expression of H. cumingii by infectivity trials. We showed that bacterial infections resulted in tubular necrosis of the hepatopancreas and induced the acute immune response in H. cumingii. The transcriptomic study identified a total of 5957 differentially expressed genes (DEGs) after A. veronii challenge. These DEGs were implicated in 302 KEGG pathways, notably in Apoptosis, Phagosome and Lysosome. The results showed that the relative expressions of all six immune-related DEGs were effectively stimulated with A. veronii, accompanied by tissue differences. Overall, these findings will contribute to an analysis of the immune response of H. cumingii to bacterial infection at the transcriptomic level and its genomic resource for research.

Dorsal regulates the expression of two phage lysozymes acquired via horizontal gene transfer in triangle sail mussel Hyriopsis cumingii.

Dorsal is a Rel/NF-κB transcription factor, which forms a key part of the Toll pathway. Lysozyme is a ubiquitous enzyme that degrades bacterial cell walls. In this study, a Dorsal homolog was cloned and characterized from triangle sail mussel Hyriopsis cumingii, namely, HcDorsal. Dorsal consisted of 3041 bp, including a 1938 bp open reading frame encoding a 645 amino acid protein. The deduced HcDorsal protein contained a Rel homology domain and an Ig-like, plexin, transcription factor domain. Analysis of expression patterns showed that HcDorsal was highly expressed in the hepatopancreas of H. cumingii. The expression level of HcDorsal continuously increased after Vibrio parahaemolyticus stimulation. When HcDorsal was knocked down by siRNA interference, two phage lysozyme genes (HcLyso1 and HcLyso2) obtained by horizontal gene transfer were significantly downregulated in hemocytes of mussels. Furthermore, knockdown of HcLyso1 and HcLyso2 could weaken V. parahaemolyticus clearance ability. Recombinant HcLyso1 and HcLyso2 proteins accelerated the bacterial clearance in vivo in mussels and evidently inhibited the growth of V. parahaemolyticus. These results suggested that HcDorsal could be activated after V. parahaemolyticus stimulation and then modulate the immune response through the transcriptional regulation of HcLyso1 and HcLyso2, thereby playing a protective role in mussels.

The gut microbiome of freshwater Unionidae mussels is determined by host species and is selectively retained from filtered seston.

Freshwater mussels are a species-rich group of aquatic invertebrates that are among the most endangered groups of fauna worldwide. As filter-feeders that are constantly exposed to new microbial inoculants, mussels represent an ideal system to investigate the effects of species or the environment on gut microbiome composition. In this study, we examined if host species or site exerts a greater influence on microbiome composition. Individuals of four co-occurring freshwater mussel species, Cyclonaias asperata, Fusconaia cerina, Lampsilis ornata, and Obovaria unicolor were collected from six sites along a 50 km stretch of the Sipsey River in Alabama, USA. High throughput 16S rRNA gene sequencing revealed that mussel gut bacterial microbiota were distinct from bacteria on seston suspended in the water column, and that the composition of the gut microbiota was influenced by both host species and site. Despite species and environmental variation, the most frequently detected sequences within the mussel microbiota were identified as members of the Clostridiales. Sequences identified as the nitrogen-fixing taxon Methylocystis sp. were also abundant in all mussel species, and sequences of both bacterial taxa were more abundant in mussels than in water. Site physicochemical conditions explained almost 45% of variation in seston bacterial communities but less than 8% of variation in the mussel bacterial microbiome. Together, these findings suggest selective retention of bacterial taxa by the freshwater mussel host, and that both species and the environment are important in determining mussel gut microbiome composition.

Utilization of freshwater mussel (Lamellidens marginalis) for the isolation of proteins through pH shift processing: characterization of isolates.

Study was conducted to use underutilized freshwater mussel (Lamellidens marginalis) for the recovery of proteins using pH shift method and to study the functionality and characteristics of the recovered isolates. From the pH range tested (pH 2.0-13.0), maximum protein yields were obtained during solubilization at pH 2.0 and pH 13.0 (p < 0.05). During the protein recovery process, pH 13.0 was found to have minimal effect on proteins resulting in higher protein yields compared to pH 2.0. Isolates obtained by both acidic and alkaline solubilization processes had low stability and poor gel network. Total lipid content, total myoglobin, and pigment contents were reduced significantly (p < 0.05) during pH shift processing, resulting in whiter protein isolates and protein gels. All the essential amino acids were present in the isolates recovered by acid and alkaline solubilization, indicating the complete recovery of amino acids. No microbial counts were observed in any of the isolates prepared using acid and alkaline-aided processing. Acid and alkaline solubilization (pH shift) process was found to be promising for the recovery of proteins from underutilized freshwater mussel thus by reducing the supply demand gap.

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.

Trematode Aspidogastrea found in the freshwater mussels in the Yangtze River basin / Trematodos Aspidogastrea encontrados en los mejillones de agua dulce en la cuenca del río Yangtze

Objective: To investigate the prevalence of trematode Aspidogastrea in the freshwater mussels in the Yangtze River basin within Anhui province, China. Methods: We initially harvested the freshwater mussels living in the Yangtze River running through Anhui area, and labeled them with corresponding number. Then the samples were dissected for isolating the flukes, which were identified by conventional staining. Results: Infection rate of trematode Aspidogastrea in freshwater mussels in the Yangtze River basin within the territory of Anhui province was 30.38% (103/339) in general, and a total of 912 flukes of Aspidogastrea were detected in the 103 mussels, with average infection rate of 8.85 for each mussel. Conclusion: Trematode Aspidogastrea is prevalent in the freshwater bivalves living in the Yangtze River basin running through Anhui area, and the treamatode was identified as Aspidogaster sp. belong to Aspidogaste under Aspidogastridae of Aspidogastrea (AU)

Objetivo: investigar la prevalencia de trematodos Aspidogastrea en mejillones de agua dulce en la cuenca del río Yangtze en la provincia de Anhui, China. Métodos: se recogieron mejillones de agua dulce en el río Yangtze a su paso por la provincia de Anhui y se etiquetaron con su número correspondiente. Posteriormente se disecaron para aislar los trematodos por medio de tinción convencional. Resultados: la tasa de infección de trematodos en mejillones de agua dulce en la cuenca del río Yangtze, en el territorio de la provincia de Anhui fue 30,38% (103/339), en general, y un total de 912 trematodos fueron detectados en 103 mejillones, con tasa promedio de infección de 8,85 por cada mejillón. Conclusión: el trematodo Aspidogastrea es frecuente en los bivalvos de agua dulce que viven en la cuenca del río Yangtze, en la región de Anhui, y el trematodo fue identifi cado como Aspidogaster sp. pertenecen a la familia Aspidogaste bajo el género Aspidogastridae de Aspidogastrea (AU)

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.

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.

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.

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.

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.

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.

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.

Molecular cloning and characterization of cathepsin L from freshwater mussel, Cristaria plicata.

Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. The Cathepsin L cDNA and genome of Cristaria plicata（CpCL） was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends (RACE) PCR. The genomic DNA was 9353 bp long and had a total of six introns and seven exons. The full-length cDNA of CpCL was 1144 bp, the cDNA contained a 5' untranslated region (UTR) of 34 nucleotides, the 3' UTR of 108 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1002 bp, encoding 333 amino acid residues with 37.65 kDa predicted molecular weight. The theoretical isoelectric point was 8.61. The prepro-cathepsin L was consisted of a typical signal peptide (Met1-Gly20), a pro-region peptide (Leu21-Glu116) and a mature peptide (Tyr117-Val333). Many members of the papain family possessed of a proline residue at position 2 in the mature enzymem, this was also observed in CpCL. The preproprotein included an oxyanion hole (Gln 135), the active center formed by Cys141, His280 and Asn 300, the potential N-glycosylation site (Asn38, Asn 113 and Asn 272) and the conserved GCXGG motifs, which was characteristic of cathepsin, the conserved ERWNIN and GNFD motifs, which were characteristic for cathepsin L. Homology analysis revealed that the CpCL shared 49-87% identity to other known cathepsin L sequences. The phylogenetic tree showed that the CpCL clustered with the invertebrate cathepsin L cysteine proteases, and was closely related to the cathepsin L of Hyriopsis cumingii. The expression of CpCL mRNA was detected in hepatopancreas, hemocytes, mantle, gills and adductor muscle, and the higher expression level was in hepatopancreas. After A. hydrophila stimulation, the expression of the CpCL mRNA was up-regulated in hemocytes and hepatopancreas, and the expression level was significantly lower in gill than one after PBS challenge group.

A novel Toll like receptor with two TIR domains (HcToll-2) is involved in regulation of antimicrobial peptide gene expression of Hyriopsis cumingii.

Animal Toll-like receptors (TLRs) are involved in innate immunity. Toll proteins are generally transmembrane proteins. In this study, an atypical Toll-like receptor (HcToll-2) was identified from the triangle-shell pearl mussel Hyriopsis cumingii, which belongs to phylum Mollusca. Unlike the typical Toll like receptors with extracellular leucine-rich repeats (LRRs), transmembrane, and intracellular Toll/interleukin-1 receptor (TIR) domains, HcToll-2 has two homologous TIR domains located at the C-terminal (designated as HcTIR1 and HcTIR2) and lacks a transmembrane domain. Phylogenetic analysis showed that HcTIR1 was clustered with TIR of sea anemone Toll, and HcTIR2 was clustered with TIR of Drosophila Toll. HcToll-2 mRNA could be detected in the hepatopancreas and was upregulated after challenge with Escherichia coli and Staphylococcus aureus. Recombinant HcLRR protein with GST tag could bind to bacteria and also to LPS and PGN. Over-expression of both HcTIR1 and HcTIR2 induced drosomycin genes in Drosophila S2 cells. RNAi analysis showed that HcToll-2 was required for the expression of theromacin, which is a cysteine-rich antimicrobial peptide (AMP) gene. This research is the first report of an atypical Toll-like receptor HcToll-2 involved in antibacterial immunity through induction of AMP expression.

Identification and function of two myeloid differentiation factor 88 variants in triangle-shell pearl mussel (Hyriopsis cumingii).

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter protein that participates in the activation of the Toll-like receptor (TLR)/interleukin-1 receptor-mediated signaling pathway. In this study, two MyD88 genes (HcMyD88-1 and HcMyD88-2) were identified from triangle-shell pearl mussel (Hyriopsis cumingii). Both HcMyD88-1 and HcMyD88-2 proteins were determined to have a death domain at the N-terminal and a TIR domain at the C-terminal. Both HcMyD88-1 and HcMyD88-2 genes were mainly expressed in the hepatopancreas of healthy mussels. HcMyD88-1 and HcMyD88-2 slightly responded to Gram-negative bacterial challenge. Upon bacterial challenge with Gram-positive Staphyloccocus aureus, HcMyD88-1 and HcMyD88-2 transcription levels remarkably increased at 2 and 6h, respectively. Overexpression of HcMyD88-1 and HcMyD88-2 proteins in Drosophila Schneider 2 cells led to the activation of antimicrobial peptide genes. These results indicated that HcMyD88-2 had higher activity than HcMyD88-1 during the activation of attacin A, drosomycin, and metchnikowin genes, suggesting that HcMyD88 genes may play a role in antibacterial innate immune defense.

Association between SNPs in interferon regulatory factor 2 (IRF-2) gene and resistance to Aeromonas hydrophila in freshwater mussel Hyriopsis cumingii.

Interferon regulatory factor 2 (IRF-2) is a multi-functional transcription factor in the IRF family exhibiting both transcriptional activating and repressing activities. In this study, an IRF-2 gene (HcIRF-2) from Hyriopsis cumingii was identified and characterized. The cDNA sequence consisted of 2688 bp, encoding a 329 amino acid-protein. The amino acid sequence had a highly conserved N-terminal DBD structure, containing characteristic repeats of six tryptophan residues. The 5'-flanking region contained several transcription regulation elements such as AP1, CdxA, HSF, NIT2 and HNF-3b. Nine SNPs were obtained through direct sequencing of HcIRF-2 from resistant and susceptible stock. Only +2365T/C SNP was significantly associated with resistance/susceptibility of H. cumingii to Aeromonas hydrophila both in genotype (P = 0.021) and allele (P = 0.006) analysis. The SNPs +2248T/C and +2365T/C were in high linkage disequilibrium, and haplotype analysis revealed that haplotype TT frequency in the resistant group was significantly higher than in the susceptible group. The mortality in +2248CC genotype individuals was significantly higher than in CT and TT genotype individuals. These results indicated that haplotype TT and genotype +2248CT and +2248GT individuals were resistant to A. hydrophila, which could make them potential markers in selective breeding of H. cumingii.

Molecular properties and immune defense of two ferritin subunits from freshwater pearl mussel, Hyriopsis schlegelii.

Ferritin is a conserved iron-binding protein involved in cellular iron metabolism and host defense. In the present study, two distinct cDNAs for ferritins in the freshwater pearl mussel Hyriopsis schlegelii were identified (designated as HsFer-1 and HsFer-2) by SMART RACE approach and expressed sequence tag (EST) analysis. The full-length cDNAs of HsFer-1 and HsFer-2 were of 760 and 877 bp, respectively. Both of the two cDNAs contained an open reading frame (ORF) of 522 bp encoding for 174 amino acid residues. Sequence characterization and homology alignment indicated that HsFer-1 and HsFer-2 had higher similarity to H-type subunit of vertebrate ferritins than L-type subunit. Analysis of the HsFer-1 and HsFer-2 untranslated regions (UTR) showed that both of them had an iron response element (IRE) in the 5'-UTR, which was considered to be the binding site for iron regulatory protein (IRP). Quantitative real-time PCR (qPCR) assays were employed to examine the mRNA expression profiles. Under normal physiological conditions, the expression level of both HsFer-1 and HsFer-2 mRNA were the highest in hepatopancreas, moderate in gonad, axe foot, intestine, kidney, heart, gill, adductor muscle and mantle, the lowest in hemocytes. After stimulation with bacteria Aeromonas hydrophila, HsFer-1 mRNA experienced a different degree of increase in the tissues of hepatopancreas, gonad and hemocytes, the peak level was 2.47-fold, 9.59-fold and 1.37-fold, respectively. Comparatively, HsFer-2 showed up-regulation in gonad but down-regulation in hepatopancreas and hemocytes. Varying expression patterns indicate that two types of ferritins in H. schlegelii might play different roles in response to bacterial challenge. Further bacteriostatic analysis showed that both the purified recombinant ferritins inhibited the growth of A. hydrophila to a certain degree. Collectively, our results suggest that HsFer-1 and HsFer-2 are likely to be functional proteins involved in immune defense against bacterial infection.

Six defensins from the triangle-shell pearl mussel Hyriopsis cumingii.

Antimicrobial peptides (AMPs) are the first line of defense of invertebrates against invading pathogens. Defensins, unique AMPs, have a cysteine-stabilized α-helix and ß-sheet (CSαß) motif. In invertebrates, defensins have been reported in arthropods and mussels. Recently, six defensins were identified from Hyriopsis cumingii for the first time, and were designated as HcDef1, HcDef2, HcDef3, HcDef4, HcDef5, and HcDef6. HcDef1 and HcDef2 encode a protein containing 61 and 60 amino acids, respectively. HcDef3, HcDef4, and HcDef6 have 65 amino acids each. HcDef5 is longer than the other five defensins, comprising 83 amino acids. HcDef3 and HcDef4 have three pairs of disulfide bonds. HcDef1, HcDef5, and HcDef6 are exceptions; each has four pairs of disulfide bonds. Evolutionary analysis revealed that only purifying selection and no positive selection could be detected in defensin genes; purifying selection might be the major evolutionary driving force in the evolution of defensin genes. The present study reveals for the first time that the defensins from H. cumingii are diverse and phylogenetic analysis showed that these 6 defensins from H. cumingii were clustered into one group. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that HcDef1-HcDef4 could be detected in the hepatopancreas and gills whereas HcDef5-HcDef6 could only be detected in gills. In addition, the expression levels of HcDef2, HcDef3, and HcDef5 in H. cumingii with pearls were higher than that in H. cumingii without pearls. Quantitative RT-PCR analysis showed that HcDef1, HcDef2, HcDef3, and HcDef5 were downregulated by Vibrio anguillarum challenge whereas HcDef4 and HcDef6 were upregulated under Vibrio challenge. Our results suggest the roles of defensins in the innate immunity of H. cumingii.