A Rhodopsin-Like Gene May Be Associated With the Light-Sensitivity of Adult Pacific Oyster Crassostrea gigas.
ABSTRACT: Light-sensitivity is important for mollusc survival, as it plays a vital role in reproduction and predator avoidance. Light-sensitivity has been demonstrated in the adult Pacific oyster Crassostrea gigas, but the genes associated with light-sensitivity remain unclear. In the present study, we designed experiments to identify the genes associated with light-sensitivity in adult oysters. First, we assessed the Pacific oyster genome and identified 368 genes annotated with the terms associated with light-sensitivity. Second, the function of the four rhodopsin-like superfamily member genes was tested by using RNAi. The results showed that the highest level of mRNA expression of the vision-related genes was in the mantle; however, this finding is not true for all oyster genes. Interestingly, we also found four rhodopsin-like superfamily member genes expressed at an very high level in the mantle tissue. In the RNAi experiment, when one of rhodopsin-like superfamily member genes (CGI_1001253) was inhibited, the light-sensitivity capacity of the injected oysters was significantly reduced, suggesting that CGI_10012534 may be associated with light-sensitivity in the adult Pacific oyster.
Project description:BACKGROUND:The pearl oyster Pinctada fucata martensii (Pfu.), widely cultured in the South China Sea, is a precious source of sea pearls and calcifying materials. A yellow shell variety of Pfu. was obtained after years of artificial breeding. To identify differentially expressed genes between yellow shell and normal black shell pearl oysters, we performed transcriptomic sequencing and proteomic analyses using mantle edge tissues. RESULTS:A total of 56,969 unigenes were obtained from transcriptomic, of which 21,610 were annotated, including 385 annotated significant up-regulated genes and 227 significant down-regulated genes in yellow shell oysters (| log2 (fold change) | ?2 and false discovery rate?<?0.001). Tyrosine metabolism, calcium signalling pathway, phototransduction, melanogenesis pathways and rhodopsin related Gene Ontology (GO) terms were enriched with significant differentially expressed genes (DEGs) in transcriptomic. Proteomic sequencing identified 1769 proteins, of which 51 were significantly differentially expressed in yellow shell oysters. Calmodulin, N66 matrix protein, nacre protein and Kazal-type serine protease inhibitor were up-regulated in yellow shell oysters at both mRNA and protein levels, while glycine-rich protein shematrin-2, mantle gene 4, and sulphide: quinone oxidoreductase were down-regulated at two omics levels. Particularly, calmodulin, nacre protein N16.3, mantle gene 4, sulphide: quinone oxidoreductase, tyrosinase-like protein 3, cytochrome P450 3A were confirmed by quantitative real-time PCR. Yellow shell oysters possessed higher total carotenoid content (TCC) compared than black shell oyster based on spectrophotography. CONCLUSIONS:The yellow phenotype of pearl oysters, characterised by higher total carotenoids content, may reflect differences in retinal and rhodopsin metabolism, melanogenesis, calcium signalling pathway and biomineralisation. These results provide insights for exploring the relationships between calcium regulation, biomineralisation and yellow shell colour pigmentation.
Project description:Light-sensitivity is an important aspect of mollusk survival as it plays a vital role in reproduction and predator avoidance. In the Pacific oyster Crassostrea gigas light sensitivity has been demonstrated in the larval stage but has not yet been conclusively demonstrated in adult oysters. In this paper we describe an experiment which was undertaken to determine if adult Pacific oysters were sensitive to light. One LED flashlight was used to shine light onto adult oysters while they were filtering seawater through their shell openings. We found that the degree of opening increased gradually during the light period but rapidly decreased when the flashlight was turned off in the treated group but not in the control group. These results suggest that adult Pacific oyster may be sensitive to light.
Project description:The Pacific oyster (Crassostrea gigas) is a kind of marine bivalve of great economic and ecological importance and is among the animals possessing the highest level of genome DNA variations. Despite large efforts made for the discovery of Pacific oyster SNPs in many research groups, challenge still remains as how to utilize SNPs in a high-throughput, transferable and economical manner. In the study, we constructed an oyster 190K SNP array with Affymetrix Axiom genotyping technology. A total of 190,420 SNPs were designed on the chip, which were selected from 54 M SNPs identified by re-sequencing of more than 400 Pacific oysters. Genotyping results from 96 wild oysters indicated that 133,984 (70.4%) SNPs were polymorphic and successfully converted on the chip. Carrying 133K polymorphic SNPs, the oyster 190K SNP array is the first high density SNP chip with the largest throughput currently in mollusc and is commercially available to the worldwide research community. Overall design: A total of 96 Pacific oysters were genotyped according to the manufacturer's directions to assess the performance of the oyster SNP array. All DNA were extracted from mantle tissues using a standard phenol-chloroform method.
Project description:Cell culture provides useful model systems used in a wide range of biological applications, but its utility in marine invertebrates is limited due to the lack of immortalised cell lines. Primary cell and tissue cultures are typically used but remain poorly characterised for oysters, which can cause issues with experimental consistency and reproducibility. Improvements to methods of repeatable isolation, culture, and characterisation of oyster cells and tissues are required to help address these issues. In the current study, systematic improvements have been developed to facilitate the culture of primary cells from adult Pacific oyster tissues and identify novel cell morphologies that have not been reported previously. Cultures analysed by light microscopy, qPCR, and live cell imaging demonstrated maintenance of live, metabolically active Pacific oyster cells for several weeks post-explant. Interestingly, whole hearts dissected from adult oysters were found to continue contracting rhythmically up to 8 weeks after being transferred to a tissue culture system. Mantle tissue explants were also actively moving in the culture system. These improvements in primary cell culture of bivalves may be beneficial for research in ecotoxicology, virology, immunology, and genetic resistance to disease.
Project description:The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.
Project description:Fast growth is one of the most desired traits for all food animals, which affects the profitability of animal production. The Pacific oyster, Crassostrea gigas, is an important aquaculture shellfish around the world with the largest annual production. Growth of the Pacific oyster has been greatly improved by artificial selection breeding, but molecular mechanisms underlying growth remains poorly understood, which limited the molecular integrative breeding of fast growth with other superior traits. In this study, comparative transcriptome analyses between the fast-growing selectively bred Pacific oyster and unselected wild Pacific oysters were conducted by RNA-Seq. A total of 1,303 protein-coding genes differentially expressed between fast-growing oysters and wild controls were identified, of which 888 genes were expressed at higher levels in the fast-growing oysters. Functional analysis of the differentially expressed genes (DEGs) indicated that genes involved in microtubule motor activity and biosynthesis of nucleotides and proteins are potentially important for growth in the oyster. Positive selection analysis of genes at the transcriptome level showed that a significant number of ribosomal protein genes had undergone positive selection during the artificial selection breeding process. These results also indicated the importance of protein biosynthesis and metabolism for the growth of oysters. The alternative splicing (AS) of genes was also compared between the two groups of oysters. A total of 3,230 differential alternative splicing events (DAS) were identified, involved in 1,818 genes. These DAS genes were associated with specific functional pathways related to growth, such as "long-term potentiation," "salivary secretion," and "phosphatidylinositol signaling system." The findings of this study will be valuable resources for future investigation to unravel molecular mechanisms underlying growth regulation in the oyster and other marine invertebrates and to provide solid support for breeding application to integrate fast growth with other superior traits in the Pacific oyster.
Project description:Noroviruses are the major cause of foodborne outbreaks of acute gastroenteritis, which are often linked to raw oyster consumption. Previous studies have suggested histo-blood group antigens (HBGA)-like structures in the oyster tissues as ligands for norovirus binding and persistence. To better understand how oysters function as vectors for the most common human noroviruses, we first tested the ability of the norovirus strains GI.1 West Chester, the pandemic GII.4 Sydney, and the epidemic GII.17 Kawasaki308 strains to interact with oyster tissues. Secondly, we explored how the HBGA preferences of these strains can affect their persistence in oyster tissues. We found limited HBGA expression in oyster tissues. HBGAs of A and H type 1 were present in the digestive tissues and palps of the Pacific oyster Crassostrea gigas, while the gills and mantle lacked any HBGA structures. By using Virus-like particles (VLPs), which are antigenically and morphologically similar to native virions, we were able to demonstrate that VLPs of GI.1 West Chester norovirus reacted with the digestive tissues and palps. Despite of the lack of HBGA expression in mantle, dominant GII.4 Sydney strain readily bound to all the oyster tissues, including the digestive tissues, gills, palps, and mantle. In contrast, no binding of the epidemic GII.17 Kawasaki308 VLPs to any of the investigated oyster tissues was observed. In synthetic HBGA and saliva-binding assays, GI.1 reacted with A type, H type, and Leb (Lewis b) HBGAs. GII.4 Sydney VLPs showed a broad binding pattern and interacted with various HBGA types. Compared to GI.1 and GII.4 VLPs, the GII.17 Kawasaki308 VLPs only weakly associated with long-chain saccharides containing A type, B type, H type, and Leb blood group epitopes. Our findings indicate that GI.1 and GII.4 noroviruses are likely to be concentrated in oysters, by binding to HBGA-like glycans, and therefore potentially leading to increased long term transmission. In regards to the GII.17 Kawasaki308 strain, we suggest that oysters can only function as short term transmission vector in periods of high environmental virus concentrations.
Project description:The aim of this investigation was to identify the parasites present in the largely understudied pleasure oyster Crassostrea corteziensis in Sinaloa state in the northwestern Mexican Pacific coast. Inspection of twenty-eight oysters collected on "Ceuta" lagoon revealed the presence of the digenean Stephanostomum sp. (Digenea: Acanthocolpidae) cysts. Metacercariae were found encapsulated and embedded in the digestive gland and mantle tissue of oysters. The prevalence of infection revealed that 84.6 % were infected, the abundance was 13.62, with a mean intensity of 16.09 per host. The members of this genus are characterized by a double crown of spines in the cephalic region surrounding the buccal opening of the worm. Significantly, we report the first incidence of the digenean Stephanostomum sp of the family Acanthocolpidae parasitizing Crassostrea corteziensis. Further we report that this bivalve is now considered a new intermediate host, and the northwestern Mexican Pacific coast is a new geographical distribution area for this digenean. The findings contribute to our understanding of the biology, biodiversity and host preference of these parasites, with implications for health risks posed by human consumption of the pleasure oyster.
Project description:The Pacific oyster Crassostrea gigas is an important marine fishery resource, which contains high levels of glycogen that contributes to the flavor and the quality of the oyster. However, little is known about the molecular and chemical mechanisms underlying glycogen content differences in Pacific oysters. Using a homogeneous cultured Pacific oyster family, we explored these regulatory networks at the level of the metabolome and the transcriptome.Oysters with the highest and lowest natural glycogen content were selected for differential transcriptome and metabolome analysis. We identified 1888 differentially-expressed genes, seventy-five differentially-abundant metabolites, which are part of twenty-seven signaling pathways that were enriched using an integrated analysis of the interaction between the differentially-expressed genes and the differentially-abundant metabolites. Based on these results, we found that a high expression of carnitine O-palmitoyltransferase 2 (CPT2), indicative of increased fatty acid degradation, is associated with a lower glycogen content. Together, a high level of expression of phosphoenolpyruvate carboxykinase (PEPCK), and high levels of glucogenic amino acids likely underlie the increased glycogen production in high-glycogen oysters. In addition, the higher levels of the glycolytic enzymes hexokinase (HK) and pyruvate kinase (PK), as well as of the TCA cycle enzymes malate dehydrogenase (MDH) and pyruvate carboxylase (PYC), imply that there is a concomitant up-regulation of energy metabolism in high-glycogen oysters. High-glycogen oysters also appeared to have an increased ability to cope with stress, since the levels of the antioxidant glutathione peroxidase enzyme 5 (GPX5) gene were also increased.Our results suggest that amino acids and free fatty acids are closely related to glycogen content in oysters. In addition, oysters with a high glycogen content have a greater energy production capacity and a greater ability to cope with stress. These findings will not only provide insights into the molecular mechanisms underlying oyster quality, but also promote research into the molecular breeding of oysters.
Project description:Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.