Oysters accumulate high concentrations of zinc (Zn) and copper (Cu), which can be transferred to human due to sea food consumption. Breeding new oyster varieties with low Zn and Cu accumulations is ...one important way to improve food safety. However, the genetic basis for metal accumulation in mollusks is not well understood. To address this issue, oysters collected in the field were used for genome-wide association study (GWAS) and then the identified genes were used for mRNA expressions analysis in laboratory. First, GWAS were conducted for Zn and Cu accumulation in 288 wild Pacific oysters (Crassostrea gigas) farmed in the same ocean environment. The oysters did not show obvious population structure or kinship but exhibited 8.43- and 10.0- fold changes of Zn and Cu contents respectively. GWAS have identified 11 and 12 single nucleotide polymorphisms (SNPs) associated with Zn and Cu, respectively, as well as 16 genes, which were Zn-containing proteins or participated in caveolae-dependent endocytosis. Second, the mRNA expressions of these 16 genes were observed under Zn and Cu exposure. After 9 days of Zn exposure, Zn contents increased 3.1-fold, while the mRNA expression of cell number regulator 3 increased 1.65-fold. Under 9 days of Cu exposure, Cu contents increased 1.97-fold, while the mRNA expression of caveolin-1 decreased 0.61-fold. These provide the evidence for their roles in regulating physiological levels of these two metals. The findings advance our understanding of the genetic basis of Zn and Cu accumulation in mollusks, which can be useful for breeding new, less toxic varieties of oysters.
•Genome-wide association analysis were conducted in oyster for Zn and Cu contents.•A total of 11 and 12 SNPs associated with Zn and Cu contents were identified.•Clustered SNPs and haplotypes were further identified within caveolin-1 and CNR3 for Cu and Zn accumulation.•High expressions of caveolin-1 and CNR3 revealed their important roles for Cu and Zn accumulation.
The Pacific oyster Crassostrea gigas is an important cultivated shellfish. As a euryhaline species, it has evolved adaptive mechanisms responding to the complex and changeable intertidal environment ...that it inhabits. To investigate the genetic basis of this salinity adaptation mechanism, we conducted a genome-wide association study using phenotypically differentiated populations (hyposalinity and hypersalinity adaptation populations, and control population), and confirmed our results using an independent population, high-resolution melting, and mRNA expression analysis. For the hyposalinity adaptation, we determined 24 genes, including Cg_CLCN7 (chloride channel protein 7) and Cg_AP1 (apoptosis 1 inhibitor), involved in the ion/water channel and transporter mechanisms, free amino acid and reactive oxygen species metabolism, immune responses, and chemical defence. Three SNPs located on these two genes were significantly differentiated between groups, as was Cg_CLCN7. For the hypersalinity adaptation, the biological process for positive regulating the developmental process was enriched. Enriched gene functions were focused on transcriptional regulation, signal transduction, and cell growth and differentiation, including calmodulin (Cg_CaM) and ficolin-2 (Cg_FCN2). These genes and polymorphisms possibly play an important role in oyster hyposalinity and hypersalinity adaptation. They not only further our understanding of salinity adaptation mechanisms but also provide markers for highly adaptable oyster strains suitable for breeding.
Studies of DNA methylomes in a wide range of eukaryotes have revealed both conserved and divergent characteristics of DNA methylation among phylogenetic groups. However, data on invertebrates ...particularly molluscs are limited, which hinders our understanding of the evolution of DNA methylation in metazoa. The sequencing of the Pacific oyster Crassostrea gigas genome provides an opportunity for genome-wide profiling of DNA methylation in this model mollusc.
Homologous searches against the C. gigas genome identified functional orthologs for key genes involved in DNA methylation: DNMT1, DNMT2, DNMT3, MBD2/3 and UHRF1. Whole-genome bisulfite sequencing (BS-seq) of the oyster's mantle tissues revealed that more than 99% methylation modification was restricted to cytosines in CpG context and methylated CpGs accumulated in the bodies of genes that were moderately expressed. Young repeat elements were another major targets of CpG methylation in oysters. Comparison with other invertebrate methylomes suggested that the 5'-end bias of gene body methylation and the negative correlation between gene body methylation and gene length were the derived features probably limited to the insect lineage. Interestingly, phylostratigraphic analysis showed that CpG methylation preferentially targeted genes originating in the common ancestor of eukaryotes rather than the oldest genes originating in the common ancestor of cellular organisms.
Comparative analysis of the oyster DNA methylomes and that of other animal species revealed that the characteristics of DNA methylation were generally conserved during invertebrate evolution, while some unique features were derived in the insect lineage. The preference of methylation modification on genes originating in the eukaryotic ancestor rather than the oldest genes is unexpected, probably implying that the emergence of methylation regulation in these 'relatively young' genes was critical for the origin and radiation of eukaryotes.
The proteins of inhibitor of apoptosis (IAP) family play important roles in regulation of apoptosis, immunological response and cell proliferation. Here we reported two IAP genes (named CfIAP1 and ...CfIAP2) in Zhikong scallop Chlamys farreri. The full-length CfIAP1 cDNA contained 1552 nucleotides, encoding a predicted protein of 251 amino acids with two BIR domains. The full-length CfIAP2 cDNA contained 1243 nt, encoding a 356-aa protein with one BIR domain and one RING domain. The two genes are ubiquitously expressed in six types of tissue of C. farreri. The expression levels of CfIAP1 and CfIAP2 were significantly up-regulated after challenged with acute viral necrobiotic disease virus, lipopolysaccharide and exposure to air. Subcellular localization assay showed that CfIAP1 was mainly distributed in cytoplasm and CfIAP2 was in cytoplasm and nucleus. As assessed using a kit designed to test Caspase3 function in mammalian cells, the activity of CfCaspase3 was enhanced as a result of the down-regulation of CfIAP2 expression by dsRNA-mediated gene silencing. Our study indicated that CfIAP1 and CfIAP2 may participate in the innate immunity and stress responses and that CfIAP2 might block apoptosis via inhibiting CfCaspase3 indirectly through an unexplored mechanism in C. farreri.
•The full-length cDNAs of two IAP genes were first characterized in Chlamys farreri.•The two genes were ubiquitously expressed in a wide variety of tissues.•The two genes are both involved in innate immunity and stress responses.•CfIAP1 was probably distributed in cytoplasm and CfIAP2 was in cytoplasm and nucleus.•CfIAP2 might block apoptosis via inhibiting CfCaspase3 indirectly through an undiscovered way.
The economic and ecological importance of the oyster necessitates further research on the molecular mechanisms, which both regulate the commercially important traits of the oyster and help it to ...survive in the variable marine environment. Single nucleotide polymorphisms (SNPs) have been widely used to assess genetic variation and identify genes underlying target traits. In addition, high‐resolution melting (HRM) analysis is a potentially powerful method for validating candidate SNPs. In this study, we adopted a rapid and efficient pipeline for the screening and validation of SNPs in the genic region of Crassostrea gigas based on transcriptome sequencing and HRM analysis. Transcriptomes of three wild oyster populations were sequenced using Illumina sequencing technology. In total, 50–60 million short reads, corresponding to 4.5–5.4 Gbp, from each population were aligned to the oyster genome, and 5.8 × 105 SNPs were putatively identified, resulting in a predicted SNP every 47 nucleotides on average. The putative SNPs were unevenly distributed in the genome and high‐density (≥2%), nonsynonymous coding SNPs were enriched in genes related to apoptosis and responses to biotic stimuli. Subsequently, 1,671 loci were detected by HRM analysis, accounting for 64.7% of the total selected candidate primers, and finally, 1,301 polymorphic SNP markers were developed based on HRM analysis. All of the validated SNPs were distributed into 897 genes and located in 672 scaffolds, and 275 of these genes were stress inducible under unfavourable salinity, temperature, and exposure to air and heavy metals. The validated SNPs in this study provide valuable molecular markers for genetic mapping and characterization of important traits in oysters.
In aquatic ecosystems, the interaction between heavy metals and dissolved organic carbon (DOC) plays a pivotal role in modifying the bioavailability of these metals. This study, employing a ...toxicokinetic–toxicodynamic model, delves into the interactive effects of humic acid (HA), a significant component of DOC, on the bioaccumulation and toxicity of copper (Cu) in the estuarine economic bivalve Sinonovacula constricta. Utilizing the stable isotope sup.65 Cu as a tracer, we evaluated Cu uptake in S. constricta under varied DOC concentrations in a controlled laboratory setting. Our findings reveal that at DOC concentrations below 3.05 mg Lsup.−1 , the bioavailability of Cu is reduced due to shifts in the speciation distribution of Cu, resulting in decreased bioaccumulation within S. constricta. Conversely, at DOC levels exceeding 3.05 mg Lsup.−1 , the formation of colloidal Cu–HA complexes allows its entry into the bivalves’ digestive system. Moreover, toxicity assays demonstrate an increase in S. constricta survival rates with higher DOC concentrations, suggesting a protective effect of DOC against Cu toxicity. The integration of accumulation and toxicity data infers that Cu–HA complexes, when ingested via the digestive tract, exhibit lower toxicity compared to Cu directly assimilated from the water phase. These findings emphasize the need to consider environmental DOC levels in assessing Cu pollution risks and provide insights for managing heavy metal toxicity in estuarine aquaculture.
Nuclear receptors (NRs) belong to the transcription factor superfamily that regulates development, homeostasis, differentiation, and reproduction in metazoans via control of gene expression. ...Recently, rapid advances in genome projects on various metazoans have provided new opportunities for studying the evolution and function of NRs. Typically structured NRs are divided into six subfamilies. Here, the gene for a typically structured NR (CgNR8A1) was cloned from the Pacific oyster Crassostrea gigas. However, this novel receptor could not be assigned to a known NR subfamily. By data mining, nine other CgNR8A1 gene homologs were identified in metazoans such as cnidarians, mollusks, annelids, echinoderms, hemichordates, and cephalochordates. Phylogenetic analysis showed that these receptors belonged to a novel NR subfamily, hereafter designated as NR8. Evolutionary analysis revealed that the NR8 subfamily was phylogenetically the third-oldest NR subfamily, and it originated from a common ancestor of Eumetazoa; several gene loss events occurred independently in ancestors of vertebrates, ecdysozoans, and platyhelminths, which do not have NR8 members. Furthermore, the function of CgNR8A1 was investigated to provide an insight into the functions of this novel NR subfamily. A nuclear localization signal peptide, GKHRNKKPRLD, was identified in CgNR8A1, and a recombinant full-length protein of CgNR8A1 was localized in the nuclei of HeLa cells. The mRNA expression profile of CgNR8A1 suggested that it might be involved in the embryogenesis of C. gigas. The electrophoretic mobility shift assay showed that CgNR8A1 binds strongly to conserved DNA core motifs DR0, DR2, and DR4 and weakly to DR1, DR3, DR5, Half, and Pal0. In summary, the novel NR8 subfamily identified in this study improves our understanding of NR evolution, and the functional analysis of CgNR8A1 provided further insights into the functions of NR8A1s.
•A novel nuclear receptor subfamily was identified from various metazoans.•The NR8 nuclear receptor subfamily originated from a common ancestor of Eumetazoa.•CgNR8A1 cDNA and genomic sequences from Crassostrea gigas were characterized.•CgNR8A1 mRNA was highly expressed in C. gigas larvae during embryogenesis.•CgNR8A1 bound conserved DNA core motifs as monomer.
Apoptosis plays significant roles in maintenance of homeostasis, immune defense and development. The Bcl-2 family proteins are important regulators of the intrinsic apoptosis. In the study, we have ...characterized a Bcl-2-like gene (named CfBcl-2) and a Bax-like gene (named CfBax) from the Zhikong scallop Chlamys farreri. The full-length of the CfBcl-2 cDNA is 944 nucleotides (nt) encoding a putative protein of 225 amino acid residues (aa) that contains four Bcl-2 homology (BH) domains, and the CfBax cDNA is 505 nt encoding a putative protein of 115 aa that contains three Bcl-2 BH domains. Sequence and phylogenetic analysis demonstrate that CfBcl-2 and CfBax present typical domain organization of the corresponding Bcl-2 related proteins and are more similar and clustered with their homologues of other molluscs. The two genes are ubiquitously expressed in six tissues of C. farreri, with the highest expression level of CfBcl-2 in adductor muscle and highest expression level of CfBax in gill. The expressions of CfBcl-2 and CfBax in hemocytes were both significantly up-regulated after an in vivo exposure of scallops to air, injection with lipopolysaccharide and infection with acute viral necrobiotic disease virus, and the expression patterns of the two genes after the three treatments vary in different change magnitude and up-regulation timespan. Yeast two-hybrid assay reveals a direct interaction between the CfBcl-2 and CfBax proteins. These results indicate that the CfBcl-2 and CfBax may participate in the apoptosis-based stress and immune responses against noxious stimulation.
•The CfBcl-2 cDNA is 944 nt encoding a putative protein of 225 aa.•The CfBax cDNA is 505 nt encoding a putative protein of 115 aa.•The two genes are ubiquitously expressed in six tissues of Chlamys farreri.•The two genes are involved in the apoptosis-based stress and immune responses.•There is a direct interaction between the CfBcl-2 and CfBax proteins.
Two calcium carbonate crystal polymorphs, aragonite and calcite, are the main inorganic components of mollusk shells. Some fossil evidences suggest that aragonite shell is more ancient than calcite ...shell for the Bivalvia. But, the molecular biology evidence for the above deduction is absent. In this study, we searched for homologs of bivalve aragonite-related and calcite-related shell proteins in the oyster genome, and found that no homologs of calcite-related shell protein but some homologs of aragonite-related shell proteins in the oyster genome. We explained the results as the new evidence to support that aragonite shells are more ancient than calcite shells in bivalves combined the published biogeological and seawater chemistry data.
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