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Aquatic animals are subject to varying degrees of starvation stress in their natural habitats due to food limitations. Consequently, starvation is a crucial environmental factor for sex determination in many species; however, limited research has been conducted on the effects of starvation on sex determination in shellfish. Here, four full sibling families of Pacific oysters were established and subjected to starvation stress. The results demonstrated that starvation caused the sex ratio (female to male) to change from 1:0.78 to 1:1.44 and resulted in a delay in gonadal development. Further studies revealed that the expression levels of DNA methylation-related genes Dnmt1 (DNA methyltransferase 1), Dnmt3a /b (DNA methyltransferase 3a/b) and Tet3 (tet methylcytosine dioxygenase 3) decreased under starvation stress. Conversely, the upregulation of Dmrt1 (doublesex and mab-3 related transcription factor 1), a gene typically associated with males, in females suggests that the increased proportion of males may be linked to starvation-induced high expression of this particular gene. In addition, the gene Dgkd (diacylglycerol kinase delta), which is involved in the regulation of second messenger protein kinase C, was differentially methylated between males and females, with the methylation level of this gene gradually increasing with male development, while the methylation level of this gene decreased under starvation stress. Correlation analysis of Dgkd methylation levels with expression levels showed a negative correlation between DNA methylation and gene expression. Finally dual fluorescence reporter experiments confirmed that DNA methylation suppressed Dgkd expression in vitro. In summary, the results suggest that starvation may regulate Dgkd gene expression through DNA methylation and thus affect Dmrt1 expression, thereby causing sex reversal in the Pacific oyster. The outcomes resolved how environmental factors are involved in sex reversal from an epigenetic perspective and provided a theoretical basis for sex control breeding in the Pacific oyster. [Display omitted] • Starvation stress causes male bias and delayed gonadal development in the oyster. • Starvation stress alters the expression of sex-associated genes Foxl2 , Dmrt1 and Dgkd in the oyster. • Sex bias is associated with DNA methylation in response to starvation stress. [ABSTRACT FROM AUTHOR]

In molluscs, muscle contraction is triggered by a direct binding of Ca 2 + to myosin. The myosin essential light chain (MELC) provides the Ca 2 + binding site and is of importance for motor function and regulation. In this study, the complete cDNA sequence of MELC gene of the Pacific oyster, Crassostrea gigas , was obtained, and the expression profiles were performed in different tissues and different embryo-larval development stages. The results showed that the full length of C. gigas MELC (CgMELC) cDNA was 659 bp, containing a 5′-untranslated region of 73 bp, a 3′-untranslated region of 112 bp. The open reading frame encoded a 157 amino acid peptide. The protein sequence of CgMELC contained a conserved EF-hand calcium binding motif, and showed a high sequence identity (68.4–100%) with other bivalves. Quantitative analysis of CgMELC mRNA in tissues indicated that CgMELC was expressed at the highest level in the striated adductor muscle, followed by the smooth adductor muscle and mantle. During the development of embryos and larvae, quantitative analysis and whole-mount in situ hybridization revealed that CgMELC was expressed starting from the blastula stage and abundantly expressed in trochophore and D-shaped larvae, indicating that CgMELC might also be involved in regulation of larval muscle system development. Our data provided valuable information for further researches on the function of MELC and regulation of muscle contraction in oysters. [ABSTRACT FROM AUTHOR]

The Pacific oyster (Crassostrea gigas), as the most productive cultured shellfish in the world, has considerable economic importance and value. Yet research on its sex determination remains lacking. Here, a novel HMG-box-containing homolog of the sox gene was uncovered in C. gigas (named CgSox-like). Alignment with the HMG-box of sox genes from other animals revealed that CgSox-like had a high similarity to the Sox gene of scallops. Among different adult tissues, CgSox-like was mainly expressed in the gonads. qPCR analysis revealed that CgSox-like and CgSox8 , a sox gene family gene mainly expressed in male gonads, shared similar expression patterns, with elevated expression as the male gonads developed. RNA interference and dual luciferase reporter assays demonstrated that CgSox-like was located upstream of CgSox8 and promoted the expression of CgSox8. In addition, analysis of CgSox-like and CgSox8 methylation patterns indicated that both genes were in hypomethylation state during the gonadal development. Finally, demethylase inhibitors resulted in elevated expression levels of CgSox-like and CgSox8 genes, suggesting that epigenetics was engaged in modulating the expression of sex-related genes in the Pacific oyster. Taken together, the novel gene CgSox-like was implicated in the sex determination or gonadal development process in male oysters. These results will provide valuable information for the investigation of sex determination mechanism and sex control breeding. • CgSox-like is involved in sex determination or gonadal development in oysters. • CgSox-like was located upstream of CgSox8 and promoted the expression of CgSox8. • DNA methylation participates in the regulation of sex determination in Crassostrea gigas. [ABSTRACT FROM AUTHOR]

Reproductive mechanisms in molluscs have been targets for biological research because of the diverse reproductive strategies exhibited in this phylum. However, little is known about the molecular mechanisms of molluscan reproduction. The whole genome sequence of the Pacific oysterCrassostrea gigastogether with the transcriptomic data-sets provides a powerful platform for studies on the characterization of the genes involved in bivalve reproduction. Reproduction-related genes were screened in this study through analyzing the Pacific oyster genome. Previously identified molluscan reproduction-related genes were used as queries to a BLASTP search against theC. gigasgenome to identify gene candidates encoding similar proteins. Transcriptomic data-sets were analyzed to profile gene candidate expression patterns. We obtained 39 gene candidates with high accuracy encoding reproduction-related genes in theC. gigasgenome, of which 17 are reported in detail, and the rest are identified as new candidate genes involved in reproduction ofC. gigas,includingnanos,piwi,dax1, and5-hydroxytryptamine(5-HT) receptors. Among 22 gene candidates, seven were gonad-specifically expressed. The set of reproduction-related genes ofC. gigasidentified in this study constitutes a new tool for further research on molecular mechanisms of reproduction inC. gigas. [ABSTRACT FROM PUBLISHER]

Triploid oysters generate fewer gametes than diploid oysters and exhibit varying degrees of gonad development. The expression of related genes, such as those involved in gametogenesis and energy metabolism, may influence reproductive potential. In addition, microRNAs (miRNAs) are important regulators of gene expression. However, the contribution of miRNAs has not yet been assessed in sterile triploid Pacific oysters (Crassostrea gigas). We therefore obtained extensive miRNA and gene expression data to identify potentially critical miRNA-mRNA interactions that are linked to sterility in triploid C. gigas. Here, we found miR-263-x to be significantly downregulated in the gonads of sterile females. The upregulated target genes were associated with lipid droplet formation, which revealed the likely involvement of nutrient accumulation during gonadal development as regulated by miR-263-x in female sterile triploids. In males, the upregulated miRNAs (miR-1992-y and miR-2001-x) were shown to regulate spermatogenesis-related gene expression, including flagellar formation, and cell proliferation and migration. Our results suggest that energy redistribution regulated by miRNAs may be critical for gonad development in females, indicating that sterile triploids undergo a significant reduction in gamete number and show an increased accumulation of nutrients in the gonads. Our findings further indicate that decreased expression of genes related to spermatogenesis regulated by miRNAs is important in gonadal dysgenesis in sterile triploids. These results shed light on the epigenetic regulatory mechanisms governing sterility in triploid bivalves. • miRNAs played a vital role in the impaired gametogenesis of triploid Pacific oysters. • miR-263-x was associated with the regulation of lipogenesis in female triploid gonads. • Spermatogenesis-related genes were negatively regulated by miR-1992-y and miR-2001-x. [ABSTRACT FROM AUTHOR]

• Molecular characteristics and expressions of POU family genes in C. gigas were examined. • CgPOU3F4-like was a transcriptional factor in activating CgB-aat1 gene promoter. • CgPOU3F4-like positively regulated CgB-aat1 gene expression. • CgPOU3F4-like knockdown led to decreases of CgB-aat1 and CgCbs expressions. Previous study has found that b (0, +) -type amino acid transporter 1 (CgB-aat1) plays an essential role on mantle pigmentation in the Pacific oyster Crassostrea gigas. However, the molecular regulation of CgB-aat1 gene expression remains unclear. Herein, three POU domain family members, CgPOU2F1 , CgPOU3F4-like and CgPOU4F3-X1 were characterized and they all had POUs and HOX domains, respectively, which were important in transcriptional regulation. CgPOU3F4-like gene expression was the highest in mantle edge. Subsequently, the dual-luciferase reporter result showed that the core regulatory region of CgB-aat1 gene was from −632 to −350 bp of promoter. In transient co-transfection assays, the strongest activity was activated only by CgPOU3F4-like, suggesting CgPOU3F4-like was a valid transcriptional activator of CgB-aat1 gene promoter. And the structural integrity of CgPOU3F4-like was essential for its activation function. In addition, site directed mutagenesis assay was applied to detect three key binding sites between CgPOU3F4-like and core region of CgB-aat1 gene promoter, and this interaction was verified by ChIP test. Furthermore, CgPOU3F4-like knockdown by RNA interference led to obvious decreases in CgB-aat1 and cystathionine beta-synthase (CgCbs) expressions at both mRNA and protein levels. Collectively, these results indicate that CgPOU3F4-like positively regulate Cg B-aat1 gene expression and it may be a critical upstream transcriptional regulation factor in pheomelanin synthesis in C. gigas. [ABSTRACT FROM AUTHOR]

Shell formation is a dynamic process involving organic matrix secretion and calcification. In this study, we characterized shell morphogenesis during larval development in Crassostrea gigas. Using scanning electron microscopy (SEM) and fluorescence staining, we demonstrated that shell field, the first morphologically distinguishable shell-forming tissue, became visible soon after enlargement of the blastopore at the anterior end of the trochophore. Shell organic matrix namely protein polysaccharides and calcified structure appeared as a slit at the dorsal side of the embryo. The early shell field began to extend along the dorsal side of the trochophore larvae, and became a saddle shaped shell field that gave rise to the prodissoconch I embryonic shell in the early D-shaped larvae. Subsequently, prodissoconch II shell was formed in the late D-shaped larvae with a characteristic appearance of growth lines. To identify gene expression markers for studying shell formation, we isolated three potential larval shell formation genes CgPOU2F1 , CgSox5 , and CgPax6 and analyzed their expression during shell morphogenesis. The three potential shell formation genes possessed a similar pattern of expression. Their expression was detected in the shell gland and shell field regions in early D-shaped larvae, hereafter, their expression was detected at the larval mantle edge in the calcified shell stages. Together, these studies provide knowledge of shell morphogenesis in pacific oyster and molecular markers for studying the molecular regulation of biomineralization and shell formation. [Display omitted] • Molecular characteristics and gene expressions of POU2F1 , Sox5 , and Pax6 were studied in Crassostrea gigas. • CgPOU2F1 , CgSox5 , and CgPax6 were involved in larval shell formation. • Calcofluor white and calcein might be an ideal marker to visualize and trace the larval shell formation. [ABSTRACT FROM AUTHOR]

Background: Shell color polymorphisms of Mollusca have contributed to development of evolutionary biology and population genetics, while the genetic bases and molecular mechanisms underlying shell pigmentation are poorly understood. The Pacific oyster (Crassostrea gigas) is one of the most important farmed oysters worldwide. Through successive family selection, four shell color variants (white, golden, black and partially pigmented) of C. gigas have been developed. To elucidate the genetic mechanisms of shell coloration in C. gigas and facilitate the selection of elite oyster lines with desired coloration patterns, differentially expressed genes (DEGs) were identified among the four shell color variants by RNA-seq. Results: Digital gene expression generated over fifteen million reads per sample, producing expression data for 28,027 genes. A total number of 2,645 DEGs were identified from pair-wise comparisons, of which 432, 91, 43 and 39 genes specially were up-regulated in white, black, golden and partially pigmented shell of C. gigas, respectively. Three genes of Abca1, Abca3 and Abcb1 which belong to the ATP-binding cassette (ABC) transporters super-families were significantly associated with white shell formation. A tyrosinase transcript (CGI_10008737) represented consistent up-regulated pattern with golden coloration. We proposed that white shell variant of C. gigas could employ “endocytosis” to down-regulate notch level and to prevent shell pigmentation. Conclusion: This study discovered some potential shell coloration genes and related molecular mechanisms by the RNA-seq, which would provide foundational information to further study on shell coloration and assist in selective breeding in C. gigas. [ABSTRACT FROM AUTHOR]

• Carotenoid metabolism of Crassostrea gigas was studies for the first time with a beta-carotene feeding method. • The function of two apolipoprotein genes in carotenoid storage was validated by RNAi in the oyster. • A negative feedback regulation mechanism was proposed to regulate carotenoid metabolism in the oyster. Carotenoids are essential micronutrients for animals, and they can only be obtained from the diet for mollusk as well as other animals. In the body, carotenoids undergo processes including absorption, transport, deposition, and metabolic conversion; however, knowledge of the involved genes is still limited. To elucidate the molecular mechanisms of carotenoid processing and identify the related genes in Pacific oyster (Crassostrea gigas), we performed a comparative transcriptome analysis using digestive gland tissues of oysters on a beta-carotene supplemented diet or a normal diet. A total of 718 differentially expressed genes were obtained, including 505 upregulated and 213 downregulated genes in the beta-carotene supplemented group. Function Annotation and enrichment analyses revealed enrichment in genes possibly involved in carotenoid transport and storage (e.g., LOC105342035), carotenoid cleavage (e.g., LOC105341121), retinoid homeostasis (e.g., LOC105339597) and PPAR signaling pathway (e.g., LOC105323212). Notably, down-regulation of mRNA expressions of two apolipoprotein genes (LOC105342035 and LOC105342186) by RNA interference significantly decreased the carotenoid level in the digestive gland, supporting their role in carotenoid transport and storage. Based on these differentially expressed genes, we propose that there may be a negative feedback mechanism regulated by nuclear receptor transcription factors controlling carotenoid oxygenases. Our findings provide useful hints for elucidating the molecular basis of carotenoid metabolism and functions of carotenoid-related genes in the oyster. [ABSTRACT FROM AUTHOR]

One of the most important physiological process in the life cycle of most bivalves is the sexual reproduction required for species propagation. Understanding the molecular regulatory mechanisms of gender determination and gonad development have potential application in bivalve aquaculture, but there is still limited knowledge about this issue. The Pacific oyster Crassostrea gigas is a commercially important bivalve in aquaculture worldwide, as well as a representative specie to study the sexual development in bivalves for its complicated sexuality, including dioecy, sex change and rare hermaphroditism. In this study, an integrated analysis of miRNA and mRNA expression profiles of oyster gonads at different gametogenesis stages was performed to explore their possibly regulatory patterns in sexual development of C. gigas. Based on small RNA sequencing, a total of 122 miRNAs were identified, including 53 conserved and 69 novel miRNAs. Six miRNAs exhibited sex-specific pattern that expressed exclusively from either male or female gonads with increasing abundance throughout the gonadal development. Moreover, 287 putative targets of the six sex-biased miRNAs were predicted by combining previous transcriptome data. GO and KEGG enrichment analysis of target genes revealed that these sex-biased miRNAs might participate in the regulation of gametogenesis. Further, potential miRNA-mRNA interactions based on six sex-biased miRNAs were collected. The homolog of a key participator in testis development, meiotic recombination protein SPO11 in C. gigas (CgSPO11), was predicted as a target of female-biased miRNA lgi-miR-96a. This negative miRNA-mRNA expression relationship was validated in additional samples by quantitative real-time PCR (qRT-PCR) and the regulation of CgSPO11 by lgi-miR-96a was preliminarily confirmed by a dual-luciferase reporter assay. This study provides information regarding the gonad miRNAs of C. gigas and leads to the discovery of miRNA-mRNA interactions in gonad development, which will facilitate further investigations in molecular mechanisms of bivalve sex differentiation. • A total of 122 miRNAs were identified during the Pacific oyster gonad development. • Six sex-biased miRNAs might participate in the regulation of oyster gametogenesis. • The CgSPO11 gene is a target of lgi-miR-96a. [ABSTRACT FROM AUTHOR]

Paramyosin is a key component of thick filaments in invertebrate muscles. In this study, we isolated the full length cDNA of paramyosin from Pacific oyster (Crassostrea gigas) , and determined its pattern of expression during myogenesis. The full length paramyosin (CgPM) cDNA contains an open reading frame (ORF) of 2586 bp encoding a 861-amino acid protein. Sequence analysis revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. These data indicate that CgPM is strongly expressed during larval myogenesis in C. gigas , which provides the basis for further functional studies of paramyosin in oyster to better understand the molecular and cellular mechanisms of muscle formation in mollusks. [Display omitted] • Here, the sequence characteristics and expression profiles of paramyosin in Crassostrea gigas (CgPM) were investigated. The main results are as follows: • Sequence analysis of CgPM revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. • Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. • Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. • Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. [ABSTRACT FROM AUTHOR]