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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]

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]

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]