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Colorful shells in mollusks are commonly attributable to the presence of biological pigments. In Pacific oysters, the inheritance patterns of several shell colors have been investigated, but little is known about the molecular mechanisms of melanogenesis and pigmentation. cAMP-response element binding proteins (CREB) are important transcription factors in the cAMP-mediated melanogenesis pathway. In this study, we characterized two CREB genes (CREB3L2 and CREB3L3) from Pacific oysters. Both of them contained a conserved DNA-binding and dimerization domain (a basic-leucine zipper domain). CREB3L2 and CREB3L3 were expressed highly in the mantle tissues and exhibited higher expression levels in the black-shell oyster than in the white. Masson-Fontana melanin staining and immunofluorescence analysis showed that the location of CREB3L2 protein was generally consistent with the distribution of melanin in oyster edge mantle. Dual-luciferase reporter assays revealed that CREB3L2 and CREB3L3 could activate the microphthalmia-associated transcription factor (MITF) promoter and this process was regulated by the level of cAMP. Additionally, we found that cAMP regulated melanogenic gene expression through the CREB-MITF-TYR axis. These results implied that CREB3L2 and CREB3L3 play important roles in melanin synthesis and pigmentation in Pacific oysters. [Display omitted] • Two transcription factors (CREB3L2 and CREB3L3) were characterized from Pacific oysters. • CREB3L2 and CREB3L3 regulated MITF expression under cAMP regulation. • A cAMP-CREB-MITF axis regulated melanogenesis in Pacific oysters. [ABSTRACT FROM AUTHOR]

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]

As the second largest phylum in the zoological kingdom next to arthropods, the mechanism of gonadal differentiation in mollusca is quite complex. Currently, although much has been carried out on gonadal differentiation in the Pacific oyster, there is still unknown information that needs to be further explored. Here, analysis of the Foxl2 and Dmrt1l expression in samples at different development periods of male and female gonads as well as in annual gonad samples revealed that Log 10 (Foxl2 / Dmrt1l) values were an effective method for sex identification in oysters. In differentiated gonadal tissue, Log 10 (Foxl2 / Dmrt1l) values greater than 2 were females and less than 1 for males. Subsequent sequential sampling of the same individuals verified that Log 10 (Foxl2 / Dmrt1l) values greater than 2 for resting gonads would develop as females and less than 1 would develop as males in the future. Relative expression analysis of Foxl2 and Dmrt1l in the annual samples revealed a negative correlation between Log 10 (Foxl2) and Log 10 (Dmrt1l). Double fluorescence reporter validation results showed that DMRT1L protein was able to bind the Foxl2 promoter and repress its activity with a weak dosage effect. Antagonism between Dmrt1l and Foxl2 is therefore not restricted to vertebrates, and the competing regulatory networks are of great significance in the maintenance of gonadal sex in oysters after sexual differentiation. This study provides novel ideas and insights into the study of early gonadal differentiation in the adult oyster. [Display omitted] • Log 10 (Foxl2 / Dmrt1l) values can distinguish oyster sex at transcriptional level. • Log 10 (Foxl2 / Dmrt1l) values can predict the direction of resting sex development. • Competing mechanisms are of great importance for the maintenance of gonadal sex. [ABSTRACT FROM AUTHOR]

Abstract Pacific oyster (Crassostrea gigas) is a sessile bivalve living in the intertidal zone. It has become an attractive model for developmental studies due to its metamorphic transition from a mobile planktonic larvae to a sessile adults. To determine the effect of metamorphosis on muscle development in oyster larvae, we characterized myogenesis during larval development and metamorphosis by phalloidin staining which labels filamentous actin filaments. Our data revealed a dynamic pattern of myogenesis during embryonic and larval development. It appears that simple "U-shaped" muscle ring first developed at the trochophore stage. This was followed by a more complex musculature including an anterior adductor, velum ventral retractors at the veliger stage, and the addition of posterior adductors and foot retractors at the veliger and pediveliger stages. During metamorphosis, muscle structures in the anterior adductor, velum retractors and ventral retractors were degenerated. At the same time, mantle and gill musculature appeared and became the primary muscle system in juveniles together with the posterior adductor. In addition, indirect immunofluorescence with the monoclonal antibody against C. gigas muscle proteins (myosin heavy chains (MYHC) and α-actinin) were used to monitor changes in the developing musculature at different larval stages. The immunofluorescence staining results of muscle proteins were consistent with phalloidin staining. The expression locations of two muscle proteins were similar and mainly located in larval velum retractor and adductor muscle. The α-actinin expression positions were located in Z -lined of velum striated retractors. Data from these studies provide a comprehensive description of myogenesis in C. gigas embryos and larvae. Moreover, our data showed that metamorphosis has a significant impact on remolding the musculature after transition from a mobile planktonic larvae to a sessile mollusk, associated with certain muscle group degradation. [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]

Molluscs exhibit diverse shell colors. The molecular regulation of shell coloration is however not well understood. To investigate the connection of shell coloration with pigment synthesis, we analyzed the distribution of porphyrins, a widespread group of pigments in nature, in four Pacific oyster strains of different shell colors including black, orange, golden, and white. The porphyrin distribution was analyzed in oyster mantles and shells by fluorescence imaging and UV spectrophotometer. The results showed that red fluorescence emitted by porphyrins under the UV light was detected only on the nacre of the orange-shell strain and mantles of orange, black and white-shell strains. Extracts from newly deposit shell, nacre and mantle tissue from orange-shell specimens showed peaks in UV–vis spectra that are characteristic of porphyrins, but these were not observed for the other shell-color strains. In addition, genes of the haem synthetic pathway were isolated and characterized. Phylogenetic analysis of CgALAS , CgALAD , CgPBGD , CgUROS , and CgUROD provide further evidence for a conserved genetic pathway of haem synthesis during evolution. Differential expression of the haem genes expressed in mantle tissues support these findings and are consistent with porphyrins being produced by the orange strain only. Tissue in situ hybridization demonstrated the expression of these candidate genes at the outer fold of C. gigas mantles where shell is deposited. Our studies provide a better understanding of shell pigmentation in C. gigas and candidate genes for future mechanistic analysis of shell color formation in molluscs. • Distribution of porphyrin pigments in the outer mantle edge and the shell of Crassostrea gigas was observed by spectral analysis. • The coding sequences of key haem pathway genes were identified and characterized in C. gigas. • The shell color of C. gigas may be affected by the accumulation and degradation of porphyrin caused by the expression of key haem pathway genes. [ABSTRACT FROM AUTHOR]

The paired-box 7 (Pax7) is a transcription factor crucial for skin color polymorphism. However, the mechanism underlying the pigmentation associated with Pax7 in mollusks have yet to be elucidated. In this study, the cDNA sequence of Pax7 in the Pacific oyster Crassostrea gigas (CgPax7) was characterized. Phylogenetically, the identity of deduced amino acid sequence was similar to that of other mollusks and contained 463 amino acids, with conserved features of paired domain (PRD), homeobox domain (HD) and octapeptide. Gene expression analysis revealed that CgPax7 was markedly increased at D-shaped larvae stage and ubiquitously expressed in six examined tissues in adult oyster. The result of whole-mount in situ hybridization (WMISH) showed a restricted pattern of CgPax7 expression on margins of shell valves at D-shaped and umbo larvae stages. Additionally, although CgPax7 silencing had no significant effect on CgMitf expression, it significantly inhibited the expressions of CgPax7 , CgTyr , CgTyrp1 , CgTyrp2 and CgCdk2 , genes involved in Tyr-mediated melanin synthesis. Furthermore, CgPax7 knockdown obviously decreased the tyrosinase activity. Less brown-granules at mantle edge was detected by micrographic examination and melanosomes defect was observed by transmission electron microscopy. It was demonstrated that CgPax7 play a key role in melanin synthesis by regulating Tyr-pathway in C. gigas. These findings indicated the potential framework by which mollusks pigmentation. [Display omitted] • Molecular characteristic and gene expression of Pax7 were studied in C. gigas. • Pax7 silencing significantly decreased melanin synthesis-related genes. • Pax7 knockdown led to melanosomes defect in mantle of C. gigas. • Pax7 play a vital role in Tyr pathway of C. gigas. [ABSTRACT FROM AUTHOR]