The Yesso scallop, Patinopecten (Mizuhopecten) yessoensis, is a commercially important bivalve in the coastal countries of Northeast Asia. It has complex modes of sex differentiation, but knowledge of the mechanisms underlying this sex determination and differentiation is limited. In this study, the gonad tissues from females and males at three developmental stages were used to investigate candidate genes and networks for sex differentiation via RNA-Req. A total of 901,980,606 high quality clean reads were obtained from 18 libraries, of which 417 expressed male-specific genes and 754 expressed female-specific genes. Totally, 10,074 genes differentially expressed in females and males were identified. Weighted gene co-expression network analysis (WGCNA) revealed that turquoise and green gene modules were significantly positively correlated with male gonads, while coral1 and black modules were significantly associated with female gonads. The most important gene for sex determination and differentiation was Pydmrt 1, which was the only gene discovered that determined the male sex phenotype during early gonadal differentiation. Enrichment analyses of GO terms and KEGG pathways revealed that genes involved in metabolism, genetic and environmental information processes or pathways are sex-biased. Forty-nine genes in the five modules involved in sex differentiation or determination were identified and selected to construct a gene co-expression network and a hypothesized sex differentiation pathway. The current study focused on screening genes of sex differentiation in Yesso scallop, highlighting the potential regulatory mechanisms of gonadal development in P. yessoensis. Our data suggested that WCGNA can facilitate identification of key genes for sex differentiation and determination. Using this method, a hypothesized P. yessoensis sex determination and differentiation pathway was constructed. In this pathway, Pydmrt 1 may have a leading function.

The circadian rhythm has a great impact on the growth, metabolism and development of animals, but little is known about the circadian rhythm of marine bivalves. Understanding of the feeding rhythm is of great significance to increase the yield of razor clam Sinonovacula constricta, an economically important bivalve mollusk. The aim of this experiment was to study the effects of circadian rhythm of feeding time on digestive enzymes activities and their gene expression in S. constricta within a light (ZT8-ZT20)/dark (ZT20-ZT8) cycle. The present results showed that circadian rhythm of feeding rate (FR) was highly associated with digestive enzyme activities and relative expression of their genes. The highest values of FR were basically observed in the night from ZT0-ZT2 and ZT6-ZT8, which were significantly higher than those values in the daytime from ZT12-ZT14 and ZT18-ZT20 (P < 0.05). The digestive enzymes activities displayed the highest values at ZT2 and ZT8, and the lowest at ZT14 and ZT20. Among them, cellulase and pepsin were found to have significantly different activities (P < 0.05), rather than amylase and lipase. Notably, the relative expression of digestive enzyme genes shared the similar pattern with the activities of digestive enzymes. The highest values of relative gene expression of amylase (AMY), lipase (LIP), cellulase (CEL), and pepsin (PEP) were found at ZT2 and ZT8 in the night, while the lowest values were found at ZT14 during the day. It is therefore suggested that the biological clock may regulate the process from feeding to digestion. Furthermore, it might be better to feed at night to reduce cultivating cost and increase economic benefits in the farming industry of S. constricta.

The Chinese razor clam (Sinonovacula constricta) is an important for Chinese aquaculture marine bivalve that naturally occurs across intertidal and estuarine areas subjected to significant changes in salinity level. However, the information on the molecular mechanisms related to high salinity stress in the species remain limited. In this study, nine gill samples of S. constricta treated with 20, 30, and 40 ppt salinity for 24 h were used for whole-transcriptome RNA sequencing, and a regulatory network of competing endogenous RNAs (ceRNAs) was constructed to better understand the mechanisms responsible for adaptation of the species to high salinity. A total of 83,262 lncRNAs, 52,422 mRNAs, 2890 circRNAs, and 498 miRNAs were identified, and 4175 of them displayed differential expression pattern among the three groups examined. The KEGG analyses of differentially expressed RNAs evidenced that amino acid synthesis and membrane transport were the dominant factors involved in the adaptation of the Chinese razor clam to acute salinity increase, while lipid metabolism and signaling played only a supporting role. In addition, lncRNA/circRNA-miRNA-mRNA regulatory networks (ceRNA network) showed clearly regulatory relationships among different RNAs. Moreover, the expression of four candidate genes, including tyrosine aminotransferase (TAT), hyaluronidase 4 (HYAL4), cysteine sulfinic acid decarboxylase (CSAD), and ∆¹-pyrroline-5-carboxylate synthase (P5CS) at different challenge time were detected by qRT-PCR. The expression trend of TAT and HYAL4 was consistent with that of the ceRNA network, supporting the reliability of established network. The expression of TAT, CSAD, and P5CS were upregulated in response to increased salinity. This might be associated with increased amino acid synthesis rate, which seems to play an essential role in adaptation of the species to high salinity stress. In contrast, the expression level of HYAL4 gene decreased in response to elevated salinity level, which is associated with reduction Hyaluronan hydrolysis to help maintain water in the cell. Our findings provide a very rich reference for understanding the important role of ncRNAs in the salinity adaptation of shellfish. Moreover, the acquired information may be useful for optimization of the artificial breeding of the Chinese razor clam under aquaculture conditions.

Introduction: The razor clam, Sinonvacula constricta, one of the important mariculture bivalves in China, has unique flavor and wide salinity adaptability. The unique flavor mainly depends on the content of free amino acids and other umami substances in vivo. However, the flavor divergence of razor clam caused by the variable salinity breeding environment, while the mechanisms remain unclear.Methods: Here, the razor clams were cultured in high salinity (30 ppt) and normal salinity (20 ppt) for eight weeks, and the effects of salinity on free amino acids and related genes expression in S. constricta were investigated by transcriptomics and metabolomics method.Results: The results showed the free amino acid content under high salinity environment was significantly higher than normal salinity environment through the duration of the experiment (P < 0.05). The combination of transcriptomic and metabolomic data also indicated that high salinity environment resulted in enhanced metabolism of free amino acids. Furthermore, eight genes such as RALDH2, ACOX1, ALDH-E2 were potentially important for enhancing free amino acids metabolism under high salinity environment.Discussion: This study preliminarily explained the regulation processes of high salinity environment on the metabolism of free amino acids in razor clams, providing a reference for the flavor regulation mechanism.

Bone morphogenetic proteins (BMPs) not only play essential roles in bone development but also are involved in embryonic growth, organogenesis cell proliferation and differentiation. However, the previous studies on the functions of shellfish BMPs genes are still very limited. To better understand its molecular structure and biological function, BMP7 of the razor clam Sinonovacula constricta (Sc-BMP7) was cloned and characterized in this study. The full length of Sc-BMP7 is 2252 bp, including an open reading frame (ORF) of 1257 bp encoding 418 amino acids. The protein sequence included a signal peptide (1–32 aa), a prodomain (38–270 aa) and a TGF-β domain (317–418 aa). The quantitative expression of eleven adult tissues showed that Sc-BMP7 was significantly higher expressed in the gill, foot, and mantle (P < 0.05), but lower in hemocytes and hepatopancreas. In the early development stages, low expression was detected in the stages of unfertilized mature eggs, fertilized eggs, 4-cell embryos, blastula, gastrulae, whereas it increased after the stage of trochophore and demonstrated the highest expression in umbo larvae (P < 0.01). In shell repair experiment, Sc-BMP7 showed increasing expression level after 12 h. The higher expression of Sc-BMP7 was detected while Ca²⁺ concentration was reduced in seawater. After inhibiting Sc-BMP7 expression using RNA interference (RNAi) technology, expression of Sc-BMP7 mRNA and protein were significantly down-regulated (P < 0.05) in the central zone of mantle (nacre formation related tissue) and the pallial zone of mantle (prismatic layer formation related tissue). Association analysis identified two shared SNPs in exon of Sc-BMP7 gene from 246 individuals of two groups. These results indicated that BMP7 might be involved in shell formation and growth. These results would contribute to clarify the role of Sc-BMP7 in the regulation of growth and shell formation, and provide growth-related markers for molecular marker assisted breeding of this species.