Abstract:The razor clam, Sinonovacula constricta, has become a major economic mollusk in coastal East Asia because of its short culture cycle, rapid growth, and high yield, which have profoundly impacted the growth and development of mollusks in the aquaculture industry. However, compared with other mollusks, razor clam reproduction presents several challenges such as asynchronous spawning, poorly regulated reproduction, and a high rate of embryonic deformities, which greatly affect artificial breeding and the development of new varieties. Therefore, it is important to study the mechanisms underlying gonadal development. Gonadal development is the basis of animal breeding and reproductive control, and its mechanisms vary across different species. Therefore, a deeper understanding of gonadal development can provide a theoretical basis for genetic breeding and gender control. Gonadal development is affected by external factors (e.g., temperature and food) and endogenous gender-related genes. The Dmrt (double-sex and mab-3 related transcription factor) gene family is a class of transcription factors involved in sex determination and gonadal development in animals, with all encoded proteins containing highly conserved zinc-finger DM domains. Dmrt5 is an important member of this family, and its encoded protein is somewhat different from that of other family members because it includes conserved DM and DMA domains. It plays important roles in sex determination, gonadal development, and organ function maintenance in animals. In this study, two Dmrt5 genes were identified through whole-genome and gonadal transcriptome analyses of razor clams. To investigate the roles of Dmrt5 genes (ScDmrt5) in gonadal development of S. constricta, the full-length cDNA sequences of the two ScDmrt5 genes were cloned using the rapid amplification of cDNA ends (RACE) technique. The proteins encoded by the ScDmrt5 genes were analyzed for sequence comparison, conserved structural domains, and phylogenetic relationships using various bioinformatics tools. The expression patterns of ScDmrt5 genes/proteins in different tissues and gonadal development stages were investigated by qRT-PCR and immunofluorescence techniques. The open reading frame (ORF) sequence of ScDmrt5-1 was 1,170 bp, encoding 389 amino acids, and that of ScDmrt5-2 was 1,125 bp, encoding 374 amino acids. Both possessed conserved DM and DMA structural domains. The phylogenetic analysis showed that ScDmrt5-1 and ScDmrt5-2 first converged into a small clade with molluscan Dmrt5 proteins. Their motif locations, types, and quantity were similar, and their amino acid identities with other molluscan Dmrt5 proteins exceeded 50%, suggesting that the two Dmrt5 proteins of S. constricta were highly similar to Dmrt5 in other mollusks. ScDmrt5-1 and ScDmrt5-2 were localized on the same chromosome (chr12), but their positions were far apart without tandem duplication. Based on sequence comparisons, conserved domains, phylogenetic analysis, and chromosome localization, the two Dmrt5 genes were designated ScDmrt5-1 and ScDmrt5-2. The qRT-PCR analysis revealed that ScDmrt5-1 and ScDmrt5-2 were expressed in seven tissues (gill, mantle, testis, ovary, siphon, hepatopancreas, and foot), with significantly higher expression levels in the gills than in other tissues (P<0.05). In the mature stage of the gonads, ScDmrt5-1 and ScDmrt5-2 expression levels were significantly higher in the testes than in the ovaries (P<0.05). Based on the morphological characteristics of gonad cells of razor clams using paraffin sections and HE staining, the gonadal development process of the testis and ovary was categorized into four stages: proliferative, growing, mature, and spawning. During these stages, the expression level of ScDmrt5-1 gradually increased with testis development, peaking at the mature stage (P<0.05). In contrast, its expression exhibited an opposite trend in the ovary. ScDmrt5-2 expression also gradually increased with testis development, reaching higher levels in the mature and spawning stages, where it was significantly higher than in the ovary at the same stage (P<0.05). However, ScDmrt5-2 expression in the ovary remained relatively low and did not differ significantly between developmental stages. The immunofluorescence analysis revealed that ScDmrt5-1 and ScDmrt5-2 proteins were mainly located in the germ cells of female and male gonads, including oocytes and mature oocytes in the ovaries, and spermatogonia, spermatocytes, and mature sperm in the testis. In conclusion, ScDmrt5-1 and ScDmrt5-2 may be primarily involved in testis development and functional maintenance with potential roles in respiration and neuroperception. This study elucidates the expression characteristics of Dmrt5 in razor clam gonadal development. It also preliminarily explores the role of Dmrt5 in the gonadal development of razor clams, which could provide theoretical guidance for reproductive regulation, artificial breeding, and the development of new S. constricta varieties.
