Identification of fruitless1 isoforms and their molecular function in gonadal development and courtship in the mud crab Scylla paramamosain

The fruitless (fru) gene is a key regulator of courtship behavior and the sex determination pathway in crustaceans. The present study identified two isoforms of fru1, Sp-fru1a and Sp-fru1b in the mud crab Scylla paramamosain. Sequence analysis revealed conserved BTB and C2H2-ZnF domains in their deduced amino acid sequences. Genomic analysis suggested that both isoforms originated from a single genomic locus. Fluorescence in situ hybridization confirmed the co-localization of both isoforms in the cytoplasm of oocytes and spermatids of the gonad. Sp-fru1 showed sexually dimorphic expression in well-developed gonads, with Sp-fru1a mRNA highly expressed in ovaries and Sp-fru1b mRNA predominantly in testis. Sp-fru1a mRNA expression remained high and consistent at various stages, with no significant changes observed, while Sp-fru1b mRNA expression was increased from unmated to the mating stage, and after mating, the expression level overall showed a downward trend. Moreover, both isoforms showed significantly higher expression in pre-mated testis. RNAi-mediated knockdown of Sp-fru1a resulted in lower mRNA expression of vasa, piwi2, and Vg in the ovary, leading to incomplete and fragmented egg cells. Similarly, knockdown of Sp-fru1b resulted in lower mRNA expression of vasa, piwi2, and dmrt1 in the testis, leading to a reduction in spermatozoa within the seminiferous tubules. Present findings suggest that Sp-fru1 plays key roles in sex-specific development and courtship behavior, providing valuable insights into the sex determination mechanisms and reproductive biology in crustaceans. This study emphasizes the significance of Sp-fru1 in the reproductive physiology of S. paramamosain, offering potential applications for molecular breeding, monosex population production, unisex farming, and aquaculture management through the control of unintended reproduction.