A testis-specific Doublesex gene is involved in spermatogenesis of the oriental river prawn, Macrobrachium nipponense.

The oriental river prawn, Macrobrachium nipponense, is an economically important species exhibiting significant sexual dimorphism, with males growing faster than females. Therefore, elucidating the mechanisms of sex determination and differentiation is crucial for developing monosex culture technologies to enhance aquaculture yield. The Doublesex (Dsx) gene is recognized as a conserved regulator of sex determination and differentiation across diverse species. Despite its crucial role, the regulatory mechanisms of Dsx remain inadequately characterized in decapod crustaceans. In this study, we cloned a Dsx homolog (MnDsx) from the freshwater prawn Macrobrachium nipponense and comprehensively investigated its tissue-specific expression and testicular distribution pattern, potential target genes, role in testis development, and protein interaction partners. MnDsx mRNA is predominantly expressed in the testis and localized to both the nucleus and cytoplasm of spermatogonia, spermatocytes, and spermatozoa. Notably, eyestalk ablation was found to negatively regulate MnDsx mRNA expression levels in the testis. RNAi-mediated knockdown of MnDsx significantly reduced the number of spermatozoa within seminiferous tubules. Through integrated RNA-seq and CUT&Tag analyses, we identified 1140 potential target genes of MnDsx in the testis, many of which are related with testis development and spermatogenesis. Additionally, pull-down assays combined with LC-MS/MS identified 63 MnDsx-interacting proteins in the testis, including several histones. These results suggest that MnDsx plays vital roles in spermatogenesis, potentially modulating the transcription of development-related genes through chromatin remodeling. Collectively, this study provides novel insights into the molecular network of sex determination and differentiation and offers potential gene targets for sex control strategies in crustacean aquaculture.