Small RNA Sequencing Reveals Dynamic Expression Profiles and Regulatory Network of miRNAs During Testis Development...

Aquaculture of the giant river prawn Macrobrachium rosenbergii faces ongoing challenges associated with aberrant gonadal development, precocious puberty and inefficient sex manipulation techniques. Although microRNAs (miRNAs) serve as critical regulators of spermatogenesis across diverse taxa, their roles in modulating testicular development of M. rosenbergii remain largely uncharacterized. In this study, we performed small RNA sequencing on testicular tissues of M. rosenbergii at three representative developmental stages to profile genome-wide miRNA expression dynamics during spermatogenesis. A total of 173 miRNAs (including 12 novel miRNAs) were identified, and several key miRNAs exhibiting significant temporal expression differences were screened. GO and KEGG enrichment analyses revealed that target genes of these miRNAs were primarily involved in pathways related to sex differentiation, hormone synthesis, and energy metabolism. Further analysis indicated that miRNAs such as miR-1-3p and miR-317-3p may activate the testis developmental program during early developmental stages, while the downregulation of negative regulators like miR-124-3p and miR-210-3p may contribute to the release of developmental inhibition. A subset of miRNAs including miR-8-3p and miR-315-5p may exert regulatory effects on the IAG signaling axis through targeting genes such as ferritin and Fem-1c, thereby modulating testicular maturation. Additionally, the observed downregulation of miRNAs implicated in innate immune response and chitin metabolism points to a reconfiguration of regulatory priorities during the rapid phase of testicular growth to support reproductive development. The study contributes to a deeper understanding of miRNA-mediated regulatory mechanisms underlying crustacean development and reproduction.