MicroRNA (miRNA) is a posttranscriptional downregulator that plays a vital role in a wide variety of biological processes. In this study, we constructed five ovarian and testicular small RNA libraries using two somatic libraries as reference controls for the identification of sex‐biased miRNAs and gonadal differentially expressed miRNAs (DEMs) of the Chinese mitten crab, Eriocheir sinensis. A total of 535 known and 243 novel miRNAs were identified, including 312 sex‐biased miRNAs and 402 gonadal DEMs. KEGG pathway analysis showed that DEM target genes were statistically enriched in MAPK, Wnt, and GnRH signaling pathway, and so on. A number of the sex‐biased miRNAs target genes associated with sex determination/differentiation, such as IAG, Dsx, Dmrt1, and Fem1, while others target the genes related to gonadal development, such as P450s, Wnt, Ef1, and Tra‐2c. Dual‐luciferase reporter assay in vitro further confirmed that miR‐34 and let‐7b can downregulate IAG expression, miR‐9‐5p, let‐7d, let‐7b, and miR‐8915 can downregulate Dsx. Taken together, these data strongly suggest a potential role for the sex‐biased miRNAs in sex determination/differentiation and gonadal development in the crab.

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