Recent studies have shown that crustacean female sex hormone (CFSH) is involved in the development of reproductive phenotype. In the present study, observation of sexually dimorphic traits revealed that gender could be distinguished from the third stage juveniles onwards in the mud crab, Scylla paramamosain. Sp-cfsh expression levels were analyzed in early juveniles. The results showed that, Sp-cfsh expression levels differed among individuals at post-molt of the first stage and second stage, and significantly different between the two sexes at post-molt of the third stage, which suggested that Sp-cfsh might participate in the sex differentiation in early juveniles. The expression of Sp-cfsh was examined during the molting cycle at the third stage juveniles, and the results showed that it was highest at the pre-molt stage. Based on the results, the expression of Sp-cfsh at pre-molt stage was further analyzed between the sexes from the third stage to the fifth stage, and it was found that the expression of Sp-cfsh was similar between two sexes at the third stage and the fourth stage; whereas at the fifth stage, when the gonopores occurred, the expression of Sp-cfsh significantly increased in females but decreased in males; suggesting that the expression of Sp-cfsh could influence the formation of gonopores. Finally, the role of Sp-cfsh in the reproductive phenotypes was confirmed through RNA interference knockdown. The combined results suggest that CFSH is involved in the regulation of sex differentiation of early juvenile in S. paramamosain.
