Identification and Characterization of MicroRNAs Involving in Initial Sex Differentiation of Chlamys farreri Gonads

SIMPLE SUMMARY: Sex formation of gonads encompasses two ancient and highly conserved biological processes, sex determination and sex differentiation. The processes are strictly regulated by a complex of gene networks. There is increasing evidence that miRNAs play key roles in many biological processes. however, information is limited in their contribution to sex differentiation in animals. In the present study, we identified the novel miRNAs involved in sex-related genes regulation and explored the miRNA–mRNA networks underlying the posttranscriptional regulation during the initial sex differentiation in Zhikong scallop, Chlamys farreri. Our findings provide an important basis for studying the sex differentiation mechanisms, as well as developing sex control techniques in bivalves. ABSTRACT: Research on expressional regulation of genes at the initial sex differentiation of gonads will help to elucidate the mechanisms of sex determination and differentiation in animals. However, information on initial sex differentiation of gonads is limited in bivalves. MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that can regulate the target gene expression at the posttranscription level by degrading the mRNA or repressing the mRNA translation. In the present study, we investigated the small RNAs transcriptome using the testes and ovaries of Zhikong scallop Chlamys farreri juveniles with a shell height of 5.0 mm, a critical stage of initial sex differentiation of gonads. A total of 75 known mature miRNAs and 103 novel miRNAs were identified. By comparing the expression of miRNAs between the ovary and testis, 11 miRNAs were determined to be differentially expressed. GO annotations and KEGG analyses indicated that many putative target genes that matched to these differentially expressed miRNAs participated in the regulation of sex differentiation. Furthermore, two selected miRNAs, cfa-novel_miR65 and cfa-miR-87a-3p_1, were confirmed to downregulate expressions of Foxl2 (a female-critical gene) and Klf4 (a male-critical gene), respectively, using a dual-luciferase reporter analysis. Our findings provided new insights into the initial sex differentiation of gonads regulated by miRNAs in bivalves.