Identification and Sexually Dimorphic Expression of a FoxL2-like Gene in Mud Crab (Scylla paramamosain): Potential Roles in Male Differentiation and Development

Abstract

Monosex breeding technology has received enormous attention for the Scylla paramamosain, an economic crustacean species with distinctive sexual dimorphism. However, the molecular mechanism of gonadal development and sexual differentiation still remain inconclusive. In this study, we reported the cloning, characterization of a FoxL2-like gene with male-biased expression in the mud crab (SpFoxL2). The SpFoxL2 encoded a putative forkhead-box protein with 352 amino acids. Phylogenetic tree revealed the SpFoxL2 was clustered with the invertebrates FoxL2 homologues, most closed to E. sinensis. By quantitative real-time PCR, it was shown that the SpFoxL2 was expressed in intestine, ovary and testis, with the highest expression level in testis. During gonadal development, the expression level of SpFoxL2 in ovary was lower than that in testis at all stages. The expression level of SpFoxL2 constantly declined from stage I to stage V in ovary. While, there is a rising tendency from stage I to stage IV in testis. Notably, this male-biased expression pattern could be detected as early as the juvenile crab stage I (CI stage) before morphological sexual differentiation in the mud crab. By fluorescence in-situ hybridization, SpFoxL2 mRNA was found located in both somatic and germ cells in gonads. In testis, it was most strongly localized in the meiosis germ cells (spermatocytes). While in ovary, the signal became weaker in the meiosis germ cells (oocytes). The results imply that the SpFoxl2 may play an important role in male differentiation and development, laying a foundation for future research on sexual differentiation of the species.