Identifying sex-chromosome-linked determinants of sexual differentiation in a WZ-ZZ sex inheritance crustacean

Sex determination triggers transcriptional cascades, leading to the development of male or female phenotypes. The aim of this study was to shed light on the linkage between sex determination and sexual differentiation by identifying key W/Z-linked elements that control sexual differentiation in crustaceans with the WZ-ZZ mode of sex inheritance. To this end, we leveraged our previous studies on the insulin-like androgenic gland (IAG) hormone, which serves as a master switch-termed the "IAG-switch"-in the freshwater prawn Macrobrachium rosenbergii, in which manipulations of the IAG-switch afforded functional WZ males, ZZ females, and even WW males and females. To uncover the link between sex determination and sexual differentiation, males and females lacking either the W or the Z chromosome were used to establish a transcriptomic library of early developmental stages (embryo, larva, and post-larva), followed by clustering analysis of differentially expressed genes. Mapping of these genes to the M. rosenbergii sex chromosomes yielded W/Z-linked candidates with a male-biased expression pattern culminating at day 10 post-larvae (PL10), which marks the phenotypic sexual differentiation stage. Among these genes, two leading candidates were identified, namely, a W/Z-associated coding gene (cytochrome P450 4c3-like, MrCYP4), and a Z-specific long noncoding RNA (MrZlnc). Silencing of these two candidates caused a reduction in MrIAG expression and vice versa, clearly indicating crosstalk with the IAG-switch. The study thus constitutes the first step in unraveling key elements that control sexual differentiation in crustaceans exhibiting WZ-ZZ sex inheritance.