The sex determination mechanism in large-scale loach (Paramisgurnus dabryanus) follows a ZZ/ZW system, with sexual differentiation regulated by both genotypic factors and temperature effects (GSD+TSD), where elevated temperatures result in a higher proportion of males. Currently, research on the sex determination mechanisms in large-scale loach is limited, and the specific gene expression profiles and the role of temperature in influencing sex remain largely unknown. This study investigated the impact of temperature on the sex ratio in cultured populations of the large-scale loach, and then identified a female-specific genetic marker by whole genome sequencing, facilitating the distinguishing of females, males, and pseudo-males within this population. Transcriptomic analysis was subsequently performed on these groups, and the data revealed a similar expression pattern between pseudo-males and true-males. The research combined differential expression analysis with WGCNA to construct a regulatory network of nine sex differentiation-related genes (SDG) (map3k4, trpv4, hsd17b12a, wt1, ar, dmrt1, bcar1, sox9a, cyp17a1), indicating that sex differentiation in large-scale loach is probably driven by the regulation of male-related genes. The transcriptomic analysis suggested that temperature significantly modified the expression of SDG in the ovaries, while in the testes, it predominantly affects metabolism-related pathways. We established a temperature-sensitive gene network in females, based on the correlation between gene expression and temperature, as well as the number of co-regulated genes in female data. We propose that, with increasing temperature, wt1 serves as a central regulator, leading to the down-regulation of foxl2a, cyp19a1a, and the cholesterol biosynthesis-related gene sqlea, ultimately resulting in the development of pseudo-males.
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