Temperature influences sex determination in the giant spiny frog (Quasipaa spinosa) via alteration of DNA methylation and transcriptional profiles

Temperature is a critical environmental factor influencing DNA epigenetic modifications. While the role of DNA methylation in gene regulation is well-established, its interplay with environmental signals in amphibians remains inadequately understood. In this study, Quasipaa spinosa tadpoles were reared at 18 ℃ (low), 23℃ (normal), and 28 ℃ (high). Sex ratios were recorded, and transcriptome and whole-genome methylation sequencing were performed. The normal-temperature group exhibited a balanced 1:1 sex ratio, while high temperature induced masculinization and low temperature promoted feminization. Although no significant differences in overall DNA methylation levels were observed, key pathways involved in sex-hormone synthesis were altered in a temperature-dependent manner. Specifically, low temperature activated CACN family genes and upregulated CYP19A1 , thereby enhancing estrogen synthesis and promoting female differentiation. In contrast, high temperature suppressed CYP19A1 activity, resulting in testosterone accumulation and masculinization. These results elucidate a pathway through which temperature influences sex determination in Q. spinosa via epigenetic regulation, providing a theoretical basis for the precise control of sex ratios in aquaculture. • Integrated methylome and transcriptome analyses reveal temperature effects on sex determination in Quasipaa spinosa tadpoles. • Low (18 ℃) feminized and high (28 ℃) masculinized tadpoles; the normal 23 ℃ group showed an approximately 1:1 sex ratio. • Temperature had minimal effects on global DNA methylation but altered key sex-related genes in a tissue-specific manner. • Revealed a negative link between promoter methylation and transcription, and a temperature-dependent sex hormone pathway.