A cyprinid fish, Onychostoma macrolepis, display a unique pattern of temperature-dependent sex determination

Current evidence supports high temperature-induced masculinization as the primary temperature-dependent sex determination (TSD) pattern in fish. Bidirectional TSD characterized by low temperature-induced masculinization and high temperature-induced feminization has never been reported in any fish species. This study revealed a unique TSD pattern in <i>Onychostoma macrolepis</i>: a balanced 1:1 sex ratio at 24°C (moderate temperature), over 80% female development at 28°C (high temperature), and 75% male development at 20°C (low temperature). <i>kdm6bb</i> and calcium channel genes were identified as key genes involved in TSD among the differentially expressed genes between high and low temperature groups. Consistently, administration of calcium influx inhibitors and Kdm6bb inhibitor effectively blocked high temperature-induced feminization and low temperature-induced masculinization, respectively. These findings suggest that <i>O. macrolepis</i> mediates temperature perception through calcium signaling, thereby regulating the expression of epigenetic modifier <i>kdm6bb</i> and modulating its sex determination. Surprisingly, the known male pathway genes (including <i>dmrt1</i> and <i>gsdf</i>) showed higher expressions in the high temperature group compared to the low temperature group at 50 daf. Aromatase inhibitors administration failed to induce sex reversal at both high and low temperatures, while very low concentration of T/MT and E2 treatment successfully induced sex reversal at high and low temperatures, respectively. These findings suggest that the downstream female and male pathways are unique for such a unique TSD pattern. Our study provides an exceptional model for investigating the impact of temperature on fish sex determination, thereby raising awareness of the harm of global warming on aquatic organisms.