Onychostoma macrolepis, a cyprinid fish, displays 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. To date, no study has reported bidirectional TSD in a fish species, in which low temperature induces masculinization while high temperature induces feminization. In this study, Onychostoma macrolepis exhibited a distinctive bidirectional TSD pattern, with a balanced 1:1 sex ratio at 24°C (moderate temperature), over 80% female development at 28°C (high temperature), and approximately 75% male development at 20°C (low temperature). Transcriptomic analyses revealed kdm6bb and calcium channel genes as key regulators of TSD among the differentially expressed genes between high and low temperature groups. Consistently, administration of calcium influx and Kdm6bb inhibitors effectively blocked high temperature-induced feminization and low temperature-induced masculinization, respectively. These findings indicate that temperature perception in O. macrolepis is mediated through calcium signaling, which regulates the expression of the epigenetic modifier kdm6bb and consequently modulates sex determination. Unexpectedly, canonical male pathway genes such as dmrt1 and gsdf showed higher expression in the high temperature group than in the low temperature group at 50 days after fertilization (daf). Administration of aromatase inhibitors failed to induce sex reversal at either temperature, whereas extremely low concentrations of testosterone/methyltestosterone (T/MT) and 17β-estradiol (E2) successfully induced sex reversal under high and low temperature conditions, respectively. These results suggest that downstream regulatory pathways controlling sexual differentiation in O. macrolepis are distinct from conventional TSD models. The discovery of this bidirectional mechanism establishes O. macrolepis as an exceptional model for elucidating the molecular and physiological bases of temperature-driven sex determination and underscores the ecological risks of global warming for aquatic vertebrates.