Histological chronology and thermosensitive period of gonadal sex differentiation in turbot (Scophthalmus maximus)

The turbot ( Scophthalmus maximus ), an economically significant marine flatfish with a ZW/ZZ sex determination system, exhibits marked sexual growth dimorphism favoring females, making all-female culture a key aquaculture objective. However, effective sex control requires precise understanding of gonadal differentiation timing and environmental influences. This study combined detailed histological analysis with systematic temperature manipulation to establish the chronology of gonadal development and identify the thermosensitive window for sex determination. Histological examination revealed that ovarian differentiation, marked by cavity formation, commenced at 45 days post-hatching (dph) (37.4 mm TL), preceding the appearance of meiotic oocytes at 65 dph. Testicular differentiation occurred later at 65 dph (50.4 mm TL), demonstrating delayed development compared to ovaries. Through controlled temperature exposures at different developmental stages, we identified a critical thermosensitive window spanning 28–54 dph (20.05–43.00 mm TL), with the most sensitive period confined to 28–40 dph (20.05–30.11 mm TL). Exposure to 25°C during this window induced significant masculinization, yielding up to 74.3 % phenotypic males, while maintaining 18°C throughout this period not only preserved a balanced 1:1 sex ratio but also supported optimal growth performance. These findings establish that the thermosensitive period precedes histological gonadal differentiation, indicating that sex determination in turbot occurs earlier than previously recognized and is strongly influenced by temperature during a defined developmental window. Our results provide crucial insights for developing temperature-based sex control protocols, enabling reliable production of all-female populations through precise thermal management during early juvenile stages. • Ovarian differentiation (37.4 mm TL) precedes testicular differentiation (50.4 mm TL) in turbot. • A thermosensitive window for sex determination (20.05–30.11 mm TL) precedes gonadal differentiation. • 25°C exposure induces masculinization (74.3 % males); 18°C maintains a balanced sex ratio. • This offers a practical thermal management strategy to secure all-female turbot production.