Time of day-dependent effects of heat treatment on thermal tolerance and sex differentiation in Nile tilapia (Oreochromis niloticus)

The present research aimed to determine the influence of the time of day at which heat treatment (HT) is applied to sexual differentiation and thermal tolerance mechanisms in Nile tilapia (Oreochromis niloticus). To this end, fish larvae were subjected to HT (36 degrees C) for 12 days at different times of the day during the thermosensitive period (i.e. 11-23 days post-fertilization, dpf). The heat treatment was applied during the light phase (L-HT); during the dark phase (D-HT), or during both light-dark phases (LD-HT). A control group maintained at constant rearing temperature (28 degrees C, CTRL) was also used. At 25 dpf, thermal tolerance was determined (survival) and whole larvae samples were collected for the mRNA expression analysis (qPCR). There after, all the groups remained at constant temperature until 270 dpf, when some fish were sacrificed and each individual's gonadal lobules were collected for histological and qPCR analyses. At the same time, each experimental group was subjected to acute heat shock (HS; 36 degrees C for 2 h) in either the mid-light (ML) or the mid-dark (MD) phase. When HS exposure finished, adults' brains were extracted to determine the HS response by qPCR. The expression of the genes involved in female (cyp19a1a, foxl2, era) and male (amh, ara, sox9a, dmrt1) sexual differentiation and thermal tolerance (hsp70, hsp90a, hsp27) in the 25 dpf larvae and adults was studied. The results revealed that all the HT-treated larvae showed an up-regulation of testicular differentiation gene expression and inhibition of ovarian differentiation gene expression, which resulted in a higher percentage of males. The time of day of HT during development influenced thermotolerance in the larval and adult stages, with the lowest survival and highest cellular stress (higher HSPs expression) observed in the larvae of the D-HT and LD-HT groups. These findings reveal the need to further investigate the link between sexual differentiation and thermotolerance daily rhythms in thermal biology research. These results may be useful for optimizing masculinization protocols in the tilapia industry, while ensuring animal welfare and minimizing negative HT effects on fish physiology.