Exposure of zebrafish to elevated temperature induces sex ratio shifts and alterations in the testicular epigenome of unexposed offspring.

Accumulating evidence shows that environmental changes can affect population sex ratios through epigenetic regulation of gene expression in species where sex depends on both genetic and environmental cues. Sometimes, altered sex ratios persist in the next generation even when the environmental cue is no longer present (a multigenerational effect). However, evidence of transgenerational effects (i.e., beyond the first non-exposed generation), which tend to be paternally transmitted, is scarce and a matter of debate. Here, we used the AB strain of zebrafish, where sex depends on both genetic and environmental influences, to study possible multi- (to the F1) and transgenerational (to the F2) effects of elevated temperature during the critical period of sex differentiation. From eight initial different families, five were selected in order to capture sufficient variation between the sex ratio of the control group (28 °C) and the group exposed to elevated (35 °C) temperature only at the parental (P) generation. Results showed a consistent increase in the proportion of males in the P generation in all five families as a result of heat treatment. Sex ratios were then determined in the F1 and F2 offspring derived from both above groups, which were all raised at 28 °C. A persisting male-skewed sex ratio in the 35°C-derived, unexposed offspring of the F1 generation was observed in three families, denoting family-dependent multigenerational effects. However, no transgenerational effects were observed in the F2 generation of any family. DNA methylation was also assessed in the testis of P, F1 and F2 males derived from exposed and non-exposed fathers and grandfathers. DNA methylation was significantly decreased only in the testis of the 35°C-derived males in the F1 generation but not of the F2 generation and, surprisingly, neither in the 35°C-exposed males of the P generation. Taken together, our results show great interfamily variation, not only in sex ratio response to elevated temperature, but also on its multigenerational effects, denoting a strong influence of genetics. Alterations in the testicular epigenome in F1 males calls for attention to possible, previously unnoticed, effects of temperature in the unexposed offspring of heat-exposed parents in a global warming scenario.