Introduction
Environmental changes can trigger epigenetic modifications that influence gene expression and, consequently, the phenotype. However, key questions remain about the mechanisms underlying epigenetic inheritance and the extent to which environmentally induced epigenetic marks are transmitted across generations. In particular, the potential role of epigenetic mechanisms —especially DNA methylation—in facilitating short-term adaptation is still under investigation. To address these questions, we examined how temperature influences genome-wide DNA methylation patterns, with a specific focus on the sex phenotype and genes involved in sex determination and differentiation, and assessed the potential transmission of these modifications to the offspring.
Materials and Methods
We used the European sea bass (Dicentrarchus labrax), a fish species with a polygenic and temperature-sensitive sex determination system, to investigate the effects of temperature on DNA methylation. Genome-wide profiles were obtained at single-nucleotide resolution using reduced representation bisulfite sequencing in 66 individuals from five families across two generations (F₀ and F₁). Individuals were exposed to control (16 °C, C) or elevated (21 °C, T) temperatures between 12 and 60 days post-fertilization in a multigenerational experimental design. Four temperature exposure regimes were applied to the F₁ generation: control (CC), ancestral exposure via sires (TC), developmental exposure in offspring (CT), and dual exposure (TT).
To assess the effects of temperature and sex on DNA methylation, we performed the following analyses: First, for each region covered, we applied a generalized linear model with a binomial distribution to methylation count data, using F₀ and F₁ temperature treatments as fixed effects and correction for multiple testing. Analyses were performed separately for testes and ovaries. This approach allowed us to classify regions into five categories: no effect, F₀-only effect, F₁-only effect, additive effect (same direction of change in F₀ and F₁ without interaction), and interactive effect (significant interaction between F₀ and F₁, regardless of direction of change). These regions were further filtered based on differential methylation, using sex and sire as covariates. Second, w e investigated temperature effects, specifically focusing on a panel of sex-related genes. And, third, we assessed the proportion of stable (constitutive) versus environmentally-induced epigenetic marks between generations to evaluate the potential for inheritance of methylation changes.
Results and Discussion
In terms of phenotypic sex, we observed a significant increase in the proportion of males in temperature-treated groups, as expected. However, when comparing the dual-exposure group (TT) with the control group (CC), differences in sex ratios approached but did not reach statistical significance (P = 0.051). Regarding other traits, males exhibited significantly higher body weight and length in response to temperature treatment when compared to CC males , with the TT group showing the highest values. In contrast, temperature-resistant females also displayed an increase in body size under temperature treatment, but this effect was not significant when comparing CC and TT groups.
Analysis of DNA methylation data revealed marked sex-specific differences in the distribution of regions across model-defined categories. Notably, only 5.1% of regions in males and 3.7% in females exhibited an additive temperature effect. In contrast, a substantial proportion of regions —37.0% in males and 31.1% in females— showed qualitative interactions, where the direction of the temperature effect differed between generations (F₀ and F₁). This pattern suggests the presence of compensatory epigenetic mechanisms in response to repeated temperature exposure.
We are currently working on analyses on neomales (genetic females that developed as phenotypic males); results from these individuals will provide further insight into temperature- and sex-specific methylation dynamics.
Additionally, we identified several key sex-related genes whose methylation patterns were significantly affected by temperature in a regime-dependent manner. Furthermore, we found that while a substantial portion of epigenetic marks were stably transmitted from sires to offspring, a small but noteworthy fraction (~5%) of inherited marks were temperature-induced and transmitted to the following generation. These findings offer new insights into the extent of environmentally-driven epigenetic inheritance.
Conclusions
Overall, our study underscores the influence of temperature on the epigenetic landscape, with important implications for understanding plasticity, adaptation, and epigenetic effects transmitted across generations in the context of global climate change.
Funding: Research funded by Spanish Ministry of Science and Innovation ‘Epipure ’ (PID2019-108888RB-I00) and the EU H2020 Grant 652831 (AQUAEXCEL2020, Transnational Access project “Transsexbass”) to FP.