Multigenerational temperature effects on paternal epigenetic inheritance in European sea bass

Environmental changes can induce epigenetic modifications, influencing gene expression, phenotype, and species adaptation. This study investigates how temperature affects genome-wide DNA methylation patterns, particularly in genes crucial for sex development and whether these modifications can be transmitted across generations. Using the European sea bass -a fish model with both genetic and environmental sex determination- we analyzed DNA methylation at single nucleotide resolution using reduced representation bisulfite sequencing in 64 individuals from five families across two generations (F0 and F1). Parental fish (F0) were exposed to either control (16 °C, C) or elevated (21 °C, T) temperatures from 12 to 60 days post-fertilization. Their offspring (F1) were then subjected to four thermal regimes: control (CC), ancestral exposure via sires (TC), developmental exposure in offspring (CT), and dual exposure (TT). We determined the length of differentially methylated regions (DMRs) using a conservative, reproducible, and species-specific method adapted from plant epigenetics. To disentangle ancestral and developmental temperature effects, DMRs were classified according to their association with F0, F1, or F0 x F1 interaction effects. This allowed us to quantify the relative contribution of each treatment, separately for testes and ovaries in the F1 generation. While the proportion of additive DMRs showing cumulative temperature effects (e.g., 2.1% in testes, 1.4% in ovaries) was relatively rare, a substantial proportion of DMRs (37% in testes, 31.1% in ovaries), exhibited opposing methylation changes with F0 and F1 treatments, indicative of compensatory epigenetic interactions. These interactions were also reflected at the phenotypic level: TT individuals showed body weights comparable to CC, and the sex ratio in TT approached statistical significance when compared to CC (P = 0.051), suggesting a link between epigenetic regulation and phenotypic plasticity under elevated temperatures. Finally, we also investigated the inheritance of epimarks from sires to offspring. While most epimarks remained stable across generations, ~ 5% of all DMRs were both temperature-induced and inherited, offering direct evidence for environmentally responsive multigenerational epigenetic inheritance. This study demonstrates the role of temperature in shaping the epigenome and highlights the potential of epigenetic plasticity and inheritance in species adaptation and conservation amid global warming.