Acute heat stress induces short- and long-term epigenetic changes in the buff-tailed bumblebee (Bombus terrestris)

Global warming and the increasing frequency of extreme weather events threaten organisms worldwide, with cold-adapted species like bumblebees being especially vulnerable. Although heat stress has been shown to affect bumblebee physiology and behaviour, whether it also impacts their epigenome remains unknown. In this study, we addressed this gap by investigating the short- and long-term effects of acute heat stress experienced early in adult life on DNA methylation profiles in the buff-tailed bumblebee ( Bombus terrestris ). The DNA methylomes of males exposed either to control conditions (25°C) or to acute heat stress (42 °C for 60 min) were sequenced 1 day and 15 days after exposure to disentangle the short- and long-term effects of thermal stress. Heat stress induced differential methylation at hundreds to thousands of sites in both the short- and long-term, yet these signatures were largely distinct across time points. Accordingly, random forest classification failed to identify a persistent long-term DNA methylation signature of heat stress, indicating that heat-induced DNA methylation patterns change over time. Similarly, epigenetic clock analyses revealed that epigenetic age was transiently increased in the short-term, but not in the long-term. By uncovering the temporal dynamics of DNA methylation profiles following heat stress in B. terrestris males, our findings suggest that DNA methylation is actively regulated rather than passively maintaining environmentally induced alterations. These results shed new light on how environmental signals may dynamically shape the insect epigenome.