Simulated heat waves aggravate the immune response to sublethal acetamiprid exposure in the honey bee (Apis mellifera)

Simultaneous exposure to thermal stress and agrochemicals represents a realistic yet understudied toxicological scenario for pollinators. Honey bees (Apis mellifera) increasingly face intense heat waves while foraging in pesticide-treated landscapes. Understanding the cumulative impact of these stressors is therefore critical for predicting pollinator health. We investigated the combined effects of simulated heat waves (exposed to 42 °C for two hours over three consecutive days) and chronic sublethal exposure to the neonicotinoid acetamiprid on honey bee immunity and physiology. By subjecting bees to a subsequent mechanical challenge, we assessed both their baseline physiological state and their capacity to mount an active immune defense under stress. Using a comprehensive panel of biomarkers - including antimicrobial peptides (AMPs: abaecin, apidaecin, defensin-1, hymenoptaecin), heat shock proteins, and oxidative stress markers - we evaluated the bees' immune and physiological resilience. Strikingly, while heat stress alone significantly reduced total hemocyte counts, indicating compromised cellular immunity, it did not trigger a humoral immune response. In contrast, combining heat stress with acetamiprid led to a significant overexpression of all tested AMP genes and elevated AMP concentrations in the hemolymph. Furthermore, this combined treatment upregulated catalase expression, signaling enhanced oxidative stress, and increased total hemolymph protein levels, suggesting altered physiological homeostasis. These results indicate that the interplay between thermal stress and pesticide exposure lowers the threshold for immune activation. We conclude that even sublethal pesticide doses can become immunologically burdensome during heat events. This stressor interaction risks physiological exhaustion and energy trade-offs, potentially compromising colony resilience against ubiquitous parasites in a warming world.