Bioenergetics Underpins Both Heat-Pesticide Synergisms and Their Transgenerational Offsetting in Water Fleas

Freshwater ecosystems are increasingly threatened by interacting stressors simultaneously or sequentially, such as pesticide contamination and extreme heat events. Although synergistic interactions between these stressors within a generation are well documented, their transgenerational consequences for sublethal, fitness-related traits, as well as the underlying bioenergetic mechanisms, remain poorly understood. Using a three-generation full-factorial experiment with Daphnia magna, we examined how parental exposure to environmentally relevant levels of a heat spike and the pesticide chlorpyrifos (0.5 μg/L) shapes grand-offspring responses to these stressors. Within generations, sequential exposure to a heat spike and chlorpyrifos produced strong synergistic effects, resulting in pronounced reductions in body size, growth, swimming performance, dry mass, delayed maturation, and overall energy budget. In contrast, parental exposure to chlorpyrifos elicited adaptive transgenerational effects that reduced grand-offspring sensitivity to chlorpyrifos and, notably, mitigated the synergistic interaction between heat and chlorpyrifos in the grand-offspring generation. Both the within-generation synergisms and their transgenerational mitigation were associated with shifts in net energy budget, identifying bioenergetic reprogramming as a key mechanistic basis of adaptive transgenerational responses. Our findings demonstrate that transgenerational effects can fundamentally modify the nature of interactions between major anthropogenic stressors, and further highlight bioenergetics as a predictive framework for understanding such context-dependent shifts. Neglecting the transgenerational dimension and its energetic basis may therefore lead to systematic overestimation of the vulnerability of previously exposed populations under ongoing climate warming.