Combined exposure to mitochondrial-targeting fungicide formulations and Nosema ceranae affects survival and midgut transcriptomic responses in the honeybee Apis mellifera

Honeybees are chronically exposed to complex mixtures of chemical and biological stressors in agricultural environments, yet the combined effects of fungicide formulations and pathogens remain insufficiently understood. Fungicides targeting mitochondrial respiration and the widespread microsporidian parasite Nosema ceranae are of particular concern, as both directly interfere with host energy metabolism. In this study, we investigated the effects of chronic oral exposure to three commercial fungicide formulations, Luna Privilege® (fluopyram), Flint® (trifloxystrobin), and Luna Sensation® (fluopyram + trifloxystrobin), administered at environmentally relevant concentrations, alone or in combination with N. ceranae infection, in newly emerged Apis mellifera workers under controlled laboratory conditions. We assessed survival, lipid reserves, parasite load, and midgut transcriptomic responses. While fungicide exposure alone had limited effects on uninfected bees, N. ceranae infection markedly increased sensitivity to all formulations, resulting in significantly elevated mortality under co-exposure conditions. Transcriptomic analyses revealed formulation-dependent patterns of gene deregulation, with the strongest effects observed for Luna Sensation®, including downregulation of genes involved in energy metabolism, mitochondrial function, cofactor metabolism, and stress responses. Flint® co-exposure with N. ceranae was associated with deregulation of distinct mitochondrial gene subsets, whereas Luna Privilege® induced more limited transcriptional changes. Co-exposure was associated with reduced parasite loads, suggesting that host mitochondrial disruption may indirectly affect N. ceranae development. Infection strongly depleted lipid reserves, with no additional effect of fungicides. Overall, these findings indicate that co-exposure to fungicide formulations and N. ceranae can increase honeybee mortality and metabolic disruption, highlighting the need to integrate multi-stressor and formulation-based approaches into pollinator risk assessment.