Role of the expanded Bombus terrestris CYP6AQ cytochrome P450s subfamily in thiacloprid metabolism and their interaction with SBI fungicides

Bumblebees are important pollinators widely used to meet crop pollination demands worldwide. In such cropping systems, the use of different active ingredients for pest control may result in pollinator exposure to a variety of pesticide mixtures. Traditional risk assessment has focused on single-pesticide toxicity to honeybees. However, it is now known that responses vary among bee species, and that interactions between pesticide mixtures can occur, highlighting the need to identify pesticide combinations that are safer for bees. To this end, we evaluated the acute toxicity of three pesticide mixtures -consisting on the neonicotinoid thiacloprid in combination with three different SBI fungicides- on Bombus terrestris bumblebees. Subsequently, their transcriptomic response to dietary exposure to thiacloprid and tebuconazole both individual and in combination was evaluated. Our experiments revealed synergistic toxic effects in bumblebee mortality, as well as on their gene expression profiles. Particularly, we found that the expanded CYP6AQ subfamily of P450 detoxification enzymes changed its expression in response to fungicide treatment. Moreover, recombinant expression of the members of this enzyme subfamily revealed that at least three of them interact in vitro with thiacloprid and that their activity was differentially inhibited depending on the SBI fungicide tested. Finally, the metabolism of thiacloprid by recombinantly expressed P450s was assessed, revealing that CYP6AQ26 generates 4-hydroxy-thiacloprid and six additional tentative thiacloprid metabolites, while CYP6AQ27 and CYP6AQ28 also contribute to its metabolism, but the specific mechanisms remain unresolved. Our findings provide evidence of the different molecular interactions and regulatory responses underlying multiple-pesticide exposure in bees.