Toxicogenomic and structural determinants of cytochrome P450-mediated insecticide detoxification and selectivity in bee pollinators

Bee pollinators provide essential ecosystem services but face multiple threats, including exposure to insecticides. Current pesticide risk assessments rely on empirical toxicity testing of a limited number of bee species (primarily Apis mellifera), which inadequately predict sensitivity across more than 20,000 bee species globally. Understanding the molecular mechanisms underlying differential pesticide sensitivity in bees is essential for effective pollinator protection. Cytochrome P450 monooxygenases (P450s), particularly the CYP9Q subfamily in honey bees (A. mellifera), have emerged as key determinants of insecticide selectivity through their capacity to metabolize xenobiotics, with recent phylogenomic analyses demonstrating conservation of this detoxification capacity across major bee families. However, the conservation of these detoxification mechanisms across bee species, the molecular basis of substrate specificity, and the complexity of P450-mediated metabolism remain incompletely understood. This cumulative thesis applied toxicogenomics approaches to characterize the functional properties of bee P450 enzymes and their role in differential pesticide sensitivity. Chapter 2 investigated the conservation of flupyradifurone (FPF) metabolism across eight bee species, including stingless bees (Tribe: Meliponini). Phylogenetic analysis identified functional orthologs of the honey bee CYP6AQ1 in all examined species, showing greater than 61% sequence identity. Heterologous expression in insect cells revealed that all orthologs exhibited similar coumarin substrate profiles and metabolized FPF via hydroxylation, demonstrating conserved detoxification capacity across phylogenetically diverse bee taxa. Chapter 3 elucidated the molecular mechanisms underlying coumaphos tolerance in western honey bees (A. mellifera). Synergist bioassays and biochemical characterization revealed that CYP9Q2 plays a pivotal role in coumaphos detoxification through rapid hydroxylation of the active metabolite coumaphos-oxon. Importantly, the study demonstrated suicide substrate properties of coumaphos, whereby desulfuration during P450-mediated activation leads to enzyme inactivation, revealing a complex balance between metabolic activation and detoxification. Transgenic Drosophila ectopically expressing CYP9Q2 and CYP9Q3 exhibited significantly increased tolerance to both coumaphos and coumaphos-oxon, functionally validating the role of these P450 enzymes in coumaphos selectivity. Chapter 4 employed site-directed mutagenesis to identify structural determinants of CYP9Q3 substrate specificity. Subfamily-discriminating position analysis and structural modeling identified nine residues differentiating CYP9Q-type P450s from other CYP3 clan members. Alanine-scanning mutagenesis across substrate recognition site 4 (SRS4) revealed that the FD dipeptide (Phe308-Asp309) is essential for CYP9Q3 catalytic activity. Reciprocal indel experiments with Megachile rotundata CYP9DMs, which lack this dipeptide, confirmed that while FD deletion abolished CYP9Q3 activity, FD insertion alone was insufficient to confer CYP9Q-like detoxification capacity to CYP9DM1, indicating that multiple structural features collectively determine enzyme function. This thesis advances the molecular understanding of insecticide metabolism in bees and demonstrates the role of P450 enzymes in mediating species-specific sensitivity. The findings provide a mechanistic basis for predicting pesticide-bee interactions, inform risk assessment strategies, and support the rational development of bee-selective insecticides that leverage or circumvent specific metabolic pathways.