Phylogenomic and functional characterization of an evolutionary conserved cytochrome P450-based insecticide detoxification mechanism in bees.

The regulatory process for assessing the risks of pesticides to bees relies heavily on the use of the honeybee, Apis mellifera, as a model for other bee species. However, the validity of using A. mellifera as a surrogate for other Apis and non-Apis bees in pesticide risk assessment has been questioned. Related to this line of research, recent work on A. mellifera has shown that specific P450 enzymes belonging to the CYP9Q subfamily act as critically important determinants of insecticide sensitivity in this species by efficiently detoxifying certain insecticide chemotypes. However, the extent to which the presence of functional orthologs of these enzymes is conserved across the diversity of bees is unclear. Here we used a phylogenomic approach to identify > 100 putative CYP9Q functional orthologs across 75 bee species encompassing all major bee families. Functional analysis of 26 P450s from 20 representative bee species revealed that P450-mediated detoxification of certain systemic insecticides, including the neonicotinoid thiacloprid and the butenolide flupyradifurone, is conserved across all major bee pollinator families. However, our analyses also reveal that CYP9Q-related genes are not universal to all bee species, with some Megachilidae species lacking such genes. Thus, our results reveal an evolutionary conserved capacity to metabolize certain insecticides across all major bee families while identifying a small number of bee species where this function may have been lost. Furthermore, they illustrate the potential of a toxicogenomic approach to inform pesticide risk assessment for nonmanaged bee species by predicting the capability of bee pollinator species to break down synthetic insecticides.

A toxicogenomics approach reveals characteristics supporting the honey bee (Apis mellifera L.) safety profile of the butenolide insecticide flupyradifurone.

Flupyradifurone, a novel butenolide insecticide, selectively targets insect nicotinic acetylcholine receptors (nAChRs), comparable to structurally different insecticidal chemotypes such as neonicotinoids and sulfoximines. However, flupyradifurone was shown in acute toxicity tests to be several orders of magnitude less toxic to western honey bee (Apis mellifera L.) than many other insecticides targeting insect nAChRs. The underlying reasons for this difference in toxicity remains unknown and were investigated here. Pharmacokinetic studies after contact application of [14C]flupyradifurone to honey bees revealed slow uptake, with internalized compound degraded into a few metabolites that are all practically non-toxic to honey bees in both oral and contact bioassays. Furthermore, receptor binding studies revealed a lack of high-affinity binding of these metabolites to honey bee nAChRs. Screening of a library of 27 heterologously expressed honey bee cytochrome P450 enzymes (P450s) identified three P450s involved in the detoxification of flupyradifurone: CYP6AQ1, CYP9Q2 and CYP9Q3. Transgenic Drosophila lines ectopically expressing CYP9Q2 and CYP9Q3 were significantly less susceptible to flupyradifurone when compared to control flies, confirming the importance of these P450s for flupyradifurone metabolism in honey bees. Biochemical assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-(trifluoromethyl)-coumarin (BOMFC) indicated a weak, non-competitive inhibition of BOMFC metabolism by flupyradifurone. In contrast, the azole fungicides prochloraz and propiconazole were strong nanomolar inhibitors of these flupyradifurone metabolizing P450s, explaining their highly synergistic effects in combination with flupyradifurone as demonstrated in acute laboratory contact toxicity tests of adult bees. Interestingly, the azole fungicide prothioconazole is only slightly synergistic in combination with flupyradifurone - an observation supported by molecular P450 inhibition assays. Such molecular assays have value in the prediction of potential risks posed to bees by flupyradifurone mixture partners under applied conditions. Quantitative PCR confirmed the expression of the identified P450 genes in all honey bee life-stages, with highest expression levels observed in late larvae and adults, suggesting honey bees have the capacity to metabolize flupyradifurone across all life-stages. These findings provide a biochemical explanation for the low intrinsic toxicity of flupyradifurone to honey bees and offer a new, more holistic approach to support bee pollinator risk assessment by molecular means.

A conserved hymenopteran-specific family of cytochrome P450s protects bee pollinators from toxic nectar alkaloids.

Many plants produce chemical defense compounds as protection against antagonistic herbivores. However, how beneficial insects such as pollinators deal with the presence of these potentially toxic chemicals in nectar and pollen is poorly understood. Here, we characterize a conserved mechanism of plant secondary metabolite detoxification in the Hymenoptera, an order that contains numerous highly beneficial insects. Using phylogenetic and functional approaches, we show that the CYP336 family of cytochrome P450 enzymes detoxifies alkaloids, a group of potent natural insecticides, in honeybees and other hymenopteran species that diverged over 281 million years. We linked this function to an aspartic acid residue within the main access channel of CYP336 enzymes that is highly conserved within this P450 family. Together, these results provide detailed insights into the evolution of P450s as a key component of detoxification systems in hymenopteran species and reveal the molecular basis of adaptations arising from interactions between plants and beneficial insects.

A mechanism-based approach unveils metabolic routes potentially mediating chlorantraniliprole synergism in honey bees, Apis mellifera L., by azole fungicides.

BACKGROUND: Almond production in California is an intensively managed agroecosystem dependent on managed pollination by honey bees, Apis mellifera L. A recent laboratory study reported synergism in honey bees between chlorantraniliprole, a common diamide insecticide used in almond orchards, and the fungicide propiconazole. Indeed, there is an emerging body of evidence that honey bee cytochrome P450 monooxygenases of the CYP9Q subfamily are involved in the detoxification of insecticides across a diverse range of chemical classes. The objective of the present study was to unveil the molecular background of the described synergism and to explore the potential role of CYP9Q enzymes in diamide detoxification. RESULTS: Our study confirmed the previously reported synergistic potential of propiconazole on chlorantraniliprole in acute contact toxicity bioassays, whereas no synergism was observed for flubendiamide. Fluorescence-based biochemical assays revealed an interaction of chlorantraniliprole, but not flubendiamide, with functionally expressed CYP9Q2 and CYP9Q3. These findings were validated by an increased chlorantraniliprole tolerance of transgenic Drosophila lines expressing CYP9Q2/3, and an analytically confirmed oxidative metabolism of chlorantraniliprole by recombinantly expressed enzymes. Furthermore, we showed that several triazole fungicides used in almond orchards, including propiconazole, were strong nanomolar inhibitors of functionally expressed honey bee CYP9Q2 and CYP9Q3, whereas other fungicides such as iprodione and cyprodinil did not inhibit these enzymes. CONCLUSION: Honey bee CYP9Q enzymes are involved in chlorantraniliprole metabolism and inhibited by triazole fungicides possibly leading to synergism in acute contact toxicity bioassays. Our mechanistic approach has the potential to inform tier I honey bee pesticide risk assessment.

The leafcutter bee, Megachile rotundata, is more sensitive to N-cyanoamidine neonicotinoid and butenolide insecticides than other managed bees.

Recent research has shown that several managed bee species have specific P450 enzymes that are preadapted to confer intrinsic tolerance to some insecticides including certain neonicotinoids. However, the universality of this finding across managed bee pollinators is unclear. Here we show that the alfalfa leafcutter bee, Megachile rotundata, lacks such P450 enzymes and is >2,500-fold more sensitive to the neonicotinoid thiacloprid and 170-fold more sensitive to the butenolide insecticide flupyradifurone than other managed bee pollinators. These findings have important implications for the safe use of insecticides in crops where M. rotundata is used for pollination, and ensuring that regulatory pesticide risk assessment frameworks are protective of this species.