Understanding the molecular and biochemical basis of insecticide selectivity against solitary bee pollinators

Certain eusocial bee pollinators have been found to exhibit profound differences in their sensitivity to different chemical insecticides within the same class (e.g. the N-nitroguanidine neonicotinoid imidacloprid and the N-cyanoamidine thiacloprid). Recent work on honey bees and bumblebees has shown that this variation in sensitivity is due, at least in part, to differences in the capacity of cytochromes P450 belonging to the CYP9Q subfamily to detoxify different insecticides. The solitary red mason bee, Osmia bicornis, is the most economically important solitary pollinator in Europe, yet its sensitivity to insecticides is not well characterised. Topical insecticide bioassays revealed that like honey bees and bumblebees, O. bicornis exhibits significant differences in sensitivity to insecticides within the same class, demonstrated by a >2,000-fold difference in sensitivity to imidacloprid and thiacloprid. Radioligand competition assays revealed no significant differences in the binding affinity of imidacloprid and thiacloprid to nicotinic acetylcholine receptors (nAChRs) isolated from head membrane preparations, demonstrating that differences in the binding affinity of imidacloprid and thiacloprid for nAChRs does not explain the marked variation in bee sensitivity to these compounds. Furthermore, cuticular penetration assays revealed no difference in the rate of penetration of these insecticides. The differential sensitivity observed during toxicity bioassays was found to be greatly suppressed on addition of the P450 inhibitor, PBO, prior to thiacloprid application but not imidacloprid application, suggesting that P450s play a role in determining the sensitivity of O. bicornis to neonicotinoid insecticides. Sequencing of the transcriptome and genome of O. bicornis along with the annotation of other bee genomes revealed that O. bicornis and most other solitary bee species lack the cyp9q subfamily. Subsequently, the most closely related O. bicornis P450s to this subfamily were selected as potential thiacloprid-detoxifying orthologs for further characterisation. Kinetic studies revealed that six of the recombinantly expressed P450s were able to metabolise both imidacloprid and thiacloprid. However, all of the catalytically active P450s displayed a greater affinity for thiacloprid compared to imidacloprid, which could enable its rapid metabolism before any detrimental effects can occur, explaining, at least in part, its comparatively low toxicity. The most effective neonicotinoid metaboliser was found to be CYP9BU1, which also has the capacity to metabolise a number of other insecticides, suggesting that it may be a key detoxification enzyme of O. bicornis. O. bicornis microsomes displayed substantial ability to metabolise the secondary plant metabolite nicotine and incubation of recombinant P450s with nicotine identified CYP6AQ55 as a major nicotine metabolising enzyme. Taken together these findings illustrate that the CYPome of O. bicornis contains P450s that can metabolise both natural and synthetic insecticides. Insecticide-metabolising P450s were found to be highly expressed in the Malpighian tubules, a primary site of xenobiotic detoxification, and the brain of O. bicornis, which contains high concentrations of nAChRs. Exposure of O. bicornis to sublethal doses of either imidacloprid or thiacloprid did not induce the expression of any P450s, suggesting constitutive expression of insecticide-metabolising P450s provides protective effects. The knowledge generated in this thesis can be leveraged to help avoid negative insecticide impacts on this important solitary bee pollinator and provide tools to aid the design of bee-safe insecticides