| dc.description.abstract | Recent research on three managed bee pollinators, namely the Western honeybee (Apis mellifera), buff-tailed bumblebee (Bombus terrestris) and red mason bee (Osmia bicornis), has demonstrated that cytochrome P450 enzymes belonging to the CYP9Q and CYP9BU lineages provide protection to certain insecticides from three different mode of action classes. The alfalfa leafcutter bee (Megachile rotundata) is the world’s most economically important solitary bee species. Nonetheless, it is unclear whether this species has a CYP9Q/BU ortholog that could afford similar levels of protection against insecticides, to those seen in other managed bee pollinators.
To address this question, the M. rotundata CYPome was curated and examined using phylogenetic and syntenic analyses. These investigations revealed that this species lacks a CYP9Q/BU ortholog or closely related P450 enzyme. Topical insecticide bioassays using M. rotundata determined that the species exhibits high sensitivity to all the compounds known to be detoxified by the CYP9Q/BU lineage. For example, M. rotundata is >2,500-fold more sensitive to thiacloprid than A. mellifera. Functional studies, using M. rotundata native microsomes, revealed no significant level of metabolism of any of the insecticides known to be detoxified by the CYP9Q/BU lineages. Radioligand competition assays, on head membrane preparations, showed no significant difference in binding affinity of neonicotinoid and butanolide insecticides at the nicotinic acetylcholine receptor. Taken together these findings indicate that the lack of a CYP9Q/BU ortholog, or closely related P450, in M. rotundata correlates with an inability of microsomal P450s to metabolise certain insecticides in vitro and a high sensitivity to these compounds in vivo.
To understand how wide spread the lack of a CYP9Q/BU ortholog might be, genomic and transcriptomic CYP9 sequences from 75 bee species were examined, using phylogenetic analyses. Five of the six bee families included in the phylogeny had genes that were closely related to, or shared a recent ancestor with CYP9Q/BU lineage (~97%). The Megachilidae showed a lower prevalence of CYP9Q/BU orthologs (50%), most notably in the two species of Megachile, where, based on phylogenetic and syntenic analyses, CYP9DM genes have evolved in place of the CYP9Q/BU lineage. Sequencing of the transcriptomes of three UK and one Canadian Megachile species further substantiated that this entire genus (~1500 species) may have CYP9DM rather than CYP9Q/BU genes. Functional expression of this P450 suggested that, in contrast to several Megachilidae CYP9BU-like enzymes, CYP9DM P450s lack the capacity to bind neonicotinoids.
From the results generated in this thesis it is clear that the use of other managed bee species as a proxy for M. rotundata in ecotoxicological testing is unreliable. This has important implications for regulatory risk assessments. The data also illustrate the utility of using phylogenetic analyses, with targeted functional studies, as a tool to predict the level of sensitivity to insecticides of a bee species. | en_GB |
