The Characterisation Of Bee Nicotinic Acetylcholine Receptors

The effects of insecticides such as neonicotinoids on beneficial insect species such as pollinators has become a matter of considerable interest. Localised bee colony losses and losses of wild bee numbers are believed to be related to the use of these insecticides, resulting in a European ban on three commercially important compounds. As it stands, the model insect Apis mellifera is used for most toxicity testing in relation to insecticide efficacy on beneficiary species, but we are learning that a species-specific approach may be far more informative and conducive to understanding insecticide toxicity and beneficiary species preservation as several studies have found that different species of bees have differential sensitivity to neonicotinoids. The target receptor of several categories of commercially important insecticides are the nicotinic acetylcholine receptors. In this study we clone the majority of the Apis mellifera nAChR gene family and putative nAChR chaperones in an attempt to functionally characterise the pharmacological effects of these insecticides. Through the use of the Drosophila melanogaster α1 chimera + Gallus gallus β2 nAChR expressed in the Xenopus oocyte expression system and two-electrode voltage-clamping we show that species-specific target site variation in the α1 subunit between three species of bees (Apis mellifera, Bombus terrestris and Bombus impatiens) causes a significant change in the efficacious properties of clothianidin, without effecting the pharmacology of the endogenous agonist acetylcholine. In addition species-specific variation in the α1 subunits tested did not affect the antagonist actions of imidacloprid and thiacloprid. By creating a homology model of this hybrid receptor we show that this polymorphism is out of range of the orthosteric site, suggesting an allosteric mechanism. These findings highlight that we should not just reply on A. mellifera when assessing the effects of insecticides on different bee species and that variation in nAChRs may differentially impact on the actions of neonicotinoids. Utilising a molecular chaperone, NACHO, we have for the first time expressed an insect homomeric α6 subunit in the Xenopus oocyte expression system. We demonstrate that this construct possesses an α-bungarotoxin binding site, and is sensitive to the insecticide spinosad. This novel approach of using the NACHO chaperone to express homomeric insect α6 subunits will allow for the pharmacology of the receptor and its splice and edit variances to be explored in further depths, also expression of α6 will allow for the study of actions of compounds on this receptor and potentially provide a valuable tool for the counter-screening of novel insecticides