Resistance to insecticides by modification of their molecular targets is a serious problem in the chemical control of many arthropod pests. Neonicotinoids target the nicotinic acetylcholine receptor (nAChR) of arthropods for which the spectrum of possible resistance-conferring mutations is poorly understood. Prediction of these is complicated by the existence of ten nAChR subunit genes, some of which are capable of a generating a number of splice isoforms.

Guided by an initial resistance screen on deficiencies of different nAChR subunits, we focused on the cluster of three Drosophila melanogaster nAChR subunit genes at cytological region 96A. EMS mutagenesis and selection for resistance to nitenpyram in hybrids carrying a deficiency for this chromosomal region identified four recessively resistant mutants. Complementation analysis identified two groups, and mutations were found in two different nAChR subunit genes, Dα1 (encoding an α-type subunit) and Dβ2 (β-type).

Mutations conferring resistance in β-type receptors have not previously been reported and we found a variety of different lesions in the predicted Dβ2 protein, including locations distant from the residues predicted to be involved in the insecticide binding site. This work clearly demonstrates that mutations in a single receptor subunit can confer neonicotinoid resistance which was not clear in previous studies.

Interestingly, mutations were found that may protect the insect against nitenpyram by interfering with subunit assembly or channel activation, rather than blocking binding of insecticide to the channel and also two nonsense mutations were found which would result in non-functional subunits. This has parallels to studies on spinosad resistance, where the knockout of nAChR subunit Dα6, confers high levels of resistance. The ability of an insect to survive without particular subunits and the redundancy of the insect nervous system are issues worth considering in future insecticide design.