Nitenpyram resistance in Drosophila melanogaster

© 2012 Dr. Judith MitchellThe neonicotinoid insecticides are effective in controlling a variety of insect pests in the field. They target the nicotinic acetylcholine receptor, a cation channel in the central nervous system. After years of usage, high levels of resistance to these insecticides are beginning to emerge. This requires greater understanding of the activity of these insecticides and the function of these channels. The bulk of this study made use of the model organism, Drosophila melanogaster, and the well-defined family of ten nicotinic acetylcholine receptor subunits encoded by its genome. Mutations and null alleles of insect nicotinic acetylcholine receptors have been described but, thus far, all of these are homozygous viable. This is surprising given the high level of conservation of these genes. Part of this study examines viability with loss of function of individual subunits through the use of stably integrated RNAi constructs. Ubiquitous knockdown of any one gene had a significant effect on mortality prior to the adult stage. Knockdown of Dα1, Dα5 or Dβ2 produced the most severe increases in mortality. However, genomic deletions of either Dα1 or Dβ2 were seen to be viable. Deletion of Dα5 resulted in a completely penetrant larval lethality phenotype with all the hallmarks of a possible role in hormonal control of development. Further studies of knockdown flies highlighted possible roles in mating for Dα1, Dα2, Dα6 and Dβ1. RNAi knockdown of individual receptors was also used to investigate the possible involvement of each subunit in mortality induced by the neonicotinoids. RNAi knockdown larvae were exposed to nitenpyram and survival to adulthood scored. Resistance was seen for knockdown of known nitenpyram targets Dα1 and Dβ2, confirming this approach provides enough sensitivity to reveal nitenpyram targets. Resistance was also seen upon knockdown of either Dα3 or Dβ1. In addition to this, knockdown of either Dα6 or Dβ3 produced hyper-sensitivity to nitenpyram. Results from this study are juxtaposed to published associations of nicotinic acetylcholine receptor subunit resistance alleles to propose likely components of the neonicotinoid binding sites. The neonicotinoid insecticides are known to bind at the acetylcholine binding site, which occurs at a cleft formed between two subunits in the mature receptor. The information gained from published work and the RNAi experiments using nitenpyram suggested two likely interfaces that could make up the insecticide binding site. Homology modeling based on the structure of the Aplysia californica acetylcholine binding protein was used to generate three dimensional structures of the two likely interfaces. Binding of several neonicotinoids was simulated and produced a similar binding orientation in a Dα1/Dβ2 interface. Comparison of imidacloprid binding to acetylcholine binding and imidacloprid resistance mutations strongly supports the model presented here. The final part of this study is the genetic mapping of a novel resistance mechanism to nitenpyram isolated in a field collected strain. A previously identified strain of D. melanogaster collected at Cape Tribulation, Australia, had been shown to carry a resistance factor on the third chromosome. P-element mapping and screening of molecular markers identified a resistance locus on chromosome 3L between cytological bands 68c4 and 68d2 that contains 50 genes. The known functions and expression patterns of these genes are considered and possible resistance mechanisms are discussed