Nicotinic acetylcholine receptors (nAChRs) are a conserved gene family that facilitates synaptic signalling through the conversion of a chemical signal (acetylcholine) to an electrical signal (cation influx). The functional nAChR is a pentamer of subunits, with the many possible combinations as yet unknown and uncharacterised. Additionally, while mutations in several insect nAChR subunits have been associated with resistance, a systematic analysis of the gene family to identify all of the receptor subunits that respond to insecticides has not been performed. Taking advantage of CRISPR/CAS9 gene editing techniques available in Drosophila melanogaster, we systematically examined each of the nAChR subunit genes to identify those targeted by several insecticide classes. Our in vivo approach avoids some of the issues associated with heterologous expression of the receptor subunits. Testing compounds from three insecticide classes, the neonicotinoids, sulfoximines and spinosyns, we identified 5 nAChR subunits targeted by at least one of these different insecticide classes. We have also identified two nAChR subunits without which, the flies cannot survive. Interestingly, we also see evidence of compensatory changes in nAChR expression in deletion mutants for individual subunits. One of the insecticide classes, the spinosyns, specifically target the α6-like invertebrate receptor. There have been reports of mutations in α6 leading to &gt;1000 fold resistance from multiple insect orders. Although the major target appears clear, the binding and impact of the insecticide is not. Utilising both a Dα6 native antibody and transgenic flies expressing a Dα6:YFP fusion protein we were able to examine and visualise the spinosynA/Dα6 interaction under both acute, lethal doses and chronic, sub-lethal doses. We also identified a compound that almost completely blocks this effect on the receptor and reduces the response to spinosad. The findings from our research help to define the receptor subunits involved in binding of insecticides and developing in vivo models has enhanced our understanding of the molecular impacts of the spinosyns on their receptor target. Our results also highlight how the sophisticated genetic tools available in model organisms can provide insights into the mode of action of insecticides.

Genetic analysis of nicotinic acetylcholine receptors and their interactions with insecticides

2019 Fall National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.