Limitations of RNAi of a6 nicotinic acetylcholine receptor subunits for assessing the in vivo sensitivity to spinosad

Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor. The a6 nicotinic acetylcholine receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking a6. RNAi was performed against the Da6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Gal4-UAS system to examine if RNAi would yield results similar to those of Da6 null mutants. These Da6-deficient flies were subject to spinosad contact bioassays to evaluate the role of the Da6 nicotinic acetylcholine receptor subunit on spinosad sensitivity. The expression of Da6 was reduced 60%75% as verified by quantitative polymerase chain reaction. However, there was no change in spinosad sensitivity in D. melanogaster. We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasa6. RNAi of Tcasa6 did not result in changes in spinosad sensitivity, similar to results obtained with D. melanogaster. The lack of change in spinosad sensitivity in both D. melanogaster and T. castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the a6 subunit in both species. Potential causes for the lack of change in spinosad sensitivity are discussed.