Inhibiting The Proteasome Reduces Molecular And Biological Impacts Of The Natural Product Insecticide, Spinosad

BACKGROUND Insecticide targets are often identified by mutations that confer resistance, but the intricacies of insecticide binding and downstream processes leading to insect death often remain obscure. Mutations in alpha 6-like nicotinic acetylcholine receptor subunit genes have been associated with high levels of resistance to spinosad in many insect species, including Drosophila melanogaster. Here, we aimed to expand our understanding of the effects of the natural product insecticide spinosad on its protein target, the alpha 6 subunit, using genetic tools available in D. melanogaster.RESULTS Functional, fluorescently tagged D alpha 6 subunits (D alpha 6(YFP)) were developed to allow observation of the protein in vivo. Larvae expressing D alpha 6(YFP) were exposed to a sub-lethal concentration of spinosyn A (0.025 ppm) for 6 days, leading to a 64% reduction in fluorescence relative to unexposed larvae. Direct application of high doses of spinosyn A to dissected larval brains resulted in a visible 38.25% decrease in D alpha 6(YFP) within 20 min, indicating that degradation of the D alpha 6 protein occurred in response to spinosyn A exposure. Chemical inhibition of the proteasome system using the multiple myeloma treatment drug, PS-341 reduced loss of D alpha 6(YFP) in response to spinosyn A at the 20-min time point to 6.35%. In addition, in vivo administration of PS-341 prior to spinosad exposure reduced the effect of spinosad on larval activity.CONCLUSION Based on these data, we propose that exposure to spinosad leads to degradation of the alpha 6-like target protein, a potentially novel element in the mode of action of spinosyns that may contribute to their toxicity towards insects.