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Mutations in \u03b16\u2010like nicotinic acetylcholine receptor subunit genes have been associated with high levels of resistance to spinosad in many insect species, including <jats:italic>Drosophila melanogaster<\/jats:italic>. Here, we aimed to expand our understanding of the effects of the natural product insecticide spinosad on its protein target, the \u03b16 subunit, using genetic tools available in <jats:italic>D. melanogaster<\/jats:italic>.<\/jats:p><\/jats:sec><jats:sec><jats:title>RESULTS<\/jats:title><jats:p>Functional, fluorescently tagged D\u03b16 subunits (D\u03b16<jats:sup>YFP<\/jats:sup>) were developed to allow observation of the protein <jats:italic>in vivo<\/jats:italic>. Larvae expressing D\u03b16<jats:sup>YFP<\/jats:sup> were exposed to a sub\u2010lethal concentration of spinosyn A (0.025\u2009ppm) for 6\u2009days, 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\u03b16<jats:sup>YFP<\/jats:sup> within 20\u2009min, indicating that degradation of the D\u03b16 protein occurred in response to spinosyn A exposure. Chemical inhibition of the proteasome system using the multiple myeloma treatment drug, PS\u2010341 reduced loss of D\u03b16<jats:sup>YFP<\/jats:sup> in response to spinosyn A at the 20\u2010min time point to 6.35%. In addition, <jats:italic>in vivo<\/jats:italic> administration of PS\u2010341 prior to spinosad exposure reduced the effect of spinosad on larval activity.<\/jats:p><\/jats:sec><jats:sec><jats:title>CONCLUSION<\/jats:title><jats:p>Based on these data, we propose that exposure to spinosad leads to degradation of the \u03b16\u2010like target protein, a potentially novel element in the mode of action of spinosyns that may contribute to their toxicity towards insects. \u00a9 2021 Society of Chemical Industry<\/jats:p><\/jats:sec>","DOI":"10.1002\/ps.6290","type":"journal-article","created":{"date-parts":[[2021,1,22]],"date-time":"2021-01-22T23:17:59Z","timestamp":1611357479000},"page":"3777-3786","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Inhibiting the proteasome reduces molecular and biological impacts of the natural product insecticide, 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