Dissecting the Insect Metabolic Machinery Using Twin Ion Mass Spectrometry: A Single P450 Enzyme Metabolizing the Insecticide Imidacloprid <i>in Vivo</i>

Insecticide resistance is one of the most prevalent examples of anthropogenic genetic change, yet our understanding of metabolic-based resistance remains limited by the analytical challenges associated with rapidly tracking the <i>in vivo</i> metabolites of insecticides at nonlethal doses. Here, using twin ion mass spectrometry analysis of the extracts of whole <i>Drosophila larvae</i> and excreta, we show that (i) eight metabolites of the neonicotinoid insecticide, imidacloprid, can be detected when formed by susceptible larval genotypes and (ii) the specific overtranscription of a single gene product, <i>Cyp6g1</i>, associated with the metabolic resistance to neonicotinoids, results in a significant increase in the formation of three imidacloprid metabolites that are formed in C–H bond activation reactions; that is, <i>Cyp6g1</i> is directly linked to the enhanced metabolism of imidacloprid <i>in vivo</i>. These results establish a rapid and sensitive method for dissecting the metabolic machinery of insects by directly linking single gene products to insecticide metabolism.