Partitioning the roles of CYP6G1 and gut microbes in the metabolism of the insecticide imidacloprid in Drosophila melanogaster

Resistance to insecticides through enhanced metabolism is a worldwide problem. The Cyp6g1 gene of the vinegar fly, Drosophila melanogaster, is a paradigm for the study of metabolic resistance. Constitutive overexpression of this gene confers resistance to several classes of insecticides, including the neonicotinoid imidacloprid (IMI). The metabolism of IMI in this species has been previously shown to yield oxidative and nitro-reduced metabolites. While levels of the oxidative metabolites are correlated with CYP6G1 expression, nitro-reduced metabolites are not, raising the question of how these metabolites are produced. Some IMI metabolites are known to be toxic, making their fate within the insect a second question of interest. These questions have been addressed by coupling the genetic tools of gene overexpression and CRISPR gene knock-out with the mass spectrometric technique, the Twin-Ion Method (TIM). Analysing axenic larvae indicated that microbes living within D. melanogaster are largely responsible for the production of the nitro-reduced metabolites. Knock-out of Cyp6g1 revealed functional redundancy, with some metabolites produced by CYP6G1 still detected. IMI metabolism was shown to produce toxic products that are not further metabolized but readily excreted, even when produced in the Central Nervous System (CNS), highlighting the significance of transport and excretion in metabolic resistance

Multiple P450s and Variation in Neuronal Genes Underpins the Response to the Insecticide Imidacloprid in a Population of Drosophila melanogaster

Insecticide resistance is an economically important example of evolution in response to intense selection pressure. Here, the genetics of resistance to the neonicotinoid insecticide imidacloprid is explored using the Drosophila Genetic Reference Panel, a collection of inbred Drosophila melanogaster genotypes derived from a single population in North Carolina. Imidacloprid resistance varied substantially among genotypes, and more resistant genotypes tended to show increased capacity to metabolize and excrete imidacloprid. Variation in resistance level was then associated with genomic and transcriptomic variation, implicating several candidate genes involved in central nervous system function and the cytochrome P450s Cyp6g1 and Cyp6g2. CRISPR-Cas9 mediated removal of Cyp6g1 suggested that it contributed to imidacloprid resistance only in backgrounds where it was already highly expressed. Cyp6g2, previously implicated in juvenile hormone synthesis via expression in the ring gland, was shown to be expressed in metabolically relevant tissues of resistant genotypes. Cyp6g2 overexpression was shown to both metabolize imidacloprid and confer resistance. These data collectively suggest that imidacloprid resistance is influenced by a variety of previously known and unknown genetic factors