The putative synergistic action of target-site mutations and enhanced detoxification in pyrethroid resistance in insects has been hypothesized as a major evolutionary mechanism responsible for dramatic consequences in malaria incidence and crop production. Combining genetic transformation and CRISPR/Cas9 genome modification, we generated transgenic Drosophila lines expressing pyrethroid metabolizing P450 enzymes in a genetic background along with engineered mutations in the voltage-gated sodium channel ( para ) known to confer target-site resistance. Genotypes expressing the yellow fever mosquito Aedes aegypti Cyp9J28 while also bearing the para ^V1016G mutation displayed substantially greater resistance ratio (RR) against deltamethrin than the product of each individual mechanism (RR _combined : 19.85 > RR _Cyp9J28 : 1.77 × RR _V1016G : 3.00). Genotypes expressing Brassicogethes aeneus pollen beetle Cyp6BQ23 and also bearing the para ^L1014F ( kdr ) mutation, displayed an almost multiplicative RR (RR _combined : 75.19 ≥ RR _Cyp6BQ23 : 5.74 × RR _L1014F : 12.74). Reduced pyrethroid affinity at the target site, delaying saturation while simultaneously extending the duration of P450-driven detoxification, is proposed as a possible underlying mechanism. Combinations of target site and P450 resistance loci might be unfavourable in field populations in the absence of insecticide selection, as they exert some fitness disadvantage in development time and fecundity. These are major considerations from the insecticide resistance management viewpoint in both public health and agriculture.

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.

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.

Background: Caterpillars from the insect order Lepidoptera are some of the most widespread and destructive agricultural pests. Most of their impact is at the larval stage, where the midgut epithelium mediates the digestion and absorption of an astonishing amount of food. Although this tissue has been the subject of frequent investigation in Lepidoptera, a comprehensive expression atlas has yet to be generated. Results: Here, we perform RNA-sequencing and proteomics on the gut of the polyphagous pest Helicoverpa armigera across, life stages, diet types, and compartments of the anterior-posterior axis. A striking relationship between the structural homology and expression pattern of a group of sugar transporters was observed in the early larval stages. Further comparisons were made among the spatial compartments of the midgut, which suggested a putative role for vATPases and SLC9 transporters in the generation of alkaline conditions in the H. armigera midgut. Conclusions: This comprehensive resource will aid the scientific community in understanding lepidopteran gut physiology in unprecedented resolution. It is hoped that this study advances the understanding of the lepidopteran midgut and also facilitates functional work in this field.