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.

Voltage-gated sodium channels are the target of several insecticides including DTT, pyrethroids, and SCBIs like indoxacarb and metaflumizone. SCBIs are an alternative insecticide resistance management (IRM) strategy against several pests resistant to other compounds. However, resistance to SCBIs has been reported in several pests, in most cases implicating metabolic resistance mechanisms, although in certain indoxacarb resistant populations of Plutella xylostella and Tuta absoluta, two mutations in the domain IV S6 segment of the voltage-gated sodium channel, F1845Y and V1848I have been identified, and have been postulated through in vitro electrophysiological studies to contribute to target-site resistance.In order to functionally validate in vivo each mutation in the absence of confounding resistance mechanisms, we have employed a CRISPR/Cas9 strategy to generate strains bearing homozygous F1845Y or V1848I mutations in the para (voltage-gated sodium channel) gene of Drosophila melanogaster. We performed toxicity bioassays of these strains compared to wild-type controls of the same genetic background. Our results indicate both mutations confer moderate resistance to indoxacarb (RR: 6 -10.2), and V1848I to metaflumizone (RR: 8.4). However, F1845Y confers very strong resistance to metaflumizone (RR: >3400), a finding that may be related to the specific binding of each insecticide to its target.

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