There is an on-going need to develop new insecticides that are not compromised by resistance and that have improved environmental profiles. However, the cost of developing novel compounds has increased significantly over the last two decades. This is in part due to increased regulatory requirements, including the need to screen both pest and pollinator insect species to ensure that pre-existing resistance will not hamper the efficacy of a new insecticide via cross-resistance, or adversely affect non-target insect species. To add to this problem the collection and maintenance of toxicologically relevant pest and pollinator species and strains is costly and often difficult. Here we present Fly-Tox, a panel of publicly available transgenic Drosophila melanogaster lines each containing one or more pest or pollinator P450 genes that have been previously shown to metabolise insecticides. We describe the range of ways these tools can be used, including in predictive screens to avoid pre-existing cross-resistance, to identify potential resistance-breaking inhibitors, in the initial assessment of potential insecticide toxicity to bee pollinators, and identifying harmful pesticide-pesticide interactions.

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 paraV1016G mutation displayed substantially greater resistance ratio (RR) against deltamethrin than the product of each individual mechanism (RRcombined: 19.85 > RRCyp9J28: 1.77 × RRV1016G: 3.00). Genotypes expressing Brassicogethes aeneus pollen beetle Cyp6BQ23 and also bearing the paraL1014F (kdr) mutation, displayed an almost multiplicative RR (RRcombined: 75.19 ≥ RRCyp6BQ23: 5.74 × RRL1014F: 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.

Culex pipiens mosquitoes cause severe nuisance and transmit human diseases including West Nile. Vector control by insecticides is the main tool to prevent these diseases and diflubenzuron is one of the most effective mosquito larvicides used in many places. Here, high levels of resistance were identified in Cx. pipiens from Italy, with a Resistance Ratio of 128 fold. The phenotype was associated with mutations at amino acid I1043 (I1043M and I1043L) of the Chitin synthase gene, which showed significantly higher frequency in bioassay survivors. Both mutations have been introduced in the Drosophila melanogaster chitin synthase gene using the genome editing method CRISPR/Cas9 and validated to confer significant levels of resistance, although at different levels. The I→M mutation results in a Resistance Ratio >2,900 fold and the I→L mutation >20 fold. Two PCR based diagnostics were developed for monitoring of the resistant mutations in field populations. The findings are of major concern for public health given the importance of diflubenzuron in mosquito control in many places, the intensity of the resistance phenotype and the limited availability of alternative larvicides.

Stink bugs are an emerging threat to crop security in many parts of the globe, but there are few genetic resources available to study their physiology at a molecular level. This is especially true for tissues such as the midgut, which forms the barrier between ingested material and the inside of the body.

Pyrethroid-impregnated bed nets have driven considerable reductions in malaria-associated morbidity and mortality in Africa since the beginning of the century1. The intense selection pressure exerted by bed nets has precipitated widespread and escalating resistance to pyrethroids in African Anopheles populations, threatening to reverse the gains that been made by malaria control2. Here we show that expression of a sensory appendage protein (SAP2), which is enriched in the legs, confers pyrethroid resistance to Anopheles gambiae. Expression of SAP2 is increased in insecticide-resistant populations and is further induced after the mosquito comes into contact with pyrethroids. SAP2 silencing fully restores mortality of the mosquitoes, whereas SAP2 overexpression results in increased resistance, probably owing to high-affinity binding of SAP2 to pyrethroid insecticides. Mining of genome sequence data reveals a selective sweep near the SAP2 locus in the mosquito populations of three West African countries (Cameroon, Guinea and Burkina Faso) with the observed increase in haplotype-associated single-nucleotide polymorphisms mirroring the increasing resistance of mosquitoes to pyrethroids reported in Burkina Faso. Our study identifies a previously undescribed mechanism of insecticide resistance that is likely to be highly relevant to malaria control efforts.