Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance inAnopheles gambiaes.l.

Abstract

Resistance to insecticides inAnophelesmosquitoes threatens the effectiveness of the most widespread tools currently used to control malaria. The genetic underpinnings of resistance are still only partially understood, with much of the variance in resistance phenotype left unexplained. We performed a multi-country large scale genome-wide association study of resistance to two insecticides widely used in malaria control: deltamethrin and pirimiphos-methyl. Using a bioassay methodology designed to maximise the phenotypic difference between resistant and susceptible samples, we sequenced 969 phenotyped femaleAn. gambiaeandAn. coluzziifrom ten locations across four countries in West Africa (Benin, Côte d'Ivoire, Ghana and Togo), identifying single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) segregating in the populations. The patterns of resistance association were highly multiallelic and variable between populations, with different genomic regions contributing to resistance, as well as different mutations within a given region. While the strongest and most consistent association with deltamethrin resistance came from the region aroundCyp6aa1, this resistance was based on a combination of several independent CNVs inAn. coluzzii, and on a non-CNV bearing haplotype inAn. gambiae. Further signals involved a range of cytochrome P450, mitochondrial, and immunity genes. Similarly, for pirimiphos-methyl, while the strongest signal came from the region ofAce1, more widespread signals included cytochrome P450s, glutathione S-transferases, and a subunit of the nAChR target site of neonicotinoid insecticides. The regions aroundCyp9k1and theTepfamily of immune genes were associated with resistance to both insecticide classes, suggesting possible cross-resistance mechanisms. These locally-varying, multigenic and multiallelic patterns highlight the challenges involved in genomic monitoring and surveillance of resistance, and form the basis for improvement of methods used to detect and predict resistance. Based on simulations of resistance variants, we recommend that yet larger scale studies, exceeding 500 phenotyped samples per population, are required to better identify associated genomic regions.

Widespread occurrence of copy number variants and fixation of pyrethroid target site resistance in Anopheles gambiae (s.l.) from southern Côte d’Ivoire

“…Although we found no significant difference in resistance status, the P -value was close to significance (smaller than 0.1) despite the relatively low power of the test, and the direction of difference (higher resistance for Vgsc- 402L compared to Vgsc- 995F) was the same as observed in another recent study of An. coluzzii from Benin ( Lucas et al., 2023 ). Combined with the high frequency of Vgsc- 402L which we observed compared to previous data from Côte d’Ivoire ( Clarkson et al., 2021 ), our study lends further weight to the emerging picture that Vgsc- 402L is gradually replacing Vgsc- 995F as a more highly resistant allele with lower fitness costs ( Williams et al., 2022 ; Lucas et al., 2023 ).…”

“…coluzzii from Benin ( Lucas et al., 2023 ). Combined with the high frequency of Vgsc- 402L which we observed compared to previous data from Côte d’Ivoire ( Clarkson et al., 2021 ), our study lends further weight to the emerging picture that Vgsc- 402L is gradually replacing Vgsc- 995F as a more highly resistant allele with lower fitness costs ( Williams et al., 2022 ; Lucas et al., 2023 ). Currently, the Vgsc- 402L and Vgsc- 995F alleles appear to be in perfect negative linkage disequilibrium, but it remains unknown whether a combination of the two mutations on the same haplotype would provide yet higher levels of resistance.…”

“…gambiae ( s.s. ), while several different CNV alleles together reach high frequencies in An. coluzzii ( Lucas et al., 2019a , 2023 ). Our results show that while no single CNV allele can be identified as the cause of metabolic resistance, as is the case in East Africa, increased copy number brought about by the CNVs in general provides resistance to deltamethrin, adding to the mounting body of evidence that CNVs in the C yp6aa cluster of genes provide deltamethrin resistance.…”

“…mutually exclusive) with Vgsc- 995F ( Vgsc- 402L). These additional Vgsc SNPs have received limited attention so far ( Clarkson et al., 2021 ; Williams et al., 2022 ; Lucas et al., 2023 ). In Ace1 , the SNP Ace1 -280S (previously known as Ace1- 119S) and a copy number variant (CNV) that increases the number of copies of Ace1 in the genome, combine to provide resistance to organophosphates ( Weetman et al., 2015 ; Grau-Bové et al., 2021 ).…”

The role of P450 enzymes in malaria and other vector‐borne infectious diseases

“…66 Allelic variants or point mutations in insect P450 could be another mechanism of insect auto-protection against insecticides. Some studies were done in Anopheles mosquitos, [67][68][69][70] but much more data are available for Drosophila's CYPs. 56,62 Mathematical models and available 3D structures were applied for the study of allelic variation relative to catalytic regions of P450s in different insects, 71 nonetheless further studies with focus on disease vectors are needed.…”