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Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation is associated with pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing a putative retro-transposon in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98% in 2021 in Cameroon. A strong association was established between this SV and pyrethroid resistance in field populations and is reducing pyrethroid-only nets' efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the expression of CYP6P9a/b but not CYP6P5. Within this structural variant (SV), we identified putative binding sites for transcription factors associated with the regulation of detoxification genes. An inverse correlation was observed between the 4.3kb SV and malaria parasite infection, indicating that mosquitoes lacking the 4.3kb SV were more frequently infected compared to those possessing it. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance. Author summary: Anopheles funestus is an important malaria transmitting mosquito whose control relies on pyrethroid insecticides. The ability to control malaria transmitting mosquitoes is threatened by the emergence of insecticide resistance. Developing effective strategies to prevent, slow, or overcome resistance require an understanding of the underlying genetic mechanisms. In this study, we identified a transposon containing insertion in Anopheles funestus mosquitoes in Central/East Africa but absent in other regions. Tracking its distribution and frequency over the years revealed a strong selection with frequencies reaching fixation in less than 3 years. Phenotype/genotype association studies revealed a strong correlation between pyrethroid resistance and genetic variant genotype but negative correlation with plasmodium parasite infection in mosquitoes. These findings shed light on the often-overlooked role of structural variations in the evolution of insecticide resistance, offering valuable insights and tools for monitoring and addressing this critical issue in malaria control. [ABSTRACT FROM AUTHOR]

Assessing the genetic diversity of metabolic resistance genes, such as cytochrome P450s, helps to understand the dynamics and evolution of resistance in the field. Here, we analyzed the polymorphisms of CYP6M2 and CYP6P4, associated with pyrethroid resistance in Anopheles coluzzii and Anopheles gambiae, to detect potential resistance markers. Field-caught resistant mosquitos and susceptible lab strains were crossed, and F4 was exposed to permethrin for 15 min and 90 min to discriminate highly susceptible (HS) and highly resistant (HR) mosquitos, respectively. Significant permethrin mortality reduction was observed after pre-exposure to PBO, suggesting the gene involvement of P450s. qPCR analysis revealed significant overexpression of CYP6M2 (FC = 19.57 [95% CI 13.96–25.18] for An. coluzzii; 10.16 [7.86–12.46] for An. gambiae) and CYP6P4 (FC = 6.73 [6.15–7.30] An. coluzzii; 23.62 [26.48–20.76] An. gambiae). Full-gene and ≈1 kb upstream were sequenced. For CYP6M2, the upstream region shows low diversity in HR and HS (overall Hd = 0.49, π = 0.018), whereas the full-gene shows allelic-variation but without evidence of ongoing selection. CYP6P4 upstream region showed a lower diversity in HR (Hd = 0.48) than HS (Hd = 0.86) of An. gambiae. These results highlighted that CYP6P4-associated resistance is potentially driven by modification in upstream region. However, further work is needed to determine the real causative variants that will help design rapid detection tools. [ABSTRACT FROM AUTHOR]

Resistance to pyrethroids (the ingredients in bed net insecticides) in the major malaria vector Anopheles funestus is threatening recent gains in the fight against malaria. Here, we established the role of an over-expressed P450, A. funestus CYP6AA1 in insecticides resistance. Transcription profiling of CYP6AA1 across Africa using microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that it is significantly more over-expressed in southern African populations compared to West (Benin) and East African (Uganda). Heterologous expression in Escherichia coli coupled with metabolism assays demonstrated that CYP6AA1 metabolises type I (permethrin) and type II (deltamethrin) pyrethroids, as well as bendiocarb (a carbamate). Transgenic Drosophila melanogaster flies over-expressing CYP6AA1 were significantly more resistant to pyrethroid insecticides, permethrin and deltamethrin compared with control flies not expressing the gene, validating the role of this gene in pyrethroid resistance. In silico modelling and docking simulations predicted the intermolecular receptor-ligand interactions which allow this P450 to metabolise the pyrethroids and bendiocarb. Validation of CYP6AA1 as a pyrethroid resistance gene makes it possible to monitor the spread of resistance in the field where this P450 is over-expressed. Its potential cross-resistance role makes it necessary to monitor the gene closely to inform control programs on molecular basis of multiple resistance in the field. [ABSTRACT FROM AUTHOR]

Insecticide-based interventions, notably long-lasting insecticidal nets, against mosquito vectors of malaria are currently threatened by pyrethroid resistance. Here, we contrasted RNAseq-based gene expression profiling of laboratory-resistant (FUMOZ) and susceptible (FANG) strains of the major malaria vector Anopheles funestus. Cytochrome P450 genes were the predominant over-expressed detoxification genes in FUMOZ, with high expression of the duplicated CYP6P9a (fold-change of 82.23 vs FANG) and CYP6P9b (FC 11.15). Other overexpressed P450s belonged to the same cluster of P450s corresponding to the resistance to pyrethroid 1 (rp1) quantitative trait loci (QTL) on chromosome 2R. Several Epsilon class glutathione S-transferases were also over-expressed in FUMOZ, as was the ATP-binding cassette transporter AFUN019220 (ABCA) which also exhibited between-strain alternative splicing events at exon 7. Significant differences in single-nucleotide polymorphism frequencies between strains occurred in resistance QTLs rp1 (CYP6P9a/b and CYP6AA1), rp2 on chromosome 2L (CYP6Z1, CYP6M7, and CYP6Z3), and rp3 on chromosome 3R (CYP9J5, CYP9J4, and CYP9J3). Differences were also detected in CYP4G17 and CYP4G16 genes on the X chromosome, both of which are associated with cuticular resistance in Anopheles gambiae. A close analysis of nonsynonymous diversity at the CYP6P9a/b loci revealed a drastic loss of diversity in FUMOZ with only a single polymorphism and 2 haplotypes vs 18 substitutions and 8 haplotypes in FANG. By contrast, a lowly expressed cytochrome P450 (CYP4C36) did not exhibit diversity differences between strains. We also detected the known pyrethroid resistance conferring amino acid change N384S in CYP6P9b. This study further elucidates the molecular bases of resistance in An. funestus, informing strategies to better manage widespread resistance across Africa. [ABSTRACT FROM AUTHOR]

Metabolic mechanisms conferring pyrethroid resistance in malaria vectors are jeopardizing the effectiveness of insecticide-based interventions, and identification of their markers is a key requirement for robust resistance management. Here, using a field-lab-field approach, we demonstrated that a single mutation G454A in the P450 CYP9K1 is driving pyrethroid resistance in the major malaria vector Anopheles funestus in East and Central Africa. Drastic reduction in CYP9K1 diversity was observed in Ugandan samples collected in 2014, with selection of a predominant haplotype (G454A mutation at 90%), which was completely absent in the other African regions. However, six years later (2020) the Ugandan 454A-CYP9K1 haplotype was found predominant in Cameroon (84.6%), but absent in Malawi (Southern Africa) and Ghana (West Africa). Comparative in vitro heterologous expression and metabolism assays revealed that the mutant 454A-CYP9K1 (R) allele significantly metabolises more type II pyrethroid (deltamethrin) compared with the wild G454-CYP9K1 (S) allele. Transgenic Drosophila melanogaster flies expressing 454A-CYP9K1 (R) allele exhibited significantly higher type I and II pyrethroids resistance compared to flies expressing the wild G454-CYP9K1 (S) allele. Furthermore, laboratory testing and field experimental hut trials in Cameroon demonstrated that mosquitoes harbouring the resistant 454A-CYP9K1 allele significantly survived to pyrethroids exposure (Odds ratio = 567, p < 0.0001). This study highlights the rapid spread of pyrethroid resistant CYP9K1 allele, under directional selection in East and Central Africa, contributing to reduced bed net efficacy. The newly designed DNA-based assay here will add to the toolbox of resistance monitoring and improving its management strategies., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Pyrethroid resistance in malaria vector, An. funestus is increasingly reported across Africa, threatening the sustainability of pyrethroid-based control interventions, including long lasting insecticidal nets (LLINs). Managing this problem requires understanding of the molecular basis of the resistance from different regions of the continent, to establish whether it is being driven by a single or independent selective events. Here, using a genome-wide transcription profiling of pyrethroid resistant populations from southern (Malawi), East (Uganda), and West Africa (Benin), we investigated the molecular basis of resistance, revealing strong differences between the different African regions. The duplicated cytochrome P450 genes ( CYP6P9a and CYP6P9b ) which were highly overexpressed in southern Africa are not the most upregulated in other regions, where other genes are more overexpressed, including GSTe2 in West (Benin) and CYP9K1 in East (Uganda). The lack of directional selection on both CYP6P9a and CYP6P9b in Uganda in contrast to southern Africa further supports the limited role of these genes outside southern Africa. However, other genes such as the P450 CYP9J11 are commonly overexpressed in all countries across Africa. Here, CYP9J11 is functionally characterized and shown to confer resistance to pyrethroids and moderate cross-resistance to carbamates (bendiocarb). The consistent overexpression of GSTe2 in Benin is coupled with a role of allelic variation at this gene as GAL4-UAS transgenic expression in Drosophila flies showed that the resistant 119F allele is highly efficient in conferring both DDT and permethrin resistance than the L119. The heterogeneity in the molecular basis of resistance and cross-resistance to insecticides in An. funestus populations throughout sub-Saharan African should be taken into account in designing resistance management strategies., (Copyright © 2017 Riveron et al.)