Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of malaria control, but the genetics of resistance are only partially understood. We performed a large scale multi-country genome-wide association study of resistance to two widely used insecticides: deltamethrin and pirimiphos-methyl, using sequencing data from An. gambiae and An. coluzzii from ten locations in West Africa. Resistance was highly multi-genic, multi-allelic and variable between populations. While the strongest and most consistent association with deltamethrin resistance came from Cyp6aa1, this was based on several independent copy number variants (CNVs) in An. coluzzii, and on a non-CNV haplotype in An. gambiae. For pirimiphos-methyl, signals included Ace1, cytochrome P450s, glutathione S-transferases and the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes showed evidence of cross-resistance to both insecticides. These locally-varying, multi-allelic patterns highlight the challenges involved in genomic monitoring of resistance, and may form the basis for improved surveillance methods.

Big Data Institute Li Ka Shing Centre for Health Information and Discovery University of Oxford Oxford UK

Biology Centre of the Czech Academy of Sciences Institute of Entomology Branišovská 31 370 05 České Budějovice Czech Republic

Centre Suisse de Recherches Scientifiques en Côte d'Ivoire 01 BP 1303 Abidjan Côte d'Ivoire

Department of Biomedical Sciences University of Cape Coast Cape Coast Ghana

Department of Parasitology Noguchi Memorial Institute for Medical Research University of Ghana Accra Ghana

Department of Vector Biology Liverpool School of Tropical Medicine Pembroke Place Liverpool L3 5QA UK

Laboratory of Ecology and Ecotoxicology Department of Zoology Faculty of Sciences Université de Lomé 01 B P 1515 Lomé Togo

Tropical Infectious Diseases Research Centre 01 B P 526 Cotonou Benin

Wellcome Sanger Institute Hinxton Cambridge CB10 1SA UK

Před aktualizací

bioRxiv. 2023 Jan 14:2023.01.13.523889. doi: 10.1101/2023.01.13.523889. PubMed

Zobrazit více v PubMed

Roth GA, et al. Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2018;392:1736–1788. doi: 10.1016/S0140-6736(18)32203-7. PubMed DOI PMC

Bhatt S, et al. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015;526:207–211. doi: 10.1038/nature15535. PubMed DOI PMC

Hancock PA, et al. Mapping trends in insecticide resistance phenotypes in African malaria vectors. PLoS Biol. 2020;18:e3000633. doi: 10.1371/journal.pbio.3000633. PubMed DOI PMC

Clarkson CS, et al. Adaptive introgression between Anopheles sibling species eliminates a major genomic island but not reproductive isolation. Nat. Commun. 2014;5:4248. doi: 10.1038/ncomms5248. PubMed DOI PMC

Grau-Bové X, et al. Evolution of the insecticide target Rdl in African Anopheles is driven by interspecific and interkaryotypic introgression. Mol. Biol. Evol. 2020;37:2900–2917. doi: 10.1093/molbev/msaa128. PubMed DOI PMC

Grau-Bové X, et al. Resistance to pirimiphos-methyl in West African Anopheles is spreading via duplication and introgression of the Ace1 locus. PLoS Genet. 2021;17:e1009253. doi: 10.1371/journal.pgen.1009253. PubMed DOI PMC

Weetman D, Wilding CS, Steen K, Pinto J, Donnelly MJ. Gene flow–dependent genomic divergence between Anopheles gambiae M and S forms. Mol. Biol. Evol. 2012;29:279–291. doi: 10.1093/molbev/msr199. PubMed DOI PMC

Donnelly MJ, Isaacs AT, Weetman D. Identification, validation, and application of molecular diagnostics for insecticide resistance in malaria vectors. Trends Parasitol. 2016;32:197–206. doi: 10.1016/j.pt.2015.12.001. PubMed DOI PMC

Lindsay SW, Thomas MB, Kleinschmidt I. Threats to the effectiveness of insecticide-treated bednets for malaria control: thinking beyond insecticide resistance. Lancet Glob. Health. 2021;9:e1325–e1331. doi: 10.1016/S2214-109X(21)00216-3. PubMed DOI

Oxborough RM. Trends in US President’s Malaria Initiative-funded indoor residual spray coverage and insecticide choice in sub-Saharan Africa (2008-2015): urgent need for affordable, long-lasting insecticides. Malar. J. 2016;15:146. doi: 10.1186/s12936-016-1201-1. PubMed DOI PMC

Weetman D, Djogbenou LS, Lucas E. Copy number variation (CNV) and insecticide resistance in mosquitoes: evolving knowledge or an evolving problem? Curr. Opin. Insect Sci. 2018;27:82–88. doi: 10.1016/j.cois.2018.04.005. PubMed DOI PMC

Lucas ER, et al. Whole genome sequencing reveals high complexity of copy number variation at insecticide resistance loci in malaria mosquitoes. Genome Res. 2019;29:1250–1261. doi: 10.1101/gr.245795.118. PubMed DOI PMC

The Anopheles Gambiae 1000 Genomes Consortium. Ag1000G phase 3 CNV data release. https://www.malariagen.net/data/ag1000g-phase3-cnv (2021).

Yi X, et al. Sequencing of 50 human exomes reveals adaptation to high altitude. Science. 2010;329:75–78. doi: 10.1126/science.1190371. PubMed DOI PMC

Garud NR, Messer PW, Buzbas EO, Petrov DA. Recent selective sweeps in North American Drosophila melanogaster show signatures of soft sweeps. PLoS Genet. 2015;11:e1005004. doi: 10.1371/journal.pgen.1005004. PubMed DOI PMC

Anopheles gambiae 1000 Genomes Consortium. Genetic diversity of the African malaria vector Anopheles gambiae. Nature. 2017;552:96–100. doi: 10.1038/nature24995. PubMed DOI PMC

Lucas, E. R. & Nagi, S. C. Analysis scripts for ‘Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance in Anopheles gambiae and Anopheles coluzzii’. zenodo 10.5281/zenodo.8102919 (2023). PubMed PMC

Choi HW, et al. The effectiveness of insecticide-impregnated bed nets in reducing cases of malaria infection: a meta-analysis of published results. Am. J. Trop. Med. Hyg. 1995;52:377–382. doi: 10.4269/ajtmh.1995.52.377. PubMed DOI

Martinez-Torres D, et al. Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae ss. Insect Mol. Biol. 1998;7:179–184. doi: 10.1046/j.1365-2583.1998.72062.x. PubMed DOI

Hancock PA, et al. Modelling spatiotemporal trends in the frequency of genetic mutations conferring insecticide target-site resistance in African mosquito malaria vector species. BMC Biol. 2022;20:46. doi: 10.1186/s12915-022-01242-1. PubMed DOI PMC

Clarkson CS, et al. The genetic architecture of target-site resistance to pyrethroid insecticides in the African malaria vectors Anopheles gambiae and Anopheles coluzzii. Mol. Ecol. 2021;30:5303–5317. doi: 10.1111/mec.15845. PubMed DOI PMC

Williams J, Cowlishaw R, Sanou A, Ranson H, Grigoraki L. In vivo functional validation of the V402L voltage gated sodium channel mutation in the malaria vector An. gambiae. Pest Manag. Sci. 2022;78:1155–1163. doi: 10.1002/ps.6731. PubMed DOI

Njoroge H, et al. Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for antimalarial intervention deployment. Mol. Ecol. 2022;31:4307–4318. doi: 10.1111/mec.16591. PubMed DOI PMC

Lycett GJ, et al. Anopheles gambiae P450 reductase is highly expressed in oenocytes and in vivo knockdown increases permethrin susceptibility. Insect Mol. Biol. 2006;15:321–327. doi: 10.1111/j.1365-2583.2006.00647.x. PubMed DOI

Ingham VA, et al. Integration of whole genome sequencing and transcriptomics reveals a complex picture of the reestablishment of insecticide resistance in the major malaria vector Anopheles coluzzii. PLoS Genet. 2021;17:e1009970. doi: 10.1371/journal.pgen.1009970. PubMed DOI PMC

Essandoh J, Yawson AE, Weetman D. Acetylcholinesterase (Ace-1) target site mutation 119S is strongly diagnostic of carbamate and organophosphate resistance in Anopheles gambiae ss and Anopheles coluzzii across southern Ghana. Malar. J. 2013;12:1–10. doi: 10.1186/1475-2875-12-404. PubMed DOI PMC

Mitchell SN, et al. Metabolic and target-site mechanisms combine to confer strong DDT resistance in Anopheles gambiae. PLoS ONE. 2014;9:e92662. doi: 10.1371/journal.pone.0092662. PubMed DOI PMC

Riveron JM, et al. A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector. Genome Biol. 2014;15:R27. doi: 10.1186/gb-2014-15-2-r27. PubMed DOI PMC

Adolfi A, et al. Functional genetic validation of key genes conferring insecticide resistance in the major African malaria vector, Anopheles gambiae. Proc. Natl Acad. Sci. USA. 2019;116:25764–25772. doi: 10.1073/pnas.1914633116. PubMed DOI PMC

Lucas ER, et al. A high throughput multi-locus insecticide resistance marker panel for tracking resistance emergence and spread in Anopheles gambiae. Sci. Rep. 2019;9:13335. doi: 10.1038/s41598-019-49892-6. PubMed DOI PMC

Riveron JM, et al. Directionally selected cytochrome P450 alleles are driving the spread of pyrethroid resistance in the major malaria vector Anopheles funestus. Proc. Natl Acad. Sci. USA. 2013;110:252–257. doi: 10.1073/pnas.1216705110. PubMed DOI PMC

Saavedra-Rodriguez K, et al. Differential transcription profiles in Aedes aegypti detoxification genes after temephos selection. Insect Mol. Biol. 2014;23:199–215. doi: 10.1111/imb.12073. PubMed DOI PMC

Enayati AA, Ranson H, Hemingway J. Insect glutathione transferases and insecticide resistance. Insect Mol. Biol. 2005;14:3–8. doi: 10.1111/j.1365-2583.2004.00529.x. PubMed DOI

Bass C, Denholm I, Williamson MS, Nauen R. The global status of insect resistance to neonicotinoid insecticides. Pestic. Biochem. Physiol. 2015;121:78–87. doi: 10.1016/j.pestbp.2015.04.004. PubMed DOI

Grigoraki L, et al. CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes. PLoS Genet. 2021;17:e1009556. doi: 10.1371/journal.pgen.1009556. PubMed DOI PMC

Main BJ, et al. Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali. Mol. Ecol. 2015;24:5145–5157. doi: 10.1111/mec.13382. PubMed DOI PMC

Vontas J, et al. Rapid selection of a pyrethroid metabolic enzyme CYP9K1 by operational malaria control activities. Proc. Natl Acad. Sci. USA. 2018;115:4619–4624. doi: 10.1073/pnas.1719663115. PubMed DOI PMC

Hearn J, et al. Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa. Mol. Ecol. 2022;31:3642–3657. doi: 10.1111/mec.16497. PubMed DOI PMC

Ibrahim, S. S. et al. Molecular drivers of insecticide resistance in the Sahelo-Sudanian populations of a major malaria vector. BMC Biol. 21, 125 (2022). PubMed PMC

Blandin S, et al. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell. 2004;116:661–670. doi: 10.1016/S0092-8674(04)00173-4. PubMed DOI

Cirimotich CM, Dong Y, Garver LS, Sim S, Dimopoulos G. Mosquito immune defenses against Plasmodium infection. Dev. Comp. Immunol. 2010;34:387–395. doi: 10.1016/j.dci.2009.12.005. PubMed DOI PMC

Saddler A, Burda P-C, Koella JC. Resisting infection by Plasmodium berghei increases the sensitivity of the malaria vector Anopheles gambiae to DDT. Malar. J. 2015;14:134. doi: 10.1186/s12936-015-0646-y. PubMed DOI PMC

Mulamba C, et al. Widespread pyrethroid and DDT resistance in the major malaria vector Anopheles funestus in East Africa is driven by metabolic resistance mechanisms. PLoS One. 2014;9:e110058. doi: 10.1371/journal.pone.0110058. PubMed DOI PMC

Weedall GD, et al. A cytochrome P450 allele confers pyrethroid resistance on a major African malaria vector, reducing insecticide-treated bednet efficacy. Sci. Transl. Med. 2019;11:eaat7386. doi: 10.1126/scitranslmed.aat7386. PubMed DOI

Mugenzi LMJ, et al. Cis-regulatory CYP6P9b P450 variants associated with loss of insecticide-treated bed net efficacy against Anopheles funestus. Nat. Commun. 2019;10:4652. doi: 10.1038/s41467-019-12686-5. PubMed DOI PMC

Ffrench-Constant, R. H. The molecular genetics of insecticide resistance. Genetics194, 807–815 (2013). PubMed PMC

Opiyo MA, Paaijmans KP. ‘We spray and walk away’: wall modifications decrease the impact of indoor residual spray campaigns through reductions in post-spray coverage. Malar. J. 2020;19:30. doi: 10.1186/s12936-020-3102-6. PubMed DOI PMC

Mechan F, et al. LLIN Evaluation in Uganda Project (LLINEUP): The fabric integrity, chemical content and bioefficacy of long-lasting insecticidal nets treated with and without piperonyl butoxide across two years of operational use in Uganda. Curr. Res. Parasitol. Vector Borne Dis. 2022;2:100092. doi: 10.1016/j.crpvbd.2022.100092. PubMed DOI PMC

Sherrard-Smith E, et al. Systematic review of indoor residual spray efficacy and effectiveness against Plasmodium falciparum in Africa. Nat. Commun. 2018;9:4982. doi: 10.1038/s41467-018-07357-w. PubMed DOI PMC

Mavridis K, et al. Rapid multiplex gene expression assays for monitoring metabolic resistance in the major malaria vector Anopheles gambiae. Parasit. Vectors. 2019;12:9. doi: 10.1186/s13071-018-3253-2. PubMed DOI PMC

Santolamazza F, et al. Insertion polymorphisms of SINE200 retrotransposons within speciation islands of Anopheles gambiae molecular forms. Malar. J. 2008;7:163. doi: 10.1186/1475-2875-7-163. PubMed DOI PMC

Chabi J, et al. Rapid high throughput SYBR green assay for identifying the malaria vectors Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae s.s. Giles. PLoS ONE. 2019;14:e0215669. doi: 10.1371/journal.pone.0215669. PubMed DOI PMC

Jun G, et al. Detecting and estimating contamination of human DNA samples in sequencing and array-based genotype data. Am. J. Hum. Genet. 2012;91:839–848. doi: 10.1016/j.ajhg.2012.09.004. PubMed DOI PMC

Manichaikul A, et al. Robust relationship inference in genome-wide association studies. Bioinformatics. 2010;26:2867–2873. doi: 10.1093/bioinformatics/btq559. PubMed DOI PMC

Hanghøj K, Moltke I, Andersen PA, Manica A, Korneliussen TS. Fast and accurate relatedness estimation from high-throughput sequencing data in the presence of inbreeding. Gigascience. 2019;8:giz034. doi: 10.1093/gigascience/giz034. PubMed DOI PMC

MalariaGEN. The Anopheles gambiae 1000 Genomes Consortium. Ag1000G phase 3 SNP data release. https://www.malariagen.net/data/ag1000g-phase3-snp (2021).

Lu, J., Breitwieser, F. P., Thielen, P. & Salzberg, S. L. Bracken: estimating species abundance in metagenomics data. PeerJ Comput. Sci.3, e104 (2017).

Klaus, B. & Strimmer, K. fdrtool: Estimation of (local) false discovery rates and higher criticism. http://CRAN.R-project.org/package=fdrtool (2015).

Cingolani P, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly. 2012;6:80–92. doi: 10.4161/fly.19695. PubMed DOI PMC

Copy number variants underlie major selective sweeps in insecticide resistance genes in Anopheles arabiensis

Copy number variants underlie the major selective sweeps in insecticide resistance genes in Anopheles arabiensis from Tanzania

Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance in Anopheles gambiae and Anopheles coluzzii