Elucidating the mechanisms of insecticide resistance in malaria vector populations from four endemic countries in Sub-Saharan Africa

Malaria mortality has fallen since 2010, largely due to the scale-up of treatment, diagnostics, and insecticide-based vector control interventions, principally insecticide treated nets and indoor residual spraying. However, global gains in malaria control have begun to stall. The use of insecticides is a critical part of reducing disease transmission. However, resistance to the most important public health insecticides is now widespread and now a major threat to malaria control. New tools are needed to ensure the long term sustainability of vector control interventions to combat malaria in the face of insecticide resistance. The development of novel vector control interventions is dependent on an understanding of the mechanisms by which resistance arises. Furthermore, local, high quality entomological data are essential to inform selection of optimal mixes of interventions to maximise impact. This thesis characterises phenotypic resistance to pyrethroids in eleven study sites across four countries in West, Central and East Africa and uses Next Genera:on Sequencing to elucidate mechanisms of resistance. We investigate microbial mediated insec:cide resistance in deltamethrin resistant An. coluzzii from Côte d’Ivoire, target site mutations in pyrethroid resistant An. gambiae s.l. from the Democratic Republic of the Congo (DRC), gene expression profiles of permethrin resistant An. arabiensis of varying ages from Tanzania and gene and microbial expression profiles of permethrin resistant An. coluzzii displaying reduced PBO synergy from Guinea. Resistance to the diagnostic dose of each pyrethroid tested was reported in every study site. Reduced PBO synergy was observed in permethrin resistant An. coluzzii from Guinea, and to alphacypermethrin, deltamethrin and permethrin in all but two sites across the DRC. In all populations tested, resistance appeared to be partially mediated by elevated expression of metabolic detoxification enzymes. We also identified upregulation of cuticular resistance genes in An. arabiensis from Tanzania, and An. coluzzii from Guinea. Comparisons between deltamethrin-resistant and -susceptible An. coluzzii demonstrated significant differences in microbiota diversity. Ochrobactrum, Lysinibacillus, and Stenotrophomonas genera, each of which comprised insec:cide-degrading species, were significantly enriched in resistant mosquitoes. We also present the most comprehensive overview to date of target site mutations in An. gambiae s.s. from the DRC. The use of NGS in this thesis identified novel SNPs, bacterial species, and a wide range of detoxification, cuticular, salivary and ABC transporter genes associated with phenotypic pyrethroid resistance. Their identification would not have been possible using standard PCR screening assays, which only detect known mutations. Where resources are available, NGS methods should be used more widely in entomological surveillance to beZer inform malaria vector control tools.