The genomics of insecticide resistance in Anopheles funestus: insights from a large bed-net trial

In response to the emerging threat of insecticide resistance in malaria vectors, insecticides are being repurposed for vector control or developed de novo. The longevity of disease control programs will require stewardship of these tools, including the early detection of resistance variants. Genomic surveillance has emerged as a pivotal tool for this purpose. We embedded whole-genome sequencing within a large-scale cluster-randomised control trial (cRCT), the Long-Lasting Insecticidal Net Evaluation in Uganda Project (LLINEUP), which compared pyrethroid-PBO to pyrethroid-only insecticide-treated nets (ITNs). We show that An. funestus populations were relatively unaffected by the interventions, compared to An. gambiae s.l., which were markedly reduced six months following ITN deployment. Genetic diversity and population structure analyses were suggestive of a single large An. funestus population in Uganda with little differentiation between geographic regions. Genome-wide selection scans revealed strong signals of selection at the RP1 and Cyp9k1 loci, both previously implicated in pyrethroid resistance, and two additional loci containing a diacyl-glycerol kinase gene and a TMC-like gene. Over the trial, we observed significant changes in the frequencies of swept haplotypes in the Dgk, Cyp6p9a, Gste2 and Cyp9k1 loci, and found larger changes in haplotype frequencies in the PBO arm. Our findings reveal the differential impact of the trial on An. gambiae s.l. and An. funestus, and the differing responses of An. funestus populations to pyrethroid and pyrethroid-PBO selection pressure. By embedding genomic surveillance in cRCTs, we facilitate the early discovery of resistance variants and can provide evidence of their impact on vector control tool efficacy- crucial for evidence-based deployment of vector control. Trial registration This trial was registered on 14th February 2017 with ISRCTN: ISRCTN17516395.