Signatures of selection and mechanisms of insecticide resistance in Ugandan Anopheles funestus: Insights from embedding translational genomics into the LLINEUP cluster randomised trial.

In response to the emerging threat of insecticide resistance in malaria vectors, insecticides are being repurposed for vector control or developed de novo. Good stewardship of these finite new resources is essential if disease control programmes are to remain effective. This is dependent on timely data to help guide evidence-based decision-making for National Malaria Control programmes (NMCPs). By embedding genomics into cluster randomized control trials (cRCTs), we can perform surveillance and early detection of insecticide resistance variants to new and repurposed chemicals in natural field conditions, supporting effective stewardship. The LLIN Evaluation Uganda Project (LLINEUP) trial evaluated the efficacy of pyrethroid-piperonyl butoxide (PBO) and pyrethroids-only long-lasting insecticidal nets (LLINs). It was conducted in Uganda between 2017-2020 and was the largest cRCT to date, covering 40% of the country in 104 health sub-districts. We embedded genomic surveillance within LLINEUP to detect and track insecticide resistance variants. At baseline and throughout the trial, we sampled Anopheles mosquitoes with Prokopack aspirators and performed Illumina whole-genome sequencing. 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 LLIN deployment. Standard approaches for describing genetic diversity and population structure e.g. fixation index (FST), Principal Component Analysis (PCA) and Neighbour-Joining (NJ) trees, were consistent with the density observations and suggestive of a single large An. funestus population in Uganda with little genetic differentiation. Genome-wide selection scans revealed strong signals of selection at the Resistance to pyrethroid-1 (RP1) locus and Cyp9k1, both loci previously implicated in pyrethroid resistance. We report two additional loci, eye diacylglycerol kinase (Dgk) ({cong}13.5Mb on the X chromosome) and O-mannosyl-transferase (TMC-like) ({cong}67.9Mb on 3RL) that showed signals of selection. Known DDT and permethrin resistance-associated variants at the Gste2 locus, L119F and L119V, were also identified. Over the trial period, changes in haplotype frequencies were observed in regions under selection, with more pronounced shifts in the PBO arm. Notably, there were significant reductions in the frequencies of swept haplotypes (measured by delta ({Delta}) H12) in the Dgk and Cyp6p9a regions, while significant increases in haplotype frequency were observed at Gste2 and Cyp9k1 loci. Our findings reveal the differential impact of the trial on An. gambiae s.l. and An. funestus densities and the differing responses of An. funestus populations to pyrethroid and pyrethroid-PBO selection pressure. These insights underscore the potential value of tailored, species- and region-specific vector control strategies, supported by regional genetic surveillance, to better control insecticide resistance evolution and spread. By embedding genomic surveillance in cRCTs we can facilitate the discovery of putative resistance variants and can provide evidence of their impact on vector control tool efficacy; both of crucial importance to evidence-based deployment of vector control tools by NMCPs.