Novel insecticide resistance mutations associated with variable PBO synergy in Anopheles gambiae s.l. from the Democratic Republic of Congo.

Over-reliance on pyrethroid insecticides in insecticide-treated nets (ITNs) has imposed significant selection pressure for the evolution of insecticide resistance among major malaria vector species. In the Democratic Republic of Congo (DRC), the country with the second highest malaria burden globally, pyrethroid resistance is pervasive, but there is a paucity of information regarding the molecular mechanisms driving resistance. A clear understanding of the specificity of resistance mechanisms to individual insecticides and the likelihood of selecting for cross-resistance mechanisms is crucial for the development of new vector control tools. Anopheles gambiae s.l. populations from eight study sites across the DRC were phenotyped for resistance to alpha-cypermethrin, deltamethrin and permethrin, with and without pre-exposure to the synergist piperonyl butoxide (PBO), followed by multiplex amplicon sequencing. Phenotypic pyrethroid resistance and loss of PBO synergy was confirmed in all sites across the DRC. In An. gambiae s.s. four non-synonymous SNPs which have been previously associated with insecticide resistance were detected: gste2-L119V, vgsc-L995F, vgsc-L995S and rdl-A296G, while three were novel: gste2-T154S, ace1-N246T and ace1-P265L. Nationwide geographical trends in insecticide resistance mutation distribution, prevalence and selection were evident. In the West, near fixation of vgsc-L995F and almost complete absence of vgsc-L995S was observed, alongside low-moderate frequencies of rdl-A296G and gste2-L119V. Further East, the converse was apparent. Gste2-L119V was significantly associated with resistance to deltamethrin following PBO-pre-exposure, warranting functional validation to determine its putative role in reduced PBO synergy. Furthermore, gste2-T154S was implicated in deltamethrin and permethrin resistance but susceptibility to alpha-cypermethrin after PBO pre-exposure. Study findings comprise the most comprehensive overview of the prevalence of genetic markers of Anopheles insecticide resistance across the DRC and provide an important baseline for improved malaria vector control and the design of proactive insecticide resistance management strategies. Given the significant scale up in PBO-ITNs, with more than 58% of all ITNs delivered to sub-Saharan Africa in 2023 containing PBO, there is an urgent need to identify novel molecular markers to monitor changes in PBO synergy, which may be predicative of loss of intervention operational efficacy.