Genetic Markers Associated with the Widespread Insecticide Resistance in Malaria Vector Anopheles funestus Populations across Tanzania

Abstract Background: Anopheles funestus is a leading vector of malaria in most parts of East and Southern Africa, yet its ecology, and responses to vector control remain poorly understood compared to other vectors such as Anopheles gambiaeand Anopheles arabiensis. This study presents the first large-scale survey of the genetic and phenotypic expression of insecticide resistance in An. funestus populations in Tanzania. Methods:We performed insecticide susceptibility bioassays on An. funestusmosquitoes in nine districts with moderate to high malaria prevalence in Tanzania, followed by genotyping for resistance-associated mutations (CYP6P9a, CYP6P9b, L119F-GSTe2) and structural variants (SV4.3kb, SV6.5kb). Generalized linear models were used to assess relationships between genetic markers and phenotypic resistance. An interactive R Shiny tool was created to visualize the data and support evidence-based interventions. Results:Pyrethroid resistance was universal but reversible by piperonyl-butoxide (PBO). However, carbamate resistance was observed in only five of the nine districts, and DDT resistance was found only in the Kilombero valley, south-eastern Tanzania. Conversely, there was universal susceptibility to the organophosphate, pirimiphos-methyl in all sites. Genetic markers of resistance had distinct geographical patterns, with CYP6P9a-R and CYP6P9b-R alleles, and the SV6.5kb structural variant absent or undetectable in the northwest but prevalent in all other sites, while SV4.3kb was prevalent in the northwestern and western regions but absent elsewhere. Emergent L119F-GSTe2, associated with deltamethrin resistance, was detected in heterozygous form in districts bordering Mozambique, Malawi, and the Democratic Republic of Congo. The resistance landscape was most complex in western Tanzania, in Tanganyika district, where all five genetic markers were detected. There was a notable south-to-north spread of resistance genes, especially CYP6P9a-R, though this appears to be interrupted, possibly by geographical features such as the Rift Valley. Conclusion:This study underscores the need to expand resistance monitoring to include An. funestus alongside other vector species; and to screen for both the genetic and phenotypic signatures of resistance. The findings can be visualized online via an interactive user interface and could inform data-driven decision-making for resistance management and vector control. Since this was the first large-scale survey of insecticide resistance in Tanzania’s An. funestus, we recommend regular updates with greater geographical and temporal coverage.