Temporal evolution of insecticide resistance and bionomics in Anopheles funestus, a key malaria vector in Uganda.

Insecticide resistance escalation is decreasing the efficacy of vector control tools. Monitoring vector resistance is paramount in order to understand its evolution and devise effective counter-solutions. In this study, we monitored insecticide resistance patterns, vector population bionomics and genetic variants associated with resistance over 3 years from 2021 to 2023 in Uganda. Anopheles funestus s.s was the predominant species in Mayuge but with evidence of hybridization with other species of the An. funestus group. Sporozoite infection rates were relatively very high with a peak of 20.41% in March 2022. Intense pyrethroid resistance was seen against pyrethroids up to 10-times the diagnostic concentration but partial recovery of susceptibility in PBO synergistic assays. Among bednets, only PBO-based nets (PermaNet 3.0 Top and Olyset Plus) and chlorfenapyr-based net (Interceptor G2) had high mortality rates. Mosquitoes were fully susceptible to chlorfenapyr and organophosphates, moderately resistant to clothianidin and resistant to carbamates. The allele frequency of key P450, CYP9K1, resistance marker was constantly very high but that for CYP6P9A/b were very low. Interestingly, we report the first detection of resistance alleles for Ace1 gene (RS =  ~ 13%) and Rdl gene (RS =  ~ 21%, RR =  ~ 4%) in Uganda. The qRT-PCR revealed that Cytochrome P450s CYP9K1, CYP6P9A, CYP6P9b, CYP6P5 and CYP6M7 were consistently upregulated while a glutathione-S-transferase gene (GSTE2) showed low expression. Our study shows the complexity of insecticide resistance patterns and underlying mechanisms, hence constant and consistent spatial and temporal monitoring is crucial to rapidly detect changing resistance profiles which is key in informing deployment of counter interventions.