The duplicated cytochrome P450 CYP6P9a/b confers cross-resistance to a mitochondrial complex I inhibitor in the African malaria vector Anopheles funestus

Metabolic resistance to pyrethroids driven by cytochrome P450s is threatening malaria control interventions and may provide cross-resistance to insecticides with unrelated modes of action. Here, we show that cytochrome P450 genes CYP6P9a/b associated with pyrethroid resistance in Anopheles funestus also confer cross-resistance to a novel mitochondrial complex I inhibitor insecticide, code-named Sherlock. Using standard bioassays (CDC bottle bioassays, WHO cone bioassays, and WHO tunnel tests), the Sherlock and pyrethroid insecticides were tested against pyrethroid-resistant An. gambiae s.s. (Nkolondom, Cameroon) and An. funestus s.s. (Mibellon, Cameroon) and FUMOZ-R). Molecular assays (genotyping of P450 markers and qRT-PCR expression) were performed to investigate the underlying resistance mechanisms and cross-resistance in An. funestus. Field sampled strains of An. gambiae s.s. and An. funestus s.s. from Cameroon were fully susceptible to Sherlock, whereas moderate resistance was observed in the FUMOZ-R An. funestus strain. Genotypic analysis of hybrid mosquitoes demonstrated a correlation between pyrethroid-resistance markers and reduced susceptibility to Sherlock. Individuals carrying one CYP6P9a_R allele had significantly higher odds of surviving exposure to Sherlock compared to those lacking this allele, as evidenced by CDC bottle bioassays (1xDC: OR = 5.3, CI = 2.7-9.8, p < 0.0001; 5xDC: OR = 18.6, CI = 7.8-46.4, p < 0.0001)), cone bioassays (OR = 5.1, CI = 2.7-9.8, p < 0.0001), and tunnel tests (OR = 6.6, CI = 3.4-12.6 p < 0.0001). qRT-PCR analysis revealed elevated expression of CYP6P9a in surviving hybrid mosquitoes exposed to Sherlock and permethrin, as observed in CDC bottle bioassays (1xDC: FC = 24.7; 5xDC: FC = 45.6; permethrin: FC = 35.4) and cone bioassays (FC = 9.8; FC = 4.8, respectively). These findings were consistent with the patterns of CYP6P9b and the 6.5 kb insertion. The L119F_GSTe2 pyrethroid resistance marker did not confer cross-resistance to Sherlock. These findings highlight the importance of considering cross-resistance patterns in the development and deployment of new insecticides for malaria vector control.