Functional Validation of Endogenous Redox Partner Cytochrome P450 Reductase Reveals the Key P450s CYP6P9a /- b as Broad Substrate Metabolizers Conferring Cross-Resistance to Different Insecticide Classes in Anopheles funestus .

The versatility of cytochrome P450 reductase ( CPR ) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR ( AgCPR ), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR ( AfCPR ) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR , e.g., the coordinates of the second FAD stacking residue AfCPR -Y 456 differ from that of AgCPR -His 456 . While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR , the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b , capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management.