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 metab-olism 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 validate their role in 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-Y456 differs from that of AgCPR-His456. 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 del-tamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only CYP6P9a-AfCPR membrane significantly metabolize DDT (producing dicofol), bendiocarb, clo-thianidin and chlorfenapyr (bioactivation into tralopyril). These demonstrate 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.