# 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

## TL;DR

This study shows that specific P450 enzymes in Anopheles funestus mosquitoes can break down multiple types of insecticides, contributing to resistance.

## Contribution

The study demonstrates the broad insecticide metabolism capability of CYP6P9a and CYP6P9b when paired with their endogenous CPR in An. funestus.

## Key findings

- CYP6P9a and CYP6P9b with AfCPR metabolize multiple insecticide classes, including DDT and deltamethrin.
- CYP6P9b-AfCPR metabolizes α-cypermethrin, while CYP6P9a-AfCPR metabolizes DDT, bendiocarb, and chlorfenapyr.
- Variations in CPR domains affect P450 metabolism, highlighting the importance of using endogenous CPR for accurate validation.

## Abstract

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-Y456 differ 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 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.