NADPH-cytochrome P450 reductase mediates P450-dependent metabolic resistance to deltamethrin in Aedes albopictus

Abstract

The cytochrome P450 monooxygenase system is a key detoxification pathway involved in insecticide metabolism in insects and plays a central role in the evolution of insecticide resistance. NADPH-cytochrome P450 reductase (CPR), a crucial electron transfer partner in cytochrome P450-mediated detoxification of xenobiotics, is an essential component of this system. However, its role in insecticide resistance in Aedes albopictus remains unclear. In this study, two CPR gene isoforms in Aedes albopictus , AalCPR-X1 and AalCPR-X2 , were cloned and characterized. qPCR was used to determine their expression levels and spatiotemporal expression patterns in laboratory-resistant strains. The effects of deltamethrin exposure on AalCPR expression were then examined. RNA interference (RNAi) was employed to silence the CPR gene, and the susceptibility of Aedes albopictus to deltamethrin following gene knockdown was evaluated. In addition, AalCPR-X1 and AalCPR-X2 were heterologously expressed in Sf9 cells to assess cytochrome c reductase activity, and cytotoxicity assays were performed to compare the survival of cells overexpressing the two isoforms. Sequence analysis showed that AalCPR-X1 contained all conserved domains of CPR, whereas AalCPR-X2 lacked the N-terminal membrane anchor and had a shortened FMN-binding domain. qPCR analysis indicated that both isoforms were significantly overexpressed in resistant strains. Spatiotemporal expression profiling revealed that AalCPR-X1 was highly expressed in third- and fourth-instar larvae, adults, and detoxification tissues, whereas AalCPR-X2 was predominantly expressed in adults and malpighian tubules. In addition, deltamethrin exposure significantly upregulated AalCPR-X1 mRNA levels in resistant strains. RNAi-mediated silencing of the CPR gene significantly increased the susceptibility of Aedes albopictus to deltamethrin. Heterologous expression assays confirmed that both AalCPR-X1 and AalCPR-X2 exhibited cytochrome c reductase activity, and cytotoxicity assays demonstrated that the survival rate of cells overexpressing AalCPR-X1 was significantly higher than that of cells overexpressing AalCPR-X2 . This study demonstrates that AalCPR plays an important role in P450-mediated metabolic resistance in Aedes albopictus and suggests that CPR may serve as a potential candidate marker for insecticide resistance management strategies.