Establishing a fluorescence-based technique for ABC transporters functional analysis in metabolism of insecticides in a Lepidopteron

Abstract

The insecticide resistance development poses challenges to the pest control effectiveness. As phase III detoxification enzyme, ATP-binding cassette (ABC) transporters play a crucial role in conferring resistance by facilitating the elimination of insecticides. However, the precise involvement of ABCs in insecticides metabolism remains unclear. In this study, a competitive assay utilizing microinjection of the ABC substrate Texas Red (TR) was established in Lepidoptera for the first time to investigate the contribution of ABCs to the detoxification process of Cydia pomonella towards lambda-cyhalothrin (LCT) and abamectin (ABM). Results revealed that the elimination of TR was hindered by ABC inhibitor verapamil, LCT, or a combination of both, implying a shared subsets of ABCs involved in the transportation of TR and LCT. Moreover, the application of ABM alone led to marked inhibition of TR elimination, with verapamil not further exacerbating this inhibitory effect, indicating that ABM exhibited a highly competitive ABC substrate characteristic and has a strong interaction with them. Furthermore, silencing key resistance associated ABC genes through RNAi resulted in increased mortality of larvae under LCT or ABM exposure, affecting TR elimination and ATP hydrolysis. These findings imply that ABC genes are critical for the active transport function of ABCs energized by ATP, ultimately reducing larvae tolerance to insecticides. In conclusion, our findings not only validated the feasibility of a competitive bioassay using fluorescent ABC substrate in lepidopteran larvae but also highlighted that ABCs in enhancing the efflux capacity of C. pomonella larvae, facilitating the binding and transportation of insecticides and their metabolites.