Establishment of a fluvalinate resistance monitoring protocol using the RCV method

The Varroa mite, Varroa destructor, is an ectoparasitic mite that feeds on honey bees and is considered to be one of the main causes of colony collapse disorder because it depletes the honey bees' nutritional stores and transmits a number of viruses. Beekeepers have used synthetic acaricides such as fluvalinate, coumaphos, and amitraz to control Varroa mites for decades. However, the continued use of such acaricides has led to the development of acaricide resistance in Varroa mites. In order to effectively combat Varroa mites and ensure honey bee health worldwide, therefore, it is crucial to evaluate the level of acaricide resistance in Varroa mites. In this study, an efficient approach for monitoring fluvalinate resistance in Varroa mites on-site was established utilizing the RCV (Residual Contact Vial) bioassay method, which has already been successfully employed in several other insect resistance monitoring studies. After treatment with fluvalinate using RCV, knockdown or dead Varroa mites were sampled every two hours, and the L925V/M/I mutation in the voltage-sensitive sodium channel, which is known to confer fluvalinate resistance in Varroa mites, was validated by genotyping each sample. As a result, the optimal dose and time point for the RCV bioassay to distinguish fluvalinate-susceptible Varroa mites from -resistant Varroa mites were established. Furthermore, the established method was implemented for monitoring fluvalinate resistance in Varroa mites in Korea. The current protocol should facilitate efficient field monitoring of fluvalinate resistance in Varroa mites.