Establishment and application of bioassay- and molecular marker-based methods for monitoring fluvalinate resistance of Varroa mites.

The Varroa mite, Varroa destructor, is an ectoparasite that infests honey bees. The extensive use of acaricides, including fluvalinate, has led to the emergence of resistance in Varroa mite populations worldwide. This study's objective is to monitor fluvalinate resistance in field populations of Varroa mites in Korea through both bioassay-based and molecular marker-based methods. To achieve this, a residual contact vial (RCV) bioassay was established for on-site resistance monitoring. A diagnostic dose of 200 ppm was determined based on the bioassay using a putative susceptible population. In the RCV bioassay, early mortality evaluation was effective for accurately discriminating mites with the knockdown resistance (kdr) genotype, while late evaluation was useful for distinguishing mites with additional resistance factors. The RCV bioassay of 14 field mite populations collected in 2021 indicated potential resistance development in four populations. As an alternative approach, quantitative sequencing was employed to assess the frequency of the L925I/M mutation in the voltage-gated sodium channel (VGSC), associated with fluvalinate kdr trait. While the mutation was absent in 2020 Varroa mite populations, it emerged in 2021, increased in frequency in 2022, and became nearly widespread across the country by 2023. This recent emergence and rapid spread of fluvalinate resistance within a span of three years demonstrate the Varroa mite's significant potential for developing resistance. This situation further underscores the urgent need to replace fluvalinate with alternative acaricides. A few novel VGSC mutations potentially involved in resistance were identified. Potential factors driving the rapid expansion of resistance were further discussed.

Molecular and kinetic properties of three acetylcholinesterases in the Varroa mite, Varroa destructor.

The Varroa mite, Varroa destructor, poses one of the most serious threats to honey bees worldwide. Although coumaphos, an anticholinesterase pesticide, is widely used for varroa mite control, little information is available on the properties of Varroa mite acetylcholinesterases (VdAChEs). In this study, three putative VdAChEs were annotated and named VdAChE1, VdAChE2, and VdAChE3. All VdAChEs possessed most of the functionally important signature domains, suggesting that they are catalytically active. Phylogenetic analysis revealed that VdAChE1 was clustered into a clade containing most arthropod AChE1s, whereas VdAChE2 and VdAChE3 formed a unique clade with other arachnid AChEs. VdAChE1 was determined to be membrane-anchored, but both VdAChE2 and VdAChE3 are soluble, as judged by electrophoresis in conjunction with western blotting. Tissue-specific transcription profiling revealed that VdAChE1 was most predominantly expressed in the synganglion. In contrast, VdAChE2 was most predominantly expressed in the legs and cuticle. VdAChE3 showed negligible expression levels in all the tissues examined. In a kinetic analysis using recombinant VdAChEs, VdAChE1 exhibited the highest catalytic efficiency, followed by VdAChE2 and VdAChE3. Inhibition experiments revealed that VdAChE1 was most sensitive to all tested inhibitors. Taken together, VdAChE1 appears to be the major synaptic enzyme with a more toxicological relevance, whereas VdAChE2 is involved in other noncatalytic functions, including chemical defense against xenobiotics. Current findings contribute to a more detailed understanding of the evolutionary and functional traits of VdAChEs and to the design of novel anticholinesterase varroacides.