The Varroa mite, Varroa destructor, a parasitic mite that afflicts honey bees, has become increasingly resistant to acaricides like fluvalinate due to its widespread use. The target site insensitivity mechanism, mediated by the L925V/M/I mutations in the voltage-gated sodium channel, plays a major role in resistance. Additionally, cytochrome P450 monooxygenases (Cyp450s) appear to function as a metabolic resistance factor; however, no Cyp450-mediated resistance mechanism has been reported to date. The aim of this study was to identify and characterize Cyp450s associated with fluvalinate resistance. A synergistic bioassay confirmed the involvement of Cyp450s in conferring tolerance or resistance to fluvalinate. Correlation analysis between mortality data and the expression levels of Cyp450 genes led to the identification of several candidates that may play a crucial role in fluvalinate resistance. Analysis of tissue distribution patterns revealed that these genes were most abundantly expressed in the cuticle and synganglion. This suggests that, despite their relatively low expression level, they may play a critical role in protecting the target site from fluvalinate due to its predominant expression in neuronal tissues. Functional analysis, in conjunction with baculovirus expression, demonstrated that fluvalinate has high inhibition rates against the recombinant candidate Cyp450s, suggestive of their strong interaction with fluvalinate. We discussed the potential utilization of their expression levels as a molecular marker for diagnosing metabolic resistance in field-collected Varroa mites.

The Varroa mite, Varroa destructor is an ectoparasite that parasitizes honey bees. The widespread usage of acaricides, particularly fluvalinate, has resulted in the emergence of resistance in Varroa mite populations all over the world. The goal of this study is to track fluvalinate resistance in Varroa mite field populations in Korea using both bioassay and molecular markers. To accomplish this, a residual contact vial (RCV) bioassay for on-site resistance monitoring was developed. Early mortality evaluation in the RCV bioassay was effective for reliably separating mites with the knockdown resistance (kdr) genotype, but late mortality evaluation was useful for distinguishing mites with additional resistance factors. The RCV bioassay of 14 field mite populations collected in 2021 revealed potential resistance development in four populations. Quantitative sequencing was used as an alternate method to examine the frequency of the L925I/M mutation in the voltage-gated sodium channel (vgsc), which is related with the fluvalinate kdr phenotype. While the mutation was not present in Varroa mite populations in 2020, it appeared in 2021, rose in frequency in 2022, and was practically ubiquitous 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 emphasizes the critical necessity to replace fluvalinate with alternate acaricides, such as fenpyroximate, coumaphos, and amitraz. A few novel vgsc mutations potentially involved in resistance were identified. Potential factors driving the rapid expansion of resistance were further discussed.

Varroa destructor is an ecto-parasite mite and worldwide pest of the honey bee Apis mellifera L. The pyrethroid tau-fluvalinate (Apistan), an acaricide that is tolerated by honey bees, has been used for varroa mite control since the mid 1980s. Even though various resistances to tau-fluvalinate in varroa mites have been reported from Europe, Israel, and USA, the nature of tau-fluvalinate resistance in varroa mites in Korea has never been investigated. To investigate and understand tau-fluvalinate resistance in varroa mites in Korea, we conducted bioassay in several apiaries located different regions in Korea. To understand the molecular mechanisms underlying the difference of tau-fluvalinate resistances in varroa mites, partial genomic DNA fragments of a voltage-sensitive sodium channel gene from varroa mites were cloned and sequenced, since tau-fluvalinate is known to act on the sodium channels directly. Two novel mutations in sodium channels of varroa mites were present in eight apiaries. Two mutations might be a geographical polymorphism of sodium channel of varroa mites in Korea.