Pyrethroid resistance in varroa destructorinvestigating the role of mutations in the voltage-gated sodium channel

Resumen

One of the largest challenges facing beekeepers today is controlling Varroa destructor mites, a problem exacerbated by the limited number of control products available and the evolution of resistance in mite populations. Pyrethroid compounds have been among the most common treatments for controlling Varroa, strongly used over many generations, even after resistance evolved in the mid 1990s. The mechanism of resistance to pyrethroids in V. destructor has been associated with amino acid substitutions in the voltage-gated sodium channel (VGSC), the target protein of pyrethroids. In particular, the mutations found associated with resistance to pyrethroids are substitutions at position 925 of the VGSC (numbered after the housefly para-type sodium channel protein). To date, three different resistant alleles have been described at this position, replacing wild-type Leucine by Valine (L925V), Isoleucine (L925I) or Methionine (L925M). These three resistant alleles are found in different mite populations worldwide, showing certain geographical distribution, with the L925V mutation found in European mite populations, while L925I and L925M were first detected in some hives in the Southeastern USA. In this thesis, we have evidenced the implication of these mutations in the resistance to pyrethroids in V. destructor, by electrophysiological and pharmacological characterization of the wild-type and 925-mutant VGSC. Our study on the distribution of these mutations in the USA territory, have revealed that, although with varying incidence, both L925I and L925M resistant alleles are widespread throughout the USA, being the most plausible cause of pyrethroid treatment failure in the country. In addition, we have found for the first time in V. destructor the M918L substitution, associated with pyrethroid resistance in other arthropod species, in mites collected from apiaries in Spain. Phylogenetic analyses of the gene region linked to position 925 of the V. destructor VGSC revealed a single origin for each resistant mutation and a close relationship between the L925M and L925I alleles. We also developed a simple and affordable detection method of 925-mutant alleles based on PCR-RFLP that can be adapted as a routine assay in any laboratory. Accurate pre-treatment detection of resistant mites opens a window for a resistance management strategy aimed at achieving sufficient control of the mite while protecting the efficacy of pyrethroids for the longest possible time. Knowing the expected efficacy of a product beforehand would undoubtedly improve the outcome of Varroa control in the short term. In the same way, having such information would cut down on unnecessary acaricide treatments. Therefore, they minimise their accumulation in hive matrixes, and also reduce the likelihood of new resistant mutations developing in a mite population. In addition, regular monitoring will allow the detection of fluctuations in resistant alleles and allow us to act accordingly, e.g., by scheduling a pyrethroid-free period when pyrethroids become less effective. The intention of this research study was to shed light on the implications of these mutations on pyrethroid resistance, their evolutive relationship, and present status, with the eventual purpose of establishing some basic keystones to develop an effective Integrated Pest Management (IPM) approach. Integrated Pest Management is considered by experts to be the most successful and environmentally friendly approach to deal with arthropod pests. It integrates the use of chemical pesticides in combination with better cultural and biological techniques, minimizing the management impact on the environment. In an IPM approach, pesticide treatments only shall be applied when pest damage suppose an economic loss for the beekeeper, following a phased treatment plan according to need.