Grzegorz Borsuk
Morphometric, genetic, epigenetic, and biochemical variability in tau-fluvalinate resistant and sensitive Varroa destructor 
zeszyt 368, ss. 80
The primary genetic diversity of mites from the genus Varroa resulted in development of adaptive variability, which ensured their survival for over 100 years. The mites have adapted to various bee species and diverse environments; they have also developed resistance to drugs used in mite control. They are able to develop cross-resistance to acaricides in a short time. In order to prevent colony collapse disorder, attempts are being made to identify the causes of pesticide resistance in mites. The resistance might be associated with enzymatic changes in the hemolymph or point mutations in the sodium channel of brain cells. Variation in mitochondrial genes has facilitated identification of mite haplogroups and haplotypes. The variation of Varroa destructor mites is the subject of this dissertation as it facilitates development of drug-resistant mite forms and can serve as a basis for characterization thereof.
The aim of the work was to investigate the prevalence of resistance of V. destructor to tau-fluvalinate in south-eastern Poland as well as to verify the existence morphometric variation, mtDNA sequence variation, and biochemical variation between tau-fluvalinate resistant and sensitive V. destructor females and to determine whether their resistance may be associated with epigenetic mechanisms. An additional objective was to classify the mites occurring in Poland into haplogroups identified worldwide and to check whether the affinity is related to their resistance to tau-fluvalinate.
The investigations were conducted in the years 2010–2012 and the material was collected from 47 apiaries in south-eastern Poland. A total number of 1439 Varroa destructor mites were collected. The Milani method was applied to differentiate between tau-fluvalinate resistant and sensitive mites. Three V. destructor female groups were formed: a control, resistant, and sensitive mite group. Morphometric measurements and genetic analyses, which included RFLP-PCR, identification of mutations in four sequenced fragments of mitochondrial genes (COI 320bp, COI 929bp, ATP6-CO III 818bp, Cyt B 958bp) were performed. A phylogenetic analysis of V. destructor female was performed and the proportion of methylated genes was determined based on the gene sequences. Additionally, the variability of surface proteins was assessed and biochemical analyses were performed.
The highest per cent proportion of tau-fluvalinate resistant V. destructor females was found in samples from apiaries examined in 2012 (34.17%), and the lowest was reported from samples examined in 2011 (12.71%). The maximum per cent proportion of tau-fluvalinate resistant V. destructor females in the entire population was 55%.
The highest per cent proportion of tau-fluvalinate resistant V. destructor females was reported from apiaries in which Bayvarol (36.92%) and Klartan (25.09%) were applied, and the lowest proportion was found in apiaries where Gabon (15.34%) and Apiwarol (13.93%) were used. A significant positive significant correlation was found between the per cent proportion of tau-fluvalinate resistant V. destructor females and the quantity of active substance administered to honeybees in Bayvarol (0.71), Klartan (0,90) and Gabon (0.94). A significant negative correlation was found between the per cent proportion of tau-fluvalinate resistant V. destructor females and the quantity of active substance received by honeybees with Apiwarol (-0.74).
A tendency toward higher resistance was exhibited by V. destructor females with smaller body sizes.
The control, tau-fluvalinate resistant and sensitive V. destructor females displayed the same of restriction enzyme cleavage pattern; hence, they may be assumed to belong to the same haplogroup. It was evident after sequencing four gene fragments that the V. destructor females from south-eastern Poland belong to the same first Korean haplogroup 1 (AmK1). The highest mutational variability was found for the Cyt B gene fragment. The percentage of missense mutation in Cyt B was higher in the sensitive V. destructor females and lower in the tau-fluvalinate resistant V. destructor females. In turn, a greater proportion of silent mutations were found in the resistant V. destructor females and a lower percentage was characteristic for the tau-fluvalinate sensitive mites.
The resistant V. destructor females displayed the lowest per cent proportion of total genomic methylation in comparison to the control and tau-fluvalinate sensitive V. destructor females.
The resistant V. destructor females exhibited a lower concentration of protein on the body surface, but the protein itself had a higher proteolytic activity. Hence, administration of acaricides, which act on mites through contact, may cause coagulation of proteins on mite body surface thereby producing a “protein shell” that prevents acaricides from penetration of the mite body and ensures their resistance.
The tau-fluvalinate resistant V. destructor females exhibited the highest levels of alanine aminotransferase (ALAT) and the total oxidative potential (FRAP) as well as the lowest levels of alkaline phosphatase (ALP), Ɣ-glutamyltransferase (GGT) and glucose.
Based on the present investigations, differences were found between the resistant and tau-fluvalinate sensitive V. destructor females in terms of morphometric features, genetic and epigenetic variability, variability of surface proteins, and basic biochemical characteristics. The differences reported may be the cause of drug resistance in V. destructor.
The increased acaricide resistance of V. destructor females may lead to substantial loss of honeybee colonies in apiaries. V. destructor mites in south-eastern Poland belong to the Korean haplogroup 1-AmK1. The mutated regions in Cyt B may alter protein conformation in the mitochondrial membrane, thereby ensuring V. destructor resistance. The resistance may be regulated by epigenetic mechanisms. The theses developed in this work concerning the reduced body sizes in the resistant mites, surface proteins, and basic biochemical analyses provide new directions of research into the mechanism of resistance in V. destructor.