Mitocidal resistance in the ectoparasitic mite,Varroa destructor, and the relationship with its host Apis mellifera

The Western honeybee, Apis mellifera, is an economically important insect, responsible for a large portion of global pollination services. They live in densely populated colonies, which give the optimum chance for opportunistic pathogens and parasites to spread. Honeybees, like all organisms, are subject to a wide range of threats, from viruses to parasites. The immune response of A. mellifera to these threats relies on a fast acting non-adaptive immunity, with effectors such as cellular defences and the release of antimicrobial peptides. The defence against invading pathogens and parasites is important not only for the individual bee, but for the colony as a whole. Many bee diseases have been shown as capable of causing collapse of honeybee colonies. The first section of this thesis examines the occurrence and prevalence of four honeybee viruses (Deformed wing virus, Chronic bee paralysis virus, Acute bee paralysis virus, Israeli acute paralysis virus), the microsporidian parasite Nosema, and the parasitic tracheal mite, Acarapis woodi. The results from all seasons indicated a very low prevalence of the tracheal mite, with only 2% of the colonies testing postive in the Spring sampling, with none positive in the Summer or Autumn samples. Deformed wing virus was detected at very high levels throughout the year, with Israeli acute paralysis virus detected in the Autumn round of sampling in 3% of the tested colonies. The other two viruses were not detected. Levels of Nosema were also high throughout the year, at 18%, 6% and 12% of the colonies testing positive in the Spring, Summer and Autumn respectively. The results indicated no obvious disease variations present in the colonies tested from apiaries that lost more than 20% of their hives during the Winter post sampling than those that had lost less than 20%. The next section was to examine the mechanisms by which one of the most serious threats to the honeybee, the parasitic mite V. destructor, has developed resistance to pyrethroid chemicals. Varroa are thought to have a negative impact in the overall health and vitality of the bee, transmitting viruses through haemolymph feeding and possibly weakening the immune response of the bee. Proteomic analysis was used to compare the proteomic profile of sensitive and resistant mites, in order to observe any variations that may be conferring the resistant phenotype. The comparison showed that a number of proteins were detected at higher levels of abundance in the resistant mites, such as heat shock proteins and detoxifying enzymes such as aldehyde dehydrogenase. A number of proteins present at lower levels include cuticle proteins involved in cuticle structure. The altered levels of these proteins in the resistant Varroa could be conferring resistance through decreased penetration and increased metabolism of the pyrethroid. In the final section, the full effect that parasitization by Varroa has on the bee was examined. Parasitized Winter bees were compared to unparasitized and the proteomic profiles were analysed for changes. Hexmerin was present at lower levels in the bees that were parasitized, as was enolase-like protein. The decreased level of these proteins indicates Varroa parasitisation could lead to insufficient energy metabolism. Drone pupae that were parasitized by Varroa were compared to unparasitized drones using proteomic analysis. Cuticle proteins decreased in abundance which could indicate a compromised healing response following parasitization. A number of proteins involved in energy and nutrition such as hexamerin were also present at lower levels of abundance in the parasitized drone pupae. Similar proteins decreased in abundance in parasitized workers. Cuticle structure proteins were present at lower levels of abundance, with proteins involved in the stress response present at higher levels in the parasitized workers. Quantitative PCR analysis of parasitized drone and worker pupae indicated a reduced level of two immune genes – Abaecin and Defensin, with two other immune related genes increased in expression: Phenoloxidase and Hymenoptaecin. Changes in the expression of immune related genes following parasitization indicates that Varroa are affecting how the immune reposnse functions. To idenitify whether or not this change in the immune reponse was caused by salivary effectors secreted by the mite during feeding, the haemolymph from parasitized pupae was compared using label free proteomics to haemolymph from unparasitized pupae. A number of proteins were found exclusive to the parasitized haemolymph, including a mettalloendopeptidase which is found in other blood feeding insects and could be functioning in the digestion of haemolymph. Sox 14, a regulator of transcription, was also exclusively present in the parasitized haemolymph. The work presented throughout this thesis offers a comprehensive analysis of the diseases found in honeybee colonies, the effect that parasitization by Varroa has on adult and developing pupae, and analysis of the pyrethoid resistant phenotype. The results offer an explanation as to why Varroa are considered one of the most serious honeybee threats, and highlights the importance of controlling infestation levels in colonies