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Title: Transmission of deformed wing virus (DWV) between Varroa destructor and the European honeybee (Apis mellifera) : in vitro and in vivo studies
Author: Bradford, Emma Louise
ISNI:       0000 0004 7659 0879
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2019
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The European honeybee (Apis mellifera) is a managed insect pollinator of global economic importance. Over the last few decades honeybees have been undergoing a major health crisis, with one of the biggest causes the parasitic mite, Varroa destructor and its role in changing the viral landscape of deformed wing virus (DWV), which consists of two major variants: DWV-A and DWV-B. Prior to the start of this project there was limited information known about the mechanisms behind the relationships between Varroa, DWV and honeybees. The overarching aim of this project was to further enhance our understanding of these complex relationships, focusing on the impact of Varroa DWV transmission and differences between the main DWV variants. One of the initial obstacles to understanding these complex interactions was the inability to accurately quantify DWV variants. Prior to the start of this project, there was a need for an accurate assay for the quantification of DWV-A, DWV-B and total DWV, allowing the role of both variants in viral transmission and establishment to be investigated. While primers did exist for DWV quantification, the majority did not distinguish between variants, or provide accurate levels of DWV. Given these challenges in variant detection, a new assay for the quantification of DWV-A, DWV-B and total DWV was designed and validated. The assay consists of an external plasmid standard with distinct sections, for the detection of variants and total DWV. This DWV variant plasmid assay was essential for further transmission studies in this project. DWV variant transmission was explored using a variety of different methods. A new in vitro feeding system was used, to allow investigations into Varroa DWV variant transmission in isolation. The feeding system utilises locust haemolymph, allowing changes in DWV transmission to be detected. In multiple feeding experiments significant changes in DWV transmission were detected. Significant changes in DWV composition within feeding Varroa were detected with decreased levels of DWV, and changing variant levels. Switches in variant composition within mites and transmission rates occurred during Varroa in vitro feeding. These variant switches occurred in both directions from DWV-A to DWV-B, and DWV-B to DWV-A dominance. These changes in mite variant composition corresponded to changes in levels of replicating strands. iv These changes in DWV transmission, composition and replicating strand detection were only seen due to the use of this in vitro feeding system. The in vitro work provided valuable information about Varroa variant transmission and composition changes during feeding but this is not a natural system. Honeybee pupae from a Varroa-free area with extremely low DWV titres provided the opportunity to investigate Varroa variant transmission and pupal DWV establishment. Over 96 hours total DWV levels underwent a 1339408X fold increase, within pupae following Varroa feeding, with a sharp increase after 12 hours, followed by a plateau after 60 hours. Within this time period, DWV-A underwent a similar increase, while DWV-B increased at a much slower rate (33X fold change). In contrast to the in vitro work, mite DWV levels did not decrease during feeding. The impact of natural Varroa cell infestation on L5 larvae was investigated, showing no significant effects between pupal total DWV levels and mite density, and DWV levels between infested and none-infested larvae. However, this lack of significance could be attributed to the use of L5 larvae, which had only undergone a maximum of 24 hours Varroa feeding within the cell. Additionally, the use of two drug treatments (ribavirin and hydroxyurea) to reduce DWV levels was explored. Both drug treatments were tested against Varroa and honeybees, using a variety of methods: immersion (Varroa), injections (honeybees) and feeding (both). While neither drug treatment resulted in consistent DWV decreases, some reduction in DWV levels were seen following Varroa soaking in drug solutions. A significant decrease in DWV was seen in honeybees following bolus and ad libitum feeding of drug treatments. Overall, information and insights have been gained regarding the complex relationship between Varroa, honeybees and DWV. A new DWV variant qPCR assay was developed and utilised in subsequent studies. DWV variant switches in both transmission rates and mite composition were found to occur in in vitro studies. Differences in DWV variant establishment within honeybees were detected following Varroa in vivo feeding, in low DWV pupae. Though the tested drug treatments did not affect DWV levels, this highlights the difficultly facing the establishment of any DWV treatment.
Supervisor: Bowman, Alan S. ; Campbell, Ewan Sponsor: Biotechnology and Biological Science Research Council (BBSRC) ; Morle Agricultural Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Honeybee ; Varroa destructor ; RNA viruses