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An adult worker honey bee. This image is credited to Jim Kalisch at The Department of Entomology, University of Nebraska-Lincoln.
An adult worker honey bee. This image is credited to Jim Kalisch at The Department of Entomology, University of Nebraska-Lincoln.

Inducible honey bee viruses associated with Varroa jacobsoni

Colin Hawthorn Denholm B.Sc. (Hons) (Glasgow) M.Sc. D.I.C. (London)


Thesis submitted for the degree of Doctor of Philosophy, 1999. Department of Entomology & Nematology, IACR-Rothamsted, Hertfordshire

All figures are copyright Colin Denholm unless stated otherwise. This online thesis is comprehensively cross-referenced using the contents section below.

The specific name of the parasitic mite studied has changed from 'jacobsoni' to 'destructor' (this note was inserted January 2001).

Adobe Acrobat versions of the thesis documents were made in 1999. However, due for formatting differences, the page numbers may not exactly match the listed contents.

PDFs  Contents »  Chapter 1 » Chapter 2 » Chapter 3 » Chapter 4 » Chapter 5 » Chapter 6 » Appendix

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Contents

Chapter 1

Introduction

Chapter 2

General materials and methods

Chapter 3

Virus induction

Chapter 4

Humoral responses to induction stimuli, viruses and mite parasitism

Chapter 5

Spatial and temporal distribution of inducible honey bee viruses

Chapter 6

General discussion

Bibliography


List of figures

Chapter1

1.1 An adult worker honey bee
1.2 The castes and stages of development of the honey bee
1.3 Yearly distribution from 1992 to 1996 of Varroa jacobsoni within England and Wales
1.4 Varroa jacobsoni. Scanning electron micrographs of an adult female, an adult male and a schematic of an infested worker brood cell
1.5 Daily development of Varroa jacobsoni offspring in both worker and drone Apis mellifera cells
1.6 Generalised viral life cycle
1.7 Electron micrograph of acute paralysis virus (APV)

Chapter 2

2.1 Photograph of a frame box used to transport samples from honey bees colonies to the laboratory
2.2 Photograph of cages used to maintain adult honey bees in the laboratory
2.3 Photograph of a Burkard microapplicator
2.4 Schematic of the internal anatomy of an adult honey bee
2.5 Output generated from an ISCO fractionator
2.6 Dose response curve of APV with respect to number of virus particles injected and percentage mortality
2.7 Immunoglobulin G (IgG) before and after pepsin digestion
2.8 Theory and practice of the F(ab')2 ELISA
2.9 Quantitative ELISA plate layout
2.10 Schematic representation of an ELISA dilution response curve

Chapter 3

3.1 A theoretical model of virus induction
3.2 ELISA data for experiment I
3.3 Percentage acute paralysis virus associated mortality (APVAM) in experiment I
3.4 ELISA data for experiment II
3.5 Percentage slow paralysis virus associated mortality (SPVAM) in experiment II
3.6 APV ELISA data for experiment III
3.7 SPV ELISA data for experiment III
3.8 APV expressed as percent positive by ELISA in experiment III

Chapter 4

4.1 Anti-bacterial (Gram +ve) peptide activity in the haemolymph of differently treated adult honey bees
4.2 Electrophoresis gels of adult honey bee haemolymph 24 and 48 hours post-treatment in experiment IV
4.3 Haemolymph protein profiles of differently treated adult honey bees 24 hours post-treatment in experiment IV
4.4 Haemolymph protein profiles of differently treated adult honey bees 48 hours post-treatment experiment IV
4.5 ELISA data for experiment V
4.6 Percentage APV associated mortality in experiment V

Chapter 5

5.1 Monthly percentage of test bees killed by APV extracted from several samples of 20 live adults from each of two uninfested colonies at two sites
5.2 Percentage APV associated mortality in rank order in experiment VI
5.3 APV ELISA data for experiment VII with respect to time post-treatment
5.4 APV ELISA data for experiment VII with respect to treatment
5.5 SPV ELISA data for experiment VII with respect to time post-treatment
5.6 SPV ELISA data for experiment VII with respect to treatment
5.7 Percentage SPV associated mortality in experiment VII
5.8 The standard curves used in the quantitative ELISA
5.9 Estimated yield of APV in tissue samples from adult honey bees
5.10 Estimated yield of SPV in tissue samples from adult honey bees
5.11 Data presentation style for tissue tropism results. APV yield data for adult honey bees injected with APV
5.12 APV yield data for adult honey bees injected with APV, with tissue weight taken into account
5.13 APV yield data for adult honey bees injected with buffer
5.14 APV yield data for adult honey bees injected with buffer, with tissue weight taken into account
5.15 SPV yield data for adult honey bees injected with SPV
5.16 SPV yield data for adult honey bees injected with SPV, with tissue weight taken into account
5.17 SPV yield data for adult honey bees injected with buffer
5.18 SPV yield data for adult honey bees injected with buffer, with tissue weight taken into account
5.19 APV ELISA data for experiment IX
5.20 SPV ELISA data for experiment IX
5.21 Percentage induction of APV, SPV and both viruses by the injection of apyrase in experiment IX
5.22 Time of death of bees dying from induced APV, SPV or mixed infections in experiment IX
5.23 ELISA results from buffer injected adult honey bees over time

List of tables

Chapter 1

1.1 Parasitic mites of Apis mellifera
1.2 Increase in the number of countries with reported cases of Varroa jacobsoni infestation between 1981 and 1996
1.3 Some characteristics of viruses isolated from Apis mellifera

Chapter 2

2.1 Raw data for LD50 determination
2.2 Estimations of LD50 parameters using probit analysis

Chapter 3

3.1 Results of published honey bee virus induction experiments
3.2 Rough calculations concerning mite feeding and virus induction
3.3 Biochemistry of insect haemolymph with respect to virus-inducing stimuli and virus induction

Chapter 4

4.1 Scores for 'Api zym' enzyme assays of haemolymph from adult bees subjected to different treatments

Chapter 5

5.1 Colonies sampled for experiment VI
5.1 Dry weights of tissues dissected from adult honey bees
5.1 Results of statistical analyses in experiment VIII
5.1 Estimated acute paralysis virus (APV) particle concentrations in extracts used in infectivity tests in experiment VI

Chapter 6

6.1 Review of possible induction mechanisms

Acknowledgements

This work was funded by the Ministry of Agriculture, Fisheries and Food of the United Kingdom. IACR receives grant-aided support from the Biotechnology & Biological Sciences Research Council of the United Kingdom.


Abstract

Acute paralysis virus (APV) and slow paralysis virus (SPV) are only known to cause mortality of European honey bees, Apis mellifera L. (Hymenoptera: Apidae), in colonies infested with Varroa jacobsoni Oudemans (Acari: Varroidae). The hypothesis that this parasitic mite can induce inapparent, sublethal virus infections to become overt and lethal was tested by subjecting bees to treatments that mimicked mite feeding. Direct evidence of mite-mediated virus induction was not obtained due to difficulties in obtaining virus-free mites. APV and SPV were found to be inducible in both adult and pupal honey bees in the laboratory by the injection of apyrase, an enzyme common to the saliva of many haematophagous arthropods, suggesting that V. jacobsoni saliva contains virus-inducing substances; physical stimuli were unsuccessful. The proportion of inducible infections of both viruses in adult honey bees was found to peak during the brood-rearing season and both viruses could be induced in the same individual. There is some evidence that virus induction is more easily achieved in parasitised bees. However, experiments that attempted to separate the effects of virus inducing stimuli from the physiological state of the individual were unsuccessful. The injection of relatively large volumes of potassium phosphate buffer achieved 100% APV induction in pupae. The humoral response of adult honey bees to the injection of APV or mite parasitism appears to be an activation of the phenoloxidase system. APV infection appears to inhibit the production of antimicrobial peptides. The injection of potassium phosphate buffer or apyrase induced the synthesis of such peptides, but neither activated the phenoloxidase system. Using infectivity tests, APV displayed significant intercolony variation and both APV and SPV showed intracolony variation. SPV shows less tissue specificity that APV. Both viruses can be found in tissues concerned with oral secretion, suggesting an oral route of normal transmission.

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