The Potential Impact of Pathogens on Honey bee, Apis mellifera L., Colonies and Possibilities for

245 Pages·2014·1.07 MB·English

The Potential Impact of Pathogens on Honey bee, Apis mellifera L., Colonies and
Possibilities for their Control

By

Suresh D. Desai

A Thesis Submitted to the Faculty of Graduate Studies of
The University of Manitoba
in partial fulfillment of the requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department of Entomology
University of Manitoba
Winnipeg, Manitoba
Canada

Copyright © 2014 by Suresh D. Desai
ACKNOWLEDGEMENTS
This dissertation would not have been possible without the guidance and help of
several individuals who in one way or another contributed and extended their valuable
assistance in the preparation and completion of this study.
First and foremost, I would like to express my sincere gratitude towards my
supervisor, Dr. Robert W Currie, for his inspiration, guidance, and support. His
encouragement was the divine push that helped me sail through my Ph.D program. Rob’s
honest scientific approach and his ideas inspired and enriched my growth as a student and
as an aspiring researcher. I am very indebted to him. I am grateful to my committee
members Dr. Neil Holliday, Dr. Fouad Daayf and Dr. Mahmood Iranpour for their
helpful advice and discussion. I am grateful to Dr. Steve Whyard for the helpful advice
and I enjoyed his interest in my research as well as the fruitful discussions. Special
thanks to Dr. Dave Rosenberg, for his comments and help with editing the thesis.
I appreciate all the past and present members of the Bee Lab for creating a
dynamic and enjoyable team environment. I take this opportunity to thank especially
Lisa Babey and Dave Holder, who worked mostly behind the scenes and were just a call
away for help. My sincere thanks go to Rhéal Lafrenière, David Ostermann, Rasoul
Bahreini, Paul Kozak, Young-Jae Eu and Doug Durnin for assisting at various stages.
My thanks also go to all the members of Department of Entomology, Department of
Animal Science and Department of Plant Science who have helped me in one way or the
other. Special thanks to all the awesome summer students, Erica Lowe, Sara Braun,
Jaclyn Deonarine, Derek Micholson and Lindsay Geisel. I would like to thank Dr. Rob
Hill and Dr. Denis Krause and their lab members for providing excellent lab facilities to
conduct my experiments. I am grateful to Dr. Gary Crow and Dr. Norm Kenkel for
i
statistical advice, especially for chapter 3. Thanks to my friends, Santosh Kumar, Raju
S.Y, and Sravankumar Jami, Shiling Jiang and all others in Canada and India for their
constant support.
I thank the beekeepers from Manitoba and Canada, and the University of
Manitoba for donating bee samples and honey bee colonies and providing logistic
support. I am grateful to Agriculture and Agri-Food Canada, Advancing Canadian
Agriculture and Agri-Food (ACAAF), Manitoba Rural Adaptation Council (MRAC), the
Canadian Bee Research Fund, Manitoba Beekeepers Association, Manitoba Queen
Breeders Association, Boone Hodgson Wilkinson Trust Fund, Saskatchewan Beekeepers
Development Commission, NSERC-CANPOLIN and Genome Canada for funding this
research through grants provided to Dr. Currie. I also acknowledge financial support in
the form of a University of Manitoba Graduate Fellowship (UMGF).
Very special thanks to my father and mother, brother and sister for their love and
never-ending support. Heartfelt thanks to my wife, Priya for her love, patience, endless
support and encouragement and my beautiful daughter Neha and dear son, Rishabh for
brightening my life.
ii
TABLE OF CONTENTS
Acknowledgments……….……….………………….…………………………………….i
List of figures………………..….……….……………….….………………………...….iv
List of tables………………....….…….……………………..……………………………vi
List of appendices ….……....….…….……………………..………………………...….vii
Abbreviations…….…….…..…….……………………….………………………………ix
Abstract…………………………………………………...……………………………….1
General introduction…………………...………………………………………………….3
Chapter 1. Literature review..…. ........................................................................................ 6
Introduction..…. .............................................................................................................. 6
Honey bee parasites and pathogens..…. ......................................................................... 9
Genetic diversity..…. .................................................................................................... 25
RNA interference..…. ................................................................................................... 27
Rationale of this study ................................................................................................... 28
Chapter 2. Occurrence and quantification of economically important viruses in healthy
and unhealthy honey bee, Apis mellifera L. colonies in Canada…………….…..30
Chapter 3. Seasonal dynamics of honey bee, Apis mellifera L., virus infections and
effects of their associations with other diseases and parasites on colony losses in
different wintering environments...…...…..…………………..………….………54
Chapter 4. Genetic diversity within honey bee colonies affects pathogen load and
relative virus levels in honey bees, Apis mellifera L.………………...………...101
Chapter 5. Reduction in deformed wing virus infection in larval and adult honey bees
(Apis mellifera L.) by double-stranded RNA ingestion…………....………...…140
Chapter 6. General discussion..…. ................................................................................. 167
Summary..…. ................................................................................................................... 182
The need for future work..…. .......................................................................................... 185
Appendices..…………...……………………………………………………………..…186
Literature cited..…. .......................................................................................................... 195

iii
LIST OF FIGURES
Figure 1.1 Honey bee Apis mellifera with mite Varroa destructor on the abdomen…….9
Figure 1.2 (A) an adult bee exhibiting typical wing and body deformity symptoms, (B) an
adult bee with normal wings…………..……………………..……………….….14
Figure 2.1. Prevalence of seven bee viruses in healthy colonies in eight provinces of
Canada n = 80 and in unhealthy colonies from Manitoba n = 23….…………….52
Figure 2.2. Relative viral concentration of DWV, BQCV, and IAPV among samples
collected from healthy colonies in eight provinces in Canada and unhealthy
colonies from Manitoba……………….….……………………………………...53
Figure 3.1. Map showing regions from which bee samples were collected. Dots represent
the locations of samples collected from beekeepers (5 beekeepers per region)...88
Figure 3.2. Change in colony population score (frames of bees, 1 frame= ~ 2,430 bees)
from fall to spring, for indoor (- -) and outdoor (─ ─ ) wintered colonies…...90
Figure 3.3. Interactions between season (spring and fall) and wintering method (indoor
and outdoor) for DWV and BQCV concentrations (left axis) and mean abundance
of Nosema (right axis) (± standard error) (see Table 3.3)……………..…..……94
Figure 3.4. Interaction between season (spring and fall), wintering method (indoor and
outdoor) and sampling location (brood area and entrance) for BQCV
concentrations (± standard error) (see Table 3.3)……………………………..…95
Figure 3.5. Interaction between season (fall and spring) and sampling location (brood area
and entrance) on mean abundance of Nosema (± standard error) (see Table
3.3)….....................................................................................................................96
Figure 3.6. Effect of season (fall and spring) on mean abundance of Varroa (left axis) and
levels of viruses (SBV, KBV and CBPV) (right axis) (± standard error) (Table
3.3)……………………………………………………………………..………...97
Figure 4.1. Wooden bioassay cages used for the grooming experiments. Below each cage
is a screened floor with a drawer that allows fallen mites to be removed from the
cage……………………………………………………………………………..131
Figure 4.2. Changes in the bee population score over winter (A) and changes in the total
Varroa population in the hive over winter (B)………………………….………132
iv
Figure 4.3. Mean abundance of Varroa in honey bee colonies collected from genetically
diverse (n = 24), genetically similar (n = 11), and open-mated (n = 8) hives in fall
2007…………………………...………………………………….......................133
Figure 4.4. Mean daily mite mortality rate (±standard error) over winter (January – March
2008) in GDCs (n = 24) and GSCs (n = 11) differed significantly (F = 30.72; df =
1, 33; p < 0.0001).……………...……………………………………..……..….134
Figure 4.5. Mean daily bee mortality rate (±standard error) over winter (January – March
2008) in GDCs (n = 24) and GSCs (n = 11) differed significantly (F = 4.92; df =
1, 33; p < 0.034)……………………………………..….………………………135
Figure 4.6. Bee and mite mortality as assessed in caged bees in the laboratory……….136
Figure 4.7. Proportion of honey bee colonies (GDC, GSC, OMC) with detectable level of
viruses in fall 2007…………...……………………..…………………..………137
Figure 4.8. Relative change in mean (±SE) concentration of DWV, BQCV, and IAPV
among samples collected from GDCs (n = 24), GSCs (n = 11) and OMCs (n = 8)
in (A) fall 2007 and (B) spring 2008…..…………..……………………………138
Figure 4.9. Prevalence of Nosema infection in GDCs (n = 15) and GSCs (n = 8) in fall
2007……………………………………………………………………..………139
Figure 5.1. The effect of treatments on survivorship of laboratory-reared bee larvae
during the 21-day rearing period…………………………………...…………..161
Figure 5.2. The effect of treatments on survivorship of adult bees during the 8-day
experiment…………………………………………..…………..………………162
Figure 5.3. The effect of treatments on proportion of adult bees with deformed wings
(SE) that emerged from laboratory reared bees on day 21..……….……….….163
Figure 5.4. Effect of the DWV-dsRNA on symptoms of wing deformity….………….164
Figure 5.5. The effect of deformed wing virus (DWV)-double-stranded (ds)RNA on
relative DWV concentration in different treatments when larvae were fed with
DWV………………………………………………..……………………….….165
Figure 5.6. Relative deformed wing virus (DWV) concentration (log) in adult honey bees
exposed to different treatments……………………...……………………….…166
v
LIST OF TABLES
Table 1.1 Economically important honey bee viruses and their global distribution
showing life stages affected, major symptoms and impacts on bees…………….12
Table 2.1. PCR primers used in the study……………….……………….…..…….…….50
Table 2.2. qPCR primers used in the study…....................................................................50
Table 2.3. Frequencies of simultaneous virus infections in healthy honey bee samples (n
= 80)…………….………………………………………………..………………51
Table 2.4. Frequencies of simultaneous virus infections in unhealthy honey bee samples
(n = 23) from MB………………………..………………………………..……...51
Table 3.1. Primers used for the used for PCR analysis and qPCR analysis for
quantification…………………………………………………………………….89
Table.3.2 Proportion of colonies (in two wintering methods) with detectable levels of
parasites or pathogens as measured by the alcohol wash method for Varroa in
brood area (250 bees) and entrance samples (200 bees approximately), spore
count for Nosema spp (from 100 bees), thoracic slice method for tracheal mite
(100 bees), and RT-PCR for viruses using 50 bees for brood area and 10 bees for
entrance samples…………………….………………………………..………….91
Table 3.3. Effect of wintering method, regions, sampling location in the hive and season
on the relative levels of parasites and pathogens in honey bee colonies.…....92-93
Table 3.4. Simple and partial correlations (Pearson’s) between colony parasite (mean
abundance of Varroa and prevalence of tracheal mite) and pathogen (mean
abundance of Nosema, log 2-ΔCt of DWV, BQCV, IAPV, SBV, KBV, CBPV and
ABPV) levels, percent bee loss over winter and spring colony population size
across all samples in fall and spring for indoor and outdoor wintering
colonies…...…………………………………………………………………..98-99
Table 3.5. Pearson’s correlation analyses of nosema levels in entrance and brood area
samples collected in fall, mid-winter and spring for colonies wintered
indoors..……….…………………………………………………………..…….100
Table 4.1. Primers used in the study for Nosema (PCR) and viruses (qPCR and PCR)..130
Table 5.1. Primers used in the experiment………………..…………………………….160

vi
LIST OF APPENDICES
Appendix 1. Simple correlations (Pearson’s) for fall indoor-wintered colonies between
colony parasites (mean abundance of Varroa and prevalence of tracheal mite),
pathogens (mean abundance of Nosema, log 2-ΔCt of DWV, BQCV, IAPV, SBV,
KBV, CBPV, and ABPV) levels, bee population score and bee loss…...…...…186
Appendix 2. Simple correlations (Pearson’s) for fall outdoor-wintered colonies between
colony parasites (mean abundance of Varroa and prevalence of tracheal mite),
pathogens (mean abundance of Nosema, log 2-ΔCt of DWV, BQCV, IAPV, SBV,
KBV, CBPV, and ABPV) levels, bee population score and bee loss. ..…….…187
Appendix 3 Simple correlations (Pearson’s) for spring indoor-wintered colonies between
colony parasite (mean abundance of Varroa and prevalence of tracheal mite),
pathogen (mean abundance of Nosema, log 2-ΔCt of DWV, BQCV, IAPV, SBV,
KBV, CBPV, and ABPV) levels, bee population score and bee loss…………..188
Appendix 4. Simple correlations (Pearson’s) for spring outdoor-wintered colonies
between colony parasite (mean abundance of Varroa and prevalence of tracheal
mite), pathogen (mean abundance of Nosema, log 2-ΔCt of DWV, BQCV, IAPV,
SBV, KBV, CBPV, and ABPV) levels, bee population score and bee loss…...189
Appendix 5. Partial correlations (Pearson’s) for fall indoor-wintered colonies between
colony parasite (mean abundance of Varroa and prevalence of tracheal mite),
pathogen (mean abundance of Nosema, log 2-ΔCt of DWV, BQCV, IAPV, SBV,
KBV, CBPV, and ABPV) levels, bee population score and bee loss…...……...190
Appendix 6. Partial correlations (Pearson’s) for fall outdoor-wintered colonies between
colony parasite (mean abundance of Varroa and prevalence of tracheal mite),
pathogen (mean abundance of Nosema, log 2-ΔCt of DWV, BQCV, IAPV, SBV,
KBV, CBPV, and ABPV) levels, bee population score and bee loss…....….....191
Appendix 7. Partial correlations (Pearson’s) for spring indoor-wintered colonies between
colony parasite (mean abundance of Varroa and prevalence of tracheal mite),
pathogen (mean abundance of Nosema, log 2-ΔCt of DWV, BQCV, IAPV, SBV,
KBV, CBPV, and ABPV) levels, bee population score and bee loss......……....192
Appendix 8. Partial correlations (Pearson’s) for spring outdoor-wintered colonies
between colony parasite (mean abundance of Varroa and prevalence of tracheal
vii
mite) and pathogen (mean abundance of Nosema, log 2-ΔCt of DWV, BQCV,
IAPV, SBV, KBV, CBPV, and ABPV) levels, bee population score and bee
loss………………………………………………………………………………193
Appendix 9. Results for the binary logistic regression analysis on the spring parasites and
pathogens compared with live and dead colonies. ………………………….…194

viii
ABBREVIATIONS
Δ Ct delta Cycle threshold
ABPV Acute Bee Paralysis Virus
AFB American Foulbrood
BLAST Basic Local Alignment Search Tool
BPMS Bee Parasitic Mite Syndrome
BQCV Black Queen Cell Virus
CBPV Chronic Bee Paralysis Virus
CCD Colony Collapse Disorder
cDNA Complementary DNA
CSBV Chinese SacBrood Virus
DEPC Diethylpyrocarbonate
DNA Deoxyribonucleic Acid
dNTP Deoxyribonucleotide Triphosphate
dsRNA double stranded RNA
DWV Deformed Wing Virus
EFB European Foulbrood
GFP-dsRNA Green fluorescent protein-dsRNA
HBTM Honey bee tracheal mite
IAPV Israeli Acute Paralysis Virus
IBDS Idiopathic brood disease syndrome
IRES Internal Ribosome Entry Site
KBV Kashmir Bee Virus
M-MLV RT Moloney Murine Leukemia Virus Reverse Transcriptase
mRNA messenger RNA
ORF Open Reading Frame
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
qPCR quantitative Polymerase Chain Reaction
RdRp RNA Dependent RNA Polymerase
RISC RNA Induced Silencing Complex
RNA Ribonucleic Acid
RNAi RNA Interference
RT-PCR Real-Time Polymerase Chain Reaction
SBV Sac Brood Virus
SID-1 Systemic Interference Defective
siRNA small interfering RNA
TSBV Thai Sac Brood Virus
UTR Untranslated Region

ix