NOSEMA CERANAE IN WESTERN HONEY BEES (APIS MELLIFERA): BIOLOGY AND MANAGEMENT by Geoffrey R. Williams Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Dalhousie University Halifax, Nova Scotia March 2013 © Copyright by Geoffrey R. Williams, 2013 DALHOUSIE UNIVERSITY DEPARTMENT OF BIOLOGY The undersigned hereby certify that they have read and recommend to the Faculty of Graduate Studies for acceptance a thesis entitled “NOSEMA CERANAE IN WESTERN HONEY BEES (APIS MELLIFERA): BIOLOGY AND MANAGEMENT” by Geoffrey R. Williams in partial fulfilment of the requirements for the degree of Doctor of Philosophy. Dated: March 27, 2013 External Examiner: _________________________________ Research Co-supervisors: _________________________________ _________________________________ Examining Committee: _________________________________ _________________________________ Departmental Representative: _________________________________ i i DALHOUSIE UNIVERSITY DATE: March 27, 2013 AUTHOR: Geoffrey R. Williams TITLE: NOSEMA CERANAE IN WESTERN HONEY BEES (APIS MELLIFERA): BIOLOGY AND MANAGEMENT DEPARTMENT OR SCHOOL: Department of Biology DEGREE: PhD CONVOCATION: May YEAR: 2013 Permission is herewith granted to Dalhousie University to circulate and to have copied for non-commercial purposes, at its discretion, the above title upon the request of individuals or institutions. I understand that my thesis will be electronically available to the public. The author reserves other publication rights, and neither the thesis nor extensive extracts from it may be printed or otherwise reproduced without the author’s written permission. The author attests that permission has been obtained for the use of any copyrighted material appearing in the thesis (other than the brief excerpts requiring only proper acknowledgement in scholarly writing), and that all such use is clearly acknowledged. _______________________________ Signature of Author ii i For honey bees, and their keepers And for the bee’s knees iv Table of Contents LIST OF TABLES .......................................................................... ix LIST OF FIGURES ......................................................................... x ABSTRACT ................................................................................. xii LIST OF ABBREVIATIONS & SYMBOLS USED ................................. xiii ACKNOWLEDGEMENTS ................................................................ xv CHAPTER 1 INTRODUCTION ........................................................ 1 1.1 GENERAL BIOLOGY OF NOSEMA IN HONEY BEES .............................. 2 1.2 DISTRIBUTION OF NOSEMA IN HONEY BEES .................................. 2 1.3 MANAGEMENT OF NOSEMA IN HONEY BEES ................................... 3 1.4 VIRULENCE OF NOSEMA IN HONEY BEES ...................................... 4 1.5 CONGENER INTERACTIONS OF NOSEMA IN HONEY BEES ..................... 4 1.6 OBJECTIVES AND THESIS STRUCTURE ........................................ 5 1.7 REFERENCES ..................................................................... 7 CHAPTER 2 COLONY COLLAPSE DISORDER IN CONTEXT .............. 10 2.1 COMMENTARY .................................................................. 10 2.2 REFERENCES ................................................................... 12 CHAPTER 3 FIRST DETECTION OF NOSEMA CERANAE, A MICROSPORIDIAN PARASITE OF EUROPEAN HONEY BEES (APIS MELLIFERA), IN CANADA AND CENTRAL U.S.A. .............................. 14 3.1 ABSTRACT ...................................................................... 14 3.2 INTRODUCTION ................................................................ 14 3.3 METHODS ....................................................................... 15 3.4 RESULTS & DISCUSSION ..................................................... 17 3.5 ACKNOWLEDGEMENTS ......................................................... 20 3.6 REFERENCES ................................................................... 20 CHAPTER 4 DOES FUMAGILLIN CONTROL THE RECENTLY DETECTED INVASIVE PARASITE NOSEMA CERANAE IN WESTERN HONEY BEES (APIS MELLIFERA)? ................................................. 23 4.1 ABSTRACT ...................................................................... 23 4.2 INTRODUCTION ................................................................ 23 4.3 METHODS ....................................................................... 24 v 4.4 RESULTS & DISCUSSION ..................................................... 26 4.5 ACKNOWLEDGEMENTS ......................................................... 29 4.6 REFERENCES ................................................................... 29 CHAPTER 5 THE MICROSPORIDIAN NOSEMA CERANAE, THE ANTIBIOTIC FUMAGILIN-B®, AND WESTERN HONEY BEE (APIS MELLIFERA) COLONY STRENGTH .................................................. 32 5.1 ABSTRACT ...................................................................... 32 5.2 INTRODUCTION ................................................................ 32 5.3 METHODS ....................................................................... 34 5.3.1 EXPERIMENT 1 ....................................................... 35 5.3.2 EXPERIMENT 2 ....................................................... 36 5.3.3 NOSEMA SPECIES IDENTIFICATION................................. 37 5.4 RESULTS ........................................................................ 37 5.4.1 EXPERIMENT 1 ....................................................... 37 5.4.2 EXPERIMENT 2 ....................................................... 40 5.4.3 NOSEMA SPECIES IDENTIFICATION................................. 40 5.5 DISCUSSION ................................................................... 41 5.6 ACKNOWLEDGEMENTS ......................................................... 43 5.7 REFERENCES ................................................................... 43 CHAPTER 6 EFFECTS AT NEARCTIC NORTH-TEMPERATE LATITUDES OF INDOOR VERSUS OUTDOOR OVERWINTERING ON THE MICROSPORIDIUM NOSEMA CERANAE AND WESTERN HONEY BEES (APIS MELLIFERA).............................................................. 46 6.1 ABSTRACT ...................................................................... 46 6.2 INTRODUCTION ................................................................ 46 6.3 METHODS ....................................................................... 48 6.4 RESULTS ........................................................................ 51 6.5 DISCUSSION ................................................................... 53 6.6 ACKNOWLEDGEMENTS ......................................................... 55 6.7 REFERENCES ................................................................... 55 v i CHAPTER 7 INFRA-POPULATION AND -COMMUNITY DYNAMICS OF NOSEMA PARASITE CONGENERS, AND CONSEQUENCES FOR HONEY BEE (APIS MELLIFERA) HOSTS .......................................... 58 7.1 ABSTRACT ...................................................................... 58 7.2 INTRODUCTION ................................................................ 59 7.3 METHODS ....................................................................... 62 7.3.1 EXPERIMENTAL DESIGN ............................................. 62 7.3.2 OBTAINING WORKERS FOR EXPERIMENTS ......................... 62 7.3.3 INOCULATION ........................................................ 63 7.3.4 HOST MORTALITY & FOOD CONSUMPTION ......................... 63 7.3.5 PARASITE FECUNDITY ................................................ 64 7.3.6 CONGENERIC PARASITE INTERACTIONS ........................... 64 7.3.7 NOSEMA QUANTIFICATION - MICROSCOPY ........................ 64 7.3.8 NOSEMA QUANTIFICATION - SIMPLEX REAL-TIME PCR .......... 65 7.3.9 STATISTICAL ANALYSES ............................................. 66 7.4 RESULTS ........................................................................ 66 7.4.1 Host mortality & food consumption ........................ 66 7.4.2 Parasite fecundity ................................................ 68 7.4.3 Congener parasite interactions .............................. 71 7.5 DISCUSSION ................................................................... 72 7.6 ACKNOWLEDGEMENTS ......................................................... 77 7.7 REFERENCES ................................................................... 79 CHAPTER 8 CONCLUSIONS AND FUTURE DIRECTIONS ................. 86 8.1 CONCLUSIONS ................................................................. 86 8.1.1 N. CERANAE DISTRIBUTION ......................................... 86 8.1.2 N. CERANAE MANAGEMENT .......................................... 88 8.1.3 N. CERANAE VIRULENCE ............................................. 88 8.1.4 N. CERANAE INTER-SPECIFIC INTERACTIONS ..................... 89 8.2 FUTURE DIRECTIONS .......................................................... 90 8.2.1 ADOPTION OF APPROPRIATE EXPERIMENTAL APPROACHES FOR N. CERANAE RESEARCH ....................................................... 90 vi i 8.2.2 DEVELOPMENT OF AN IPM STRATEGY FOR N. CERANAE CONTROL........................................................................91 8.2.3 IDENTIFICATION OF THE CAUSES OF VARIATION IN N. CERANAE DISTRIBUTION, VIRULENCE, AND INTER-SPECIFIC INTERACTIONS ................................................................. 92 8.3 FINAL REMARKS ............................................................... 94 8.4 REFERENCES ................................................................... 95 BIBLIOGRAPHY ........................................................................ 101 APPENDIX A PUBLICATIONS ................................................. 114 APPENDIX B COPYRIGHT PERMISSIONS ................................. 115 vi ii LIST OF TABLES Table 4.1 Median intensity (number of spores/bee) and prevalence (percent of colonies) of Nosema in spring (20 April-4 May) and late summer (20-26 August) 2007 in western honey bee (Apis mellifera) colonies (n) from 8 beekeeping operations in Nova Scotia, Canada that had been treated or untreated with Fumagilin-B® in September 2006. ................................................... 26 Table 5.1 Summary statistics of Nosema spore counts (in millions) for control and fumagillin-treated honey bee colonies from seven beekeepers in Nova Scotia during fall 2007 and spring 2008, and corresponding repeated measures ANOVA results (significant differences in bold). .................................... 40 Table 6.1 Indoor over-wintering management conditions for participating beekeeping operations in Maritime Canada, and sampling regime for Nosema spp. quantification. .................................................................................................... 50 Table 6.2 Summary statistics of mean Nosema ceranae intensity (in millions of spores/bee) for indoor (Ind.) - and outdoor (Out.) - wintered western honey bee colonies, as well as colony mortality, from three beekeeping operations in Maritime Canada................................................................................................... 52 Table 7.1 Summary of experimental treatment groups, including quantity of Nosema spores inoculated orally to newly emerged workers in 5 µl of 75% (weight/volume) sucrose-water solution at 0 d. ........................................................ 62 ix LIST OF FIGURES Figure 3.1 Phylogenetic tree illustrating the position of Nosema collected from European honey bees (Apis mellifera) in the Canadian Maritime Provinces (Nova Scotia, New Brunswick, and Prince Edward Island) and Minnesota USA, with previous Nosema GenBank submissions (denoted by *). Vairimorpha imperfecta is used as the outgroup reference. GenBank accession numbers are in parentheses. Bootstrap support values above the branch (minimum evolution/maximum parsimony) are from 1000 replicates. Support for the maximum likelihood analysis, below the branch, has also been obtained from 1000 bootstrapped replicates. ................................................... 18 Figure 4.1 Comparison of western honey bee (Apis mellifera) colonies in Nova Scotia, Canada treated (n = 94, from 5 beekeepers) and untreated (n = 51, from 3 beekeepers) with Fumagilin-B® in September 2006 in spring (20 April-4 May) and late summer (20-26 August) 2007 (n = 15-21 colonies per operation). Boxplots show interquartile range (box), median (black line within interquartile range), data range (vertical lines), and outliers (asterisks). Black dots represent means. ...................................................................................... 28 Figure 5.1 Comparisons between control and fumagillin-treated honey bee colonies in fall 2007 and spring 2008 for number of frames out of 10 of: (a) adult bees; (b) honey; (c) pollen; (d) capped brood; and (e) uncapped brood. Boxplots show interquartile range (box), median (black line within interquartile range), data range (dashed vertical lines), and outliers (open dots). Asterisks represent means. Estimates were made for every other frame in each colony. ................................................................................................ 39 Figure 6.1 Comparisons of Nosema ceranae intensity (in millions of spores/bee) between indoor- and outdoor-wintered western honey bee colonies in September 2008, April 2009, and May 2009 for: (a) all beekeeping operations, (b) beekeeping operation 1, (c) 2, and (d) 3. Boxplots show interquartile range (box), median (black line within interquartile range), data range (dashed vertical lines), and outliers (open dots). Asterisks represent means. ....................................................................................................................... 53 Figure 7.1 Effect of Nosema infection on adult worker western honey bee mortality. Mortality is shown as the cumulative percentage of dead individuals per treatment (Table 7.1) per day. The experiment was terminated at 30 d post inoculation when no living workers remained from the N. ceranae treatment. Treatments with different letters had significant differences in mortality.. ........................................................................................... 67 x