Quality of commercially reared queen and drone honey bees (Apis mellifera L.) in eastern Australia By John W. Rhodes A thesis prepared for the degree of Doctor of Philosophy at the University of Western Sydney, New South Wales, Australia. August, 2011. Declaration The research presented in this thesis is my own original work with assistance and contributions by other persons identified in the text and in the Acknowledgements. John Rhodes. August 2011. Acknowledgements I would like to express my appreciation to my Principal Supervisor, Assoc. Professor Robert Spooner-Hart, Centre for Plants and the Environment, University of Western Sydney, and to Dr. Denis Anderson, CSIRO Entomology, Canberra, for guidance and assistance during the progress of this study. I would like to thank the Rural Industries Research and Development Corporation, Honeybee Research and Development Program, Canberra; the University of Western Sydney, Hawkesbury Campus; and the New South Wales Department of Primary Industries for providing financial assistance and facilities. For the experiments on queen bee introduction and survival: I would like to acknowledge the following persons for their support which allowed these diverse range of experiments to come together successfully. Dr. Doug Somerville, NSW Department of Primary Industries, Goulburn for managing the collection of data at one of the field apiaries, and for providing input during the course of these experiments. Mr. Steven Harden, Biometry Branch, NSW Department of Primary Industries, Tamworth, for data analysis and comments on presentation. Laurie, Paula and Robert Dewar, Aratula, Queensland, commercial queen bee breeders, for the production of queen bees. Noel and Neil Bingley, Queanbeyan, NSW; Bob and Marie Michey, and Rob and Raelene Michie, Tamworth, NSW, commercial honey producers, for providing commercial apiaries for field evaluation of queen bees and for assisting with the collection of data from their apiaries. Dr. Denis Anderson and Ms. Kerry Medveczky; and Dr. Mike Lacey, CSIRO Entomology Laboratories, Canberra, ACT, for laboratory examinations of queen bees; and for chemical extraction and identification from queen head glands, respectively. Mr. Bruce White, former Apiary Officer, NSW Department of Primary Industries, Windsor, for providing comments during the course of the experiments. For the experiments on drones, the following people provided major support and assistance: Ms. Gretchen Wheen, Richmond, NSW, for collection of samples, use of apiary and laboratory facilities, and interpretation of results; Ms. Liz. Kabanoff, University of Western Sydney, Hawkesbury, for support and laboratory use for sperm viability experiments; Mr. Michael Duncan, Apiary Manager, University of Western Sydney, Hawkesbury, for assistance and use of apiary facilities; Ms. Sue Rees, Laboratory Technician, University of Western Sydney, Richmond, determination of sperm motility ratings; Mr. Steven Harden and Mr. Bruce McCorkell, Biometricians, NSW DPI, Tamworth, for statistical analysis of data; Ms. Sharon Nielsen, Biometrician, NSW DPI, provided comments on sperm viability and motility data; Dr. Bruce Tier, Animal Genetics and Breeding Unit, University of New England, Armidale, NSW, for assistance in developing experimental methodology; and Dr. Adrian Nicholas, NSW DPI, Tamworth, for use of laboratory facilities. Amino acid analysis of drone semen samples was carried out by the Australian Proteome Analysis Facility Ltd., Macquarie University, Sydney, NSW, and fatty acid analysis by the NSW Department of Primary Industries, Oil Testing Service, Wagga Wagga, NSW. I would like to thank Dr. Rod Mailer and staff at Wagga Wagga and staff at the Macquarie University Facility for their efforts in analysing samples of small volumes. Commercial beekeepers who contributed with queen bees, use of facilities and information were: Mr. Col Wilson, Kurri Kurri, NSW; Mr. Ken Olley, Clifton, Qld.; Mr.Linton Briggs, Glenrowan, Victoria; Mr.Frank Malfroy, Freemans Reach, NSW; Mr.Greg Mulder, Murrays Run, NSW; Mr.Greg Tyson, Inverell, NSW and Mr. David Weik, Tamworth, NSW. I would like to thank members of the beekeeping section, NSW DPI, for support, in particular Mr. Tim Burfitt, Manager, Intensive Livestock Industry Development, Orange; Mr. Bruce White, former Technical Specialist, Richmond; Dr. Doug Somerville, Technical Specialist, Goulburn; and Mr. Nick Annand, Livestock Officer, Bathurst. I would like to thank support staff at the Tamworth DPI office, particularly the librarians who have been very helpful over the past years. Table of Contents Table of Contents i List of Tables vii List of Figures xii Thesis summary xiii Chapter 1 Literature review, thesis aims and objectives. 1.1 Literature review 1 1.2 Research objectives 36 1.3 Thesis structure 37 Chapter 2 Queen bee introduction and early performance success 2.1 Introduction 39 2.2 Objectives 40 2.3 Materials and methods 42 2.3.1 Year 1 (1999- 42 2000) Investigations 2.3.2 Year 2 (2000-1) 47 Investigations 2.3.3 Year 3 (2001-2) 54 Investigations 2.4 Statistical Analyses 58 2.4.1 Introduction 58 Success Rate and Early Performance Success Rate. 2.4.2 Sperm counts 59 and number of i ovarioles 2.4.3 Amounts of 59 mandibular and head gland chemicals 2.5 Results 59 2.6 Discussion 83 2.6.1 Effects of queen 83 age on Introduction Success, Early Performance Success and Satisfactory Performance Success rates. 2.6.2 The number of 85 sperm in queen spermathecae 2.6.3 Chemicals 87 present in the mandibular and head glands of queen honey bees. 2.6.4 Physical 90 characteristics and disease status of queen bees and hive bees as a measure of queen quality. 2.7 Conclusions 95 Chapter 3 General materials and methods for drone studies 3.1 Introduction 97 3.2 Determination of drone sample size 97 3.3 Rearing and sampling drone honey 99 bees ii 3.3.1 Breeding lines 100 3.3.2 Location of the 100 experimental work 3.3.3 Experimental 101 bee hives 3.3.4 Colony 102 nutrition 3.3.5 Rearing drones 102 to a known age 3.3.6 Collection of 105 drone bees from the apiary 3.3.7 Laboratory 106 collection of data Chapter 4 Investigating drone semen characteristics and the possible role of genetics. 4.1 Introduction 114 4.2 Volume of semen and number of 115 sperm per drone 4.2.1 Introduction 115 4.2.2 Materials and 117 methods 4.2.3 Statistical 118 analysis 4.2.4 Results 118 4.3 Sperm viability assessment 124 4.3.1 Introduction 124 4.3.2 Materials and 125 methods 4.3.3 Statistical 125 analysis 4.3.4 Results 126 4.4 Sperm motility rating 128 4.4.1 Introduction 128 4.4.2 Materials and 130 iii methods 4.4.3 Statistical 130 analysis 4.4.4 Results 131 4.5 Investigating the role of genetics in 133 drone semen production 4.5.1 Introduction 133 4.5.2 Materials and 135 methods 4.5.2.1 Two-way cross 135 methodology 4.5.2.2 Confirmation of 139 High and Low breeding lines 4.5.2.3 Disease 139 identification 4.5.3 Statistical 140 analysis 4.5.4 Results 140 4.5.4.1 Confirmation of 140 High Line and Low Breeding Lines 4.5.4.2 Disease 141 examination 4.6 Drones not releasing semen after 145 manual eversion 4.6.1 Initial 145 examination 4.6.1.1 Introduction 145 4.6.1.2 Materials and 145 methods 4.6.1.3 Results 146 4.6.2 Manual 145 eversion – comparison between two operators 4.6.2.1 Introduction 147 iv 4.6.2.2 Materials and 147 methods 4.6.2.3 Statistical 149 analysis 4.6.2.4 Results 149 4.6.3 Effects of age 149 and genetics on sperm release 4.6.3.1 Introduction 149 4.6.3.2 Materials and methods151 4.6.3.3 Statistical 152 analysis 4.6.3.4 Results 152 4.6.4 Semen 161 movement 4.6.4.1 Introduction 161 4.6.4.2 Materials and 161 methods 4.6.4.3 Statistical 162 analysis 4.6.4.4 Results 163 4.7 Discussion 164 4.8 Conclusions 177 Chapter 5 Drone honey bee semen – amino acid and fatty acid content 5.1 Introduction 180 5.2 Materials and Methods 183 5.3 Results 184 5.3.1 Amino acids 184 5.3.2 Fatty acids 189 5.4 Discussion 5.4.1 Amino acids 189 5.4.2 Fatty acids 190 5.4.3 General 190 discussion 5.5 Conclusions 191 Chapter 6 General Discussion and Recommendations v 6.1 Introduction 192 6.2 General Discussion 193 6.3 Industry impacts 196 6.4 Further research 201 6.5 Final conclusion 202 References 204 vi List of Tables Table Table Title Page Number Number 1.1 Numbers of registered beekeepers and hives by State or Territory, 2006- 3 7. 1.2 Gross value of Australian honey and beeswax production. 5 1.3 Portion of queen bees purchased by beekeepers each year. 8 2.1 Sampling methods used to assess disease incidence in honey bee 49 colonies. 2.2 Introduction success rate. The number of queen bees present in hives 14 61 days after introduction at each apiary, for each of the ages queen bees were caught from their mating nucleus or queen bank. Unless otherwise indicated, 20 queen bees were introduced into each apiary at each date. 2.3 Early performance success. The number of queen bees present in hives 62 15 weeks after introduction, for each apiary, for each of the ages queen bees were caught from their mating nucleus or queen bank. Unless otherwise indicated, 20 queen bees were introduced into each apiary at each date. 2.4 Satisfactory performance success. The number of queen bees present 64 present 15 weeks after introduction displaying satisfactory performance for each of the ages that queens were caught from their mating nucleus or queen bank.