UUnniivveerrssiittyy ooff PPrreettoorriiaa eettdd –– LLuubbbbee,, AA ((22000055)) The phenomenon of Apis mellifera capensis laying workers in Apis mellifera scutellata colonies in the summer rainfall region of South Africa by Annelize Lubbe Submitted in fulfillment of the requirements for the degree Magister Scientiae In the Department of Zoology and Entomology Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria October 2005 UUnniivveerrssiittyy ooff PPrreettoorriiaa eettdd –– LLuubbbbee,, AA ((22000055)) ABSTRACT African honeybee workers, Apis mellifera scutellata can activate their ovaries under queenless conditions to produce male (haploid) offspring. In contrast, laying workers of the Cape honeybee, Apis mellifera capensis, produce female (diploid) offspring via thelytokous parthenogenesis. In the early 1990’s colonies of A. m. capensis were transported into the distribution area of A. m. scutellata (corresponding to the summer rainfall region of South Africa), leading to the “capensis calamity”. Laying workers of A. m. capensis invaded and killed colonies of A. m. scutellata leading to losses of thousands of commercial colonies. A survey of the apiaries in the A. m. scutellata region was conducted over 18 months from 1997 to 1998, to determine the extent of the problem. It was found that the parasites were established in many apiaries throughout the distribution range of A. m. scutellata. As the problem seemed to be more severe with commercial and migratory beekeepers, the apiaries surveyed were divided into risk groups related to beekeeping practices. The low risk group included apiaries of beekeepers in areas that are separated from commercial beekeepers and their high risk activities. These low risk colonies were sedentary vs the migration to high risk ares eg. Aloes, sunflower pollination areas, citrus and other fruit pollination areas of the high risk apairies. The apiaries were monitored and records of the colonies’ condition were taken. Samples of workers were collected for dissection. It was found that the low risk group had a lower rate of infection, a higher production of brood and honey and a higher rate of survival over a 12 month period. The significant characteristics for identifying infection of a colony were determined as being the colour of the workers, the brood pattern, the presence of multiple eggs in cells and the presence of the queen. Indeed, the presence of dark workers with a black scutellum, an irregular brood pattern, the presence of ii UUnniivveerrssiittyy ooff PPrreettoorriiaa eettdd –– LLuubbbbee,, AA ((22000055)) multiple eggs in cells and the absence of queen were all prevalent in infected colonies. As sample of workers from all inspected colonies were dissected and the average ovariole counts as well of the development stage of the ovaries proved to be significant variables in the diagnosis. Other variables eg. Ovariole counts, spermatheca size and aggression proved to be not significant, but in conjunction with other variables, could be used for diagnosis. The genetic nature of the invasive parasitic population was determined using polymerase chain reaction (PCR) analysis. Nine loci were tested and the DNA fingerprints of all individuals sampled throughout the summer rainfall region were proved to be identical. This genetic identity led to the descripter of these individuals as a pseudoclone. In contrast, workers of A. m. scutellata were tested with the same loci and showed the normal distribution of an out-breeding population. In order to investigate the spread of the parasite within an apiary, colonies were exposed to heavily infected hives and inspected regularly. Ninety five percent of the colonies had either died or absconded within 12 months. It is concluded that this phenomenon of social parasitism is the consequence of apicaultural activities and that it can be managed by adopting low risk beekeeping practices. iii UUnniivveerrssiittyy ooff PPrreettoorriiaa eettdd –– LLuubbbbee,, AA ((22000055)) FRONTICEPIECE Figure showing Apis mellifera scutellata workers with a queen. iv UUnniivveerrssiittyy ooff PPrreettoorriiaa eettdd –– LLuubbbbee,, AA ((22000055)) CONTENTS ABSTRACT.....................................................................................................ii FRONTICPIECE..............................................................................................iv CONTENTS.....................................................................................................v LIST OF FIGURES..........................................................................................ix LIST OF TABLES............................................................................................xii PUBLICATIONS ARISING FROM THIS STUDY.............................................xiv ACKNOWLEDGEMENTS................................................................................xv CHAPTER 1 INTRODUCTION.............................................................................................1 1.1 SHORT NOTES ON BEEKEEPING HISTORY IN SOUTH AFRICA .....................................................................1 1.2 BACKGROUND TO THE CAPENSIS CALAMITY.......................3 1.3 HISTORY AND CONSEQUENCES OF THE ‘CAPENSIS CALAMITY’..............................................................6 1.4 ACTION AGAINST THE ‘CAPENSIS CALAMITY’.......................8 1.5 RESEARCH PROJECTS ON THE CAPE PROBLEM BEE.........9 1.5.1 Information transfer................................................................9 1.5.2 Survey of capensis laying workers and diseases...................9 1.5.3 Penetration of the wild population..........................................10 1.5.4 Laying worker characteristic – inheritance..............................10 1.5.5 Controlling element ................................................................10 1.5.6 Management practices...........................................................11 1.5.7 A. m. scutellata reservoir........................................................11 1.5.8 Modes of infestation/invasion of A. m. scutellata colonies......11 1.5.9 Absconding swarms...............................................................11 1.5.10 Other projects resulting from original Working Group Projects..................................................................................11 1.5.10.1 Hybridisation of Apis mellifera capensis and Apis mellifera scutellata: v UUnniivveerrssiittyy ooff PPrreettoorriiaa eettdd –– LLuubbbbee,, AA ((22000055)) Does it occur and contribute to the capensis problem?..........11 1.5.10.2 Dispersal of the pseudoclone in survey apiaries....................12 1.6 SCOPE OF THIS THESIS...........................................................12 1.7 REFERENCES............................................................................13 CHAPTER 2 SURVEY FOR CAPENSIS LAYING WORKERS IN APIARIES.....................15 2.1 INTRODUCTION.........................................................................15 2.2 MATERIALS & METHODS..........................................................16 2.2.1 Regions and background .......................................................16 2.2.2 Observations..........................................................................19 2.2.2.1 External observations.........................................................19 2.2.2.2 Internal observations ..........................................................21 2.2.3 Sampling bees for dissection..................................................25 2.2.4 Dissection of bees..................................................................26 2.2.5 Classification of apiaries by risk of capensis infection............27 2.2.6 Data Analysis.........................................................................28 2.3 RESULTS......................................................................................28 2.3.1 Colony and apiary classification.............................................28 2.3.2 Observations..........................................................................29 2.3.2.1 Colour of worker bees.........................................................29 2.3.2.2 Quality of brood pattern......................................................30 2.3.2.3 Colony defensive behaviour................................................31 2.3.2.4 Numbers of ovarioles/ovary................................................31 2.3.2.5 Ovariole development.........................................................32 2.3.2.6 Queen survival....................................................................33 2.3.2.7 Colony size.........................................................................33 2.3.3 Infection rates of colonies in three risk situations...................34 2.3.4 Productivity of colonies in three risk situations.......................36 2.3.5 Survival of colonies in three risk situations.............................38 2.4 DISCUSSION..............................................................................40 2.4.1 Characteristics used for diagnosis..........................................41 2.4.1.1 Colour of worker bees.........................................................41 vi UUnniivveerrssiittyy ooff PPrreettoorriiaa eettdd –– LLuubbbbee,, AA ((22000055)) 2.4.1.2 Brood pattern......................................................................41 2.4.1.3 Colony defensive behaviour ...............................................41 2.4.1.4 Number of ovarioles/ovary..................................................41 2.4.1.5 Ovariole development.........................................................42 2.4.1.6 Queen survival....................................................................43 2.4.1.7 Colony size.........................................................................43 2.4.2 Infection rates.........................................................................43 2.4.3 Productivity.............................................................................44 2.4.4 Survival ..................................................................................44 2.4.5 Shortcomings of the survey....................................................44 2.5 CONCLUSIONS..........................................................................45 2.6 REFERENCES............................................................................46 CHAPTER 3 GENETIC BACKGROUND OF CAPENSIS LAYING WORKERS..................49 3.1 INTRODUCTION.........................................................................49 3.2 MATERIALS & METHODS..........................................................50 3.2.1 DNA extraction procedure......................................................51 3.2.2 Polymerase chain reaction (PCR) procedures.......................52 3.2.3 DNA sequencing....................................................................54 3.3 RESULTS....................................................................................55 3.4 DICSUSSION..............................................................................61 3.5 REFERENCES............................................................................63 CHAPTER 4 EFFECT OF THE PSEUDOCLONE ON COLONIES IN AN APIARY ............66 4.1 INTRODUCTION.........................................................................66 4.2 MATERIALS & METHODS..........................................................66 4.2.1 Observations..........................................................................67 4.2.1.1 External observations.........................................................68 4.2.1.2 Internal observations ..........................................................68 4.2.2 Sampling bees for dissection..................................................68 4.2.3 Dissection of bees..................................................................68 vii UUnniivveerrssiittyy ooff PPrreettoorriiaa eettdd –– LLuubbbbee,, AA ((22000055)) 4.3 RESULTS....................................................................................69 4.4 DISCUSSION..............................................................................74 4.5 REFERENCES............................................................................75 CHAPTER 5 SUMMARY......................................................................................................77 5.1 SUMMARY OF RESULTS OBTAINED........................................77 5.2 POSSIBLE CONCLUSIONS........................................................78 5.3 RECOMMENDATIONS TO BEEKEEPERS ................................79 5.4 REFERENCES............................................................................80 APPENDIX A..................................................................................................82 viii UUnniivveerrssiittyy ooff PPrreettoorriiaa eettdd –– LLuubbbbee,, AA ((22000055)) LIST OF FIGURES Figure 1.2 Geographical distribution of A. m. capensis (South of mountain ranges 1 - 4), A. m. scutellata (North of mountain ranges 5 - 10) and the hybrid zone between mountain ranges 1 - 4 and 5 - 10 as drawn from Hepburn & Crewe (1991). The numbers are described in the paragraph above. Figure 2.2.1 Map of South Africa showing the regions and sites where the surveys were conducted. The sites are indicated with letters close to where the apiaries were geographically located. Figure 2.2.2 The Inspector, on the left, inspects a brood frame while the recorder, (the author of this thesis) on the right, recorded the observations. Figure 2.2.3 Laying worker brood showing most of the signs of infection. The arrows point to the following characteristics: a. multiple eggs, b. raised cappings, c. & d. larvae of different ages in a group, e. uncapped prepupa. Figure 2.2.4 Yellow (level 3) honeybees, note the yellow scutellum indicated by the arrow marked a. A single black capensis bee (level 5) with a black abdomen and black scutellum, indicated by the arrow marked b. Figure 2.2.5 Equipment used to mark queens, TippEx, queen marker lid and queen cage. Figure 2.2.6 Sample bottles that were used to take samples of worker bees. ix UUnniivveerrssiittyy ooff PPrreettoorriiaa eettdd –– LLuubbbbee,, AA ((22000055)) Figure 2.2.7 Stages of ovariole development: a. Stage I ovarioles showing no development and no spermatheca. b. Stage IV ovarioles showing mature oocytes and the large spermatheca is indicated with the arrow. Figure 2.3.1 Colour ranking of worker bees in infected and non-infected colonies in apiaries classified into high, medium and low risk situations. Figure 2.3.2 Ranking of the quality of the brood pattern for infected and non- infected colonies in apiaries classified into high, medium and low risk situations. Figure 2.3.3 Level of colony defensiveness in the three risk groupings divided into infected and non infected colonies. Figure 2.3.4 Level of ovariole development in the workers of infected and non-infected colonies in apiaries classified into high, medium and low risk situations. Figure 2.3.5 Percentage infected colonies in three risk situations over time using the less conservative method of classifying colonies as infected. Figure 2.3.6 Brood production in colonies for three risk groups over time. Vertical lines denote 95% confidence intervals. Figure 2.3.7 Honey production in colonies of the three risk groups over time. Vertical lines denote 95% confidence intervals. Figure 2.3.8. Percentage survival of colonies in apiaries of the three risk groups during the three surveys. Figure 3.2.1 Two ml Eppendorf tubes that were used for DNA extraction. x