Hygienic behaviour in the honeybee (Apis mellifera) has been shown to be an effective control mechanism against brood diseases such as chalkbrood and AFB. Chalkbrood has proven to be problematic for the Australian honey industry since it was identified here in 1993. Hygienic behaviour is a much studied trait. Rothenbuhler investigated the genetic basis of hygienic behaviour, proposing a two-gene model to explain the uncapping and removal of dead brood. His elegant experiment remains the textbook example of a behavioural genetic study. Although this model has been challenged, it is still generally agreed that a small number of unlinked genes produce a large effect on hygienic behaviour, that hygienic alleles are recessive and are inherited in a Mendelian manner. Experimental backcross colonies were produced from an inbred hygienic line and an inbred non-hygienic line, both provided by Dr. Marla Spivak, University of Minnesota. These backcross colonies were assessed for hygienic behaviour using a standard assay. Statistical analyses of the field data indicated that the genetic basis of the trait was more complex than either the simple Mendelian and widely accepted two-gene or three-gene models that have been proposed previously. Molecular techniques, linkage mapping and QTL analysis then were employed to determine how many loci directly influence hygienic behaviour and the relative level of influence and location of each locus within the genome of A. mellifera. Full multipoint linkage analysis by Mapmaker v3.0 software produced a new genetic map of the honeybee comprised of 358 marker loci ordered over 25 linkage groups spanning a total distance of 3406.2 cM. The average distance between each marker was 9.5 cM. QTL analysis of the experimental data identified seven putative genetic markers associated with hygienic behaviour. QTLs located on linkage groups 2, 4, 6 and 22 were detected for both overall hygienic behaviour and uncapping behaviour only. Individually, each QTL is of relatively small effect with each explaining only 9% � 15% of the variance in hygienic levels observed. Collectively, the putative QTLs identified here explain 79.4% of the observed variance in the expression of hygienic behaviour. These results indicate that there are many genes of low to moderate effect rather than few genes of large effect involved in this complex behavioural trait. This is typical of inherited quantitative traits which do not exhibit Mendelian phenotypic ratios. DNA extracted from the brood samples taken during testing of commercial stock, and from individual bees identified as either highly hygienic or non-hygienic in a reciprocal backcross experiment, were screened with the candidate markers associated with putative QTLs to test their diagnostic power. Unfortunately, none have produced reliably diagnostic DNA profiles. As we have now shown that hygienic behaviour is a polygenic, quantitative trait, simple diagnostic markers for Rothenbuhler's 'uncapping' and 'removal' genes are unlikely to be achieved. Our results show that the most likely way to improve disease resistance in Australian stock is via traditional methods of recurrent selection. The project was responsible for the importation of new genetic material into Australia from the United States. This hygienic stock has been well received by industry, has been widely disseminated, and incorporated into local breeding programs. We hope that it has lead to a general improvement in the level of disease resistance in Australian commercial bees.

Thesis (PhD.(Zoology))--University of Pretoria, 2006. / Abstract in English. Includes bibliographical references.

Philosophiae Doctor - PhD / The South African isolate of the Black Queen-Cell Virus (BQCV), a honeybee virus, was previously found to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a 5'-proximal ORF encoding a putative replicase protein and a 3'-proximal ORF encoding a capsid polyprotein.A reverse transcriptase PCR (RT -PCR) assay was developed for the detection of BQCV and acute bee-paralysis virus (ABPV). Complete genomes sequences w ere used to design unique PCR primers within a l-kb region from the 3' end of both genomes to amplify a fragment of 70.0 bp from BQCV and 900 bp from ABPV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing BQCV and ABPV. Sensitivities were of the order of 130 genome equivalents of purified BQCV and 1600 genome equivalents of ABPV. / South Africa

Philosophiae Doctor - PhD / Propagation methods for honeybee viruses have not changed since these viruses were discovered. There are no suitable cell lines or cell culture techniques available for honeybee viruses. Honeybee viruses have to be manually injected with virus in order for the virus to multiply and be extracted. With the presence of inapparent viruses which could co-infect pupae, a method for pure virus propagations needs to be found. Recombinant baculovirus systems have been used extensively to produce foreign proteins from different viruses using vectors and recombinant technology. In this chapter we inserted the capsid gene from BQCV into a transfer vector under the control of the p10 promoter of Autographa californica. Fractions of the sucrose gradient containing the virus like particles (VLPs) were seen under the electron microscope. A Western blot showed the four capsid proteins at the expected sizes for BQCV capsid. This study therefore has shown that a heterologous system such as baculovirus can be used for virus like particle production. Infectious virus technology has helped gain insight into how viruses work. Using this technology altering honeybee viruses could be used to observe different functionalities of the viruses. An attempt was made to interchange the open reading frames of ABPV and BQCV to observe any changes in virus assembly and infectivity. A fusion PCR strategy was employed to interchange the 5’ and 3’ ORFs of APBV and BQCV. The strategy however was unsuccessful. Alternative strategies could improve the chances of obtaining a chimeric virus. / South Africa

Honeybee queens produce a number of pheromones that influence the behaviour and physiology of worker bees. The mandibular gland secretion of queens, the major pheromone source, suppresses the formation of emergency queen cells, worker reproduction and coordinates the social organisation of the colony. A study of analytical procedures for honeybee queen mandibular gland pheromone was undertaken, with the aim of doing multiple analyses of the same individual over a period of time. Attention was given to developing new non-destructive sampling methods that would help to characterize signal changes. This study involves the characterisation of non-destructive sampling devices that are highly selective and sensitive towards extraction of mandibular pheromone. Two polymer based sampling techniques, solid phase micro extraction and silicone rubber tubing, compatible with gas chromatography were studied. A solvent extract, of mandibular pheromone was analysed by gas chromatography (GC) and employed as a tested reference method for the two newly developed techniques. Direct sampling with solid phase micro extraction fibres at the glandular openings at the base of the mandibles is a non-destructive method that met our objectives. Mandibular gland secretions from living honeybee queens were sampled with polar and non-polar fibres. Non-polar fibres were saturated with Bis(trimethylsilyl)triflouroacetamide (BSTFA) prior to mandibular pheromone extraction. Treatment of the polymer devices with derivatising agent enhances extraction of polar components of the mandibular pheromone. BSTFA saturated non-polar fibres with a low-polarity column gave consistent results compared to polar fibres with a mid-polar column. The results confirmed that the solid phase micro extraction technique is a sensitive and non-destructive method that can ideally be used to analyse insect secretions particularly in tracking temporal changes in the secretion composition during an individual’s life. Silicone rubber tubing consisting of polydimethylsiloxane was explored as an alternative sampling technique for pheromones from living individuals. Prepared One cm long silicone rubber tubing was saturated with BSTFA prior to mandibular pheromone extraction to enhance extraction of polar components. Preliminary studies done on mandibular pheromone standards sampled with this method showed promising results. However, queen mandibular secretion analyses were characterized by low recovery of pheromonal compounds. The new polymer based techniques that we employed isolated the mandibular pheromones from living honeybee queens directly from the mandibles. The pheromonal components of the mandibular gland secretion were successfully analysed. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Chemistry / unrestricted

Thesis (MSc (Botany and Zoology))--University of Stellenbosch, 2007. / Honeybee queens typically inhibit the reproductive development of workers in the colony. However, African, Apis mellifera scutellata, honeybee queens seem to have little effect on neighbouring A. m. capensis honeybee workers as is evident in the huge losses of African honeybee colonies due to the invasion by ‘social parasitic’ Cape honeybees (pseudoclones). Certain factors; such as queen and brood presence, the level of colony defence and food availability may render host colonies more vulnerable to invasion by the Cape worker honeybees. In this study host African colonies were split to determine whether a “window of opportunity” existed for Cape honeybee infiltration and thus critical to the capensis problem. Nine African colonies were infected with native and pseudoclone Cape workers over different time periods; before, during and after splitting (treatments). I measured survival rates, as well as reproductive and pheromone development of introduced workers. The effect of brood pheromones on Cape worker reproduction was also examined. Approximately 70% of all workers were removed within 72 hours, a critical period to avoid detection by Cape workers. Queen absence significantly affected the success rate of intrusion and establishment by Cape honeybee workers (GLZ; Wald χ² = 4.49, df = 1, P = 0.033). 21% of 21-day old pseudoclones survived African queenless colonies and only 6% queenright colonies. Native Cape workers showed no difference in survival rates between African queenless (12%) and queenright (11%) colonies. Looking at introduction time, considerably more pseudoclone honeybee workers survived in treatment 1 than did native Cape honeybee workers while for treatment 3 the converse was true. These data show no obvious ‘window of opportunity’ surrounding the swarming process promoting Cape honeybee infiltration and establishment of African honeybee colonies, however the period immediately prior to colony fission represents the best opportunity for invasion by pseudoclones. As for ovary and mandibular gland secretion development, all surviving pseudoclones, irrespective of A. m. scutellata queen presence, fully developed their ovaries and concomitantly produced a mandibular gland secretion dominated by 9- oxo-2-decenoic acid (9ODA). Native Cape workers showed low levels of ovary development in queenright host colonies (8-17%) but this was not true for queenless colonies, with all but one worker developing their ovaries when introduced during and after splitting. Only 40% of native Cape workers introduced before splitting developed their ovaries suggesting that queen pheromones in the three days before splitting retarded ovary development in native Cape workers. These data strengthens the suggestion that the pseudoclone honeybee workers have advanced along the queen-worker developmental continuum. Preliminary studies on brood pheromones, an important factor regulating worker reproduction, indicated that Cape workers reproduce quicker and more eggs when exposed to African brood pheromones, compared to both A. m. capensis brood pheromones and no brood pheromones. Pheromones produced by African larvae therefore do not simply inhibit Cape worker reproductive development but accelerate the commencement of egg laying by these workers. On the whole, host African colonies, especially in the absence of their queen, appear vulnerable surrounding colony fission to invasion by both Cape honeybee worker populations even though there are low survival rates. I conclude that these two Cape honeybee worker populations do differ significantly regarding their reproductive capacity and ability in becoming social parasites.

Abstract available in PDF file. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1989.

The occurrence of Nosema disease, tracheal mites and the “pseudo-parasitic” behaviour of Cape honeybee workers when placed amongst African honeybees – known as the Cape Bee Problem – were studied over a 18 month period. Three surveys, approximately 6 months apart were done. The aims of this study were to establish the distribution and severity of the diseases and compare the disease with the presence of the Cape Bee Problem. Before this survey commenced European Foul Brood disease, Sacbrood (virus), Nosema, Brood nosema, and Tracheal mite have sporadically been reported in the summer rainfall region of South Africa. In the first survey 1005 colonies in 61 apiaries were surveyed, 803 colonies in 57 apiaries in the second, and 458 colonies in 41 apiaries in the third. Samples for disease and parasite analysis were taken at 4 colonies per apiary. Ten colonies per apiary were inspected for Cape Problem Bees, and samples of workers were collected and dissected at each of these colonies. Even with the addition of apiaries to 'fill-up' lost colonies during the second survey, 63% of all colonies were lost by the third survey. There was only a small difference in colony loss between sedentary and migratory beekeepers of 22% compared to 27%. Nosema was more prevalent amongst commercial beekeepers and increased in migratory operations during the survey period. The percentage of colonies infected increased during the survey period from 23% to 32% to 34%. The placement of colonies in Eucalyptus plantations may boost infection. Trachea mites seem to have spread quite rapidly in South Africa since its discovery. This parasitic mite was present in all regions, although in low numbers in three most northern regions. Sedentary colonies had higher levels of infestation than migratory colonies. The number of colonies infested diminished over the survey period, which may be a result of general colony loss. The Cape Problem Bee was less of a problem than anticipated. Colonies succumbed to Cape Problem Bees in all regions. When beekeepers reported high levels of infestation in their bee stocks the colonies would be dead within six months. In apiaries with low infestation the die-out was slower.

The honeybees Apis cerana and Apis mellifera are derived from the same ancestral base about two million years ago. With speciation and evolution, they have acquired many advanced living skills in common, but have also evolved very different living strategies due to different distributions. This thesis is an intensive study of the biology of the mixed-species colonies of these species, the aims of which were to investigate their behavioural relationships and uncover the evolutionary conserved features of their behaviours subsequent to speciation. The results show that the two species can form a stable society to perform normal tasks. First, workers of both species in the mixed-colonies could form the typical retinue behaviour to hetero-species queens, thus indicating that queen pheromones could be spread to and by both species. Secondly, both species did not show significantly different ovarian activation under hetero-species queens, suggesting that the queen pheromones more likely play a role of "honest signal" rather than a "repression" substance in the honeybee colonies. Thirdly, both species could mutually decode each other‘s waggle dances, with unexpectedly low misunderstanding; revealing that the dance language in a dark environment is quite adaptive for cavity-nesting honeybees. Fourthly, workers of both species could cooperate with each other in comb construction, although the combs they built contain many irregular cells. Interestingly, A. cerana workers could be stimulated by A. mellifera workers to perform this task, thus confirming self-organization theory in the colony. Fifthly, A. mellifera workers behaved more "defectively" in thermoregulation, but perhaps because A. cerana workers are more sensitive to changes in hive temperature. Given these differences in strategy, A. mellifera workers‘ performance might in fact reduce conflicts. Lastly, when faced with threats of predatory wasps, both species engaged in aggressive defence. Although they did not learn from each other‘s responses, species-specific strategies were adopted by each of them so that the defence of the mixed-colonies is very effective. I conclude that the two species can adapt to each other‘s efforts and task allocation is reasonably organized allowing mixed-species colonies to reach stability. These results suggest that all of the social behaviours discussed here were highly conserved following speciation. This thesis could provide some clues for the study of honeybee evolution from open-nesting to the transition of cavity-nesting.

One of the benefits of colonial living in insect societies is the ability to build a nest which enables the maintenance of a homeostatic microenvironment. The detrimental and uncertain effects of fluctuating ambient conditions are thus avoided. An extensive amount of work has documented the regulation of respiratory gases and temperature by honeybee (Apis mellifera) colonies but relatively little is known of their water relations. Nest humidity influences the fitness of the honeybee colony by affecting adult and brood mortality, microbial and parasitic growth, nectar concentration and thermoregulation. This study aims at determining whether honeybee colonies are able to actively regulate humidity within their nest or whether humidity is stabilised merely as consequence of other socially regulated parameters. As a first step in understanding water relations in a hive, the daily, seasonal and two-dimensional humidity patterns are described in diverse contexts: various subspecies, nest architectures, ambient climates and colony conditions. The humidity in the brood nest of a healthy honeybee colony does not show a daily pattern: mean hourly RH remains between 50 and 60 % and high vapour pressure deficit results in a large evaporative capacity. Two-dimensional humidity patterns show that a vapour pressure gradient exists from the central brood area to the periphery of a hive. This finding suggests possible active regulation by workers and to test this idea we determined the behavioural response of a group of workers to a humidity gradient. Young honeybee workers in the absence of brood exhibit a weak hygropreference for approximately 75% RH. When brood is present the expression of this preference is further weakened, suggesting that workers tend to the brood by distributing evenly in the gradient. In addition, fanning behaviour is shown to be triggered by increasing humidity adding to our understanding of this behaviour. Although these results suggest that humidity in honeybee colonies is actively controlled by workers, passive mechanisms are also involved in the observed patterns. Cocoons that are spun by the larvae accumulate in cells and these hygroscopic cocoons contribute to passive stabilisation of humidity. Old comb containing cocoons absorb 11 % of its own mass in water when placed in high humidity and this water can readily evaporate into the atmosphere when humidity decreases. This buffering effect may increase brood survivorship by maintaining a high and stable humidity in the brood cells. This study contributes to our understanding of the complex mechanisms that govern microclimatic regulation in social insect nests and specifically the active and passive mechanisms that ensure homeostasis of honeybee nest humidity. Copyright / Dissertation (MSc)--University of Pretoria, 2008. / Zoology and Entomology / unrestricted