BACKGROUND: The honey bee is an important model system for increasing understanding of molecular and neural mechanisms underlying social behaviors relevant to the agricultural industry and basic science. The western honey bee, Apis mellifera, has served as a model species, and its genome sequence has been published. In contrast, the genome of the Asian honey bee, Apis cerana, has not yet been sequenced. A. cerana has been raised in Asian countries for thousands of years and has brought considerable economic benefits to the apicultural industry. A cerana has divergent biological traits compared to A. mellifera and it has played a key role in maintaining biodiversity in eastern and southern Asia. Here we report the first whole genome sequence of A. cerana. RESULTS: Using de novo assembly methods, we produced a 238 Mbp draft of the A. cerana genome and generated 10,651 genes. A.cerana-specific genes were analyzed to better understand the novel characteristics of this honey bee species. Seventy-two percent of the A. cerana-specific genes had more than one GO term, and 1,696 enzymes were categorized into 125 pathways. Genes involved in chemoreception and immunity were carefully identified and compared to those from other sequenced insect models. These included 10 gustatory receptors, 119 odorant receptors, 10 ionotropic receptors, and 160 immune-related genes. CONCLUSIONS: This first report of the whole genome sequence of A. cerana provides resources for comparative sociogenomics, especially in the field of social insect communication. These important tools will contribute to a better understanding of the complex behaviors and natural biology of the Asian honey bee and to anticipate its future evolutionary trajectory.

Honey bees are fundamental for maintaining biodiversity in our ecosystems, but a recent decline in honey bee colonies has caused a growing concern for honey bee health worldwide. One component of colony collapses is Nosema (Microsporidia), which is associated with colony collapses in many subtropical regions. However, infection by Nosema is also known to accumulate within the honey bee hive during overwintering in colder climates. In this study, the prevalence of Nosema is compared between four honey bee subspecies during fall and winter and is focused on two hypotheses: 1) infection by Nosema is more prevalent in honey bees during winter and 2) infection by Nosema differs between different honey bee subspecies. Bees were dissected, and their guts were analysed for Nosema spores using a light microscope. Results showed a difference in amount of Nosema infected colonies between winter and fall. Also, results showed a difference between Buckfast bee (A. mellifera hybrid) and Carniolan bee (A. mellifera carnica) in Nosema infected colonies during the fall period. These results indicate that infection by Nosema in cold climates might be more prevalent than previously thought. Additionally, there might be differences in resilience between honey bee subspecies, but infection of Nosema seem to depend less on subspecies than season. More research is needed on Nosema in cold regions to assess the effect of Nosema on honey bees in Sweden and worldwide to prevent future colony collapses of honey bees.

Most animals contain chronic microbial infections that inflict no harm on their hosts. Recently, the gut microflora of humans and other animals have been characterized. However, little is known about the forces that shape the diversity of these bacterial communities. In this work, comparative genomics was used to investigate the evolutionary dynamics of host-adapted bacterial communities, using Wolbachia infecting arthropods and Lactobacteria infecting bees as the main model systems. Wolbachia are maternally inherited bacteria that cause reproductive disorders in arthropods, such as feminization, male killing and parthenogenesis. These bacteria are difficult to study because they cannot be cultivated outside their hosts. We have developed a novel protocol employing multiple displacement amplification to isolate and sequence their genomes. Taxonomically, Wolbachia is classified into different supergroups. We have sequenced the genomes of Wolbachia strain wHa and wNo that belong to supergroup A and B, respectively, and are present as a double-infection in the fruit-fly Drosophila simulans. Together with previously published genomes, a supergroup comparison of strains belonging to supergroups A and B indicated rampant homologous recombination between strains that belong to the same supergroup but were isolated from different hosts. In contrast, we observed little recombination between strains of different supergroups that infect the same host. Likewise, phylogenetically distinct members of Lactic acid bacteria co-exist in the gut of the honeybee, Apis mellifera, without transfer of genes between phylotypes. Nor did we find any evidence of co-diversification between symbionts and hosts, as inferred from a study of 13 genomes of Lactobacillus kunkeei isolated from diverse bee species and different geographic origins. Although Lactobacillus kunkeii is the most frequently isolated strain from the honey stomach, we hypothesize that the primary niche is the beebread where the bacteria are likely to contribute to the fermentation process. In the human gut, the microbial community has been shown to interact with the immune system, and likewise the microbial communities associated with insects are thought to affect the health of their host. Therefore, a better understanding of the role and evolution of endosymbiotic communities is important for developing strategies to control the health of their hosts.

This dissertation examines some important physiological and pheromonal factors regulating foraging and colony growth in honey bee colonies. The first study analyzed effects of soybean trypsin inhibitor (SBTI) on the development of hypopharyngeal gland, midgut enzyme activity and survival of the honey bee. In this study newly emerged caged bees were fed pollen diets containing three different concentrations of SBTI. Bees fed 1% SBTI had significantly reduced hypopharyngeal gland protein content. This study indicated that nurse bees fed a pollen diet containing at least 1% SBTI would be poor producers of larval food. In the second study nurse bee biosynthesis of brood food was manipulated using SBTI, and the resulting effects on pollen foraging were measured. Experimental colonies were given equal amounts of SBTI treated and untreated pollen. SBTI treatments had significantly lower hypopharyngeal gland protein content than controls. There was no significant difference in the ratio of pollen to non-pollen foragers and pollen load weights collected between the treatments. These results supported the pollen foraging effort predictions generated from the direct independent effects hypothesis. In the third study we tested whether brood pheromone (BP) regulated queen egg laying via modulation of worker-queen interactions and nurse bee rearing behaviors. This experiment had BP and control treatments. Queens in the BP treatment laid greater number of eggs, were fed for a greater amount of time and were less idle. Significantly more time was spent in cell cleaning by the bees in BP treatments. The results suggest that brood pheromone regulated queen egg-laying rate by modulating worker-queen interactions and nurse bee rearing behavior. The final study of this dissertation focused on how dose-dependent BP-mediated division of labor affected the partitioning of non-foraging and foraging work forces and the amount of brood reared. Triple cohort colonies were used and there were three treatments, Low BP, High BP and Control. Low BP treatments had significantly higher ratio of pollen to non-pollen foragers and greater pollen load weights. Low BP treatment bees foraged at a significantly younger age. This study has shown that BP elicits dose-dependent modulation of foraging and brood rearing behaviors.

To enhance the scientific utility and practical application of the honey bee genome and assign the linkage groups to specific chromosomes, I identified chromosomes and characterized the karyotype of the sequenced strain DH4 of the honey bee. The primary analysis of the karyotype and ideogram construction was based on banding and Fluorescence In Situ Hybridization (FISH) for rDNA detection. FISH confirmed two locations for the NOR on telomeric regions of chromosomes 6 and 12 plus an additional less frequent signal on chromosome 1, all three of which were confirmed with silver staining (AgNO3). 4’6-diamidino-2phenylindole (DAPI), and CBanding methods were used to construct the primary ideograms that served as a basis to further identify the chromosomes and locate important structures. The primary map was compared with Giemsa banding, AgNO3-banding, Trypsin banding, and R-banding. The karyotype of the honey bee was established as two metacentric chromosomes (1 and 10), two submetacentric with ribosomal organizer (6 and 12), four submetacentric heterochromatic chromosomes (16, 15, 4 and 13), four euchromatic subtelocentric chromosomes (2, 8, 11 and 14) and four acrocentric chromosomes (3, 5, 7 and 9). In situ nick-translation banding methods were used to verify the heterochromatin distribution. The cytogenetic maps of the honey bee karyotype represented in the ideograms were subsequently used to place 35 mapped BACs (Solignac et. al. 2004) of Solignac’s BAC library. As the BACs hybridized to multiple sites, the mapping was based on strength and frequency of the signals. Location and position of the BACs was compared with those published in the different version of Map Viewer of the NCBI and BeeBase web sites. 10 BACs were confirmed with the last version of Map Viewer V4, 12 BACs were mapped based on high frequency and agreement with the earlier version of Map Viewer. 14 BACs were mapped as confirmed based on moderate frequency of the signal and agreement with the last version of MVV, most of these BACs hits as a secondary signal.

This dissertation examines some important physiological and pheromonal factors regulating foraging and colony growth in honey bee colonies. The first study analyzed effects of soybean trypsin inhibitor (SBTI) on the development of hypopharyngeal gland, midgut enzyme activity and survival of the honey bee. In this study newly emerged caged bees were fed pollen diets containing three different concentrations of SBTI. Bees fed 1% SBTI had significantly reduced hypopharyngeal gland protein content. This study indicated that nurse bees fed a pollen diet containing at least 1% SBTI would be poor producers of larval food. In the second study nurse bee biosynthesis of brood food was manipulated using SBTI, and the resulting effects on pollen foraging were measured. Experimental colonies were given equal amounts of SBTI treated and untreated pollen. SBTI treatments had significantly lower hypopharyngeal gland protein content than controls. There was no significant difference in the ratio of pollen to non-pollen foragers and pollen load weights collected between the treatments. These results supported the pollen foraging effort predictions generated from the direct independent effects hypothesis. In the third study we tested whether brood pheromone (BP) regulated queen egg laying via modulation of worker-queen interactions and nurse bee rearing behaviors. This experiment had BP and control treatments. Queens in the BP treatment laid greater number of eggs, were fed for a greater amount of time and were less idle. Significantly more time was spent in cell cleaning by the bees in BP treatments. The results suggest that brood pheromone regulated queen egg-laying rate by modulating worker-queen interactions and nurse bee rearing behavior. The final study of this dissertation focused on how dose-dependent BP-mediated division of labor affected the partitioning of non-foraging and foraging work forces and the amount of brood reared. Triple cohort colonies were used and there were three treatments, Low BP, High BP and Control. Low BP treatments had significantly higher ratio of pollen to non-pollen foragers and greater pollen load weights. Low BP treatment bees foraged at a significantly younger age. This study has shown that BP elicits dose-dependent modulation of foraging and brood rearing behaviors.

Brood pheromone of the honey bee, (Apis mellifera) has been shown to elicit a wide array of primer and releaser effects on non‐foragers and foragers leading to the regulation of nursing, pollen foraging, and behavioral development such that the behavior of the colony may be regulated by the amount and condition of the larvae. To date, all studies into the effects of brood pheromone have either used uncharacterized whole extracts or a single blend of brood pheromone characterized from a population of honey bees in France. The variation in the relative proportions of the ten fatty‐acid ester components that characterize brood pheromone and some effects of this variation on pollen foraging and sucrose response thresholds were therefore observed. The objectives met in this dissertation were to determine whether changes in brood pheromone component proportions (blend) or amount communicates larval nutritional status and reports the results of observations of nurses and foragers in response to blends of brood pheromone from deprived and‐non deprived larvae, to measure how changes in brood pheromone blend changed pollen foraging behavior and if such changes could account for the pollen foraging differences between Africanized and European bees, and finally to observe the effects of exposure time on brood pheromone blend and to observe whether non‐foragers made contact with the pheromone. Brood pheromone was found to vary by larval rearing environment, but did not elicit the expected behaviors that would support a cue of nutritional status. Brood pheromone also varied significantly by mitochondrial lineage/population source and responses to brood pheromone appeared to be coadapted to blend, suggesting that brood pheromone may be important in race recognition. Finally, brood pheromone varied significantly over time and was found to be removed from sources by bees, suggesting possible mechanisms for loss of effect. Combined the results of this research indicate that brood pheromone blend differences lead to profound changes in colony behavior related to pollen foraging and food provisioning, providing novel tools for colony manipulation and mechanisms for understanding brood rearing division of labor and chemical communication.

Honey bees (Apis mellifera) are the most economically important insect pollinator of agricultural crops in the United States. Honey bee colonies are required for pollination of approximately one-third of the nation’s fruit, vegetable, nut, and forage crops, with an estimated annual value in the billions of dollars. The economic value of a honey bee colony comes from its population size, as large colonies provide the necessary foraging force required for large-scale crop pollination services. A major component of colony strength is its genetic diversity, a consequence of the reproductive mating strategy of the queen known as polyandry. Despite some inherent risks of multiple mating, several studies have demonstrated significant advantages of intracolonial genetic diversity for honey bee colony productivity. Colony-level benefits include better disease resistance, more stable brood nest thermoregulation, and greater colony growth. Instrumental insemination of honey bee queens is a technique to precisely control queen mating, and thereby creates the opportunity to investigate the effects of intracolonial genetic diversity on colony performance. In this dissertation, I first consider the effects of intracolonial genetic diversity on pollen foraging using colonies headed by queens which were instrumentally inseminated with either one or twenty drones to generate colonies of very high or very low intracolonial genetic diversity, respectively. I found that colonies with high intracolonial genetic diversity amass significantly more pollen and rear more brood than colonies with low intracolonial genetic diversity. Of particular interest, colonies with low intracolonial genetic diversity collected a significantly greater variety of pollen types. I discuss these results in the context of scouting and recruiting, and suggest a more efficient foraging strategy exists among genetically diverse colonies. While intracolonial genetic diversity is positively correlated with collected pollen, its effect on the colony’s ability to process and distribute inbound protein resources is unknown. Again using colonies headed by queens instrumentally inseminated with either one or twenty drones, I studied the effects of intracolonial genetic diversity on pollen consumption and digestion by nurse bees, as well as protein allocation among nestmates by assessing total soluble protein concentration of late instar larvae, and total soluble hemolymph protein concentration in both nurses and pollen foragers. I found that nurse bees from colonies with high intracolonial genetic diversity consume and process more protein than nurses from colonies with low intracolonial genetic diversity, even when given equal access to protein resources. Further, both forager hemolymph protein concentrations and larval total protein concentrations were higher among the colonies with high intracolonial genetic diversity. My findings suggest that protein processing and distribution within a honey bee colony is affected by the social context of the hive. I discuss “worker policing”, and the role of nurse bees in modulating the foraging effort. Finally, I assess the standing genetic variability among several colonies sourced from different genetic and geographic locations. Using microsatellite DNA from workers sampled from each colony, I determined allelic richness, gene diversity, and effective mating frequency for each genetic line. I found differences in all three metrics between lines, and for one line in particular, there was no correlation with genetic variation and effective mating frequency, suggesting non-random mating. My results showed very different levels of intracolonial genetic diversity among naturally mated queens. Because of its impact on colony performance, the importance of maintaining genetic diversity in breeding populations is discussed.

No description available.

Excessive honey bee colony losses all over the world are believed to be caused by multiple stressors. In this thesis, I characterized and quantified pathogen levels in honey bee colonies, studied their interactions with each other and with their associated parasite vectors, examined factors that influence their combined impacts on honey bees and developed methods to manage honey bee viruses so that colony losses can be minimized. My baseline study of virus prevalence and concentration in healthy and unhealthy (showing visible signs of disease) colonies in Canada showed that seven economically important viruses (DWV, BQCV, IAPV, KBV, SBV, ABPV, and CBPV) were all widely distributed in Canada. Differences in concentration and prevalence of some viruses were found between unhealthy and healthy colonies but these differences may have been due in part to seasonal or regional effects. Studies of the impact of viruses on worker bee populations over winter showed different factors were correlated with bee loss in different environments. Spring concentrations of DWV and mean abundance of Varroa (Varroa destructor) were positively correlated with bee loss and negatively correlated with spring population size in outdoor-wintered colonies. Fall concentration of IAPV was negatively correlated with spring population size of colonies in indoor-wintering environments but not in outdoor-environments. My study showed that it is important to consider location of sampling when associating pathogen loads with bee loss with Nosema and BQCV. Seasonal patterns of parasites and pathogens were characterized for each wintering methods (indoor and outdoor). My results revealed lower ABPV and Nosema ceranae prevalence and lower DWV concentration in genetically diverse than genetically similar colonies. I showed that within colony genetic diversity may be an important evolutionary adaptation to allow honey bees to defend against a wide range of diseases. In laboratory studies, I showed that feeding DWV to larvae in the absence of Varroa causes wing deformity and decreased survival rates of adult bees relative to bees not fed DWV. Finally, I showed that RNA silencing can be used to reduce DWV concentrations in immature and adult bees, reduce wing deformity in emerging adults, and increase their longevity relative to controls.