Honey bees (Apis mellifera) may serve as bioindicators of habitat quality for themselves and also other insect pollinators because we can observe, decode, map, and analyze the information encoded in the waggle dance communication behavior, which allows us to know where and when bees are collecting high quality forage. Previously we measured honey bee foraging dynamics for two years (2018-2019) by waggle dance decoding at three geographically distanced sites in Virginia (Blacksburg, Winchester, Suffolk), consisting of different dominant landcover types. Here we use those data on where and when honey bees were finding profitable resources throughout the season to predict the success of a non-Apis bee in these same landscapes. Alfalfa leafcutting bees (Megachile rotundata) are managed, polylectic, solitary, cavity-nesting bees that are widely naturalized in North America. We selected M. rotundata as a model organism to validate the honey bee foraging data because they share some characteristics with other cavity nesting wild bees, but they are a tractable study system because they are commercially reared and can be purchased for study. At each of the three sites, we installed 15 nest box stations, each stocked with nesting materials and 160 M. rotundata cocoons, at varying distances and directions from the original honey bee hive locations. Most importantly, nest box stations were distributed across a range of honey bee foraging propensities, calculated as the mean foraging probability determined from our honey bee waggle dance decoding data, within a 300m buffer around each nest box. We hypothesized that honey bee foraging probability would positively correlate with M. rotundata cocoon production and survival. For two years (2021-2022) from May-August, we monitored the nest boxes and also collected data on the relative abundance of floral resources at each of the 15 stations per site. At the end of each season, we collected nesting materials and counted both M. rotundata along with incidental (i.e., non-M. rotundata) wild bee cocoons. M. rotundata cocoon productivity varied by location (log-likelihood ratio test: χ2 = 311.0, df = 2, p < 0.001), with Winchester as the most productive location (mean cocoon count (95% CI): 26.2 (23.7 to 28.9)), followed by Blacksburg (20.4 (18.2 to 22.9)), and Suffolk (4.4 (3.5 to 5.5)). The abundance of clover, both red and white, had a significant positive effect on ALCB productivity (log-likelihood ratio test: χ2 = 778.36, < 0.001). On the other hand, the number of ALCB cocoons decreased significantly with the count of Trypoxylon wasp cocoons present in the nest boxes (log-likelihood ratio test: χ2 = 54.37, < 0.001). Most importantly, we found that there was an overall negative relationship between honey bee foraging probability and alfalfa leafcutting bee cocoon productivity ((log-likelihood ratio test: χ2 = 55.42, < 0.001), where areas of higher honey bee foraging probability were associated with lower levels of alfalfa leafcutting bee productivity. This surprising result is in the opposite direction to our original hypothesis that preferred honey bee foraging areas in the landscape, as indicated by decoded waggle dance data, would be positively correlated with alfalfa leafcutting bee productivity. These data demonstrate that while honey bees may indeed act as bioindicators to other insect pollinators, this indication will likely be species and context specific and may even specify the opposite direction. / Master of Science in Life Sciences / Recent challenges facing the beekeeping industry have laid bare the fragility of honey bee pollination services, highlighted the role of other bee pollinators, and sparked widespread concern over the effect of declining bee populations on food security and continued ecosystem function and services. Both honey bees and wild bees face similar challenges including pesticides, parasites, pests, and poor nutrition from a lack of flowers in the landscape. Therefore, it is critical that we develop methods to evaluate the landscape's ability to feed bees in order to help them and other pollinators continue providing essential pollination services. There are many ways to measure the quality of a landscape for pollinators, but honey bees offer a unique opportunity to do the work for us: honey bees communicate the location of where they find food to their nestmates through a behavior called the waggle dance. Waggle dances can be observed and the dance language decoded so that we can determine the location of high-quality food sources. Previously, we used honey bee waggle dance data to map where bees are collecting food in three geographically distinct sites (Blacksburg, Winchester, and Suffolk, Virginia). These data allow us to understand where, when, and on what flowers the honey bees were feeding. The goal of this project was to investigate the relationship between honey bee foraging and non-honey bee success across the same three landscapes to determine if honey bees can be used as bioindicators of habitat quality for other bees. We chose Alfalfa leafcutting bees (Megachile rotundata) as a model organism because they are solitary, cavity-nesting bees, like the majority of wild bees. However, as managed pollinators, Alfalfa leafcutting bees (ALCBs) can be purchased commercially and retained in nesting boxes to allow us to gather productivity and survival data, which we can then compare to what the dancing honey bees previously told us about where and when they can collect good food. We hypothesized that areas of the landscape that honey bees had indicated where higher quality would correlate to better ALCB cocoon production and survival. We placed wooden nest boxes, 15 per site, stocked with ALCBs across the same landscapes for which honey bee data had been collected in the previous years and measured their productivity in terms of cocoons produced at each site. ALCB productivity varied by location, with Winchester as the most productive location (mean ALCB count (95% CI): 26.2 (23.7 to 28.9)), followed by Blacksburg (20.4 (18.2 to 22.9)), and Suffolk (4.4 (3.5 to 5.5)). The abundance of clover, both red and white, had a significant positive effect on ALCB productivity (log-likelihood ratio test: χ2 = 778.36, < 0.001). On the other hand, the number of ALCB cocoons decreased significantly with the count of a non ALCB nest box resident, Trypoxylon wasp cocoons, present in the nest box (log-likelihood ratio test: χ2 = 54.37, < 0.001). Surprisingly, we found that there was an overall negative relationship between honey bee foraging probability and alfalfa leafcutting bee cocoon production (log-likelihood ratio test: χ2 = 55.42, < 0.001). In this study, across three different field sites with varying landscapes in Virginia, areas of higher honey bee foraging probability were associated with lower levels of alfalfa leafcutting bee productivity.

Microbiomes are an extremely vast and complex network of microorganisms. Bacteriophages are a key factor in the microbial health of an ecological system and impact the evolution of pathogenic bacterial strains. Bacteria and the phages that infect them have an intricate relationship due to the dependency on the bacterial host for phage replication, the ability of the phage to lyse and kill its host, and the horizontal gene transfer between the host and phage. This thesis aimed to understand how bacteria and the bacteriophages that infect them impact an ecological system, with a focus on disease states. By analyzing all bacteriophages targeting a specific host, genomic properties, physical similarities and differences a better understanding of how a group of tailed phages have evolved numerous mechanisms and tools to infect host bacteria was understood. The microbiome study of the Western honey bee Apis mellifera, comparing the microbial communities of colonies infested with the external parasite Varroa destructor against those not infested revealed a need for more directed treatment of Varroa infestations. Through our study we discovered that the honey bee microbiome is much more complex than previously reported, consisting of hundreds of bacterial species. In addition, through comparing infested and healthy colonies, we discovered that infestation of Varroa destructor mites negatively impact the colony microbiome in part by reducing microbes key in digestion and immune health of honey bees. Results in this thesis indicate that two microbes which have not been previously established as part of the key microbes in honey bee guts, Xenorhabdus and Sodalis, may in fact be key to honey bee health as they were both effected negatively by the presence of Varroa mite infestations. These bacteria have been shown to be involved in immune health in other insects, supporting this hypothesis. The final stage of this thesis involved the development of an acaricide bacteriophage therapy designed to target key microbes in the gut of V. destructor. Our therapy was shown to safely treat honey bee colonies infested with this parasite. It can be concluded that while phages are a normal part of the microbial ecosystem of the intestinal tract of organisms, altering that balance by increasing the phage load on the microbiome to target specific beneficial microbes drastically reduces the overall fitness of the organism. Results from this study indicate that multi-target cocktail and single-target phage therapy are an effective low impact biological Varroacide. The discovery of an effective therapy is important and to better understand the results of this thesis, further investigation is required, including a study of the impact of the phage therapy on the mite microbiome, as well as the safety of the therapy to humans.

Accepted theories of honey bee foraging state that foragers are active at only 1 time of day. It has been shown that a few foragers can be trained to forage at multiple times of day and at many locations. The purpose of the current study was to further investigate the phenomenon of foragers maintaining multiple time-memories. It was found that in small and large sample populations, a minority of foragers could be trained to 2 or more times and places. Within the hive, the foragers that do not fly to the stations also tend to exhibit a persistent time-memory. Remaining experienced foragers cluster at the dance floor at the approach of a training time and remain dispersed throughout the hive at other times. Because foragers can only be recruited from the dance floor, these foragers that stay behind are also exhibiting a time-memory with respect to the proper training time.

The small hive beetle (Aethina tumida) is an invasive pest from sub-Saharan Africa that has posed increasing threats to European honey bee (Apis mellifera) colonies in the United States over the past two decades. While control has been attempted, consistently effective management strategies still not been developed. This study sought to explore novel experimental methods to better understand and use A. tumida biology to target this pest. One aspect of A. tumida biology that has emerged as potential basis for improved control is olfactory manipulation, which could be used to disrupt beetles as they seek out A. mellifera colonies. Through olfactometry and electroantennography, key volatiles in A. tumida attraction and repulsion were tested and sensitivity of A. tumida to several attractants and repellents was quantified on behavioral and physiological levels. An additional source of attractive volatiles is the A. tumida fungal symbiont Kodamaea ohmeri, which ferments larval waste and is present throughout the A. tumida lifecycle, both externally and in the GI tract. This study explored the development of feeding and soil bioassays to test the effects of several insecticides on A. tumida larvae. Feeding and injection bioassays were also used to deliver a fungicide with the goal of repressing K. ohmeri, which was expected to detrimentally impact A. tumida health. The results of this work enhance our current knowledge or A. tumida biology and provide a useful basis for development of safe and selective management A. tumida management options for the future. / Doctor of Philosophy / The small hive beetle is an invasive European honey bee pest that poses a significant threat to apiaries in the United States. These beetles feed on hive products and brood, pollute the hive with fermenting waste, and, in severe infestations, cause colonies to abandon their hives. This project investigated previously unexplored control options that take small hive beetle biology into account. Small hive beetles have an exceptional sense of smell compared to other beetles, and this ability helps them to locate honey bee hives. Therefore, behavioral responses to attractants and repellants were tested through olfactometry, in which beetles were given a choice to travel toward or away from specific odors or odor blends. Responses to these odors on a physiological level were also quantified through electrical recordings of beetle antennae. Small hive beetles are also known to have a yeast-like symbiont, which is present throughout the small hive beetle lifecycle, both externally and internally. Feeding bioassasy for small hive beetle larvae, along with soil bioassays for the delivery of insecticides were also developed and used to test several compounds against small hive beetle larvae. Feeding bioassays were also used to deliver a fungicide to larval and adult beetles with the goal of repressing internal fungal activity The results of these studies help expand the knowledge of small hive beetle olfaction and provide a background for the development of novel control options to effectively manage this destructive pest.

Honey bees are economically important pollinators of a wide variety of crops that have attracted the attention of both researchers and the public alike due to unusual declines in the numbers of managed colonies in some parts of the world. Viral infections are thought to be a significant factor contributing to these declines, along with exposure to agricultural and apicultural pesticides, but viruses have proven a challenging pathogen to study in a bee model and interactions between viruses and the bee antiviral immune response remain poorly understood. Recent studies have demonstrated an important role for inwardly-rectifying ATP-sensitive potassium (KATP) channels in the cardiac regulation of the fruit fly antiviral immune response, but no information is available on their role in the heart-specific regulation of bee immunity. The results of this work demonstrate that KATP channel modulators have an observable effect on honey bee heart rate that supports their expected physiological role in bee cardiac function. Here, it is also reported that the entomopathogenic flock house virus (FHV) infects adult bees, causing rapid onset of mortality and accumulation of viral RNA. Furthermore, infection-mediated mortality can be altered by pre-exposure to KATP channel modulators. Finally, this work shows that exposure to environmental stressors such as commonly used in-hive acaricides can impact bee cardiac physiology and tolerance to viral infection. These results suggest that KATP channels provide a significant link between cellular metabolism and the antiviral immune response in bees and highlight the significant impact of environmental stressors on pollinator health. / Ph. D.

The varroa mite is a primary driver behind periodical losses of honey bee colonies. The mite requires bees for food and reproduction and, in turn, elicits physiological deficiencies and diseases that compromise bee colony health. The mite nervous system is a target site for existing acaricides. These acaricides not only have adverse health effects on bees, but resistance limits their use to reduce mites and diseases in bee colonies. Voltage-gated chloride channels are involved in the maintenance of nerve and muscle excitability in arthropod pests, which suggests that these channels might be exploited as targets for acaricides. Apistan® (the pyrethroid tau-fluvalinate), Checkmite+® (the organophosphate coumaphos), and Apivar® (the formamidine amitraz) are control products for mite management. The effectiveness of these chemistries has diminished as a result of the increasing incidence resistance in mite populations. I report a toxicological analysis of stilbene products against acaricide-susceptible and -resistant mites. My results find a significant increase in metabolic detoxification enzyme activities in acaricide-resistant mites compared to susceptible mites. Acetylcholinesterase of coumaphos-resistant mites was significantly less sensitive to the toxic coumaphos metabolite compared to susceptible mites, which suggests target-site insensitivity as a mechanism of acaricide resistance. The stilbene product DIDS had significantly higher field efficacy to acaricide-resistant mites compared to Apistan®- and CheckMite+®. These data suggest that DIDS, and other stilbene products, might serve as candidate chemistries to continue field efficacy testing of alternative acaricides for Apistan® and CheckMite+® resistant mites. / Master of Science in Life Sciences

Honey bees, Apis mellifera, play an important role in many ecosystems, pollinating a wide variety of native, agricultural, and exotic plants. The recent decline in the number of feral and managed honey bee colonies in North America, as well as the arrival of Africanized honey bees, have caused concern about adequate pollination for agricultural crops and natural plant communities. However, little is known about feral colonies, and the feral population is the source for Africanized honey bees as they spread and infiltrate managed populations. The goal of my dissertation was to examine the ecology of feral honey bee colonies, adding the spatial context necessary to understand the population ecology and patterns of resource use by feral honey bees on the Welder Wildlife Refuge. I defined the functional heterogeneity of feral honey bee habitat by identifying the suitability of different habitats for feral colonies based on the distribution and abundance of important resources (cavities, nectar, and pollen). I evaluated the distribution and abundance of feral colonies by examining nest site characteristics, population trends, and spatial and temporal patterns in cavity use. Lastly, I examined resource use by evaluating patterns in pollen collection and identifying where and when honey bees searched for resources. Overall, the Welder Wildlife Refuge provided excellent habitat for feral honey bees, supporting a high density of feral colonies. The dense live oak habitat was the best overall source for cavities, nectar, and pollen. Nectar and pollen were abundant throughout the year, with the exception of December and January, when a large number of honey bees searched for resources. Cavities did not appear to vary in their suitability for feral colonies based on measured structural and environmental attributes, since no cavity attributes were correlated with indices of cavity quality. However, the cavity quality indices varied between cavities, suggesting some cavities were more suitable for feral honey bees than others. Colonies were aggregated within the study area, probably due to the distribution of resources. The invasion of Africanized honey bees appeared to fragment the existing European population, with Africanized colonies aggregated in distribution and European colonies random in distribution.

BACKGROUND:Widely considered probiotic organisms, Bifidobacteria are common inhabitants of the alimentary tract of animals including insects. Bifidobacteria identified from the honey bee are found in larval guts and throughout the alimentary tract, but attain their greatest abundance in the adult hind gut. To further understand the role of Bifidobacteria in honey bees, we sequenced two strains of Bifidobacterium cultured from different alimentary tract environments and life stages.RESULTS:Reflecting an oxygen-rich niche, both strains possessed catalase, peroxidase, superoxide-dismutase and respiratory chain enzymes indicative of oxidative metabolism. The strains show markedly different carbohydrate processing capabilities, with one possessing auxiliary and key enzymes of the Entner-Doudoroff pathway.CONCLUSIONS:As a result of long term co-evolution, honey bee associated Bifidobacterium may harbor considerable strain diversity reflecting adaptation to a variety of different honey bee microenvironments and hive-mediated vertical transmission between generations.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Insect pollination contributes in various degrees toward the production of a variety of agricultural crops that ensure diversity and nutritional value in the human diet. Although managed honeybees (Apis mellifera L.) are still the most economically valuable pollinators of monoculture crops cultivated globally, wild pollinator communities can contribute substantially toward crop pollination through pollination ecosystem services sourced from neighbouring natural habitats. Pollination ecosystem services are thus valuable and can motivate for the protection of natural ecosystems hosting diverse insect pollinator communities. F1 onion hybrid seed production is entirely dependent on high insect pollinator activity to ensure cross pollination, seed set and profitable seed yields. Data was collected on 18 onion hybrid seed crops grown in the semi‐arid Klein Karoo and southern Karoo regions of the Western Cape, South Africa. These two main production regions are located within the Succulent Karoo biome, recognized as a global biodiversity hotspot of especially high plant diversity. It is also habitat to the indigenous Cape honeybee (Apis mellifera capensis Esch.). Sites selected varied in the percentages of available natural habitat and managed honeybee hives stocking density. Diverse anthophile assemblages were sampled with pan traps within all the onion fields, regardless of the percentage of available natural habitat near the crop. Crop management practices significantly affected the diversity of anthophile species caught within onion fields, although less than 20% of this diversity was observed actually visiting onion flowers. The honeybee (managed and wild) was by far the most important pollinator because of its high visitation frequency and regular substantial onion pollen loads carried on their bodies. Honeybee visitation significantly increased onion hybrid seed yield, while anthophile diversity and non‐Apis visitation had no effect on seed yield. Neither managed hive density, nor percentage natural habitat were important in determining honeybee visitation or seed yield. Total annual rainfall was the only significant factor determining honeybee visitation. Secondary factors caused by rainfall variability, such as wild flower abundance or soil moisture, may have significantly affected honeybee visitation. In addition, the positive correlation between honeybee visitation and the diversity of hand‐sampled insects from onion flowers; indicate that either or both onion varietal attractiveness and/or pollinator population size may have had significant effects on overall insect visitation. Honeybees showed marked discrimination between hybrid onion parental lines and preferred to forage on one or the other during single foraging trips. Hybrid onion parents differed significantly in nectar characteristics and onion flower scent which would encourage selective foraging through floral constancy. Interspecies interactions were insignificant in causing increased honeybee pollination because of the scarcity of non‐Apis visitors. Most farming practices are subjected to favourable environmental conditions for successful production. However, and especially in the South African context, the dependence of onion hybrid seed crops on insect pollination for successful yields, increase its reliance on natural ecosystem dynamics that may deliver abundant wild honeybee pollinators, or attract them away from the crops. Nevertheless, this dependence can be mitigated effectively by the use of managed honeybee colonies to supplement wild honeybee workers on the flowers. / AFRIKAANSE OPSOMMING: Insek bestuiwing dra in verskillende grade by tot die produksie van landbou gewasse wat variteit en voedingswaarde in die mens se dieet verseker. Al is die heuningby (Apis mellifera L.) steeds die waardevolste ekonomiese bestuiwer van verboude enkelgewasse, kan wilde bestuiwers wesenlik bydra tot gewasbestuiwing deur middel van ekosisteem dienste afkomstig van natuurlike habitatte. Bestuiwing ekosisteem dienste is daarom waardevol en kan dus die bewaring van natuurlike ekosisteme, wat diverse gemeenskappe huisves, regverdig. F1 basterui saadproduksie is totaal afhanklik van hoë insek‐bestuiwer aktiwiteit om kruisbestuiwing, saadvorming en winsgewende saadopbrengste te verseker. Data is ingesamel op 18 basterui saad aanplantings in die half‐droë Klein Karoo en suid‐Karoo streke van die Weskaap, Suid‐Afrika. Hierdie twee hoof produksie streke is geleë binne die Sukkulente Karoo bioom wat erken word as ʼn globale biodiversiteits “hotspot” met hoë plant diversiteit. Dit is ook die habitat van die inheemse Kaapse heuningby (Apis mellifera capensis Esch.). Aanplantings is gekies om verskillende grade van beskikbare natuurlike habitat en bestuurde heuningby korf digthede te verteenwoordig. Diverse versamelings blom‐besoekers is versamel met water‐wippe in al die aanplantings, ongeag die persentasie natuurlike habitat beskikbaar by elke aanplanting. Gewas bestuurspraktyke het die diversiteit van blombesoekers betekenisvol beïnvloed. Tog is minder as 20% van hierdie diversiteit as aktiewe besoekers op die uiekoppe waargeneem. Heuningbye (bestuur of wild) was oorwegend die belangrikste bestuiwers as gevolg van hoë besoek frekwensies en wesenlike ladings uiestuifmeel op hulle liggame. Heuningby besoeke het saadopbrengs betekenisvol verhoog, maar blom‐besoeker diversiteit en nie‐Apis besoeke het geen effek op saadopbrengs gehad nie. Bestuurde korf digtheid en persentasie natuurlike habitat was nie belangrik in die bepaling van heuningby besoeke of basterui saadopbrengste nie. Totale jaarlikse reënval was die enigste betekenisvolle faktor wat heuningby besoeke bepaal het. Sekondêre faktore wat versoorsaak word deur reënval veranderlikheid, soos veldblom volopheid of grondvog, kon betekenisvolle effekte op die aantal heuningby besoeke gehad het. Bykomend, dui die positiewe korrelasie tussen heuningby besoeke en die diversiteit van hand‐versamelde insekte vanaf die uiekoppe op die moontlike betekenisvolle effek van elk of beide basterui variteit aantreklikheid en/of bestuiwer populasie grote op algehele insek besoeke. Heuningbye het noemenswaardige diskriminasie getoon tussen die basterui ouerlyne en het verkies om op een of die ander te wei tydens enkele weidingstogte. Basterui ouerlyne het betekenisvol verskil in nektar eienskappe en blomgeur wat die selektiewe weiding van heuningbye, toegepas deur blomkonstantheid, sal aanmoedig. Tussen‐spesie interaksies was onbetekenisvol in die verhoging van heuningby bestuiwing omdat nie‐Apis besoekers baie skaars was. Meeste boerdery praktyke is onderhewig aan gunstige omgewings toestande vir suksesvolle produksie. Maar, en veral in die Suid‐Afrikaanse konteks, omdat basterui saad aanplantings afhanklik is van insek bestuiwing vir suksesvolle opbrengste, word daar meer staat gemaak op natuurlike ekosisteem dinamika wat volop wilde heuningby bestuiwers kan voorsien, of selfs bestuiwers van die aanplanting kan weg lok. Nietemin, hierdie afhanklikheid kan effektief verlaag word deur die gebruik van bestuurde heuningby kolonies om die aantal wilde heuningby werkers op die blomme aan te vul.

It has been observed that returning honey bee foragers congregate with unemployed foragers and food receiver bees in a localized region of the hive known as the dance floor. Here, the returning foragers advertise food sources via the waggle dance. It was hypothesized that the close proximity of the dance floor to the hive entrance was related to foragers minimizing time and travel inside the hive. The hive entrance is conventionally located at the bottom of the hive. It was suggested that this location was ideal for easy removal of debris. This correlation between dance floor location and hive entrance location invokes further examination of the relationship. Is the hive entrance location used to establish dance floor location? Using scan sampling- the hive was visually scanned along rows in a descending fashion from the top right corner to hive entrance. The location of each observed waggle dance was recorded for 30 minutes. Observations were conducted for three consecutive days, then the hive entrance location was displaced. The observation hive was altered to contain three hive entrances located adjacent to the bottom first frame, adjacent to the center of the second frame, and adjacent to the middle of the third frame. Only one hive entrance was open at a given time. For the last three days of the experiment, the bottom hive entrance was made accessible again. Regardless of entrance position, the dance floor was seen to be established adjacent to the hive entrance.