Neonicotinoids are primarily used in agriculture where they are applied as seed coatings, foliar sprays, and soil drenches or through drip irrigation. In urban areas neonicotinoids are used in home garden products and tree treatments. The maximum foraging ranges of honey bees are usually 10 – 15 km (median distances are 1 – 6 km) from the hive. Hence bee exposure to neonicotinoids is dependent upon the land use type within limited foraging distances from the hive. However, there are virtually no data showing levels of neonicotinoid use in urban areas and few studies have been done to compare urban and agricultural exposure. Several neonicotinoids have shown various toxic effects on pollinators and particularly honey bees. Honey bees have a limited arsenal of detoxification proteins to withstand neonicotinoid exposure, which makes them more sensitive and less able to develop tolerance to these insecticides compared to other insects. Sublethal exposure of honey bees to neonicotinoids can cause behavioral disturbances, orientation difficulties, impairment of social activities, and respiratory pattern changes. These behavioral changes can cause insufficient foraging behavior in honey bees due to the sublethal effect of neonicotinoids, thus putting the colony at risk of food shortage and eventually collapse. My objectives were to (1) develop a highly sensitive and selective, multi-residual analytical method for neonicotinoids in honey bee and pollen samples, (2) investigate the impacts of land use type (agriculture vs. urban) on the exposure of honey bees to neonicotinoid, (3) investigate the sublethal effect of imidacloprid on honey bees’ behavioral performance, and (4) investigate the sublethal effect of field-realistic concentrations of imidacloprid on honey bees’ metabolism at different ambient temperatures.To address my first objective (Chapter 2), I tested three sample cleanup methods (silica SPE, NH2-silica SPE, and Z-Sep SPE) based on solid phase extraction (SPE), which were investigated for determination of neonicotinoid insecticides and selected metabolites in honey bee and pollen samples by LC-MS/MS. Samples were extracted by hexane and ethyl acetate and then cleaned up with a SPE cartridge packed with silica gel, which showed a better cleanup efficiency compared to the aminopropyl silica SPE and zirconium-based sorbents method. Matrix effects of the three cleanup methods were evaluated and compared. Silica gel showed the highest analyte recoveries and method detection limit for this method were 2.0 to 9.1 μg/kg for honey bees and 2.4 to 4.7 μg/kg for pollen. Recovery studies were performed at three spiking levels (10, 60, and 120 μg/kg) and ranged from 78 to 140% with RSDs between 3 to 18% in honey bees and 83 to 124% with RSDs between 3 to 17% in pollen. The silica gel SPE cleanup method was then applied using honey bee and pollen samples that were collected from different apiaries. To address my second objective (Chapter 3), I analyzed honey bee and beebread (pollen) samples from apiaries in agricultural, developed, and undeveloped areas that were collected during two years in Virginia to assess if landscape type or county pesticide use were predictive of honey bee colony exposure to neonicotinoid insecticides. Trace concentrations of the neonicotinoid imidacloprid were detected in honey bees (3 out of 84 samples, 2.02 – 3.97 ng/g), while higher levels were detected in beebread (5 out of 84 samples, 4.68 – 11.5 ng/g) and pollen (3 out of 5 pollen trap samples, 7.86 – 12.6 ng/g). Imidacloprid was only detected in samples collected during July and August and were not detected in honey bees from hives where neonicotinoids were detected in pollen or beebread. Number of hives sampled at a site, county pesticide use, and landscape characteristics were not predictive of neonicotinoid detections in honey bees or beebread (all P>0.05). Because of the low detection rates, field surveys may underestimate honey bee exposure to field realistic levels of pesticides or the risk of exposure in different landscapes. Undetectably low levels of exposure or high levels of exposure that go undetected raise questions with regard to potential threats to honey bees and other pollinators. To address my third objective (Chapter 4), I investigated the effects of sub-lethal concentrations of imidacloprid on late fall forager honey bees’ behavior by accessing their activity levels and walking performance after being fed ad libitum with six different concentrations (2 – 125 μg/kg) of imidacloprid-dosed syrup for up to 48 hours in laboratory. Honey bee activity levels and motivation to move after being released into a UV light illuminated tunnel decreased significantly as dosages of neonicotinoid in their diet increased. However, their walking speeds were not significantly affected by imidacloprid. The behavioral changes I observed in honey bees chronically exposed to neonicotinoid via diet could negatively affect individual honey bee performance of their hive duties and consequently, colony survival during late fall and winter. To address my fourth objective (Chapter 5), I measured honey bee (Apis mellifera) foragers’ CO2 production rates at different temperatures (25, 30, or 35°C) after they consumed syrup dosed with a field realistic (5 μg/L) or high (20 μg/L) concentration of a neonicotinoid insecticide (i.e. imidacloprid) for 48h. We found that imidacloprid exposure significantly disrupted honey bees’ non-flight metabolic rates and there was a significant interaction between imidacloprid dosage and ambient temperature. Honey bee foragers dosed with 5 μg/L imidacloprid displayed higher average metabolic rates and those dosed with 20 μg/L imidacloprid displayed similar average metabolic rates compared to the corresponding control group across all temperatures. Exposure to field realistic concentrations of neonicotinoid may have a higher energetic cost for honey bees at 25℃ than at higher ambient temperatures. Disrupted energy costs in honey bees fed imidacloprid might be due to the thermoregulation, nerve excitation, or detoxification processes. Metabolic rate changes caused by pesticide exposure could result in less available energy for honey bees to perform hive duties and forage, which could negatively affect colony health.

Even though honey is one of the oldest known sweets, there is a general lack of knowledge concerning its chemical and physical properties. This is particularly true of those honeys produced in the intermountain region. Beekeepers of this region have long felt that their product was sweeter than those honeys produced in other sections of the country. This, of course, implies that honey produced in the intermountain area is higher in sugar concentration and lower in percent moisture. The present study was made in order to supply some data concerning honeys of this area.

No description available.

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 honey bee is one of the most important crop pollinating insects in the world. Researchers have recently identified a disease that has begun to impact the honey bee population. Colony Collapse Disorder results in the death of many bee colonies every year, but the cause for this remains unknown. Investigating the cause, harmonic radars are being considered to track the foraging patterns of honey bees. This research endeavors to find an optimized waveform for use in tracking foraging bees. Harmonic oscillators were developed for a transmit frequency of 1.2 GHz and various waveforms were tested against the oscillators. Ultimately, the waveform was found to be arbitrary. The amount of power that the harmonic oscillator receives is the determining factor. Given this, a general pulsed waveform can be developed that attempts to provide the maximum possible return for a predetermined maximum range of interest.

No description available.

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.

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