The waggle dance of the honey bee (Apis mellifera L.) is perhaps the most celebrated animal communication behavior. With a waggle dance, a forager bee who has discovered a profitable resource on the landscape, usually floral nectar or pollen, can inform her nestmates of its location and recruit them to exploit it by communicating both a distance and a direction. Since Karl von Frisch described the waggle dance in 1942, scientific exploration of the dance has exploded into the realms of its structure, function, role in the regulation of collective foraging in the context of the hive as a super-organism, and even its utility as a study system for understanding sublethal behavioral effects of pesticide exposure. This dissertation presents three novel studies of the waggle dance. In the first, we asked whether consistent inter-bee differences (i.e., individuality) in a waggle dance distance - duration calibrations could affect communication success. In the second, we characterized the networks of recruitment arising from waggle dance communications and explored the role of the aforementioned individuality in network formation. In the third, we tested whether sublethal exposure to glyphosate (GLY), the most-applied herbicide in the world, could affect foraging, recruitment, or the levels and balance of biogenic amines in the bee brain. In each of these experiments, we housed bees in clear-walled observation colonies and trained cohorts of bees to visit artificial feeders to record both foraging and recruitment data. In our first experiment, we found that individuality in waggle dance behavior does shape communication outcomes, indicating that individual-level behavioral differences should not be discounted as factors at work in eusocial insect societies. In the second, we present the first network density and dance burstiness data from in vivo bee networks, revealing that recruitment networks are sparse, and waggle dancers are partitioned into bursty and non-bursty behavioral types. In the third, we show that not only can sublethal GLY exposure reduce foraging, but it can also produce significant correlations between levels of the important insect neurotransmitter octopamine and its two biosynthetic precursors, tyramine and tyrosine, where levels in control bees were unrelated. The results of this dissertation research, while distinct by experiment, together emphasize the continuing usefulness and tractability of the honey bee colony as a system in which to study the role of individuality in animal communication and to better understand the threat posed by non-insecticidal pesticide chemistries to the planet's most economically impactful pollinator. / Doctor of Philosophy / One of the most famous and well-studied animal behaviors is the waggle dance of the honey bee. A honey bee's waggle dance works similarly to a Yelp review for a restaurant: a bee who has found a good food source, like a flower patch offering especially sweet nectar or high-quality pollen, can come back to the colony and recommend it to her nestmates with a dance. The waggle dance is even more specific than a Yelp review, however, in that it also gives instructions to find the food source, communicating both a distance and a direction so that dance followers can go out into the landscape and look for the food source themselves. Even though the waggle dance has been studied extensively since it was first described by Karl von Frisch in the 1940s, there are still unknowns about how it works, and how it might be impacted by certain stressors. This dissertation presents three different experiments aimed at shedding light on these unknowns. First, it has recently been shown that there are consistent differences between bees in the way they communicate distance in the dance, and we tested whether that between-bee individuality can affect the likelihood that two bees will communicate successfully. Second, we studied how information about a food source moves from bee to bee via the waggle dance to form a communication network. Specifically, we described how efficiently information moved from bee to bee, patterns of dancing behavior, and the role of that individuality in its formation. Third and lastly, we looked to see whether exposure to a weedkiller called glyphosate (GLY) could affect either honey bees' waggle dance or food-collecting behavior, as well as levels of certain neurotransmitters in their brains that are involved in those behaviors. In all three experiments, we collected our data by housing bees in a clear-walled observation hives that let us view and film their waggle dance behavior, and then training groups of bees to collect artificial nectar from a feeder station that we provided, so we could also observe them as they collected food. We found that individuality in waggle dance communication can indeed affect the likelihood of communication success between two given bees, where the likelihood of communication success is greater when the dancer communicates a farther distance to the food source than the follower would. In the second experiment, our study of the waggle dance communication network showed that (1) information does not flow from bee to bee very efficiently, and (2) bees either dance quite regularly or sporadically. As far as we know, we are the first to describe these aspects of the waggle dance communication network, which may be useful in the field of computing algorithms inspired by living organisms. Finally, our third experiment showed that mild GLY exposure not only reduced how frequently bees collected food from our feeder, but also changed the relative amounts of certain neurotransmitters in their brains. This result emphasizes the importance of understanding how weedkillers that are not meant to target beneficial insects like honey bees are actually affecting them, so that we can make better-informed decisions to protect honey bees and other good insects.

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.

The western honey bee, Apis mellifera, provide invaluable economic and ecological services while simultaneously facing stressors that may compromise their health. For example, agricultural landscapes, such as a row crop system, are necessary for our food production, but they may cause poor nutrition in bees from a lack of available nectar and pollen. Row crops are largely wind or self-pollinated, and while previous studies have focused on the impact of bees to row crops, fewer studies have examined the reciprocal relationship of the row crops on honey bees. Here we investigated the foraging dynamics of honey bees in a row crop environment. We decoded, mapped, and analyzed 3460 waggle dances, which communicate the location of where bees collected food, for two full foraging seasons (April – October, 2018-2019), and concurrently collected pollen from returning foragers. We found that bees foraged mostly locally (< 2 km) throughout the season. The shortest communicated median distances (0.48 and 0.32 km), indicating abundant food availability, occurred in July in both years, which was when our row crops were in full bloom. We determined, by plotting and analyzing the communicated locations, that most mid-summer foraging was in row crops, with at least 40% of honey bee recruitment dances indicating either cotton or soybean fields. Bees also largely foraged for nectar when visiting row crop fields, only returning to the hive with Glycine spp. pollen, and foraging on nearby trees and weeds for pollen. Foragers were exposed to thirty-five different pesticides throughout the foraging season, based on pesticide residues in collected pollen. Overall, row crop fields are contributing a surprising majority of mid-summer forage to honey bee hives and suggests that similar agricultural landscapes may also provide abundant, mid-summer forage opportunities for honey bees, however, at the risk of pesticide exposure. / Master of Science in Life Sciences / Declines in the number of honey bee hives have been observed in the United States and western Europe throughout the last century, driven by environmental stressors such as poor nutrition caused by anthropogenic landscape change and pesticide exposure. Agricultural landscapes, for example, contain monocultures and often necessitate pesticide use, which may be detrimental to bee health. Because of these effects, it is necessary to understand how honey bees forage in these systems and what potential health risks they face. We investigated honey bees foraging dynamics in a row crop environment, observing honey bee waggle dance recruitment behavior and gathering forager-collected pollen to better understand when, where, and what honey bees forage on throughout the season (April – October). We found that bees largely foraged near the hive throughout the season, indicating that sufficient resources were available, particularly in July when crops were in full bloom. During full bloom bees considerably foraged in cotton and soybean fields. We found that bees collected minimal row crop pollen, apart from soybean pollen, largely foraging on trees and flowering weeds for pollen. Through pollen foraging bees were exposed to thirty-five pesticides, ranging in toxicity and mode of action. Overall, honey bees foraging in a row crop system foraged substantially in row crop fields during the mid-summer. Row crops systems may be able to provide abundant forage during the mid-summer, but could come at the risk of exposure to pesticides.

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.

Anthropogenic landscape changes threaten our ecologically and economically critical honey bees by decreasing the availability of quality foraging resources. Importantly, waggle dance analysis provides a versatile and relatively cost-effective tool for investigating the obstacles that honey bees face, such as habitat loss, in our changing landscapes. While this emerging tool has improved our understanding of honey bee foraging in specific landscape contexts, additional research is needed to identify broad trends that span across landscapes. For this dissertation, I used waggle dance decoding and analysis to investigate honey bee foraging, and sublethal stress responses, across three ecologically distinct landscapes in Virginia. In Chapter 1, I introduce waggle dances as a model study system for investigating honey bee foraging and sublethal stress responses by summarizing modern methodological advances in its analysis and emerging research gaps. In Chapter 2, I tested the effects of sublethal imidacloprid exposure on honey bee foraging and recruitment using a semi-field feeder experiment. In doing so, I report that honey bees decreased their foraging, but not recruitment, to an imidacloprid-laced sucrose solution, compared to a control solution. Together, these effects could potentially harm honey bee health by increasing their exposure to pesticides and decreasing their food intake. In Chapter 3, I compared the foraging distances communicated by waggle dancing nectar and pollen foragers across landscapes to explore the economic forces driving foraging to these resources. I observed higher overall and monthly nectar foraging distances compared to pollen foraging distances. Such results suggest that nectar foraging cost dynamics are driven by supply, while pollen foraging cost dynamics are driven by demand. In Chapter 4, I used waggle dance decoding to map and quantify foraging to agricultural grasslands in a mixed-use landscape. In doing so, I demonstrate that honey bees recruit to agricultural grasslands throughout the season, but that this land type was not more attractive than the broader landscape after correcting for foraging distance, which is a relevant cost that flying bees must consider. Additionally, I qualitatively observe a foraging hot spot, representing high honey bee interest, over a highly heterogenous section of the landscape. The collective results of this chapter identify agricultural grasslands as a potential management target and support the importance of landscape heterogeneity to honey bees/pollinators. In Chapter 5, I used waggle dance decoding to investigate honey bee foraging spatial patterns in the context of optimal foraging theory. In particular, I explore whether co-localized honey bee colonies forage optimally by converging on the same resource patches, or by partitioning the landscape in to distinct foraging territories. Spatial analysis revealed that the colonies widely distributed their foraging at the landscape-scale, with dances from the same and different colonies being similarly distributed, while also establishing distinct, patch-scale, colony-specific, foraging aggregations. Together, these results suggest that the honey bee foraging system produces an emergent foraging pattern that may decrease both within- and among-colony foraging competition. Finally, in Chapter 6, I place my research findings in the context of historical and current trends in honey bee behavioral ecology. Overall, my dissertation improves our understanding of honey bee foraging ecology across landscape contexts using waggle dance analysis, while demonstrating its versatility and effectiveness as a tool for ecologists. / Doctor of Philosophy / Honey bees collect nectar (carbohydrate source) and pollen (protein source) from flowers as their food for survival and reproduction. Human activities, such urbanization, change landscapes and threaten our critically important honey bees by decreasing the availability of flower-rich habitats. Importantly, honey bees share the location of good food sources with their nest mates using a communication behavior called the waggle dance. Interestingly, scientists can estimate the approximate location of the food sources communicated by waggle dancing bees through close observation and cutting-edge analysis. Therefore, we can "decode" honey bees' waggle dances to map their food collection, or foraging, patterns and investigate the obstacles that they face in our changing landscapes. For this dissertation, I used waggle dance decoding and analysis to investigate honey bee foraging across three different landscapes in Virginia. In Chapter 1, I introduce waggle dances as a tool for investigating honey bee behavior by summarizing the modern improvements in its analysis and areas where research is needed. In Chapter 2, I tested the effects of a sublethal exposure to a pesticide, imidacloprid, by observing the foraging and waggle dance behavior of bees visiting feeders with artificial food. I report that honey bees decreased their foraging, but not recruitment, while collecting an imidacloprid-laced sugar solution, compared to a solution without imidacloprid. In Chapter 3, I compared the foraging distances communicated by waggle dancing nectar and pollen foragers across landscapes to explore the economic forces driving foraging to these resources. I observed higher overall and monthly nectar foraging distances compared to pollen foraging distances. Such results suggest that nectar foraging is driven by supply, while pollen foraging is more driven by demand. In Chapter 4, I used waggle dance decoding to map and quantify foraging to agricultural grasslands (pastures and hay fields) in a landscape characterized by diverse land uses. In doing so, I demonstrate that honey bees recruit to agricultural grasslands throughout the season, but that this land type was not more attractive than the broader landscape after correcting for foraging distance. Additionally, I qualitatively observe a foraging hot spot, representing high honey bee interest, over a highly heterogenous section of the landscape. The collective results of this chapter identify agricultural grasslands as a potential management target and support the importance of landscape heterogeneity to honey bees/pollinators. In Chapter 5, I used waggle dance decoding to investigate the spatial patterns of honey bee foraging in the context of optimal foraging theory, which attempts to explain efficient resource collection strategies. In particular, I explore whether neighboring honey bee colonies forage optimally by converging on the same resource patches, or by dividing the landscape in to distinct foraging territories. We found that colonies distributed their foraging widely at the landscape-scale, with dances locations from the same and different colonies being similarly distributed, while also establishing distinct, patch-scale, colony-specific, foraging areas. Together, these results suggest that honey bees use a foraging strategy that decreases both within- and among-colony foraging competition. Finally, in Chapter 6, I place my research findings in the context of historical and current trends in honey bee behavioral ecology. Overall, my dissertation uses waggle dance analysis to improve our understanding of honey bee foraging behavior, while demonstrating its versatility and effectiveness as a tool for ecologists.

Honey bees, Apis mellifera Linn., provide vital economic and ecological services via pollination while concurrently facing multiple interconnected stressors impacting their health. Many crops like apples, peaches, and cherries that add diversity and nutrition to our diet are wholly or partially dependent upon the pollination services of insects. Orchard crops are self-incompatible and commonly regarded as crops reliant on the pollination services of insects, and while previous studies have focused on the impact of bees to orchard crops during bloom, fewer studies have examined the reciprocal relationship of the orchards on honey bees, particularly across the entire foraging season. Here we investigated the foraging dynamics of honey bees in an orchard crop environment in Northern Virginia, United States. We decoded, mapped, and analyzed 3,710 waggle dances, which communicate the location of a valuable resource in the environment, for two full foraging seasons (April-October, 2018-2019), and, concurrent to the dance filming, collected pollen from returning foragers. We found that bees forage locally the majority of the time (< 2 km) throughout the season, with some long-range distances occurring in May after bloom (both 2018 and 2019) and in fall (2019). The shortest communicated median distances (0.50 km and 0.53 km), indicating abundant food availability, occurred during September in both years, paralleling the bloom of an important late season resource, goldenrod (Solidago). We determined, through plotting and analyzing the communicated forage locations and from the collected pollen from returning foragers, that honey bees forage more within apple orchards after the bloom (29.4% and 28.5% foraging) compared to during bloom (18.6% and 21.4% foraging) on the understory of clover and plantain. This post bloom foraging also exposes honey bees to the highest concentration of pesticides across the entire foraging season (2322.89 ppb pesticides versus 181.8 during bloom, 569.84 in late summer, and 246.24 in fall). Therefore, post bloom apple orchards supply an abundance of forage, but also the highest risk of pesticide exposure, which may have important implications for management decisions of bees in orchards. / Master of Science in Life Sciences / Honey bee hives have been declining significantly in the United States, driven by a multitude of issues and stressors including pesticide exposure, disease, pests such as varroa mites, and poor nutrition caused by natural land being converted into development or agriculture. Agricultural landscapes, in particular, are often monocultures are saturated with pesticides creating a potentially hazardous environment, yet reliant on bees to provide pollination for crops. Because of this interconnected relationship between bees and flowers and the effects of stressors agricultural systems cause have with pollinators, it is necessary to understand how honey bees forage in these environments and what potential health risks they face. We investigated honey bees foraging dynamics in an apple orchard in Northern Virginia, United States by observing honey bee waggle dance behavior, where bees literally waggle back and forth for a certain time and at a certain angle telling their nestmates where a resource is, and collecting pollen from returning forager bees to better understand when, where, and upon what honey bees forage throughout the foraging season, which is when flowers are available and the weather warm enough (April – October). We found that bees mostly forage locally near the hive throughout the season, indicating that sufficient amount of food was available even after short bloom time of the apple (April to mid-May). We determined, through plotting and analyzing the waggle dance locations, that honey bees forage more within apple orchards after the bloom on mostly clover and plantain. This abundance of post bloom foraging also exposes honey bees to the highest amounts of pesticides across the entire foraging season. Post bloom apple orchards supply an abundance of forage, but also the highest risk of pesticide exposure to honey bees.

Sleep is a behavioral condition fraught with mystery. Its definition—either a suite of diagnostic behavioral characters, electrophysiological signatures, or a combination of the two—varies in the literature and lacks an over-arching purpose. In spite of these vagaries, sleep supports a large and dynamic research community studying the mechanisms, ontogeny, possible functions and, to a lesser degree, its evolution across vertebrates and in a small number of invertebrates. Sleep has been described and examined in many social organisms, including eusocial honey bees (Apis mellifera), but the role of sleep within societies has rarely been addressed in non-human animals. I investigated uniquely social aspects of sleep within honey bees by asking basic questions relating to who sleeps, when and where individuals sleep, the flexibility of sleep, and why sleep is important within colonies of insects. First, I investigated caste-dependent sleep patterns in honey bees and report that younger workers (cell cleaners and nurse bees) exhibit arrhythmic and brief sleep bouts primarily while inside comb cells, while older workers (food storers and foragers) display periodic, longer sleep bouts primarily outside of cells. Next, I mapped sleep using remote thermal sensing across colonies of honey bees after introducing newly eclosed workers to experimental colonies and following them through periods of their adult lives. Bees tended to sleep outside of cells closer to the edge of the hive than when asleep inside cells or awake, and exhibited caste-dependent thermal patterns, both temporally and spatially. Wishing to test the flexibility of sleep, I trained foragers to a feeder and made a food resource available early in the morning or late in the afternoon. The bees were forced to shift their foraging schedule, which consequently also shifted their sleep schedule. Finally, I sleep-deprived a subset of foragers within a colony by employing a magnetic “insominator” to test for changes in their signaling precision. Sleep-deprived foragers exhibited reduced precision when encoding direction information to food sources in their waggle dances. These studies reveal patterns and one possible purpose of sleep in the context of a society. / text

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