Honey bee foragers form time memories that enable them to match their foraging activity to the time of day when a particular food source is most productive. Persistent foragers show food-anticipatory activity by making reconnaissance flights to the previously productive food source and may continue to inspect it for several days. In contrast, reticent foragers do not investigate the source but wait for confirmation from returning persistent foragers. To determine how persistent and reticent foragers might contribute to the colony's ability to rapidly reallocate foragers among sources, we trained foragers to collect sucrose from a feeder at a restricted time of day for several days and then observed their behavior for three consecutive days during which the feeder was empty. In two separate trials, video monitoring of the hive entrance during unrewarded test days in parallel with observing reconnaissance visits to the feeder revealed a high level of activity, in both persistent and reticent foragers, thought to be directed at other food sources. This 'extracurricular' activity showed a high degree of temporal overlap with reconnaissance visits to the feeder. In some cases, inspection flights to the unrewarded feeder were made within the same trip to an extracurricular source, indicating that honey bees have the ability to manage at least two different time memories despite coincidence with respect to time of day. The results have major implications for understanding flower fidelity throughout the day, flower constancy within individual foraging excursions, and the sophisticated cognitive management of spatiotemporal memories in honey bees.

Honey bee foragers form time memories that enable them to match their foraging activity to the time of day when a particular food source is most productive. Persistent foragers show food-anticipatory activity by making reconnaissance flights to the previously productive food source and may continue to inspect it for several days. In contrast, reticent foragers do not investigate the source but wait for confirmation from returning persistent foragers. To determine how persistent and reticent foragers might contribute to the colony's ability to rapidly reallocate foragers among sources, we trained foragers to collect sucrose from a feeder at a restricted time of day for several days and then observed their behavior for three consecutive days during which the feeder was empty. In two separate trials, video monitoring of the hive entrance during unrewarded test days in parallel with observing reconnaissance visits to the feeder revealed a high level of activity, in both persistent and reticent foragers, thought to be directed at other food sources. This 'extracurricular' activity showed a high degree of temporal overlap with reconnaissance visits to the feeder. In some cases, inspection flights to the unrewarded feeder were made within the same trip to an extracurricular source, indicating that honey bees have the ability to manage at least two different time memories despite coincidence with respect to time of day. The results have major implications for understanding flower fidelity throughout the day, flower constancy within individual foraging excursions, and the sophisticated cognitive management of spatiotemporal memories in honey bees.

Pollination syndromes refer to stereotyped floral characteristics (flower colour, shape, etc.) that are associated with a functional group of pollinators (bee, bird, etc.). The trumpet creeper Campsis radicans, endemic to the southeast and mid-west United States, has been assigned to the hummingbird-pollination syndrome, due mainly to its red, trumpet-shaped flowers. Previous studies demonstrated that the ruby-throated hummingbird Archilochus colubris is C. radicans' primary pollinator, but anecdotal data suggest various bee species may provide pollination service when hummingbirds are absent. This study characterised C. radicans nectar volume and concentration by time of day. Nectar volume was suitable for hummingbirds, but concentration was higher than typical hummingbird-pollinated plants (∼20% w/w); at ∼30% w/w, it approached the concentration expected in bee-pollinated plants (∼50% w/w). We also found substantial amounts of nectar at night. Two C. radicans populations received virtually no hummingbird visits, but the number of bees were markedly higher than in the populations previously described. Interestingly, there were no night-time visitors despite the large quantity of nocturnal nectar. Based on previously published pollen delivery per visit by various species, this study estimated that cumulative deposition by bees routinely reached pollen deposition thresholds for setting fruit in C. radicans. They are, unequivocally, the predominant pollinators in these populations, thus providing pollination service in the absence of hummingbirds. These results highlight C. radicans as a food source for native bees and add to the understanding of how floral phenotypes can facilitate pollination by disparate functional groups.

Honey bee (Apis mellifera) foragers can remember both the location and time of day food is collected and, even in the absence of a reward, reconnoiter the food source at the appropriate time on subsequent days. This spatiotemporal memory (time-memory) is linked to the circadian clock and enables foragers to synchronize their behavior with floral nectar secretion rhythms, thus eliminating the need to rediscover productive food sources each day. Here, we asked whether the establishment of one time-memory influences the formation of another time-memory at the same time of day. In other words, can two time-place memories with the same ‘time-stamp’ coexist? We simultaneously trained two groups of foragers from a single hive to two separate feeders at the same restricted time of day. After 5 days of training, one feeder was shut off. The second feeder continued being productive 4 more days. Our results showed that (1) foragers with high experience levels at the first source were significantly more likely than low-experience foragers to maintain fidelity to their original source and resist recruitment to the alternative source, (2) nearly one-third of foragers demonstrated multiple, overlapping time-memories by visiting both feeders at the correct time and (3) significantly more high-experience than low-experience foragers exhibited this multitasking behavior. The ability to maintain and act upon two different, yet contemporaneous, time-memories gives the forager bee a previously unknown level of versatility in attending to multiple food sources. These findings have major implications for understanding the formation and management of circadian spatiotemporal memories.

The mushroom bodies (MBs) are insect brain regions important for sensory integration, learning, and memory. In adult worker honey bees (Apis mellifera), the volume of neuropil associated with the MBs is larger in experienced foragers compared with hive bees and less experienced foragers. In addition, the characteristic synaptic structures of the calycal neuropils, the microglomeruli, are larger but present at lower density in 35-day-old foragers relative to 1-day-old workers. Age- and experience-based changes in plasticity of the MBs are assumed to support performance of challenging tasks, but the behavioral consequences of brain plasticity in insects are rarely examined. In this study, foragers were recruited from a field hive to a patch comprising two colors of otherwise identical artificial flowers. Flowers of one color contained a sucrose reward mimicking nectar; flowers of the second were empty. Task difficulty was adjusted by changing flower colors according to the principle of honey bee color vision space. Microglomerular volume and density in the lip (olfactory inputs) and collar (visual inputs) compartments of the MB calyces were analyzed using anti-synapsin I immunolabeling and laser scanning confocal microscopy. Foragers displayed significant variation in microglomerular volume and density, but no correlation was found between these synaptic attributes and foraging performance.

Little is known about the behavioral patterns of honey bee queens. To determine if mated honey bee queens possess diel rhythmicity in behavior, we observed them in glass-sided observation hives using three types of observation regimes: focal studies consisting of 2-hour and 24-hour continuous observations as well as scan-sampling of multiple queens. All behaviors (active: walking, inspecting, egg-laying, begging for food, feeding, and grooming self; inactive: standing) occurred at all times of day and night, but no queen showed consistent diel rhythmicity in any of the individual behaviors. There were no consistent diel differences in active versus inactive behaviors or the number of bees in the queen's retinue. This arrhythmicity was unchanged despite daily changes in both light and temperature levels. The arrhythmic behavior observed by most of the honey bee queens inside the colony appears to be similar to that exhibited by worker bees before they initiate foraging behavior.

Experiments were performed to investigate the influence of water availability on the diel patterns of nectar secretion (volume, concentration, sugar production) in male squash flowers as well as to discover what physical component of nectar honey bees use to trigger their time-memory. Squash plants were grown in the greenhouse and in the field under both constant and variable watering regimes. Throughout anthesis, nectar volume and sugar concentration were recorded. In the field, the temporal distribution of arrivals to squash was observed with and without blossoms present. In the greenhouse and in the field, squash flowers exhibit a consistent diel pattern of nectar secretion that does not significantly alter during drought conditions; flowers open just before sunrise (with low volume and sugar and high concentration) and close at midday (with high volume and sugar and low concentration). Honey bees preferentially arrived early in anthesis possibly cueing on either the sugar concentration or the first availability of nectar.

Experiments were performed to determine the influence of the honey bee time-memory on a forager bee's sensitivity to recruitment. Two groups of foragers from one colony were trained to separate food stations at the same restricted time of day for several consecutive days. Feeding then was canceled at one station but continued for four more days at the other. Bees with more days of training at a non-productive source were significantly less likely than foragers with less training to be recruited to an alternative food source presented at the same time of day. Furthermore, the ability of a forager to be recruited recovered after several days, but this recovery period was longer for bees with greater experience. These findings demonstrate a long-term influence of time-memory on subsequent foraging behavior, in contrast to currently accepted models for the allocation and re-allocation of honey bee foragers to food patches in the environment.

In the early twentieth century, Karl von Frisch performed seminal work on the organization of social behavior of honey bees. Much of his work involved training individual foragers to distant artificial feeders. Similar training methods have been used in research laboratories for the better part of a century, and these methods lend themselves well to advanced undergraduate biology classes in animal behavior. In recent years, students have used these methods in group projects to study color preference and time-memory. In this Technical Paper, we describe the basic steps of training honey bees to a distant feeder. We also provide alternative methods for answering specific types of questions that students in animal behavior classes might wish to address.

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.