Behavioral Syndromes in Individual Honeybees

Behavior can be defined as a response to a stimulus due to an individual’s unique genotype and environment. As behaviors are expressed across contexts and over time it becomes personality. While personality has been studied extensively in vertebrates, it is only beginning to be investigated in insects. Considering the ecological importance of honeybees (Apis mellifera), it is important to learn whether or not, and how behavior influences their personality. In light of current concern regarding the welfare of honeybees, studies regarding their behavior may provide vital information about their responses to environmental stressors. In my study, I used a series of assays to quantify behavioral patterns. A shy-bold continuum of personality is recognized in many animals. I measured behaviors along this continuum in honeybees to determine if there are behavioral differences among individuals within a colony. I found correlations that suggest that individual bees may display behavioral differences. I was able to investigate differences among bees, but not behavioral trajectories over time. This study provides an initial look into how behavioral syndromes differ between individual honeybees within the same colony. Potential future research that follows bees from their first day as adults and throughout their lives will be necessary to augment the data collected here and provide information that may lead to the idea of personality in honeybees. As we continue to learn more about behavior and personality in honeybees it may become possible to provide ecosystem services that increase the fitness of the colony. Background: Honeybee Biology The western honeybee, Apis mellifera, evolved in Africa, Europe, and the Middle East and has been transported by humans to nearly all temperate and tropical terrestrial habitats (Breed, 2010). In their native range, bees are important pollinators and food sources for a variety of animals. In the habitats to which they have been introduced, bees have displaced native pollinators and are critical in the pollination of fruits and vegetables (Breed, 2010). Honeybees generally live in colonies of about 30,000 bees, but in mid-summer colonies can reach populations of 100,000 bees (Winston, 1987). Each colony contains a single queen and a large number of female workers. Males, or drones, are specifically raised to mate with the queen and do not contribute to the work within the colony. During the first few days of a queen’s life, she mates with 10-15 drones and stores the sperm for the rest of her life (Winston, 1987). This generates genetic diversity among the workers as they are still all halfor full sisters. Full sisters are sometimes called super-sisters as they are, on average, related by 75% as all sperm from one male are identical. After new queen eggs are laid, the old queen departs from the hive with half of the workers in a process called colony fission or swarming (Winston, 1987). This allows for the spread of bees and may be repeated as new queens hatch until the parent hive reaches a low population. Honeybees generally forage in a circular range with a 2-3 km radius around their hive (Winston, 1987). When a single worker bee finds an area that is rich in pollen or nectar it returns to the hive and very efficiently gathers recruits to help harvest the food source. Bees do this through a waggle dance that portrays angle relative to the sun and distance (Winston, 1987). Floral odor is also important and is carried from the flowers. Recruited worker bees then follow the directions from the dances to the feeding site. Bees consume a diet of carbohydrates, provided by nectar, and protein, provided by pollen (Winston, 1987). During the summer, food sources wax and wane as weather changes interact with the plants’ natural growing seasons. During times of low food supply, bees become less picky about the sources from which they harvest. Bees also stockpile honey for use during the winter when it is too cold to fly. Bees are able to create a microclimate within the hive during these cold seasons by using their flying muscles to “shiver” in a giant ball with an interior temperature of about 30° C and that warms up the outside bee to at least 10° C (Winston, 1987). Efficient work within the colony is achieved by dividing labor according to age and genetically determined thresholds for performing particular tasks (Page, 2013). Throughout their lives, honeybees differentiate into different tasks according to age group. During their first few days of adult life, a bee primarily works as a cell cleaner of brood cells (Seeley, 1995). At this time, a worker will spend approximately 20% of her day resting, and 20% walking through the combs. By day three of adult life, a bee develops fully functioning hypopharyngeal glands which secret brood food. For the next ten days, the adult bee functions as a nurse. During this time, a bee also develops a sting reflex that will aid her as she ventures outside the nest (Breed et al., 2004). At an age of approximately 12 days, the adult bee leaves the broodnest to begin work in food storage. Here, she will use evaporation to turn nectar into honey. She also packs pollen, ventilates the hive, helps with guarding the hive entrance, and builds comb (Winston, 1987). At an age of approximately 20 days, a bee begins the dangerous work outside of the hive as a forager involved in gathering pollen, nectar, water, and resin. This work continues until death, which usually occurs after about four weeks of adult life (Winston, 1987). Bees are also able to transition through the worker castes depending on colony need. When the hive is functioning normally, bees may become elite in their role. Elitism can be defined as a situation where a small proportion of workers perform a large proportion of specific work for the colony. When a disturbance wipes out these elite members, the hive is able to quickly recover by rearranging the division of labor to accommodate for the loss (Tenczar, 2014). While bees progress through castes based on age, they can also differentiate based on colony need; at other times genetic behavioral differences may dictate the caste a particular bee belongs to. A study by Whitfield et al. (2003) showed that nurses and foragers differ in gene expression for 39% of DNA. Based on these differences, scientists were able to identify the job of a bee with up to 92% accuracy regardless of age, colony, or genetic source. Using the researcher’s definition of behavior (an individual’s unique product of genotype and environment), it is impossible to tell if the genotype of the bee determines which job it will move into, or if the environment the bee encounters (either before moving to a new task, or once assigned to a certain role) determines which genes will be expressed. While all bees pass through some jobs, such as nursing larvae, only some bees will participate in guarding (Winston, 1987). Temporal polyethism, or the division of labor between morphologically similar individuals in an insect community, has long interested scientists (Johnson, 2008; Tofts, 1993). In other eusocial insects, such as ants, there seems to be an algorithm for division of labor that has three assumptions: (1) individuals seek out opportunities to work; (2) tasks exist both within and outside of the nest but are organized “centrifugally,” radiating from the center of the colony; (3) workers start work at the center point of the nest and move to outer positions as they age. This creates a model in which a colony displays a pattern of age polyethism (Tofts, 1993). While the second and third assumption are easily seen in honeybees, as workers emerge in the center of the hive, immediately become a nurse bees, and then start working outward until finally becoming foragers, the first assumption is less certain. It is not yet understood if workers switch task in response to the environment or due to some internal cue such as age, genetics, or personality (Seeley, 1995). Introduction: Personality in Animals Personality can be defined as a consistent set of behaviors across contexts, over time. Within the eusocial insect world, personality has been studied extensively. An individual’s personality may affect its ability to survive (Jandt et al., 2013). Because of this, it may be easy to assume that one personality type would be favored over another. However, if a personality does not exhibit some plasticity, an individual may not be able to adapt to new situations, leading to a decreased ability to survive (Jandt et al., 2013). In these eusocial insects, it is sometimes easier to observe the personality of a colony rather than an individual (Jandt et al., 2013). Because colonies reproduce by swarming when additional queens are reared, natural selection works at the colony level rather than on variation among workers. While selection at this larger level might create differences in personalities between hives as each hive employs different strategies to survive, it is still the sum of individual contributions that determines which hive will be successful enough to undergo colonial fission (swarming). Individuals and the colonies in which they live may vary in their personality across situations (Pinter-Wollman, 2012). When studying honeybees, it is important to note not only the obvious difference among colonies, but also the differences within a colony that contribute to its success over another colony. A broad range of studies suggest that individual animals differ in their behavior across contexts and in response to social and environmental variation (Bergmuller et al., 2010; Whitfield et al., 2003; Jandt et al., 2013). Studies of Apis mellifera have shown that genes related to behavior help to determine specific tasks in the colony that the bee may perform (Whitfield et al., 2003). While personality can be seen easily in various contexts, it is still unknown if the bee exhibits a predictable personality across its lifespan (Jandt et al., 2013). For workers to make the greatest contribution within their hive, it would seem that unique personalities among the individuals could be important in facilitating division of labor. Bees of the same age often differentiate across jobs such as guarding and hive maintenance; additionally, colonies encounter changing situations during their active season. To be adaptable, differing personalities among workers should exist in order to allow hives to respond appropriately to new environmental stressors. To measure personality on the hive level, scientists have used a myriad of experimental designs. The majority of these look at easy to recognize traits such as shy-bold or aggression. In honeybees specifically, behaviors such as a “defensive response” has been correlated to aggression (Wray et al., 2011). If these behavioral responses were to be studied over the lifespan of a colony, an investigator could estimate personality of the colony. Wray et al. (2011) assessed collective personality across hives on honeybees. The results of this study found two principal components for behavior: the first being composed of defensive response, activity level, and comb repair; the second being composed of defensive response, foraging activity, and undertaking. The first component corresponded with more excitable bees that were warier of disturbances and less likely to repair their hives. The second component related to more flexibility but also riskier behaviors. This study provides another look at behavioral syndromes in honeybees. While most studies choose to look at differences between the hives (as selection acts at the hive level), this experiment is one of the beginning steps in observing how individual differences might create the behavioral plasticity that is necessary for a hive to survive. Previous studies have looked at how environmental and genetic changes may affect colony personality, but it has remained difficult to relate an individual’s behavior to that of the group (Bengston and Jandt, 2014). Previous studies have explored the phenomenon of individual personality in other social insects. In Myrmica ants, it was found that colonies that were bold in responsiveness were made up of highly social individuals. Furthermore, within a colony, certain behavioral traits were correlated with task allocation (Chapman, 2011). In the social spider Anelosimus studiosus, within group variation has been found to aid fitness. In this species, task specialization and behavior have a positive association with individual and group level task efficiency (Pruitt, 2011). In paper wasps (Polistes dominulus), aggression creates hierarchy as individual bees compete to gain breeding status (Cant et al. 2006). With other social insects displaying such differences in personality, honeybees may also benefit from individual differences within the colony. In this study, I examined individual honeybees of the same age range and recorded time to calm, alarm response, and simulated predation response in order to determine how individuals differ from each other behaviorally. I arrived at these assays after extensive library research, field observation, and preliminary experiments. I hypothesized bees which took longer to calm and were more active after exposure to alarm pheromone could be seen as more active and this trait could be correlated with nervousness (Wray et al., 2011). The final assays explored how shy or bold a bee was by determining how quickly they were able to regain composure after a disorienting experience. Background Information for the Behavioral Assays While some eusocial insects divide labor based on fixed morphological differences, honeybees remain uniform in body shape and size throughout their adult life and have more temporary jobs within the hive (Seeley, 1995). One such job is “guarding.” Generally, bees in this group are between the ages of 12 and 25 days (Winston, 1987). Guard workers are easily distinguishable by the posture the bee assumes when on the entrance, or porch, of the hive. With their wings up, antenna alert, and forelegs lifted, these bees patrol the porch and ensure that any bee entering the hive is a member of the colony by looking at their odor and behavior (Winston, 1987). By the time bees have reached the point of engaging in guarding behavior, they are producing the maximum amount of alarm pheromone they will ever produce and their mandibular glands have switched from producing brood food to producing a second type of alarm pheromone, 2-heptanone (Winston, 1987). Isopentyl acetate (also called amyl acetate) is one of two alarm pheromones used by honeybees. It is emitted either when a bee takes a stinging posture and fans, or when the stinger is ripped from the body of the worker (Winston, 1987). This acts as a chemical signal that can coordinate a defensive response against danger (Seeley, 1995). When pheromone is placed on the porch of a hive, bees will emerge from inside the hive to provide an army against any potential enemy (Boch et al., 1969). Even though a defensive group is gathered, bees will still refrain from stinging unless the potential target moves (Boch et al., 1969). This allows the bees to be prepared to defend themselves without losing members of the group unnecessarily. When bees are exposed to alarm pheromone, this causes an increase in activity. In order to determine if different bees have different personalities, it is necessary to see if activity levels in response to alarm pheromone vary among individuals. Because it would not be fortuitous to have an entire hive place itself in danger, I aimed to test whether there would be differences among individuals. In A. mellifera, workers attempt to mitigate the risks of predation by flying away from the colony to sting and bite vertebrates. They have a small defensive perimeter that extends just 50 meters away from the nest (Breed et al., 2004). In a colony level test that measured the speed with which bees ran across the comb (runniness) following a brick drop, Wray et al. (2011) found significant differences between hives. I attempted to run a similar experiment with the hypothesis that if colonies differ, individuals within the hive probably also differed. Methods From July to August of 2014, worker A. mellifera were tested from hives located on the East Campus of the University of Colorado at Boulder. Two hives that were not involved in any other experiments were sampled in to ensure that hive-wide personality traits were accounted for. Tests alternated frequently between the two hives in order to ensure that times of year were not a factor. In an effort to standardize any personality that may come about from assigned jobs, only guard bees were tested. Guard bees were determined by their presence on the porch of the hive and their guarding stance (Winston, 1987). Bees were collected and placed in individual glass petri dishes with filter paper on the bottom during the morning hours and were run through a series of 3 assays. The average length of time a bee spent in captivity was just over an hour.