Effects of worker genotypic diversity on honey bee colony development and behavior (Apis mellifera L.)

There have been numerous reports of genetic influences on division of labor in honey bee colonies, but the effects of worker genotypic diversity on colony behavior are unclear. We analyzed the effects of worker genotypic diversity on the phenotypes of honey bee colonies during a critical phase of colony development, the nest initiation phase. Five groups of colonies were studied (n = 5–11 per group); four groups had relatively low genotypic diversity compared to the fifth group. Colonies were derived from queens that were instrumentally inseminated with the semen of four different drones according to one of the following mating schemes: group A, 4 A-source drones; group B, 4 B-source drones; group C, 4 C-source drones; group D, 4 D-source drones; and group E, 1 drone of each of the A-D drone sources. There were significant differences between colonies in groups A-D for 8 out of 19 colony traits. Because the queens in all of these colonies were super sisters, the observed differences between groups were primarily a consequence of differences in worker genotypes. There were very few differences (2 out of 19 traits) between colonies with high worker genotypic diversity (group E) and those with low diversity (groups A-D combined). This is because colonies with greater diversity tended to have phenotypes that were average relative to colonies with low genotypic diversity. We hypothesize that the averaging effect of genotypic variability on colony phenotypes may have selective advantages, making colonies less likely to fail because of inappropriate colony responses to changing environmental conditions.     

The influence of pollen quality on foraging behavior in honeybees (Apis mellifera L.)

Individual and colony-level foraging behaviors were evaluated in response to changes in the quantity or nutritional quality of pollen stored within honeybee (Apis mellifera L.) colonies. Colonies were housed in vertical, three-frame observation hives situated inside a building, with entrances leading to the exterior. Before receiving treatments, all colonies were deprived of pollen for 5 days and pollen foragers were marked. In one treatment group, colony pollen reserves were quantitatively manipulated to a low or high level, either by starving colonies of pollen or by providing them with a fully provisioned frame of pollen composed of mixed species. In another treatment group, pollen reserves were qualitatively manipulated by removing pollen stores from colonies and replacing them with low- or high-protein pollen supplements. After applying treatments, foraging rates were measured four times per day and pollen pellets were collected from experienced and inexperienced foragers to determine their weight, species composition, and protein content. Honeybee colonies responded to decreases in the quantity or quality of pollen reserves by increasing the proportion of pollen foragers in their foraging populations, without increasing the overall foraging rate. Manipulation of pollen stores had no effect on the breadth of floral species collected by colonies, or their preferences for the size or protein content of pollen grains. In addition, treatments had no effect on the weight of pollen loads collected by individual foragers or the number of floral species collected per foraging trip. However, significant changes in foraging behavior were detected in relation to the experience level of foragers. Irrespective of treatment group, inexperienced foragers exerted greater effort by collecting heavier pollen loads and also sampled their floral environment more extensively than experienced foragers. Overall, our results indicate that honeybees respond to deficiencies in the quantity or quality of their pollen reserves by increasing the gross amount of pollen returned to the colony, rather than by specializing in collecting pollen with a greater protein content. Individual pollen foragers appear to be insensitive to the quality of pollen they collect, indicating that colony-level feedback is necessary to regulate the flow of protein to and within the colony. Colonies may respond to changes in the quality of their pollen stores by adjusting the numbers of inexperienced to experienced foragers within their foraging populations.     

Brood pheromone modulates honeybee (Apis mellifera L.) sucrose response thresholds

Foraging behavior and the mechanisms that regulate foraging activity are important components of social organization. Here we test the hypothesis that brood pheromone modulates the sucrose response threshold of bees. Recently the honeybee proboscis extension response to sucrose has been identified as a ”window” into a bee’s perception of sugar. The sucrose response threshold measured in the first week of adult life, prior to foraging age, predicts forage choice. Bees with low response thresholds are more likely to be pollen foragers and bees with high response thresholds are more likely to forage for nectar. There is an associated genetic component to sucrose response thresholds and forage choice such that bees selected to hoard high quantities of pollen have low response thresholds and bees selected to hoard low quantities of pollen have higher response thresholds. The number of larvae in colonies affects the number of bees foraging for pollen. Hexane-extractable compounds from the surface of larvae (brood pheromone) significantly increase the number of pollen foragers. We tested the hypothesis that brood pheromone decreases the sucrose response threshold of bees, to suggest a pheromone- modulated sensory-physiological mechanism for regulating foraging division of labor. Brood pheromone significantly decreased response thresholds as measured in the proboscis extension response assay, a response associated with pollen foraging. A synthetic blend of honeybee brood pheromone stimulated and released pollen foraging in foraging bioassays. Synthetic brood pheromone had dose-dependent effects on the modulation of sucrose response thresholds. We discuss how brood pheromone may act as a releaser of pollen foraging in older bees and a primer pheromone on the development of response thresholds and foraging ontogeny of young bees. Received: 24 May 2000 / Revised: 26 September 2000 / Accepted: 15 October 2000     

Social regulation of egg-laying by queenless honeybee workers (Apis mellifera L.)

Summary If a honeybee (Apis mellifera L.) colony loses its queen and remains queenless, a small percentage of the workers will develop into egg-layers and subsequently will produce males. The process of differentiation into laying and non-laying workers is accompanied by a great deal of aggression within the colony. In this study, I tried to establish the relationship between the potential to become egg-layers and behavioural differences at the individual level. To eight observation hives, I introduced 200 individually marked workers of similar age and observed their behavioural differentiation during a queenless period. Shortly after the first egg-laying worker appeared, the marked workers were dissected in order to determine their level of ovarian activation. The future laying-workers seemed to be slightly more involved in the rearing of new queens than other workers. As reported by other authors, aggression was mostly directed towards workers with activated ovaries. Only a very small number of aggressive workers were aggressive; on an average, slightly fewer of these marked workers had activated ovaries. Aggression resulted in physical damage in only a small number of cases. The possible disadvantage of aggression for the bees under attack is discussed, as well as the possible benefit for the aggressors. Evidence is presented that the most detrimental effect of aggression for bees under attack is the fact that they lose considerable quantities of food through trophallaxis to other workers. Workers with activated ovaries withdrew inside empty cells significantly more often than other workers, possibly to avoid attacks. Aggressive workers were (almost) never seen to receive food from the bees they attacked. It is suggested that by preventing other workers from becoming egg-layers, aggressors increase their own chances of future reproduction.     

Pathogen-associated self-medication behavior in the honeybee Apis mellifera

Honeybees, Apis mellifera, have several prophylactic disease defense strategies, including the foraging of antibiotic, antifungal, and antiviral compounds of plant products. Hence, honey and pollen contain many compounds that prevent fungal and bacterial growth and inhibit viral replication. Since these compounds are also fed to the larvae by nurse bees, they play a central role for colony health inside the hive. Here, we show that honeybee nurse bees, infected with the microsporidian gut parasite Nosema ceranae, show different preferences for various types of honeys in a simultaneous choice test. Infected workers preferred honeys with a higher antibiotic activity that reduced the microsporidian infection after the consumption of the honey. Since nurse bees feed not only the larvae but also other colony members, this behavior might be a highly adaptive form of therapeutic medication at both the individual and the colony level.     

Colony-level selection effects on individual and colony foraging task performance in honeybees, Apis mellifera L.

In honeybees, as in other highly eusocial species, tasks are performed by individual workers, but selection for worker task phenotypes occurs at the colony level. We investigated the effect of colony-level selection for pollen storage levels on the foraging behavior of individual honeybee foragers to determine (1) the relationship between genotype and phenotypic expression of foraging traits at the individual level and (2) how genetically based variation in worker task phenotype is integrated into colony task organization. We placed workers from lines selected at the colony level for high or low pollen stores together with hybrid workers into a common hive environment with controlled access to resources. Workers from the selected lines showed reciprocal variation in pollen and nectar collection. High-pollen-line foragers collected pollen preferentially, and low- pollen-line workers collected nectar, indicating that the two tasks covary genetically. Hybrid workers were not intermediate in phenotype, but instead showed directional dominance for nectar collection. We monitored the responses of workers from the selected strains to changes in internal (colony) and external (resource) stimulus levels for pollen foraging to measure the interaction between genotypic variation in foraging behavior and stimulus environment. Under low-stimulus conditions, the foraging group was over-represented by high-pollen-line workers. However, the evenness in distribution of the focal genetic groups increased as foraging stimuli increased. These data are consistent with a model where task choice is a consequence of genetically based response thresholds, and where genotypic diversity allows colony flexibility by providing a range of stimulus thresholds. Received: 3 May 1999 / Received in revised form: 22 December 1999 / Accepted: 23 January 2000     

Phenotype interactions in group behavior of honey bee workers (Apis mellifera L.)

Summary The alarm reaction of groups of honey bee workers was quantified using a metabolic bioassay. The genetic structure of these groups was varied in order to estimate the effects of worker interactions. Though the group phenotype was mainly determined by additive interactions, nonlinear effects were also found. Mixed worker groups, combined from colonies with similar reactivity in the bioassay, showed a stronger response than pure groups. This phenomenon, analogous to the overdominance model for individuals in classical genetics, has implications for mechanisms of natural and artificial selection in social populations and for the evolution of polyandry in social Hymenoptera.     

Effect of queen quality on interactions between workers and dueling queens in honeybee (Apis mellifera L.) colonies

The fitness of a social insect colony depends greatly on the quality (i.e., mating ability, fecundity, and offspring viability) of its queen(s). In honeybees, there is marked variation in the quality of young queens that compete in a series of lethal duels to replace a colonys previous queen. Workers interact with queens during these duels and could increase their inclusive fitness by biasing the outcomes of the duels in favor of high-quality queens. We predicted that workers will have more antagonistic interactions (chasing, grabbing, clamping) and fewer beneficent interactions (feeding, grooming) with low-quality than high-quality queens. To test this prediction, we reared queens from 0-day-old, 2-day-old, and 3-day-old worker larvae in observation colonies undergoing queen replacement, thus producing high-quality, low-quality, and very low-quality queens, respectively. Immediately after each queen emerged, we observed her for 1 h to record her interactions with the workers. Subsequent morphological measurement of the queens confirmed that initial larval age had a significant effect on queen quality. However, there was no consistent effect of queen quality on the rates of worker–queen interactions, thus falsifying our hypothesis. The mean power of our tests was high (0.599), therefore the probability of a type II error (a false negative) is low. We conclude that if workers actively select high-quality queens, then they do so prior to queen duels, during queen development. We suggest that each worker–queen interaction has a distinct adaptive significance rather than forming a suite of behavior that favors particular queens (e.g., chasing repels any queen that approaches a queen cell, thus protecting all queen cells from destruction).Communicated by M. Giurfa     

Evolution of extreme polyandry in the honeybee Apis mellifera L.

A number of hypotheses have been proposed to explain the evolution of multiple mating in the honeybee queen. In particular, the consequences of reduced intracolonial relatedness provide plausible explanations for multiple mating with up to ten drones, but fail to account for the much higher mating frequencies observed in nature. In this paper, we propose an alternative mechanism which builds on non-linear relationships between intracolonial frequencies in genotypic worker specialization and colony fitness. If genes for any worker specialization confer an advantage on colony fitness only when they are rare, this would require a stable mix of sperm from a few drones which contribute that trait, and many which do not. To ensure both specific, low within-colony proportions of “rare specialist” genes, and to reduce random variation of these proportions would require mating with high numbers of drones. The quantitative implementation shows that moderate to very high numbers of matings are required to exploit colony advantages from genotypic allocation of workers to rare tasks. Extreme polyandry thus could result from colony selection dependent on the intracolonial frequency of rare genetic specialists. Received: 30 January 1998 / Accepted after revision: 7 October 1998     

Reproduction by worker honey bees (Apis mellifera L.)

Summary Genetic markers were used to study the reproductive behavior of worker honey bees. Five experiments were conducted that demonstrate the significance of worker reproduction. Biases were found in the egg-laying success of workers belonging to different subfamilies within queenless colonies, however, members of all subfamilies laid eggs. These biases were probably not a consequence of direct reproductive competition among subfamily members but most likely represent genetic variability for the timing of the onset of oviposition. Workers preferentially oviposit in drone-sized cells, demonstrating a caste-specific adaptation for oviposition behavior. Drone brood production is highly synchronous within colonies and can result in the production of more than 6000 drones before colonies die. Workers reproduce in queenright colonies but at a very low frequency.     

Ovariole number—a predictor of differential reproductive success among worker subfamilies in queenless honeybee (Apis mellifera L.) colonies

A honeybee queen normally mates with 10–20 drones, and reproductive conflicts may arise among a colony’s different worker patrilines, especially after a colony has lost its single queen and the workers commence egg laying. In this study, we employed microsatellite markers to study aspects of worker reproductive competition in two queenless Africanized honeybee colonies. First, we determined whether there was a bias among worker patrilines in their maternity of drones and, second, we asked whether this bias could be attributed to differences in the degree of ovary activation of workers. Third, we relate these behavioral and physiological factors to ontogenetic differences between workers with respect to ovariole number. Workers from each of three (colony A) and one (colony B) patrilineal genotypes represented less than 6% of the worker population, yet each produced at least 13% of the drones in a colony, and collectively they produced 73% of the drones. Workers representing these genotypes also had more developed follicles and a greater number of ovarioles per ovary. Across all workers, ovariole development and number were closely correlated. This suggests a strong effect of worker genotype on the development of the ovary already in the postembryonic stages and sets a precedent to adult fertility, so that “workers are not born equal”. We hypothesize a frequency-dependent or “rare patriline” advantage to queenless workers over the parentage of males and discuss the maintenance of genetic variance in the reproductive capacity of workers.Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Reproductive competition in queenless honey bee colonies (Apis mellifera L.)

Previously we reported that there are subfamily differences in drone production in queenless honey bee colonies, but these biases are not always explained by subfamily differences in oviposition behavior. Here we determine whether these puzzling results are best explained by either inadequate sampling of the laying worker population or reproductive conflict among workers resulting in differential treatment of eggs and larvae. Using colonies composed of workers from electrophoretically distinct subfamilies, we collected samples of adult bees engaged in the following behavior: true egg laying, false egg laying, indeterminate egg laying, egg cannibalism, or nursing (contact with larvae). We also collected samples of drone brood at four different ages: 0 to 2.5-h-old eggs, 0 to 24-h-old eggs, 3 to 8-day-old larvae, and 9 to 14-day-old larvae and pupae. Allozyme analyses revealed significant subfamily differences in the likelihood of exhibiting egg laying, egg cannibalism, and nursing behavior, as well as significant subfamily differences in drone production. There were no subfamily differences among the different types of laying workers collected from each colony, suggesting that discrepancies between subfamily biases in egg-laying behavior and drone production are not due to inadequate sampling of the laying worker population. Subfamily biases in drone brood production within a colony changed significantly with brood age. Laying workers had significantly more developed ovaries than either egg cannibals or nurses, establishing a physiological correlate for the observed behavioral genetic differences. These results suggest there is reproductive conflict among subfamilies and individuals within queenless colonies of honey bees. The implications of these results for the evolution of reproductive conflict, in both queenright and queenless contexts, are discussed.     

Self-organization of circadian rhythms in groups of honeybees (Apis mellifera L.)

Workers in social groups of honeybees (Apis mellifera L.) synchronize their individual free-running circadian rhythms to an overall group rhythm. By monitoring the activity of bees by recording the oxygen consumption and intragroup temperature, it is shown that the rhythm coordination is in part achieved by temperature fluctuations as an intragroup Zeitgeber. Trophallaxis was shown to have only a minor (if any) effect on circadian rhythm synchronization. A model incorporating a feed back loop between temperature and activity can plausibly explain the observed synchronization of individual rhythms in social groups as a self-organization phenomenon. Correspondence to: R.F.A. Moritz     

Brood pheromone stimulates pollen foraging in honey bees (Apis mellifera)

Foraging and the mechanisms that regulate the quantity of food collected are important evolutionary and ecological attributes for all organisms. The decision to collect pollen by honey bee foragers depends on the number of larvae (brood), amount of stored pollen in the colony, as well as forager genotype and available resources in the environment. Here we describe how brood pheromone (whole hexane extracts of larvae) influenced honey bee pollen foraging and test the predictions of two foraging-regulation hypotheses: the indirect or brood-food mechanism and the direct mechanism of pollen-foraging regulation. Hexane extracts of larvae containing brood pheromone stimulated pollen foraging. Colonies were provided with extracts of 1000 larvae (brood pheromone), 1000 larvae (brood), or no brood or pheromone. Colonies with brood pheromone and brood had similar numbers of pollen foragers, while those colonies without brood or pheromone had significantly fewer pollen foragers. The number of pollen foragers increased more than 2.5-fold when colonies were provided with extracts of 2000 larvae as a supplement to the 1000 larvae they already had. Within 1 h of presenting colonies with brood pheromone, pollen foragers responded to the stimulus. The results from this study demonstrate some important aspects of pollen foraging in honey bee colonies: (1) pollen foragers appear to be directly affected by brood pheromone, (2) pollen foraging can be stimulated with brood pheromone in colonies provided with pollen but no larvae, and (3) pollen forager numbers increase with brood pheromone as a supplement to brood without increasing the number of larvae in the colony. These results support the direct-stimulus hypothesis for pollen foraging and do not support the indirect-inhibitor, brood-food hypothesis for pollen-foraging regulation. Received: 5 March 1998 / Accepted after revision: 29 August 1998     

Sperm limitation and the evolution of extreme polyandry in honeybees (Apis mellifera L.)

Honeybee queens (Apis mellifera) show extreme levels of polyandry, but the evolutionary mechanisms underlying this behaviour are still unclear. The sperm-limitation hypothesis, which assumes that high levels of polyandry are essential to get a lifetime sperm supply for large and long-lived colonies, has been widely disregarded for honeybees because the semen of a single male is, in principle, sufficient to fill the spermatheca of a queen. However, the inefficient post-mating sperm transfer from the queens lateral oviducts into the spermatheca requires multiple matings to ensure an adequate spermatheca filling. Males of the African honeybee subspecies A. m. capensis have fewer sperm than males of the European subspecies A. m. carnica. Thus, given that sperm limitation is a cause for the evolution of multiple mating in A. mellifera, we would expect A. m. capensis queens to have higher mating frequencies than A. m. carnica. Here we show that A. m. capensis queens indeed exhibit significantly higher mating frequencies than queens of A. m. carnica, both in their native ranges and in an experiment on a North Sea island under the same environmental conditions. We conclude that honeybee queens try to achieve a minimum number of matings on their mating flights to ensure a sufficient lifetime sperm supply. It thus seems premature to reject the sperm-limitation hypothesis as a concept explaining the evolution of extreme polyandry in honeybees.Communicated by R.E. Page     

Production and transmission of honey bee queen (Apis mellifera L.) mandibular gland pheromone

Summary The social cohesiveness of eusocial insect colonies is maintained primarily through the utilization of pheromones. In this study we quantitatively elucidated the production, secretion, and transmission of 9-keto2(E)-decenoic acid (9-ODA), one of the components of the mandibular gland pheromone of the honey bee queen Apis mellifera; this is the only identified primer pheromone complex in the eusocial insects. Mated queens produce 12–400 g of 9-ODA/day, or between 10% and 170% the average amount found in the glands at any one time. Approximately 0.5 g of 9-ODA is maintained on the body surface of queens by an equilibrium between exudation, internalization, tracking on the comb, and removal by workers. Retinue bees, attending the queen, remove the greatest amount, although the role of the wax as both a sink and a medium for pheromone transfer has been previously underestimated. Only about 1 in 10 retinue workers pick up substantial quantities of pheromone while attending the queen and, within seconds, most of the acquired 9-ODA is found externally on the abdomen, or in the gut. These attendants, also called messenger bees, transfer 9-ODA to other workers, mostly through direct contacts, but also via the wax. A model evaluating the pathways and relative quantities of 9-ODA transferred throughout the nest is presented. As well as being important for a basic understanding of the system, the results have implications for the proper design and use of pheromones in bee management.Offprint requests to: K. Naumann     

Worker reproductive parasitism and drift in the western honeybee Apis mellifera

When a honeybee (Apis spp.) colony loses its queen and is unable to rear a new one, some of the workers activate their ovaries and produce eggs. When a colony has a queen (i.e., it is queenright) almost all worker-laid eggs are eaten, but when hopelessly queenless, the workers become more tolerant of worker-laid eggs and rear some of them to adult drones. This increased tolerance renders a queenless colony vulnerable to worker reproductive parasitism, wherein unrelated workers enter the colony and lay eggs. Here, we show that the proportion of unrelated (non-natal) workers significantly decreases after an Apis mellifera colony becomes queenless. The remaining non-natal workers are as likely to have activated ovaries as natal workers, yet they produce more eggs than natal workers, resulting in significantly higher reproductive success for non-natal workers. In a second experiment, we provided queenless and queenright workers with a choice to remain in their own colony or to join a queenless or queenright colony nearby. The experiment was set up such that worker movement was unlikely to be due to simple orientation errors. Very few workers joined another colony, and there was no preference for workers to drift into or out of queenless or queenright colonies, in accordance with the proportion of non-natal workers declining significantly after becoming queenless in the first experiment.     

Colony state and regulation of pollen foraging in the honey bee,Apis mellifera L.

Summary To place social insect foraging behavior within an evolutionary context, it is necessary to establish relationships between individual foraging decisions and parameters influencing colony fitness. To address this problem, we examined interactions between individual foraging behavior and pollen storage levels in the honey bee, Apis mellifera L. Colonies responded to low pollen storage conditions by increasing pollen intake rates 54% relative to high pollen storage conditions, demonstrating a direct relationship between pollen storage levels and foraging effort. Approximately 80% of the difference in pollen intake rates was accounted for by variation in individual foraging effort, via changes in foraging activity and individual pollen load size. An additional 20% resulted from changes in the proportion of the foraging population collecting pollen. Under both high and low pollen storage treatments, colonies returned pollen storage levels to pre-experimental levels within 16 days, suggesting that honey bees regulate pollen storage levels around a homeostatic set point. We also found a direct relationship between pollen storage levels and colony brood production, demonstrating the potential for cumulative changes in individual foraging decisions to affect colony fitness. Offprint requests to: J.H. Fewell at the current address     

The role of the queen in circadian rhythms of honeybees (Apis mellifera L.)

Summary Colonies and smaller social groups of honeybees (Apis mellifera carnica L.) show distinct free-running circadian rhythms similar to that of individual organisms. The workers of a colony synchronize their individual rhythms to one overall group rhythm. Caste plays an important role in this synchronization process. Queens were introduced into worker groups which were entrained to a phase-shifted light/dark cycle. The introduction of the queen caused a shift in the free-running phase under constant dark conditions. Single introduced workers had no effect on the free-running rhythms. This indicates that the queen plays an important role in the synchronization of circadian rhythms of honeybee colonies.