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.     

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.     

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     

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.     

Kin structure and the swarming behavior of the honey bee Apis mellifera

Summary Experimental hives obtained from cordovan queens that were instrumentally inseminated with semen from one cordovan and one Italian drone were set up and allowed to swarm. Cordovan provides a resessive genetic marker system (cuticle color) so that the workers from the cordovan and Italian male lines are distinguishable. Our results show that these patrilineal worker groups segregate non-randomly during colony fission and this segregation cannot be explained by observed age structure. Evidence of innate kin recognition in bees has been previously established. We argue that kin recognition could be responsible for the observed non-random grouping of kin during swarming.     

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.     

Queen and young larval pheromones impact nursing and reproductive physiology of honey bee (Apis mellifera) workers

Several insect pheromones are multifunctional and have both releaser and primer effects. In honey bees (Apis mellifera), the queen mandibular pheromone (QMP) and e-beta-ocimene (eβ), emitted by young worker larvae, have such dual effects. There is increasing evidence that these multifunctional pheromones profoundly shape honey bee colony dynamics by influencing cooperative brood care, a fundamental aspect of eusocial insect behavior. Both QMP and eβ have been shown to affect worker physiology and behavior, but it has not yet been determined if these two key pheromones have interactive effects on hypopharyngeal gland (HPG) development, actively used in caring of larvae, and ovary activation, a component of worker reproductive physiology. Experimental results demonstrate that both QMP and eβ significantly suppress ovary activation compared to controls but that the larval pheromone is more effective than QMP. The underlying reproductive anatomy (total ovarioles) of workers influenced HPG development and ovary activation, so that worker bees with more ovarioles were less responsive to suppression of ovary activation by QMP. These bees were more likely to develop their HPG and have activated ovaries in the presence of eβ, providing additional links between nursing and reproductive physiology in support of the reproductive ground plan hypothesis.     

Complex pleiotropy characterizes the pollen hoarding syndrome in honey bees (Apis mellifera L.)

The pollen hoarding syndrome consists of a large suite of correlated traits in honey bees that may have played an important role in colony organization and consequently the social evolution of honey bees. The syndrome was first discovered in two strains that have been artificially selected for high and low pollen hoarding. These selected strains are used here to further investigate the phenotypic and genetic links between two central aspects of the pollen hoarding syndrome: sucrose responsiveness and pollen hoarding. Sons of hybrid queen offspring of these two strains were tested for sucrose responsiveness and used to produce colonies with either a highly responsive or an unresponsive father. These two colony groups differed significantly in the amount of pollen stored on brood combs and with regard to their relationship between brood and pollen amounts. Additionally, four quantitative trait loci (QTL) for pollen hoarding behavior were assessed for their effect on sucrose responsiveness. Drone offspring of two hybrid queens were phenotyped for responsiveness and genotyped at marker loci for these QTL, identifying some pleiotropic effects of the QTL with significant QTL interactions. Both experiments thus provided corroborating evidence that the distinct traits of the pollen hoarding syndrome are mechanistically and genetically linked and that these links are complex and dependent on background genotype. The study demonstrates genetic worker–drone correlations within the context of the pollen hoarding syndrome and establishes that an indirect selection response connects pollen hoarding and sucrose responsiveness, regardless of which trait is directly selected.     

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     

Measurement of nest cavity volume by the honey bee (Apis mellifera)

Behavioral Ecology and Sociobiology -     

Behavioral and life-history components of division of labor in honey bees (Apis mellifera L.)

Variability exists among worker honey bees for components of division of labor. These components are of two types, those that affect foraging behavior and those that affect life-history characteristics of workers. Variable foraging behavior components are: the probability that foraging workers collect (1) pollen only; (2) nectar only; and (3) pollen and nectar on the same trip. Life history components are: (1) the age the workers initiate foraging behavior; (2) the length of the foraging life of a worker; and (3) worker length of life. We show how these components may interact to change the social organization of honey bee colonies and the lifetime foraging productivity of individual workers. Selection acting on foraging behavior components may result in changes in the proportion of workers collecting pollen and nectar. Selection acting on life-history components may affect the size of the foraging population and the distribution of workers between within nest and foraging activities. We suggest that these components define possible sociogenic pathways through which colony-level natural selection can change social organization. These pathways may be analogous to developmental pathways in the morphogenesis of individual organisms because small changes in behavioral or life history components of individual workers may lead to major changes in the organizational structure of colonies. Correspondence to: R.E. Page, Jr.     

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     

Maternity of replacement queens in the thelytokous Cape honey bee Apis mellifera capensis

Unlike workers of all other honey bee (Apis mellifera) subspecies, workers of the Cape honey bee of South Africa (A. mellifera capensis) reproduce thelytokously and are thus able to produce female offspring that are pseudoclones of themselves. This ability allows workers to compete with their queen over the maternity of daughter queens and, in one extreme case, has led to a clonal lineage of workers becoming a social parasite in commercially managed populations of A. mellifera scutellata. Previous work (Jordan et al., Proc R Soc Lond B Biol Sci 275:345, 2008) showed that, in A. mellifera capensis, 59% of queen cells produced during swarming events contained the offspring of workers and that, of these, 65% were the offspring of non-natal workers. Here, we confirm that a substantial proportion (38.5%) of offspring queens is worker-laid. We additionally show that: (1) Although queens produce most diploid female offspring sexually, we found some homozygous or hemizygous queen offspring, suggesting that queens also reproduce by thelytoky. These parthenogenetic individuals are probably nonviable beyond the larval stage. (2) Worker-laid offspring queens are viable and become the resident queen at the same frequency as do sexually produced queen-laid offspring queens. (3) In this study, all but one of the worker-derived queens were laid by natal workers rather than workers from another nest. This suggests that the very high rates of social parasitism observed in our previous study were enhanced by beekeeping manipulations, which increased movement of parasites between colonies.     

The effects of colony-level selection on the social organization of honey bee (Apis mellifera L.) colonies: colony-level components of pollen hoarding

Two-way selection for quantities of stored pollen resulted in the production of high and low pollen hoarding strains of honey bees (Apis mellifera L.). Strains differed in areas of stored pollen after a single generation of selection and, by the third generation, the high strain colonies stored an average 6 times more pollen than low strain colonies. Colony-level organizational components that potentially affect pollen stores were identified that varied genetically within and between these strains. Changes occurred in several of these components, in addition to changes in the selected trait. High strain colonies had a significantly higher proportion of foragers returning with loads of pollen, however, high and low strain colonies had equal total numbers of foragers Colony rates of intake of pollen and nectar were not independent. Selection resulted in an increase in the number of pollen collectors and a decrease in the number of nectar collectors in high strain colonies, while the reciprocal relationship occurred in the low strain. High and low strain colonies also demonstrated different diurnal foraging patterns as measured by the changing proportions of returning pollen foragers. High strain colonies of generation 3 contained significantly less brood than did low strain colonies, a consequence of a constraint on colony growth resulting from a fixed nest volume and large quantities of stored pollen. These components represent selectable colony-level traits on which natural selection can act and shape the social organization of honey bee coloniesCommunicated by R.F.A. Moritz     

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.     

Heritable variation in learning performance affects foraging preferences in the honey bee (Apis mellifera)

There has now been an abundance of research conducted to explore genetic bases that underlie learning performance in the honey bee (Apis mellifera). This work has progressed to the point where studies now seek to relate genetic traits that underlie learning ability to learning in field-based foraging problems faced by workers. Accordingly, the focus of our research is to explore the correlation between laboratory-based performance using an established learning paradigm and field-based foraging behavior. To evaluate learning ability, selected lines were established by evaluating queens and drones in a proboscis extension reflex (PER) conditioning procedure to measure learning in a laboratory paradigm—latent inhibition (LI). Hybrid queens were then produced from our lines selected for high and low levels of LI and inseminated with semen from many drones chosen at random. The genetically diverse worker progeny were then evaluated for expression of LI and for preference of pollen and/or nectar during foraging. Foragers from several different queens, and which had resulted from fertilization by any of several different drone fathers, were collected as they returned from foraging flights and analyzed for pollen and nectar contents. They were subsequently evaluated for expression of LI. Our research revealed that pollen foragers exhibited stronger learning, both in the presence (excitatory conditioning) and absence (LI) of reinforcement. The heightened overall learning ability demonstrated by pollen foragers suggests that pollen foragers are in general more sensitive to a large number of environmental stimuli. This mechanism could contribute toward explanations of colony-level regulation of foraging patterns among workers.Communicated by R. Page     

Genotype and colony environment affect honeybee (Apis mellifera L.) development and foraging behavior

We examined the interaction of genotype and environment on foraging-behavior development and forage choice in honeybees. High- and low-pollen-hoarding strains and unselected wild-type bees were co-fostered in pairs of colonies manipulated to differentially stimulate high and low pollen foraging. The high-pollen-foraging stimulus consisted of high amounts of larvae, a known stimulus for pollen foraging, plus low amounts of pollen, known to induce pollen foraging. The low-pollen-foraging stimulus consisted of low amounts of larvae plus high amounts of pollen. We estimated the median age at which bees initiated foraging, determined forage choice, and the quality and quantity of resources collected. High-strain bees consistently foraged at younger ages than workers from the other sources. High-strain bees appeared to be more sensitive to the pollen-foraging-stimulus treatments, showing greater differences in foraging age and behavior. Three-way interactions of genotype, pollen foraging stimulus, and colony pair (replicate) were statistically significant for most foraging variables measured suggesting that additional, unknown environmental factors also affect foraging behavior. Our results suggest there is a functional relationship between age of first foraging and forage choice with a strong genetic component that is modulated by colony environment.