Juvenile hormone, reproduction, and worker behavior in the neotropical social wasp Polistes canadensis

Previous studies of the division of labor in colonies of eusocial Hymenoptera (wasps and bees) have led to two hypotheses regarding the evolution of juvenile hormone (JH) involvement. The novel- or single-function hypothesis proposes that the role of JH has changed from an exclusively reproductive function in primitively eusocial species (those lacking morphologically distinct queen and worker castes), to an exclusively behavioral function in highly eusocial societies (those containing morphologically distinct castes). In contrast, the split-function hypothesis proposes that JH originally functioned in the regulation of both reproduction and behavior in ancestral solitary species. Then, when reproductive and brood-care tasks came to be divided between queens and workers, the effects of JH were divided as well, with JH involved in regulation of reproductive maturation of egg-laying queens, and behavioral maturation, manifested as age-correlated changes in worker tasks, of workers. We report experiments designed to test these hypotheses. After documenting age-correlated changes in worker behavior (age polyethism) in the neotropical primitively eusocial wasp Polistes canadensis, we demonstrate that experimental application of the JH analog methoprene accelerates the onset of guarding behavior, an age-correlated task, and increases the number of foraging females; and we demonstrate that JH titers correlate with both ovarian development of queens and task differentiation in workers, as predicted by the split-function hypothesis. These findings support a view of social insect evolution that sees the contrasting worker and queen phenotypes as derived via decoupling of reproductive and brood-care components of the ancestral solitary reproductive physiology.     

Reproduction in worker honey bees is associated with low juvenile hormone titers and rates of biosynthesis.

Three experiments were performed to determine the role of juvenile hormone (JH) in worker reproduction in queenless colonies of honey bees. In Experiment 1, egg-laying workers had low hemolymph titers of JH, as did bees engaged in brood care, while foragers had significantly higher titers. Experiment 2 confirmed these findings by demonstrating that laying workers have significantly lower rates of JH biosynthesis than foragers do. In Experiment 3, ovary development was inhibited slightly by application of the JH analog methoprene to 1-day-old bees, but was not affected by application to older bees, at least some already displaying egg-laying behavior. These results, which are consistent with earlier findings for queen honey bees, are contrary to a common model of insect reproduction, in which elevated JH titers trigger ovary development, which then leads to oviposition. Previous experiments have demonstrated that JH regulates nonreproductive behavior in workers that is associated with colony division of labor; perhaps this function is incompatible with a traditional role for JH in reproduction.     

Pleiotropy of segregating genetic variants that affect honey bee worker life expectancy

In contrast to many other complex traits, the natural genetic architecture of life expectancy has not been intensely studied, particularly in non-model organisms, such as the honey bee (Apis mellifera L.). Multiple factors that determine honey bee worker lifespan have been identified and genetic analyses have been performed on some of those traits. Several of the traits are included in a suite of correlated traits that form the pollen hoarding syndrome, which was named after the behavior to store surplus pollen in the nest and is tied to social evolution. Here, seven quantitative trait loci that had previously been identified for their effects on different aspects of the pollen hoarding syndrome were studied for their genetic influence on the survival of adult honey bee workers. To gain a more comprehensive understanding of the genetic architecture of worker longevity, a panel of 280 additional SNP markers distributed across the genome was also tested. Allelic distributions were compared between young and old bees in two backcross populations of the bi-directionally selected high- and low-pollen hoarding strain. Our results suggest a pleiotropic effect of at least one of the behavioral quantitative trait loci on worker longevity and one significant and several other putative genetic effects in other genomic regions. At least one locus showed evidence for strong antagonistic pleiotropy and several others suggested genetic factors that influence pre-emergence survival of worker honey bees. Thus, the predicted association between worker lifespan and the pollen hoarding syndrome was supported at the genetic level and the magnitude of the identified effects also strengthened the view that naturally segregating genetic variation can have major effects on age-specific survival probability in the wild.     

Regulation of behavioral maturation by a primer pheromone produced by adult worker honey bees

Previous research showed that the presence of older workers causes a delayed onset of foraging in younger individuals in honey bee colonies, but a specific worker inhibitory factor had not yet been identified. Here, we report on the identification of a substance produced by adult forager honey bees, ethyl oleate, that acts as a chemical inhibitory factor to delay age at onset of foraging. Ethyl oleate is synthesized de novo and is present in highest concentrations in the bee's crop. These results suggest that worker behavioral maturation is modulated via trophallaxis, a form of food exchange that also serves as a prominent communication channel in insect societies. Our findings provide critical validation for a model of self-organization explaining how bees are able to respond to fragmentary information with actions that are appropriate to the state of the whole colony.     

Kin discrimination by worker honey bees in genetically mixed groups

We tested the hypothesis that in a genetically mixed assemblage of worker honey bees, individual workers would behave differently toward unfamiliar sisters than toward unfamiliar nonsisters. Groups of worker honey bees of mixed genetic composition were assembled by collecting pupae from separate colonies and placing the worker bees together on eclosion. A total of 10 workers, 5 from each of two kin groups, were used to form each group. When the workers were 5 days old, a worker of one of the two kin groups was introduced into the mixed group. This worker had previously been held in a group of its sisters, without contact with queen or nonsister bees. The interactions with the introduced bee indicate that in a mixed kin group, individual workers learn the composite identity of the group and do not attack unfamiliar bees differentially on the basis of kinship. However, kinship does influence the total number of interactions in which an introduced bee engages when placed in a genetically mixed group; bees interacted significantly more often with sisters than with nonsisters. There was a trend for bees to be involved in more feeding interactions with sisters. This finding indicates an ability of a bee to learn and use its own cues. In mixed groups, each bee maintains its genotypically correlated identity; the bees' odors do not comingle into a “group” or “gestalt” odor. The significance of these results is discussed in light of the genetic structure of natural colonies of honey bees.     

A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid

By using a functional genomics approach, we have identified a honey bee [Apis mellifera (Am)] odorant receptor (Or) for the queen substance 9-oxo-2-decenoic acid (9-ODA). Honey bees live in large eusocial colonies in which a single queen is responsible for reproduction, several thousand sterile female worker bees complete a myriad of tasks to maintain the colony, and several hundred male drones exist only to mate. The "queen substance" [also termed the queen retinue pheromone (QRP)] is an eight-component pheromone that maintains the queen's dominance in the colony. The main component, 9-ODA, acts as a releaser pheromone by attracting workers to the queen and as a primer pheromone by physiologically inhibiting worker ovary development; it also acts as a sex pheromone, attracting drones during mating flights. However, the extent to which social and sexual chemical messages are shared remains unresolved. By using a custom chemosensory-specific microarray and qPCR, we identified four candidate sex pheromone Ors (AmOr10, -11, -18, and -170) from the honey bee genome based on their biased expression in drone antennae. We assayed the pheromone responsiveness of these receptors by using Xenopus oocytes and electrophysiology. AmOr11 responded specifically to 9-ODA (EC50=280+/-31 nM) and not to any of the other seven QRP components, other social pheromones, or floral odors. We did not observe any responses of the other three Ors to any of the eight QRP pheromone components, suggesting 9-ODA is the only QRP component that also acts as a long-distance sex pheromone.     

Egg viability and worker policing in honey bees

In many species of social Hymenoptera, unmated workers can lay eggs that will produce males by parthenogenesis. Nevertheless, in queenright honey bee colonies (Apis mellifera), worker reproduction is low. One possible mechanism for this difference is worker policing, the removal of worker-laid eggs by other workers. This behavior can evolve in species in which queens are multiply mated, where workers are more closely related to the sons of their mother than those of their sisters. Another possible mechanism of the low level of worker reproduction is worker-laid eggs being less viable than queen-laid eggs. We show that this difference in quality is the case for honey bees.     

Cellular senescence in honey bee brain is largely independent of chronological age

Accumulation of oxidative stress-induced damage in brain tissue plays an important role in the pathogenesis of normal aging and neurodegenerative diseases. Neuronal oxidative damage typically increases with age in humans, and also in the invertebrate and vertebrate model species most commonly used in aging research. By use of quantitative immunohistochemistry and Western blot, we show that this aspect of brain senescence is largely decoupled from chronological age in the honey bee (Apis mellifera). The bee is a eusocial insect characterized by the presence of a reproductive queen caste and a caste of functionally sterile female workers that performs various alloparental tasks such as nursing and foraging. We studied patterns of oxidative nitration and carbonylation damage in the brain of worker bees that performed nurse tasks as 8- and 200-day-olds and foraging tasks as 20- and 200-day-olds. In addition, we examined 180-day-old diutinus bees, a stress-resistant temporal worker form that survives unfavorable periods. Our results indicate that nitration damage occurs only at low levels in vivo, but that a 60-kDa protein from honey bee brain is selectively nitrated by peroxynitrite in vitro. Oxidative carbonylation is present at varying levels in the visual and chemosensory neuropiles of worker bees, and this inter-individual variation is better explained by social role than by chronological age.     

Evolution and mechanisms of long life and high fertility in queen honey bees

Honey bees (Apis mellifera) are eusocial insects that exhibit striking caste-specific differences in longevity. Queen honey bees live on average 1–2 years whereas workers live on average 15–38 days in the summer and 150–200 days in the winter. Previous studies of senescence in the honey bee have focused on establishing the importance of extrinsic mortality factors (predation, weather) and behavior (nursing and foraging) in worker bee longevity. However, few studies have tried to elucidate the mechanisms that allow queen honey bees to achieve their long lifespan without sacrificing fecundity. Here, we review both types of studies and emphasize the importance of understanding both proximate and ultimate causes of the unusual life history of honey bee queens.     

Age, caste, and behavior determine the replicative activity of intestinal stem cells in honeybees (Apis mellifera L.)

Honeybees (Apis mellifera L.) display a pronounced natural aging plasticity. The differences in aging rates between the alternative phenotypes and behavioral classes could reflect differences in protection against damage or in the ability to repair vulnerable tissues. As in other animals, including humans, the gut is continually exposed to environmental insults and harbors a large population of replicating stem cells that maintain the intestinal epithelium. Through studies of the major internal organs using incorporation and immunodetection of the mitotic marker bromo-deoxyuridine, the intestine was determined to be the main site of tissue renewal in adult honeybees. Proliferative activity of the intestinal stem cells was compared among queens, workers, and males of different ages. Simultaneous attempts to assess intestinal cell loss via apoptosis yielded inconclusive results. The relationship between intestinal cell proliferation and worker life-history was evaluated in greater depth by studying diutinus winter workers, reproductive workers, and by decoupling worker behavioral status from chronological age in a single-cohort colony. Intestinal cell proliferation was abundant in all groups and showed an age-related decline in workers, queens, and males. At young ages, workers exhibited relatively more intestinal cell proliferation than did queens and queens more than drones, but the caste and sex differences decreased with age. Cell proliferation did not decrease beyond 6 weeks of age in older queens and in diutinus workers. Ovary activation did not correlate with the amount of intestinal stem cell proliferation in workers, although the queenless hive condition was associated with lower overall counts. In the single-cohort colony, nurse bees exhibited more cell proliferation than foragers, regardless of age. The overall results do not support our hypothesis that longer-lived phenotypes exhibit increased somatic repair in the form of higher replicative activity of intestinal stem cells. Instead, the observed proliferation patterns reflect differential demands for digestive activity in the different groups, which result in different requirements for replacement of lost intestinal cells. The maintenance of proliferative capacity for over 1 year suggests that queen intestinal stem cells have a relatively high replicative potential, but further studies are needed to relate honeybee lifespan differences to cellular aging.     

Regulation of life history determines lifespan of worker honey bees (Apis mellifera L.)

Life expectancy of honey bees (Apis mellifera L.) is of general interest to gerontological research because its variability among different groups of bees is one of the most striking cases of natural plasticity of aging. Worker honey bees spend their first days of adult life working in the nest, then transition to foraging and die between 4 and 8 weeks of age. Foraging is believed to be primarily responsible for the early death of workers. Three large-scale experiments were performed to quantitatively assess the importance of flight activity, chronological age, extrinsic mortality factors and foraging specialization. Forager mortality was higher than in-hive bee mortality. Most importantly however, reducing the external mortality hazards and foraging activity did not lead to the expected strong extension of life. Most of the experimental effects were attributable to an earlier transition from hive tasks to foraging. This transition is accompanied by a significant mortality peak. The age at the onset of foraging is the central variable in worker life-history and behavioral state was found more important than chronological age for honey bee aging. However, mortality risk increased with age and the negative relation between pre-foraging and foraging lifespan indicate some senescence irrespective of behavioral state. Overall, honey bee workers exhibit a logistic mortality dynamic which is mainly caused by the age-dependent transition from a low mortality pre-foraging state to a higher mortality foraging state.     

Chronic Bee Paralysis Virus in Honeybee Queens: Evaluating Susceptibility and Infection Routes

Chronic bee paralysis virus (CBPV) is known as a disease of worker honey bees. To investigate pathogenesis of the CBPV on the queen, the sole reproductive individual in a colony, we conducted experiments regarding the susceptibility of queens to CBPV. Results from susceptibility experiment showed a similar disease progress in the queens compared to worker bees after infection. Infected queens exhibit symptoms by Day 6 post infection and virus levels reach 10¹¹ copies per head. In a transmission experiment we showed that social interactions may affect the disease progression. Queens with forced contact to symptomatic worker bees acquired an overt infection with up to 10¹¹ virus copies per head in six days. In contrast, queens in contact with symptomatic worker bees, but with a chance to receive food from healthy bees outside the cage appeared healthy. The virus loads did not exceed 10⁷ in the majority of these queens after nine days. Symptomatic worker bees may transmit sufficient active CBPV particles to the queen through trophallaxis, to cause an overt infection.     

Aging and development in social insects with emphasis on the honey bee, Apis mellifera L

Honey bee colonies typically consist of about 20-40 thousand workers, zero to few thousand males (drones), depending on the time of year, and a single queen, the mother of the colony. Workers typically live 3-6 weeks during the spring and summer and can live about 4months during the winter. Queens are longer lived. Anecdotes of queens living 2-3years are not unusual, though they normally live less than a year in commercial hives. Little is known about the life span of drones. Queens develop from fertilized eggs that are not different from the eggs that develop into workers. Queens are, however, twice as large, have specialized anatomy, live much longer, and develop faster from egg to adult. All of these differences are derived from differences in larval rearing environment, primarily nutrition. The developmental trajectory of a female larva from worker into a queen can be determined as late as the third day of larval development, after this time the developmental pathway is fixed for a worker phenotype. The total time of larval development is only 5-6 days, therefore, just 2-3 days of differential feeding can lead to profound differences in development, and longevity. Workers undergo age development after they become adults. Workers usually initiate foraging behavior when they are 2-3 weeks old. The age at which a worker initiates foraging is a strong determinant of her length of life. This is presumed to be a result of the hazards of foraging, but natural senescence also occurs. Some bees remain in the nest and are never observed to forage, thereby outliving their forager sisters. Corresponding to this behavioral development are changes in the sizes of glands and the production of glandular products, increases in biogenic amine titers within the brain, an increase in the volume of specific regions of the brain, and changes in the neural system that affect perception of stimuli, and learning and memory. These age-related changes in behavior are regulated by intrinsic and extrinsic factors. Genetic variation has been demonstrated for many of these life history and behavioral traits. Selection and genome mapping studies have demonstrated relationships between the neural system, behavior, and life history traits.     

Vitellogenin, juvenile hormone, insulin signaling, and queen honey bee longevity

In most animals, longevity is achieved at the expense of fertility, but queen honey bees do not show this tradeoff. Queens are both long-lived and fertile, whereas workers, derived from the same genome, are both relatively short-lived and normally sterile. It has been suggested, on the basis of results from workers, that vitellogenin (Vg), best known as a yolk protein synthesized in the abdominal fat body, acts as an antioxidant to promote longevity in queen bees. We explored this hypothesis, as well as related roles of insulin-IGF-1 signaling and juvenile hormone. Vg was expressed in thorax and head fat body cells in an age-dependent manner, with old queens showing much higher expression than workers. In contrast, Vg expression in worker head was much lower. Queens also were more resistant to oxidative stress than workers. These results support the hypothesis that caste-specific differences in Vg expression are involved in queen longevity. Consistent with predictions from Drosophila, old queens had lower head expression of insulin-like peptide and its putative receptors than did old workers. Juvenile hormone affected the expression of Vg and insulin-IGF-1 signaling genes in opposite directions. These results suggest that conserved and species-specific mechanisms interact to regulate queen bee longevity without sacrificing fecundity.     

Reproductive ground plan may mediate colony-level selection effects on individual foraging behavior in honey bees

The colony-level phenotype of an insect society emerges from interactions between large numbers of individuals that may differ considerably in their morphology, physiology, and behavior. The proximate and ultimate mechanisms that allow this complex integrated system to form are not fully known, and understanding the evolution of social life strategies is a major topic in systems biology. In solitary insects, behavior, sensory tuning, and reproductive physiology are linked. These associations are controlled in part by pleiotropic networks that organize the sequential expression of phases in the reproductive cycle. Here we explore whether similar associations give rise to different behavioral phenotypes in a eusocial worker caste. We document that the pleiotropic genetic network that controls foraging behavior in functionally sterile honey bee workers (Apis mellifera) has a reproductive component. Associations between behavior, physiology, and sensory tuning in workers with different foraging strategies indicate that the underlying genetic architectures were designed to control a reproductive cycle. Genetic circuits that make up the regulatory "ground plan" of a reproductive strategy may provide powerful building blocks for social life. We suggest that exploitation of this ground plan plays a fundamental role in the evolution of social insect societies.     

Cognitive aging is linked to social role in honey bees (Apis mellifera)

Aging is associated with cognitive impairment in numerous animal species. Across taxa, decline in learning performance is linked to chronological age. The honey bee (Apis mellifera), in contrast, offers an opportunity to study such aspects of aging largely independent of age per se. This is because foraging onset can be decoupled from chronological age, although workers typically first perform tasks inside the nest and later forage outside the hive. Further, early phases of foraging are characterized by growth of specific brain neuropiles, whereas late phases of the forager life-stage are accompanied by accelerated rates of physiological senescence. Yet, it is unclear if these patterns of senescence include cognitive function. The flexibility of worker ontogeny, however, suggests that the bee can become an attractive model for studies of plasticity in cognitive aging that ultimately may lead to insight into mechanisms that govern age-related cognitive decline. To address this potential, we studied effects of honey bee chronological age and of social role on sensory sensitivity and associative olfactory learning performance. Our results show a decline in olfactory acquisition performance that is linked to social role, but not to chronological age. This decline occurs only in foragers with long foraging duration, but at the same time the foragers show less generalization of odors, which is indicative of more precise learning. Foragers that are reversed from foraging to nest tasks, furthermore, do not show deficits in olfactory acquisition. These results point to complex effects of aging on associative learning in honey bees.     

Reproductive protein protects functionally sterile honey bee workers from oxidative stress

Research on aging shows that regulatory pathways of fertility and senescence are closely interlinked. However, evolutionary theories on social species propose that lifelong care for offspring can shape the course of senescence beyond the restricted context of reproductive capability. These observations suggest that control circuits of aging are remodeled in social organisms with continuing care for offspring. Here, we studied a circuit of aging in the honey bee (Apis mellifera). The bee is characterized by the presence of a long-lived reproductive queen caste and a shorter-lived caste of female workers that are life-long alloparental care givers. We focus on the role of the conserved yolk precursor gene vitellogenin that, in Caenorhabditis elegans, shortens lifespan as a downstream element of the insulin/insulin-like growth factor signaling cascade. Vitellogenin protein is synthesized at high levels in honey bee queens and is abundant in long-lived workers. We establish that vitellogenin gene activity protects worker bees from oxidative stress. Our finding suggests that one mechanistic explanation for patterns of longevity in bees is that a reproductive regulatory pathway has been remodeled to extend life. This perspective is of considerable relevance to research on longevity regulation that builds largely on inference from solitary model species.     

Hormonal control of the yolk precursor vitellogenin regulates immune function and longevity in honeybees

A striking example of plasticity in life span is seen in social insects such as ants and bees, where different castes may display distinct ageing patterns. In particular, the honeybee offers an intriguing illustration of environmental control on ageing rate. Honeybee workers display a temporal division of labour where young bees (or 'hive bees') perform tasks within the brood nest, and older bees forage for nectar, pollen propolis and water. When bees switch from the hive bee to the forager stage, their cellular defence machinery is down-regulated by a dramatic reduction in the number of functioning haemocytes (immunocytes). This study documents that the yolk precursor vitellogenin is likely to be involved in a regulatory pathway that controls the observed decline in somatic maintenance function of honeybee foragers. An association between the glyco-lipoprotein vitellogenin and immune function has not previously been reported for any organism. Honeybee workers are functionally sterile, and via the expression of juvenile hormone, a key gonotrophic hormone in adult insects, their vitellogenin levels are influenced by social interactions with other bees. Our results therefore suggest that in terms of maintenance of the cellular immune system, senescence of the honeybee worker is under social control.     

The development of an ethanol model using social insects I: behavior studies of the honey bee (Apis mellifera L.)

BACKGROUND: The purpose of this experiment was to test the feasibility of creating an animal model of ethanol consumption using social insects. Honey bees were selected as the model social insect because much is known about their natural history, physiology, genetics, and behavior. They are also inexpensive to procure and maintain. Of special interest is their use of communication and social organization. METHODS: Using both between- and within-experiment designs, studies were conducted with harnessed foragers to determine whether honey bees would consume ethanol mixed with sucrose (and, in some cases, water). Shuttle-box and running-wheel studies were conducted to examine the effect of ethanol on locomotion. The effect of ethanol on stinging behavior in harnessed foragers was investigated. The effect of ethanol on Pavlovian conditioning of proboscis extension was also investigated. Finally, in a self-administration study, foraging honey bees were trained to fly to an artificial flower containing ethanol. RESULTS: (1) Harnessed honey bees readily consume 1%, 5%, 10%, and 20% ethanol solutions; (2) 95% ethanol will also be consumed as long as the antennae do not make contact with the solution; (3) with the exception of 95% ethanol, consumption as measured by contact time or amount consumed does not differ in animals that consume 1%, 5%, 10%, and 20% ethanol solutions; (4) exposure to a lesser (or greater) concentration of ethanol does not influence consumption of a greater (or lesser) concentration; (5) consumption of 10% and 20% ethanol solutions decreases locomotion when tested in both a shuttle-box and running-wheel situation; (6) consumption of 1%, 5%, 10%, and 20% ethanol does not influence stinging behavior in harnessed foragers; (7) ethanol solutions greater than 5% significantly impair Pavlovian conditioning of proboscis extension; and (8) free-flying honey bee foragers will readily drink from an artificial flower containing 5% ethanol. CONCLUSIONS: The experiments on consumption, locomotion, and learning suggest that exposure to ethanol influences behavior of honey bees similar to that observed in experiments with analogous vertebrates. The honey bee model presents unique research opportunities regarding the influence of ethanol in the areas of language, social interaction, development, and learning. Although the behavioral results are interesting, similarity between the physiologic effects of ethanol on honey bees and vertebrates has not yet been determined.