Behavior and molecular physiology of nurses of worker and queen larvae in honey bees (Apis mellifera)

In a honey bee colony, worker bees rear a new queen by providing her with a larger cell in which to develop and a large amount of richer food (royal jelly). Royal jelly and worker jelly (fed to developing worker larvae) differ in terms of sugar, vitamin, protein and nucleotide composition. Here we examined whether workers attending queen and worker larvae are separate specialized sub-castes of the nurse bees. We collected nurse bees attending queen larvae (AQL) and worker larvae (AWL) and compared gene expression profiles of hypopharyngeal gland tissues, using Solexa/Illumina digital gene expression tag profiling (DGE). Significant differences in gene expression were found that included a disproportionate number of genes involved in glandular secretion and royal jelly synthesis. However behavioral observations showed that these were not two entirely distinct populations. Nurse workers were observed attending both worker larvae and queen larvae, and there was no evidence of a specialized group of workers that preferentially or exclusively attended developing queens. Nevertheless, AQL attended larvae more frequently compared to AWL, suggesting that nurses sampled attending queen larvae may have been the most active nurses. This study serves as another example of the relationship between differences in gene expression and behavioral specialisation in honey bees.     

Queen Dominance May Reduce Worker Mushroom Body Size in a Social Bee

The mushroom body (MB) is an area of the insect brain involved in learning, memory, and sensory integration. Here, we used the sweat bee Megalopta genalis (Halictidae) to test for differences between queens and workers in the volume of the MB calyces. We used confocal microscopy to measure the volume of the whole brain, MB calyces, optic lobes, and antennal lobes of queens and workers. Queens had larger brains, larger MB calyces, and a larger MB calyces:whole brain ratio than workers, suggesting an effect of social dominance in brain development. This could result from social interactions leading to smaller worker MBs, or larger queen MBs. It could also result from other factors, such as differences in age or sensory experience. To test these explanations, we next compared queens and workers to other groups. We compared newly emerged bees, bees reared in isolation for 10 days, bees initiating new observation nests, and bees initiating new natural nests collected from the field to queens and workers. Queens did not differ from these other groups. We suggest that the effects of queen dominance over workers, rather than differences in age, experience, or reproductive status, are responsible for the queen–worker differences we observed. Worker MB development may be affected by queen aggression directly and/or manipulation of larval nutrition, which is provisioned by the queen. We found no consistent differences in the size of antennal lobes or optic lobes associated with differences in age, experience, reproductive status, or social caste.     

Venom replenishment,as indicated by histamine,in honey bee (Apis mellifera) venom

Venom resynthesis, as indicated by histamine, has been investigated in the venom system of worker honey bees.After depletion of the venom contained in the venom reservoir, by electrical “milking”, histamine resynthesis was most active between 18 and 24 hr after “milking”.The ability of a bee to resynthesise histamine is age dependent. Young bees show some resynthetic ability, two week old bees show maximal ability to replace histamine. This capability is gradually lost as bees age and is virtually absent in 40 day old bees.     

THE EVOLUTION OF WORKER STERILITY IN HONEY BEES: AN INVESTIGATION INTO A BEHAVIORAL MUTANT CAUSING FAILURE OF WORKER POLICING

Normally, worker honey bees (Apis mellifera) only lay eggs when their colony is queenless. When a queen is present, worker egg-laying is controlled by mutual “policing” behavior in which any rare worker-laid eggs are eaten by other workers. However, an extremely rare behavioral phenotype arises in which workers develop functional ovaries and lay large numbers of eggs despite the presence of the queen. In this study, microsatellite analysis was used to determine the maternity of drones produced in such a colony under various conditions. One subfamily was found to account for about 90% of drone progeny, with the remainder being laid by other subfamilies or the queen. No evidence of queen policing was found. After a one-month period of extreme worker oviposition in spring, the colony studied reverted to normal behavior and showed no signs of worker oviposition. However, upon removal of the queen, workers commenced oviposition very quickly. Significantly, the subfamily that laid eggs when the queen was present did not contribute to the drone production when the colony was queenless. However, another subfamily contributed a disproportionately large number of drones. The frequency of worker oviposition appears to be determined by opposing selective forces. Individual bees benefit from personal reproduction, whereas other bees and the colony are disadvantaged by it. Thus a behavioral polymorphism can be maintained in the population in which some workers can escape worker policing, with balancing selection at the colony level to detect and eliminate these mutations.     

Conflict over male parentage in stingless bees

Summary Stingless bees usually have one, singly-mated queen. This can lead to a genetic conflict of interest between the queen and the non-laying workers over who should produce the males. In many stingless bee species workers have developed ovaries and can produce male-destined eggs. In this study we compile the available data on who produces the males in stingless bees. Worker reproduction is common but less frequent than expected from predictions built on relatedness-based preferences of non-laying workers. We tested whether the pattern in worker reproduction can be explained best by queen control, by an arms race between workers and their queen, by the costs of losing workers to reproductive competition, or by phylogenetic constraints. The data are consistent with the view that there is ongoing conflict over male production that is resolved differently depending on the specific dynamics of costs and benefits of worker reproduction. There was also a role for phylogeny; Melipona workers often reproduced while Plebeia and Australian stingless bee workers seldom or never did. The high worker reproduction in Melipona may reflect low costs, because many of the replaced queen-laid eggs would become excess queens.Received 17 April 2003; revised 2 September 2003; accepted 5 September 2003.     

Differential proteomics in dequeened honeybee colonies reveals lower viral load in hemolymph of fertile worker bees

The eusocial societies of honeybees, where the queen is the only fertile female among tens of thousands sterile worker bees, have intrigued scientists for centuries. The proximate factors, which cause the inhibition of worker bee ovaries, remain largely unknown; as are the factors which cause the activation of worker ovaries upon the loss of queen and brood in the colony. In an attempt to reveal key players in the regulatory network, we made a proteomic comparison of hemolymph profiles of workers with completely activated ovaries vs. rudimentary ovaries. An unexpected finding of this study is the correlation between age matched worker sterility and the enrichment of Picorna-like virus proteins. Fertile workers, on the other hand, show the upregulation of potential components of the immune system. It remains to be investigated whether viral infections contribute to worker sterility directly or are the result of a weaker immune system of sterile workers.     

Effect of honey bee queen mating condition on worker ovary activation

The presence of the honey bee queen reduces worker ovary activation. When the queen is healthy and fecund, this is interpreted as an adaptive response as workers can gain fitness from helping the queen raise additional offspring, their sisters. However, when the queen is absent, workers activate their ovaries and lay unfertilized eggs that become males. Queen pheromones are recognised as a factor affecting worker ovary activation. Recent work has shown that queen mandibular pheromone composition changes with queen mating condition and workers show different behavioural responses to pheromone extracts from these queens. Here, we tested whether workers reared in colonies with queens of different mating condition varied in level of ovary activation. We also examined the changes in the chemical composition of the queen mandibular glands to determine if the pheromone blend varied among the queens. We found that the workers activated their ovaries when queens were unmated and had lower ovary activation when raised with mated queens, suggesting that workers detect and respond adaptively to queens of differing mating status. Moreover, variation in queen mandibular gland’s chemical composition correlated with the levels of worker ovary activation. Although correlative, this evidence suggests that queen pheromone may act as a signal of queen mating condition for workers, in response to which they alter their level of ovary activation.     

Optimal colony fissioning in social insects: testing an inclusive fitness model with honey bees

In most species of social insects, when a queen departs from her parental nest to found a new colony, she leaves on her own. In some species, however, the departing queen leaves accompanied by a portion of the parental colony’s workers and there is a permanent fissioning of the worker force. Little is known about how the adult workers in colonies of fissioning species distribute themselves between the old and the new colonies. We examined this problem, building on Bulmer’s (J Theor Biol 100: 329–339, 1983) model for the optimal splitting of a colony’s adult workforce during colony reproduction. We first created an inclusive fitness model of optimal colony fissioning that applies to species in which fissioning gives rise to two autonomous colonies. The model predicts the optimal “swarm fraction”, which we define as the proportion of the adult workers in a fissioning colony that join the departing queen. We then tested the model by comparing the predicted and observed swarm fractions in honey bees. We found a close match between predicted (0.76–0.77) and observed (0.72 ± 0.04) swarm fractions. Evidently, worker honey bees distribute themselves between the old and new colonies in a way that jointly maximizes the inclusive fitness of each worker. We conclude by discussing additional ways to test the model.     

Breeding behaviour, relatedness and sex-investment ratios inLeptothorax tuberum Fabricius

The ant speciesLeptothorax tuberum was shown to be predominantly monogynous. Queens usually mate once only but some nests may have a multiply-mated queen or are partially or serially polygynous. As expected from these results, within nest relatedness between workers and between workers and alate queens was found to be high. Almost fifty percent of nests had no nest queen which may indicate high queen mortality, queens leaving to found new nests or nest fragmentation. Observed female investment frequencies (IF _o ) were not significantly different from those expected on the basis of worker control of sexual production and the relatedness estimates of workers to alate queens and workers to males calculated from isozyme data (IF _E ). These values were not consistent with queen control. There was no evidence for lower IF _o s in queenless nests nor for higher IF _o s in larger nests classified by worker number. When nests were classified by sexual productivity, however, there was a strong rank correlation between productivity and female bias. This is the first study of an ant species to test observed IF against expected IF calculated without inferring between caste relatedness from worker data or pooling of data from different castes.     

Male production in stingless bees: variable outcomes of queen-worker conflict

The genetic structure of social insect colonies is predicted to affect the balance between cooperation and conflict. Stingless bees are of special interest in this respect because they are singly mated relatives of the multiply mated honeybees. Multiple mating is predicted to lead to workers policing each others' male production with the result that virtually all males are produced by the queen, and this prediction is borne out in honey bees. Single mating by the queen, as in stingless bees, causes workers to be more related to each others' sons than to the queen's sons, so they should not police each other. We used microsatellite markers to confirm single mating in eight species of stingless bees and then tested the prediction that workers would produce males. Using a likelihood method, we found some worker male production in six of the eight species, although queens produced some males in all of them. Thus the predicted contrast with honeybees is observed, but not perfectly, perhaps because workers either lack complete control or because of costs of conflict. The data are consistent with the view that there is ongoing conflict over male production. Our method of estimating worker male production appears to be more accurate than exclusion, which sometimes underestimates the proportion of males that are worker produced.     

Age and Behavior of Honey Bee Workers,Apis mellifera,that Interact with Drones

Colony reproduction in honey bees involves complex interactions between sterile workers and reproductive castes. Although worker–queen interactions have been studied in detail, worker–drone interactions are less well understood. We investigated caste interactions in honey bees by determining the age and behavior of workers that perform vibration signals, trophallaxis, and grooming with drones. Workers of all ages could engage in the different interactions monitored, although workers that performed vibration signals on drones were significantly older than those engaging in trophallaxis and grooming. Only 3–8% of workers engaged in the different behaviors were monitored. Compared with workers that performed vibration signals only on workers (‘worker vibrators’), those that performed signals on both workers and drones (‘drone vibrators’) had greater movement rates inside the nest, higher vibration signaling rates, and were more likely to have an immediate association with foraging. Both worker vibrators and drone vibrators contacted drones of all ages as they moved through the nest. However, drone vibrators contacted drones at higher rates, contacted slightly, but significantly younger drones, and were more likely to engage in trophallaxis and grooming with drones, in addition to vibrating them. Taken together, our results suggest that tiny proportions of workers belonging to separate, but overlapping age groups provide most of the care received by adult drones, and that drone vibrators comprise a subset of signalers within a colony that have an increased tendency to contact and interact with drones. Vibratory, tactile signals are involved in colony reproductive and movement decisions in a number of species of bees, wasps and ants, and may provide valuable tools for investigating caste interactions in many insect societies.     

Social Modulation of Stress Reactivity and Learning in Young Worker Honey Bees

Alarm pheromone and its major component isopentylacetate induce stress-like responses in forager honey bees, impairing their ability to associate odors with a food reward. We investigated whether isopentylacetate exposure decreases appetitive learning also in young worker bees. While isopentylacetate-induced learning deficits were observed in guards and foragers collected from a queen-right colony, learning impairments resulting from exposure to this pheromone could not be detected in bees cleaning cells. As cell cleaners are generally among the youngest workers in the colony, effects of isopentylacetate on learning behavior were examined further using bees of known age. Adult workers were maintained under laboratory conditions from the time of adult emergence. Fifty percent of the bees were exposed to queen mandibular pheromone during this period, whereas control bees were not exposed to this pheromone. Isopentylacetate-induced learning impairments were apparent in young (less than one week old) controls, but not in bees of the same age exposed to queen mandibular pheromone. This study reveals young worker bees can exhibit a stress-like response to alarm pheromone, but isopentylacetate-induced learning impairments in young bees are suppressed by queen mandibular pheromone. While isopentylacetate exposure reduced responses during associative learning (acquisition), it did not affect one-hour memory retrieval.     

Hemolymph juvenile hormone titers in worker honey bees under normal and preswarming conditions

Swarming is an important mechanism by which honey bee, Apis mellifera L., colonies reproduce, yet very little is known about the physiological changes in workers that are preparing to swarm. In this study, we determined the endocrine status of worker honey bees in preswarming colonies and in normal (nonswarming) colonies. Juvenile hormone (JH) titers in worker bees were similar in both groups before queen cells were present, but they became significantly lower in preswarming colonies compared with normal colonies when queen cells occurred in preswarming colonies. The lower JH titers in the preswarming colonies suggest that behavioral development is delayed in these colonies, consistent with previous reports that preswarming colonies have reduced foraging activities. Understanding the endocrine status of bees preparing for swarming will help us to better understand the biology of swarming.     

Effects of insemination quantity on honey bee queen physiology

Mating has profound effects on the physiology and behavior of female insects, and in honey bee (Apis mellifera) queens, these changes are permanent. Queens mate with multiple males during a brief period in their early adult lives, and shortly thereafter they initiate egg-laying. Furthermore, the pheromone profiles of mated queens differ from those of virgins, and these pheromones regulate many different aspects of worker behavior and colony organization. While it is clear that mating causes dramatic changes in queens, it is unclear if mating number has more subtle effects on queen physiology or queen-worker interactions; indeed, the effect of multiple matings on female insect physiology has not been broadly addressed. Because it is not possible to control the natural mating behavior of queens, we used instrumental insemination and compared queens inseminated with semen from either a single drone (single-drone inseminated, or SDI) or 10 drones (multi-drone inseminated, or MDI). We used observation hives to monitor attraction of workers to SDI or MDI queens in colonies, and cage studies to monitor the attraction of workers to virgin, SDI, and MDI queen mandibular gland extracts (the main source of queen pheromone). The chemical profiles of the mandibular glands of virgin, SDI, and MDI queens were characterized using GC-MS. Finally, we measured brain expression levels in SDI and MDI queens of a gene associated with phototaxis in worker honey bees (Amfor). Here, we demonstrate for the first time that insemination quantity significantly affects mandibular gland chemical profiles, queen-worker interactions, and brain gene expression. Further research will be necessary to elucidate the mechanistic bases for these effects: insemination volume, sperm and seminal protein quantity, and genetic diversity of the sperm may all be important factors contributing to this profound change in honey bee queen physiology, queen behavior, and social interactions in the colony.     

Experimentally induced variation in the physical reproductive potential and mating success in honey bee queens

In honeybee colonies, reproduction is monopolized by the queen while her daughter workers are facultatively sterile. Caste determination is a consequence of environmental conditions during development, during which female larvae may become either queens or workers depending on their larval diet. This bipotency introduces significant variation in the reproductive potential of queen bees, with queens raised from young worker larvae exhibiting high reproductive potential and queens raised from older worker larvae exhibiting lower reproductive potential. We verify that low-quality queens are indeed produced from older worker larvae, as measured morphometrically (e.g., body size) and by stored sperm counts. We also show, for the first time, that low-quality queens mate with significantly fewer males, which significantly influences the resultant intracolony genetic diversity of the worker force of their future colonies. These results demonstrate a reproductive continuum of honeybee queens and provide insights into the reproductive constraints of social insects.     

Regulation of oogenesis in honey bee workers via programed cell death

Reproductive division of labour characterises eusociality. Currently little is known about the mechanisms that underlie the ‘sterility’ of the worker caste, but queen pheromone plays a major role in regulating the reproductive state. Here we investigate oogenesis in the young adult honey bee worker ovary in the presence of queen pheromone and in its absence. When queen pheromone is absent, workers can activate their ovaries and have well-developed follicles. When queen pheromone is present, even though workers have non-activated ovaries, they continually produce oocytes which are aborted at an early stage. Therefore, irrespective of the presence of the queen, the young adult worker ovary contains oocytes. By this means young workers retain reproductive plasticity. The degeneration of the germ cells in the ovarioles of workers in the presence of queen pheromone has the morphological hallmarks of programmed cell death. Therefore the mechanistic basis of ‘worker sterility’ relies in part on the regulation of oogenesis via programmed cell death. Our results suggest that honey bees have co-opted a highly conserved checkpoint at mid-oogenesis to regulate the fertility of the worker caste.     

Anarchistic queen honey bees have normal queen mandibular pheromones

Summary. Anarchistic honey bees are a line developed by recurrent selection in which workers frequently lay eggs. In unselected colonies, workers refrain from reproduction in response to pheromonal signals that indicate the presence of brood and a queen. We show that queen type (anarchistic or wild type) has no effect on rates of ovary activation of anarchistic or wild type workers. In addition, we show that an important component of the queens signalling system, the queen mandibular gland pheromone, is similar in wild type and anarchistic queens. Anarchistic larvae do not inhibit worker ovary development to the same degree as wild type larvae, however all colonies in this experiment contained only wild type larvae. Anarchistic workers had greater rates of ovary activation than wild type workers in colonies headed by either queen type. We therefore conclude that there must be differences in the transmission or reception of queen pheromones, or worker sensitivity to these compounds. These results clearly demonstrate that anarchy is a complex syndrome, not simply the result of reduced pheromone production by anarchist queens and larvae.Received 23 December 2003; revised 14 May 2004; accepted 1 June 2004.