Transfer of pheromone from immature queen honeybees, Apis mellifera

ABSTRACT. Immediately after visiting cells containing immature queens, workers of Apis mellifera L. were observed to engage in prolonged cleaning, particularly of their tongues when they had visited larvae, and of their antennae when they had visited pupae. Thereafter other workers usually initiated and made antennal contacts with them. During such antennal contact the bee that had visited the queen larva often donated food. The implication of these findings on the distribution of pheromone produced by immature queens is discussed. Workers were stimulated to make antennal contact with the excised heads of bees from a queen's court, providing further evidence that queen pheromone is transferred between workers' antennae.     

The behaviour of queen honeybees and their attendants

Abstract. The behaviour of queen and worker honeybees (Apis mellifera L.) was observed using small colonies in observation hives. Workers paid more attention to queens which had been mated for 2 months or more than to those which were newly mated; virgin queens received least attention. Queens received most attention when they were stationary and least when they were walking over the comb; virgin queens were most active. Queen cells had as many attendants as virgin queens and queen larvae were inspected almost continuously. The queen pheromone component 9–oxo-trans-2–decenoic acid stimulated 'court' behaviour when presented on small polyethylene blocks, but workers responded aggressively to complete extracts of queens' heads. Both the heads and abdomens of mated queens received much attention from court workers but the abdomens were palpated by more workers for longer and were licked much more. The queens' thoraces were least attended. Abdominal tergites posterior to tergite glands were licked for longer than those anterior to the glands. Only worker bees very near to the queen reacted to her and joined her 'court'.
No evidence was found of a diel periodicity in the behaviour of a queen or her 'court'. During the winter the queen's court was smaller than in summer and she walked less and laid fewer eggs. When colonies were fed with sucrose syrup in winter, their queens laid more eggs and workers reared more brood but there was no change in the attention received by the queens.
The implications of these findings for the secretion and distribution of queen pheromones are discussed.     

Queens, not workers, produce the males in the stingless bee Schwarziana quadripunctata quadripunctata

Most stingless bee colonies have one singly mated queen, resulting in a potential conflict between workers and queen over male production, because workers are more closely related to the sons of other workers than they are to the queen's sons. Furthermore, workers in the majority of stingless bee species have ovarian development, can produce haploid eggs, and show apparently agonistic behaviour towards their queen, suggesting a real conflict. We investigated whether genetic conflict over male production resulted in reproductive and behavioural conflict in Schwarziana q. quadripunctata. DNA microsatellite loci showed that, even though workers are more related to other workers' sons than to queen's sons, it is the queen who produces the males. Behavioural interactions between workers and their queen were not uniformly more aggressive during male production than at times with only worker production, although some differences might have been obscured by the fact that food supply was greater during male production. The potential conflict over male production inS. q. quadripunctata seems not to lead to an observable conflict between the workers and their queen. Workers might refrain from reproduction because of the costs involved for the colony or because of queen control.     

Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees

The mandibular glands of queen honeybees produce a pheromone that modulates many aspects of worker honeybee physiology and behavior and is critical for colony social organization. The exact chemical blend produced by the queen differs between virgin and mated, laying queens. Here, we investigate the role of mating and reproductive state on queen pheromone production and worker responses. Virgin queens, naturally mated queens, and queens instrumentally inseminated with either semen or saline were collected 2 days after mating or insemination. Naturally mated queens had the most activated ovaries and the most distinct chemical profile in their mandibular glands. Instrumentally inseminated queens were intermediate between virgins and naturally mated queens for both ovary activation and chemical profiles. There were no significant differences between semen- and saline-inseminated queens. Workers were preferentially attracted to the mandibular gland extracts from queens with significantly more activated ovaries. These studies suggest that the queen pheromone blend is modulated by the reproductive status of the queens, and workers can detect these subtle differences and are more responsive to queens with higher reproductive potential. Furthermore, it appears as if insemination substance does not strongly affect physiological characteristics of honeybee queens 2 days after insemination, suggesting that the insemination process or volume is responsible for stimulating these early postmating changes in honeybee queens.     

Swarming generates rebel workers in honeybees

According to kin selection theory, the colony kin structure of eusocial insects motivates workers' altruistic behaviors and therefore their sterility or restricted reproduction [1]. Indeed, theory and cross-species comparison confirm that workers engage in their own reproduction depending on relatedness among colony members [2, 3]. We show that in a honeybee colony, the workers switch from their typical altruistic role to a more selfish one if at their larval stage there are environmental cues of an upcoming decline in intracolony relatedness. This happens inevitably when a colony multiplies by swarming and replaces the mother queen with her daughter, because the mother queen's workers are faced with rearing the sister queen's offspring related to them half as much as between sisters. Workers developing from the mother queen's eggs immediately after swarming, in a temporarily queenless colony, had more ovarioles in their ovaries and less-developed hypopharyngeal glands producing brood food than control workers reared in queenright conditions. These "rebel" workers were more engaged in laying their own male-determined eggs than in rearing offspring, whether or not the sister queen was present in the colony. The finding of this previously unknown rebel strategy confirms that kin selection shapes both cooperation and conflict in honeybee societies.     

How do workers of the primitively eusocial wasp Ropalidia marginata detect the presence of their queens?

Queens in primitively eusocial insect societies are morphologically indistinguishable from their workers, and occupy the highest position in the dominance hierarchy. Such queens are believed to use aggression to maintain worker activity and reproductive monopoly in the colony. However, in the primitively eusocial wasp Ropalidia marginata, the queen is a strikingly docile individual, who interacts rarely with her workers. If the queen is experimentally removed, one of the workers becomes extremely aggressive within minutes, and eventually becomes the new queen of the colony. We designate her as the potential queen. Experimental evidence suggests that the queen probably uses a non-volatile pheromone to signal her presence to her workers. Here we attempt to identify the mechanism by which the queen transmits information about her presence to the workers. We designate the time taken for the potential queen to realize the absence of the queen as the realization time and model the realization time as a function of the decay time of the queen's signal and the average signal age. We find that the realization time obtained from the model, considering only direct interactions (193.5 min) is too large compared to the experimentally observed value of 30 min. Hence we consider the possibility of signal transfer through relay. Using the Dijkstra's algorithm, we first establish the effectiveness of relay in such a system and then use experimental data to fit the model. We find that the realization time obtained from the model, considering relay (237.1 min) is also too large compared to the experimentally observed value of 30 min. We thus conclude that physical interactions, both direct and indirect (relay), are not sufficient to transfer the queen's signal in R. marginata. Finally, we discuss the possibility that the queen applies her pheromone on the nest material from where the workers can perceive it without having to physically interact with the queen.     

The queen in relation to wax secretion and comb building in honeybees

Summary The role of the queen in relation to wax secretion and comb building in honeybees was analyzed with respect to queen status (mated, virgin and dead queens and queenlessness), and pheromones of the head and abdominal tergite of queens. Worker variables considered were colony size, percentage of bees bearing wax scales, wax scale weight, and weight of constructed combs.The amount of wax recovered from festoon bees and the percentage of festoon bees bearing wax were independent of queen status, the pheromones of queens and access to the queen. Colonies with full access to freely moving mated queens always constructed significantly more comb than those headed by virgin or dead queens as well as all permutations of caged and division board queens whose mandibular glands and/or abdominal tergite glands were operative or not.Despite pheromonal similarity of virgin queens to mated ones, colonies headed by virgin queens constructed as little comb as did queenless colonies. The bouquets of the mandibular glands did not differ significantly among queens nor was the amount of comb constructed correlated with pheromonal bouquet. Comb building is greatest among colonies having full access to freely moving queens but the stimulus for such building is not attributable to the 90DA, 9HDA and 10HDA components of the queen's mandibular gland secretions.     

Factors determining the release of Nasonov pheromone by honeybees at the hive entrance

ABSTRACT. Worker bees recently denied access to their colony expose their Nasonov glands, thereby releasing pheromone, at the entrance to their hive. Odours of the following induced this response: empty comb, purified beeswax, honey, pollen, propolis, a live queen, the (E)-9-hydroxy-2-decenoic acid component of a queen's mandibular glands, live drones and workers, inert material on which workers had walked inside the hive, and synthetic Nasonov pheromone. The total odour of a foreign colony also induced worker bees to expose their Nasonov glands but was less effective than the odour of their own colony. Odours of the following were not effective: the (E)-9-oxo-2-decenoic acid component of a queen's mandibular glands, recently killed drones and workers, worker brood (eggs, larvae, pupae).     

Effects of honey bee (Apis mellifera L.) queen insemination volume on worker behavior and physiology

Honey bee colonies consist of tens of thousands of workers and a single reproductive queen that produces a pheromone blend which maintains colony organization. Previous studies indicated that the insemination quantity and volume alter queen mandibular pheromone profiles. In our 11-month long field study we show that workers are more attracted to high-volume versus low-volume inseminated queens, however, there were no significant differences between treatments in the number of queen cells built by workers in preparation for supersedure. Workers exposed to low-volume inseminated queens initiated production of queen-like esters in their Dufour's glands, but there were no significant difference in the amount of methyl farnesoate and juvenile hormone in worker hemolymph. Lastly, queen overwintering survival was unexpectedly lower in high-volume inseminated queens. Our results suggest that the queen insemination volume could ultimately affect colony health and productivity.     

How honeybee queen attendants become ordinary workers

Honeybee queen attendants disperse queen pheromones to supplement pheromone dispersal by direct queen-worker contacts. With time they lose their dispersal function exponentially due mainly to volatilization of queen pheromones carried on their bodies. The elimination of those airborne pheromones together with the air while ventilating the hive is balanced by pheromone release by the queen. This equilibrium results in a certain level of queen pheromones in the broodnest. The change of the pheromone level (for example, due to loss of the colony of its queen) can serve as a signal to alter the behaviour of the workers and the state of the colony.     

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.     

Chemical communication in Ropalidia marginata: Dufour's gland contains queen signal that is perceived across colonies and does not contain colony signal

Queens of the primitively eusocial wasp Ropalidia marginata appear to maintain reproductive monopoly through pheromone rather than through physical aggression. Upon queen removal, one of the workers (potential queen, PQ) becomes extremely aggressive but drops her aggression immediately upon returning the queen. If the queen is not returned, the PQ gradually drops her aggression and becomes the next queen of the colony. In a previous study, the Dufour's gland was found to be at least one source of the queen pheromone. Queen-worker classification could be done with 100% accuracy in a discriminant analysis, using the compositions of their respective Dufour's glands. In a bioassay, the PQ dropped her aggression in response to the queen's Dufour's gland macerate, suggesting that the queen's Dufour's gland contents mimicked the queen herself. In the present study, we found that the PQ also dropped her aggression in response to the macerate of a foreign queen's Dufour's gland. This suggests that the queen signal is perceived across colonies. This also suggests that the Dufour's gland in R. marginata does not contain information about nestmateship, because queens are attacked when introduced into foreign colonies, and hence PQ is not expected to reduce her aggression in response to a foreign queen's signal. The latter conclusion is especially significant because the Dufour's gland chemicals are adequate to classify individuals correctly not only on the basis of fertility status (queen versus worker) but also according to their colony membership, using discriminant analysis. This leads to the additional conclusion (and precaution) that the ability to statistically discriminate organisms using their chemical profiles does not necessarily imply that the organisms themselves can make such discrimination.     

Dose-dependent inhibition of emergency queen rearing by synthetic 9-ODA in the honey bee,Apis mellifera carnica

In the honey bee colony queen rearing is usually suppressed by releaser effects of the queen's pheromone. This is part of the dominance hierarchy maintaining the monogynous homeostasis. Under queenless conditions, the queen's control over the construction of emergency queen cells by the workers can be substitued by exposure to only one component of the mandibular pheromone secretion of a queen, the main compound (E)-9-oxo-2-decenoic acid. A novel and simple synthesis of (E)-9-oxo-2-decenoic acid is described, and a bioassay was developed by which a dose-dependent effect of synthetic (E)-9-oxo-2-decenoic acid presented on a dummy bee was evaluated.Abbreviation 9-ODA (E)-9-oxo-2-decenoic acid In memoriam Viktor Schwartz (1907–1992), Professor of Zoology and Developmental Biology, University of Tübingen, who introduced smoothened bee stings into microsurgery     

Colony strength and queen replacement in Melipona marginata (Apidae: Meliponini).

Physogastric queens of Melipona marginata were removed from their colonies in order to verify the acceptance of a new queen by workers. Colony strength was evaluated according to queen oviposition rate and comb diameters. Replacement was observed seven times. Its occurrence and speed related positively to colony strength, independently of queen's age. In weak colonies, queen replacement was observed only once, following colony population increase that occurred after introduction of combs from another colony. Worker oviposition after queen removal was observed three times: in a strong colony with virgin queens and males, and in two of the weak colonies. In the first two or three days of new queen oviposition, during which most of the eggs were eaten by the queen, worker oviposition preceded almost all provisioning and oviposition processes (POPs). After this period, worker oviposition decreased until it reached around 25% of the POPs. Daily oviposition rate of young queens decreased or was even interrupted by hatching of their first brood.     

Exocrine glands and the attractiveness of the ergatoid queen in the ponerine antMegaponera foetens

Summary Queens inMegaponera foetens are permanently wingless. In a colony from Kenya we observed that the ergatoid queen was often surrounded by a large number of workers, all conspicuously facing her. To investigate the nature of this attraction, we divided the colony in half. When the queen was transferred from one group to the other, a large court of workers formed immediately. When marked workers were transferred from the queenright group to the other, they attracted much attention and were licked repeatedly. These data suggest that the queen produces a chemical signal which attracts workers, and this signal is transferred onto the workers. In a histological investigation of queen and workers, we found many intersegmental glands in both castes. However, only the queen had a thick glandular epithelium lining her entire body, and this may be the source of her signal. The numerous erect setae covering the queen (but not the workers) may help to dispense the secretions. Our results confirm that ergatoid queens in the Ponerinae are morphologically specialized, despite their external similarity with workers.     

Worker interests and male production in Polistes gallicus, a Mediterranean social wasp

The resolution of social conflict in colonies may accord with the interests of the most numerous party. In social insect colonies with single once-mated queens, workers are more closely related to the workers' sons than they are to the queens' sons. Therefore, they should prefer workers to produce males, against the queen's interests. Workers are capable of producing males as they arise from unfertilized eggs. We found Polistes gallicus to have colonies of single, once-mated queens, as determined by microsatellite genotyping of the workers, so worker interests predict worker male production. In colonies lacking queens, workers produced the males, but not in colonies with original queens. Thus worker interests were expressed only when the queen was gone. The high fraction of missing queens and early end to the colony cycle relative to climate so early in the season is surprising and may indicate a forceful elimination of the queen.     

Complex sociogenetic organization and reproductive skew in a primitively eusocial sweat bee,Lasioglossum malachurum,as revealed by microsatellites

The sweat bees (Family Halictidae) are a socially diverse taxon in which eusociality has arisen independently numerous times. The obligate, primitively eusocial Lasioglossum malachurum, distributed widely throughout Europe, has been considered the zenith of sociality within halictids. A single queen heads a colony of smaller daughter workers which, by mid-summer, produce new sexuals (males and gynes), of which only the mated gynes overwinter to found new colonies the following spring. We excavated successfully 18 nests during the worker- and gyne-producing phases of the colony cycle and analysed each nest's queen and either all workers or all gynes using highly variable microsatellite loci developed specifically for this species. Three important points arise from our analyses. First, queens are facultatively polyandrous (queen effective mating frequency: range 1-3, harmonic mean 1.13). Second, queens may head colonies containing unrelated individuals (n = 6 of 18 nests), most probably a consequence of colony usurpation during the early phase of the colony cycle before worker emergence. Third, nonqueen's workers may, but the queen's own workers do not, lay fertilized eggs in the presence of the queen that successfully develop into gynes, in agreement with so-called 'concession' models of reproductive skew.     

Changes in the Content of Brain Biogenic Amine Associated with Early Colony Establishment in the Queen of the Ant, Formica japonica

We examined changes in the content of biogenic amines in the brains of ant queen associated with early colony establishment. In ants, including Formica japonica, winged virgin queens lose their wings following copulation, and then start establishing a colony. Significant changes in brain biogenic amine content in the queen are associated with transition from winged virgin queen to wingless mated queen. The levels of serotonin (5HT), octopamine (OA) and dopamine (DA) decreased significantly in the brain of the queen after starting a colony. On the other hand, tyramine (TA) increased significantly in the brain following colony establishment. Catabolized substances of the biogenic amines in the brain were also measured. The levels of N-acetyloctopamine (Nac-OA) and N-acetyltyramine (Nac-TA) in the brain did not show a significant change after the queen established a colony. However, the levels of N-acetylserotonin (Nac-5HT) in the brain were significantly higher in wingless mated queens than in winged virgin queens, whereas levels of N-acetyldopamine (Nac-DA) in the brain were significantly lower in wingless mated queens than winged virgin queens. These results suggest that serotonergic and octopaminergic systems in the brain of the queen change when the mated queen starts to establish a new colony.     

Chemical Profiles of Two Pheromone Glands Are Differentially Regulated by Distinct Mating Factors in Honey Bee Queens (Apis mellifera L.)

Pheromones mediate social interactions among individuals in a wide variety of species, from yeast to mammals. In social insects such as honey bees, pheromone communication systems can be extraordinarily complex and serve to coordinate behaviors among many individuals. One of the primary mediators of social behavior and organization in honey bee colonies is queen pheromone, which is produced by multiple glands. The types and quantities of chemicals produced differ significantly between virgin and mated queens, and recent studies have suggested that, in newly mated queens, insemination volume or quantity can affect pheromone production. Here, we examine the long-term impact of different factors involved during queen insemination on the chemical composition of the mandibular and Dufour''s glands, two of the major sources of queen pheromone. Our results demonstrate that carbon dioxide (an anesthetic used in instrumental insemination), physical manipulation of genital tract (presumably mimicking the act of copulation), insemination substance (saline vs. semen), and insemination volume (1 vs. 8 µl) all have long-term effects on mandibular gland chemical profiles. In contrast, Dufour''s gland chemical profiles were changed only upon insemination and were not influenced by exposure to carbon dioxide, manipulation, insemination substance or volume. These results suggest that the chemical contents of these two glands are regulated by different neuro-physiological mechanisms. Furthermore, workers responded differently to the different mandibular gland extracts in a choice assay. Although these studies must be validated in naturally mated queens of varying mating quality, our results suggest that while the chemical composition of Dufour''s gland is associated with mating status, that of the mandibular glands is associated with both mating status and insemination success. Thus, the queen appears to be signaling both status and reproductive quality to the workers, which may impact worker behavior and physiology as well as social organization and productivity of the colony.     

Differential antennal proteome comparison of adult honeybee drone, worker and queen (Apis mellifera L.)

To understand the olfactory mechanism of honeybee antennae in detecting specific volatile compounds in the atmosphere, antennal proteome differences of drone, worker and queen were compared using 2-DE, mass spectrometry and bioinformatics. Therefore, 107 proteins were altered their expressions in the antennae of drone, worker and queen bees. There were 54, 21 and 32 up-regulated proteins in the antennae of drone, worker and queen, respectively. Proteins upregulated in the drone antennae were involved in fatty acid metabolism, antioxidation, carbohydrate metabolism and energy production, protein folding and cytoskeleton. Proteins upregulated in the antennae of worker and queen bees were related to carbohydrate metabolism and energy production while molecular transporters were upregulated in the queen antennae. Our results explain the role played by the antennae of drone is to aid in perceiving the queen sexual pheromones, in the worker antennae to assist for food search and social communication and in the queen antennae to help pheromone communication with the worker and the drone during the mating flight. This first proteomic study significantly extends our understanding of honeybee olfactory activities and the possible mechanisms played by the antennae in response to various environmental, social, biological and biochemical signals.