Queen-signal modulation of worker pheromonal composition in honeybees

Worker sterility in honeybees is neither absolute nor irreversible. Whether under queen or worker control, it is likely to be mediated by pheromones. Queen-specific pheromones are not exclusive to queens; workers with activated ovaries also produce them. The association between ovarian activation and queen-like pheromone occurrence suggests the latter as providing a reliable signal of reproductive ability. In this study we investigated the effect of queen pheromones on ovary development and occurrence of queen-like esters in workers' Dufour's gland. Workers separated from the queenright compartment by a double mesh behaved like queenless workers, activating their ovaries and expressing a queen-like Dufour's gland secretion, confirming that the pheromones regulating both systems are non-volatile. Workers with developed ovaries produced significantly more secretion than sterile workers, which we attribute primarily to increased ester production. Workers separated from the queenright compartment by a single mesh displayed a delayed ovarian development, which we attribute to interrupted transfer of the non-volatile pheromone between compartments. We suggest that worker expression of queen-like characters reflects a queen-worker arms race; and that Dufour's gland secretion may provide a reliable signal for ovarian activation. The associative nature between ovary development and Dufour's gland ester production remains elusive.     

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.     

Egg marking pheromones of anarchistic worker honeybees (Apis mellifera)

In honeybees, worker policing via egg eating enforces functionalworker sterility in colonies with a queen and brood. It is thoughtthat queens mark their eggs with a chemical signal, indicatingthat their eggs are queen-laid. Worker-laid eggs lack this signaland are, therefore, eaten by policing workers. Anarchistic workerhoneybees have been hypothesized to circumvent worker policingby mimicking the queen egg-marking signal. We investigated thisphenomenon by relating chemical profiles of workers and theireggs to egg acceptability. We found that the ability of someworkers (anarchistic workers in queenright colonies and deviantworkers from a queenless colony) to lay more acceptable eggsis due to them producing significant amounts of queen-like estersfrom their Dufour's gland. These esters appear to be transferredto eggs during laying and increase egg survival. However, theseesters cannot be the normal queen egg-marking signal, as theyare generally absent from queen-laid eggs and only increasethe short-term persistence of worker-laid eggs, because only7–30% of anarchistic worker-laid eggs persisted to hatchingversus 91–92% of queen-laid eggs. All workers can producesome esters, but only workers that greatly increase their esterproduction lay more acceptable eggs. The production of estersappears to be a flexible response, as anarchistic workers rearedin queenless colonies did not increase their ester production,while some deviant workers in queenless colonies did increasetheir ester production.     

The Releaser Effects of the Tergal Gland Secretion of Queen Honeybees (Apis mellifera)

Queen honeybees are attractive to their workers, due partially to the pheromonal bouquet they secrete. Queen mandibular gland pheromone is a powerful attractant to worker honeybees but it is not solely responsible for eliciting retinue behavior. The attractiveness of virgin queen tergal gland secretions and queen mandibular pheromone to worker honeybees was tested using a retinue bioassay. The number of workers attending the treated pseudoqueen lures was videorecorded in order to allow for the quantification of attractiveness. Queen mandibular gland secretions were more attractive than tergal gland secretions (P 0.008), and both queen tergal gland secretions (P 0.0002) and mandibular gland secretions (P 0.0001) were significantly more attractive than the control treatment. This laboratory bioassay indicates that queen tergal gland secretions have a releaser effect that evokes retinue behavior from worker honeybees.     

Plasticity in caste-related exocrine secretion biosynthesis in the honey bee (Apis mellifera)

Plasticity of Dufour's gland secretion in the honey bee is correlated with the individual's plasticity. Queens and queenless (QL) egg-laying workers possess a bouquet of esters and hydrocarbons, whereas queenright (QR) workers produce exclusively hydrocarbons. The effects of social environment (QR vs. QL conditions) and possible physiological constraints on the gland were studied by following the biosynthesis of these classes of compounds in vivo and in vitro. Biosynthesis in vivo followed the prediction based on glandular chemistry. Queens and QL egg-laying workers, but not QR workers or QL foragers, showed incorporation of sodium acetate into both hydrocarbons and esters. In contrast, the in vitro studies revealed that, in addition to queens and QL egg-laying workers, QR nurses retained their ability to produce the queen characteristic esters. Although there was some ester production in foragers, it occurred to a lesser extent. It is possible that the glands in the older foragers undergo irreversible changes. The in vitro incubation also revealed a temporal activation of ester biosynthesis in QR workers. In these glands alcohols, corresponding to the alcohol moiety of the esters, predominated in short-term incubations but decreased as the amount of newly synthesized esters increased. In contrast, queens and QL egg-laying workers showed predominant incorporation into esters from the onset of incubation. Thus, expression within the workers' Dufour's gland is regulated. In the presence of a queen, ester production is inhibited. Once the queen is removed the physiologically unconstrained gland starts to biosynthesize the queen-specific esters after a certain lag needed for the build-up of precursors and the enzymatic machinery.     

Mate number,kin selection and social conflicts in stingless bees and honeybees

Microsatellite genotyping of workers from 13 species (ten genera) of stingless bees shows that genetic relatedness is very high. Workers are usually daughters of a single, singly mated queen. This observation, coupled with the multiple mating of honeybee queens, permits kin selection theory to account for many differences in the social biology of the two taxa. First, in contrast to honeybees, where workers are predicted to and do police each other''s male production, stingless bee workers are predicted to compete directly with the queen for rights to produce males. This leads to behavioural and reproductive conflict during oviposition. Second, the risk that a daughter queen will attack the mother queen is higher in honeybees, as is the cost of such an attack to workers. This explains why stingless bees commonly have virgin queens in the nest, but honeybees do not. It also explains why in honeybees the mother queen leaves to found a new nest, while in stingless bees it is the daughter queen who leaves.     

Dufour's gland secretion as a repellent used during usurpation by the slave-maker ant Rossomyrmex minuchae

In slave-making ants, the invasion of the host colony by newly mated queens is a critical stage. We studied the strategy used by Rossomyrmex minuchae queens to invade their host Proformica longiseta. Field observations revealed that queens enter the host nest unchallenged by the host workers in the vicinity of the nest entrance. Pre-usurpation queens were found to possess a highly inflated Dufour's gland, which considerably reduces in size after successful usurpation. Chemical analysis of these queen glands revealed tetradecanal to be the major product in pre-usurpation Rossomyrmex queens, but to be almost absent in queens that have been adopted by P. longiseta. We consequently hypothesized that tetradecanal is a repellent that is used by queens to prevent host worker aggression. We tested its repellent effect by attempting to deter starved, highly motivated workers from a droplet of honey. Tetradecanal indeed proved to be highly repellent both to host worker P. longiseta and non-host worker Formica selysi. It was even more powerful than limonene, a reported general ant repellent. These results are consistent with the hypothesis that R. minuchae queens use Dufour's gland secretion as a weapon during nest usurpation. The general use of tetradecanal as a defensive compound, and its seemingly non-specific repellent effect on ants, indicate that it may act as a general ant repellent. Its adoption by R. minuchae queens thus provides them with an efficient defensive and offensive chemical weapon during their long and risky search for new host nests.     

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.     

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.     

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.     

The biosynthesis of Dufour's gland constituents in queens of the honeybee (Apis mellifera)

Chemical analysis of queen Dufour's gland by GC/MS revealed the presence of long chain esters and hydrocarbons. To evaluate whether the biosynthesis of Dufour's gland components is modulated by physiological factors, we studied thede novo biosynthesis of esters and hydrocarbons in virgin and mated queen glandsin vivo andin vitro. Using [1-¹⁴C] sodium acetate as a precursor, it was shown thatin vivo, esters and hydrocarbons comprised the majorde novo products. The relative proportion of the hydrocarbon fraction was significantly higher in mated queens than in virgins, while in esters the situation was reversed. Comparision between reproductive and non reproductive virgin queens revealed that mating and not egg laying induced changes in glandular activity. Glands incubatedin vitro, on the other hand, synthesized mainly esters and alcohols but not hydrocarbons. These results suggest that either hydrocarbons are produced elsewhere and subsequently sequestered by Dufour's gland, or that the gland biosynthetic expressionin vivo is modulated. Differences between the gland activityin vivo andin vitro further support the hypothesis of a physiological mechanism in regulation of glandular performance.     

Source of the host marking pheromone in the egg parasitoid Trissolcus basalis (Hymenoptera: Scelionidae)

After oviposition, Trissolcus basalis females always mark the host's surface, depositing host marking substances for herself and to warn other ovipositing females. The perception of these host marking substances, probably through the antennae, can induce the female to leave and seek healthy hosts. Parasitoid females exposed to conspecific parasitized egg masses left the host egg masses significantly more often than when exposed to non-parasitized egg masses. More egg mass leaving behavior also was observed when the egg masses were treated with Dufour's gland secretion but not when treated with secretion from the common oviducts. The common oviduct has a secretory epithelium that produces electron-dense vesicles, probably containing proteinaceous substances. The secretory cells of the accessory gland, Dufour's gland, contain electron-lucid vesicles, whose secretion appears to be a lipid similarly to that found in pheromone secreting glands. Ultrastructural and behavioral evidence suggests that Dufour's gland is the host marking pheromone source.     

Chemical signals of queens in kin recognition of honeybees,Apis mellifera L.

Summary The volatile signals of live queens as well as head and tergite extracts were analyzed by capillary gas chromatography. Multivariate analysis revealed a significant decrease in the variability of the volatile signals of queens the more closely related they were. Though extracts of the tergal glands showed the same phenomenon, head extracts of closely related queens had no significantly reduced variabilities compared to extracts of unrelated queens. Workers, tested in a metabolic bioassay, could not discriminate among head extracts of related and unrelated queens. Therefore the classical queen substance and the semiochemicals of the mandibular glands are unlikely to be used as kin recognition labels by the workers. Workers could, however, discriminate among the odours of tergite extracts and the volatile signals of queens, indicating that the signal of the tergal gland secretions is a possible source for kin recognition labels which the mandibular gland secretions are not. Furthermore the results show that learning is strongly involved in the kin recognition system of honeybees.     

The role of Dufour's gland secretions in bees

ABSTRACT. Dufour's gland of bees is often hypertrophied and an extremely rich source of diverse natural products. In the ground-dwelling bees the secretion is mostly used for lining the brood cell with a hydrophobic lining. Whereas in several species the secretion is smeared on the cell walls without any modification, in others the hydrophobic lining is formed only after a chemical transformation of the secretion. The diversity of the chemical means by which hydrophobicity of the brood cells is achieved suggests that it has evolved repeatedly in the bees. In addition to lining their brood cells, several species use Dufour's gland secretion to mark their nest entrance. Analyses of single glands from various species reveal that each bee possesses its own individual composition, expressed in specific relative amounts of each component. Interestingly in the social halictid Evylaeus malachurum , nestmates (considered as sisters) are more similar to each other than non-related bees. The possible functional evolution of the glandular exudate from structural functions to communication and its implications for our understanding of eusociality in bees, is discussed.     

Use of Dufour's gland secretion in nest defence and brood nutrition by hover wasps (Hymenoptera, Stenogastrinae)

Social wasps of the subfamily Stenogastrinae produce an abdominal secretion that is used in two distinct biological contexts. First, the secretion plays an important role in larval nutrition where it serves as a substrate in which food is placed by the adults for eventual consumption by the larvae. Second, in several species, females apply the same secretion to the substrate on which their nests are constructed, where it constitutes a sticky barrier that defends the immature brood from predation by ants. This paper describes for the first time ant guard construction behaviour of three species of stenogastrine wasps belonging to the genera Eustenogaster and Liostenogaster. The identification of compounds making up these secretions was also performed by gas chromatography-mass spectrometry. Ant guards and brood secretions were similar, with saturated and unsaturated long chain hydrocarbons and alcohols as major components. We further confirm that the glandular source of abdominal secretion is the Dufour's gland. This gland contains the same hydrocarbons, and in the same proportions as ant guards and brood secretion. We discuss the fundamental importance of Dufour's gland secretion in the social life of these wasps by comparing species with and without ant guards within the subfamily.     

Multiple glandular origins of queen pheromones in the fire ant Solenopsis invicta

The poison sac of the fire ant Solenopsis invicta is the only identified glandular source of pheromones produced by a functional ant queen. This structure, which contains the poison gland, has previously been shown to be the source of a releaser pheromone that mediates queen recognition and tending by workers. The poison sac has also been demonstrated to be the source of a primer pheromone that inhibits winged, virgin queens from shedding their wings (dealating) and developing their ovaries. To determine if the poison sac was the only source of these pheromones, we excised the poison sac from queens and observed whether operated queens retained their pheromonal effects. In a first experiment, the poison sac was removed from functional (egg-laying) queens which were then paired with unoperated nestmate queens in small colonies. Counts of the workers surrounding each queen two weeks after the operation showed that queens without poison sac were as effective as their unoperated nestmates in attracting worker retinues. In a second experiment, we removed the poison sacs of virgin queens which had not yet begun laying eggs and thus had not begun producing queen pheromone. After allowing them to develop their ovaries, these individuals produced amounts of queen recognition pheromone comparable to those secreted by unoperated or sham operated virgin queens as determined by bioassay. Testing the head, thorax and abdomens of functional queens separately revealed that the head was the most attractive region in relation to its relative surface area. Bioassays of extracts of two cephalic glands-the mandibular gland and postpharyngeal gland-showed that the postpharyngeal gland is a second source of the queen recognition pheromone. Finally, we found that virgin queens whose poison sacs were removed before they began producing queen pheromone initiated production of a primer pheromone that inhibits winged virgin queens from dealating, indicating that this pheromonal effect also has an additional but as yet undetermined source. These results parallel those on the honey bee in which several of the pheromonal effects of functional queens appear to have multiple glandular sources.     

Queen pheromones affecting the production of queen-like secretion in workers

The honeybee queen pheromones promote both worker sterility and worker-like pheromone composition; in their absence workers become fertile and express the queen pheromones. Which of the queen pheromones regulate worker pheromone expression and how, is still elusive. Here we investigated how two queen pheromones, the mandibular and Dufour’s, singly or combined, affect worker ovarian activation and occurrence of queen-like Dufour’s esters. Although queen mandibular pheromone (QMP) alone, or combined with Dufour’s secretion, inhibited to some extent worker reproduction, neither was as effective as the queen. The effect of the queen pheromones on worker pheromone expression was limited to workers with developed ovaries. Here too, QMP and Dufour’s combined had the greatest inhibitory effect. In contrast, treatment with Dufour’s alone resulted in augmentation of esters in the workers. This is another demonstration that a pheromone emitted by one individual affects the rates of its production in another individual. Ester production was tightly coupled to ovarian development. However fertile workers from queenright or QMP-treated colonies had significantly higher amounts of esters in their Dufour’s gland than untreated queenless colonies. The fact that the queen or QMP exert greater suppression on signal production than on ovary activation, suggests disparate regulatory pathways, and presents a challenging ultimate as well as proximate questions.     

蜜蜂群内生殖分工体系的形成及其维持

本文对蜜蜂群内生殖分工体系的形成及其维持机制进行综述。蜜蜂群体具有完善的劳动分工(包括生殖分工)体系,蜂王垄断生殖权力,而工蜂生殖器官发育不完全,在蜂王信息素和幼虫信息素的作用下产卵受到抑制。蜂王的多雄交配机制降低了群内个体间的亲缘关系,但也促进了工蜂间相互监督机制的形成。工蜂间的相互监督,结合蜂王和幼虫信息素对工蜂卵巢发育的影响,解决了蜂王与工蜂、工蜂与工蜂间的生殖利益冲突,保障了蜂群内的生殖分工体系,提高了群体效率,维护了蜂群的真社会性。     

Behavioral responses to the alarm pheromone of the ant Camponotus obscuripes (Hymenoptera: Formicidae)

The alarm pheromone of the ant Camponotus obscuripes (Formicinae) was identified and quantified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Comparisons between alarm pheromone components and extracts from the major exocrine gland of this ant species revealed that the sources of its alarm pheromone are Dufour's gland and the poison gland. Most components of Dufour's gland were saturated hydrocarbons. n-Undecane comprised more than 90% of all components and in a single Dufour's gland amounted to 19 microg. n-Decane and n-pentadecane were also included in the Dufour's gland secretion. Only formic acid was detected in the poison gland, in amounts ranging from 0.049 to 0.91 microl. This ant species releases a mixture of these substances, each of which has a different volatility and function. When the ants sensed formic acid, they eluded the source of the odor; however, they aggressively approached odors of n-undecane and n-decane, which are highly volatile. In contrast, n-pentadecane, which has the lowest volatility among the identified compounds, was shown to calm the ants. The volatilities of the alarm pheromone components were closely related to their roles in alarm communication. Highly volatile components vaporized rapidly and spread widely, and induced drastic reactions among the ants. As these components became diluted, the less volatile components calmed the excited ants. How the worker ants utilize this alarm communication system for efficient deployment of their nestmates in colony defense is also discussed herein.     

Primer effect of queen pheromone on juvenile hormone biosynthesis in adult worker honey bees

Juvenile hormone synthesis in adult worker honey bees was measured by an in vitro corpora allata bioassay. Adult queenless workers exhibit higher rates of juvenile hormone biosynthesis than queenright workers. Hormone synthesis is not correlated with the volume of the glands. Extract of queen mandibular glands, applied to a dummy, reduces juvenile hormone biosynthesis in caged queenless workers to the level of queenright workers. The same result was obtained with synthetic (E)-9-oxo-2-decenoic acid, the principal component of the queen mandibular gland secretion. This pheromonal primer effect may function as a key regulating element in maintaining eusocial colony homeostasis. The presence of brood does not affect the hormone production of the corpora allata.Abbreviations BSA bovine serum albumin - CA Corpora allata - JH juvenile hormone - 9-ODA (E)-9-oxo-2-decnoic acid