Honeybee egg-laying workers mimic a queen signal

Summary. In the honeybee, Dufour's gland secretion is caste specific and constitutes a component of the multi-sourced queen signal. As predicted, it is attractive to workers, which form a retinue around the scented source. Bioassays reveal the ester fraction and not the hydrocarbons to be the active constituents. This function of the esters was corroborated by assays with the synthetic queen-esters mixture, which successfully mimicked the queen's secretion. As predicted from the queen-like secretion exhibited by egg-laying workers, their glandular secretion was also attractive to nestmates, albeit to a lesser degree than that of the queen; while that of non-egg-laying workers was totally inactive. The evolution of the multiple queen signals in honeybees can be regarded as a component in an arms race between queen and workers. We hypothesize that in response to a reduced sensitivity to a certain queen signal, queen honeybees were selected to develop an alternative signaling-source. Dufour's gland seems to be one of these sources.     

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.     

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.     

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.     

Reproductive allocation in multinest colonies of the ponerine ant Pachycondyla goeldii

Abstract.  1.  Pachycondyla goeldii constitutes the only recorded case of a monogynous (i.e. one queen per colony) polydomous (i.e. several nests per colony) species in the Ponerinae subfamily. This study examines the impact of polydomy on reproductive allocation between nests (also called 'calies' in polydomous society) in Pachycondyla goeldii Forel, by: (i) recording the number of workers and sexuals in 67 nests belonging to 21 colonies; (ii) dissection of workers in nine nests containing a queen (QR nests), nine nests without a queen but associated to a QR nest (QL nests) and five nests belonging to colonies that permanently lost the queen (OR nests); and (iii) measuring the length of all eggs present in the nests (our laboratory study shows that queen-laid eggs were significantly longer than worker-laid eggs).
2. The number of workers was significantly higher in QR nests than in QL nests, while the number of virgin queens was significantly higher in QL nests compared with QR nests.
3. Worker ovarian activity is inversely related to queen proximity: highest in OR nests, intermediate in QL nests, and lowest in QR nests.
4. Egg length was highest in QR nests, where the queen was most likely the primary egg-layer, intermediate in QL nests, where eggs could have originated from both the queen and workers, and lowest in OR nests, where workers were the sole egg-layers.
5. We postulate that the proximal mechanism explaining differences between QR and QL nests is the pheromonal absence of the queen from QL nests and that the evolutionary reasons of these divergences between nest types are likely to originate from the different conflicts occurring in ant colonies.     

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.     

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.     

Queen signal should be honest to be involved in maintenance of eusociality: chemical correlates of fertility in <Emphasis Type="Italic">Ropalidia marginata</Emphasis>

Queens of many social insect species are known to maintain reproductive monopoly by pheromonal signalling of fecundity. Queens of the primitively eusocial wasp Ropalidia marginata appear to do so using secretions from their Dufour’s glands, whose hydrocarbon composition is correlated with fertility. Solitary nest foundresses of R. marginata are without nestmates; hence expressing a queen signal can be redundant, since there is no one to receive the signal. But if queen pheromone is an honest signal inextricably linked with fertility, it should correlate with fertility and be expressed irrespective of the presence or absence of receivers of the signal, by virtue of being a byproduct of the state of fertility. Hence we compared the Dufour’s gland hydrocarbons and ovaries of solitary foundresses with queens and workers of post-emergence nests. Our results suggest that queen pheromone composition in R. marginata is a byproduct of fertility and hence can honestly signal fertility. This provides important new evidence for the honest signalling hypothesis.     

Exocrine gland secretions of virgin queens of five bumblebee species (Hymenoptera: Apidae, Bombini)

Secretions of three different glands (mandibular gland, labial gland, and Dufour's gland) of virgin queens of five bumblebee species (Bombus lucorum, B. lapidarius, B. hypnorum, B. pascuorum, and B. terrestris) were analysed. Around 200 compounds were identified in the secretions. The compositions of the secretions of labial and mandibular glands were species-specific. Dufour's gland of all species produced mainly hydrocarbons, both saturated and unsaturated, the proportions of which differed quantitatively between the species studied.     

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.     

Queen power in relation to age and mating status in the Argentine antIridomyrmex humilis (Mayr)

Summary Queen power inIridomyrmex humilis during six arbitrarily chosen physiological stages of queens (virgin winged queens at the time of emergence, the same 4–5 days old, mated winged queens 5–6 days old, mated queens at the time of dealation, young egg-laying queens, and old egg-laying queens) was tested with regard to workers. Tested workers and queens were nestmates. The results were as follows. 1) Power of the old queens remained rather constant throughout the reproductive season. 2) Young queens were always less attractive than older ones. No changes were observed from the time of emergence to dealation. 3) At the time of egg-laying, these young queens became markedly attractive for workers but never as much as old egg-laying queens. Therefore, insemination and dealation do not trigger the increase in queen power. In contrast, oogenesis ending by egg-laying produces a significant increase in queen power.     

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.     

Ontogenetic patterns of volatiles identified in Dufour's gland extracts from queens and workers of the primitively eusocial halictine bee,Lasioglossum malachmum (Hymenoptera: Halictidae)

Summary Ontogenetic patterns of volatile compounds identified in Dufour's gland extracts from queens and workers of the primitively eusocial sweat beeLasioglossum malachurum (K.) were compared. Only young unmated queens showed high proportions of isopentenyl esters, while macrocyclic lactones were dominant in old breeding queens, spring queens, and workers. In young queens the relative and absolute amounts of volatiles changed one day after mating. A discriminant analysis revealed significant differences in odor patterns of unmated and mated young queens. The fat body was the largest in young females, while eggs could be recorded only in breeding queens. Possible functions of different odor components in the investigated female groups are discussed.     

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.     

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.     

Independent colony founding by ergatoid queens in the ant genus Pogonomyrmex: queen foraging provides an alternative to dependent colony founding

Ant queens exhibit two primary strategies to initiate nests, independent colony founding (ICF) by solitary queens and dependent colony founding (DCF) when the queen starts a nest with a group of workers that disperse on foot from the parent nest. Numerous ant species have wingless (ergatoid) queens, and it is generally assumed that these species exhibit obligate DCF because their lack of wing musculature provides them with few resources to divert towards producing their first brood of workers. Thus, ICF by ergatoid queens is viewed as maladaptive because these queens need to take additional dangerous foraging trips to garner sufficient food to rear their first brood of workers. Contrary to this prediction, I document ICF by ergatoid queens for three species of harvester ants in the genus Pogonomyrmex (subfamily Myrmicinae), P. cunicularius cunicularius, P. cunicularius pencosensis, and P. huachucanus. Queens of P. huachucanus were obligate foragers, i.e., no minim workers could be produced without external food, and one queen of P. cunicularius pencosensis was observed foraging in the field. Abundant and/or predictable food resources likely select for the evolution of semi-claustral nest founding and ICF by these ergatoid queens. Under these conditions, foraging time would be minimized and the number and size of minim workers would be maximized. These benefits should increase founding success, which could compensate for loss of long-range dispersal. Overall, this study demonstrates that care should be taken before concluding that ant colonies employ DCF based solely on queen morphology.     

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.     

Competition over workers: fertility signalling in wingless queens of <Emphasis Type="Italic">Hypoponera opacior</Emphasis>

The ant Hypoponera opacior exhibits alternative reproductive morphs associated with distinct sexual behaviours. Wingless, worker-like gynes mate in fall in their mother nest. Subsequently, these polygynous nests divide and wingless queens disperse on foot accompanied by workers, which help during nest foundation. Local resource competition amongst wingless queens was indicated by male-biased sex ratios. Queens compete over access to workers, and this competition manifests in dominance interactions. We colour-marked queens and workers to study this behaviour. Marked queens were treated aggressively and frequently killed by nestmates, whilst similarly labelled workers were readopted. We hypothesise that mated wingless queens—although they resemble workers in external morphology—differ chemically and that fertile queens with divergent odours are not tolerated. Thereby, sterile H. opacior workers avoid the risk to raise offsprings of unrelated queens. However, when nests split, workers should base their decision which queen to follow not only on relatedness, but also on queen fertility. Cuticular hydrocarbon profiles of queens could serve as an honest signal for workers. Indeed, gas chromatography showed that hydrocarbon composition differs between workers and mated queens and is linked to ovary development in queens. Workers and unmated queens exhibit complex profiles of short-chained hydrocarbons, whilst fertile queens carry few, long-chained alkenes and branched alkanes. Furthermore, ovary length was correlated to the relative amounts of characteristic hydrocarbons of queens. The chemical profile was also associated with eye size—an unalterable trait in adult insects—suggesting that larval nutrition and consequently body size affects adult ovary development.     

No evidence that reproductive bumblebee workers reduce the production of new queens

Kin selection theory predicts potential conflict between queen and workers over male parentage in hymenopteran societies headed by one, singly mated queen, because each party is more closely related to its own male offspring. In ‘late-switching’ colonies of the bumblebee Bombus terrestris, i.e. colonies whose queens lay haploid eggs relatively late in the colony cycle, workers start to lay male eggs shortly after the queen lays the female eggs that will develop into new queens. It has been hypothesized that this occurs because workers recognize, via a signal given by the queen instructing female larvae to commence development as queens, that egg laying is now in their kin-selected interest. This hypothesis assumes that aggressive behaviour in egg-laying workers does not substantially reduce the production of new queens, which would decrease the workers' fitness payoff from producing males. We tested the hypothesis that reproductive activity inB. terrestris workers does not reduce the production of new queens. We used microsatellite genotyping to sex eggs and hence to select eight size-matched pairs of ‘late-switching’ colonies from a set of commercial colonies. From one colony of each pair we removed every egg-laying or aggressive worker observed. From the other colony, we simultaneously removed a nonegg-laying, nonaggressive worker. Removed workers were replaced with young workers from separate colonies at equal frequencies within the pair. There was no significant difference in queen productivity between colonies with reduced or normal levels of egg-laying or aggressive workers. Therefore, as predicted, reproductive B. terrestris workers did not significantly reduce the production of new queens.