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.     

Suppression of queen rearing in European and Africanized honey beesApis mellifera L. by synthetic queen mandibular gland pheromone

Summary Queen rearing is suppressed in honey bees (Apis mellifera L.) by pheromones, particularly the queen's mandibular gland pheromone. In this study we compared this pheromonally-based inhibition between temperate and tropically-evolved honey bees. Colonies of European and Africanized bees were exposed to synthetic queen mandibular gland pheromone (QMP) for ten days following removal of resident queens, and their queen rearing responses were examined. Queen rearing was suppressed similarly in both European and Africanized honey bees with the addition of synthetic QMP, indicating that QMP acts on workers of both races in a comparable fashion. QMP completely suppressed queen cell production for two days, but by day six, cells containing queen larvae were present in all treated colonies, indicating that other signals play a role in the suppression of queen rearing. In queenless control colonies not treated with QMP, Africanized bees reared 30% fewer queens than Europeans, possibly due to racial differences in response to feedback from developing queens and/or their cells. Queen development rate was faster in Africanized colonies, or they selected older larvae to initiate cells, as only 1 % of queen cells were unsealed after 10 days compared with 12% unsealed cells in European colonies.     

Conditions favoring queen execution in young social insect colonies

Summary. Previous attempts to explain worker aggression against extra queens in young social insect colonies have used kin selection arguments. These have been inconsistent with experimental evidence demonstrating aggression against extra queens without strong evidence of kin discrimination. Using a game theoretical model, I suggest a series of decision rules that are consistent with the current experimental evidence from study of young colonies of the fire ant Solenopsis invicta. These decision rules are: 1) When workers cannot directly determine which queen is their mother, they should behave in favor of a queen that maximizes the product of the chance that a queen is the workers mother multiplied by the chance that it will survive to colony maturity. In some cases, the survival potential of the different queens may be the only character that influences the workers decision. 2) Workers should delay aggression against extra queens until the workers can gain their greatest advantage through such aggression. 3) Queens may adopt strategies that allow them to dominate rivals, either by gaining an advantage in fights among the queens or by increasing their attractiveness to workers.Received 2 June 2004; revised 5 July 2004; accepted 14 July 2004.     

Effect of queen excluders on ovary activation in workers of the Eastern honeybee Apis cerana

Honeybee workers generally refrain from personal reproduction when a queen is present. Workers discern the presence and fecundity of the queen via volatile pheromones that permeate throughout the colony. Pheromones are emitted both by the queen herself and by the brood that she produces. If pheromone production is disrupted, some workers initiate egg laying. The Eastern honeybee Apis cerana is unusual in that workers have high levels of ovary activation even in the presence of a queen. To investigate the effect of disruption to pheromone dispersal, we fitted three A. cerana colonies with vertical queen excluders, thus splitting the colonies into a half containing a queen and a half without a queen. We regularly sampled adult workers from both sides of the excluder for 3 weeks. We also sampled workers from three control colonies that did not contain excluders. We found a significant increase in worker ovary activation 3 days after addition of excluders, suggesting that the reduced dispersal of pheromones allowed some workers to become reproductively active. Workers attempted to rear queen cells on the queenless halves of all three colonies. Queen-rearing ceased on day 9, at which time no queen-laid brood remained on the queenless halves of the colonies. Ovary activation rates continued to climb until day 9 and then gradually began to decline. With the exception of one egg, we did not observe worker-laid brood on the queenless side of the colonies, suggesting that workers continued policing eggs laid by workers. We conclude that if the distribution of brood pheromone is impeded, workers prepare to supersede their queen, accompanied by high levels of worker ovary activation. However, because workers continue to police each other, high ovary activation does not result in worker-produced drones.     

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 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.     

Development and behaviour of anarchistic honeybees

Anarchistic honeybees result from extremely rare behavioural mutations which allow workers to lay eggs despite the presence of the queen. We investigated the behavioural development of bees derived from a line in which ca. 5% of workers have developed ovaries and lay viable eggs. Other than their developed ovaries and proclivity to lay eggs, the anarchistic workers we studied are apparently normal, performing normal worker-like behaviour. Unlike many laying workers in queenless colonies, they are not queen-like and are apparently not the objects of aggression. When day-old workers from anarchistic colonies were cross-fostered into anarchistic and wild-type host colonies, the frequency of ovary development was an order of magnitude higher in the anarchistic host (9.1%) than in the wild-type host (0.7%). This suggests that there is a policing mechanism that affects ovary development in honeybees. Thus, worker reproduction is probably suppressed at the level of ovary development as well as by oophagy of worker-laid eggs. Other mechanisms, such as aggression towards individuals with developed ovaries, may also exist, but we found no evidence for this.     

Reproductive development and ontogeny of queen pheromone production in the fire ant Solenopsis invicta

Queens of the fire ant, Solenopsis invicta Buren, produce several releaser and primer pheromones. Using bioassays, the ontogeny of three of these pheromones related to reproductive development was investigated. Virgin queens, in which the process of wing-shedding (dealation) serves as an indicator of the initiation of reproductive development, were studied. First, the production of two queen pheromones produced in the poison gland was examined. The pheromone responsible for initiation and maintenance of retinue formation, a releaser effect, was found to be produced in detectable quantities 2 days after dealation, at which time queens showed significant ovary development and many (30%) had started laying eggs. A primer pheromone that inhibits alate virgin queens from dealating was detected in queens 3 days following wing-shedding, when 80% of the queens were ovipositing. Second, I examined the onset of a pheromone of unknown glandular origin produced by reproductively active virgin queens which leads to their destruction in queenright colonies by stimulating workers to attack and kill them. This pheromone is secreted in quantities detectable by bioassay 2 days after dealation. Thus, in S. invicta, the ontogeny of three distinct queen pheromones is tightly linked with ovary development and initiation of egg laying. These results demonstrate reproductive and communicative functions are closely associated during the transition from potential to functional queen.     

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.     

Absence of reproductive conflict during queen rearing in Apis cerana

Polyandry in honeybee queens (Apis) causes many patrilines (subfamilies) within a colony, which may lead to a potential conflict of interest among workers. This may be most apparent during queen rearing when nepotistic worker behavior could influence the genetics of future generations. Several studies have searched for such conflict in European honeybees (A. mellifera), but studies on other Apis species remain lacking. We investigated the presence of reproductive conflict in A. cerana japonica by comparing the patriline proportion of queen larvae to that of adult workers. We determined the patrilines of 272 workers and 57 queen larvae using four polymorphic microsatellite markers that were sampled from queenless colonies originally derived from four naturally mated queen-right colonies. The number of patrilines in each colony was 9, 12, 8, and 7, respectively, which is lower than that observed in continental Asia. We found no difference in patriline proportion between adult workers and queen larvae. Our data support neither genetic variance for royalty or existence of worker nepotism in A. cerana japonica.     

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.     

A sensitive and reliable assay for queen discrimination ability in laboratory-reared workers of the ant Camponotus japonicus

The queen discrimination abilities of laboratory-reared Camponotus japonicus workers were examined individually by allowing them to carry their nestmate larvae toward either the mother queen or an alien queen. Source colonies had been reared under controlled conditions from founding queens and maintained at small size (< or = 10 workers each). Fifty-two of fifty-four workers raised in these eight different colonies carried nestmate larvae to the mother queen, and never carried them to the alien queen. Most of them attended nestmate larvae but never alien larvae. These results clearly demonstrate that the tested workers discriminate the nestmate queen and larvae from non-nestmate conspecifics. The assay used in this study is novel and sensitive, and may be suitable for neuroethological and molecular studies of social discrimination mechanisms.     

Patriline differences in emergency queen rearing in the honey bee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

Summary In the polyandrous honey bee, Apis mellifera, workers can potentially increase their inclusive fitness by rearing full-sister queens. If the mother queen dies suddenly, workers feed a few larvae in worker cells with royal jelly and rear them into queens (emergency queen rearing). Using DNA microsatellite markers we determined the patriline of emergency queens reared in two colonies headed by naturally-mated queens before being made queenless. We found that some patrilines were reared more than others in one colony, but not in the other. These differences between colonies suggest that selective rearing is not always present and this might explain the mixed results of previous nepotism studies in the honey bee.Received 10 February 2003; revised 7 March 2003; accepted 17 March 2003.     

Workers of the primitively eusocial wasp <Emphasis Type="Italic">Ropalidia marginata</Emphasis> do not perceive their queen across a wire mesh partition

Queens of primitively eusocial wasps generally have active and behaviourally dominant queens who use physical aggression to suppress worker reproduction. Although a Ropalidia marginata queen is strikingly docile and behaviourally non-dominant, she is completely successful in maintaining reproductive monopoly. R. marginata queens must achieve such reproductive monopoly by some means other than overt physical aggression. Upon loss or removal of the queen, one of the workers (referred to as the potential queen) becomes extremely aggressive and will eventually go on to become the next queen of the colony, if the original queen is not returned. The fact that potential queens are not discernible in the presence of the queen but become obvious within minutes after removal of the queen raises the question of how workers in general and the potential queens in particular, perceive the presence or absence of their queens. Here, we have conducted experiments in which we separate half of the workers from their queen by a wire mesh screen and study their behavioural response to such separation. We demonstrate that the presence of the queen is not perceived across the wire mesh screen, which suggests that if the queen uses a pheromone to signal her presence, then that pheromone is not very volatile.     

Elimination of sexual brood in the Argentine antLinepithema humile: queen effect and brood recognition

Mated queens of the antLinepithema humile (Iridomyrmex humilis Mayr) introduced into dequeened colony fragments rearing sexual brood elicited worker aggression resulting in queen larvae being bitten and eliminated. By contrast, male larvae were spared. Regarding queen brood, killing mainly concerned small and medium sized larvae. A large proportion of the large larvae escaped extermination, and prepupae and pupae were spared. These data suggest that workers were able to discriminate sex, caste and age of the brood. That a queen pheromone may be involved was shown by experiments using whole or cut corpses that were either rinsed or not rinsed in pentane. The pheromone eliciting worker aggressive behaviour was shown to act over a short distance, suggesting that it is somewhat volatile. Similarities and differences between this new queen pheromone and other known queen pheromones acting on queen production or worker attraction are discussed as well as the origin of the signals underlying the recognition of the larval classes.     

The effect of queen ants on the survival of Maculinea arion larvae in Myrmica ant nests

Summary The mortality of Maculinea arion caterpillars was measured in both laboratory and wild Myrmica nests, and found to be nearly 3 times higher in nests that had queen ants present. This is attributed to queen effect, which causes worker ants in nests with queens to attack large ant larvae (gynes) that would otherwise develop into new queens. Maculinea arion caterpillars mimic Myrmica larvae, and are usually attacked during the first 10 days after adoption, when they pass through the size range of ant gyne larvae. Caterpillars are also likely to be attacked during this period because their nethod of feeding brings them into close contact with the skins of large ant larvae, which contain gyne larval pheromones; older caterpillars are large enough to eat larvae without their exposed surfaces contacting the larval skin. In the wild, many caterpillars of Maculinea arion die in ant nests, and this has been shown in previous work to be the key factor that determines changes in their abundance from year to year. It is suggested that queen effect can be an important cause of these deaths, and one that particularly affects populations of butterflies that breed on sites with long-established plagioclimaxes of short turf rather than short-lived grass-land successions.     

Making a queen: an epigenetic analysis of the robustness of the honeybee (Apis mellifera) queen developmental pathway

Specialized castes are considered a key reason for the evolutionary and ecological success of the social insect lifestyle. The most essential caste distinction is between the fertile queen and the sterile workers. Honeybee (Apis mellifera) workers and queens are not genetically distinct, rather these different phenotypes are the result of epigenetically regulated divergent developmental pathways. This is an important phenomenon in understanding the evolution of social insect societies. Here, we studied the genomic regulation of the worker and queen developmental pathways, and the robustness of the pathways by transplanting eggs or young larvae to queen cells. Queens could be successfully reared from worker larvae transplanted up to 3 days age, but queens reared from older worker larvae had decreased queen body size and weight compared with queens from transplanted eggs. Gene expression analysis showed that queens raised from worker larvae differed from queens raised from eggs in the expression of genes involved in the immune system, caste differentiation, body development and longevity. DNA methylation levels were also higher in 3‐day‐old queen larvae raised from worker larvae compared with that raised from transplanted eggs identifying a possible mechanism stabilizing the two developmental paths. We propose that environmental (nutrition and space) changes induced by the commercial rearing practice result in a suboptimal queen phenotype via epigenetic processes, which may potentially contribute to the evolution of queen–worker dimorphism. This also has potentially contributed to the global increase in honeybee colony failure rates.     

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.     

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.     

Host colony usurpation by the queen of the Japanese pirate ant, Polyergus samurai (hymenoptera: formicidae)

Colony usurpations by newly mated queens of Polyergus samurai were observed under artificial conditions. Newly mated queens of P. samurai were introduced into three kinds of Formica japonica host colonies: queenright, queenless (artificially orphaned), and workerless (only a queen remaining) colonies. In the queenright condition, the P. samurai queen intruded into the host nest and killed the host queen, and was subsequently adopted by the host workers. In all queenright and queenless host colonies, seven of 13 queens of P. samurai succeeded in colony usurpation, although the starting time of grooming, a nestmate behavior, by host workers in the queenright condition occurred earlier than in the queenless condition. In workerless conditions, four of five P. samurai queens ignored the F. japonica queen. The results suggest that while host-queen killing is not necessary, it is important to win acceptance by host workers.