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.     

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.     

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.     

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.     

Estimation of intracolonial worker relationship in a honey bee colony (Apis mellifera L.) using DNA fingerprinting

Summary The number and frequencies of subfamilies in a honey bee colony were determined by DNA fingerprinting. Queen and brood samples were taken from three colonies with artificially inseminated queens and from one colony with a naturally mated queen. UsingHae III restriction enzyme and (GATA)₄ oligonucleotide, the number of subfamilies in the colonies with artificially inseminated queens corresponded with the number of drones used for insemination. In the colony with the naturally mated queen, 12 subfamilies were found in a random sample of 104 workers. Considering that subfamily frequencies range from 1 to 26%, introcolonial worker relationship was estimated to be 0.328, corresponding to a genetical effective number of 6.4 matings.     

Queen substances from the abdomen of the honey bee queen

Summary The secretion of the mandibular glands of a honey bee queen enables the worker bees to react to the presence of their queen. Extirpating the mandibular glands of the queen does not prevent that she is accepted by her colony. Hitherto this was attributed to contamination of the queen's body by mandibular gland substances during or preceding the extirpation. When, however, these glands are extirpated before they have secreted any material and the queens are inseminated artificially, the colonies still accept these queens. A normal-sized retinue, the absence of emergency cell building and the absence of activation of the worker's ovaries indicate that such a queen is still able to maintain her social position. This supports Verheijen-Voogd's (1959) conclusion that the queen's influence on her workers has a behavioural basis (chemoreception) rather than a biochemical one.Laboratory experiments reveal that apart from the mandibular gland substances other queen pheromones are produced in glands on the abdomen, most probably in the glands described by Renner and Baumann (1964).     

Cuticular lipids correlate with age and insemination status in queen honeybees

Eusocial insects exhibit reproductive division of labour, in which one or a few queens perform almost all of the reproduction, while the workers are largely sterile and assist in rearing their siblings. Consequently, many of the colony’s tasks (e.g. nest construction and brood rearing) should be modulated depending on whether the queen is fertile. Here, we tested whether queens’ cuticular lipids could provide reliable signals of fertility in the honeybee Apis mellifera, as they do in other social Hymenoptera. Specifically, we tested whether cuticular lipids differ between virgin queens of different ages, and between queens exposed to different artificial insemination treatments being semen (sperm and seminal fluid), seminal fluid only or saline control. Using gas chromatography–mass spectrometry, we found 27 lipids: 21 different hydrocarbons, namely alkanes, alkenes and dienes, as well as six wax esters. The lipid profile changed dramatically in the first 10 days after eclosion, and there were differences in lipid composition between virgin and artificially inseminated queens. Insemination with semen, seminal fluid or saline did not result in distinct chemical profiles. Our findings indicate that the physical stimulus of insemination was responsible for the observed changes in the cuticular profile in honeybee queens. Our results demonstrate that cuticular lipid profiles encode information on queen age, fertility and mating status, which could in principle be utilised by workers and rival queens.     

Kin structure and colony male reproduction in the hornet<Emphasis Type="Italic"> Vespa crabro</Emphasis> (Hymenoptera: Vespidae)

We estimated queen mating frequency, genetic relatedness among workers, and worker reproduction in Vespa crabro flavofasciata using microsatellite DNA markers. Of 20 colonies examined, 15 contained queens inseminated by a single male, 3 colonies contained queens inseminated by two males, and 2 colonies contained queens inseminated by three males. The genetic relatedness among workers was estimated to be 0.73±0.003 (mean±SE). For this high relatedness, kin selection theory predicts a potential conflict between queens and workers over male production. To verify whether males are derived from queens or workers, 260 males from 13 colonies were genotyped at four microsatellite loci. We found that all of the males were derived from the queens. This finding was further supported by the fact that only 33 of 2,990 workers dissected had developed ovaries. These workers belonged to 2 of the 20 colonies. There was no relationship between queen mating frequency and worker reproduction, and no workers produced male offspring in any of the colonies. These results suggest that male production dominated by queens in V. crabro flavofasciata is possibly due to worker policing.     

Age at Which Larvae Are Orphaned Determines Their Development into Typical or Rebel Workers in the Honeybee (Apis mellifera L.)

In the honeybee, diploid larvae fed with royal jelly develop into reproductive queens, whereas larvae fed with royal jelly for three days only and subsequently with honey and pollen develop into facultatively sterile workers. A recent study showed that worker larvae fed in a queenless colony develop into another female polyphenic form: rebel workers. These rebel workers are more queenlike and have greater reproductive potential than normal workers. However, it was unclear whether larvae orphaned at any time during their feeding period can develop into rebels. To answer this question, the anatomical features of newly emerged workers reared in queenless conditions at different ages during the larval period were evaluated. Our results showed that larvae orphaned during the final four or more days of their feeding life develop into rebel workers with more ovarioles in their ovaries, smaller hypopharyngeal glands, and larger mandibular and Dufour’s glands compared with typical workers with low reproductive potential that were reared with a queen or orphaned at the third to last or a later day of feeding life.     

利用VNTR分子标记鉴定蜜蜂群内蜂王交配次数和雄蜂母系来源

如何准确测定蜂王交配次数和雄蜂母系来源,是研究蜜蜂亚家系行为生物学的关键。本研究利用王浆主蛋白（MRJPs）的串联重复序列多态性（VNTR）分子标记分别鉴定了蜂王单雄人工授精、双雄人工授精和自然交尾的中华蜜蜂Apis cerana cerana蜂群中的蜂王交配次数和雄蜂母系来源。结果表明： 在蜂王单雄人工授精和双雄人工授精蜂群中,蜂王的交配次数分别为1和2;在蜂王自然交尾的2个蜂群中,蜂王的交配次数分别为8和5。另外,经鉴定发现：在以上实验蜂群中,所有雄蜂都是由蜂王产的未受精卵发育而来。因此,作为一种分子标记,蜜蜂MRJPs VNTR能简单、有效地鉴定蜂群内蜂王的交配次数和雄蜂母系来源。     

Queen promiscuity lowers disease within honeybee colonies

Most species of social insects have singly mated queens, but in some species each queen mates with numerous males to create a colony with a genetically diverse worker force. The adaptive significance of polyandry by social insect queens remains an evolutionary puzzle. Using the honeybee (Apis mellifera), we tested the hypothesis that polyandry improves a colony's resistance to disease. We established colonies headed by queens that had been artificially inseminated by either one or 10 drones. Later, we inoculated these colonies with spores of Paenibacillus larvae, the bacterium that causes a highly virulent disease of honeybee larvae (American foulbrood). We found that, on average, colonies headed by multiple-drone inseminated queens had markedly lower disease intensity and higher colony strength at the end of the summer relative to colonies headed by single-drone inseminated queens. These findings support the hypothesis that polyandry by social insect queens is an adaptation to counter disease within their colonies.     

Assessing the mating 'health' of commercial honey bee queens

Honey bee queens mate with multiple males, which increases the total genetic diversity within colonies and has been shown to confer numerous benefits for colony health and productivity. Recent surveys of beekeepers have suggested that 'poor queens' are a top management concern, thus investigating the reproductive quality and mating success of commercially produced honey bee queens is warranted. We purchased 80 commercially produced queens from large queen breeders in California and measured them for their physical size (fresh weigh and thorax width), insemination success (stored sperm counts and sperm viability), and mating number (determined by patriline genotyping of worker offspring). We found that queens had an average of 4.37 +/- 1.446 million stored sperm in their spermathecae with an average viability of 83.7 +/- 13.33%. We also found that the tested queens had mated with a high number of drones (average effective paternity frequency: 17.0 +/- 8.98). Queen "quality" significantly varied among commercial sources for physical characters but not for mating characters. These findings suggest that it may be more effective to improve overall queen reproductive potential by culling lower-quality queens rather than systematically altering current queen production practices.     

Mating structure and male production in Vespa analis and Vespa simillima (Hymenoptera: Vespidae)

We estimated queen mating frequency, genetic relatedness between workers and worker reproduction in the hornets Vespa analis and Vespa simillima using microsatellite DNA genotyping. The 20 V. analis colonies studied each contained a queen inseminated by a single male. Of the 15 V. simillima colonies studied, nine had a queen inseminated by a single male, four had a queen inseminated by two males, and two had a queen inseminated by three males. The estimated effective number of matings was 1.33 ± 0.74 (mean ± SD), with 75–85% of the offspring of the six multiply mated queens sired by single males. The values for genetic relatedness between the workers of V. analis and V. simillima were 0.739 ± 0.004 and 0.698 ± 0.013 (mean ± SD), respectively. We conclude that V. analis and V. simillima colonies are genetically monogynous and monandrous. When high relatedness between the workers occurs within colonies, kin selection theory predicts a potential conflict between queens and workers over male production. To determine whether males were derived from queens or workers, males from V. analis and V. simillima colonies were genotyped at four microsatellite loci and the level of ovary activation in workers was determined. None of the 787 V. analis workers and only 15 of 3520 V. simillima workers had developed ovaries. Furthermore, the genotyping identified no worker‐produced males in any colony. The presence of reproductive workers correlated positively with the number of workers within the colony. These results suggest that eusocial colonies with an annual life cycle tend to break down socially when they become large and are close to dying.     

The occurrence of vitellogenin in workers and queens of Apis mellifica and the possibility of its transmission to the queen

Immuno-diffusion tests show that worker and queen haemolymph contains a protein fraction which does not occur in the haemolymph of drones. Its immunological and electrophoretical properties are identical with those of the main soluble fraction in the ovaries of queens in oviposition. It therefore is a vitellogenin.The titre of this vitellogenin in the haemolymph of 0- to 28-day-old workers was determined by rocket-immunoelectrophoresis. It attains a maximum on day 12. Its changes seem to be positively correlated with the volume of the corpora allata during the first 12 days of adult life.The hypopharyngeal, mandibular, and salivary glands and the content of the honey stomach of workers were immunologically examined. Vitellogenin could not be found in these organs nor in worker or royal jelly. It is also absent from the digestive tract of queens.¹⁴C-labelled amino acids were injected into 5-day-old workers. Later the uptake of radioactive proteins by the queen was examined. Autoradiography of immuno-diffusion plates showed that within 72 hr active material passed from the injected workers into the eggs laid by the queen. The soluble proteins were extracted from the ovaries and the thorax of the queens and their radioactivity determined. The ratio of ovary to thorax radioactivity of queens directly injected was significantly different from that of queens kept with injected workers.Several proteins of the homogenized hypopharyngeal glands of workers showed precipitation reactions with the antiserum against homogenate of queen ovaries. This together with the results of the tracer experiments indicates that the proteins of the worker hypopharyngeal glands may be precursors of queen yolk components.     

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.     

Reproductive and worker castes in the primitively eusocial wasp Belonogaster petiolata (DeGeer) (Hymenoptera: Vespidae): evidence for pre-imaginal differentiation

Until recently, morphological differences between castes in independent-founding polistine wasps were considered absent. This paper investigates the extent of morphological and physiological differences between reproductive (foundress and gyne) and worker castes of Belonogaster petiolata, and tests the hypothesis that caste differentiation in this species occurs pre-imaginally.Foundresses were significantly larger than workers, to the extent that foundress/worker ratios were comparable with those between queens and workers in some swarm-founding Polistinae. Early emerging workers were small, but body size increased over the colony cycle such that late-season workers were similar in size to gynes. In proportion to body size, workers possessed broader heads while foundresses and gynes had broader thoraces and gasters. All queens, 98% of subordinate foundresses, and 95% of over-wintering gynes were inseminated. Workers were never inseminated and lacked mature ovaries in colonies with active queens. However, in the absence of the queen (and other foundresses), 11% of workers developed mature ovaries. Ovarian size and fat content of foundresses and gynes was significantly greater than that of workers. The differences in external morphology and reproductive physiology between castes support the hypothesis that differentiation occurs pre-imaginally. However, imaginal factors, in particular social dominance of the queen, maintain the reproductive subordinance of workers.     

Colony structure and reproduction in the thelytokous parthenogenetic ant Platythyrea punctata (F. Smith) (Hymenoptera, Formicidae)

Summary: An important evolutionary characteristic of the formicine subfamily Ponerinae is the occurrence of various alternative reproductive tactics within single species. In Platythyrea punctata Smith, 1858, queens, gamergates and parthenogenetic workers co-occur in the same species. Morphological queens, both alate and dealate, were present in only 29 percent of the colonies collected in Florida, but absent from colonies collected in Barbados and Puerto Rico. One of the six queens which were dissected (three alate and three dealate) was found to be inseminated but not fertile. Instead, in most queenless colonies, a single uninseminated worker monopolized reproduction by means of thelytokous parthenogenesis, i.e., it produced female offspring from unfertilized eggs. A single mated, reproductive worker (gamergate) was found dominating reproduction in the presence of an inseminated alate queen in one of the Florida colonies. Thelytokous parthenogenesis was examined in artificial groups of virgin laboratory-reared workers, where one worker typically monopolized reproduction despite the presence of several individuals with elongated ovaries. In 16 colonies collected in Florida, a total of 66 individuals differed morphologically from queens and workers. Their thorax morphology varied from a worker-like to an almost queen-like structure. We refer to these individuals as "intercastes" (sensu Peeters, 1991a). The remarkable complexity of reproductive strategies renders P. punctata unique within ants.     

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

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

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.     

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.