Regulation of oogenesis in honey bee workers via programed cell death

Reproductive division of labour characterises eusociality. Currently little is known about the mechanisms that underlie the ‘sterility’ of the worker caste, but queen pheromone plays a major role in regulating the reproductive state. Here we investigate oogenesis in the young adult honey bee worker ovary in the presence of queen pheromone and in its absence. When queen pheromone is absent, workers can activate their ovaries and have well-developed follicles. When queen pheromone is present, even though workers have non-activated ovaries, they continually produce oocytes which are aborted at an early stage. Therefore, irrespective of the presence of the queen, the young adult worker ovary contains oocytes. By this means young workers retain reproductive plasticity. The degeneration of the germ cells in the ovarioles of workers in the presence of queen pheromone has the morphological hallmarks of programmed cell death. Therefore the mechanistic basis of ‘worker sterility’ relies in part on the regulation of oogenesis via programmed cell death. Our results suggest that honey bees have co-opted a highly conserved checkpoint at mid-oogenesis to regulate the fertility of the worker caste.     

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.     

蜜蜂无政府主义蜂群的研究进展

西方蜜蜂Apis mellifera作为典型的社会性昆虫, 最重要的特征是生殖劳动分工。蜂王垄断蜂群的生殖权利, 工蜂生殖功能受到抑制, 从事除产卵和交配以外的所有职能。而在无政府主义蜂群中, 即使蜂王存在, 也有较多工蜂的卵巢激活并产卵, 蜂群中大多数雄蜂是工蜂的后代。这些特殊蜂群为正常蜂群工蜂不育机制研究提供了绝佳的反例材料。本文对无政府主义蜂群的行为特征、 产生条件、 遗传基础等研究进行了综述。无政府主义蜂群中有较多的工蜂产卵, 且工蜂所产卵能够逃避工蜂监督, 这种行为的产生受环境、 遗传组成、 基因表达等多种因素的影响, 并且遗传结构体系复杂, 参与调控的基因数量多。无政府主义蜂群行为机制的研究为工蜂不育机制的揭示及其他社会性昆虫工职不育基因的筛选和功能研究提供借鉴。     

Agonistic behaviours in colonies of the bumblebeeBombus terrestris

The first aim of this study was to investigate whether specific changes in the frequencies of behavioural characteristics are correlated with the cause or reflect the onset of the laying of haploid eggs by the queen, and whether these changes cause the onset of egg laying and aggressive behaviour by workers. The second aim was to obtain a better understanding of the occurrence of the behaviours in relation to the physiology and function of the queen and the workers. No behavioural characteristics of workers which could predict the occurrence of the queen's switch to haploid egg laying was found nor did any such behaviour indicate that the switch had already occurred. The onset of worker oviposition was not found to be the consequence of the level or the type of agonistic actions between the workers and the queen. With the onset of worker oviposition the agonistic behaviours called trembling and buzzing, which are directed at the queen, disappear. At the same time the frequency of a non-directed behaviour, called humming, increases. This behaviour is seen particularly in the small housebees with developed ovaries. Before worker oviposition begins, the frequencies with which individual workers perform agonistic behaviour are not correlated with the conditions of their ovaries nor correlated with other characteristics of the workers. However, these frequencies are related to the sequence in which workers emerge. The correlations with age are due to the fact that workers of the first brood have higher activity levels than those of the later broods. The first egg layers are to be found among the first workers. In the period of worker oviposition, humming and the aggressive behaviours of darting and attack are performed by workers with developed ovaries. These behaviours are directed particularly towards workers who also have active ovaries. If the queen has disappeared 1 worker usually becomes dominant. This worker is characterized by the relatively high frequency with which she engages in darting and attacks. In addition, this worker is characterized by a specific behaviour, called pumping, which is directed at workers that have mature eggs in their ovaries. Behavioural domination, however, is no guarantee that the bee concerned will eventually become the most productive one. Although at an apparently pre-determined moment in the ontogeny of the colony the queen loses control over worker oviposition and related behaviours, she remains dominant in various other behavioural hierarchies. It is therefore concluded that the queen never loses dominance completely.     

Durability of information concerning the presence of a gamergate in Diacamma sp. from Japan

In social insects, information on the presence of a queen is known to affect the reproductive behaviour of workers. In the queenless ant Diacamma sp., information of the presence of the gamergate, the functional queen, is transmitted exclusively by direct physical contact between the gamergate and workers. Periodic contacts between the gamergate and each worker are therefore necessary to maintain worker sterility. However, how the difference in the contact interval influences reproductive behaviour of workers is unclear. In the present study, by artificially controlling the exact interval of physical contact between workers with the gamergate, it is shown that the inter‐contact interval influences the worker's reproductive condition (i.e. a longer interval leads to more developed ovaries). This system provides a good opportunity to study the mechanisms underling the colony size dependence of individual as well as colony characteristics.     

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。     

Genome-wide analysis reveals differences in brain gene expression patterns associated with caste and reproductive status in honey bees (Apis mellifera)

A key characteristic of eusocial species is reproductive division of labour. Honey bee colonies typically have a single reproductive queen and thousands of sterile workers. Adult queens differ dramatically from workers in anatomy, physiology, behaviour and lifespan. Young female workers can activate their ovaries and initiate egg laying; these 'reproductive' workers differ from sterile workers in anatomy, physiology, and behaviour. These differences, however, are on a much smaller scale than those observed between the queen and worker castes. Here, we use microarrays to monitor expression patterns of several thousand genes in the brains of same-aged virgin queens, sterile workers, and reproductive workers. We found large differences in expression between queens and both worker groups (~2000 genes), and much smaller differences between sterile and reproductive workers (221 genes). The expression patterns of these 221 genes in reproductive workers are more queen-like, and may represent a core group of genes associated with reproductive physiology. Furthermore, queens and reproductive workers preferentially up-regulate genes associated with the nurse bee behavioural state, which supports the hypothesis of an evolutionary link between worker division of labour and molecular pathways related to reproduction. Finally, several functional groups of genes associated with longevity in other species are significantly up-regulated in queens. Identifying the genes that underlie the differences between queens, sterile workers, and reproductive workers will allow us to begin to characterize the molecular mechanisms underlying the evolution of social behaviour and large-scale remodelling of gene networks associated with polyphenisms.     

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.     

The evolution of worker sterility in honeybees: the genetic basis of failure of worker policing

Worker honeybees (Apis mellifera) usually only lay eggs when their colony is queenless. However, an extremely rare ''anarchistic'' phenotype occurs, in which workers develop functional ovaries and lay large numbers of haploid eggs which develop into adult drones despite the presence of the queen. Studies of such colonies can give important insights into the mechanisms by which worker sterility is maintained in normal colonies. Here we report on the results of a breeding programme which enhanced the frequency of the anarchistic phenotype. Colonies derived from queens inseminated only by worker-laid males showed up to 9% of workers with highly developed ovaries. In these colonies a large proportion of males arose from worker-laid eggs. Colonies headed by queens inseminated with 50% worker-laid drones and 50% queen-laid drones showed variable phenotypes. In most such colonies there was no worker reproduction. In some, many workers had highly developed ovaries, but no worker-laid eggs were reared. In one colony, many worker-laid eggs were reared to maturity. The results suggest that the anarchy phenotype results from a complex interaction of queen genotype, the worker genotype of subfamilies that successfully reproduce and of those that do not, and the external environment.     

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.     

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.     

Modifications in the oviducts of workers and queens of <Emphasis Type="Italic">Melipona quadrifasciata anthidioides</Emphasis> (Hymenoptera: Apidae) with different ages

The study of morphological features of highly eusocial bees has helped to answer a series of questions concerning their biology. The labor division places the females into two castes, queen with reproductive function and worker with a wide variety of tasks. However, in different species and under different conditions, workers can develop ovaries and lay trophic eggs that are eaten by the queen or used to originate males. In this work, the development of the oviducts was monitored in workers and virgin queens of Melipona quadrifasciata anthidioides specimens of different ages to verify whether there is permanent sterility of these queens and workers due to aging. Lateral and common oviducts of virgin queens aged <7, 10, 15, 20, and 25 days old, physogastric queens, nurse, and forager workers were analyzed for histology and ultrastructure. Although the structural organization of the lateral and common oviducts were very similar, differences in width of the cuticle and the degree of chromatin condensation were observed, indicating differences in the development of this organ of the reproductive system between the castes. It was also demonstrated that electron-lucent vesicles appear to be related to the formation of the cuticle that lines the oviducts. Because no cellular death characteristics were found, it can be inferred that the absence of mating of the queens as old as of 25 days of age does not cause permanent sterility.     

Identification of an ant queen pheromone regulating worker sterility

The selective forces that shape and maintain eusocial societies are an enduring puzzle in evolutionary biology. Ordinarily sterile workers can usually reproduce given the right conditions, so the factors regulating reproductive division of labour may provide insight into why eusociality has persisted over evolutionary time. Queen-produced pheromones that affect worker reproduction have been implicated in diverse taxa, including ants, termites, wasps and possibly mole rats, but to date have only been definitively identified in the honeybee. Using the black garden ant Lasius niger, we isolate the first sterility-regulating ant queen pheromone. The pheromone is a cuticular hydrocarbon that comprises the majority of the chemical profile of queens and their eggs, and also affects worker behaviour, by reducing aggression towards objects bearing the pheromone. We further show that the pheromone elicits a strong response in worker antennae and that its production by queens is selectively reduced following an immune challenge. These results suggest that the pheromone has a central role in colony organization and support the hypothesis that worker sterility represents altruistic self-restraint in response to an honest quality signal.     

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 potential for worker reproduction in the ant <Emphasis Type="Italic">Aphaenogaster cockerelli</Emphasis> and its absence in the field

A reproductive division of labor between subordinates and established reproductives is a hallmark of eusociality. In most groups, however, workers retain some reproductive capabilities. Across insect societies, measures of successful worker reproduction in the presence of a queen, with few exceptions, indicate that worker reproduction is kept at very low levels. There are, however, certain colony-level characteristics that may influence the degree to which worker reproduction is promoted, such as queen number, queen mating frequency, and physical presence of a queen in species with multiple nesting sites (polydomy). In this study, the level of worker reproduction in field colonies of the ant species Aphaenogaster cockerelli was measured. A. cockerelli is a monogynous and polydomous species, so worker reproduction across nesting sites was investigated. None of the 297 males sampled provided any evidence of worker reproduction. Worker reproduction would have been detectable if it was present at or above a level of 1.5 % of the total males per colony. An effective mating frequency for queens of this species was found to be 1.03. Although A. cockerelli colonies have many colony-level factors potentially promoting worker reproduction (workers with active, trophic egg-producing ovaries, a single singly-mated queen, workers who are physically separated from the queen), it is evident that worker reproduction is highly regulated. Synthesizing the extensive amount of policing and fertility signaling data previously reported on this species, A. cockerelli is presented as case study for how worker reproduction is repressed and cooperation is maintained in insect societies.     

Juvenile hormone effect on DNA synthesis and apoptosis in caste-specific differentiation of the larval honey bee (Apis mellifera L.) ovary

Caste-specific differentiation of the honey bee ovary commences in the last larval instar. In this process, formation of germ cell clusters by synchronous and incomplete mitoses occurs in the queen ovary, whereas in the worker ovary programmed cell death is the dominant feature. BrdU and TUNEL labeling were used to study dynamics of cell proliferation and apoptosis-dependent DNA degradation in ovaries of naturally developing queens and workers, as well as in juvenile hormone-treated worker larvae. Cell proliferation in ovaries of last-instar queen larvae generally exceeded that in workers, except for the late feeding phase. This inversion in cell proliferation patterns coincided with the onset of apoptosis in worker ovaries, as evidenced by TUNEL labeling. Juvenile hormone application to early-fifth-instar worker larvae had two noticeable effects. First, it diminished the number of S-phase nuclei in ovaries of late feeding-phase workers, bringing them to queen-like levels. Second, it prevented the induction of apoptotic DNA degradation. Caste-specific regulation of cell division in connection with programmed cell death can thus be attributed to the previously described differences in juvenile hormone titer in queen and worker larvae, adding a new facet to this hormone's multiple functions.     

Gene expression patterns associated with caste and reproductive status in ants: worker‐specific genes are more derived than queen‐specific ones

Variation in gene expression leads to phenotypic diversity and plays a central role in caste differentiation of eusocial insect species. In social Hymenoptera, females with the same genetic background can develop into queens or workers, which are characterized by divergent morphologies, behaviours and lifespan. Moreover, many social insects exhibit behaviourally distinct worker castes, such as brood‐tenders and foragers. Researchers have just started to explore which genes are differentially expressed to achieve this remarkable phenotypic plasticity. Although the queen is normally the only reproductive individual in the nest, following her removal, young brood‐tending workers often develop ovaries and start to reproduce. Here, we make use of this ability in the ant Temnothorax longispinosus and compare gene expression patterns in the queens and three worker castes along a reproductive gradient. We found the largest expression differences between the queen and the worker castes (~2500 genes) and the smallest differences between infertile brood‐tenders and foragers (~300 genes). The expression profile of fertile workers is more worker‐like, but to a certain extent intermediate between the queen and the infertile worker castes. In contrast to the queen, a high number of differentially expressed genes in the worker castes are of unknown function, pointing to the derived status of hymenopteran workers within insects.     

Are workers of Atta leafcutter ants capable of reproduction?

Summary. Workers of most eusocial Hymenoptera can produce sons after queen loss, which (posthumously) benefits the queen and increases worker inclusive fitness. However, the evolutionary loss of worker ovaries has occurred in several lineages, while workers in other taxa may be infertile despite having ovaries. Workers of Atta leafcutter ants only lay trophic eggs in queenright colonies. Although Atta colonies are commonly kept at universities, museums, and zoos, no reports of worker sons in orphaned colonies exist, suggesting that Atta workers are infertile. To explicitly test this, we created eleven orphaned laboratory nests of Atta cephalotes, A. sexdens, and A. colombica, and maintained them for 3–6 months after queen loss. Eight colonies did not produce any brood, but three nests produced 1–4 worker-derived male larvae and pupae. Microsatellite genotyping indicated that these were worker sons. However, all males were tiny (3.5–9 mm long) compared to normal queen sons (16 mm long), and would almost certainly be unable to mate. We also found reproductive eggs, but most of these had no yolk and were thus inviable. We conclude that Atta workers are not completely infertile, but that worker fertility is low compared to the sister genus Acromyrmex, where workers routinely produce normally-size males after queen loss in the laboratory. We hypothesize that worker reproduction in orphaned Atta field colonies is almost never successful because the last workers die before their sons can be raised to adulthood, but that the importance of worker-laid trophic eggs for queen feeding has precluded the evolutionary loss of worker ovaries. Received 17 January 2005; revised 12 September 2005; accepted 3 October 2005.     

Self-restraint and sterility in workers of Acromyrmex and Atta leafcutter ants

Summary. Queens of leafcutter ants (Acromyrmex and Atta) are highly multiply mated, resulting in a potential queenworker and worker-worker conflict over who should produce the males in the colony. We studied whether this conflict is expressed, by determining the amount of reproductive egg-laying by workers in queenright colonies of Acromyrmex echinatior, Acromyrmex octospinosus, Atta cephalotes, and Atta sexdens through ovary dissections. Worker sons are absent or rare in queenright Acromyrmex colonies, but can be produced in orphaned colonies. In Atta, most workers have rudimentary ovaries that never produce eggs, but a few (mostly small and medium workers that form a retinue around the queen) lay many trophic eggs for consumption by the queen. These eggs are large, flaccid, and lacking in yolk compared to queen-laid eggs, and appear to be always inviable. In Acromyrmex, many workers (especially young large workers) lay eggs that are similar in size to queen-laid eggs, but mostly with a reduced amount of yolk. Trophic eggs appear to be an important source of food for larvae in Acromyrmex (especially in Ac. echinatior), but not in Atta. Five (0.8) of 616 dissected Ac. echinatior workers but no Ac. octospinosus workers (n = 552), had ready-to-lay reproductive eggs. Old workers in all four species are incapable of laying eggs due to ovary resorption. We conclude that Atta workers are sterile, while Acromyrmex workers display reproductive self-restraint, possibly representing an earlier stage in the evolution towards worker sterility. Worker selfrestraint in Acromyrmex may be maintained by a queen or worker policing mechanism, but individual cost-benefit explanations may also apply.Received 1 March 2004; revised 28 June 2004; accepted 1 July 2004.