Lack of interspecific parasitism between the dwarf honeybees Apis andreniformis and Apis florea

The dwarf honeybees Apis florea and Apis andreniformis are sympatric in Southeast Asia. We examined undisturbed nests of both species finding that heterospecific workers are present in some nests at low frequency. This suggested that workers may enter heterospecific nests as a prelude to reproductive parasitism. To test this hypothesis, we created mixed-species colonies and determined the reproductive response of workers within them based on molecular markers. In queenless colonies, workers of both species activated their ovaries at equal frequency. However, the majority species, A. florea, had complete reproductive dominance over A. andreniformis, most likely because the A. florea workers recognised and removed heterospecific larvae. In queenright mixed-species colonies, workers responded to heterospecific signals of the presence of the queen and did not activate their ovaries. Thus, despite predictions from kin selection theory that workers would benefit from parasitising heterospecific nests, we find no evidence that selection has established a parasitic strategy in these sibling species.     

Worker reproductive parasitism and drift in the western honeybee Apis mellifera

When a honeybee (Apis spp.) colony loses its queen and is unable to rear a new one, some of the workers activate their ovaries and produce eggs. When a colony has a queen (i.e., it is queenright) almost all worker-laid eggs are eaten, but when hopelessly queenless, the workers become more tolerant of worker-laid eggs and rear some of them to adult drones. This increased tolerance renders a queenless colony vulnerable to worker reproductive parasitism, wherein unrelated workers enter the colony and lay eggs. Here, we show that the proportion of unrelated (non-natal) workers significantly decreases after an Apis mellifera colony becomes queenless. The remaining non-natal workers are as likely to have activated ovaries as natal workers, yet they produce more eggs than natal workers, resulting in significantly higher reproductive success for non-natal workers. In a second experiment, we provided queenless and queenright workers with a choice to remain in their own colony or to join a queenless or queenright colony nearby. The experiment was set up such that worker movement was unlikely to be due to simple orientation errors. Very few workers joined another colony, and there was no preference for workers to drift into or out of queenless or queenright colonies, in accordance with the proportion of non-natal workers declining significantly after becoming queenless in the first experiment.     

Worker policing and worker reproduction in Apis cerana

Workers of the Asian hive bee, Apis cerana, are shown to have relatively high rates of worker ovary activation. In colonies with an active queen and brood nest, 1-5% of workers have eggs in their ovarioles. When A. cerana colonies are dequeened, workers rapidly activate their ovaries. After 4 days 15% have activated ovaries and after 6 days, 40%. A cerana police worker-laid eggs in the same way that A. florea and A. mellifera do, but are perhaps slightly more tolerant of worker-laid eggs than the other species. Nevertheless, no worker's sons were detected in a sample of 652 pupal males sampled from 4 queenright colonies. A cerana continue to police worker-laid eggs, even after worker oviposition has commenced in a queenless colony.     

Differential reproductive success among subfamilies in queenless honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.) colonies

With very rare exceptions, queenright worker honeybees (Apis mellifera L.) forego personal reproduction and suppress reproduction by other workers, preferring to rear the queens sons. This is in stark contrast to colonies that have lost their queen and have failed to rear a replacement. Under these conditions workers activate their ovaries and lay many eggs that develop parthenogenetically into a final brood of males (drones) before the colony perishes. Interestingly, not all workers contribute equally to this final generation of drones in queenless colonies. Some subfamilies (workers that share the same father) contribute a disproportionately greater number of offspring than other subfamilies. Here we explore some of the mechanisms behind this reproductive competition among subfamilies. We determined the relative contribution of different subfamilies present in colonies to laying workers, eggs, larvae and pupae by genotyping samples of all life stages using a total of eight microsatellite loci. Our colonies were headed by free-mated queens and comprised 8–17 subfamilies and therefore differed significantly from colonies used in an earlier study investigating the same phenomena where colonies comprised an artificially low number of subfamilies. We show that, first, subfamilies vary in the speed with which they activate their ovaries after queen-loss and, second, that the survival of eggs to the larval stage is unequal among subfamilies suggesting that some subfamilies lay eggs that are more acceptable than others. However, there is no statistically significant difference among subfamilies in the survival of larvae to pupae, indicating that ovary activation and egg survival are the critical components to reproductive competition among subfamilies of queenless honeybee workers.Communicated by R. Page     

Worker policing in the bee Apis florea

Apis florea is a single-combed, open-nesting, dwarf honeybee indigenous to Asia. In common with other species of this genus, A. florea is highly polyandrous, and is therefore predicted to curtail worker reproduction by mutual policing mechanisms that keep worker reproduction at an extremely low level. Policing mechanisms could involve destruction of workers' eggs or offspring, or aggression toward those workers that are reproductively active. We show that in A. florea, worker-laid eggs are eliminated approximately twice as fast as queen-laid eggs, indicating that A. florea uses oophagy of worker-laid eggs as a mechanism of worker policing. Genetic analysis of four colonies indicated that all males produced were sons of queens, not workers. Dissections of 800 workers, from four colonies, did not reveal any significant levels of ovary activation. These results suggest that worker policing is an effective component of the mechanisms that maintain worker sterility in this species. Furthermore, they suggest that worker policing via oophagy of worker-laid eggs is pleisiomorphic for the genus.     

Social parasitism of queens and workers in the Cape honeybee (<Emphasis Type="Italic">Apis mellifera capensis</Emphasis>)

Workers of a queenless honeybee colony can requeen the colony by raising a new queen from a young worker brood laid by the old queen. If this process fails, the colony becomes hopelessly queenless and workers activate their ovaries to lay eggs themselves. Laying Cape honeybee workers (Apis mellifera capensis) produce female offspring as an additional pathway for requeening. We tested the frequency of successful requeening in ten hopelessly queenless colonies. DNA genotyping revealed that only 8% of all queens reared in hopelessly queenless colonies were the offspring of native laying worker offspring. The vast majority of queens resulted from parasitic takeovers by foreign queens (27%) and invading parasitic workers (19%). This shows that hopelessly queenless colonies typically die due to parasitic takeovers and that the parasitic laying workers are an important life history strategy more frequently used than in providing a native queen to rescue the colony. Parasitism by foreign queens, which might enter colonies alone or accompanied by only a small worker force is much more frequent than previously considered and constitutes an additional life history strategy in Cape honeybees.     

Reproductive conflict in honey bees: a stalemate of worker egg-laying and policing

 Using electrophoretic markers, eggs laid by workers were identified in honey bee (Apis mellifera) colonies with a queen. Based on extrapolation, these represented about 7% of the unfertilized (male) eggs laid in the colonies. A very small proportion of workers (of the order of 0.01%) lay these eggs. Worker-laid eggs are rapidly removed, so that very few sons of workers are reared. Thus the reproductive cooperation in bee colonies is maintained by ongoing antagonistic interactions among the members of the colony, with worker laying and egg removal policing by other workers being relatively common. Received: 24 November 1995/Accepted after revision: 25 May 1996     

Changes in the cuticular hydrocarbons of incipient reproductives correlate with triggering of worker policing in the bulldog ant Myrmecia gulosa

In social insects, conflicts over male parentage can be resolved by worker policing. However, the evolution of policing behavior is constrained by the ability of individuals to identify reproductive nestmates, or their eggs. We investigated the occurrence of worker policing and its underlying chemical communication in the bulldog ant Myrmecia gulosa. Although workers have functional ovaries and can lay male-destined eggs, they do not reproduce in queenright colonies. To determine if their sterility is a consequence of worker policing, we experimentally induced worker reproduction in the presence of a queen. Some individuals were seized and immobilized by nestmates, and sometimes killed as a consequence. Although the ovarian development of immobilized individuals was variable, their cuticular hydrocarbon profiles were intermediate between reproductive and nonreproductive workers, indicating they were in the process of starting to reproduce. Approximately 29% of these incipient reproductive workers were successfully policed. To test for policing on eggs, we transferred viable worker eggs to queenright colonies and monitored their acceptance. Furthermore, we compared the surface hydrocarbons of the different types of eggs to determine whether these chemicals could be involved in egg recognition. We found that although there were differences in hydrocarbon profiles and discrimination between queen and worker-laid eggs, viable eggs were not destroyed. Our results strongly support the idea that cuticular hydrocarbons are involved in the policing of reproductive workers. A low level of worker policing appears sufficient to select for self-restraint in workers when few fitness benefits are gained by selfish reproduction. Policing of eggs may thus be unnecessary.     

Social regulation of ovary activation in ’anarchistic’ honey-bees (Apis mellifera)

Honey-bee (Apis mellifera) colonies exhibit extreme reproductive division of labour. Workers almost always have inactive ovaries and the queen monopolises egg laying. Although extremely rare, ’anarchistic’ colonies exist in which workers produce male offspring despite the presence of the queen. By comparing the rates of ovary activation in anarchistic and wild-type bees fostered to host colonies of different genotype (i.e. anarchist and non-anarchist) and queen status (i.e. queenless and queenright), we investigated the factors involved in inhibiting ovary activation. Fostered anarchist workers always had a higher level of ovary development than fostered wild-type bees in both anarchist and non-anarchist host colonies. Fostered workers of both genotypes had more active ovaries in anarchistic than in wild-type hosts. Fostered workers of both strains also had more active ovaries in queenless than in queenright hosts. The results suggest that selection for worker reproduction in the anarchistic line has both reduced the effects of brood and queen pheromones on worker ovary inhibition and increased the likelihood that workers of the anarchistic line will develop ovaries compared to wild-type workers. Received: 14 June 2000 / Revised: 26 September 2000 / Accepted: 7 October 2000     

Colony level and within colony level selection in honeybees

Summary Although honeybee workers are usually infertile, in queenless colonies some workers can develop ovaries and produce offspring. Therefore the classical Darwinian fitness of workers is not zero. Experimental studies in the Cape honey bee (Apis mellifera capensis) reveal a huge genetic variation for individual fitness of workers. The present study with a one locus, two allele model for reproductive dominance of workers shows that a balanced system between colony level and individual within colony selection plausibly explains the phenomenon of a high genetic variance of worker fitness. In particular, a frequent occurrence of queenless colonies in the population leads to stable polymorphic equilibria. Also the multiple mating system of the honey bee queen supports the propagation of alleles causing reproductive dominance of workers.     

Worker reproduction in honey-bees (Apis) and the anarchic syndrome: a review

Honey-bees, Apis, are an important model system for investigating the evolution and maintenance of worker sterility. The queen is the main reproductive in a colony. Workers cannot mate, but they can lay unfertilized eggs, which develop into males if reared. Worker reproduction, while common in queenless colonies, is rare in queenright colonies, despite the fact that workers are more related to their own sons than to those of the queen. Evidence that worker sterility is enforced by 'worker policing' is reviewed and worker policing is shown to be widespread in Apis. We then discuss a rare behavioural syndrome, 'anarchy', in which substantial worker production of males occurs in queenright colonies. The level of worker reproduction in these anarchic colonies is far greater than in a normal queenright honey-bee colony. Anarchy is a counterstrategy against worker policing and an example of a 'cheating' strategy invading a cooperative system.     

Different policing rates of eggs laid by queenright and queenless anarchistic honey-bee workers (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.)

Worker-reproduction is rare in queenright honey-bee colonies. When workers do lay eggs, their eggs are normally eaten by other workers presumably because they lack the queen's egg-marking signal. Workers use the absence of this queen signal to enforce the queen's reproductive monopoly by policing any worker-laid eggs. In contrast, in anarchistic colonies, the majority of the males arise from worker-laid eggs. Anarchistic worker-laid eggs escape policing because workers perceive anarchistic eggs as queen-laid. However, in this study, we show that eggs laid by queenless anarchistic workers do not escape policing and have very similar removal rates to worker-laid eggs from queenless wild-type (i.e. non-anarchistic) colonies. This suggests that, under queenless conditions, eggs laid by anarchistic workers lose their chemical protection and are therefore no longer perceived as queen-laid. Hence, the egg-marking signal seems to be only applied to eggs when queen and brood are present. This suggests that in the absence of queen and brood, the biosynthetic pathway that produces the egg-marking signal is switched off.Communicated by L. Keller     

Reproductive competition in queenless honey bee colonies (Apis mellifera L.)

Previously we reported that there are subfamily differences in drone production in queenless honey bee colonies, but these biases are not always explained by subfamily differences in oviposition behavior. Here we determine whether these puzzling results are best explained by either inadequate sampling of the laying worker population or reproductive conflict among workers resulting in differential treatment of eggs and larvae. Using colonies composed of workers from electrophoretically distinct subfamilies, we collected samples of adult bees engaged in the following behavior: true egg laying, false egg laying, indeterminate egg laying, egg cannibalism, or nursing (contact with larvae). We also collected samples of drone brood at four different ages: 0 to 2.5-h-old eggs, 0 to 24-h-old eggs, 3 to 8-day-old larvae, and 9 to 14-day-old larvae and pupae. Allozyme analyses revealed significant subfamily differences in the likelihood of exhibiting egg laying, egg cannibalism, and nursing behavior, as well as significant subfamily differences in drone production. There were no subfamily differences among the different types of laying workers collected from each colony, suggesting that discrepancies between subfamily biases in egg-laying behavior and drone production are not due to inadequate sampling of the laying worker population. Subfamily biases in drone brood production within a colony changed significantly with brood age. Laying workers had significantly more developed ovaries than either egg cannibals or nurses, establishing a physiological correlate for the observed behavioral genetic differences. These results suggest there is reproductive conflict among subfamilies and individuals within queenless colonies of honey bees. The implications of these results for the evolution of reproductive conflict, in both queenright and queenless contexts, are discussed.     

Evolution of worker sterility in honey-bees (Apis mellifera): how anarchistic workers evade policing by laying eggs that have low removal rates

In anarchistic honey-bee colonies, many workers’ sons are reared despite the presence of the queen. Worker-laid eggs are normally eaten by other workers in queenright colonies. Workers are thought to discriminate between queen-laid and worker-laid eggs by the presence or absence of a queen-produced egg-marking pheromone. This study compared the survival of three classes of eggs (worker-laid eggs from anarchistic colonies, worker-laid eggs from non-anarchistic queenless colonies, and queen-laid eggs) in both queenright normal colonies and queenright anarchistic colonies, in order to test the hypothesis that anarchistic workers evade policing by laying more acceptable eggs. As expected, few worker-laid eggs from non-anarchistic colonies survived more than 2 h. In contrast, worker-laid eggs from anarchistic colonies had much greater acceptability, which in some trials equalled the acceptability of queen-laid eggs. Anarchistic colonies were generally less discriminatory than normal queenright colonies towards worker-laid eggs, whether these originated from anarchistic colonies or normal queenless colonies. This indicates that the egg-removal aspect of the anarchistic syndrome involves both worker laying of eggs with greater acceptability and reduced discriminatory behaviour of policing workers. Received: 19 July 1999 / Received in revised form: 3 November 1999 / Accepted: 20 November 1999     

“Selfish worker policing” controls reproduction in a Temnothorax ant

Animal societies, including those of humans, are under constant threat by selfish individuals, who attempt to enforce their own interests at the cost of the group. In the societies of bees, wasps, and ants, such individual selfishness can be prevented by “policing,” whereby workers or queens impede the reproduction of other individuals by aggression, immobilization, or egg eating. In this study, we report on a particular kind of reproduction control in the ant Temnothorax unifasciatus, which can be considered as a selfish act itself. We experimentally induced workers to lay eggs by dividing several colonies into two halves, one with and one without a queen. In queenless colonies, workers established rank orders by aggression and several top-ranking workers started to reproduce. Upon reunification, egg-laying workers mostly stopped behaving aggressively. They were neither attacked by the queen nor by random workers, but instead received infrequent, nondestructive, targeted aggression from a few workers, most of which became fertile when the queen was later removed. The introduction of differentially stained worker-laid and queen-laid eggs in queenright fragments did not lead to a selective removal of worker-laid eggs. Hence, there appears to be no collective worker policing in T. unifasciatus. Instead, reproduction appears to be controlled mostly through a few attacks from high-ranking workers, which, in this way, might attempt to selfishly increase their chances of future reproduction.     

Policing in queenless ponerine ants

Potential reproductive conflicts are common in insect societies. One process that can reduce or suppress conflict is policing. We review worker and "queen" policing in queenless ponerine ants. Queenless ants are an important model system for the study of intracolony reproductive conflict. Policing is widespread in queenless ants because workers are totipotent, so that additional potential conflicts occur in comparison to species where workers cannot mate, and these additional conflicts are frequently reduced by policing. Policing is more diverse than suggested by the examples known in other social insects. In almost all species of social Hymenoptera it can include preventing workers from reproducing by killing worker-laid eggs, but in queenless ants it can additionally include immobilisation or mutilation of workers attempting to reproduce by replacing the gamergate (i.e. mated worker with a queen-like role) or by becoming an extra gamergate. Policing by both workers and by the gamergate are important. Policing can be facultative. Depending on the age of the gamergate, workers can prevent her replacement by immobilising challenging workers or they can favour replacement by immobilising the gamergate. The initial definition of policing was inspired by species in which workers retain ovaries but cannot mate. We broaden the definition to include species, such as queenless ants, where females are totipotent, thereby including not only conflict over male production but also over gamergate replacement and gamergate number. Finally, we compare policing with punishment and dominance hierarchy. Policing is not always punishment and it does not necessarily entail dominance relationships.     

Role of esters in egg removal behaviour in honeybee (Apis mellifera) colonies

In queen-right honeybee colonies workers detect and eat the vast majority of worker-laid eggs, a behaviour known as worker policing. However, if a colony becomes permanently queen-less then up to 25% of the worker population develops their ovaries and lay eggs, which are normally reared into a final batch of males. Ovary development in workers is accompanied by changes in the chemical secretion of the Dufour's gland with the production of queen-like esters. We show that ester production increases with the period that the colony is queen-less. The increased ester production also corresponds to an increase in persistence of worker-laid eggs in queen-right colonies, since the esters somehow mask the eggs true identity. However, in a rare queen-less colony phenotype, workers always eat eggs indiscriminately. We found that the egg-laying workers in these colonies were unusual in that they were unable to produce esters. This apparently maladaptive egg eating behaviour is also seen in queen-less colonies prior to the appearance of egg-laying workers, a period when esters are also absent. However, the indiscriminate egg eating behaviour stops with the appearance of ester-producing egg-laying workers. These observations suggest that esters are providing some contextual information, which affects the egg eating behaviour of the workers.     

Levels of polyandry and intracolonial genetic relationships in Apis florea

DNA was extracted from worker and drone pupae of each of five colonies of the dwarf honey bee Apis florea. Polymerase chain reactions (PCR) were conducted on DNA extracts using five sets of primers known to amplify microsatellite loci in A. mellifera. Based on microsatellite allele distributions, queens of the five colonies mated with at least 5–14 drones. This is up to 3 times previous maximum estimates obtained from sperm counts. The discrepancy between sperm count and microsatellite estimates of the number of matings in A. florea suggests that despite direct injection of semen into the spermatheacal duct, either A. florea drones inject only a small proportion of their semen, or queens are able to rapidly expel excess semen after mating. A model of sexual selection (first proposed by Koeniger and Koeniger) is discussed in which males attempt to gain reproductive dominance by increasing ejaculate volume and direct injection of spermatozoa into the spermatheca, while queens attempt to maintain polyandry by retaining only a small fraction of each male's ejaculate. It is shown, at least in this limited sample, that the effective number of matings is lower in A. florea than in A. mellifera.     

Reproductive plasticity in bumblebee workers (<Emphasis Type="Italic">Bombus terrestris</Emphasis>)—reversion from fertility to sterility under queen influence

Worker sterility in the bumblebee Bombus terrestris is conditional and is linked to the social development of the colony. Workers refrain from reproducing or overtly challenging the queen until gyne production has initiated, at the so-called competition point (CP). It is not known whether this behavior is hard-wired or workers show reproductive plasticity. It also remains unclear whether worker reproductive decision is under queen and/or worker control. In this study, we tested worker reproductive plasticity in an attempt to assess whether and under which conditions worker sterility/fertility are reversible. We introduced egg-laying workers into colonies with different social structures for 1 week then monitored their reproductive status. We revealed a remarkable reproductive plasticity in the introduced workers that was social-condition-dependent. In the presence of a pre-CP queen, the introduced workers reverted to sterility, whereas in the presence of a post-CP queen, such workers remained egg-layer. Reversion to sterility does not occur when direct contact with the queen is prevented, as the introduced workers remained egg-layer in the queenright colonies with a confined queen. Egg-laying workers that were introduced into queenless colonies mostly maintained their fertility regardless of colony social phase. This shows that worker transition from cooperative to selfish behavior is reversible depending on the social context.     

Intracolonial behavioral variation in worker oviposition,oophagy, and larval care in queenless honey bee colonies

Summary Two experiments were performed to determine whether worker reproduction in queenless honey bee colonies is influenced by colony genetic structure. In Experiment 1, allozyme analyses of workers and worker-derived drone larvae revealed that in half the colonies, there were genotypic differences in worker egg-laying behavior (presumed to involve actual oviposition), but biases in drone production were not always consistent with biases in egg-laying behavior. In Experiment 2, allozyme analyses again revealed intracolonial differences in egg-laying behavior and in behavior patterns thought to involve oophagy and larval care. Data support the hypothesis of a genetic influence on this intracolonial behavioral variation. Differences in the genotypic distributions of worker-derived drones relative to workers engaged in oviposition behavior in queenless colonies may be a consequence of genetic variability for egg production or for treatment of eggs and larvae (possibly coupled with kin recognition), or both. Offprint requests to: G.E. Robinson