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     

Retinue attraction and ovary activation: responses of wild type and anarchistic honey bees (Apis mellifera) to queen and brood pheromones

In most social insect colonies, workers do not attempt to lay eggs in the presence of a queen. However, in the honey bee (Apis mellifera), a rare phenotype occurs in which workers activate their ovaries and lay large numbers of male eggs despite the presence of a fecund queen. We examined the proximate mechanisms by which this ‘anarchistic’ behaviour is expressed. We tested the effects of brood and queen pheromones on retinue attraction and worker ovary activation using caged worker bees. We found no difference between the anarchistic and wild type queen pheromones in the retinue response elicited in either wild type or anarchistic workers. Further, we found that anarchistic queens produce a pheromone blend that is as effective at inhibiting ovary activation as the wild type queen pheromone. However, anarchistic workers are less inhibited by queen pheromones than their wild type counterparts, in a dose-dependent manner. These results show that the anarchistic phenomenon is not due to changes in the production of queen pheromones, but rather is due in part to a shift in the worker response to these queen-produced signals. In addition, we demonstrate the dose-dependent nature of the effect of queen pheromones on honey bee worker ovary activation.     

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 drift and egg dumping by queens in wild Bombus terrestris colonies

Wild bumblebee colonies are hard to find and often inaccessible, so there have been few studies of the genetic structure of bumblebees within natural colonies, and hence, it is not clear how frequently events such as worker reproduction, worker drift and queen usurpation take place. This study aimed to quantify the occurrence of natal-worker reproduction, worker drift and drifter reproduction within 14 wild colonies of Bombus terrestris in Central Scotland. Four unlinked microsatellites were used to identify patterns of relatedness of the colonies’ adults and broods. In colonies with queens (queenright colonies), worker reproduction accounted for just 0.83 % of males, increasing to 12.11 % in queenless colonies. Four colonies contained a total of six workers which were not daughters of the queen, and were assumed to be drifters, and four male offspring of drifters. Drifting is clearly not common and results in few drifter offspring overall, although drifters produced approximately seven times more offspring per capita than workers that remained in their natal colony. Unexpectedly, two colonies contained clusters of sister workers and juvenile offspring that were not sisters to the rest of the adults or brood found in the colonies, demonstrating probable egg dumping by queens. A third colony contained a queen which was not a sister or daughter to the other bees in the colony. Although usurping of bumblebee colonies by queens in early season is well documented, this appears to be the first record of egg dumping, and it remains unclear whether it is being carried out by old queens or newly mated young queens.     

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.     

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.     

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.     

Queen and young larval pheromones impact nursing and reproductive physiology of honey bee (Apis mellifera) workers

Several insect pheromones are multifunctional and have both releaser and primer effects. In honey bees (Apis mellifera), the queen mandibular pheromone (QMP) and e-beta-ocimene (eβ), emitted by young worker larvae, have such dual effects. There is increasing evidence that these multifunctional pheromones profoundly shape honey bee colony dynamics by influencing cooperative brood care, a fundamental aspect of eusocial insect behavior. Both QMP and eβ have been shown to affect worker physiology and behavior, but it has not yet been determined if these two key pheromones have interactive effects on hypopharyngeal gland (HPG) development, actively used in caring of larvae, and ovary activation, a component of worker reproductive physiology. Experimental results demonstrate that both QMP and eβ significantly suppress ovary activation compared to controls but that the larval pheromone is more effective than QMP. The underlying reproductive anatomy (total ovarioles) of workers influenced HPG development and ovary activation, so that worker bees with more ovarioles were less responsive to suppression of ovary activation by QMP. These bees were more likely to develop their HPG and have activated ovaries in the presence of eβ, providing additional links between nursing and reproductive physiology in support of the reproductive ground plan hypothesis.     

The reproductive dilemmas of queenless red dwarf honeybee (Apis florea) workers

Honeybee (Apis) workers cannot mate, but retain functional ovaries. When colonies have lost their queen, many young workers begin to activate their ovaries and lay eggs. Some of these eggs are reared, but most are not and are presumably eaten by other workers (worker policing). Here we explore some of the factors affecting the reproductive success of queenless workers of the red dwarf honeybee Apis florea. Over a 2-year period we collected 40 wild colonies and removed their queens. Only two colonies remained at their translocated site long enough to rear males to pupation while all the others absconded. Absconding usually occurred after worker policing had ceased, as evidenced by the appearance of larvae. Dissections of workers from eight colonies showed that in A. florea, 6% of workers have activated ovaries after 4 days of queenlessness, and that 33% of workers have activated ovaries after 3 weeks. Worker-laid eggs may appear in nests within 4 days and larvae soon after, but this is highly variable. As with Apis mellifera, we found evidence of unequal reproductive success among queenless workers of A. florea. In the two colonies that reared males to pupation and which we studied with microsatellites, some subfamilies had much higher proportions of workers with activated ovaries than others. The significance of absconding and internest reproductive parasitism to the alternative reproductive strategies of queenless A. florea workers is discussed.     

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     

Higher removal rate of eggs laid by anarchistic queens—a cost of anarchy?

Anarchy, where honeybee workers produce males in the presence of a queen, is extremely rare in natural honeybee populations, suggesting that there are colony-level costs associated with being anarchistic. Yet, no significant costs have yet been identified. A possible reason for this may be that researchers have only focused on the behaviour of anarchistic workers, which have been shown to perform worker-tasks as do wild-type workers. Possibly, therefore, costs associated with anarchy should be sought in anarchistic queens and not workers. A potential cost could be a lower survival rate of eggs laid by anarchistic queens perhaps because their egg-marking signal is not as clear as that of a wild-type queen. In this paper, we determined the removal rate of eggs laid by anarchistic queens in standard worker-policing bioassays. Our results show that eggs laid by anarchistic queens are removed at a higher rate than eggs laid by wild-type queens. This does not seem to be due to differences in hydrocarbons found on the surface of eggs, as both egg types showed the same alkanes and alkenes in similar proportions. We postulate that higher removal rates of queen-laid eggs due to recognition errors may be one reason that anarchy is rare in natural honeybee populations.     

Reproductive conflicts and egg discrimination in a socially polymorphic ant

The ability to discriminate against competitors shapes cooperation and conflicts in all forms of social life. In insect societies, workers may detect and destroy eggs laid by other workers or by foreign queens, which can contribute to regulate reproductive conflicts among workers and queens. Variation in colony kin structure affects the magnitude of these conflicts and the diversity of cues used for discrimination, but the impact of the number of queens per colony on the ability of workers to discriminate between eggs of diverse origin has so far not been investigated. Here, we examined whether workers from the socially polymorphic ant Formica selysi distinguished eggs laid by nestmate workers from eggs laid by nestmate queens, as well as eggs laid by foreign queens from eggs laid by nestmate queens. Workers from single- and multiple-queen colonies discriminated worker-laid from queen-laid eggs, and eliminated the former. This suggests that workers collectively police each other in order to limit the colony-level costs of worker reproduction and not because of relatedness differences towards queens’ and workers’ sons. Workers from single-queen colonies discriminated eggs laid by foreign queens of the same social structure from eggs laid by nestmate queens. In contrast, workers from multiple-queen colonies did not make this distinction, possibly because cues on workers or eggs are more diverse. Overall, these data indicate that the ability of F. selysi workers to discriminate eggs is sufficient to restrain worker reproduction but does not permit discrimination between matrilines in multiple-queen colonies.     

Social influences on body size and developmental time in the bumblebee Bombus terrestris

In many social insects, including bumblebees, the division of labor between workers relates to body size, but little is known about the factors influencing larval development and final size. We confirmed and extend the evidence that in the bumblebee Bombus terrestris the adult bee body size is positively correlated with colony age. We next performed cross-fostering experiments in which eggs were switched between incipient (before worker emergence) and later stage colonies with workers. The introduced eggs developed into adults similar in size to their unrelated nestmates and not to their same-age full sisters developing in their mother colony. Detailed observations revealed that brood tending by the queen decreases, but does not cease, in young colonies with workers. We next showed that both worker number and the queen presence influenced the final size of the developing brood, but only the queen influence was mediated by shortening developmental time. In colonies separated by a queen excluder, brood developmental time was shorter in the queenright compartment. These findings suggest that differences in body size are regulated by the brood interactions with the queen and workers, and not by factors inside the eggs that could vary along with colony development. Finally, we developed a model showing that the typical increase in worker number and the decrease in brood contact with the queen can account for the typical increase in body size. Similar self-organized social regulation of brood development may contribute to the optimization of growth and reproduction in additional social insects.     

Colony take-over and brood survival in temporary social parasites of the ant genus Formica

Parasites reduce host fitness, and so instigate counter adaptations by their hosts. In temporary social parasitism, usurpers must not only enter the colony unharmed, but also have their eggs reared by the host workers. We introduced parasitic Formica lugubris and Formica aquilonia queens into queen right and orphaned fragments of three host species, Formica cinerea, Formica picea and Formica fusca, and show that workers of all three host species kill over 40 % of the introduced queens within 10 days, regardless of the presence/absence of a resident queen, and parasite species. More parasite queens died in F. cinerea than in F. picea and F. fusca. There were no major differences in survival between the parasite species (except that F. lugubris survived longer than F. aquilonia in F. fusca colonies compared to F. picea colonies), but parasite queens survived longer in orphaned than in queen right fragments of F. fusca. Experimental introduction of parasite (F. aquilonia) eggs into orphaned colonies of F. fusca showed that none of the parasite eggs were reared until pupation; whereas on average, 12 % of the con-specific hetero-colonial eggs introduced in the same manner were reared until pupation. In all colonies that received parasite brood, all offspring consisted of worker-laid males, whereas the corresponding value was 50 % for colonies that received con-specific hetero-colonial brood. Thus, when the risks of entering host colonies and brood failure are combined, the rate of successful colony take-over is very low. Moreover, the host workers can to some extent alleviate the costs of parasitism by producing a final batch of own offspring.     

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     

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.     

Social regulation of egg-laying by queenless honeybee workers (Apis mellifera L.)

Summary If a honeybee (Apis mellifera L.) colony loses its queen and remains queenless, a small percentage of the workers will develop into egg-layers and subsequently will produce males. The process of differentiation into laying and non-laying workers is accompanied by a great deal of aggression within the colony. In this study, I tried to establish the relationship between the potential to become egg-layers and behavioural differences at the individual level. To eight observation hives, I introduced 200 individually marked workers of similar age and observed their behavioural differentiation during a queenless period. Shortly after the first egg-laying worker appeared, the marked workers were dissected in order to determine their level of ovarian activation. The future laying-workers seemed to be slightly more involved in the rearing of new queens than other workers. As reported by other authors, aggression was mostly directed towards workers with activated ovaries. Only a very small number of aggressive workers were aggressive; on an average, slightly fewer of these marked workers had activated ovaries. Aggression resulted in physical damage in only a small number of cases. The possible disadvantage of aggression for the bees under attack is discussed, as well as the possible benefit for the aggressors. Evidence is presented that the most detrimental effect of aggression for bees under attack is the fact that they lose considerable quantities of food through trophallaxis to other workers. Workers with activated ovaries withdrew inside empty cells significantly more often than other workers, possibly to avoid attacks. Aggressive workers were (almost) never seen to receive food from the bees they attacked. It is suggested that by preventing other workers from becoming egg-layers, aggressors increase their own chances of future reproduction.     

Reproduction by worker honey bees (Apis mellifera L.)

Summary Genetic markers were used to study the reproductive behavior of worker honey bees. Five experiments were conducted that demonstrate the significance of worker reproduction. Biases were found in the egg-laying success of workers belonging to different subfamilies within queenless colonies, however, members of all subfamilies laid eggs. These biases were probably not a consequence of direct reproductive competition among subfamily members but most likely represent genetic variability for the timing of the onset of oviposition. Workers preferentially oviposit in drone-sized cells, demonstrating a caste-specific adaptation for oviposition behavior. Drone brood production is highly synchronous within colonies and can result in the production of more than 6000 drones before colonies die. Workers reproduce in queenright colonies but at a very low frequency.     

Behavioral regulation of genetic caste determination in a Pogonomyrmex population with dependent lineages

The fate of a social insect colony is partially determined by its ability to allocate individuals to the caste most appropriate for the requirements for growth, maintenance, and reproduction. In pairs of dependent lineages of Pogonomyrmex barbatus, the allocation of individuals to the queen or worker caste is constrained by genotype, a system known as genetic caste determination (GCD). In mature GCD colonies, interlineage female eggs develop into sterile workers, while intralineage eggs become reproductively capable queens. Although the population-level consequences of this system have been intensively studied, the proximate mechanisms for GCD remain unknown. To elucidate these mechanisms, we brought newly mated queens into the laboratory and allowed them to establish colonies, nearly half of which unexpectedly produced virgin queens only seven months after colony founding. We genotyped eggs, workers, and the virgin queens from these colonies. Our results showed that queens in young colonies produce both interlineage and intralineage eggs, demonstrating that queens of GCD colonies indiscriminately use sperm of at least two lineages to fertilize their eggs. Intralineage eggs were more frequent in colonies producing virgin queens. These findings suggest that intralineage eggs are predetermined to become queens and that workers may cull these eggs when colonies are not producing queens. Virgin queens produced by young GCD colonies were smaller than field-caught virgin queens, and often had developmental problems. Hence, they are probably nonfunctional and represent an intense resource drain for developing colonies, not a contribution to colony fitness.     

Sex determination and the evolution of polyandry in honey bees (Apis mellifera)

Many hypotheses attempt to explain why queens of social insects mate multiply. We tested the sex locus hypothesis for the evolution of polyandry in honey bees (Apis mellifera). A queen may produce infertile, diploid males that reduce the viability of worker brood and, presumably, adversely affect colony fitness. Polyandry reduces the variance in diploid male production within a colony and may increase queen fitness if there are non-linear costs associated with brood viability, specifically if the relationship between brood viability and colony fitness is concave. We instrumentally inseminated queens with three of their own brothers to vary brood viability from 50% to 100% among colonies. We measured the colonies during three stages of their development: (1) colony initiation and growth, (2) winter survival, and (3) spring reproduction. We found significant relationships between brood viability and most colony measures during the growth phase of colonies, but the data were too variable to distinguish significant non-linear effects. However, there was a significant step function of brood viability on winter survival, such that all colonies above 72% brood viability survived the winter but only 37.5% of the colonies below 72% viability survived. We discuss the significance of this and other "genetic diversity" hypotheses for the evolution of polyandry.