Worker-laid Eggs Research Articles

Differences between queen- and worker-laid male eggs of the honey bee (Apis mellifera)

In this study, we investigated whether differences in the reproductive biology of honey bee (Apis mellifera) queens and laying workers are reflected in their eggs. We first tested the capacity of queen- and worker-laid male eggs to withstand dry conditions, by incubating samples at 30.0, 74.9, and 98.7% relative humidity. We found that worker-laid eggs were more sensitive to desiccation. Secondly, we measured the weight and quantities of vitellin, total protein, lipid, glycogen, and free carbohydrate in queen- and workerlaid eggs. Although worker-laid eggs were found to be heavier than queen-laid eggs in two of the four replicates, no systematic differences were found regarding nutrient content. Finally, we compared the duration of embryo development in the two egg types. Worker-laid eggs developed more slowly than queen-laid eggs in two out of three replicates, suggesting that they may only be partly mature at the moment they are laid. Possible causes and consequences of the observed differences are discussed.

The evolution of extreme altruism and inequality in insect societies

In eusocial organisms, some individuals specialize in reproduction and others in altruistic helping. The evolution of eusociality is, therefore, also the evolution of remarkable inequality. For example, a colony of honeybees (Apis mellifera) may contain 50 000 females all of whom can lay eggs. But 100 per cent of the females and 99.9 per cent of the males are offspring of the queen. How did such extremes evolve? Phylogenetic analyses show that high relatedness was almost certainly necessary for the origin of eusociality. However, even the highest family levels of kinship are insufficient to cause the extreme inequality seen in e.g. honeybees via 'voluntary altruism'. 'Enforced altruism' is needed, i.e. social pressures that deter individuals from attempting to reproduce. Coercion acts at two stages in an individual's life cycle. Queens are typically larger so larvae can be coerced into developing into workers by being given less food. Workers are coerced into working by 'policing', in which workers or the queen eat worker-laid eggs or aggress fertile workers. In some cases, individuals rebel, such as when stingless bee larvae develop into dwarf queens. The incentive to rebel is strong as an individual is the most closely related to its own offspring. However, because individuals gain inclusive fitness by rearing relatives, there is also a strong incentive to 'acquiesce' to social coercion. In a queenright honeybee colony, the policing of worker-laid eggs is very effective, which results in most workers working instead of attempting to reproduce. Thus, extreme altruism is due to both kinship and coercion. Altruism is frequently seen as a Darwinian puzzle but was not a puzzle that troubled Darwin. Darwin saw his difficulty in explaining how individuals that did not reproduce could evolve, given that natural selection was based on the accumulation of small heritable changes. The recognition that altruism is an evolutionary puzzle, and the solution was to wait another 100 years for William Hamilton.

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.

Ant Queen Egg-Marking Signals: Matching Deceptive Laboratory Simplicity with Natural Complexity

BackgroundExperiments under controlled laboratory conditions can produce decisive evidence for testing biological hypotheses, provided they are representative of the more complex natural conditions. However, whether this requirement is fulfilled is seldom tested explicitly. Here we provide a lab/field comparison to investigate the identity of an egg-marking signal of ant queens. Our study was based on ant workers resolving conflict over male production by destroying each other's eggs, but leaving queen eggs unharmed. For this, the workers need a proximate cue to discriminate between the two egg types. Earlier correlative evidence indicated that, in the ant Pachycondyla inversa, the hydrocarbon 3,11-dimethylheptacosane (3,11-diMeC27) is more abundant on the surface of queen-laid eggs.MethodologyWe first tested the hypothesis that 3,11-diMeC27 functions as a queen egg-marking pheromone using laboratory-maintained colonies. We treated worker-laid eggs with synthetic 3,11-diMeC27 and found that they were significantly more accepted than sham-treated worker-laid eggs. However, we repeated the experiment with freshly collected field colonies and observed no effect of treating worker-laid eggs with 3,11-diMeC27, showing that this compound by itself is not the natural queen egg-marking pheromone. We subsequently investigated the overall differences of entire chemical profiles of eggs, and found that queen-laid eggs in field colonies are more distinct from worker-laid eggs than in lab colonies, have more variation in profiles, and have an excess of longer-chain hydrocarbons.ConclusionsOur results suggest that queen egg-marking signals are significantly affected by transfer to the laboratory, and that this change is possibly connected to reduced queen fertility as predicted by honest signaling theory. This change is reflected in the worker egg policing response under field and laboratory conditions.

Two independent mechanisms of egg recognition in worker Formica fusca ants

Informational constraints can be an important limitation on the accuracy of recognition. One potential constraint is the use of recognition information from the same sources in multiple discriminatory contexts. Worker wood ants, Formica fusca, discriminate eggs based on their maternal sources of origin in two main contexts: recognition of eggs laid by nestmate versus non-nestmate queens and recognition of worker-laid versus queen-laid eggs. We manipulated the experience of F. fusca workers in laboratory colonies to both worker-laid and queen-laid eggs by transferring eggs between colonies in order to investigate whether these two contexts of egg discrimination are independent. Experience of non-nestmate queen-laid eggs significantly increased worker acceptance of both familiar (18% accepted) and unfamiliar (10%) queen-laid eggs compared to control workers without experience of eggs other than those laid by their own colony’s queen (2%). In contrast, worker acceptance of worker-laid eggs was not affected by variation in the egg experience of workers (14% in workers from control colonies exposed only to eggs from their own colony’s queen versus 19% and 17% in workers from colonies which had received eggs laid by either a non-nestmate queen or nestmate workers, respectively). Our results suggest that these two recognition contexts do not strongly constrain each other and are different in their ontogeny. In particular, worker-laid eggs are universally discriminated against by workers from colonies with a queen whatever the egg experience of the workers, while non-nestmate queen-laid eggs are strongly discriminated against only by workers without experience of eggs laid by more than one queen.

Nestmate recognition by guards of the Asian hive bee Apis cerana

When a honey bee colony becomes queenless and broodless its only reproductive option is for some of its workers to produce sons before the colony perishes. However, for this to be possible the policing of worker-laid eggs must be curtailed and this provides the opportunity for queenless colonies to be reproductively parasitized by workers from other nests. Such reproductive parasitism is known to occur in Apis florea and A. cerana. Microsatellite analyses of worker samples have demonstrated that the proportion of non-natal workers present in an A. cerana colony declines after a colony is made queenless. This observation suggests that queenless A. cerana colonies may be more vigilant in repelling potentially parasitic non-natal workers than queenright colonies. We compared rates of nestmate and non-nestmate acceptance in both queenright and queenless A. cerana colonies using standard assays and showed that there is no statistical difference between the proportion of non-nestmate workers that are rejected in queenless and queenright colonies. We also show that, contrary to earlier reports, A. cerana guards are able to discriminate nestmate workers from non-nestmates, and that they reject significantly more non-nestmate workers than nestmate workers.

Worker policing in the German wasp Vespula germanica

In some ants, bees, and wasps, workers kill or “police” male eggs laid by other workers in order to maintain the reproductive primacy of the queen. Kin selection theory predicts that multiple mating by the queen is one factor that can selectively favor worker policing. This is because when the queen is mated to multiple males, workers are more closely related to the queen's sons than to the sons of other workers. Earlier work has suggested that reproductive patterns in the German wasp Vespula germanica may contradict this theory, because in some colonies a large fraction of the adult males were inferred to be the workers' sons, despite the effective queen mating frequency being greater than 2 (2.4). In the present study, we reexamine the V. germanica case and show that it does support the theory. First, genetic analysis confirms that the effective queen mating frequency is high, 2.9, resulting in workers being more related to the queen's sons than to other workers' sons. Second, behavioral assays show that worker-laid eggs are effectively killed by other workers, despite worker-laid eggs having the same intrinsic viability as queen-laid ones. Finally, we estimate that approximately 58.4% of the male eggs but only 0.44% of the adult males are worker derived in queenright colonies, consistent with worker reproduction being effectively policed.

Lack of physical policing and fertility cues in egg-laying workers of the ant Camponotus floridanus

In many kin groups cooperation is maintained by mutual policing and enforcement of fairness, a pattern that is predicted to change with group size. Variation in group size is most pronounced in hymenopteran societies, in which reproductive division of labour between queens and workers exists. Workers should not reproduce because of relatedness patterns and/or productivity costs. In small colonies where workers retain high reproductive potential egg laying is controlled by worker policing in the form of aggression towards incipient egg-layers or as destruction of worker-laid eggs. Egg-layers and worker eggs can be recognized in many species by differences in their surface hydrocarbons. We investigated whether these mechanisms change in species with large colonies and low worker reproductive potential. In the ant Camponotus floridanus, worker policing is based on differences in the hydrocarbon profiles of worker- and queen-laid eggs. However, in this study workers with developed ovaries were never the focus of attacks by nestmates, which contrasts with most studies on species in which workers have high reproductive potential. The lack of aggression towards egg-laying workers fits with the absence of relevant differences in the cuticular hydrocarbon profile between egg-laying and infertile C. floridanus workers, although strong differences existed between highly fertile queens and workers. Large colonies and low worker reproductive potential were thus accompanied by a lack of physical worker policing in C. floridanus, as predicted. Furthermore, we found costs of worker reproduction and we discuss their significance for worker policing.

Nestmate recognition for eggs in the honeybee (Apis mellifera L.)

Colony integrity is fundamental to social insects and is threatened by the reproduction of non-nestmates. Therefore, discrimination between eggs derived from nestmates and non-nestmates would constitute an adaptation to prevent exploitation of the entire cooperative group by unrelated individuals. The removal of nestmate and non-nestmate queen and worker-laid eggs was evaluated in honeybees using colonies of Apis mellifera capensis to test female and of A. m. scutellata to test male eggs. The data show that honeybees can distinguish between nestmate and non-nestmate eggs of both sexes. Moreover, non-nestmate female queen-laid eggs were removed significantly faster than nestmate female worker-laid eggs in A. m. capensis, indicating that nestmate recognition cues can override caste-specific ones. While the experimental manipulation accounts for 37.2% (A. m. scutellata) or 1.6% (A. m. capensis) of variance in relation to egg removal, nestmate recognition explains 33.3% for male eggs (A. m. scutellata) and 60.6% for female eggs (A. m. capensis), which is almost twice as high as the impact of caste (16.7% A. m. scutellata; 25% A. m. capensis). Our data show a stronger effect of nestmate recognition on egg removal in the honeybee, suggesting that cues other than caste-specific ones (viability/kin) can dominate egg removal behavior. In light of intraspecific social parasitism, preventing the reproduction of unrelated individuals (group selection) rather than preferring queens’ eggs (kin selection) appears to be the driving force behind the evolution of egg removal behavior in honeybees.

Specialization in policing behaviour among workers in the ant Pachycondyla inversa

Most animal societies are non-clonal and thus subject to conflicts. In social insects, conflict over male production can be resolved by worker policing, i.e. eating of worker-laid eggs (WLE) or aggression towards reproductive workers. All workers in a colony have an interest in policing behaviour being expressed, but there can be asymmetries among workers in performing the actual behaviour. Here, we show that workers of the ant Pachycondyla inversa specialize in policing behaviour. In two types of behavioural assays, workers developed their ovaries and laid eggs. In the first experiment, reproductive workers were introduced into queenright colonies. In the second experiment, WLE were introduced. By observing which individuals policed, we found that aggressive policing was highly skewed among workers that had opportunity to police, and that a similar tendency occurred in egg policing. None of the policing workers had active ovaries, so that policing did not incur a direct selfish benefit to the policer. This suggests that policing is subject to polyethism, just like other tasks in the colony. We discuss several hypotheses on the possible causes of this skew in policing tasks. This is the first non-primate example of specialization in policing tasks without direct selfish interests.

“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.

Preservation and loss of the honey bee (Apis) egg-marking signal across evolutionary time

Honey bee workers are able to distinguish queen-laid eggs from worker-laid eggs, and remove (‘police’) worker-laid eggs. The cue that police workers use is as yet unidentified but is likely to be a chemical signal. This signal benefits queens for it ensures their reproductive monopoly. It also benefits collective workers because it allows them to raise more closely related queen-laid males than the less-related sons of half sisters. Because both parties benefit from the egg-marking signal, it should be stable over evolutionary time. We show that Apis mellifera workers can distinguish queen-laid from worker-laid eggs of the dwarf honey bee A. florea, a phylogenetically distant species that diverged from the A. mellifera lineage 6–10 mya. However, A. mellifera workers are unable to distinguish worker-laid eggs of A. cerana, a much more recent divergence (2–3 mya). The apparent change in the egg-marking signal used by A. cerana may be associated with the high rates of ovary activation in this species.

Worker policing in the common wasp Vespula vulgaris is not aimed at improving colony hygiene

In insect societies, eggs laid by workers are frequently killed by other workers – a behaviour known as “worker policing”. The traditional explanation of worker policing is that it is a mechanism to resolve intracolony conflict, and maintain the reproductive monopoly of the queen. Recently, Pirk et al. (2004) proposed that worker policing instead is aimed at removing unviable worker-laid eggs and is ultimately just another example of hygienic behaviour. Here we test this hypothesis for the common wasp Vespula vulgaris, a species with highly effective worker policing. We show that worker-laid eggs from queenless colonies have a lower hatch rate (68%) than queen-laid eggs (82%). Analysis of egg laying rates of queens and workers, however, shows that the difference is not big enough to explain the apparent absence of adult worker-derived males in this species.

Nepotism and brood reliability in the suppression of worker reproduction in the eusocial Hymenoptera

In many eusocial Hymenoptera, workers prevent each other from producing male offspring by destroying worker-laid eggs. Kin selection theory predicts that such 'worker policing' behaviour can evolve by increasing the average relatedness between workers and their male brood. Alternatively, if worker-laid eggs are of low relative viability, their replacement would increase the developmental reliability of the brood. Less colony investment in terms of time and resources would be lost on poor males. This gain is independent of the relatedness of the males. Unfortunately, both nepotistic and group efficiency benefits can simultaneously accrue with the replacement of worker-laid eggs. Therefore, worker behaviour towards eggs cannot completely resolve whether both processes have been equally evolutionarily important. Adequate resolution requires the presentation of worker-produced brood of various ages. The stage at which brood are replaced can discriminate whether worker policing occurs owing to a preference for closer genetic kin, a preference for the more reliable brood or both.

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.

Are workers of Atta leafcutter ants capable of reproduction?

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.

Non-transferable signals on ant queen eggs

How biological systems resolve internal conflicts is a major evolutionary question. Social insect workers cooperate but also pursue individual interests, such as laying male eggs. The rewards of this individual selfishness can be reduced by policing, such as by killing worker-laid eggs. However, selfish individuals may evade policing. What factors prevent individuals from being able to evade policing? In the ant Pachycondyla inversa, workers kill (police) worker-laid eggs. Because the colony keeps eggs in piles and worker-laid and queen-laid eggs are chemically distinct, worker-laid eggs might become more acceptable once placed in the egg pile by odour transfer from touching queen-laid eggs. Here, we show that such "cue scrambling" does not occur. Worker-laid eggs that were sandwiched between three queen-laid eggs for 45 min were not more acceptable in a policing bioassay than control worker-laid eggs. Chemical analyses also showed that the surface hydrocarbon profile of these eggs was unchanged. Policing, therefore, is stable against this potential cheating mechanism probably because queen-laid eggs are made chemically distinct using chemicals, that are not easily transferred by physical contact.

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.

A TEST OF WORKER POLICING THEORY IN AN ADVANCED EUSOCIAL WASP, VESPULA RUFA

Mutual policing is an important mechanism for maintaining social harmony in group-living organisms. In some ants, bees, and wasps, workers police male eggs laid by other workers in order to maintain the reproductive primacy of the queen. Kin selection theory predicts that multiple mating by the queen is one factor that can selectively favor worker policing. This is because when the queen is mated to multiple males, workers are more closely related to queen's sons than to the sons of other workers. Here we provide an additional test of worker policing theory in Vespinae wasps. We show that the yellowjacket Vespula rufa is characterized by low mating frequency, and that a significant percentage of the males are workers' sons. This supports theoretical predictions for paternities below 2, and contrasts with other Vespula species, in which paternities are higher and few or no adult males are worker produced, probably due to worker policing, which has been shown in one species, Vespula vulgaris. Behavioral observations support the hypothesis that V. rufa has much reduced worker policing compared to other Vespula. In addition, a significant proportion of worker-laid eggs were policed by the queen.

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.