Worker self-restraint and policing maintain the queen’s reproductive monopoly in a pseudomyrmecine ant

Division of reproductive labor in insect societies is often based on worker self-restraint and both queen and worker policing. Workers of many hitherto studied wasps, bees and ants do not lay eggs in the presence of a queen. However, it is presently unclear how far these observations in a few select clades can be generalized. We investigated if and how queens maintain a reproductive monopoly in colonies of the elongate twig ant, Pseudomyrmex gracilis, a member of the previously unstudied ant subfamily Pseudomyrmecinae. Colonies are usually headed by a single, singly mated queen (monogyny, monandry). Workers therefore would be more closely related to males produced by other workers (r?=?0.375) than to the sons of queens (r?=?0.25). Nevertheless, workers appear to refrain from laying male-destined eggs in the presence of the queen. In queenless conditions, workers form dominance hierarchies by antennal boxing, and only one or a few high-ranking individuals readily begin to lay eggs. When returned into a queenright colony, egg-laying workers are immediately bitten, stung and expelled or killed by other workers. While the composition of cuticular hydrocarbons clearly differed between castes, it less clearly reflected worker ovarian development. An association with worker ovarian development that would allow workers to monitor the reproductive status of nestmates could only be tentatively postulated for certain substances. Our study broadens our knowledge about reproductive conflict in social Hymenoptera and shows that worker sterility in the presence of a queen is more common in monogynous, monandrous ants than expected from relatedness alone.     

Reproductive conflict in animal societies: hierarchy length increases with colony size in queenless ponerine ants

Dominance interactions determine reproductive status in many animal societies, including many cooperatively breeding vertebrates and eusocial Hymenoptera without queen-worker dimorphism. Typically, the dominant individual monopolises reproduction, and subordinates behave like helpers. In Dinoponera queenless ants, workers are totipotent females and can potentially reproduce, yet only the top-ranking worker actually reproduces. Individual workers ranked immediately below the dominant breeder worker (gamergate) are hopeful reproductives. Whether or not a worker benefits from joining the hierarchy of high-ranking workers depends on the trade-off between the probability of becoming dominant and reproducing directly, and the colony-level cost of an additional lazy high ranker. Inclusive fitness models predict that the length of the dominance hierarchy depends on relatedness, colony size, and the linearity of the hierarchy. Here, we test the effect of colony size by comparing hierarchy length among three species that differ in colony size (Dinoponera australis: median=14 workers, quartiles=10 and 19 workers; D. gigantea: median=41, quartiles=33 and 74; D. quadriceps: median=78, quartiles=55 and 90). Although difficulties in defining where the hierarchy ends hamper comparisons, the results are in broad agreement with the predictions. Hierarchies are close to the predicted lengths and are longer in species with larger colonies (one, three and three workers in the three species in order from smallest to largest colony vs two, three and four predicted). These conclusions are further supported by determining Kokko and Lindström's λ index of skew, which is smaller (i.e. characteristic of a longer hierarchy) in species with larger colonies.     

Queen and worker policing in the monogynous and monandrous ant, Diacamma sp.

Policing behavior that prevents workers from laying male eggs was examined in the monogynous and monandrous ponerine ant Diacamma sp. from Japan, in which a singly mated worker called a “gamergate” reproduces as the functional queen in each colony. Since oviposition by virgin workers is rare in the presence of a gamergate, we separated a portion of workers from the gamergates and induced their oviposition experimentally. When orphaned workers had started to oviposit, they were returned to the original colonies, where they continued to lay eggs for a while. The gamergates and other workers interfered with the laying workers by aggressively taking and finally eating the eggs. In total, 60% and 29% of the worker-derived eggs were eaten by gamergates and non-mother workers, respectively. The observed worker-worker interactions were not driven simply by competition to leave own sons, because non-laying non-orphaned workers interfered with worker reproduction. Furthermore, orphaned workers were usually attacked by non-orphaned workers soon after colony reunification. These results indicate that both queen policing by gamergates and worker policing in this species are mechanisms inhibiting worker oviposition. The gamergate contribution to policing was proportionately larger than that of workers, but among virgin workers, the relationship between dominance rank and contribution to policing was not clear. But about 11% of the eggs were not policed and were added to egg piles, especially in large colonies. Worker policing in a monandrous and monogynous eusocial Hymenoptera contrasts to other recent findings, and possible genetic, social, and ecological factors for its evolution in Diacamma sp. are discussed. Received: 16 November 1998 / Received in revised form: 9 February 1999 / Accepted: 21 February 1999     

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.     

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.     

Serial polygyny and colony genetic structure in the monogynous queenless ant Diacamma cyaneiventre

Serial polygyny, defined as the temporal succession of several reproductive females in a colony, occurs in some monogynous social insects and has so far attracted little attention. Diacamma cyaneiventre is a queenless ponerine ant found in the south of India. Colonies are headed by one singly mated worker, the gamergate. After the death of the gamergate or her absence following colony fission, the gamergate is replaced by a newly eclosed nestmate worker. After a replacement, colonies go through short-lived periods in which two matrilines of sisters co-occur. This is a situation which can be described as serial polygyny. To measure the consequences of serial polygyny, a genetic analysis was performed on 449 workers from 46 colonies of D. cyaneiventre using five microsatellite loci. The presence of more than one matriline among workers of the same nest was detected in 19% of colonies, indicating a recent change of gamergate. The average genetic relatedness among nestmate workers was 0.751 and did not significantly differ from the theoretical expectation under strict monogyny and monandry (0.75). A simple analytical model of the temporal dynamics of serial polygyny was developed in order to interpret these results. We show that the rate of gamergate turnover relative to the rate of worker turnover is the crucial parameter determining the level of serial polygyny and its effect on the genetic structure of colonies. This parameter, estimated from our data, confirms that serial polygyny occurs in D. cyaneiventre but is not strong enough to influence significantly the average genetic relatedness among workers.     

Dominance orders,worker reproduction,and queen-worker conflict in the slave-making ant Harpagoxenus sublaevis

Summary In a queenright colony of the monogynous slave-making ant Harpagoxenus sublaevis, a subset of workers formed a linear dominance order in which dominance was corrlated with ovarian development, frequency of trophallaxis, length of time spent in the nest, but not body size. Identical dominance orders occurred in queenless colonies. Experiments in which the top-ranking workers were removed from queenless colonies demonstrated that worker dominance behaviour inhibits egg-laying in subordinates. A similar removal experiment showed queens restrict dominance behaviour and egg-laying in workers, probably pheromonally. Observations of slave raids indicated ovary-developed workers spent significantly less time scouting for slaves, and tended to participate less in slave raids, than workers without ovarian development. These findings suggest that potentially fertile H. sublaevis workers aggressively compete for egg-laying rights, consume extra food for egg development, and safeguard their reproductive futures by avoiding risks outside the nest. Hence worker reproduction in this species strongly influences the colony's social structure, nutrient flow, and division of labour, even though all workers in a colony are full sisters. I hypothesize that worker reproduction was formerly even more prevalent in H. sublaevis, with workers following the strategy of raising sisters and producing sons predicted by kinship theory. Its continued existence despite queen opposition conceivably results from selection on orphaned workers to reproduce, and the inability of slave-maker workers to raise female-biased broods. The social organization of H. sublaevis therefore highlights the importance both of worker reproduction and of the concomitant queen-worker conflict over male parentage in Hymenopteran social evolution.     

Patterns of male parentage in the fungus-growing ants

Ant queens from eight species, covering three genera of lower and two genera of higher attine ants, have exclusively or predominantly single mating. The ensuing full-sib colonies thus have a strong potential reproductive conflict between the queen and the workers over male production. This is because, all other things being equal, relatedness incentives should favour traits expressed in both workers and the queen to monopolise the production of the colony's male offspring. Microsatellite genotyping of males from these attine species shows that workers in queenless colonies are able to produce males, but that no worker-produced males were found in queenright colonies. Our results suggest that worker reproduction is rare or even absent in colonies with a fertile queen. This indicates that either the queen directly prevents the workers from raising their own sons, or that worker reproduction is absent in the presence of a fertile queen due to high ergonomic costs of this behaviour.     

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.     

Worker policing and nest mate recognition in the ant <Emphasis Type="Italic">Formica fusca</Emphasis>

A conflict over male production arises in social insects where workers are able to lay unfertilized male eggs. This happens because each female (queen or worker) is most closely related to her own sons and is thus predicted to reproduce. The conflict is modulated by worker policing where workers prevent each other from reproducing by aggression or egg cannibalism. In this study, we show that in the ant Formica fusca, worker policing occurs by egg cannibalism rather than by overt aggression among workers. Furthermore, we show that, contrary to bees, wasps and other ant species, egg discrimination in F. fusca is not based only on a universal queen signature chemical and that nest mate recognition of eggs occurs.     

Reproduction versus work in queenless ants: when to join a hierarchy of hopeful reproductives?

Many animal societies contain few breeders and many helpers. In some species, individuals can become either helpers or breeders, and breeding is often restricted to high rankers in a dominance hierarchy. In social Hymenoptera with no morphological queen caste, all females have the potential to become dominant and reproduce. In our model, females involved in the hierarchy do not work and gain direct fitness if they become alpha following death of the current alpha, while females not in the hierarchy gain indirect fitness by working. Using an inclusive-fitness model combined with models of alpha replacement in slightly different types of near-linear hierarchies, we determined the critical rank (i.e., optimal hierarchy length) for which the gain in fitness by entering the hierarchy as a hopeful reproductive equals the cost caused by a reduced worker force. High relatedness decreases the hierarchy length by lowering the potential benefit of entering the hierarchy. In contrast, large colony size increases the hierarchy length because the cost of each non-working high ranker is relatively lower. The model predicts a hierarchy of five workers in the queenless ant Dinoponera quadriceps, close to empirical data which show that in a colony of about 100 females, the top 5 carry out 72.8% of the aggressive interactions. The model is also used to make several testable predictions about the effect on hierarchy length of (1) variation in colony size and (2) variation in worker reproductive options between species of the queenless ants genus Diacamma. Received: 16 March 1999 / Received in revised form: 30 June 1999 / Accepted: 11 July 1999     

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.     

Experimental evidence that workers recognize reproductives through cuticular hydrocarbons in the ant Odontomachus brunneus

Eusociality is characterized by a reproductive division of labor, wherein workers respond to the presence of reproductive individuals by refraining from reproduction themselves and restricting the reproductive efforts of others. Our understanding of how eusociality is maintained therefore depends on characterizing the mechanism by which workers detect the presence of a reproductive. Variations in cuticular hydrocarbons correspond to changes in reproductive ability in ants, and experimental studies are beginning to reveal the function of hydrocarbons as signals. In this study, we compare the cuticular hydrocarbon profiles of dominant and reproductive workers and queens of the ant Odontomachus brunneus with profiles of non-reproductive workers. Using split/reunification tests we document the existence of worker policing in both queenless and queenright colonies; supernumerary reproductives were treated aggressively by nestmates. Finally, we induce aggression and replicate queen-like submissive nestmate responses by supplementing the hydrocarbon profile of workers with (Z)-9-nonacosene, a compound that was significantly more abundant on the cuticles of reproductives. In three bioassays, we compare this manipulation to various control manipulations of the hydrocarbon profile and demonstrate that workers gauge the reproductive activity of nestmates through changes in their cuticular hydrocarbon profiles.     

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 dominance modifies behavioral rhythm in a queenless ant

Social insects provide an intriguing model system in chronobiology. Typically, an egg-laying queen exhibits arrhythmicity in activity while foraging worker has clear rhythmicity. In the queenless ant, Diacamma sp., from Japan, colony members lack morphological caste, and reproductive differentiation occurs as a consequence of dominance hierarchy formation. Their specialized dominance interaction “gemmae mutilation”, provide us a fascinating model system to investigate the effect of social dominance on rhythmic ontogeny. Measurement of individual rhythms revealed that they have clear circadian rhythm at eclosion but it is diminished by social mutilation of gemmae. Moreover, unlike highly eusocial species, mated egg-layer (i.e., gamergate) possessed a circadian rhythm even after mating in Diacamma. Measurement of colony-level rhythms revealed that gemmae mutilations are performed in the limited time of the day, but foraging occurs around-the-clock. The above finding is a novel form of temporal organization in social insects, providing a new insight in morphologically casteless species. We discuss the causes and consequences of rhythmic variability in social organization.     

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     

Ovariole number—a predictor of differential reproductive success among worker subfamilies in queenless honeybee (Apis mellifera L.) colonies

A honeybee queen normally mates with 10–20 drones, and reproductive conflicts may arise among a colony’s different worker patrilines, especially after a colony has lost its single queen and the workers commence egg laying. In this study, we employed microsatellite markers to study aspects of worker reproductive competition in two queenless Africanized honeybee colonies. First, we determined whether there was a bias among worker patrilines in their maternity of drones and, second, we asked whether this bias could be attributed to differences in the degree of ovary activation of workers. Third, we relate these behavioral and physiological factors to ontogenetic differences between workers with respect to ovariole number. Workers from each of three (colony A) and one (colony B) patrilineal genotypes represented less than 6% of the worker population, yet each produced at least 13% of the drones in a colony, and collectively they produced 73% of the drones. Workers representing these genotypes also had more developed follicles and a greater number of ovarioles per ovary. Across all workers, ovariole development and number were closely correlated. This suggests a strong effect of worker genotype on the development of the ovary already in the postembryonic stages and sets a precedent to adult fertility, so that “workers are not born equal”. We hypothesize a frequency-dependent or “rare patriline” advantage to queenless workers over the parentage of males and discuss the maintenance of genetic variance in the reproductive capacity of workers.Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

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.     

Sex ratio determination and worker reproduction in the slave-making ant Harpagoxenus sublaevis

Summary In a population of the monogynous slave-making ant Harpagoxenus sublaevis in S.E. Sweden, the mean proportion of dry weight investment in queens was 0.54. This result differed significantly from 0.75 but not from 0.5, matching the prediction from the genetic relatedness hypothesis of sex ratio applied to slave-makers, given (as confirmed by this study) single mating of queens, population-wide mate competition, and relatively low levels of worker male production. Sex investment appeared unaffected by resource availability. In the same 47 colony population sample, fertile slave-maker workers were found in every queenless colony (ca. 30% of all colonies), and in 58% of queen-right colonies. Fertile workers occurred at a significantly higher frequency in the queenless colonies (19.2%) than in the queenright ones (9.8%), confirming that queenless conditions promote worker fertility. Fertile and sterile workers were similar in size. Electrophoretic allozyme analysis of ants from 49 colonies showed that: 1) queens mated singly; 2) female nestmates were full sisters (their regression coefficient of relatedness (±SE) was 0.735±0.044); 3) inbreeding did not occur; 4) queen and worker siblings were not genetically differentiated. Worker male production in queenright colonies was neither confirmed nor ruled out by the genetic data. However, production data indicated that queenless workers produced between 4.4 and 21.6% of all males. Overall colony productivity was largely determined by slave number, itself positively correlated with the number of slave-maker workers. There was an abrupt switch from all worker to all sexual production as colony size rose, as predicted by life history models. In queenright colonies, fertile slave-makers did not discernibly reduce colony productivity. Such workers occurred in queenright colonies with most slaves, suggesting they exploited energetic surpluses. Worker reproduction in H. sublaevis therefore appears to have greater influence at the level of individual behaviour than at colony or population level.     

Dufour's gland secretion of the queen honeybee (Apis mellifera): an egg discriminator pheromone or a queen signal?

The role of Dufour's gland secretion as an egg discriminator pheromone was reevaluated by simultaneously exposing workers to two combs, one containing queen- or worker-laid eggs and the second containing treated or untreated worker-laid eggs. Treatments included extracts of Dufour's gland secretion as well as the synthetic esters that were identified in the secretion. Policing was clearly detected both in queenright and queenless colonies by the swift removal of worker, but not of queen eggs. However, neither the glandular secretion nor its synthetic ester constituents were able to protect worker-born eggs from policing. Treated worker eggs were removed significantly faster than queen eggs, and at the same rate as non-treated worker eggs. These results are not consistent with the hypothesis that the secretion serves as an egg-marking pheromone. Chemical analyses of the queen abdominal tips revealed the presence of Dufour's esters, indicating that the glandular secretion oozes out and spreads over the cuticle around the genital chamber. However, contamination while ovipositing may also explain the minute amounts of these esters that were detected on the egg surface. Dufour's gland caste-specific composition suggests that in queens it may constitute a signal that plays a role in queen-worker interactions. Attraction bioassays revealed that the queen secretion, but not that of workers, is very attractive to workers. When applied either on a glass slide or on another worker, a retinue formed around the "surrogate queen". We conclude that Dufour's gland secretion constitutes part of a complex queen signal that is the basis for the social integrity of the honeybee colony.