Male production in stingless bees: variable outcomes of queen-worker conflict

The genetic structure of social insect colonies is predicted to affect the balance between cooperation and conflict. Stingless bees are of special interest in this respect because they are singly mated relatives of the multiply mated honeybees. Multiple mating is predicted to lead to workers policing each others' male production with the result that virtually all males are produced by the queen, and this prediction is borne out in honey bees. Single mating by the queen, as in stingless bees, causes workers to be more related to each others' sons than to the queen's sons, so they should not police each other. We used microsatellite markers to confirm single mating in eight species of stingless bees and then tested the prediction that workers would produce males. Using a likelihood method, we found some worker male production in six of the eight species, although queens produced some males in all of them. Thus the predicted contrast with honeybees is observed, but not perfectly, perhaps because workers either lack complete control or because of costs of conflict. The data are consistent with the view that there is ongoing conflict over male production. Our method of estimating worker male production appears to be more accurate than exclusion, which sometimes underestimates the proportion of males that are worker produced.     

Paternity frequency and maternity of males in some stingless bee species

In monogynous hymenopteran societies, the number of mates of a queen strongly influences the potential for conflict between workers and queens over the maternity of males. Queens always 'prefer' their own sons to sons of workers, regardless of queen mating frequency. When a queen mates once, workers are more closely related to, and therefore are expected to prefer, their own sons and then sons of sisters to sons of the queen. However, if effective paternity frequency exceeds 2, workers on average should prefer queen-produced males to males produced by their sisters. We studied the queen mating frequency of seven stingless bee species: the Mexican species Scaptotrigona mexicana, S. pectoralis and the Australian species Austroplebeia symei, Trigona clypearis, T. hockingsi, T. mellipes and T. sapiens. We then determined whether males arise from eggs laid by workers or queens in A. symei, T. clypearis, T. hockingsi and T. mellipes. We show that all seven species investigated are most likely singly mated and that the queen dominates reproduction. This indicates that the queen's mating frequency alone does not determine whether workers or the queen produces the males.     

Conflict over male parentage in stingless bees

Summary Stingless bees usually have one, singly-mated queen. This can lead to a genetic conflict of interest between the queen and the non-laying workers over who should produce the males. In many stingless bee species workers have developed ovaries and can produce male-destined eggs. In this study we compile the available data on who produces the males in stingless bees. Worker reproduction is common but less frequent than expected from predictions built on relatedness-based preferences of non-laying workers. We tested whether the pattern in worker reproduction can be explained best by queen control, by an arms race between workers and their queen, by the costs of losing workers to reproductive competition, or by phylogenetic constraints. The data are consistent with the view that there is ongoing conflict over male production that is resolved differently depending on the specific dynamics of costs and benefits of worker reproduction. There was also a role for phylogeny; Melipona workers often reproduced while Plebeia and Australian stingless bee workers seldom or never did. The high worker reproduction in Melipona may reflect low costs, because many of the replaced queen-laid eggs would become excess queens.Received 17 April 2003; revised 2 September 2003; accepted 5 September 2003.     

How queen and workers share in male production in the stingless bee Melipona subnitida Ducke (Apidae, Meliponini)

Summary. Potential conflict between the queen and workers over the production of males is expected in stingless bees as a result of the higher relatedness of workers with their sons than with their brothers. This conflict was studied in Melipona subnitida by observing how the queen and the workers share in male production. The oviposition of individual cells was observed in two colonies with individually marked workers for a period of 51 and 40 days respectively. The gender that developed from these cells was then determined. The results revealed that most male production was concentrated in a 2–3-week period, during which laying workers were present. During these weeks, the queens produced twice as many males as all laying workers together. Outside this distinct period, the queens produced an occasional male. A reproductive worker either oviposited before the queen did, in which case she immediately proceeded to close the cell and thus prevented the queen from oviposition, or oviposited and sealed the cell after the queen had laid an egg. When cell construction and oviposition occured on several combs simultaneously, the workers preferentially laid male eggs on the newest combs. We discuss the proximate mechanism and ultimate cause of the way in which queen-worker male production occurred. In conclusion, we argue that overt behavioural conflict, occasionally displayed by reproductive workers of this species, can be of great cost to the colony.Received 27 February 2004; revised 6 September 2004; accepted 1 October 2004.     

No evidence that reproductive bumblebee workers reduce the production of new queens

Kin selection theory predicts potential conflict between queen and workers over male parentage in hymenopteran societies headed by one, singly mated queen, because each party is more closely related to its own male offspring. In ‘late-switching’ colonies of the bumblebee Bombus terrestris, i.e. colonies whose queens lay haploid eggs relatively late in the colony cycle, workers start to lay male eggs shortly after the queen lays the female eggs that will develop into new queens. It has been hypothesized that this occurs because workers recognize, via a signal given by the queen instructing female larvae to commence development as queens, that egg laying is now in their kin-selected interest. This hypothesis assumes that aggressive behaviour in egg-laying workers does not substantially reduce the production of new queens, which would decrease the workers' fitness payoff from producing males. We tested the hypothesis that reproductive activity inB. terrestris workers does not reduce the production of new queens. We used microsatellite genotyping to sex eggs and hence to select eight size-matched pairs of ‘late-switching’ colonies from a set of commercial colonies. From one colony of each pair we removed every egg-laying or aggressive worker observed. From the other colony, we simultaneously removed a nonegg-laying, nonaggressive worker. Removed workers were replaced with young workers from separate colonies at equal frequencies within the pair. There was no significant difference in queen productivity between colonies with reduced or normal levels of egg-laying or aggressive workers. Therefore, as predicted, reproductive B. terrestris workers did not significantly reduce the production of new queens.     

Mate number,kin selection and social conflicts in stingless bees and honeybees

Microsatellite genotyping of workers from 13 species (ten genera) of stingless bees shows that genetic relatedness is very high. Workers are usually daughters of a single, singly mated queen. This observation, coupled with the multiple mating of honeybee queens, permits kin selection theory to account for many differences in the social biology of the two taxa. First, in contrast to honeybees, where workers are predicted to and do police each other''s male production, stingless bee workers are predicted to compete directly with the queen for rights to produce males. This leads to behavioural and reproductive conflict during oviposition. Second, the risk that a daughter queen will attack the mother queen is higher in honeybees, as is the cost of such an attack to workers. This explains why stingless bees commonly have virgin queens in the nest, but honeybees do not. It also explains why in honeybees the mother queen leaves to found a new nest, while in stingless bees it is the daughter queen who leaves.     

Worker interests and male production in Polistes gallicus, a Mediterranean social wasp

The resolution of social conflict in colonies may accord with the interests of the most numerous party. In social insect colonies with single once-mated queens, workers are more closely related to the workers' sons than they are to the queens' sons. Therefore, they should prefer workers to produce males, against the queen's interests. Workers are capable of producing males as they arise from unfertilized eggs. We found Polistes gallicus to have colonies of single, once-mated queens, as determined by microsatellite genotyping of the workers, so worker interests predict worker male production. In colonies lacking queens, workers produced the males, but not in colonies with original queens. Thus worker interests were expressed only when the queen was gone. The high fraction of missing queens and early end to the colony cycle relative to climate so early in the season is surprising and may indicate a forceful elimination of the queen.     

The effect of sex-allocation biasing on the evolution of worker policing in hymenopteran societies

Mutual policing is thought to be important in conflict suppression at all levels of biological organization. In hymenopteran societies (bees, ants, and wasps), multiple mating by queens favors mutual policing of male production among workers (worker policing). However, worker policing of male production is proving to be more widespread than predicted by relatedness patterns, occurring in societies headed by single-mated queens in which, paradoxically, workers are more related to the workers' sons that they kill than the queen's sons that they spare. Here we develop an inclusive-fitness model to show that a second reproductive conflict, the conflict over sex allocation, can explain the evolution of worker policing contrary to relatedness predictions. Among ants, and probably other social Hymenoptera, workers kill males to favor their more related sisters. Importantly, males are killed at the larval stage, presumably because workers cannot determine the sex of queen-laid eggs. Sex-allocation biasing favors worker policing because policing removes some males (the workers' sons) at low cost at the egg stage rather than at higher cost at the larval stage. Our model reveals an important interaction between two reproductive conflicts in which the presence of one conflict (sex allocation) favors the suppression of the other (male production by workers).     

Regulation of queen-worker conflict in bumble-bee (Bombus terrestris) colonies

In annual colonies of bumble-bees overt queen-worker conflict is limited to a distinct 'competition phase' (CPh). In unmanipulated Bombus terrestris colonies, the queen's switch to male production (the 'switch point', SP) accounted for only-22% of the variation in the onset of the CPh. In some colonies, the CPh even began before the SP. The CPh was more strongly correlated with the transition in queen production (r=0.79). Replacing the queen eggs with male eggs or doubling the number of workers in young colonies resulted in a significantly earlier onset of the CPh and a significantly earlier transition to queen production. Replacing queen eggs with female eggs did not have this effect. These manipulations did not affect the timing of the queen's switch from female to male production. These findings show that the mechanism underlying the queen-worker conflict in insect societies is more complex than previously appreciated. The onset of queen-worker conflict cannot be attributed simply to a single factor such as the queen's switch to male production or a decrease in queen inhibition. Rather, multiple cues are important.     

Workers dominate male production in the neotropical bumblebee Bombus wilmattae (Hymenoptera: Apidae)

Background

Cooperation and conflict in social insects are closely linked to the genetic structure of the colony. Kin selection theory predicts conflict over the production of males between the workers and the queen and between the workers themselves, depending on intra-colonial relatedness but also on other factors like colony efficiency, sex ratios, cost of worker reproduction and worker dominance behaviour. In most bumblebee (Bombus) species the queen wins this conflict and often dominates male production. However, most studies in bumblebees have been conducted with only a few selected, mostly single mated species from temperate climate regions. Here we study the genetic colony composition of the facultative polyandrous neotropical bumblebee Bombus wilmattae, to assess the outcome of the queen-worker conflict over male production and to detect potential worker policing.

Results

A total of 120 males from five colonies were genotyped with up to nine microsatellite markers to infer their parentage. Four of the five colonies were queen right at point of time of male sampling, while one had an uncertain queen status. The workers clearly dominated production of males with an average of 84.9% +/- 14.3% of males being worker sons. In the two doubly mated colonies 62.5% and 96.7% of the male offspring originated from workers and both patrilines participated in male production. Inferring the mother genotypes from the male offspring, between four to eight workers participated in the production of males.

Conclusions

In this study we show that the workers clearly win the queen-worker conflict over male production in B. wilmattae, which sets them apart from the temperate bumblebee species studied so far. Workers clearly dominated male production in the singly as well the doubly mated colonies, with up to eight workers producing male offspring in a single colony. Moreover no monopolization of reproduction by single workers occurred.     

Comparative analysis of worker reproduction and policing in eusocial hymenoptera supports relatedness theory

In many bees, wasps, and ants, workers police each other in order to prevent individual workers from selfishly producing their own male offspring. Although several factors can selectively favor worker policing, genetic relatedness is considered to be of special importance. In particular, kin selection theory predicts that worker policing should be more common in species where workers are more related to the queen's sons than to other workers' sons. Here we provide strong novel support for this theory based on a comparative analysis of policing and male parentage in 109 species of ants, bees, and wasps. First, an analysis of behavioral data confirms that worker policing occurs more frequently in species where workers are more related to the queen's sons than to other workers' sons. Second, an analysis of male parentage shows that a significantly higher percentage of the males are workers' sons in species where the workers are more related to other workers' sons. Both conclusions also hold if data are analyzed using phylogenetically independent contrasts. Although our analysis provides strong overall support for the theory that relatedness affects kin conflict over male parentage, there is also significant residual variation. Several factors that may explain this variation are discussed.     

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.     

Intergenerational reproductive parasitism in a stingless bee

Insect colonies have been traditionally regarded as closed societies comprised of completely sterile workers ruled over by a single once-mated queen. However, over the past 15 years, microsatellite studies of parentage have revealed that this perception is far from the truth ( Beekman & Oldroyd 2008 ). First, we learned that honey bee queens are far more promiscuous than we had previously imagined ( Estoup et al. 1994 ), with one Apis dorsata queen clocked at over 100 mates ( Wattanachaiyingcharoen et al. 2003 ). Then Oldroyd et al. (1994) reported a honey bee colony from Queensland, where virtually all the males were sons of a single patriline of workers – a clear case of a cheater mutant that promoted intra-colonial reproductive parasitism. Then we learned that both bumble bee colonies ( Lopez-Vaamonde et al. 2004 ) and queenless honey bee colonies ( Nanork et al. 2005, 2007 ) are routinely parasitized by workers from other nests that fly in and lay male-producing eggs that are then reared by the victim colony. There is even evidence that in a thelytokous honey bee population, workers lay female-destined eggs directly into queen cells, thus reincarnating themselves as a queen ( Jordan et al. 2008 ). And let us not forget ants, where microsatellite studies have revealed equally bizarre and totally unexpected phenomena (e.g. Cahan & Keller 2003 ; Pearcy et al. 2004 ; Fournier et al. 2005 ). Now, in this issue, Alves et al. (2009) use microsatellites to provide yet another shocking and completely unexpected revelation about the nefarious goings-on in insect colonies: intergenerational reproductive parasitism by stingless bee workers.     

Dose-dependent inhibition of emergency queen rearing by synthetic 9-ODA in the honey bee,Apis mellifera carnica

In the honey bee colony queen rearing is usually suppressed by releaser effects of the queen's pheromone. This is part of the dominance hierarchy maintaining the monogynous homeostasis. Under queenless conditions, the queen's control over the construction of emergency queen cells by the workers can be substitued by exposure to only one component of the mandibular pheromone secretion of a queen, the main compound (E)-9-oxo-2-decenoic acid. A novel and simple synthesis of (E)-9-oxo-2-decenoic acid is described, and a bioassay was developed by which a dose-dependent effect of synthetic (E)-9-oxo-2-decenoic acid presented on a dummy bee was evaluated.Abbreviation 9-ODA (E)-9-oxo-2-decenoic acid In memoriam Viktor Schwartz (1907–1992), Professor of Zoology and Developmental Biology, University of Tübingen, who introduced smoothened bee stings into microsurgery     

Worker reproduction in the ant Formica fusca

We studied the kin conflict over male parentage in the ant Formica fusca. The conflict arises because each worker and queen is most related to her own sons and is thus predicted to lay eggs. Microsatellite analysis of eggs revealed that workers laid eggs in more than half the queenright experimental nests. Nevertheless, almost exclusively diploid offspring were reared in the presence of a queen. This also occurred when worker-laid haploid male eggs were experimentally introduced in to the nests. Because our experimental setup allowed us to exclude the possibility of queen policing, we conclude that worker laid eggs are removed by other workers, either as a response to their parentage or gender. Our results suggest that worker reproduction in F. fusca is ultimately an interplay of conflicts over male parentage and sex allocation and that both worker and self policing have roles as proximate mechanisms of resolution.     

Male parentage and queen mating frequency in the bumblebee <Emphasis Type="Italic">Bombus ignitus</Emphasis> (Hymenoptera: bombinae)

Kin selection theory predicts conflict between queens and workers in the social insect colony with respect to male production. This conflict arises from the haplodiploid system of sex determination in Hymenoptera that creates relatedness asymmetries in which workers are more closely related to the sons of other workers than to those of the queen. In annual hymenopteran societies that are headed by a single queen, the mating frequency of the queen is the only factor that affects the colony kin structure. Therefore, we examined the mating structure of queens and the parentage of males in a monogynous bumblebee, Bombus ignitus, using DNA microsatellites. In the seven colonies that were studied, B. ignitus queens mated once, thereby leading to the prediction of conflict between the queen and workers regarding male production. In each of the five queen-right colonies, the majority of the males (95%) were produced by the colony’s queen. In contrast, workers produced approximately 47% of all the males in two queenless colonies. These results suggest that male production in B. ignitus is a conflict between queen and workers.     

Sex ratio conflict and worker production in eusocial hymenoptera

The best known of the conflicts occurring in eusocial Hymenoptera is queen-worker conflict over sex ratio. So far, sex ratio theory has mostly focused on optimal investment in the production of male versus female sexuals, neglecting the investment in workers. Increased investment in workers decreases immediate sexual productivity but increases expected future colony productivity. Thus, an important issue is to determine the queen's and workers' optimal investment in each of the three castes (workers, female sexuals, and male sexuals), taking into account a possible trade-off between production of female sexuals and workers (both castes developing from diploid female eggs). Here, we construct a simple and general kin selection model that allows us to calculate the evolutionarily stable investments in the three castes, while varying the identity of the party controlling resource allocation (relative investment in workers, female sexuals, and male sexuals). Our model shows that queens and workers favor the investment in workers that maximizes lifetime colony productivity of sexual males and females, whatever the colony kin structure. However, worker production is predicted to be at this optimum only if one of the two parties has complete control over resource allocation, a situation that is evolutionarily unstable because it strongly selects the other party to manipulate sex allocation in its favor. Queens are selected to force workers to raise all the males by limiting the number of eggs they lay, whereas workers should respond to egg limitation by raising a greater proportion of the female eggs into sexual females rather than workers as a means to attain a more female-biased sex allocation. This tug-of-war between queens and workers leads to a stable equilibrium where sex allocation is between the queen and worker optima and the investment in workers is below both parties' optimum. Our model further shows that, under most conditions, female larvae are in strong conflict with queens and workers over their developmental fate because they value their own reproduction more than that of siblings. With the help of our model, we also investigate how variation in queen number and number of matings per queen affect the level of conflict between queens, workers, and larvae and ultimately the allocation of resource in the three castes. Finally, we make predictions that allow us to test which party is in control of sex allocation and caste determination.     

Worker policing in the German wasp Vespula germanica

In some ants, bees, and wasps, workers kill or "police" maleeggs laid by other workers in order to maintain the reproductiveprimacy of the queen. Kin selection theory predicts that multiplemating by the queen is one factor that can selectively favorworker policing. This is because when the queen is mated tomultiple males, workers are more closely related to the queen'ssons than to the sons of other workers. Earlier work has suggestedthat reproductive patterns in the German wasp Vespula germanicamay contradict this theory, because in some colonies a largefraction of the adult males were inferred to be the workers'sons, despite the effective queen mating frequency being greaterthan 2 (2.4). In the present study, we reexamine the V. germanicacase and show that it does support the theory. First, geneticanalysis confirms that the effective queen mating frequencyis high, 2.9, resulting in workers being more related to thequeen's sons than to other workers' sons. Second, behavioralassays show that worker-laid eggs are effectively killed byother workers, despite worker-laid eggs having the same intrinsicviability as queen-laid ones. Finally, we estimate that approximately58.4% of the male eggs but only 0.44% of the adult males areworker derived in queenright colonies, consistent with workerreproduction being effectively policed.     

Queen-worker conflicts over male production and sex allocation in a primitively eusocial wasp

Abstract In a colony headed by a single monandrous foundress, theories predict that conflicts between a queen and her workers over both sex ratio and male production should be intense. If production of males by workers is a function of colony size, this should affect sex ratios, but few studies have examined how queens and workers resolve both conflicts simultaneously. We conducted field and laboratory studies to test whether sex-ratio variation can be explained by conflict over male production between queen and workers in the primitively eusocial wasp Polistes chinensis antennalis.
Worker oviposition rate increased more rapidly with colony size than did queen oviposition. Allozyme and micro-satellite markers revealed that the mean frequency of workers' sons among male adults in queen-right colonies was 0.39 ± 0.08 SE (n = 22). Genetic relatedness among female nestmates was high (0.654–0.796), showing that colonies usually had a single, monandrous queen. The mean sex allocation ratio (male investment/male and gyne investments) of 46 queen-right colonies was 0.47 ± 0.02, and for 25 orphaned colonies was 0.86 ± 0.04. The observed sex allocation ratio was likely to be under queen control. For queen-right colonies, the larger colonies invested more in males and produced reproductives protandrously and/or simultaneously, whereas the smaller colonies invested more in females and produced reproductives protogynously. Instead of positive relationships between colony size and worker oviposition rate, the frequency of workers' sons within queen-right colonies did not increase with colony size. These results suggest that queens control colony investment, even though they allow worker oviposition in queen-right colonies. Eggs laid by workers may be policed by the queen and/or fellow workers. Worker oviposition did not influence the outcome of sex allocation ratio as a straightforward function of colony size.     

Colony kin structure and male production in Dolichovespula wasps

In annual hymenopteran societies headed by a single outbred queen, paternity (determined by queen mating frequency and sperm use) is the sole variable affecting colony kin structure and is therefore a key predictor of colony reproductive characteristics. Here we investigate paternity and male production in five species of Dolichovespula wasps. Twenty workers from each of 10 colonies of each of five species, 1000 workers in total, were analysed at three DNA microsatellite loci to estimate paternity. To examine the relationship between kin structure and reproductive behaviour, worker ovary activation was assessed by dissection and the maternal origin of adult males was assessed by DNA microsatellites. Effective paternity was low in all species (D. media 1.08, D. maculata 1.0, D. sylvestris 1.15, D. norwegica 1.08 and D. saxonica 1.35), leading to the prediction of queen-worker conflict over male production. In support of this, workers with full-size eggs in their ovaries (four out of five species) and adult males that were workers' sons (all five species) were found in queenright colonies. However, workers were only responsible for a minority of male production (D. media 7.4%, D. maculata 20.9%, D. sylvestris 9.8%, D. norwegica 2.6% and D. saxonica 34.6%) suggesting that the queen maintains considerable reproductive power over the workers. Kin structure and reproductive conflict in Dolichovespula contrast with their sister group Vespula. Dolichovespula is characterized by low paternity, worker reproduction, and queen-worker conflict and Vespula by high paternity, effective worker policing and absence of worker reproduction. The trend revealed by this comparison is as predicted by kin selection theory suggesting that colony kin structure has been pivotal in the evolution of the yellowjacket wasps.