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.     

Moving home: nest-site selection in the Red Dwarf honeybee (<Emphasis Type="Italic">Apis florea</Emphasis>)

The Red Dwarf honeybee (Apis florea) is one of two basal species in the genus Apis. A. florea differs from the well-studied Western Hive bee (Apis mellifera) in that it nests in the open rather than in cavities. This fundamental difference in nesting biology is likely to have implications for nest-site selection, the process by which a reproductive swarm selects a new site to live in. In A. mellifera, workers show a series of characteristic behaviors that allow the swarm to select the best nest site possible. Here, we describe the behavior of individual A. florea workers during the process of nest-site selection and show that it differs from that seen in A. mellifera. We analyzed a total of 1,459 waggle dances performed by 197 scouts in five separate swarms. Our results suggest that two fundamental aspects of the behavior of A. mellifera scouts—the process of dance decay and the process of repeated nest site evaluation—do not occur in A. florea. We also found that the piping signal used by A. mellifera scouts to signal that a quorum has been reached at the chosen site, is performed by both dancing and non-dancing bees in A. florea. Thus, the piping signal appears to serve a different purpose in A. florea. Our results illustrate how differences in nesting biology affect the behavior of individual bees during the nest-site selection process.     

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.     

Polyandry in the genus Apis, particularly Apis andreniformis

Using four polymorphic microsatellite loci, we found that four Apis andreniformis queens collected in Thailand each mated at least 10–20 times, producing an average relatedness, g _ww, of workers of 0.30 ± 0.007, and an average effective number of matings of 9.1 ± 2.2. The degrees of polyandry and intra-colonial genetic relatedness in A. andreniformis are similar to those in A. mellifera, slightly more than in A. florea, and up to 6 times less than in A. dorsata. We argue that while presently favoured hypotheses for the evolution of polyandry in monogynous social insects may adequately explain the evolution of up to five or six matings, they are inadequate to explain the extreme polyandry (10–60 matings) observed in Apis. One alternative possibility is that colony fitness is a non-additive function of the fitness of individual subfamilies. Such behavioral over-dominance may mean that queen fitness is increased by high levels of polyandry, which increase the probability of desirable combinations of worker genotypes occurring in one colony. The special attributes of honey bees which may lead to behavioral over-dominance include colony aggregation (which may increase the incidence of disease), and frequent long-distance migration. Received: 8 May 1996/Accepted after revision: 9 August 1996     

Levels of polyandry and intracolonial genetic relationships in Apis florea

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

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     

Social parasitism by honeybee workers (Apis mellifera capensis Esch.): evidence for pheromonal resistance to host queen’s signals

Social parasites exploit their host’s communication system to usurp resources and reproduce. In the honeybee, Apis mellifera, worker reproduction is regulated by pheromones produced by the queen and the brood. Workers usually reproduce when the queen is removed and young brood is absent. However, Cape honeybee workers, Apis mellifera capensis, are facultative intraspecific social parasites and can take over reproduction from the host queen. Investigating the manner in which parasitic workers compete with host queens pheromonally can help us to understand how such parasitism can evolve and how reproductive division of labour is regulated. In A. m. capensis, worker reproduction is associated with the production of queen-like pheromones. Using pheromonal contest experiments, we show that Apis mellifera scutellata queens do not prevent the production of queen-like mandibular gland compounds by the parasites. Given the importance of these pheromones in acquiring reproductive status, our data suggest that the single invasive lineage of parasitic workers occurring in the range of A. m. scutellata was selected for its superior ability to produce these signals despite the presence of a queen. Such resistance was indeed less frequent amongst other potentially parasitic lineages. Resistance to reproductive regulation by host queens is probably the key factor that facilitates the evolution of social parasitism by A. m. capensis workers. It constitutes a mechanism that allows workers to evade reproductive division of labour and to follow an alternative reproductive option by acquiring direct fitness in foreign colonies instead of inclusive fitness in their natal nests.     

Nest site selection in the open-nesting honeybee Apis florea

We studied nest site selection by swarms of the red dwarf honeybee, Apis florea. By video recording and decoding all dances of four swarms, we were able to determine the direction and distances indicated by 1,239 dances performed by the bees. The bees also performed a total of 715 nondirectional dances; dances that were so brief that no directional information could be extracted. Even though dances converged over time to a smaller number of areas, in none of the swarms did dances converge to one site. As a result, even prior to lift off, bees performed dances indicating nest sites in several different directions. Two of four swarms traveled directly in what seemed to be the general direction indicated by the majority of dances in the half hour prior to swarm lift off. The other two traveled along circuitous routes in the general direction indicated by the dances. We suggest that nest site selection in A. florea has similar elements to nest site selection in the better-studied Apis mellifera. However, the observation that many more locations are indicated by dances prior to lift off also shows that there are fundamental differences between the two species.     

Worker reproductive parasitism and drift in the western honeybee Apis mellifera

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

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.     

Preservation and loss of the honey bee (<Emphasis Type="Italic">Apis</Emphasis>) 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.     

High degree of polyandry in Apis dorsata queens detected by DNA microsatellite variability

Workers of six colonies of the giant honeybee Apis dorsata from Sabah, Malaysia (five colonies) and Java (one colony) were genotyped using single locus DNA fingerprinting. The colonies from Sabah nested in colony aggregations of 5 and 28 nests respectively on two trees. Three DNA microsatellite loci (A14, A76, A88) with a total of 27 alleles provided sufficient genetic variability to classify the workers into distinct sub-families revealing the degree of polyandry of the queens. Queens mated on average with 30.17 ± 5.98 drones with a range from 19 to 53. The average effective number of matings per queen was 25.56 ± 11.63. In the total sample of 192 workers, 22 individuals were found that were not offspring of the colony's queen. Three of these were potentially drifted offspring workers from genotyped queens of colonies nesting on the same tree.     

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     

Social discrimination tuning in ants: template formation and chemical similarity

To investigate the role of template plasticity in shaping nest-mate recognition processes in ants, we constructed experimental mixed-species groups of Manica rubida with either Myrmica rubra, Tetramorium bicarinatum or Formica selysi. Selecting Ma. rubida as the focal species, we observed the behaviour within mixed-species groups and the transfer rates of cuticular hydrocarbons (CHC) onto the focal ants, and we also tested the aggression of the focal species reared either alone or in association with each of the three different species. We show that Ma. rubida workers were always amicable towards their mixed group members, as towards members of the respective parental colonies, irrespective of the associated species. They did, however, express different levels of aggression towards single-species groups of the other species tested, depending on the species with which they were reared. The study suggests that similarity in CHC profiles in two species leads to a narrow template in mixed groups, while dissimilarity is followed by lower levels of aggression (a broader template), at least against species with similar CHC compound compositions (i.e. both a broader template in the focal ants and familiarity with the compound groups of the tested individuals operate together). This refutes the hypothesis that ants reared in mixed-species groups are systematically more tolerant. It also demonstrates that heterospecific information is not treated equally during development. We suggest that post-imaginal learning, template reforming and decision making are more precisely tuned when the two species' chemical complexes are similar.     

Interspecific aggression in colonies of the slave-making ant Harpagoxenus sublaevis

Colonies of the slave-making ant, Harpagoxenus sublaevis, may simultaneously contain workers of several Leptothorax slave species. We observed aggressive interactions among slave-makers, between slavemakers and slaves, and among slaves in 11 mixed colonies. The first two types of aggression appear to be correlated with reproductive competition for the production of males. Aggressive interactions among slaves, however, occurred mainly between slaves belonging to different species. In two colonies, in which one slave species clearly outnumbered the other, the majority attacked and finally expelled all nestmates belonging to the minority species. Our observations thus suggest that in Harpagoxenus colonies a homogeneous colony odor is not always achieved and that heterospecific slaves may occasionally be mistaken for alien ants. Gas chromatographic analyses of ants from mixed colonies similarly show that cuticular hydrocarbon profiles may differ strongly between heterospecific nestmate slaves.     

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.     

Task specialization in a wild bee,Apis florea (Hymenoptera: Apidae), revealed by RFLP banding

Workers in a wild in situ colony of the dwarf honey bee, Apis florea, were observed undertaking the following behavior: liquid foraging, pollen foraging, guarding, stinging, fanning and wagging abdomen. Bees of each behavioral class were separately collected and frozen. Collections were made over a period of 10 days. Random samples of brood and workers were also collected. DNA was extracted from each bee and fingerprinted using a probe of unknown sequence obtained from an A. mellifera genomic library. Patterns of fingerprints (Fig. 1) were dissimilar among behavioral classes (Tables 1 and 2), strongly suggesting a genetic component to division of labor in this species. This result supports similar findings in A. mellifera in a species that is not troubled by many of the experimental difficulties inherent in A. mellifera. Correspondence to: B.P. Oldroyd     

Nestmate recognition and the ontogeny of acceptability in the ant,Leptothorax curvispinosus

Summary In social insects, there is often a brief period following eclosion when workers are highly acceptable in alien nests of their own or other species. This study tested for such an acceptance period in the facultatively polygynous ant, Leptothorax curvispinosus, and compared the duration and effectiveness of this period for conspecific and heterospecific introductions. Workers that eclosed and aged for 1–70 h or 30 days in isolation were introduced into either their parental nests (n=24), alien conspecific nests (n=265), or nests of the closely related and biologically similar species, L. longispinosus (n=341). In alien conspecific nests, acceptance was maximal for workers aged 1–12 h at introduction (67.7% not attacked, 75.8% adopted) and gradually decreased until the level of nonaggression (after 60 h) and adoption (after 36 h) were not significantly different from 30-day-old workers (5.9% not attacked, 17.6% adopted). In heterospecific nests, acceptance was maximal for workers aged 1–4 h at introduction (34.8% not attacked, 37.0% adopted) but thereafter was not significantly different from 30-day-old workers (5.6% not attacked, 8.3% adopted). In their parental nests, workers were generally accepted regardless of age (4–56 h posteclosion, 95.8% not attacked, 100% adopted); a result that is consistent with previous research on older workers (38–157 days posteclosion). This study demonstrates an acceptance period that is more effective and of longer duration within than between these species but that, under uniform laboratory conditions, is often not necessary for the integration of workers into their parental colonies. Within colonies, acceptance periods might only be important during relatively brief periods in a colony's life history when eclosing workers produce genetically based nestmate recongition cues that are not already represented in the colony and must be learned by colony members (e.g., during early colony growth or following adoption of queens), or when young workers must acquire environmentally based nestmate recognition cues to achieve and maintain acceptability.     

Colony performance in honeybees (Apis mellifera capensis Esch.) depends on the proportion of subordinate and dominant workers

Summary Individual worker dominance correlated with trophallactic behavior, which affects several social behaviors related to colony fitness, shows a high genetic variance in worker bees. In a bioassay we tested trophallactic behavior of workers and selected dominant (receiving) and subordinate worker bees (offering) of Apis mellifera capensis to establish genetic lines of both kinds. Queenright test colonies were experimentally composed of 100% subordinate workers, 100% dominant workers, 50% dominant plus 50% subordinate workers, and 100% hybrid workers from the two genetic lines. The chosen test parameters were brood-rearing, comb building and hoarding behavior. In all cases, the colonies of pure subordinate bees showed the best colony performance, whereas the colonies composed of only dominant bees were nearly unproductive. The mixed colonies (50% dominant + 50% subordinate) ranked in the middle and did not differ significantly from the hybrid colonies. The results indicate that colony performance under queenright conditions depends on the proportion of subordinate workers. This result supports a selection model based on the combination of individual selection and on group selection at the colony level, which explains the high genetic variance of individual worker reproduction.