Factors influencing survival duration and choice of virgin queens in the stingless bee Melipona quadrifasciata

In Melipona quadrifasciata, about 10 % of the females develop into queens, almost all of which are killed. Occasionally, a new queen replaces or supersedes the mother queen or heads a new colony. We investigated virgin queen fate in queenright and queenless colonies to determine the effects of queen behaviour, body mass, nestmate or non-nestmate status, queenright or queenless colony status, and, when queenless, the effect of the time a colony had been queenless, on survival duration and acceptance. None of 220 virgin queens observed in four observation hives ever attacked another virgin queen nor did any of 88 virgin queens introduced into queenright colonies ever attack the resident queen. A new queen was only accepted in a queenless colony. Factors increasing survival duration and acceptance of virgin queens were to emerge from its cell at 2 h of queenlessness, to hide, and to avoid fights with workers. In this way, a virgin queen was more likely to be available when a colony chooses a new queen, 24-48 h after resident queen removal. Running, walking or resting, antennating or trophallaxis, played little or no role, as did the factors body mass or nestmate. “Queen choice” took about 2 h during which time other virgin queens were still being killed by workers. During this agitated process, the bees congregated around the new queen. She inflated her abdomen and some of the workers deposited a substance on internal nest surfaces including the glass lid of the observation hive.     

Age-related changes in biogenic amines in individual brains of the ant Pheidole dentata

The behavioral development of minor workers of the ant Pheidole dentata involves a progression of tasks beginning with brood care and culminating in foraging as individuals age. To understand the role of brain neurochemistry in age-related division of labor, we measured the levels of serotonin, dopamine and octopamine in individual brains of minor workers of different age. Serotonin and dopamine levels were significantly correlated with worker age: both increased as minor workers matured, and serotonin rose significantly in the oldest ants. In addition, the serotonin:dopamine ratio was significantly higher in the oldest workers. Octopamine levels did not change with age, although the ratios of octopamine:serotonin and octopamine:dopamine were significantly higher in the youngest workers. These age-associated changes in biogenic amine levels suggest an involvement of neuromodulators in minor worker behavioral ontogeny and temporal polyethism in P. dentata.     

Biogenic amine levels, reproduction and social dominance in the queenless ant Streblognathus peetersi

Social harmony often relies on ritualised dominance interactions between society members, particularly in queenless ant societies, where colony members do not have developmentally predetermined castes but have to fight for their status in the reproductive and work hierarchy. In this behavioural plasticity, their social organisation resembles more that of vertebrates than that of the “classic” social insects. The present study investigates the neurochemistry of the queenless ant species, Streblognathus peetersi, to better understand the neural basis of the high behavioural plasticity observed in queenless ants. We report measurements of brain biogenic amines [octopamine, dopamine, serotonin] of S. peetersi ants; they reveal a new set of biogenic amine influences on social organisation with no common features with other “primitively organised societies” (bumble bees) and some common features with “highly eusocial” species (honey bees). This similarity to honey bees may either confirm the heritage of queenless species from their probably highly eusocial ancestors or highlight independent patterns of biogenic amine influences on the social organisation of these highly derived species.     

Decision rules for egg recognition are related to functional roles and chemical cues in the queenless ant Dinoponera quadriceps

The capacity to distinguish colony members from strangers is a key component in social life. In social insects, this extends to the brood and involves discrimination of queen eggs. Chemical substances communicate colony affiliation for both adults and brood; thus, in theory, all colony members should be able to recognize fellow nestmates. In this study, we investigate the ability of Dinoponera quadriceps workers to discriminate nestmate and non-nestmate eggs based on cuticular hydrocarbon composition. We analyzed whether cuticular hydrocarbons present on the eggs provide cues of discrimination. The results show that egg recognition in D. quadriceps is related to both age and the functional role of workers. Brood care workers were able to distinguish nestmate from non-nestmate eggs, while callow and forager workers were unable to do so. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reclaiming the crown: queen to worker conflict over reproduction in <Emphasis Type="Italic">Aphaenogaster cockerelli</Emphasis>

In many social taxa, reproductively dominant individuals sometimes use aggression to secure and maintain reproductive status. In the social insects, queen aggression towards subordinate individuals or workers has been documented and is predicted to occur only in species with a small colony size and a low level of queen–worker dimorphism. We report queen aggression towards reproductive workers in the ant species Aphaenogaster cockerelli, a species with a relatively large colony size and a high level of reproductive dimorphism. Through analysis of cuticular hydrocarbon profiles, we show that queens are aggressive only to reproductively active workers. Non-reproductive workers treated with a hydrocarbon typical for reproductives are attacked by workers but not by queens, which suggests different ways of recognition. We provide possible explanations of why queen aggression is observed in this species.     

Lethal fighting between honeybee queens and parasitic workers (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Pheromonal signals associated with queen and worker policing prevent worker reproduction and have been identified as important factors for establishing harmony in the honeybee (Apis mellifera) colony. However, "anarchic workers", which can evade both mechanisms, have been detected at low frequency in several honeybee populations. Worker bees of the Cape honeybee, Apis mellifera capensis, also show this anarchistic trait but to an extreme degree. They can develop into so called "pseudoqueens", which release a pheromonal bouquet very similar to that of queens. They prime and release very similar reactions in sterile workers to those of true queens (e.g. suppress ovary activation; release retinue behavior). Here we show in an experimental bioassay that lethal fights between these parasitic workers and the queen (similar to queen–queen fights) occur, resulting in the death of either queen or worker. Although it is usually the queen that attacks the parasitic workers and kills many of them, in a few cases the workers succeeded in killing the queen. If this also occurs in a parasitized colony where the queen encounters many parasitic workers, she may eventually be killed in one of the repeated fights she engages in.     

Cross-species correlation between queen mating numbers and worker ovary sizes suggests kin conflict may influence ovary size evolution in honeybees

During social evolution, the ovary size of reproductively specialized honey bee queens has dramatically increased while their workers have evolved much smaller ovaries. However, worker division of labor and reproductive competition under queenless conditions are influenced by worker ovary size. Little comparative information on ovary size exists in the different honey bee species. Here, we report ovariole numbers of freshly dissected workers from six Apis species from two locations in Southeast Asia. The average number of worker ovarioles differs significantly among species. It is strongly correlated with the average mating number of queens, irrespective of body size. Apis dorsata, in particular, is characterized by numerous matings and very large worker ovaries. The relation between queen mating number and ovary size across the six species suggests that individual selection via reproductive competition plays a role in worker ovary size evolution. This indicates that genetic diversity, generated by multiple mating, may bear a fitness cost at the colony level.     

Brain modulation of Dufour’s gland ester biosynthesis in vitro in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Caste-specific pheromone biosynthesis is a prerequisite for reproductive skew in the honeybee. Nonetheless, this process is not hardwired but plastic, in that egg-laying workers produce a queen-like pheromone. Studies with Dufour’s gland pheromone revealed that, in vivo, workers’ gland biosynthesis matches the social status of the worker, i.e., sterile workers showed a worker-like pattern whereas fertile workers showed a queen-like pattern (production of the queen-specific esters). However, when incubated in vitro, the gland spontaneously exhibits the queen-like pattern, irrespective of its original worker type, prompting the notion that ester production in workers is under inhibitory control that is queen-dependent. We tested this hypothesis by exposing queen or worker Dufour’s glands in vitro to brain extracts of queens, queenright (sterile) workers and males. Unexpectedly, worker brain extracts activated the queen-like esters biosynthesis in workers’ Dufour’s gland. This stimulation was gender-specific; queen or worker brains demonstrated a stimulatory activity, but male brains did not. Queen gland could not be further stimulated. Bioassays with heated and filtered extracts indicate that the stimulatory brain factor is below 3,000 Da. We suggest that pheromone production in Dufour’s gland is under dual, negative–positive control. Under queenright conditions, the inhibitor is released and blocks ester biosynthesis, whereas under queenless conditions, the activator is released, activating ester biosynthesis in the gland. This is consistent with the hypothesis that queenright workers are unequivocally recognized as non-fertile, whereas queenless workers try to become “false queens” as part of the reproductive competition.     

The role of tyramine and octopamine in the regulation of reproduction in queenless worker honeybees

In honeybees, workers under queenless condition compete for reproduction and establish reproductive dominance hierarchy. Ovary activation is generally accompanied by the expression of queen-like pheromones. Biogenic amines (BAs), in particular dopamine, are believed to be involved in this process by regulating ovarian development. However, the role of BAs in establishing reproductive dominance or their effect on queen-like pheromone production was not investigated. Here, we explored the effect of octopamine (OA) and tyramine (TA) oral treatments on the propensity of treated bees to become reproductively dominant and produce queen-like pheromones in Dufour’s and mandibular glands. One bee in a pair was treated with either OA or TA while the other was fed sugar solution. TA was found to enhance ovary development and the production of esters in the Dufour’s gland and 9HDA (queen component) in the mandibular glands, thus facilitating worker reproductive dominance. OA, on the other hand, did not enhance ovarian development or ester production, but increased the production of 10HDA (worker major component) in the mandibular glands of their sugar-paired mates. OA is known to induce foraging behavior by workers, while increased production of 10HDA characterizes nursing workers. Therefore, we suggest that TA induces reproductive division of labor, while OA treatment results in caste differentiation of workers to foragers and nurses.     

Clonal structure affects the assembling behavior in the Japanese queenless ant <Emphasis Type="Italic">Pristomyrmex punctatus</Emphasis>

The queenless ant Pristomyrmex punctatus (Hymenoptera: Myrmicinae) has a unique society that differs from those of other typical ants. This species does not have a queen, and the workers lay eggs and produce their clones parthenogenetically. However, a colony of these ants does not always comprise members derived from a single clonal line. In this study, we examined whether P. punctatus changes its “assembling behavior” based on colony genetic structure. We prepared two subcolonies—a larger one comprising 200 individuals and a smaller one comprising 100 individuals; these subcolonies were established from a single stock colony. We investigated whether these subcolonies assemble into a single nest. The genetically monomorphic subcolonies (single clonal line) always fused into a single nest; however, the genetically polymorphic subcolonies (multiple clonal lines) did not tend to form a single colony. The present study is the first to demonstrate that the colony genetic structure significantly affects social viscosity in social insects.     

Surface lipids of queen-laid eggs do not regulate queen production in a fission-performing ant

In animal societies, most collective and individual decision making depends on the presence of reproductive individuals. The efficient transmission of information among reproductive and non-reproductive individuals is therefore a determinant of colony organization. In social insects, the presence of a queen modulates multiple colonial activities. In many species, it negatively affects worker reproduction and the development of diploid larvae into future queens. The queen mostly signals her presence through pheromone emission, but the means by which these chemicals are distributed in the colony are still unclear. In several ant species, queen-laid eggs are the vehicle of the queen signal. The aim of this study was to investigate whether queen-laid eggs of the ant Aphaenogaster senilis possess queen-specific cuticular hydrocarbons and/or Dufour or poison gland compounds, and whether the presence of eggs inhibited larval development into queens. Our results show that the queen- and worker-laid eggs shared cuticular and Dufour hydrocarbons with the adults; however, their poison gland compounds were not similar. Queen-laid eggs had more dimethylalkanes and possessed a queen-specific mixture of cuticular hydrocarbons composed of 3,11?+?3,9?+?3,7-dimethylnonacosane, in higher proportions than did worker-laid eggs. Even though the queen-laid eggs were biochemically similar to the queen, their addition to experimentally queenless groups did not prevent the development of new queens. More studies are needed on the means by which queen ant pheromones are transmitted in the colony, and how these mechanisms correlates with life history traits.     

Two pathways ensuring social harmony

Reproductive division of labour is a characteristic trait of social insects. The dominant reproductive individual, often the queen, uses chemical communication and/or behaviour to maintain her social status. Queens of many social insects communicate their fertility status via cuticle-bound substances. As these substances usually possess a low volatility, their range in queen–worker communication is potentially limited. Here, we investigate the range and impact of behavioural and chemical queen signals on workers of the ant Temnothorax longispinosus. We compared the behaviour and ovary development of workers subjected to three different treatments: workers with direct chemical and physical contact to the queen, those solely under the influence of volatile queen substances and those entirely separated from the queen. In addition to short-ranged queen signals preventing ovary development in workers, we discovered a novel secondary pathway influencing worker behaviour. Workers with no physical contact to the queen, but exposed to volatile substances, started to develop their ovaries, but did not change their behaviour compared to workers in direct contact to the queen. In contrast, workers in queen-separated groups showed both increased ovary development and aggressive dominance interactions. We conclude that T. longispinosus queens influence worker ovary development and behaviour via two independent signals, both ensuring social harmony within the colony.     

Nestmate recognition in ants is possible without tactile interaction

Ants of the genus Camponotus are able to discriminate recognition cues of colony members (nestmates) from recognition cues of workers of a different colony (non-nestmates) from a distance of 1 cm. Free moving, individual Camponotus floridanus workers encountered differently treated dummies on a T-bar and their behavior was recorded. Aggressive behavior was scored as mandibular threat towards dummies. Dummies were treated with hexane extracts of postpharyngeal glands (PPGs) from nestmates or non-nestmates which contain long-chain hydrocarbons in ratios comparable to what is found on the cuticle. The cuticular hydrocarbon profile bears cues which are essential for nestmate recognition. Although workers were prevented from antennating the dummies, they showed significantly less aggressive behavior towards dummies treated with nestmate PPG extracts than towards dummies treated with non-nestmate PPG extracts. In an additional experiment, we show that cis-9-tricosene, an alkene naturally not found in C. floridanus' cuticular profile, is behaviorally active and can interfere with nestmate recognition when presented together with a nestmate PPG extract. Our study demonstrates for the first time that the complex multi-component recognition cues can be perceived and discriminated by ants at close range. We conclude that contact chemosensilla are not crucial for nestmate recognition since tactile interaction is not necessary.     

Aggressions and size-related fecundity of queenless workers in the ant <Emphasis Type="Italic">Cataglyphis cursor</Emphasis>

In social hymenoptera, the reproductive division of labor is often linked to differences in individual body size with the reproductive caste (the queen) being larger than the workers. Likewise, the reproductive potential may vary with size within the worker caste and could affect the evolution of worker size in social insects. Here, we tested the relationship between worker size and reproductive potential in the facultative parthenogenetic ant Cataglyphis cursor. Colonies are headed by a multiply mated queen, but workers can produce gynes (virgin queens) and workers by thelytokous parthenogenesis after the queen's death. We observed the behaviour of workers (n = 357) until the production of gynes (212 h over 3 months) in an orphaned colony (mated queen not present). The size of workers was measured, and their paternal lineage determined using six microsatellite markers, to control for an effect of patriline. Larger workers were more likely to reproduce and lay more eggs indicating that individual level selection could take place. However, paternal lineage had no effect on the reproductive potential and worker size. From the behavioural and genetic data, we also show for the first time in this species, evidence of aggressive interactions among workers and a potential for nepotism to occur in orphaned colonies, as the five gynes produced belonged to a single paternal lineage.     

Fertility signals in the bumblebee Bombus terrestris (Hymenoptera: Apidae)

In eusocial Hymenoptera, queen control over workers is probably inseparable from the mechanism of queen recognition. In primitively eusocial bumblebees (Bombus), worker reproduction is controlled not only by the presence or absence of a dominant queen but also by other dominant workers. Furthermore, it was shown that the queen dominance is maintained by pheromonal cues. We investigated whether there is a similar odor signal released by egg-laying queens and workers that may have a function as a fertility signal. We collected cuticular surface extracts from nest-searching and breeding Bombus terrestris queens and workers that were characterized by their ovarian stages. In chemical analyses, we identified 61 compounds consisting of aldehydes, alkanes, alkenes, and fatty acid esters. Nest-searching queens and all groups of breeding females differed significantly in their odor bouquets. Furthermore, workers before the competition point (time point of colony development where workers start to develop ovaries and lay eggs) differed largely from queens and all other groups of workers. Breeding queens showed a unique bouquet of chemical compounds and certain queen-specific compounds, and the differences toward workers decrease with an increasing development of the workers' ovaries, hinting the presence of a reliable fertility signal. Among the worker groups, the smallest differences were found after the competition point. Egg-laying females contained higher total amounts of chemical compounds and of relative proportions of wax-type esters and aldehydes than nest-searching queens and workers before the competition point. Therefore, these compounds may have a function as a fertility signal present in queens and workers.     

Intrinsic worker mortality depends on behavioral caste and the queens’ presence in a social insect

According to the classic life history theory, selection for longevity depends on age-dependant extrinsic mortality and fecundity. In social insects, the common life history trade-off between fecundity and longevity appears to be reversed, as the most fecund individual, the queen, often exceeds workers in lifespan several fold. But does fecundity directly affect intrinsic mortality also in social insect workers? And what is the effect of task on worker mortality? Here, we studied how social environment and behavioral caste affect intrinsic mortality of ant workers. We compared worker survival between queenless and queenright Temnothorax longispinosus nests and demonstrate that workers survive longer under the queens’ absence. Temnothorax ant workers fight over reproduction when the queen is absent and dominant workers lay eggs. Worker fertility might therefore increase lifespan, possibly due to a positive physiological link between fecundity and longevity, or better care for fertile workers. In social insects, division of labor among workers is age-dependant with young workers caring for the brood and old ones going out to forage. We therefore expected nurses to survive longer than foragers, which is what we found. Surprisingly, inactive inside workers showed a lower survival than nurses but comparable to that of foragers. The reduced longevity of inactive workers could be due to them being older than the nurses, or due to a positive effect of activity on lifespan. Overall, our study points to behavioral caste-dependent intrinsic mortality rates and a positive association between fertility and longevity not only in queens but also in ant workers.     

Hierarchy length in orphaned colonies of the ant <Emphasis Type="Italic">Temnothorax nylanderi</Emphasis>

Workers of the ant Temnothorax nylanderi form dominance orders in orphaned colonies in which only one or a few top-ranking workers begin to produce males from unfertilized eggs. Between one and 11 individuals initiated 80% of all aggression in 14 queenless colonies. As predicted from inclusive fitness models (Molet M, van Baalen M, Monnin T, Insectes Soc 52:247–256, 2005), hierarchy length was found to first increase with colony size and then to level off at larger worker numbers. The frequency and skew of aggression decreased with increasing size, indicating that rank orders are less pronounced in larger colonies.     

A queen pheromone induces workers to kill sexual larvae in colonies of the red imported fire ant (<Emphasis Type="Italic">Solenopsis invicta</Emphasis>)

We conducted five bioassays to study how queens control the execution of sexual larvae by workers in colonies of the red imported fire ant, Solenopsis invicta. In each assay, subset colonies were made from many large polygyne colonies, and the 20 sexual larvae they contained were monitored over time. Sexual larvae mostly survived in queenless colonies, but were mostly killed in colonies with a single dealated queen, regardless of whether or not the queen was fertilized. The larvae were also killed when fresh corpses of queens were added to queenless colonies. Whereas acetone extracts of queens did not produce a significant increase in killings, extracts in buffered saline induced workers to execute most sexual larvae, indicating successful extraction of an execution pheromone. We identified the probable storage location of the chemical as the poison sac, and found both fresh (1 day) and old (21 day) extracts of poison sacs to be equally effective in inducing executions. The pheromone is stable at room temperature, perhaps because venom alkaloids also present in the extracts keep the pheromone from degrading. It is apparently either proteinaceous or associated with a proteinaceous molecule, a novel finding, as no queen pheromone of a proteinaceous nature has been previously demonstrated in ants.     

Nest- and colony-mate recognition in polydomous colonies of meat ants (Iridomyrmex purpureus)

Workers of polydomous colonies of social insects must recognize not only colony-mates residing in the same nest but also those living in other nests. We investigated the impact of a decentralized colony structure on colony- and nestmate recognition in the polydomous Australian meat ant (Iridomyrmex purpureus). Field experiments showed that ants of colonies with many nests were less aggressive toward alien conspecifics than those of colonies with few nests. In addition, while meat ants were almost never aggressive toward nestmates, they were frequently aggressive when confronted with an individual from a different nest within the same colony. Our chemical analysis of the cuticular hydrocarbons of workers using a novel comprehensive two-dimensional gas chromatography technique that increases the number of quantifiable compounds revealed both colony- and nest-specific patterns. Combined, these data indicate an incomplete transfer of colony odor between the nests of polydomous meat ant colonies.