Gamergates in the myrmicine genus <Emphasis Type="Italic">Metapone</Emphasis> (Hymenoptera:Formicidae)

Gamergates (i.e. mated reproductive workers) are mostly known from ant species within the Ponerinae. We report here the discovery of gamergates in two species of the subfamily Myrmicinae. Until now, mated reproductive females in colonies of myrmicine species have been considered morphologically distinct from the worker caste. However, in two species of the myrmicine Metapone (Metapone madagascarica and a newly discovered, not yet described Metapone species) all workers have six ovarioles and a spermatheca; and some of them are mated. There are no morphological differences between mated and non-mated workers. Field observations and laboratory studies indicate that colonies of the Metapone species can reproduce with gamergates only.     

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.     

Small queens and big-headed workers in a monomorphic ponerine ant

Evolution of caste is a central issue in the biology of social insects. Comparative studies on their morphology so far suggest the following three patterns: (1) a positive correlation between queen–worker size dimorphism and the divergence in reproductive ability between castes, (2) a negative correlation among workers between morphological diversity and reproductive ability, and (3) a positive correlation between queen–worker body shape difference and the diversity in worker morphology. We conducted morphological comparisons between castes in Pachycondyla luteipes, workers of which are monomorphic and lack their reproductive ability. Although the size distribution broadly overlapped, mean head width, head length, and scape length were significantly different between queens and workers. Conversely, in eye length, petiole width, and Weber’s length, the size differences were reversed. The allometries (head length/head width, scape length/head width, and Weber’s length/head width) were also significantly different between queens and workers. Morphological examinations showed that the body shape was different between queens and workers, and the head part of workers was disproportionately larger than that of queens. This pattern of queen–worker dimorphism is novel in ants with monomorphic workers and a clear exception to the last pattern. This study suggests that it is possible that the loss of individual-level selection, the lack of reproductive ability, influences morphological modification in ants.     

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.     

Genomewide analysis indicates that queen larvae have lower methylation levels in the honey bee (Apis mellifera)

The honey bee is a social insect characterized by caste differentiation, by which a young larva can develop into either a queen or a worker. Despite possessing the same genome, queen and workers display marked differences in reproductive capacity, physiology, and behavior. Recent studies have shown that DNA methylation plays important roles in caste differentiation. To further explore the roles of DNA methylation in this process, we analyzed DNA methylome profiles of both queen larvae (QL) and worker larvae (WL) of different ages (2, 4, and 6 day old), by using methylated DNA immunoprecipitation-sequencing (meDIP-seq) technique. The global DNA methylation levels varied between the larvae of two castes. DNA methylation increased from 2-day- to 4-day-old QL and then decreased in 6-day-old larvae. In WL, methylation levels increased with age. The methylcytosines in both larvae were enriched in introns, followed by coding sequence (CDS) regions, CpG islands, 2 kbp downstream and upstream of genes, and 5′ and 3′ untranslated regions (UTRs). The number of differentially methylated genes (DMGs) in 2-, 4-, and 6-day-old QL and WL was 725, 3,013, and 5,049, respectively. Compared to 4- and 6-day-old WL, a large number of genes in QL were downmethylated, which were involved in many processes including development, reproduction, and metabolic regulation. In addition, some DMGs were concerned with caste differentiation.     

No facultative worker policing in the honey bee (Apis mellifera L.)

Kin selection theory predicts that in colonies of social Hymenoptera with multiply mated queens, workers should mutually inhibit (“police”) worker reproduction, but that in colonies with singly mated queens, workers should favor rearing workers’ sons instead of queens’ sons. In line with these predictions, Mattila et al. (Curr Biol 22:2027–2031, 2012) documented increased ovary development among workers in colonies of honey bees with singly mated queens, suggesting that workers can detect and respond adaptively to queen mating frequency and raising the possibility that they facultative police. In a follow-up experiment, we test and reject the hypothesis that workers in single-patriline colonies prefer worker-derived males and are able to reproduce directly; we show that their eggs are policed as strongly as those of workers in colonies with multiply mated queens. Evidently, workers do not respond facultatively to a kin structure that favors relaxed policing and increased direct reproduction. These workers may instead be responding to a poor queen or preparing for possible queen loss.     

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.     

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.     

Queen movement during colony emigration in the facultatively polygynous ant Pachycondyla obscuricornis

In ants, nest relocations are frequent but nevertheless perilous, especially for the reproductive caste. During emigrations, queens are exposed to predation and face the risk of becoming lost. Therefore the optimal strategy should be to move the queen(s) swiftly to a better location, while maintaining maximum worker protection at all times in the new and old nests. The timing of that event is a crucial strategic issue for the colony and may depend on queen number. In monogynous colonies, the queen is vital for colony survival, whereas in polygynous colonies a queen is less essential, if not dispensable. We tested the null hypothesis that queen movement occurs at random within the sequence of emigration events in both monogynous and polygynous colonies of the ponerine ant Pachycondyla obscuricornis. Our study, based on 16 monogynous and 16 polygynous colony emigrations, demonstrates for the first time that regardless of the number of queens per colony, the emigration serial number of a queen occurs in the middle of all emigration events and adult ant emigration events, but not during brood transport events. It therefore appears that the number of workers in both nests plays an essential role in the timing of queen movement. Our results correspond to a robust colony-level strategy since queen emigration is related neither to colony size nor to queen number. Such an optimal strategy is characteristic of ant societies working as highly integrated units and represents a new instance of group-level adaptive behaviors in social insect colonies.     

Alternative mating behaviors of the queen polymorphic ant <Emphasis Type="Italic">Temnothorax longispinosus</Emphasis>

Mating behaviors of ants fall into two categories: female calling, in which a female alate releases pheromones that attract males, and male swarming, in which large male aggregations attract females. Female calling is common in species with queens that return to their natal nest to found colonies dependently after mating, while male swarming is common in species with queens that disperse to found independently. In some species that display both founding strategies, a queen-size polymorphism has evolved in which dependent-founding queens are smaller than independent-founding queens. Dependent founding is likely difficult if gynes (virgin queens) are mating in distant swarms. Therefore, a queen may adopt one or the other mating strategy based on its size and founding behavior. We investigated mating behaviors in the queen-polymorphic ant, Temnothorax longispinosus. Observations in laboratory mating arenas indicated that small gynes exhibited significantly lower flight activity than large gynes. Both forms mated in male swarms, and neither form exhibited female calling. The reduced flight activity of the small morph may facilitate returning to the natal nest after mating, provided the mating swarm is located nearby. Therefore, alternative colony-founding behaviors may be possible without the evolution of female-calling behavior; however, the reduced flight activity of small morphs may require that mating swarms are not distant from the natal nest.     

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.     

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.     

Insights into the role of age and social interactions on the sexual attractiveness of queens in an eusocial bee, <Emphasis Type="Italic">Melipona flavolineata</Emphasis> (Apidae,Meliponini)

The attraction of sexual partners is a vital necessity among insects, and it involves conflict of interests and complex communication systems among male and female. In this study, we investigated the developing of sexual attractiveness in virgin queens (i.e., gynes) of Melipona flavolineata, an eusocial stingless bee. We followed the development of sexual attractiveness in 64 gynes, belonging to seven age classes (0, 3, 6, 9, 15, 18 days post-emergence), and we also evaluated the effect of different social interactions (such as competition between queens and interactions with workers) on the development of attractiveness in other 60 gynes. We used the number of males that tried to mate with a focal gyne as a representative variable of its sexual attractiveness. During the essays, each gyne was individually presented to 10 sexually mature males, and during 3 min, we counted the number of males that everted their genitalia in response to the presence of a gyne. Here, we show that M. flavolineata gynes are capable to (i) maintain their sexual attractiveness for long periods through adult life, (ii) they need a minimum social interaction to trigger the development of sexual attractiveness, and (iii) that gynes express this trait only within a social context. We conclude that the effective occurrence of matings is conditional on potential social interactions that gynes experienced before taking the nuptial flight, when they are still in the nest. These findings bring insights into the factors determining reproductive success in social insects.     

Rare royal families in honeybees, Apis mellifera

The queen is the dominant female in the honeybee colony, Apis mellifera, and controls reproduction. Queen larvae are selected by the workers and are fed a special diet (royal jelly), which determines caste. Because queens mate with many males a large number of subfamilies coexist in the colony. As a consequence, there is a considerable potential for conflict among the subfamilies over queen rearing. Here we show that honeybee queens are not reared at random but are preferentially reared from rare “royal” subfamilies, which have extremely low frequencies in the colony's worker force but a high frequency in the queens reared.     

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.     

Winged queens replaced by reproductives smaller than workers in <Emphasis Type="Italic">Mystrium</Emphasis> ants

In ants, winged queens that are specialized for independent colony foundation can be replaced by wingless reproductives better adapted for colony fission. We studied this shift in reproductive strategy by comparing two Mystrium species from Madagascar using morphometry, allometry and dissections. Mystrium rogeri has a single dealate queen in each colony with a larger thorax than workers and similar mandibles that allow these queens to hunt during non-claustral foundation. In contrast, Mystrium ‘red’ lacks winged queens and half of the female adults belong to a wingless ‘intermorph’ caste smaller and allometrically distinct from the workers. Intermorphs have functional ovaries and spermatheca while those of workers are degenerate. Intermorphs care for brood and a few mate and reproduce making them an all-purpose caste that takes charge of both work and reproduction. However, their mandibles are reduced and inappropriate for hunting centipedes, unlike the workers’ mandibles. This together with their small thorax disallow them to perform independent colony foundation, and colonies reproduce by fission. M. rogeri workers have mandibles polymorphic in size and shape, which allow for all tasks from brood care to hunting. In M. ‘red’, colonial investment in reproduction has shifted from producing expensive winged queens to more numerous helpers. M. ‘red’ intermorphs are the first case of reproductives smaller than workers in ants and illustrate their potential to diversify their caste system for better colonial economy.     

Queen regulates biogenic amine level and nestmate recognition in workers of the fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Nestmate recognition is a critical element in social insect organization, providing a means to maintain territoriality and close the colony to parasites and predators. Ants detect the colony chemical label via their antennae and respond to the label mismatch of an intruder with aggressive behavior. In the fire ant, Solenopsis invicta, worker ability to recognize conspecific nonnestmates decreases if the colony queen is removed, such that they do not recognize conspecific nonnestmates as different. Here, we tested the hypothesis that the presence of the colony queen influences the concentration of octopamine, a neuromodulator, in worker ants, which in turn has an effect on nestmate recognition acuity in workers. We demonstrate that queenless workers exhibit reduced brain octopamine levels and reduced discriminatory acuteness; however, feeding queenless workers octopamine restored both. Dopamine levels are influenced by honeybee queen pheromones; however, levels of this biogenic amine were unchanged in our experiments. This is the first demonstration of a link between the presence of the colony queen, a worker biogenic amine, and conspecific nestmate recognition, a powerful expression of colony cohesion and territoriality.     

Irregular brood patterns and worker reproduction in social wasps

The potential for reproductive conflict among colony members exists in all social insect societies. For example, queens and workers may be in conflict over the production of males within colonies. Kin selection theory predicts that in a colony headed by a multiply-mated queen, worker reproduction is prevented by worker policing in the form of differential oophagy. However, few studies have demonstrated that workers actually lay eggs within queenright colonies. The purpose of this study was to determine if workers laid male eggs within unmanipulated queen-right colonies of the polyandrous social wasps Vespula maculifrons and V. squamosa. We focused our analysis on an unusual brood pattern within colonies, multiple egg cells. We were primarily interested in determining if individuals reared in these irregular circumstances were queen or worker offspring. To address this question, we genotyped 318 eggs from eight V. maculifrons and two V. squamosa colonies. No worker‑reproduction was detected in any of the queenright colonies; all of the eggs found in multiple egg cells were consistent with being queen‑produced. However, the frequency of multiple egg cells differed among colonies, suggesting that queens vary in the frequency of errors they make when laying eggs within cells. Finally, we suggest that workers may not be laying eggs within queenright colonies and that worker reproduction may be controlled through mechanisms other than differential oophagy in polyandrous Vespula wasps.     

Signaling hunger through aggression—the regulation of foraging in a primitively eusocial wasp

Primitively eusocial wasps are generally headed by behaviorally dominant queens who use their aggression to suppress worker reproduction. In contrast, queens in the primitively eusocial wasp Ropalidia marginata are strikingly docile and non-aggressive. However, workers exhibit dominance-subordinate interactions among themselves. These interactions do not appear to reflect reproductive competition because there is no correlation between the relative position of an individual in the dominance hierarchy of the colony and the likelihood that she will succeed a lost/removed queen. Based on the observation that foraging continues unaltered in the absence of the queen and the correlation between dominance behavior and foraging, we have previously suggested that dominance-subordinate interactions among workers in R. marginata have been co-opted to serve the function of decentralized, self-organized regulation of foraging. This idea has been supported by an earlier experimental study where it was found that a reduced demand for food led to a significant decrease in dominance behavior. In this study, we perform the converse experiment, demonstrate that dominance behavior increases under conditions of starvation, and thus provide further evidence in support of the hypothesis that intranidal workers signal hunger through aggression.