The effects of honey bee (Apis mellifera L.) queen reproductive potential on colony growth

Reproduction in species of eusocial insects is monopolized by one or a few individuals, while the remaining colony tasks are performed by the worker caste. This reproductive division of labor is exemplified by honey bees (Apis mellifera L.), in which a single, polyandrous queen is the sole colony member that lays fertilized eggs. Previous work has revealed that the developmental fate of honey bee queens is highly plastic, with queens raised from younger worker larvae exhibiting higher measures in several aspects of reproductive potential compared to queens raised from older worker larvae. Here, we investigated the effects of queen reproductive potential (“quality”) on the growth and winter survival of newly established honey bee colonies. We did so by comparing the growth of colonies headed by “high-quality” queens (i.e., those raised from young worker larvae, which are more queen-like morphologically) to those headed by “low-quality” queens (i.e., those raised from older worker larvae, which are more worker-like morphologically). We confirmed that queens reared from young worker larvae were significantly larger in size than queens reared from old worker larvae. We also found a significant positive effect of queen grafting age on a colony’s production of worker comb, drone comb, and stored food (honey and pollen), although we did not find a statistically significant difference in the production of worker and drone brood, worker population, and colony weight. Our results provide evidence that in honey bees, queen developmental plasticity influences several important measures of colony fitness. Thus, the present study supports the idea that a honey bee colony can be viewed (at least in part) as the expanded phenotype of its queen, and thus selection acting predominantly at the colony level can be congruent with that at the individual level.     

Making a queen: an epigenetic analysis of the robustness of the honeybee (Apis mellifera) queen developmental pathway

Specialized castes are considered a key reason for the evolutionary and ecological success of the social insect lifestyle. The most essential caste distinction is between the fertile queen and the sterile workers. Honeybee (Apis mellifera) workers and queens are not genetically distinct, rather these different phenotypes are the result of epigenetically regulated divergent developmental pathways. This is an important phenomenon in understanding the evolution of social insect societies. Here, we studied the genomic regulation of the worker and queen developmental pathways, and the robustness of the pathways by transplanting eggs or young larvae to queen cells. Queens could be successfully reared from worker larvae transplanted up to 3 days age, but queens reared from older worker larvae had decreased queen body size and weight compared with queens from transplanted eggs. Gene expression analysis showed that queens raised from worker larvae differed from queens raised from eggs in the expression of genes involved in the immune system, caste differentiation, body development and longevity. DNA methylation levels were also higher in 3‐day‐old queen larvae raised from worker larvae compared with that raised from transplanted eggs identifying a possible mechanism stabilizing the two developmental paths. We propose that environmental (nutrition and space) changes induced by the commercial rearing practice result in a suboptimal queen phenotype via epigenetic processes, which may potentially contribute to the evolution of queen–worker dimorphism. This also has potentially contributed to the global increase in honeybee colony failure rates.     

The influence of queen age and quality during queen replacement in honeybee colonies

Honeybee, Apis mellifera, colonies replace their queens by constructing many queen cells and then eliminating supernumerary queens until only one remains. The ages of the queens and the variation in their reproductive potential are important factors in the outcome of such events. Selection would favour colonies that requeen as quickly as possible to minimize the brood hiatus, therefore selecting for queens reared from older larvae. Conversely, reproductive potential (queen 'quality') is maximized by rearing queens from younger larvae. This potential trade-off was tested during two phases of queen replacement, namely queen rearing and polygyny reduction. Our results suggest that queen age is a significant element during both queen rearing and polygyny reduction, whereas queen quality, at least to the magnitude tested in this experiment, has little impact on the outcome of either process. The rate of queen replacement therefore appears to be an important factor in the honeybee life cycle, and further mechanisms of potential importance during this life history transition are discussed. Copyright 2000 The Association for the Study of Animal Behaviour.     

Absence of reproductive conflict during queen rearing in Apis cerana

Polyandry in honeybee queens (Apis) causes many patrilines (subfamilies) within a colony, which may lead to a potential conflict of interest among workers. This may be most apparent during queen rearing when nepotistic worker behavior could influence the genetics of future generations. Several studies have searched for such conflict in European honeybees (A. mellifera), but studies on other Apis species remain lacking. We investigated the presence of reproductive conflict in A. cerana japonica by comparing the patriline proportion of queen larvae to that of adult workers. We determined the patrilines of 272 workers and 57 queen larvae using four polymorphic microsatellite markers that were sampled from queenless colonies originally derived from four naturally mated queen-right colonies. The number of patrilines in each colony was 9, 12, 8, and 7, respectively, which is lower than that observed in continental Asia. We found no difference in patriline proportion between adult workers and queen larvae. Our data support neither genetic variance for royalty or existence of worker nepotism in A. cerana japonica.     

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.     

Asexually produced Cape honeybee queens (Apis mellifera capensis) reproduce sexually

Unmated workers of the Cape honeybee Apis mellifera capensis can produce female offspring including daughter queens. As worker-laid queens are produced asexually, we wondered whether these asexually produced individuals reproduce asexually or sexually. We sampled 11 colonies headed by queens known to be the clonal offspring of workers and genotyped 23 worker offspring from each queen at 5 microsatellite loci. Without exception, asexually produced queens produced female worker offspring sexually. In addition, we report the replacement of a queen by her asexually produced granddaughter, with this asexually produced queen also producing offspring sexually. Hence, once a female larva is raised as a queen, mating and sexual reproduction appears to be obligatory in this subspecies, despite the fact that worker-laid queens are derived from asexual lineages.     

Cheaters sometimes prosper: targeted worker reproduction in honeybee (Apis mellifera) colonies during swarming

Kin selection theory predicts that honeybee (Apis mellifera) workers should largely refrain from producing their own offspring, as the workers collectively have higher inclusive fitness if they rear the sons of their mother, the queen. Studies that have quantified levels of ovary activation and reproduction among workers have largely supported this prediction. We sampled pre‐emergent male pupae and adult workers from seven colonies at regular intervals throughout the reproductive part of the season. We show that the overall contribution of workers to male (drone) production is 4.2%, nearly 40 times higher than is generally reported, and is highest during reproductive swarming, when an average of 6.2% of the males genotyped are worker‐produced. Similarly, workers in our samples were 100 times more likely to have active ovaries than previously assumed. Worker reproduction is seasonally influenced and peaks when colonies are rearing new queens. Not all worker subfamilies contribute equally to reproduction. Instead, certain subfamilies are massively over‐represented in drone brood. By laying eggs within the period in which many colonies produce virgin queens, these rare worker subfamilies increase their direct fitness via their well‐timed sons.     

Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees

The mandibular glands of queen honeybees produce a pheromone that modulates many aspects of worker honeybee physiology and behavior and is critical for colony social organization. The exact chemical blend produced by the queen differs between virgin and mated, laying queens. Here, we investigate the role of mating and reproductive state on queen pheromone production and worker responses. Virgin queens, naturally mated queens, and queens instrumentally inseminated with either semen or saline were collected 2 days after mating or insemination. Naturally mated queens had the most activated ovaries and the most distinct chemical profile in their mandibular glands. Instrumentally inseminated queens were intermediate between virgins and naturally mated queens for both ovary activation and chemical profiles. There were no significant differences between semen- and saline-inseminated queens. Workers were preferentially attracted to the mandibular gland extracts from queens with significantly more activated ovaries. These studies suggest that the queen pheromone blend is modulated by the reproductive status of the queens, and workers can detect these subtle differences and are more responsive to queens with higher reproductive potential. Furthermore, it appears as if insemination substance does not strongly affect physiological characteristics of honeybee queens 2 days after insemination, suggesting that the insemination process or volume is responsible for stimulating these early postmating changes in honeybee queens.     

Age at Which Larvae Are Orphaned Determines Their Development into Typical or Rebel Workers in the Honeybee (Apis mellifera L.)

In the honeybee, diploid larvae fed with royal jelly develop into reproductive queens, whereas larvae fed with royal jelly for three days only and subsequently with honey and pollen develop into facultatively sterile workers. A recent study showed that worker larvae fed in a queenless colony develop into another female polyphenic form: rebel workers. These rebel workers are more queenlike and have greater reproductive potential than normal workers. However, it was unclear whether larvae orphaned at any time during their feeding period can develop into rebels. To answer this question, the anatomical features of newly emerged workers reared in queenless conditions at different ages during the larval period were evaluated. Our results showed that larvae orphaned during the final four or more days of their feeding life develop into rebel workers with more ovarioles in their ovaries, smaller hypopharyngeal glands, and larger mandibular and Dufour’s glands compared with typical workers with low reproductive potential that were reared with a queen or orphaned at the third to last or a later day of feeding life.     

Significant reproductive skew in the facultatively polygynous ant Pheidole pallidula

Reproductive skew - the extent to which reproduction is unevenly shared between individuals in a social group - varies greatly between and within animal species. In this study, we investigated how queens share parentage in polygynous (multiple queen) colonies of the Mediterranean ant Pheidole pallidula. We used highly polymorphic microsatellites markers to determine parentage of gynes (new queens), males and workers in P. pallidula field colonies. The comparison of the genotypes of young and adult workers revealed a very low queen turnover (less than 2%). The first main finding of the study of reproductive skew in these colonies was that there was a significant departure from equal contribution of queens to gyne, male and worker production. Reproductive skew was greater for male production than for queen and worker production. There was no relationship between the magnitude of the reproductive skew and the number of reproductive queens per colony, their relatedness and the overall colony productivity, some of the factors predicted to influence the extent of reproductive skew. Finally, our study revealed for the first time a trade-off in the relative contribution of nestmate queens to gyne and worker production. The queens contributing more to gyne production contributed significantly less to worker production.     

Caste-specific postembryonic development of primary and secondary olfactory centers in the female honeybee brain

Eusocial insects are characterized by division of labor among a sterile worker caste and a reproductive queen. In the honeybee both female castes are determined postembryonically by environmental factors, and queens develop substantially faster than workers. Since olfaction plays a crucial role in organizing honeybee behavior and social interactions, we compared the development of primary and secondary olfactory centers in the brain. Age-synchronized queen and worker pupae were raised in incubators at 34.5 degrees C, and their external morphology was characterized for all pupal stages. The development of olfactory synaptic neuropil was analyzed using anti-synapsin immunocytochemistry, f-actin-phalloidin labeling and confocal microscopy. In the antennal lobes of queens olfactory glomeruli formed approximately 4 days earlier than in workers. The adult number of olfactory glomeruli was in a similar range, but the total glomerular volume was slightly smaller in queens. Olfactory and visual subdivisions (lip, collar) of the mushroom-body calyx formed early, whereas the basal ring separated late. Synaptic microglomeruli in the olfactory lip were established approximately 3-4 days earlier in queens compared to workers. We propose that developmental heterochrony results in fewer synapses in olfactory centers (smaller glomeruli, fewer microglomeruli) in queens, which may result in poorer performance on olfactory learning tasks compared to workers.     

mRNA expression and DNA methylation in three key genes involved in caste differentiation in female honeybees(Apis mellifera)

In honeybee(Apis mellifera)colonies,queens and workers are alternative forms of the adult female honeybee that develop from genetically identical zygotes but that depend on differential nourishment.Queens and workers display distinct morphologies,anatomies and behavior,better known as caste differentiation.Despite some basic insights,the exact mechanism responsible for this phenomenon,especially at the molecular level,remains unclear although some progress has been achieved.In this study,we examined mRNA levels of the TOR(target of rapamycin)and Dnmt3(DNA methyltransferase 3)genes,closely related to caste differentiation in honeybees.We also investigated mRNA expression of the S6K(similar to RPS6-p70-protein kinase)gene linked closely to organismal growth and development in queen and worker larvae(1-day and 3-day old).Last,we investigated the methylation status of these three genes in corresponding castes.We found no difference in mRNA expression for the three genes between 1st instar queen and worker larvae;however,3rd instar queen larvae had a higher level of TOR mRNA than worker larvae.Methylation levels of all three genes were lower in queen larvae than worker larvae but the differences were not statistically significant.These findings provide basic data for broadening our understanding of caste differentiation in female honeybees.     

Effects of social conditions on Juvenile Hormone mediated reproductive development in Bombus terrestris workers

Abstract. During the annual life cycle of the bumble bee Bombus terrestris (L.) colony, there is a stage characterized by worker reproduction in the presence of the queen. It has been proposed that this is a result of a decrease in queen inhibition. This hypothesis was examined by studying the effects of queens taken from colonies at different stages of development on several aspects of worker physiology and behaviour: rates of Juvenile Hormone (JH) release in vitro , ovary development, and behaviour associated with reproduction. After optimizing and validating the radiochemical assay for JH release for bumble bee workers, we found that queenless workers had significantly more developed ovaries and higher rates of release of JH than did queenright workers, confirming and extending previous findings that suggest that bumblebee ovarian development is under JH control. Mated queens, separated from their colony and brood, can have the same inhibitory effect on the reproductive development of callow workers. In contrast, workers confined with virgin queens or in queenless groups demonstrated a significantly higher rate of release of JH, overt aggression and threatening behaviours. However, there were no differences in rates of release of JH between workers confined in groups in the laboratory with queens taken from colonies either before or after the onset of worker reproduction. Furthermore, overt aggression and threatening behaviours were similar and low in both types of groups. These results gave no support to the hypothesis that a decrease in queen inhibition is associated with the onset of worker reproduction. We also show that young workers reared in colonies either before or after worker reproduction occurs, or in queenless colonies, all demonstrated similar, low rates of release of JH. These results suggest that older workers may inhibit the corpora allata of younger workers in queenless colonies.     

Effect of honey bee queen mating condition on worker ovary activation

The presence of the honey bee queen reduces worker ovary activation. When the queen is healthy and fecund, this is interpreted as an adaptive response as workers can gain fitness from helping the queen raise additional offspring, their sisters. However, when the queen is absent, workers activate their ovaries and lay unfertilized eggs that become males. Queen pheromones are recognised as a factor affecting worker ovary activation. Recent work has shown that queen mandibular pheromone composition changes with queen mating condition and workers show different behavioural responses to pheromone extracts from these queens. Here, we tested whether workers reared in colonies with queens of different mating condition varied in level of ovary activation. We also examined the changes in the chemical composition of the queen mandibular glands to determine if the pheromone blend varied among the queens. We found that the workers activated their ovaries when queens were unmated and had lower ovary activation when raised with mated queens, suggesting that workers detect and respond adaptively to queens of differing mating status. Moreover, variation in queen mandibular gland’s chemical composition correlated with the levels of worker ovary activation. Although correlative, this evidence suggests that queen pheromone may act as a signal of queen mating condition for workers, in response to which they alter their level of ovary activation.     

Maternity of emergency queens in the Cape honey bee,Apis mellifera capensis

During reproductive swarming, some workers of the Cape honey bee, Apis mellifera capensis, lay eggs in queen cells, many of which are reared to maturity. However, it is unknown if workers are able to lay in queen cells immediately after queen loss during an episode of emergency queen rearing. In this study we experimentally de‐queened colonies and determined the maternity of larvae and pupae that were reared as queens. This allowed us to determine how soon after queen loss workers contribute to the production of new queens. We were further interested to see if workers would preferentially raise new queens from queen‐laid brood if this was introduced later. We performed our manipulations in two different settings: an apiary setting where colonies were situated close together and a more natural situation in which the colonies were well separated. This allowed us to determine how the vicinity of other colonies affects the presence of parasites. We found that workers do indeed contribute to queen cell production immediately after the loss of their queen, thus demonstrating that some workers either have activated ovaries even when their colony has a queen or are able to activate their ovaries extremely rapidly. Queen‐laid brood introduced days after queen loss was ignored, showing that workers do not prefer to raise new queens from queen brood when given a choice. We also detected non‐natal parasitism of queen cells in both settings. We therefore conclude that some A. m. capensis genotypes specialize in parasitizing queen cells.     

Genome-wide analysis reveals differences in brain gene expression patterns associated with caste and reproductive status in honey bees (Apis mellifera)

A key characteristic of eusocial species is reproductive division of labour. Honey bee colonies typically have a single reproductive queen and thousands of sterile workers. Adult queens differ dramatically from workers in anatomy, physiology, behaviour and lifespan. Young female workers can activate their ovaries and initiate egg laying; these 'reproductive' workers differ from sterile workers in anatomy, physiology, and behaviour. These differences, however, are on a much smaller scale than those observed between the queen and worker castes. Here, we use microarrays to monitor expression patterns of several thousand genes in the brains of same-aged virgin queens, sterile workers, and reproductive workers. We found large differences in expression between queens and both worker groups (~2000 genes), and much smaller differences between sterile and reproductive workers (221 genes). The expression patterns of these 221 genes in reproductive workers are more queen-like, and may represent a core group of genes associated with reproductive physiology. Furthermore, queens and reproductive workers preferentially up-regulate genes associated with the nurse bee behavioural state, which supports the hypothesis of an evolutionary link between worker division of labour and molecular pathways related to reproduction. Finally, several functional groups of genes associated with longevity in other species are significantly up-regulated in queens. Identifying the genes that underlie the differences between queens, sterile workers, and reproductive workers will allow us to begin to characterize the molecular mechanisms underlying the evolution of social behaviour and large-scale remodelling of gene networks associated with polyphenisms.     

The genetic structure of swarms and the timing of their production in the queen cycles of neotropical wasps

Kin selection theory has received some of its strongest support from analyses of within-colony conflicts between workers and queens in social insects. One of these conflicts involves the timing of queen production. In neotropical wasps, new queens are only produced by colonies with just one queen while males are produced by colonies with more queens, a pattern favoured by worker interests. We now show that new colonies, or swarms, have few queens and variable within-colony relatednesses which means that their production is not tied to new queen production. The queens in these swarms are seldom the mothers of the workers in the swarm. Therefore, either colonies producing swarms have very many queens, or queens joining daughter swarms are reproductive losers on the original colonies. As new colony production is not linked to queen production, it can occur at the ecologically optimum time, i.e. the rainy season. This disassociation between queen production and new colony production allows worker interests in sex ratios to prevail without hampering new colony production at the most favourable season, an uncoupling that may contribute to the ecological success of the Epiponini.     

Absence of nepotism in the harassment of duelling queens by honeybee workers

Nepotism shapes interactions among the members of almost every animal society. However, clear evidence of nepotism within highly cooperative insect societies, such as ant, wasp and honeybee colonies, is rare. Recent empirical findings suggest that nepotism occurs within honeybee colonies where kin-selection theory most strongly predicts its existence: during the lethal queen-queen duels that determine which of several young queens will become the colony's next queen. In this study, I test whether worker bees act nepotistically by hindering duelling queens that are distantly related to themselves. I accomplished this by observing labelled workers harassing duelling queen bees in observation hives and subsequently by determining worker-queen relatedness using DNA microsatellites. I show that the workers that harassed duelling queens were neither more-closely nor more-distantly related to them than were workers selected randomly from the colony. Thus, workers did not behave nepotistically by hindering half-sister queens more than full-sister queens. These results demonstrate that under certain conditions, natural selection limits the evolution of nepotism within animal societies despite strong theoretical predictions for its existence.     

Attraction and Repellence of Workers by the Honeybee Queen (Apis mellifera L.)

The spatial distribution of worker honeybees in colonies of two African subspecies ( Apis mellifera capensis and Apis mellifera scutellata ), as well as their natural hybrids, was determined in five observation colonies, each containing one frame. The queens were allowed to roam freely throughout the hive during the initial phase of the experiment, and were observed on both sides of the frame in all colonies. In the second phase of the experiment the queen was caged on one side of the frame in three of the observation colonies, the other two colonies serving as controls. Queen caging significantly affected the distribution of worker bees, with more A. m. scutellata workers being attracted to the queen and more A. m. capensis worker bees being repelled by the presence of the queen. The hybrid workers were also repelled, but to a lesser extent. Queens thus not only attract workers to form a retinue or during swarming but also repel workers in the nest. Evasion of the reproductive suppression by the queen pheromones may be a typical behavior for workers with a high reproductive potential.     

Influence of queen weight and colony origin on worker response in Solenopsis geminata

Abstract. The influence of weight and colony origin of the queen of Solenopsis geminata (F.) (Hymenoptera: Formicidae) on worker attraction is studied under laboratory conditions. In the first experiment, worker response to individual queens of different weight from the same colony is evaluated. Heavier queens are more attractive than smaller queens to their own workers. In subsequent experiments, the colony origin effect is investigated and worker response to a pair of queens of the same weight from the same or different colonies is compared. When queens are from the same colony, workers do not show a significant preference between queens. However, when queens are from a different colony, workers are significantly more attracted to their own queen than to the foreign queen. Finally, the response of workers to queens of different weight from the same or different colonies is investigated. In both cases, workers are significantly more attracted to a heavier queen than a lighter queen, even if the lighter queen is their own queen. A putative pheromonal component (E)‐6‐(1‐pentenyl)‐2H‐2‐pyranone, is not positively correlated with queen weight.