Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation.

The genome sequencing of several bee species, and the development of functional genomics tools, paved the way for understanding the fascinating behaviors of bees in molecular terms. Here we review recent progress in research on the hormonal regulation of bee behavior, with emphasis on two key insect hormones: Juvenile hormones (JH) and ecdysteroids (Ec). We discuss recent progress in deciphering the molecular bases for JH regulation of gene expression in the nervous system and other tissues. The patterns of JH-dependent changes in gene expression show many similarities across tissues, which are associated with the effects of JH on worker task allocation. Ec, which have previously been studied mainly in the context of insect development, now appear to also play imortant roles in the regulation of many molecular processes in the brain that are asociated with bee behavior. Finally, we discuss the possibility that JH-signaling and Ec-signaling pathways interact to shape the complex behavioral repertoire of bees.

Small cysteine-rich peptides have diverse functions in insects including antimicrobial defense, phenoloxidase activity regulation, and toxic inhibition of ion channels of prey or predator. We combined bioinformatics and measurements of transcript abundance to start characterizing AmOCLP1, a recently discovered Inhibitor Cysteine Knot peptide in the honey bee Apis mellifera. We found that the genomes of ants, bees, and the wasp Nasonia vitripennis encode orthologous sequences indicating that OCLP1 is a conserved peptide and not unique to the honey bee. Search of available EST libraries and quantitative real time PCR analyses indicate that the transcript of AmOCLP1 is ubiquitous with expression in life stages ranging from embryos to adults and in all tested tissues. In worker honey bees AmOCLP1 expression was not associated with age or task and did not show clear enrichment in any of the tested tissues. There was however a consistent trend toward higher transcript levels in the abdomen of foragers relative to levels in the head or thorax, and compared to levels in the abdomen of younger worker bees. By contrast, in drones AmOCLP1 transcript levels appeared higher in the head relative to the abdomen. Finer analyses of the head and abdomen indicated that the AmOCLP1 transcript is not enriched in the stinger and the associated venom sac or in cephalic exocrine glands. The evolutionary conservation in the Hymenoptera, the ubiquitous expression, and the lack of enrichment in the venom gland, stinger, exocrine glands, and the brain are not consistent with the hypotheses that OCLP1 is a secreted honeybee toxin or an endotoxin acting in the central nervous system. Rather we hypothesize that OCLP1 is a conserved antimicrobial or phenoloxidase inhibitor peptide. (C) 2014 Elsevier Ltd. All rights reserved.

The evolution of advanced sociality in bees is associated with apparent modifications in juvenile hormone (JH) signaling. By contrast to most insects in which JH is a gonadotropin regulating female fertility, in the highly eusocial honey bee (Apis mellifera) JH has lost its gonadotrophic function in adult females, and instead regulates age-related division of labor among worker bees. In order to shed light on the evolution of JH signaling in bees we performed allatectomy and replacement therapies to manipulate JH levels in workers of the ``primitively eusocial'' bumblebee Bombus terrestris. Allatectomized worker bees showed remarkable reduction in ovarian development, egg laying, Vitellogenin and Kruppel homolog 1 fat body transcript levels, hemolymph Vitellogenin protein abundance, wax secretion, and egg-cell construction. These effects were reverted, at least partially, by treating allatectomized bees with JH-III, the natural JH of bees. Allatectomy also affected the amount of ester component in Dufour's gland secretion, which is thought to convey a social signal relating to worker fertility. These findings provide a strong support for the hypothesis that in contrast to honey bees, JH is a gonadotropin in bumblebees and lend credence to the hypothesis that the evolution of advanced eusociality in honey bees was associated with major modifications in JH signaling.

Sibling care is a hallmark of social insects, but its evolution remains challenging to explain at the molecular level. The hypothesis that sibling care evolved from ancestral maternal care in primitively eusocial insects has been elaborated to involve heterochronic changes in gene expression. This elaboration leads to the prediction that workers in these species will show patterns of gene expression more similar to foundress queens, who express maternal care behaviour, than to established queens engaged solely in reproductive behaviour. We tested this idea in bumblebees (Bombus terrestris) using a microarray platform with approximately 4500 genes. Unlike the wasp Polistes metricus, in which support for the above prediction has been obtained, we found that patterns of brain gene expression in foundress and queen bumblebees were more similar to each other than to workers. Comparisons of differentially expressed genes derived from this study and gene lists from microarray studies in Polistes and the honeybee Apis mellifera yielded a shared set of genes involved in the regulation of related social behaviours across independent eusocial lineages. Together, these results suggest that multiple independent evolutions of eusociality in the insects might have involved different evolutionary routes, but nevertheless involved some similarities at the molecular level.

Circadian rhythms are ubiquitous in many organisms. Animals that are forced to be active around the clock typically show reduced performance, health and survival. Nevertheless, we review evidence of animals showing prolonged intervals of activity with attenuated or nil overt circadian rhythms and no apparent ill effects. We show that around-the-clock and ultradian activity patterns are more common than is generally appreciated, particularly in herbivores, in animals inhabiting polar regions and habitats with constant physical environments, in animals during specific life-history stages (such as migration or reproduction), and in highly social animals. The underlying mechanisms are diverse, but studies suggest that some circadian pacemakers continue to measure time in animals active around the clock. The prevalence of around-the-clock activity in diverse animals and habitats, and an apparent diversity of underlying mechanisms, are consistent with convergent evolution. We suggest that the basic organizational principles of the circadian system and its complexity encompass the potential for chronobiological plasticity. There may be trade-offs between benefits of persistent daily rhythms versus plasticity, which for reasons still poorly understood make overt daily arrhythmicity functionally adaptive only in selected habitats and for selected lifestyles.

Daily rhythms of physiology and behaviour are governed by an endogenous timekeeping mechanism (a circadian `clock'), with the alternation of environmental light and darkness synchronizing (entraining) these rhythms to the natural day-night cycle. Our knowledge of the circadian system of animals at the molecular, cellular, tissue and organismal levels is remarkable, and we are beginning to understand how each of these levels contributes to the emergent properties and increased complexity of the system as a whole. For the most part, these analyses have been carried out using model organisms in standard laboratory housing, but to begin to understand the adaptive significance of the clock, we must expand our scope to study diverse animal species from different taxonomic groups, showing diverse activity patterns, in their natural environments. The seven papers in this Special Feature of Proceedings of the Royal Society B take on this challenge, reviewing the influences of moonlight, latitudinal clines, evolutionary history, social interactions, specialized temporal niches, annual variation and recently appreciated post-transcriptional molecular mechanisms. The papers emphasize that the complexity and diversity of the natural world represent a powerful experimental resource.

Many behavioral and physiological processes in adult insects are influenced by both the endocrine and circadian systems, suggesting that these two key physiological systems interact. We reviewed the literature and found that experiments explicitly testing these interactions in adult insects have only been conducted for a few species. There is a shortage of measurements of hormone titers throughout the day under constant conditions even for the juvenile hormones (JHs) and ecdysteroids, the best studied insect hormones. Nevertheless, the available measurements of hormone titers coupled with indirect evidence for circadian modulation of hormone biosynthesis rate, and the expression of genes encoding proteins involved in hormone biosynthesis, binding or degradation are consistent with the hypothesis that the circulating levels of many insect hormones are influenced by the circadian system. Whole genome microarray studies suggest that the modulation of farnesol oxidase levels is important for the circadian regulation of JH biosynthesis in honey bees, mosquitoes, and fruit flies. Several studies have begun to address the functional significance of circadian oscillations in endocrine signaling. The best understood system is the circadian regulation of Pheromone Biosynthesis Activating Neuropeptide (PBAN) titers which is important for the temporal organization of sexual behavior in female moths. The evidence that the circadian and endocrine systems interact has important implications for studies of insect physiology and behavior. Additional studies on diverse species and physiological processes are needed for identifying basic principles underlying the interactions between the circadian and endocrine systems in insects. (C) 2012 Elsevier Ltd. All rights reserved.

In many social insects, including bumblebees, the division of labor between workers relates to body size, but little is known about the factors influencing larval development and final size. We confirmed and extend the evidence that in the bumblebee Bombus terrestris the adult bee body size is positively correlated with colony age. We next performed cross-fostering experiments in which eggs were switched between incipient (before worker emergence) and later stage colonies with workers. The introduced eggs developed into adults similar in size to their unrelated nestmates and not to their same-age full sisters developing in their mother colony. Detailed observations revealed that brood tending by the queen decreases, but does not cease, in young colonies with workers. We next showed that both worker number and the queen presence influenced the final size of the developing brood, but only the queen influence was mediated by shortening developmental time. In colonies separated by a queen excluder, brood developmental time was shorter in the queenright compartment. These findings suggest that differences in body size are regulated by the brood interactions with the queen and workers, and not by factors inside the eggs that could vary along with colony development. Finally, we developed a model showing that the typical increase in worker number and the decrease in brood contact with the queen can account for the typical increase in body size. Similar self-organized social regulation of brood development may contribute to the optimization of growth and reproduction in additional social insects.

During the nest-founding phase of the bumble bee colony cycle, queens undergo striking changes in maternal care behavior. Early in the founding phase, prior to the emergence of workers in the nest, queens are reproductive and also provision and feed their offspring. However, later in the founding phase, queens reduce their feeding of larvae and become specialized on reproduction. This transition is synchronized with the emergence of workers in the colony, who assume the task of feeding their siblings. Using a social manipulation experiment with the bumble bee Bombus terrestris, we tested the hypothesis that workers regulate the transition from feeding brood to specialization on reproduction in nest-founding bumble bee queens. Consistent with this hypothesis, we found that early-stage nest-founding queens with workers prematurely added to their nests reduce their brood-feeding behavior and increase egg laying, and likewise, late-stage nest-founding queens increase their brood-feeding behavior and decrease egg-laying when workers are removed from their nests. Further, brood-feeding and egg-laying behaviors were negatively correlated. We used Agilent microarrays designed from B. terrestris brain expressed sequenced tags (ESTs) to explore a second hypothesis, that workers alter brain gene expression in nest-founding queens. We found evidence that brain gene expression in nest-founding queens is altered by the presence of workers, with the effect being much stronger in late-stage founding queens. This study provides new insights into how the transition from feeding brood to specialization on reproduction in queen bumble bees is regulated during the nest initiation phase of the colony cycle.

Daily rhythms of physiology and behaviour are governed by an endogenous timekeeping mechanism (a circadian `clock'). The alternation of environmental light and darkness synchronizes (entrains) these rhythms to the natural day-night cycle, and underlying mechanisms have been investigated using singly housed animals in the laboratory. But, most species ordinarily would not live out their lives in such seclusion; in their natural habitats, they interact with other individuals, and some live in colonies with highly developed social structures requiring temporal synchronization. Social cues may thus be critical to the adaptive function of the circadian system, but elucidating their role and the responsible mechanisms has proven elusive. Here, we highlight three model systems that are now being applied to understanding the biology of socially synchronized circadian oscillators: the fruitfly, with its powerful array of molecular genetic tools; the honeybee, with its complex natural society and clear division of labour; and, at a different level of biological organization, the rodent suprachiasmatic nucleus, site of the brain's circadian clock, with its network of mutually coupled single-cell oscillators. Analyses at the `group' level of circadian organization will likely generate a more complex, but ultimately more comprehensive, view of clocks and rhythms and their contribution to fitness in nature.

Honey bee (Apis mellifera) workers emerge from the pupae with no circadian rhythms in behavior or brain clock gene expression but show strong rhythms later in life. This postembryonic development of circadian rhythms is reminiscent of that of infants of humans and other primates but contrasts with most insects, which typically emerge from the pupae with strong circadian rhythms. Very little is known about the internal and external factors regulating the ontogeny of circadian rhythms in bees or in other animals. We tested the hypothesis that the environment during early life influences the later expression of circadian rhythms in locomotor activity in young honey bees. We reared newly emerged bees in various social environments, transferred them to individual cages in constant laboratory conditions, and monitored their locomotor activity. We found that the percentage of rhythmic individuals among bees that experienced the colony environment for their first 48 h of adult life was similar to that of older sister foragers, but their rhythms were weaker. Sister bees isolated individually in the laboratory for the same period were significantly less likely to show circadian rhythms in locomotor activity. Bees experiencing the colony environment for only 24 h, or staying for 48 h with 30 same-age sister bees in the laboratory, were similar to bees individually isolated in the laboratory. By contrast, bees that were caged individually or in groups in single- or double-mesh enclosures inside a field colony were as likely to exhibit circadian rhythms as their sisters that were freely moving in the same colony. These findings suggest that the development of the circadian system in young adult honey bees is faster in the colony than in isolation. Direct contact with the queen, workers, or the brood, contact pheromones, and trophallaxis, which are all important means of communication in honey bees, cannot account for the influence of the colony environment, since they were all withheld from the bees in the double-mesh enclosures. Our results suggest that volatile pheromones, the colony microenvironment, or both influence the ontogeny of circadian rhythms in honey bees.

Following general anesthesia, people are often confused about the time of day and experience sleep disruption and fatigue. It has been hypothesized that these symptoms may be caused by general anesthesia affecting the circadian clock. The circadian clock is fundamental to our well-being because it regulates almost all aspects of our daily biochemistry, physiology, and behavior. Here, we investigated the effects of the most common general anesthetic, isoflurane, on time perception and the circadian clock using the honeybee (Apis mellifera) as a model. A 6-h daytime anesthetic systematically altered the time-compensated sun compass orientation of the bees, with a mean anticlockwise shift in vanishing bearing of 87 in the Southern Hemisphere and a clockwise shift in flight direction of 58 in the Northern Hemisphere. Using the same 6-h anesthetic treatment, time-trained bees showed a delay in the start of foraging of 3.3 h, and whole-hive locomotor-activity rhythms were delayed by an average of 4.3 h. We show that these effects are all attributable to a phase delay in the core molecular clockwork. mRNA oscillations of the central clock genes cryptochrome-m and period were delayed by 4.9 and 4.3 h, respectively. However, this effect is dependent on the time of day of administration, as is common for clock effects, and nighttime anesthesia did not shift the clock. Taken together, our results suggest that general anesthesia during the day causes a persistent and marked shift of the clock effectively inducing ``jet lag'' and causing impaired time perception. Managing this effect in humans is likely to help expedite postoperative recovery.

Age-related division of labor in honey bees is associated with plasticity in circadian rhythms. Forager bees that are typically older than 3 weeks of age show strong behavioral and molecular circadian rhythms with higher activity during the day. Younger bees that typically care for (''nurse'') the brood are active around the clock with similar brain clock gene levels throughout the day. However, nurses that are caged on brood-less combs inside or outside the hive show robust circadian rhythms with higher activity during the day, suggesting that direct contact with the brood mediates the plasticity in the circadian system. The nature of the brood signals affecting the workers' circadian system and the modalities by which they are detected are unknown. Given that the antennae are pivotal sensory organs in bees, we hypothesized that they are involved in mediating the brood influence on the plasticity in circadian rhythms. The flagella of the antennae are densely covered with diverse sensory structures able to detect a wide range of signals. To test our hypothesis, we removed the flagella of nurses and observed their behavior in isolation and in free-foraging colonies. We found that individually-isolated flagella-less bees under constant laboratory conditions show robust circadian rhythms in locomotor activity. In observation hives, flagella-less bees cared for the brood, but were more active during the day. By contrast, sham-treated bees were active around the clock as typical of nurses. Detailed video recordings showed that the brood-tending behavior of flagella-less and sham-treated bees is similar. These observations suggest that the difference in the patterns of brood care activity is not because the flagella-less bees did not contact the brood. Our results suggest that nurses are able to find the brood in the dark environment of the hive without their flagella, perhaps by using other sensory organs. The higher activity of flagella-less bees during the day further suggests that the flagella are involved in mediating the brood signals modulating plasticity in the circadian system. (C) 2012 Elsevier Ltd. All rights reserved.

Honey bee workers care for (''nurse'') the brood around the clock without circadian rhythmicity, but then they forage outside with strong circadian rhythms and a consolidated nightly rest. This chronobiological plasticity is associated with variation in the expression of the canonical ``clock genes'' that regulate the circadian clock: nurse bees show no brain rhythms of expression, while foragers do. These results suggest that the circadian system is organized differently in nurses and foragers. Nurses switch to activity with circadian rhythms shortly after being removed from the hive, suggesting that at least some clock cells in their brain continue to measure time while in the hive. We performed a microarray genome-wide survey to determine general patterns of brain gene expression in nurses and foragers sampled around the clock. We found 160 and 541 transcripts that exhibited significant sinusoidal oscillations in nurses and foragers, respectively, with peaks of expression distributed throughout the day in both task groups. Consistent with earlier studies, transcripts of genes involved in circadian rhythms, including Clockwork Orange that has not been studied before in bees, oscillated in foragers but not in nurses. The oscillating transcripts also were enriched for genes involved in the visual system, ``development'' and ``response to stimuli'' (foragers), ``muscle contraction'' and ``microfilament motor gene expression'' (nurses), and ``generation of precursor metabolites'' and ``energy'' (both). Transcripts of genes encoding P450 enzymes oscillated in both nurses and foragers but with a different phase. This study identified new putative clock-controlled genes in the honey bee and suggests that some brain functions show circadian rhythmicity even in nurse bees that are active around the clock.

The circadian clock is a core molecular mechanism that allows organisms to anticipate daily environmental changes and adapt the timing of behaviors to maximize efficiency. In social insects, the ability to maintain the appropriate temporal order is thought to improve colony efficiency and fitness. We used the newly sequenced fire ant (Solenopsis invicta) genome to characterize the first ant circadian clock. Our results reveal that the fire ant clock is similar to the clock of the honeybee, a social insect with an independent evolutionary origin of sociality. Gene trees for the eight core clock genes, period, cycle, clock, cryptochrome-m, timeout, vrille, par domain protein 1 & clockwork orange, show ant species grouping closely with honeybees and Nasonia wasps as an outgroup to the social Hymenoptera. Expression patterns for these genes suggest that the ant clock functions similar to the honeybee clock, with period and cry-m mRNA levels increasing during the night and cycle and clockwork orange mRNAs cycling approximately anti-phase to period. Gene models for five of these genes also parallel honeybee models. In particular, the single ant cryptochrome is an ortholog of the mammalian-type (cry-m), rather than Drosophila-like protein (cry-d). Additionally, we find a conserved VPIFAL C-tail region in clockwork orange shared by insects but absent in vertebrates. Overall, our characterization of the ant clock demonstrates that two social insect lineages, ants and bees, share a similar, mammalian-like circadian clock. This study represents the first characterization of clock genes in an ant and is a key step towards understanding socially-regulated plasticity in circadian rhythms by facilitating comparative studies on the organization of circadian clockwork.

We identified a predicted compact cysteine-rich sequence in the honey bee genome that we called Raalin. Raalin transcripts are enriched in the brain of adult honey bee workers and drones, with only minimum expression in other tissues or in pre-adult stages. Open-reading frame (ORF) homologues of Raalin were identified in the transcriptomes of fruit flies, mosquitoes and moths. The Raalin-like gene from Drosophila melanogaster encodes for a short secreted protein that is maximally expressed in the adult brain with negligible expression in other tissues or pre-imaginal stages. Raalin-like sequences have also been found in the recently sequenced genomes of six ant species, but not in the jewel wasp Nasonia vitripennis. As in the honey bee, the Raalin-like sequences of ants do not have an ORF. A comparison of the genome region containing Raalin in the genomes of bees, ants and the wasp provides evolutionary support for an extensive genome rearrangement in this sequence. Our analyses identify a new family of ancient cysteine-rich short sequences in insects in which insertions and genome rearrangements may have disrupted this locus in the branch leading to the Hymenoptera. The regulated expression of this transcript suggests that it has a brain-specific function.

Female bumble bee workers of the same species often show a profound body size variation that is linked to a division of labour. Large individuals are more likely to forage whereas small individuals are more likely to perform in-nest activities. A higher sensory sensitivity, stronger circadian rhythms as well as better learning and memory performances appear to better equip large individuals for outdoor activities compared to their smaller siblings. The molecular mechanisms underlying worker functional polymorphism remain unclear. Proteins are major determinants of an individual's morphology and behaviour. We hypothesized that the abundance of proteins such as metabolic enzymes as well as proteins involved in neuronal functions would differ with body size and provide insights into the mechanisms underlying size-dependent physiological specialization in bumble bee workers. We conducted protein quantification measurements based on liquid chromatography coupled with tandem mass spectrometry on tissue samples derived from small and large Bombus impatiens and Bombus terrestris workers. Proteins found to differ significantly in abundance between small and large workers belong to the categories of structure, energy metabolism and stress response. These findings provide the first proteomic insight into mechanisms associated with size-based division of labour in social insects.