Substances in the mandibular glands mediate queen effects on larval development and colony organization in an annual bumble bee.

Social organization is commonly dynamic, with extreme examples in annual social insects, but little is known about the underlying signals and mechanisms. Bumble bee larvae with close contact to a queen do not differentiate into gynes, pupate at an earlier age, and are commonly smaller than siblings that do not contact a queen. We combined detailed observations, proteomics, microRNA transcriptomics, and gland removal surgery to study the regulation of brood development and division of labor in the annual social bumble bee Bombus terrestris. We found that regurgitates fed to larvae by queens and workers differ in their protein and microRNA composition. The proteome of the regurgitate overlaps significantly with that of the mandibular (MG) and hypopharyngeal glands (HPG), suggesting that these exocrine glands are sources of regurgitate proteins. The proteome of the MG and HPG, but not the salivary glands, differs between queens and workers, with caste-specificity preserved for the MG and regurgitate proteomes. Queens subjected to surgical removal of the MG showed normal behavior, brood care, and weight gain, but failed to shorten larval development. These findings suggest that substances in the queen MG are fed to larvae and influence their developmental program. We suggest that when workers emerge and contribute to larval feeding, they dilute the effects of the queen substances, until she can no longer manipulate the development of all larvae. Longer developmental duration may allow female larvae to differentiate into gynes rather than to workers, mediating the colony transition from the ergonomic to the reproductive phase.

Integration of information from multiple sources drives and maintains the division of labor in bumble bee colonies.

Bumble bees are eusocial bees in which the division of labor (DoL) in reproduction and in task performance changes during their annual lifecycle. The queen monopolizes reproduction in young colonies, but at later stages, some workers start to challenge the queen and lay their own unfertilized eggs. The division of colony maintenance and growth tasks relates to worker body size. Reproduction and task performance are regulated by multiple social signals of the queen, the workers, and the brood. Here, we review recent studies suggesting that bumble bees use multiple sources of information to establish and maintain DoL in both reproduction and in task performance. Juvenile hormone (JH) is an important neuroendocrine signal involved in the regulation of DoL in reproduction but not in worker task performance. The reliance on multiple signals facilitates flexibility in face of changes in the social and geophysical environment.

Inhibitory signaling in collective social insect networks, is it indeed uncommon?

Individual entities across levels of biological organization interact to reach collective decisions. In centralized neuronal networks, competing neural populations commonly accumulate information over time while increasing their own activity, and cross-inhibiting other populations until one group passes a given threshold. In social insects, there is good evidence for decisions mediated by positive feedbacks, but we found evidence for similar inhibitory signals only in honey bee (Apis mellifera) stop signals, and Pharaoh's ant- (Monomorium pharaonic) repellent pheromones, with only the former occasionally being used as cross-inhibition. We discuss whether these differences stem from insufficient research effort or represent genuine differences across levels of biological organization.

Bumble Bees (Bombus terrestris) Use Time-Memory to Associate Reward with Color and Time of Day.

Circadian clocks regulate ecologically important complex behaviors in honey bees, but it is not clear whether similar capacities exist in other species of bees. One key behavior influenced by circadian clocks is time-memory, which enables foraging bees to precisely time flower visitation to periods of maximal pollen or nectar availability and reduces the costs of visiting a non-rewarding flower patch. Bumble bees live in smaller societies and typically forage over shorter distances than honey bees, and it is therefore not clear whether they can similarly associate reward with time of day. We trained individually marked bumble bee (Bombus terrestris) workers to forage for sugar syrup in a flight cage with yellow or blue feeders rewarding either during the morning or evening. After training for over two weeks, we recorded all visitations to colored feeders filled with only water. We performed two experiments, each with a different colony. We found that bees tended to show higher foraging activity during the morning and evening training sessions compared to other times during the day. During the test day, the trained bees were more likely to visit the rewarding rather than the non-rewarding colored feeders at the same time of day during the test sessions, indicating that they associated time of day and color with the sugar syrup reward. These observations lend credence to the hypothesis that bumble bees have efficient time-memory, indicating that this complex behavior is not limited to honey bees that evolved sophisticated social foraging behaviors over large distances.

Field-realistic concentrations of a neonicotinoid insecticide influence socially regulated brood development in a bumblebee.

The systemic neonicotinoid insecticides are considered as one of the key culprits contributing to ongoing declines in pollinator health and abundance. Bumblebees are among the most important pollinators of temperate zone plants, making their susceptibility to neonicotinoid exposure of great concern. We report that bumblebee (Bombus terrestris) colonies exposed to field-realistic concentrations of the commonly used neonicotinoid Imidacloprid grew slower, consumed less food, and produced fewer workers, males and gynes, but unexpectedly produced larger workers compared to control colonies. Behavioural observations show that queens in pesticide-treated colonies spend more time inactive and less time caring for the brood. We suggest that the observed effects on brood body size are driven by a decreased queen ability to manipulate the larva developmental programme. These findings reveal an intricate and previously unknown effect of insecticides on the social interactions controlling brood development in social insect colonies. Insecticide influences on the social mechanisms regulating larval development are potentially detrimental for bumblebees, in which body size strongly influences both caste differentiation and the division of labour among workers, two organization principles of insect societies.

The Influences of Illumination Regime on Egg-laying Rhythms of Honey Bee Queens.

Honey bee queens show extreme fecundity, commonly laying more than a thousand eggs in a single day. It has proven challenging to study the temporal organization of egg-laying behavior because queens are typically active around the clock in the dark cavity of a densely populated nest. To contend with this challenge, we developed two novel methods allowing detailed monitoring of queen activity and egg laying. We first adapted a high-resolution, continuous, tracking system allowing to track the position of barcode-tagged queens in observation hives with colonies foraging outside. We found that the queen is active ~96% of the day with typically no diurnal rhythm. Next, we developed a new laboratory procedure to monitor egg laying at single egg resolution under different light regimes. We found that under constant darkness (DD) and temperature conditions, queens laid eggs with no circadian rhythms. Queen fecundity was severely reduced under constant light (LL). Under a 12:12 illumination regime, queen fecundity was comparable to under constant darkness, with a higher number of eggs during the light phase. These daily rhythms in egg laying continued when these queens were released to DD conditions, suggesting that egg-laying rhythms are influenced by endogenous circadian clocks. These results suggest that honey bee queens are active and lay eggs around the clock with no diurnal rhythms. Light has complex influences on these behaviors, but more studies are needed to determine whether these effects reflect the influence of light directly on the queen or indirectly by affecting workers that interact with the queen.

Earlier Morning Arrival to Pollen-Rewarding Flowers May Enable Feral Bumble Bees to Successfully Compete with Local Bee Species and Expand Their Distribution Range in a Mediterranean Habitat.

During recent decades, bumble bees (Bombus terrestris) have continuously expanded their range in the Mediterranean climate regions of Israel. To assess their potential effects on local bee communities, we monitored their diurnal and seasonal activity patterns, as well as those of native bee species in the Judean Hills. We found that all bee species tend to visit pollen-providing flowers at earlier times compared to nectar-providing flowers. Bumble bees and honey bees start foraging at earlier times and colder temperatures compared to other species of bees. This means that the two species of commercially managed social bees are potentially depleting much of the pollen, which is typically non-replenished, before most local species arrive to gather it. Taking into consideration the long activity season of bumble bees in the Judean hills, their ability to forage at the low temperatures of the early morning, and their capacity to collect pollen at early hours in the dry Mediterranean climate, feral and range-expanding bumble bees potentially pose a significant competitive pressure on native bee fauna. Their effects on local bees can further modify pollination networks, and lead to changes in the local flora.

Krüppel-homologue 1 Mediates Hormonally Regulated Dominance Rank in a Social Bee.

Dominance hierarchies are ubiquitous in invertebrates and vertebrates, but little is known on how genes influence dominance rank. Our gaps in knowledge are specifically significant concerning female hierarchies, particularly in insects. To start filling these gaps, we studied the social bumble bee Bombus terrestris, in which social hierarchies among females are common and functionally significant. Dominance rank in this bee is influenced by multiple factors, including juvenile hormone (JH) that is a major gonadotropin in this species. We tested the hypothesis that the JH responsive transcription factor Krüppel homologue 1 (Kr-h1) mediates hormonal influences on dominance behavior. We first developed and validated a perfluorocarbon nanoparticles-based RNA interference protocol for knocking down Kr-h1 expression. We then used this procedure to show that Kr-h1 mediates the influence of JH, not only on oogenesis and wax production, but also on aggression and dominance rank. To the best of our knowledge, this is the first study causally linking a gene to dominance rank in social insects, and one of only a few such studies on insects or on female hierarchies. These findings are important for determining whether there are general molecular principles governing dominance rank across gender and taxa.

Social synchronization of circadian rhythms with a focus on honeybees.

Many animals benefit from synchronizing their daily activities with conspecifics. In this hybrid paper, we first review recent literature supporting and extending earlier evidence for a lack of clear relationship between the level of sociality and social entrainment of circadian rhythms. Social entrainment is specifically potent in social animals that live in constant environments in which some or all individuals do not experience the ambient day-night cycles. We next focus on highly social honeybees in which there is good evidence that social cues entrain the circadian clocks of nest bees and can override the influence of conflicting light-dark cycles. The current understanding of social synchronization in honeybees is consistent with self-organization models in which surrogates of forager activity, such as substrate-borne vibrations and colony volatiles, entrain the circadian clocks of bees dwelling in the dark cavity of the nest. Finally, we present original findings showing that social synchronization is effective even in an array of individually caged callow bees placed on the same substrate and is improved for bees in connected cages. These findings reveal remarkable sensitivity to social time-giving cues and show that bees with attenuated rhythms (weak oscillators) can nevertheless be socially synchronized to a common phase of activity. This article is part of the theme issue 'Synchrony and rhythm interaction: from the brain to behavioural ecology'.

Body Size and Behavioural Plasticity Interact to Influence the Performance of Free-Foraging Bumble Bee Colonies.

Specialisation and plasticity are important for many forms of collective behaviour, but the interplay between these factors is little understood. In insect societies, workers are often developmentally primed to specialise in different tasks, sometimes with morphological or physiological adaptations, facilitating a division of labour. Workers may also plastically switch between tasks or vary their effort. The degree to which developmentally primed specialisation limits plasticity is not clear and has not been systematically tested in ecologically relevant contexts. We addressed this question in 20 free-foraging bumble bee (Bombus terrestris) colonies by continually manipulating colonies to contain either a typically diverse, or a reduced ("homogeneous"), worker body size distribution while keeping the same mean body size, over two trials. Pooling both trials, diverse colonies produced a larger comb mass, an index of colony performance. The link between body size and task was further corroborated by the finding that foragers were larger than nurses even in homogeneous colonies with a very narrow body size range. However, the overall effect of size diversity stemmed mostly from one trial. In the other trial, homogeneous and diverse colonies showed comparable performance. By comparing behavioural profiles based on several thousand observations of individuals, we found evidence that workers in homogeneous colonies in this trial rescued colony performance by plastically increasing behavioural specialisation and/or individual effort, compared to same-sized individuals in diverse colonies. Our results are consistent with a benefit to colonies of large and small specialists under certain conditions, but also suggest that plasticity or effort can compensate for reduced (size-related) specialisation. Thus, we suggest that an intricate interplay between specialisation and plasticity is functionally adaptive in bumble bee colonies.

Care-giver identity impacts offspring development and performance in an annually social bumble bee.

BACKGROUND: The developmental fates of offspring have the potential to be influenced by the identity of their care-givers and by the nature of the care that they receive. In animals that exhibit both parental and alloparental care, such as the annually eusocial insects, the influence of care-giver identity can be directly assessed to yield mechanistic and evolutionary insights into the origins and elaboration of brood care. Here, we performed a comparative investigation of maternal and worker brood care in bumble bees, a pollinator group where mothers (queens) rear the first offspring in the nest, and then daughters (workers) assume this role upon their emergence. Specifically, we compared the effects of queen and worker brood care on offspring development and also offspring performance, for a set of traits related to sensory biology, learning, and stress resistance. RESULTS: We found that queen-reared workers were smaller-bodied than worker-reared offspring, suggesting that bumble bee queens influence body size determination in their offspring. We also found that queen-reared workers were more resistant to starvation, which might be beneficial for early nesting success. These maternal influences could not be explained by feeding rate, given that we detected a similar offspring feeding frequency in both queens and workers. CONCLUSION: Bumble bee queens have a unique influence on the development of the first offspring in the nest, which they rear, relative to worker-reared workers. We propose that bumble bee brood care has been shaped by a suite of evolutionary and ecological factors, which might include a maternal influence on traits that promote survival of incipient colonies.

Remarkable Sensitivity of Young Honey Bee Workers to Multiple Non-photic, Non-thermal, Forager Cues That Synchronize Their Daily Activity Rhythms.

Honey bees live in colonies containing tens of thousands of workers that coordinate their activities to produce efficient colony-level behavior. In free-foraging colonies, nest bees are entrained to the forager daily phase of activity even when experiencing conflicting light-dark illumination regime, but little is known on the cues mediating this potent social synchronization. We monitored locomotor activity in an array of individually caged bees in which we manipulated the contact with neighbour bees. We used circular statistics and coupling function analyses to estimate the degree of social synchronization. We found that young bees in cages connected to cages housing foragers showed stronger rhythms, better synchronization with each other, higher coupling strength, and a phase more similar to that of the foragers compared to similar bees in unconnected cages. These findings suggest that close distance contacts are sufficient for social synchronization or that cage connection facilitated the propagation of time-giving social cues. Coupling strength was higher for bees placed on the same tray compared with bees at a similar distance but on a different tray, consistent with the hypothesis that substrate borne vibrations mediate phase synchronization. Additional manipulation of the contact between cages showed that social synchronization is better among bees in cages connected with tube with a single mesh partition compared to sealed tubes consistent with the notion that volatile cues act additively to substrate borne vibrations. These findings are consistent with self-organization models for social synchronization of activity rhythms and suggest that the circadian system of honey bees evolved remarkable sensitivity to non-photic, non-thermal, time giving entraining cues enabling them to tightly coordinate their behavior in the dark and constant physical environment of their nests.

Juvenile hormone affects the development and strength of circadian rhythms in young bumble bee (Bombus terrestris) workers.

The circadian and endocrine systems influence many physiological processes in animals, but little is known on the ways they interact in insects. We tested the hypothesis that juvenile hormone (JH) influences circadian rhythms in the social bumble bee Bombus terrestris. JH is the major gonadotropin in this species coordinating processes such as vitellogenesis, oogenesis, wax production, and behaviors associated with reproduction. It is unknown however, whether it also influences circadian processes. We topically treated newly-emerged bees with the allatoxin Precocene-I (P-I) to reduce circulating JH titers and applied the natural JH (JH-III) for replacement therapy. We repeated this experiment in three trials, each with bees from different source colonies. Measurements of ovarian activity suggest that our JH manipulations were effective; bees treated with P-I had inactive ovaries, and this effect was fully recovered by subsequent JH treatment. We found that JH augments the strength of circadian rhythms and the pace of rhythm development in individually isolated newly emerged worker bees. JH manipulation did not affect the free-running circadian period, overall level of locomotor activity, sleep amount, or sleep structure. Given that acute manipulation at an early age produced relatively long-lasting effects, we propose that JH effects on circadian rhythms are mostly organizational, accelerating the development or integration of the circadian system.

The Complexity of Social Complexity: A Quantitative Multidimensional Approach for Studies of Social Organization.

AbstractThe rapid increase in "big data" during the postgenomic era makes it crucial to appropriately measure the level of social complexity in comparative studies. We argue that commonly used qualitative classifications lump together species showing a broad range of social complexity and falsely imply that social evolution always progresses along a single linear stepwise trajectory that can be deduced from comparing extant species. To illustrate this point, we compared widely used social complexity measures in "primitively eusocial" bumble bees with "advanced eusocial" stingless bees, honey bees, and attine ants. We find that a single species can have both higher and lower levels of complexity compared with other taxa, depending on the social trait measured. We propose that measuring the complexity of individual social traits switches focus from semantic discussions and offers several directions for progress. First, quantitative social traits can be correlated with molecular, developmental, and physiological processes within and across lineages of social animals. This approach is particularly promising for identifying processes that influence or have been affected by social evolution. Second, key social complexity traits can be combined into multidimensional lineage-specific quantitative indices, enabling fine-scale comparison across species that are currently bundled within the same level of social complexity.

Brain microRNAs among social and solitary bees.

Evolutionary transitions to a social lifestyle in insects are associated with lineage-specific changes in gene expression, but the key nodes that drive these regulatory changes are unknown. We examined the relationship between social organization and lineage-specific microRNAs (miRNAs). Genome scans across 12 bee species showed that miRNA copy-number is mostly conserved and not associated with sociality. However, deep sequencing of small RNAs in six bee species revealed a substantial proportion (20-35%) of detected miRNAs had lineage-specific expression in the brain, 24-72% of which did not have homologues in other species. Lineage-specific miRNAs disproportionately target lineage-specific genes, and have lower expression levels than shared miRNAs. The predicted targets of lineage-specific miRNAs are not enriched for genes with caste-biased expression or genes under positive selection in social species. Together, these results suggest that novel miRNAs may coevolve with novel genes, and thus contribute to lineage-specific patterns of evolution in bees, but do not appear to have significant influence on social evolution. Our analyses also support the hypothesis that many new miRNAs are purged by selection due to deleterious effects on mRNA targets, and suggest genome structure is not as influential in regulating bee miRNA evolution as has been shown for mammalian miRNAs.

Juvenile hormone regulates brain-reproduction tradeoff in bumble bees but not in honey bees.

Gonadotropic hormones coordinate processes in diverse tissues regulating animal reproductive physiology and behavior. Juvenile hormone (JH) is the ancient and most common gonadotropin in insects, but not in advanced eusocial honey bees and some ants. To start probing the evolutionary basis of this change, we combined endocrine manipulations, transcriptomics, and behavioral analyses to study JH regulated processes in a bumble bee showing a relatively simple level of eusociality. We found that in worker fat body, more JH-regulated genes were up- rather than down-regulated, and enriched for metabolic and biosynthetic pathways. This transcriptomic pattern is consistent with earlier evidence that JH is the major gonadotropin in bumble bees. In the brain, more JH-regulated genes were down- rather than up-regulated and enriched for protein turnover pathways. Brain ribosomal protein gene expression shows a similar trend of downregulation in dominant workers, which naturally have high JH titers. In other species, similar downregulation of protein turnover is found in aging brains or under stress, associated with compromised long-term memory and health. These findings suggest a previously unknown gonadotropin-mediated tradeoff. Analysis of published data reveals no such downregulation of protein turnover pathways in the brain of honey bee workers, which exhibit more complex eusociality and in which JH is not a gonadotropin but rather regulates division of labor. These results suggest that the evolution of complex eusociality in honey bees was associated with modifications in hormonal signalling supporting extended and extremely high fertility while reducing the ancient costs of high gonadotropin titers to the brain.

Colony Volatiles and Substrate-borne Vibrations Entrain Circadian Rhythms and Are Potential Cues Mediating Social Synchronization in Honey Bee Colonies.

Internal circadian clocks organize animal behavior and physiology and are entrained by ecologically relevant external time-givers such as light and temperature cycles. In the highly social honey bee, social time-givers are potent and can override photic entrainment, but the cues mediating social entrainment are unknown. Here, we tested whether substrate-borne vibrations and hive volatiles can mediate social synchronization in honey bees. We first placed newly emerged worker bees on the same or on a different substrate on which we placed cages with foragers entrained to ambient day-night cycles, while minimizing the spread of volatiles between cages. In the second experiment, we exposed young bees to constant airflow drawn from either a free-foraging colony or a similar-size control hive containing only heated empty honeycombs, while minimizing transfer of substrate-borne vibrations between cages. After 6 days, we isolated each focal bee in an individual cage in an environmental chamber and monitored her locomotor activity. We repeated each experiment 5 times, each trial with bees from a different source colony, monitoring a total of more than 1000 bees representing diverse genotypes. We found that bees placed on the same substrate as foragers showed a stronger phase coherence and a phase more similar to that of foragers compared with bees placed on a different substrate. In the second experiment, bees exposed to air drawn from a colony showed a stronger phase coherence and a phase more similar to that of foragers compared with bees exposed to air from an empty hive. These findings lend credence to the hypothesis that surrogates of activity entrain circadian rhythms and suggest that multiple social cues can act in concert to entrain social insect colonies to a common phase.

Juvenile hormone interacts with multiple factors to modulate aggression and dominance in groups of orphan bumble bee (Bombus terrestris) workers.

Juvenile hormone (JH) is a key regulator of insect development and reproduction. Given that JH commonly affects adult insect fertility, it has been hypothesized to also regulate behaviors such as dominance and aggression that are associated with reproduction. We tested this hypothesis in the bumble bee Bombus terrestris for which JH has been shown to be the major gonadotropin. We used the allatoxin Precocene-I (P-I) to reduce hemolymph JH titers and replacement therapy with the natural JH to revert this effect. In small orphan groups of workers with similar body size but mixed treatment, P-I treated bees showed lower aggressiveness, oogenesis, and dominance rank compared with control and replacement therapy treated bees. In similar groups in which all bees were treated similarly, there was a clear dominance hierarchy, even in P-I and replacement therapy treatment groups in which the bees showed similar levels of ovarian activation. In a similar experiment in which bees differed in body size, larger bees were more likely to be dominant despite their similar JH treatment and ovarian state. In the last experiment, we show that JH manipulation does not affect dominance rank in groups that had already established a stable dominance hierarchy. These findings solve previous ambiguities concerning whether or not JH affects dominance in bumble bees. JH positively affects dominance, but bees with similar levels of JH can nevertheless establish dominance hierarchies. Thus, multiple factors including JH, body size, and previous experience affect dominance and aggression in social bumble bees.

Bumble Bee Workers Give Up Sleep to Care for Offspring that Are Not Their Own.

Sleep is ubiquitous in vertebrates and invertebrates, and its loss is typically associated with reduced performance, health, or survival, for reasons that are yet unclear [1-3]. Nevertheless, some animals can reduce sleep for increasing foraging time [4], under predation risk [5-8], during seasonal migration [9-11], or for having greater mating opportunities [12, 13]. Here, we tested the hypothesis that social bumble bee (Bombus terrestris) workers give up sleep for improving brood care. We combined video recordings, detailed behavioral analyses, sleep-deprivation experiments, and response-threshold assessments to characterize the sleep behavior of worker bees and showed that immobility bouts of ≥5 min provide a reliable proxy for sleep. We next used this index to study sleep with an automated video-based activity monitoring system. We found that isolated workers severely reduce sleep time in the presence of both larvae that need to be fed and pupae that do not. Reduced sleep was also correlated with around-the-clock activity and wax-pot building, which are typical for nest-founding mother queens. Cocoons, from which we removed the pupae, elicited a similar but transient sleep loss in tending workers, suggesting that the pupa effect on sleep is mediated by pheromonal signals. Sleep time increased following brood removal but remained lower compared to control bees, suggesting that the brood modulated sleep need. This first evidence for brood modulation of sleep in an insect suggests that plasticity in sleep can evolve as a mechanism to improve care for dependent juveniles, even in social insect workers that do not care for their own offspring.

Body size variation in bees: regulation, mechanisms, and relationship to social organization.

Size polymorphism is common in bees, and is determined by environmental factors such as temperature, brood cell size, and the diet provided to developing larvae. In social bees, these factors are further influenced by intricate interactions between the queen, workers, and the developing brood which eventually determine the final size and caste of developing larvae. Environmental and social factors act in part on juvenile hormone and ecdysteroids, which are key hormonal regulators of body size and caste determination. In some social bees, body size variation is central for social organization because it structures reproductive division of labor, task allocation among workers, or both. At ecological scales, body size also impacts bee-mediated pollination services in solitary and social species by influencing floral visitation and pollination efficacy.