Division of labour in reproduction and in task performance are two hallmarks of insect societies. Bumble bees and honey bees are both classified as eusocial, but differ in the social organization of their colonies and in the hormonal regulation of division of labour. Bumble bees exhibit size-related division of labour among adult workers that is independent of juvenile hormone (JH), whereas honey bees exhibit age-related division of labour among same-size bees that is influenced by JH. We hypothesized that despite these differences, some common molecular pathways for social organization are shared between the two species. We addressed this hypothesis by analysing brain transcriptomes of bumble bee (Bombus terrestris) queenright workers differing in the performance of foraging and brood care activities, as well as of queenless workers. The brain transcriptomes of nurses, foragers and queenless bees were different, with little overlap. qPCR validation shows that some of the genes are associated with task performance even for bees with a similar body size. The set of genes influenced by JH treatment overlaps with the genes influenced by queen presence but not with those associated with division of labour. These results support and extend the evidence that in bumble bees JH regulates reproduction but not the division of labour among workers for colony tasks. Additionally, we found significant overlap between genes differentially expressed among workers performing different tasks or as a function of queen presence in the current study with bumble bees and previous studies with the honey bee Apis mellifera. These findings support the hypothesis that a common 'toolkit' of genes is involved in the regulation of division of labour in honey bees and bumble bees for both reproduction and task performance.

Bumble bees are essential pollinators that have an annual eusocial lifestyle where new cohorts of queens establish colonies each season. The early stages of colony establishment are critical for colony success and are heavily impacted by how readily queens activate their ovaries and initiate egg laying at the start of the season. Understanding the social and nesting factors that influence queen reproduction may provide key insights into early colony establishment. Here, we examine how social conditions and nest cavity size affect the onset of egg laying in early nesting Bombus impatiens queens. In the first experiment, queens were assigned to a social condition where they were placed into nesting boxes that either completely restricted contact with nestmates (and were smaller in size) or allowed direct physical contact and then housed either alone or with nestmates (males or workers). We found that contact with either workers or males accelerated egg laying in queens relative to being housed alone. Unexpectedly, queens housed in the restricted cages, regardless of whether alone or with nestmates, also initiated egg laying earlier. This led us to a second experiment, where we examined ovarian activation and hemolymph juvenile hormone (JH-III) concentrations in early queens housed in different nesting cavity sizes. Here, we found that smaller nest cavity sizes lead to a greater degree of ovarian activation in gynes (young, unmated queens), but juvenile hormone concentrations did not differ. Our findings demonstrate that both social conditions and nest cavity size influence reproductive timing in queens in early-stage bumble bee colonies.

Division of labor in reproduction in eusocial insects is governed by delicate and context- dependent mechanisms, yet the regulatory processes remain largely unclear. Here, we investigate social cues involved in the establishment of reproductive dominance in bumble bee (Bombus terrestris) workers. We reveal the roles of two alkenes, (Z)-9-nonacosene (Z9- C29) and (Z)-9-hentriacontene (Z9- C31) in the activation of ovarian development. These alkenes are found throughout the queenright bumble bee colony, including wax, workers, drones, and gynes, and their levels are positively correlated with colony size. They act as quorum-sensing pheromones that facilitate ovary maturation in a threshold-dependent manner under queenright conditions. A synergistic interaction between the perception of these two alkenes and the occurrence of dominance contests is essential for the ovary development of worker bumble bees, independent of temporal sequence. Artificial addition of these two alkenes into colonies confirms their stimulatory effects on the ovarian developmental potential of worker bees, even in the presence of a queen. Our findings highlight a quorum-sensing-like mechanism that primes the initiation of worker ovary development, providing insights into the complex regulation of context-dependent reproductive plasticity in social insects.

Hormones play a decisive role in many aspects of insect biology. To study processes controlled by hormones, one needs methods to identify and quantify hormone titers and tools to enhance or suppress hormonal signaling experimentally. In this review, we focus on the key lipidic insect hormones, the juvenile hormones (JHs), and the ecdysteroids. The lipophilic nature of JH and ecdysteroids in combination with their low endogenous titers makes handling and quantification challenging but feasible owing to the improvement of analytical detection methods. Chemical and genetic approaches to modulate hormonal homeostasis have been developed based on knowledge of hormone biosynthetic and biodegrading enzymes, transporters, and receptors and enabled by advances in reverse genetics techniques. Here, we overview contemporary methods available to detect and quantify JHs and ecdysteroids from insect samples and to manipulate endocrine homeostasis.

How social complexity evolved remains a long-standing enigma. In most animal groups, social complexity is typically classified into a few discrete classes. This approach is oversimplified and constrains our inference of social evolution to a narrow trajectory consisting of transitions between classes. Such categorical classifications also limit quantitative studies on the molecular and environmental drivers of social complexity. The recent accumulation of relevant quantitative data has set the stage to overcome these limitations. Here, we propose a data-driven, high-dimensional approach for studying the full diversity of social phenotypes. We curated and analyzed a comprehensive dataset encompassing 17 social traits across 80 species and studied the evolution of social complexity in bees. We found that honey bees, stingless bees, and bumble bees underwent a major evolutionary transition '80 mya, inconsistent with the stepwise progression of the social ladder conceptual framework. This major evolutionary transition was followed by a phase of substantial phenotypic diversification of social complexity. Other bee lineages display a continuum of social complexity, ranging from solitary to simple societies, but do not reach the levels of social complexity seen in honey bees, stingless bees, and bumble bees. Bee evolution, therefore, provides a remarkable demonstration of a macroevolutionary process in which a major transition removed biological constraints and opened novel evolutionary opportunities, driving the exploration of the landscape of social phenotypes. Our approach can be extended to incorporate additional data types and readily applied to illuminate the evolution of social complexity in other animal groups.

The seminal discovery that in adults of the highly social honey bee (Apis mellifera), juvenile hormone (JH) regulates age-related division of labor (DoL) but not adult fertility, unlike in most insects, has led to the hypothesis that the evolution of insect sociality involved modifications in JH signaling. Recent studies examining JH functions across the Hymenoptera provide two main insights: First, significant progress in studies of the bumble bee Bombus terrestris, which exhibits an intermediate level of social complexity relative to honey bees, shows that JH regulates multiple tissues involved in reproduction, but not task performance. JH also seems to function as a primary gonadotropin in bees showing solitary lifestyles or low levels of social complexity, highlighting a marked contrast with its roles in honey bees. Second, this association between JH function and social complexity in bees does not generalize to other lineages. The few studies on JH function in highly social stingless bees are not consistent with the honey bee model. In wasps and hornets, JH typically influences both fertility and age-related DoL. There is substantial variability across ant species, offering no consistent model linking JH function to social complexity. We propose that although JH signaling is commonly modified in social insects, the specific changes differ between - and sometimes within - lineages. There is no one model linking JH function to social complexity across major lineages, likely due to changes in related pathways. These modifications enable social insects to circumvent the trade-off between reproduction and maintenance.

Juvenile hormones (JHs) are key insect endocrine signals regulating pre-adult development and adult life history strategies, physiology, and behavior. The importance of this endocrine signal has led to the development of insecticides targeting JH signaling pathways. While effective against target pests, they can also affect beneficial insects, such as pollinators and natural pest enemies, many of which are hymenopterans. Research on JH, which is crucial for understanding the physiology, behavior, and organization of social insects, requires effective means to manipulate JH signaling pathways. A common method for reducing JH titers includes applying the phytotoxin precocene-I (P-I), which abolishes JH biosynthesis in the corpora allata (CA). However, achieving consistently effective topical treatments has proven challenging. Here, we investigated the dynamics of the effect of P-I manipulations on JH titers and reproductive development in orphan worker groups of a key pollinator bumble bee. A single P-I treatment effectively reduced circulating JH levels in newly emerged workers, even after 12 days. The treatment delayed but did not prevent ovarian activation. Multiple treatments failed to further reduce JH titers or ovarian activity, and were overall less effective than surgically removing the CA. Finally, we suggest guidelines for developing P-I manipulation studies, and more generally, compare methods for reducing circulating JH levels in insects.

Mating success depends on many factors, but first of all, a male and a female need to meet at the same place and time. The circadian clock is an endogenous system regulating activity and sex-related behaviors in animals. We studied bumble bees (Bombus terrestris) in which the influence of circadian rhythms on sexual behavior has been little explored. We characterized circadian rhythms in adult emergence and locomotor activity under different illumination regimes for males and gynes (unmated queens). We developed a method to monitor adult emergence from the pupal cocoon and found no circadian rhythms in this behavior for either males or gynes. These results are not consistent with the hypothesis that the circadian clock regulates emergence from the pupa in this species. Consistent with this premise, we found that both gynes and males do not show circadian rhythms in locomotor activity during the first 3 days after pupal emergence, but shortly after developed robust circadian rhythms that are readily shifted by a phase delay in illumination regime. We conclude that the bumble bees do not need strong rhythms in adult emergence and during early adult life in their protected and regulated nest environment, but do need strong activity rhythms for timing flights and mating-related behaviors. Next, we tested the hypothesis that the locomotor activity of males and gynes have a similar phase, which may improve mating success. We found that both males and gynes have strong endogenous circadian rhythms that are entrained by the illumination regime, but males show rhythms at an earlier age, their rhythms are stronger, and their phase is slightly advanced relative to that of gynes. An earlier phase may be advantageous to males competing to mate a receptive gyne. Our results are consistent with the hypothesis that sex-related variations in circadian rhythms is shaped by sexual selection.

How social complexity evolved is a long-standing enigma. In most animal groups, social complexity is typically classified into a few discrete classes. This approach is oversimplified and constrains our inference of social evolution to a narrow trajectory consisting of transitions between classes. This approach also limits quantitative studies on the molecular and environmental drivers of social complexity. However, the recent accumulation of relevant quantitative data has now set the stage to overcome these limitations. Here, we propose a data-driven approach for studying the full diversity of social phenotypes. We curated and analyzed a comprehensive dataset encompassing 17 social traits for 77 species and studied the evolution of social complexity in bees. We found that corbiculate bees — honey bees, stingless bees, and bumble bees — underwent a major evolutionary transition ∼70 mya, which is inconsistent with the stepwise progression of the social ladder conceptual framework. This major evolutionary transition was followed by a phase of substantial phenotypic diversification of social complexity. Non-corbiculate bee lineages display a continuum of social complexity, ranging from solitary to simple societies, but do not reach levels of social complexity comparable to those of corbiculate bees. Bee evolution provides a unique demonstration of a macroevolutionary process in which a major transition removed biological constraints and opened novel evolutionary opportunities, driving the exploration of the landscape of social phenotypes. Our approach can be extended to incorporate additional data types and readily applied to illuminate the evolution of social complexity in additional animal groups.### Competing Interest StatementThe authors have declared no competing interest.

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.

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.

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.

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.

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.

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.

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.

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'.

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.

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.