Hormonal gatekeeping via the blood-brain barrier governs caste-specific behavior in ants.

Here, we reveal an unanticipated role of the blood-brain barrier (BBB) in regulating complex social behavior in ants. Using scRNA-seq, we find localization in the BBB of a key hormone-degrading enzyme called juvenile hormone esterase (Jhe), and we show that this localization governs the level of juvenile hormone (JH3) entering the brain. Manipulation of the Jhe level reprograms the brain transcriptome between ant castes. Although ant Jhe is retained and functions intracellularly within the BBB, we show that Drosophila Jhe is naturally extracellular. Heterologous expression of ant Jhe into the Drosophila BBB alters behavior in fly to mimic what is seen in ants. Most strikingly, manipulation of Jhe levels in ants reprograms complex behavior between worker castes. Our study thus uncovers a remarkable, potentially conserved role of the BBB serving as a molecular gatekeeper for a neurohormonal pathway that regulates social behavior.

SLEAP: A deep learning system for multi-animal pose tracking.

The desire to understand how the brain generates and patterns behavior has driven rapid methodological innovation in tools to quantify natural animal behavior. While advances in deep learning and computer vision have enabled markerless pose estimation in individual animals, extending these to multiple animals presents unique challenges for studies of social behaviors or animals in their natural environments. Here we present Social LEAP Estimates Animal Poses (SLEAP), a machine learning system for multi-animal pose tracking. This system enables versatile workflows for data labeling, model training and inference on previously unseen data. SLEAP features an accessible graphical user interface, a standardized data model, a reproducible configuration system, over 30 model architectures, two approaches to part grouping and two approaches to identity tracking. We applied SLEAP to seven datasets across flies, bees, mice and gerbils to systematically evaluate each approach and architecture, and we compare it with other existing approaches. SLEAP achieves greater accuracy and speeds of more than 800 frames per second, with latencies of less than 3.5 ms at full 1,024 × 1,024 image resolution. This makes SLEAP usable for real-time applications, which we demonstrate by controlling the behavior of one animal on the basis of the tracking and detection of social interactions with another animal.

Developmental transcriptomes predict adult social behaviours in the socially flexible sweat bee, Lasioglossum baleicum.

Natural variation can provide important insights into the genetic and environmental factors that shape social behaviour and its evolution. The sweat bee, Lasioglossum baleicum, is a socially flexible bee capable of producing both solitary and eusocial nests. We demonstrate that within a single nesting aggregation, soil temperatures are a strong predictor of the social structure of nests. Sites with warmer temperatures in the spring have a higher frequency of social nests than cooler sites, perhaps because warmer temperatures provide a longer reproductive window for those nests. To identify the molecular correlates of this behavioural variation, we generated a de novo genome assembly for L. baleicum, and we used transcriptomic profiling to compare adults and developing offspring from eusocial and solitary nests. We find that adult, reproductive females have similar expression profiles regardless of social structure in the nest, but that there are strong differences between reproductive females and workers from social nests. We also find substantial differences in the transcriptomic profiles of stage-matched pupae from warmer, social-biased sites compared to cooler, solitary-biased sites. These transcriptional differences are strongly predictive of adult reproductive state, suggesting that the developmental environment may set the stage for adult behaviours in L. baleicum. Together, our results help to characterize the molecular mechanisms shaping variation in social behaviour and highlight a potential role of environmental tuning during development as a factor shaping adult behaviour and physiology in this socially flexible bee.

Solitary bees reduce investment in communication compared with their social relatives.

Social animals must communicate to define group membership and coordinate social organization. For social insects, communication is predominantly mediated through chemical signals, and as social complexity increases, so does the requirement for a greater diversity of signals. This relationship is particularly true for advanced eusocial insects, including ants, bees, and wasps, whose chemical communication systems have been well-characterized. However, we know surprisingly little about how these communication systems evolve during the transition between solitary and group living. Here, we demonstrate that the sensory systems associated with signal perception are evolutionarily labile. In particular, we show that differences in signal production and perception are tightly associated with changes in social behavior in halictid bees. Our results suggest that social species require a greater investment in communication than their solitary counterparts and that species that have reverted from eusociality to solitary living have repeatedly reduced investment in these potentially costly sensory perception systems.

Testing the kinship theory of intragenomic conflict in honey bees (Apis mellifera).

Sexual reproduction brings genes from two parents (matrigenes and patrigenes) together into one individual. These genes, despite being unrelated, should show nearly perfect cooperation because each gains equally through the production of offspring. However, an individual's matrigenes and patrigenes can have different probabilities of being present in other relatives, so kin selection could act on them differently. Such intragenomic conflict could be implemented by partial or complete silencing (imprinting) of an allele by one of the parents. Evidence supporting this theory is seen in offspring-mother interactions, with patrigenes favoring acquisition of more of the mother's resources if some of the costs fall on half-siblings who do not share the patrigene. The kinship theory of intragenomic conflict is little tested in other contexts, but it predicts that matrigene-patrigene conflict may be rife in social insects. We tested the hypothesis that honey bee worker reproduction is promoted more by patrigenes than matrigenes by comparing across nine reciprocal crosses of two distinct genetic stocks. As predicted, hybrid workers show reproductive trait characteristics of their paternal stock, (indicating enhanced activity of the patrigenes on these traits), greater patrigenic than matrigenic expression, and significantly increased patrigenic-biased expression in reproductive workers. These results support both the general prediction that matrigene-patrigene conflict occurs in social insects and the specific prediction that honey bee worker reproduction is driven more by patrigenes. The success of these predictions suggests that intragenomic conflict may occur in many contexts where matrigenes and patrigenes have different relatednesses to affected kin.

The risk-return trade-off between solitary and eusocial reproduction.

Social insect colonies can be seen as a distinct form of biological organisation because they function as superorganisms. Understanding how natural selection acts on the emergence and maintenance of these colonies remains a major question in evolutionary biology and ecology. Here, we explore this by using multi-type branching processes to calculate the basic reproductive ratios and the extinction probabilities for solitary vs. eusocial reproductive strategies. We find that eusociality, albeit being hugely successful once established, is generally less stable than solitary reproduction unless large demographic advantages of eusociality arise for small colony sizes. We also demonstrate how such demographic constraints can be overcome by the presence of ecological niches that strongly favour eusociality. Our results characterise the risk-return trade-offs between solitary and eusocial reproduction, and help to explain why eusociality is taxonomically rare: eusociality is a high-risk, high-reward strategy, whereas solitary reproduction is more conservative.

Transitions in social complexity along elevational gradients reveal a combined impact of season length and development time on social evolution.

Eusociality is taxonomically rare, yet associated with great ecological success. Surprisingly, studies of environmental conditions favouring eusociality are often contradictory. Harsh conditions associated with increasing altitude and latitude seem to favour increased sociality in bumblebees and ants, but the reverse pattern is found in halictid bees and polistine wasps. Here, we compare the life histories and distributions of populations of 176 species of Hymenoptera from the Swiss Alps. We show that differences in altitudinal distributions and development times among social forms can explain these contrasting patterns: highly social taxa develop more quickly than intermediate social taxa, and are thus able to complete the reproductive cycle in shorter seasons at higher elevations. This dual impact of altitude and development time on sociality illustrates that ecological constraints can elicit dynamic shifts in behaviour, and helps explain the complex distribution of sociality across ecological gradients.

Variation in season length and development time is sufficient to drive the emergence and coexistence of social and solitary behavioral strategies.

Season length and its associated variables can influence the expression of social behaviors, including the occurrence of eusociality in insects. Eusociality can vary widely across environmental gradients, both within and between different species. Numerous theoretical models have been developed to examine the life history traits that underlie the emergence and maintenance of eusociality, yet the impact of seasonality on this process is largely uncharacterized. Here, we present a theoretical model that incorporates season length and offspring development time into a single, individual-focused model to examine how these factors can shape the costs and benefits of social living. We find that longer season lengths and faster brood development times are sufficient to favor the emergence and maintenance of a social strategy, while shorter seasons favor a solitary one. We also identify a range of season lengths where social and solitary strategies can coexist. Moreover, our theoretical predictions are well-matched to the natural history and behavior of two flexibly-eusocial bee species, suggesting our model can make realistic predictions about the evolution of different social strategies. Broadly, this work reveals the crucial role that environmental conditions can have in shaping social behavior and its evolution and underscores the need for further models that explicitly incorporate such variation to study evolutionary trajectories of eusociality.

Developmental transcriptomes predict adult social behaviors in the socially flexible sweat bee, Lasioglossum baleicum.

Natural variation can provide important insights into the genetic and environmental factors that shape social behavior and its evolution. The sweat bee, Lasioglossum baleicum , is a socially flexible bee capable of producing both solitary and eusocial nests. We demonstrate that within a single nesting aggregation, soil temperatures are a strong predictor of the social structure of nests. Sites with warmer temperatures in the spring have a higher frequency of social nests than cooler sites, perhaps because warmer temperatures provide a longer reproductive window for those nests. To identify the molecular correlates of this behavioral variation, we generated a de novo genome assembly for L. baleicum , and we used transcriptomic profiling to compare adults and developing offspring from eusocial and solitary nests. We find that adult, reproductive females have similar expression profiles regardless of social structure in the nest, but that there are strong differences between reproductive females and workers from social nests. We also find substantial differences in the transcriptomic profiles of stage-matched pupae from warmer, social-biased sites compared to cooler, solitary-biased sites. These transcriptional differences are strongly predictive of adult reproductive state, suggesting that the developmental environment may set the stage for adult behaviors in L. baleicum . Together, our results help to characterize the molecular mechanisms shaping variation in social behavior and highlight a potential role of environmental tuning during development as a factor shaping adult behavior and physiology in this socially flexible bee.

NAPS: Integrating pose estimation and tag-based tracking.

1. Significant advances in computational ethology have allowed the quantification of behaviour in unprecedented detail. Tracking animals in social groups, however, remains challenging as most existing methods can either capture pose or robustly retain individual identity over time but not both. 2. To capture finely resolved behaviours while maintaining individual identity, we built NAPS (NAPS is ArUco Plus SLEAP), a hybrid tracking framework that combines state-of-the-art, deep learning-based methods for pose estimation (SLEAP) with unique markers for identity persistence (ArUco). We show that this framework allows the exploration of the social dynamics of the common eastern bumblebee (Bombus impatiens). 3. We provide a stand-alone Python package for implementing this framework along with detailed documentation to allow for easy utilization and expansion. We show that NAPS can scale to long timescale experiments at a high frame rate and that it enables the investigation of detailed behavioural variation within individuals in a group. 4. Expanding the toolkit for capturing the constituent behaviours of social groups is essential for understanding the structure and dynamics of social networks. NAPS provides a key tool for capturing these behaviours and can provide critical data for understanding how individual variation influences collective dynamics.

Convergent and complementary selection shaped gains and losses of eusociality in sweat bees.

Sweat bees have repeatedly gained and lost eusociality, a transition from individual to group reproduction. Here we generate chromosome-length genome assemblies for 17 species and identify genomic signatures of evolutionary trade-offs associated with transitions between social and solitary living. Both young genes and regulatory regions show enrichment for these molecular patterns. We also identify loci that show evidence of complementary signals of positive and relaxed selection linked specifically to the convergent gains and losses of eusociality in sweat bees. This includes two pleiotropic proteins that bind and transport juvenile hormone (JH)-a key regulator of insect development and reproduction. We find that one of these proteins is primarily expressed in subperineurial glial cells that form the insect blood-brain barrier and that brain levels of JH vary by sociality. Our findings are consistent with a role of JH in modulating social behaviour and suggest that eusocial evolution was facilitated by alteration of the proteins that bind and transport JH, revealing how an ancestral developmental hormone may have been co-opted during one of life's major transitions. More broadly, our results highlight how evolutionary trade-offs have structured the molecular basis of eusociality in these bees and demonstrate how both directional selection and release from constraint can shape trait evolution.

Regulation of behaviorally associated gene networks in worker honey bee ovaries.

Several lines of evidence support genetic links between ovary size and division of labor in worker honey bees. However, it is largely unknown how ovaries influence behavior. To address this question, we first performed transcriptional profiling on worker ovaries from two genotypes that differ in social behavior and ovary size. Then, we contrasted the differentially expressed ovarian genes with six sets of available brain transcriptomes. Finally, we probed behavior-related candidate gene networks in wild-type ovaries of different sizes. We found differential expression in 2151 ovarian transcripts in these artificially selected honey bee strains, corresponding to approximately 20.3% of the predicted gene set of honey bees. Differences in gene expression overlapped significantly with changes in the brain transcriptomes. Differentially expressed genes were associated with neural signal transmission (tyramine receptor, TYR) and ecdysteroid signaling; two independently tested nuclear hormone receptors (HR46 and ftz-f1) were also significantly correlated with ovary size in wild-type bees. We suggest that the correspondence between ovary and brain transcriptomes identified here indicates systemic regulatory networks among hormones (juvenile hormone and ecdysteroids), pheromones (queen mandibular pheromone), reproductive organs and nervous tissues in worker honey bees. Furthermore, robust correlations between ovary size and neuraland endocrine response genes are consistent with the hypothesized roles of the ovaries in honey bee behavioral regulation.

Isolation disrupts social interactions and destabilizes brain development in bumblebees.

Social isolation, particularly in early life, leads to deleterious physiological and behavioral outcomes. Here, we leverage new high-throughput tools to comprehensively investigate the impact of isolation in the bumblebee, Bombus impatiens, from behavioral, molecular, and neuroanatomical perspectives. We reared newly emerged bumblebees in complete isolation, in small groups, or in their natal colony, and then analyzed their behaviors while alone or paired with another bee. We find that when alone, individuals of each rearing condition show distinct behavioral signatures. When paired with a conspecific, bees reared in small groups or in the natal colony express similar behavioral profiles. Isolated bees, however, showed increased social interactions. To identify the neurobiological correlates of these differences, we quantified brain gene expression and measured the volumes of key brain regions for a subset of individuals from each rearing condition. Overall, we find that isolation increases social interactions and disrupts gene expression and brain development. Limited social experience in small groups is sufficient to preserve typical patterns of brain development and social behavior.

Cooperation, conflict, and the evolution of queen pheromones.

While chemical communication regulates individual behavior in a wide variety of species, these communication systems are most elaborated in insect societies. In these complex systems, pheromones produced by the reproductive individuals (queens) are critical in establishing and maintaining dominant reproductive status over hundreds to thousands of workers. The proximate and ultimate mechanisms by which these intricate pheromone communication systems evolved are largely unknown, though there has been much debate over whether queen pheromones function as a control mechanism or as an honest signal facilitating cooperation. Here, we summarize results from recent studies in honey bees, bumble bees, wasps, ants and termites. We further discuss evolutionary mechanisms by which queen pheromone communication systems may have evolved. Overall, these studies suggest that queen-worker pheromone communication is a multi-component, labile dialog between the castes, rather than a simple, fixed signal-response system. We also discuss future approaches that can shed light on the proximate and ultimate mechanisms that underlie these complex systems by focusing on the development of increasingly sophisticated genomic tools and their potential applications to examine the molecular mechanisms that regulate pheromone production and perception.

Rate variation in the evolution of non-coding DNA associated with social evolution in bees.

The evolutionary origins of eusociality represent increases in complexity from individual to caste-based, group reproduction. These behavioural transitions have been hypothesized to go hand in hand with an increased ability to regulate when and where genes are expressed. Bees have convergently evolved eusociality up to five times, providing a framework to test this hypothesis. To examine potential links between putative gene regulatory elements and social evolution, we compare alignable, non-coding sequences in 11 diverse bee species, encompassing three independent origins of reproductive division of labour and two elaborations of eusocial complexity. We find that rates of evolution in a number of non-coding sequences correlate with key social transitions in bees. Interestingly, while we find little evidence for convergent rate changes associated with independent origins of social behaviour, a number of molecular pathways exhibit convergent rate changes in conjunction with subsequent elaborations of social organization. We also present evidence that many novel non-coding regions may have been recruited alongside the origin of sociality in corbiculate bees; these loci could represent gene regulatory elements associated with division of labour within this group. Thus, our findings are consistent with the hypothesis that gene regulatory innovations are associated with the evolution of eusociality and illustrate how a thorough examination of both coding and non-coding sequence can provide a more complete understanding of the molecular mechanisms underlying behavioural evolution. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

Genomic analysis of post-mating changes in the honey bee queen (Apis mellifera).

BACKGROUND: The molecular mechanisms underlying the post-mating behavioral and physiological transitions undergone by females have not been explored in great detail. Honey bees represent an excellent model system in which to address these questions because they exhibit a range of "mating states," with two extremes (virgins and egg-laying, mated queens) that differ dramatically in their behavior, pheromone profiles, and physiology. We used an incompletely-mated mating-state to understand the molecular processes that underlie the transition from a virgin to a mated, egg-laying queen. We used same-aged virgins, queens that mated once but did not initiate egg-laying, and queens that mated once and initiated egg-laying. RESULTS: Differences in the behavior and physiology among groups correlated with the underlying variance observed in the top 50 predictive genes in the brains and the ovaries. These changes were correlated with either a behaviorally-associated pattern or a physiologically-associated pattern. Overall, these results suggest that the brains and the ovaries of queens are uncoupled or follow different timescales; the initiation of mating triggers immediate changes in the ovaries, while changes in the brain may require additional stimuli or take a longer time to complete. Comparison of our results to previous studies of post-mating changes in Drosophila melanogaster identified common biological processes affected by mating, including stress response and alternative-splicing pathways. Comparison with microarray data sets related to worker behavior revealed no obvious correlation between genes regulated by mating and genes regulated by behavior/physiology in workers. CONCLUSION: Studying the underlying molecular mechanisms of post-mating changes in honey bee queens will not only give us insight into how molecular mechanisms regulate physiological and behavioral changes, but they may also lead to important insights into the evolution of social behavior. Post-mating changes in gene regulation in the brains and ovaries of honey bee queens appear to be triggered by different stimuli and may occur on different timescales, potentially allowing changes in the brains and the ovaries to be uncoupled.

The genetic basis of a social polymorphism in halictid bees.

The emergence of eusociality represents a major evolutionary transition from solitary to group reproduction. The most commonly studied eusocial species, honey bees and ants, represent the behavioral extremes of social evolution but lack close relatives that are non-social. Unlike these species, the halictid bee Lasioglossum albipes produces both solitary and eusocial nests and this intraspecific variation has a genetic basis. Here, we identify genetic variants associated with this polymorphism, including one located in the intron of syntaxin 1a (syx1a), a gene that mediates synaptic vesicle release. We show that this variant can alter gene expression in a pattern consistent with differences between social and solitary bees. Surprisingly, syx1a and several other genes associated with sociality in L. albipes have also been implicated in autism spectrum disorder in humans. Thus, genes underlying behavioral variation in L. albipes may also shape social behaviors across a wide range of taxa, including humans.

Social behaviour in bees influences the abundance of Sodalis (Enterobacteriaceae) symbionts.

Social interactions can facilitate transmission of microbes between individuals, reducing variation in gut communities within social groups. Thus, the evolution of social behaviours and symbiont community composition have the potential to be tightly linked. We explored this connection by characterizing the diversity of bacteria associated with both eusocial and solitary bee species within the behaviourally variable family Halictidae using 16S amplicon sequencing. Contrary to expectations, we found few differences in bacterial abundance or variation between social forms; most halictid species appear to share similar gut bacterial communities. However, several strains of Sodalis, a genus described as a symbiont in a variety of insects but yet to be characterized in bees, differ in abundance between eusocial and solitary bees. Phylogenetic reconstructions based on whole-genome alignments indicate that Sodalis has independently colonized halictids at least three times. These strains appear to be mutually exclusive within individual bees, although they are not host-species-specific and no signatures of vertical transmission were observed, suggesting that Sodalis strains compete for access to hosts. The symbiosis between halictids and Sodalis therefore appears to be in its early stages.

Spatial fidelity of workers predicts collective response to disturbance in a social insect.

Individuals in social insect colonies cooperate to perform collective work. While colonies often respond to changing environmental conditions by flexibly reallocating workers to different tasks, the factors determining which workers switch and why are not well understood. Here, we use an automated tracking system to continuously monitor nest behavior and foraging activity of uniquely identified workers from entire bumble bee (Bombus impatiens) colonies foraging in a natural outdoor environment. We show that most foraging is performed by a small number of workers and that the intensity and distribution of foraging is actively regulated at the colony level in response to forager removal. By analyzing worker nest behavior before and after forager removal, we show that spatial fidelity of workers within the nest generates uneven interaction with relevant localized information sources, and predicts which workers initiate foraging after disturbance. Our results highlight the importance of spatial fidelity for structuring information flow and regulating collective behavior in social insect colonies.