Differential gene expression underlying the biosynthesis of Dufour's gland signals in Bombus impatiens.

Pheromones regulating social behavior are one of the most explored phenomena in social insects. However, compound identity, biosynthesis and their genetic basis are known in only a handful of species. Here we examined the gene expression associated with pheromone biosynthesis of two main chemical classes: esters and terpenes, using the social bee Bombus impatiens. We conducted chemical and RNA-seq analyses of the Dufour's gland, an exocrine gland producing a plethora of pheromones regulating social behavior in hymenopteran species. The Dufour's gland contains mostly long-chained hydrocarbons, terpenes and esters that signal reproductive and social status in several bee species. In bumble bees, the Dufour's gland contains queen- and worker-specific esters, in addition to terpenes and terpene-esters only found in gynes and queens. These compounds are assumed to be synthesized de novo in the gland, however, their genetic basis is unknown. A whole transcriptome gene expression analysis of the gland in queens, gynes, queenless and queenright workers showed distinct transcriptomic profiles, with thousands of differentially expressed genes between the groups. Workers and queens express genes associated with key enzymes in the biosynthesis of wax esters, while queens and gynes preferentially express key genes in terpene biosynthesis. Overall, our data demonstrate gland-specific regulation of chemical signals associated with social behavior and identifies candidate genes and pathways regulating caste-specific chemical signals in social insects.

Symptomatic Infection with Vairimorpha spp. Decreases Diapause Survival in a Wild Bumble Bee Species (Bombus griseocollis).

Vairimorpha, a microsporidian parasite (previously classified as Nosema), has been implicated in the decline of wild bumble bee species in North America. Previous studies examining its influence on colony performance have displayed variable results, from extremely detrimental effects to no observable influence, and little is known about the effects it has on individuals during the winter diapause, a bottleneck for survival in many annual pollinators. Here, we examined the effect of Vairimorpha infection, body size, and mass on diapause survival in Bombus griseocollis gynes. We demonstrate that gyne survival length in diapause is negatively affected by symptomatic Vairimorpha infection of the maternal colony but does not correlate with individual pathogen load. Our findings further indicate that increased body mass offers a protective effect against mortality during diapause in infected, but not in healthy, gynes. This suggests that access to adequate nutritional resources prior to diapause might offset the harmful effect of Vairimorpha infection.

Impacts and mechanisms of CO2 narcosis in bumble bees: narcosis depends on dose, caste and mating status and is not induced by anoxia.

Carbon dioxide (CO2) is commonly used to immobilize insects and to induce reproduction in bees. However, despite its wide use and potential off-target impacts, its underlying mechanisms are not fully understood. Here, we used Bombus impatiens to examine whether CO2 impacts are mediated by anoxia and whether these mechanisms differ between female castes or following mating in queens. We examined the behavior, physiology and gene expression of workers, mated queens and virgin queens following exposure to anoxia, hypoxia, full and partial hypercapnia, and controls. Hypercapnia and anoxia caused immobilization, but only hypercapnia resulted in behavioral, physiological and molecular impacts in bees. Recovery from hypercapnia resulted in increased abdominal contractions and took longer in queens. Additionally, hypercapnia activated the ovaries of queens, but inhibited those of workers in a dose-dependent manner and caused a depletion of fat-body lipids in both castes. All responses to hypercapnia were weaker following mating in queens. Analysis of gene expression related to hypoxia and hypercapnia supported the physiological findings in queens, demonstrating that the overall impacts of CO2, excluding virgin queen ovaries, were unique and were not induced by anoxia. This study contributes to our understanding of the impacts and the mechanistic basis of CO2 narcosis in insects and its impacts on bee physiology. This article has an associated ECR Spotlight interview with Anna Cressman.

CO2 narcosis induces a metabolic shift mediated via juvenile hormone in Bombus impatiens gynes.

Carbon dioxide (CO2) has pleiotropic effects on insect physiology and behavior. Although diverse, many impacts are related to changes in metabolism and reallocation of macronutrients. Here we examined the metabolic shift induced by CO2 and its regulation using Bombus impatiens. CO2 applied to bumble bee gynes induces bypass of diapause and transition into reproduction. We analyzed ovary activation and macronutrient amounts in four tissues/body parts (fat body, thorax, ovaries, and crop) at three timepoints following CO2 administration. To tease apart the effects of CO2 on reproduction and metabolism, we monitored the metabolic changes in gynes following ovary removal and CO2 narcosis. We also explored the role of juvenile hormone in mediating CO2 impact by feeding queens with a JH antagonist (Precocene). Gynes ovary activation was increased following CO2 treatment. Additionally, CO2-treated gynes showed lower lipid amount in the fat body and higher glycogen and protein amount in the ovary ten days after the treatment. CO2 treatment following ovary removal also resulted in decreased fat body lipids, suggesting that CO2 operates by inducing a metabolic shift independent of reproduction. Lastly, gynes fed with precocence did not show a metabolic shift following CO2, suggesting CO2 impact is mediated via juvenile hormone. Overall, these data suggest that CO2 induces transfer of macronutrients and utilization of stored reserved by accelerating metabolism. The proposed mechanism of CO2 may explain many of the pleiotropic effects of CO2 across species and can aid in understanding how this common anastatic influences insect physiology.

Examining the individual and additive effects of cold storage and CO2 narcosis on queen survival and reproduction in bumble bees.

Diapause is a pre-programmed arrest of development allowing insects to survive in unfavorable environments. In adult insects, diapause termination is often followed by a reallocation of macronutrients and a transition to reproduction, and in some insects, this transition can be achieved using narcosis with CO2. However, whether CO2narcosis and diapause act in concert to affect reproduction remains unknown. Here, we investigated the separated and combined effects of diapause and CO2on female reproduction in queens of the common eastern bumble bee Bombus impatiens. Queens were treated with CO2 following a cold storage period (zero days, two weeks, two and four months) and were compared with untreated queens at the same timepoints for survival, colony initiation, egg-laying latency, and offspring production. We found that both CO2 and a period of at least two months in cold storage induced a transition to egg laying in gynes, and as expected, survival decreased with cold storage length. When CO2 and cold storage were combined, CO2narcosis positively affected egg laying in the earlier timepoints but its impact diminished following a longer cold storage. These data suggest that the impacts of CO2narcosis and cold storage are partially additive, and application of CO2 is effective only after a short cold storage. It further demonstrates that CO2 has complex effects on insect reproduction that are independent from diapause.

A Gland of Many Uses: a Diversity of Compounds in the Labial Glands of the Bumble Bee Bombus impatiens Suggests Multiple Signaling Functions.

Communication in social insect colonies depends on signals accurately reflecting the identity and physiological state of the individuals. Such information is coded by the products of multiple exocrine glands, and the resulting blends reflect the species, sex, caste, age, task, reproductive status, and health of an individual, and may also contain caste-specific pheromones regulating the behavior and physiology of other individuals. Here we examined the composition of labial gland secretions in females of the bumble bee Bombus impatiens, of different castes, social condition, age, mating status, and ovarian activation. We show that active queens, gynes, and workers each produce caste-specific compounds that may serve different communicative functions. The composition and amounts of wax esters, mostly octyl esters produced by active queens, differed significantly between castes, mating, and social conditions, suggesting a social signaling role. Farnesyl esters were predominant in gynes and peaked at optimal mating age (6-10 days), suggesting their possible roles as sex pheromone components. Reproductive status of females and age across castes was reflected by the ratio between short- and long-chain hydrocarbons, suggesting that these compounds may serve as fertility signals. Our findings overall suggest that the labial gland composition in B. impatiens reflects different facets of female physiology. While further bioassays are required to determine the functions of these compounds, they are likely to have important roles in communication between individuals.

The effect of the brood and the queen on early gene expression in bumble bee workers' brains.

Worker reproduction in social insects is often regulated by the queen, but can be regulated by the brood and nestmates, who may use different mechanisms to induce the same outcomes in subordinates. Analysis of brain gene expression patterns in bumble bee workers (Bombus impatiens) in response to the presence of the queen, the brood, both or neither, identified 18 differentially expressed genes, 17 of them are regulated by the queen and none are regulated by the brood. Overall, brain gene expression differences in workers were driven by the queen's presence, despite recent studies showing that brood reduces worker egg laying and provides context to the queen pheromones. The queen affected important regulators of reproduction and brood care across insects, such as neuroparsin and vitellogenin, and a comparison with similar datasets in the honey bee and the clonal raider ant revealed that neuroparsin is differentially expressed in all species. These data emphasize the prominent role of the queen in regulating worker physiology and behavior. Genes that serve as key regulators of workers' reproduction are likely to play an important role in the evolution of sociality.

Optimizing Laboratory Rearing of a Key Pollinator, Bombus impatiens.

Bumble bees are key pollinators for wild and managed plants and serve as a model system in various research fields, largely due to their commercial availability. Despite their extensive use, laboratory rearing of bumble bees is often challenging, particularly during the solitary phase queens undergo before founding a colony. Using a literature survey, we demonstrate that most studies rely on commercially available species that are provided during the colony's social phase, limiting study on early phases of the life cycle and the ability to control for colony age and relatedness. Laboratory rearing is challenging since the queen solitary phase is less understood compared to the social phase. To overcome this barrier, we examined several aspects related to the queen solitary phase: the effect of age on likelihood of mating, how the timing of CO2 narcosis post-mating (a technique to bypass diapause) affects egg-laying, and whether different social cues affect the success of colony initiation. Our data show an optimum age for mating in both sexuals and decreased egg-laying latency in the presence of workers and pupae. The timing of CO2 narcosis did not significantly affect egg laying in queens. These findings can be incorporated to improve bumble bee rearing for research purposes.

Bumble bee queen pheromones are context-dependent.

Queen pheromones have long been studied as a major factor regulating reproductive division of labor in social insects. Hitherto, only a handful of queen pheromones were identified and their effects on workers have mostly been studied in isolation from the social context in which they operate. Our study examined the importance of behavioral and social context for the perception of queen semiochemicals by bumble bee workers. Our results indicate that a mature queen's cuticular semiochemicals are capable of inhibiting worker reproduction only when accompanied by the queen's visual presence and the offspring she produces, thus, when presented in realistic context. Queen's chemistry, queen's visual presence and presence of offspring all act to regulate worker reproduction, but none of these elements produces an inhibitory effect on its own. Our findings highlight the necessity to reconsider what constitutes a queen pheromone and suggest a new approach to the study of chemical ecology in social insects.

Dufour's gland analysis reveals caste and physiology specific signals in Bombus impatiens.

Reproductive division of labor in insect societies is regulated through multiple concurrent mechanisms, primarily chemical and behavioral. Here, we examined if the Dufour's gland secretion in the primitively eusocial bumble bee Bombus impatiens signals information about caste, social condition, and reproductive status. We chemically analyzed Dufour's gland contents across castes, age groups, social and reproductive conditions, and examined worker behavioral and antennal responses to gland extracts. We found that workers and queens each possess caste-specific compounds in their Dufour's glands. Queens and gynes differed from workers based on the presence of diterpene compounds which were absent in workers, whereas four esters were exclusive to workers. These esters, as well as the total amounts of hydrocarbons in the gland, provided a separation between castes and also between fertile and sterile workers. Olfactometer bioassays demonstrated attraction of workers to Dufour's gland extracts that did not represent a reproductive conflict, while electroantennogram recordings showed higher overall antennal sensitivity in queenless workers. Our results demonstrate that compounds in the Dufour's gland act as caste- and physiology-specific signals and are used by workers to discriminate between workers of different social and reproductive status.

The effect of intrinsic physiological traits on diapause survival and their underlying mechanisms in an annual bee species Bombus impatiens.

In the face of insect declines, identifying phases of the life cycle when insects are particularly vulnerable to mortality is critical to conservation efforts. For numerous annual insect groups, diapause is both a key adaptation that allows survival of inhospitable conditions and a physiologically demanding life stage that can result in high rates of mortality. As bees continue to garner attention as a group experiencing high rates of decline, improving our understanding of how annual bees prepare for diapause and identifying factors that reduce survival is imperative. Here, we studied factors affecting diapause survival length and their underlying mechanisms using an economically and ecologically important annual bee species, Bombus impatiens. We examined how age and mass upon diapause onset correlate with diapause survival length, and the mechanistic role of nutrient acquisition and oxidative stress post pupal eclosion in mediating these effects. Our findings show that both age and mass were strong predictors of diapause survival length. Heavier queens or queens in the age range of ~6-17 days survived longer in diapause. Mass gain was attributed to increases in lipid, protein and glycerol amounts following pupal eclosion, and the ability to deal with oxidative stress was significantly compromised in older pre-diapause queens. Our results demonstrate that age-related shifts in bee physiology and timing of nutrient acquisition may both be critical factors driving diapause survival.

Shift in worker physiology and gene expression pattern from reproductive to diapause-like with colony age in the bumble bee Bombus impatiens.

Insects maximize their fitness by exhibiting predictable and adaptive seasonal patterns in response to changing environmental conditions. These seasonal patterns are often expressed even when insects are kept in captivity, suggesting they are functionally and evolutionarily important. In this study, we examined whether workers of the eusocial bumble bee Bombus impatiens maintained a seasonal signature when kept in captivity. We used an integrative approach and compared worker egg laying, ovarian activation, body size and mass, lipid content in the fat body, cold tolerance and expression of genes related to cold tolerance, metabolism and stress throughout colony development. We found that bumble bee worker physiology and gene expression patterns shift from reproductive-like to diapause-like as the colony ages. Workers eclosing early in the colony cycle had increased egg laying and ovarian activation, and reduced cold tolerance, body size, mass and lipid content in the fat body, in line with a reproductive-like profile, while late-eclosing workers exhibited the opposite characteristics. Furthermore, expression patterns of genes associated with reproduction and diapause differed between early- and late-eclosing workers, partially following the physiological patterns. We suggest that a seasonal signature, innate to individual workers, the queen or the colony, is used by workers as a social cue determining the phenology of the colony and discuss possible implications for understanding reproductive division of labor in bumble bee colonies and the evolutionary divergence of female castes in the genus Bombus.

A small family business: synergistic and additive effects of the queen and the brood on worker reproduction in a primitively eusocial bee.

The mechanisms that maintain reproductive division of labor in social insects are still incompletely understood. Most studies focus on the relationship between adults, overlooking another important stakeholder - the juveniles. Recent studies show that not only the queen but also the brood regulate worker reproduction. However, how the two coordinate to maintain reproductive monopoly remained unexplored. Here, we disentangled the roles of the brood and the queen in primitively eusocial bees (Bombus impatiens) by examining their separated and combined effects on worker behavioral, physiological and brain gene expression. We found that young larvae produce a releaser effect on workers, decreasing oviposition and aggression, while the queen produces both releaser and primer effects, modifying worker behavior and reproductive physiology. The expression of reproduction- and aggression-related genes was altered in the presence of both queen and brood but was stronger or the same in the presence of the queen. We identified two types of interactions between the queen and the brood in regulating worker reproduction: (1) synergistic interactions regulating worker physiology, where the combined effect of the queen and the brood on worker physiology was greater than their separate effects; (2) additive interactions, where the combined effect of the queen and the brood on worker behavior was similar to the sum of their separate effects. Our results suggest that the queen and the brood interact synergistically and additively to regulate worker behavior and reproduction, and this interaction exists at multiple regulatory levels.

Do Bumble Bees Produce Brood Pheromones?

Reproductive division of labor, a defining feature of social insects, is often regulated by a combination of behavioral and chemical means. It is hypothesized that behavioral interactions play a more important role in regulating reproduction of primitive eusocial species, while pheromones are typically used by large sized, advanced eusocial species. Here we examined if worker reproduction in the primitively eusocial species Bombus impatiens is regulated by brood pheromones. We recently demonstrated that worker egg laying in this species is inhibited by young larvae and triggered by pupae. However, the mechanism by which the brood communicates its presence and whether brood or hunger pheromones are involved remain unknown. We found that workers were behaviorally attracted to pupae over larvae or control in a choice experiment, in line with their reproductive interests. However, odors from larvae or pupae were insufficient to inhibit worker reproduction. We further show that the youngest larvae are particularly vulnerable to starvation, however, despite a slight attraction and fewer eggs laid by workers in the presence of starved compared with fed larvae, these effects were insignificant. Our study demonstrates that workers can differentiate between larvae and pupae, but not between starved and fed larvae based on olfactory information. However, these signals alone do not explain the reduction in worker egg laying previously found. Bumble bee workers may use information from multiple sources or rely solely on behavioral interactions with brood and other females to make decisions about reproduction, in line with their small colony size and simple social organization.

Context matters: plasticity in response to pheromones regulating reproduction and collective behavior in social Hymenoptera.

Pheromones mediating social behavior are critical components in the cohesion and function of the colony and are instrumental in the evolution of eusocial insect species. However, different aspects of colony function, such as reproductive division of labor and colony maintenance (e.g. foraging, brood care, and defense), pose different challenges for the optimal function of pheromones. While reproductive communication is shaped by forces of conflict and competition, colony maintenance calls for enhanced cooperation and self-organization. Mechanisms that ensure efficacy, adaptivity and evolutionary stability of signals such as structure-to-function suitability, honesty and context are important to all chemical signals but vary to different degrees between pheromones regulating reproductive division of labor and colony maintenance. In this review, we will discuss these differences along with the mechanisms that have evolved to ensure pheromone adaptivity in reproductive and non-reproductive context.

Evolution of Olfactory Functions on the Fire Ant Social Chromosome.

Understanding the molecular evolutionary basis of social behavior is a major challenge in evolutionary biology. Social insects evolved a complex language of chemical signals to coordinate thousands of individuals. In the fire ant Solenopsis invicta, chemical signals are involved in the determination of a polymorphic social organization. Single-queen (monogyne) or multiqueen (polygyne) social structure is determined by the "social chromosome," a nonrecombining region containing ∼504 genes with two distinct haplotypes, SB and Sb. Monogyne queens are always SBB, while polygyne queens are always SBb. Workers discriminate monogyne from polygyne queens based on olfactory cues. Here, we took an evolutionary genomics approach to search for candidate genes in the social chromosome that could be responsible for this discrimination. We compared the SB and Sb haplotypes and analyzed the evolutionary rates since their divergence. Notably, we identified a cluster of 23 odorant receptors in the nonrecombining region of the social chromosome that stands out in terms of nonsynonymous changes in both haplotypes. The cluster includes twelve genes formed by recent Solenopsis-specific duplications. We found evidence for positive selection on several tree branches and significant differences between the SB and Sb haplotypes of these genes. The most dramatic difference is the complete deletion of two of these genes in Sb. These results suggest that the evolution of polygyne social organization involved adaptations in olfactory genes and opens the way for functional studies of the molecular mechanisms underlying social behavior.

Evaluating the molecular, physiological and behavioral impacts of CO2 narcosis in bumble bees (Bombus impatiens).

Exposure to carbon dioxide (CO2) has pleiotropic effects in many insect species, ranging from eliciting rapid behavioral responses such as attraction, to dramatic physiological changes, including ovary activation. In bumble bees, CO2 narcosis causes queens to bypass diapause and initiate egg laying, but its mode of action is not well-understood. Here, we evaluated the effects of CO2 narcosis on the behavior, physiology and immune function of virgin bumble bee queens (Bombus impatiens). We tested the hypothesis that CO2 induces these changes by stimulating oxidative stress response pathways. We found that CO2 stimulates ovarian activation and egg production and suppresses lipid (but not glycogen) accumulation in virgin queens. Additionally, CO2 treated queens were more active (particularly in terms of flight) and performed, but did not receive, more aggressive behaviors compared to controls. Moreover, CO2 positively affected immune function in queens, reduced transcript levels of 5/6 antioxidant enzyme genes and had no effect on longevity. Thus, although CO2 treatment stimulated reproduction, we did not observe any evidence of a trade-off in queen health parameters, aside from a reduction in lipids. Overall CO2 narcosis does not appear to stimulate a typical stress response in virgin bumble bee queens. On the contrary, CO2 narcosis appears to stimulate changes that prepare queens to cope with the nutritional, metabolic and behavioral challenges associated with reproduction and colony-founding.

Do Bumble Bee, Bombus impatiens, Queens Signal their Reproductive and Mating Status to their Workers?

Reproduction in social insect societies reflects a delicate balance between cooperation and conflict over offspring production, and worker reproduction is widespread even in species showing strong reproductive skew in favor of the queen. To navigate these conflicts, workers are predicted to develop the means to estimate the queen's fecundity - potentially through behavioral and/or chemical cues - and to adjust their reproduction to maximize their fitness. Here, we introduced bumble bee, Bombus impatiens, workers to queens of different mating and reproductive status and examined worker reproduction and expression levels of two genes which were previously shown to be sensitive to the presence of the queen, vitellogenin and Krüppel-homolog 1. We further explored whether the queen's chemical secretion alone is sufficient to regulate worker reproduction, aggression and gene expression. We found that worker ovary activation was inhibited only in the presence of egg-laying queens, regardless of their mating status. Workers reared in the presence of newly-mated queens showed intermediate vitellogenin expression levels relative to workers reared with mated egg-laying and virgin queens. However, none of the whole-body chemical extracts of any of the queen treatment groups affected ovary activation, aggressive behavior, or gene expression in workers. Our findings indicate that only the presence of a freely-behaving, egg-laying queen can fully inhibit worker reproduction. It remains to be determined if workers detect differences in queen mating status and fecundity through differences in the queens' behavior alone or through the queen's behavior in concert with fertility signals.

Chemical communication is not sufficient to explain reproductive inhibition in the bumblebee Bombus impatiens.

Reproductive division of labour is a hallmark of eusociality, but disentangling the underlying proximate mechanisms can be challenging. In bumblebees, workers isolated from the queen can activate their ovaries and lay haploid, male eggs. We investigated if volatile, contact, visual or behavioural cues produced by the queen or brood mediate reproductive dominance in Bombus impatiens. Exposure to queen-produced volatiles, brood-produced volatiles and direct contact with pupae did not reduce worker ovary activation; only direct contact with the queen could reduce ovary activation. We evaluated behaviour, physiology and gene expression patterns in workers that were reared in chambers with all stages of brood and a free queen, caged queen (where workers could contact the queen, but the queen was unable to initiate interactions) or no queen. Workers housed with a caged queen or no queen fully activated their ovaries, whereas ovary activation in workers housed with a free queen was completely inhibited. The caged queen marginally reduced worker aggression and expression of an aggression-associated gene relative to queenless workers. Thus, queen-initiated behavioural interactions appear necessary to establish reproductive dominance. Queen-produced chemical cues may function secondarily in a context-specific manner to augment behavioural cues, as reliable or honest signal.