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.

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.

Antenna movements as a function of odorants' biological value in honeybees (Apis mellifera L.).

In honeybees, the antennae are highly mobile sensory organs that express scanning movements in various behavioral contexts and toward many stimuli, especially odorants. The rules underlying these movements are still unclear. Using a motion-capture system, we analyzed bees' antennal responses to a panel of pheromonal and other biologically relevant odorants. We observed clear differences in bees' antennal responses, with opposite movements to stimuli related to opposite contexts: slow backward movements were expressed in response to alarm pheromones, while fast forward movements were elicited by food related cues as well as brood and queen related pheromones. These responses are reproducible, as a similar pattern of odor-specific responses was observed in bees from different colonies, on different years. We then tested whether odorants' attractiveness for bees, measured using an original olfactory orientation setup, may predict antenna movements. This simple measure of odorants' valence did however not correlate with either antennal position or velocity measures, showing that more complex rules than simple hedonics underlie bees' antennal responses to odorants. Lastly, we show that newly-emerged bees express only limited antennal responses compared to older bees, suggesting that a significant part of the observed responses are acquired during bees' behavioral development.

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.

Social Contact Acts as Appetitive Reinforcement and Supports Associative Learning in Honeybees.

Social learning is taxonomically widespread in the animal kingdom [1], and although it is long thought to be a hallmark of vertebrates, recent studies revealed that it also exists in insects [2-5]. The adaptive functions of social learning are well known, but its underlying mechanisms remain debated [2, 5, 6]. Social insects critically depend on the social transmission of information for successful food search and their colonies' fitness [7] and are tractable models for studying the social cues and cognitive mechanisms involved [2-5]. Besides the well-known dance language allowing them to communicate the location of food sources among nestmates [8], honeybees also learn chemosensory information about these sources both outside and within the hive [9, 10]. In the latter case, they associate the floral scent carried by returning foragers on their body with the nectar provided through mouth-to-mouth trophallaxis, similar to the manner in which foragers directly learn odorant-nectar reward associations at the foraging patch [9-11]. Strikingly, however, neither the dance nor trophallaxis is strictly necessary for foragers recruited within the hive to find the right floral source, and simple body contact between foragers may be sufficient [12]. What is the reinforcing agent in this case? We show here that simple social contact acts as appetitive reinforcement and can be used in associative olfactory learning. We demonstrate that this social reinforcement is mediated by bees' antennal movements and modulated by bees' behavioral development. These results unveil a social learning mechanism that may play a facilitating role in resource exploitation by social groups.

Virgin queen attraction toward males in honey bees.

Although the honeybee is a crucial agricultural agent and a prominent scientific model organism, crucial aspects of its reproductive behaviour are still unknown. During the mating season, honeybee males, the drones, gather in congregations 10-40 m above ground. Converging evidence suggests that drones emit a pheromone that can attract other drones, thereby increasing the size of the congregation. Virgin queens join the vicinity of the congregation after it has formed, and mate with as many as 20 males in mid-air. It is still unclear which sensory cues help virgin queens find drone congregations in the first place. Beside visual cues for long-range orientation, queens may use olfactory cues. We thus tested virgin queens' olfactory orientation on a walking simulator in which they have full control over odour stimulation. We show that sexually-mature virgin queens are attracted to the odour bouquet from a group of living drones. They are not attracted to the bouquet from a group of workers. In addition, non-sexually receptive females (workers) of the same age are not attracted to the drone odour bouquet. Interpreted in the context of mating, these results may suggest that virgin queens use volatile olfactory cues from the drones to find the congregations.

Appetitive but not aversive olfactory conditioning modifies antennal movements in honeybees.

In honeybees, two olfactory conditioning protocols allow the study of appetitive and aversive Pavlovian associations. Appetitive conditioning of the proboscis extension response (PER) involves associating an odor, the conditioned stimulus (CS) with a sucrose solution, the unconditioned stimulus (US). Conversely, aversive conditioning of the sting extension response (SER) involves associating the odor CS with an electric or thermal shock US. Each protocol is based on the measure of a different behavioral response (proboscis versus sting) and both only provide binary responses (extension or not of the proboscis or sting). These limitations render the measure of the acquired valence of an odor CS difficult without testing the animals in a freely moving situation. Here, we studied the effects of both olfactory conditioning protocols on the movements of the antennae, which are crucial sensory organs for bees. As bees' antennae are highly mobile, we asked whether their movements in response to an odorant change following appetitive or aversive conditioning and if so, do odor-evoked antennal movements contain information about the acquired valence of the CS? We implemented a tracking system for harnessed bees' antennal movements based on a motion capture principle at a high frequency rate. We observed that differential appetitive conditioning had a strong effect on antennal movements. Bees responded to the reinforced odorant with a marked forward motion of the antennae and a strong velocity increase. Conversely, differential aversive conditioning had no associative effect on antennal movements. Rather than revealing the acquired valence of an odorant, antennal movements may represent a novel conditioned response taking place during appetitive conditioning and may provide a possible advantage to bees when foraging in natural situations.