Glyphosate impairs aversive learning in bumblebees.

Agrochemicals represent prominent anthropogenic stressors contributing to the ongoing global insect decline. While their impact is generally assessed in terms of mortality rates, non-lethal effects on fitness are equally important to insect conservation. Glyphosate, a commonly used herbicide, is toxic to many animal species, and thought to impact a range of physiological functions. In this study, we investigate the impact of long-term exposure to glyphosate on locomotion, phototaxis and learning abilities in bumblebees, using a fully automated high-throughput assay. We find that glyphosate exposure had a very slight and transient impact on locomotion, while leaving the phototactic drive unaffected. Glyphosate exposure also reduced attraction towards UV light when blue was given as an alternative and, most strikingly, impaired learning of aversive stimuli. Thus, glyphosate had specific actions on sensory and cognitive processes. These non-lethal perceptual and cognitive impairments likely represent a significant obstacle to foraging and predator avoidance for wild bumblebees exposed to glyphosate. Similar effects in other species could contribute to a widespread reduction in foraging efficiency across ecosystems, driven by the large-scale application of this herbicide. The high-throughput paradigm presented in this study can be adapted to investigate sublethal effects of other agrochemicals on bumblebees or other important pollinator species, opening up a critical new avenue for the study of anthropogenic stressors.

Geosmin suppresses defensive behaviour and elicits unusual neural responses in honey bees.

Geosmin is an odorant produced by bacteria in moist soil. It has been found to be extraordinarily relevant to some insects, but the reasons for this are not yet fully understood. Here we report the first tests of the effect of geosmin on honey bees. A stinging assay showed that the defensive behaviour elicited by the bee's alarm pheromone component isoamyl acetate (IAA) is strongly suppressed by geosmin. Surprisingly, the suppression is, however, only present at very low geosmin concentrations, and disappears at higher concentrations. We investigated the underlying mechanisms at the level of the olfactory receptor neurons by means of electroantennography, finding the responses to mixtures of geosmin and IAA to be lower than to pure IAA, suggesting an interaction of both compounds at the olfactory receptor level. Calcium imaging of the antennal lobe (AL) revealed that neuronal responses to geosmin decreased with increasing concentration, correlating well with the observed behaviour. Computational modelling of odour transduction and coding in the AL suggests that a broader activation of olfactory receptor types by geosmin in combination with lateral inhibition could lead to the observed non-monotonic increasing-decreasing responses to geosmin and thus underlie the specificity of the behavioural response to low geosmin concentrations.

Extracting individual characteristics from population data reveals a negative social effect during honeybee defence.

Honeybees protect their colony against vertebrates by mass stinging and they coordinate their actions during this crucial event thanks to an alarm pheromone carried directly on the stinger, which is therefore released upon stinging. The pheromone then recruits nearby bees so that more and more bees participate in the defence. However, a quantitative understanding of how an individual bee adapts its stinging response during the course of an attack is still a challenge: Typically, only the group behaviour is effectively measurable in experiment; Further, linking the observed group behaviour with individual responses requires a probabilistic model enumerating a combinatorial number of possible group contexts during the defence; Finally, extracting the individual characteristics from group observations requires novel methods for parameter inference. We first experimentally observed the behaviour of groups of bees confronted with a fake predator inside an arena and quantified their defensive reaction by counting the number of stingers embedded in the dummy at the end of a trial. We propose a biologically plausible model of this phenomenon, which transparently links the choice of each individual bee to sting or not, to its group context at the time of the decision. Then, we propose an efficient method for inferring the parameters of the model from the experimental data. Finally, we use this methodology to investigate the effect of group size on stinging initiation and alarm pheromone recruitment. Our findings shed light on how the social context influences stinging behaviour, by quantifying how the alarm pheromone concentration level affects the decision of each bee to sting or not in a given group size. We show that recruitment is curbed as group size grows, thus suggesting that the presence of nestmates is integrated as a negative cue by individual bees. Moreover, the unique integration of exact and statistical methods provides a quantitative characterisation of uncertainty associated to each of the inferred parameters.

Olfactory Strategies in the Defensive Behaviour of Insects.

Most animals must defend themselves in order to survive. Defensive behaviour includes detecting predators or intruders, avoiding them by staying low-key or escaping or deterring them away by means of aggressive behaviour, i.e., attacking them. Responses vary across insect species, ranging from individual responses to coordinated group attacks in group-living species. Among different modalities of sensory perception, insects predominantly use the sense of smell to detect predators, intruders, and other threats. Furthermore, social insects, such as honeybees and ants, communicate about danger by means of alarm pheromones. In this review, we focus on how olfaction is put to use by insects in defensive behaviour. We review the knowledge of how chemical signals such as the alarm pheromone are processed in the insect brain. We further discuss future studies for understanding defensive behaviour and the role of olfaction.

Honeybee communication during collective defence is shaped by predation.

BACKGROUND: Social insect colonies routinely face large vertebrate predators, against which they need to mount a collective defence. To do so, honeybees use an alarm pheromone that recruits nearby bees into mass stinging of the perceived threat. This alarm pheromone is carried directly on the stinger; hence, its concentration builds up during the course of the attack. We investigate how bees react to different alarm pheromone concentrations and how this evolved response pattern leads to better coordination at the group level. RESULTS: We first present a dose-response curve to the alarm pheromone, obtained experimentally. This data reveals two phases in the bees' response: initially, bees become more likely to sting as the alarm pheromone concentration increases, but aggressiveness drops back when very high concentrations are reached. Second, we apply Projective Simulation to model each bee as an artificial learning agent that relies on the pheromone concentration to decide whether to sting or not. Individuals are rewarded based on the collective performance, thus emulating natural selection in these complex societies. By also modelling predators in a detailed way, we are able to identify the main selection pressures that shaped the response pattern observed experimentally. In particular, the likelihood to sting in the absence of alarm pheromone (starting point of the dose-response curve) is inversely related to the rate of false alarms, such that bees in environments with low predator density are less likely to waste efforts responding to irrelevant stimuli. This is compensated for by a steep increase in aggressiveness when the alarm pheromone concentration starts rising. The later decay in aggressiveness may be explained as a curbing mechanism preventing worker loss. CONCLUSIONS: Our work provides a detailed understanding of alarm pheromone responses in honeybees and sheds light on the selection pressures that brought them about. In addition, it establishes our approach as a powerful tool to explore how selection based on a collective outcome shapes individual responses, which remains a challenging issue in the field of evolutionary biology.

Complexity and plasticity in honey bee phototactic behaviour.

The ability to move towards or away from a light source, namely phototaxis, is essential for a number of species to find the right environmental niche and may have driven the appearance of simple visual systems. In this study we ask if the later evolution of more complex visual systems was accompanied by a sophistication of phototactic behaviour. The honey bee is an ideal model organism to tackle this question, as it has an elaborate visual system, demonstrates exquisite abilities for visual learning and performs phototaxis. Our data suggest that in this insect, phototaxis has wavelength specific properties and is a highly dynamical response including multiple decision steps. In addition, we show that previous experience with a light (through exposure or classical aversive conditioning) modulates the phototactic response. This plasticity is dependent on the wavelength used, with blue being more labile than green or ultraviolet. Wavelength, intensity and past experience are integrated into an overall valence for each light that determines phototactic behaviour in honey bees. Thus, our results support the idea that complex visual systems allow sophisticated phototaxis. Future studies could take advantage of these findings to better understand the neuronal circuits underlying this processing of the visual information.

Aversive Training of Honey Bees in an Automated Y-Maze.

Honeybees have remarkable learning abilities given their small brains, and have thus been established as a powerful model organism for the study of learning and memory. Most of our current knowledge is based on appetitive paradigms, in which a previously neutral stimulus (e.g., a visual, olfactory, or tactile stimulus) is paired with a reward. Here, we present a novel apparatus, the yAPIS, for aversive training of walking honey bees. This system consists in three arms of equal length and at 120° from each other. Within each arm, colored lights (λ = 375, 465 or 520 nm) or odors (here limonene or linalool) can be delivered to provide conditioned stimuli (CS). A metal grid placed on the floor and roof delivers the punishment in the form of mild electric shocks (unconditioned stimulus, US). Our training protocol followed a fully classical procedure, in which the bee was exposed sequentially to a CS paired with shocks (CS+) and to another CS not punished (CS-). Learning performance was measured during a second phase, which took advantage of the Y-shape of the apparatus and of real-time tracking to present the bee with a choice situation, e.g., between the CS+ and the CS-. Bees reliably chose the CS- over the CS+ after only a few training trials with either colors or odors, and retained this memory for at least a day, except for the shorter wavelength (λ = 375 nm) that produced mixed results. This behavior was largely the result of the bees avoiding the CS+, as no evidence was found for attraction to the CS-. Interestingly, trained bees initially placed in the CS+ spontaneously escaped to a CS- arm if given the opportunity, even though they could never do so during the training. Finally, honey bees trained with compound stimuli (color + odor) later avoided either components of the CS+. Thus, the yAPIS is a fast, versatile and high-throughput way to train honey bees in aversive paradigms. It also opens the door for controlled laboratory experiments investigating bimodal integration and learning, a field that remains in its infancy.

Seasonality, alarm pheromone and serotonin: insights on the neurobiology of honeybee defence from winter bees.

Honeybees maintain their colony throughout the cold winters, a strategy that enables them to make the most of early spring flowers. During this period, their activity is mostly limited to thermoregulation, while foraging and brood rearing are stopped. Less is known about seasonal changes to the essential task of defending the colony against intruders, which is regulated by the sting alarm pheromone. We studied the stinging responsiveness of winter bees exposed to this scent or a control (solvent). Surprisingly, winter bees, while maintaining their responsiveness in control conditions, did not increase stinging frequency in response to the alarm pheromone. This was not owing to the bees not perceiving the pheromone, as shown by calcium imaging of the antennal lobes. As the alarm pheromone is thought to act through an increase in brain serotonin levels, ultimately causing heightened defensiveness, we checked if serotonin treatments would affect the stinging behaviour of winter bees. Indeed, treated winter bees became more inclined to sting. Thus, we postulate that loss of responsiveness to the sting alarm pheromone is based on a partial or total disruption of the mechanism converting alarm pheromone perception into high serotonin levels in winter bees.

Cooperative defence operates by social modulation of biogenic amine levels in the honey bee brain.

The defence of a society often requires that some specialized members coordinate to repel a threat at personal risk. This is especially true for honey bee guards, which defend the hive and may sacrifice their lives upon stinging. Central to this cooperative defensive response is the sting alarm pheromone, which has isoamyl acetate (IAA) as its main component. Although this defensive behaviour has been well described, the neural mechanisms triggered by IAA to coordinate stinging have long remained unknown. Here we show that IAA upregulates brain levels of serotonin and dopamine, thereby increasing the likelihood of an individual bee to attack and sting. Pharmacological enhancement of the levels of both amines induces higher defensive responsiveness, while decreasing them via antagonists decreases stinging. Our results thus uncover the neural mechanism by which an alarm pheromone recruits individuals to attack and repel a threat, and suggest that the alarm pheromone of honey bees acts on their response threshold rather than as a direct trigger.

The defensive response of the honeybee Apis mellifera.

Honeybees (Apis mellifera) are insects living in colonies with a complex social organization. Their nest contains food stores in the form of honey and pollen, as well as the brood, the queen and the bees themselves. These resources have to be defended against a wide range of predators and parasites, a task that is performed by specialized workers, called guard bees. Guards tune their response to both the nature of the threat and the environmental conditions, in order to achieve an efficient trade-off between defence and loss of foraging workforce. By releasing alarm pheromones, they are able to recruit other bees to help them handle large predators. These chemicals trigger both rapid and longer-term changes in the behaviour of nearby bees, thus priming them for defence. Here, we review our current understanding on how this sequence of events is performed and regulated depending on a variety of factors that are both extrinsic and intrinsic to the colony. We present our current knowledge on the neural bases of honeybee aggression and highlight research avenues for future studies in this area. We present a brief overview of the techniques used to study honeybee aggression, and discuss how these could be used to gain further insights into the mechanisms of this behaviour.

Sustained Rhythmic Brain Activity Underlies Visual Motion Perception in Zebrafish.

Following moving visual stimuli (conditioning stimuli, CS), many organisms perceive, in the absence of physical stimuli, illusory motion in the opposite direction. This phenomenon is known as the motion aftereffect (MAE). Here, we use MAE as a tool to study the neuronal basis of visual motion perception in zebrafish larvae. Using zebrafish eye movements as an indicator of visual motion perception, we find that larvae perceive MAE. Blocking eye movements using optogenetics during CS presentation did not affect MAE, but tectal ablation significantly weakened it. Using two-photon calcium imaging of behaving GCaMP3 larvae, we find post-stimulation sustained rhythmic activity among direction-selective tectal neurons associated with the perception of MAE. In addition, tectal neurons tuned to the CS direction habituated, but neurons in the retina did not. Finally, a model based on competition between direction-selective neurons reproduced MAE, suggesting a neuronal circuit capable of generating perception of visual motion.

Appetitive floral odours prevent aggression in honeybees.

Honeybees defend their colonies aggressively against intruders and release a potent alarm pheromone to recruit nestmates into defensive tasks. The effect of floral odours on this behaviour has never been studied, despite the relevance of these olfactory cues for the biology of bees. Here we use a novel assay to investigate social and olfactory cues that drive defensive behaviour in bees. We show that social interactions are necessary to reveal the recruiting function of the alarm pheromone and that specific floral odours-linalool and 2-phenylethanol-have the surprising capacity to block recruitment by the alarm pheromone. This effect is not due to an olfactory masking of the pheromone by the floral odours, but correlates with their appetitive value. In addition to their potential applications, these findings provide new insights about how honeybees make the decision to engage into defence and how conflicting information affects this process.

Involvement of newborn neurons in olfactory associative learning? The operant or non-operant component of the task makes all the difference.

New neurons are continuously generated in the adult mammalian olfactory bulb. The role of these newborn neurons in olfactory learning has been debated. Blocking the addition of neurons has been reported either to result in memory alteration or to have no effect at all (Imayoshi et al., 2008; Breton-Provencher et al., 2009; Lazarini et al., 2009; Sultan et al., 2010). These discrepancies may have arisen from differences in the behavioral paradigms used: operant procedures indicated that neurogenesis blockade had substantial effects on long-term memory (Lazarini et al., 2009; Sultan et al., 2010) whereas other methods had little effect (Imayoshi et al., 2008; Breton-Provencher et al., 2009). Surprisingly, while operant learning is known to modulate the survival of new neurons, the effect of non-operant learning on newborn cells is unknown. Here we use mice to show that compared with operant learning, non-operant learning does not affect cell survival, perhaps explaining the current controversy. In addition, we provide evidence that distinct neural substrates at least partly underlie these two forms of learning. We conclude that the involvement of newborn neurons in learning is subtly dependent on the nature of the behavioral task.