Environmental exposure to metallic pollution impairs honey bee brain development and cognition.

Laboratory studies show detrimental effects of metallic pollutants on invertebrate behaviour and cognition, even at low levels. Here we report a field study on Western honey bees exposed to metal and metalloid pollution through dusts, food and water at a historic mining site. We analysed more than 1000 bees from five apiaries along a gradient of contamination within 11 km of a former gold mine in Southern France. Bees collected close to the mine exhibited olfactory learning performances lower by 36% and heads smaller by 4%. Three-dimensional scans of bee brains showed that the olfactory centres of insects sampled close to the mine were also 4% smaller, indicating neurodevelopmental issues. Our study raises serious concerns about the health of honey bee populations in areas polluted with potentially harmful elements, particularly with arsenic, and illustrates how standard cognitive tests can be used for risk assessment.

Natural variability in bee brain size and symmetry revealed by micro-CT imaging and deep learning.

Analysing large numbers of brain samples can reveal minor, but statistically and biologically relevant variations in brain morphology that provide critical insights into animal behaviour, ecology and evolution. So far, however, such analyses have required extensive manual effort, which considerably limits the scope for comparative research. Here we used micro-CT imaging and deep learning to perform automated analyses of 3D image data from 187 honey bee and bumblebee brains. We revealed strong inter-individual variations in total brain size that are consistent across colonies and species, and may underpin behavioural variability central to complex social organisations. In addition, the bumblebee dataset showed a significant level of lateralization in optic and antennal lobes, providing a potential explanation for reported variations in visual and olfactory learning. Our fast, robust and user-friendly approach holds considerable promises for carrying out large-scale quantitative neuroanatomical comparisons across a wider range of animals. Ultimately, this will help address fundamental unresolved questions related to the evolution of animal brains and cognition.

Memory consolidation in honey bees is enhanced by down-regulation of Down syndrome cell adhesion molecule and changes its alternative splicing.

Down syndrome cell adhesion molecule (Dscam) gene encodes a cell adhesion molecule required for neuronal wiring. A remarkable feature of arthropod Dscam is massive alternative splicing generating thousands of different isoforms from three variable clusters of alternative exons. Dscam expression and diversity arising from alternative splicing have been studied during development, but whether they exert functions in adult brains has not been determined. Here, using honey bees, we find that Dscam expression is critically linked to memory retention as reducing expression by RNAi enhances memory after reward learning in adult worker honey bees. Moreover, alternative splicing of Dscam is altered in all three variable clusters after learning. Since identical Dscam isoforms engage in homophilic interactions, these results suggest a mechanism to alter inclusion of variable exons during memory consolidation to modify neuronal connections for memory retention.

Reduction of stress responses in honey bees by synthetic ligands targeting an allatostatin receptor.

Honey bees are of great economic and ecological importance, but are facing multiple stressors that can jeopardize their pollination efficiency and survival. Therefore, understanding the physiological bases of their stress response may help defining treatments to improve their resilience. We took an original approach to design molecules with this objective. We took advantage of the previous identified neuropeptide allatostatin A (ASTA) and its receptor (ASTA-R) as likely mediators of the honey bee response to a biologically relevant stressor, exposure to an alarm pheromone compound. A first series of ASTA-R ligands were identified through in silico screening using a homology 3D model of the receptor and in vitro binding experiments. One of these (A8) proved also efficient in vivo, as it could counteract two behavioral effects of pheromone exposure, albeit only in the millimolar range. This putative antagonist was used as a template for the chemical synthesis of a second generation of potential ligands. Among these, two compounds showed improved efficiency in vivo (in the micromolar range) as compared to A8 despite no major improvement in their affinity for the receptor in vitro. These new ligands are thus promising candidates for alleviating stress in honey bees.

Honey bees cannot sense harmful concentrations of metal pollutants in food.

Whether animals can actively avoid food contaminated with harmful compounds through taste is key to assess their ecotoxicological risks. Here, we investigated the ability of honey bees to perceive and avoid food resources contaminated with common metal pollutants known to impair behaviour at low concentrations. In laboratory assays, bees did not discriminate food contaminated with arsenic, lead or zinc and ingested it readily, up to estimated doses of 929.1 µg g-1 As, 6.45 mg g-1 Pb and 72.46 mg g-1 Zn. A decrease of intake and appetitive responses indicating metal detection was only observed at the highest concentrations of lead (3.6 mM) and zinc (122.3 mM) through contact with the antennae and the proboscis. Electrophysiological analyses confirmed that only high concentrations of the three metals in a sucrose solution induced a consistently reduced neural response to sucrose in antennal taste receptors (As: >0.1 µM, Pb: >1 mM; Zn: >100 mM). Overall, cellular and behavioural responses did not provide evidence for specific mechanisms that would support selective detection of toxic metals (arsenic, lead), as compared to zinc, which has important biological functions. Our results thus show that honey bees can avoid metal pollutants in their food only at high concentrations unlikely to be encountered in the environment. By contrast, they appear to be unable to detect low, yet harmful, concentrations found in flowers. Metal pollution at trace levels is therefore a major threat for pollinators.

Dynamically expressed single ELAV/Hu orthologue elavl2 of bees is required for learning and memory.

Changes in gene expression are a hallmark of learning and memory consolidation. Little is known about how alternative mRNA processing, particularly abundant in neuron-specific genes, contributes to these processes. Prototype RNA binding proteins of the neuronally expressed ELAV/Hu family are candidates for roles in learning and memory, but their capacity to cross-regulate and take over each other's functions complicate substantiation of such links. Honey bees Apis mellifera have only one elav/Hu family gene elavl2, that has functionally diversified by increasing alternative splicing including an evolutionary conserved microexon. RNAi knockdown demonstrates that ELAVL2 is required for learning and memory in bees. ELAVL2 is dynamically expressed with altered alternative splicing and subcellular localization in mushroom bodies, but not in other brain regions. Expression and alternative splicing of elavl2 change during memory consolidation illustrating an alternative mRNA processing program as part of a local gene expression response underlying memory consolidation.

Metal pollutants have additive negative effects on honey bee cognition.

Environmental pollutants can exert sublethal deleterious effects on animals. These include disruption of cognitive functions underlying crucial behaviours. While agrochemicals have been identified as a major threat to pollinators, metal pollutants, which are often found in complex mixtures, have so far been overlooked. Here, we assessed the impact of acute exposure to field-realistic concentrations of three common metal pollutants, lead, copper and arsenic, and their combinations, on honey bee appetitive learning and memory. All treatments involving single metals slowed down learning and disrupted memory retrieval at 24 h. Combinations of these metals had additive negative effects on both processes, suggesting common pathways of toxicity. Our results highlight the need to further assess the risks of metal pollution on invertebrates.

Current permissible levels of metal pollutants harm terrestrial invertebrates.

The current decline of invertebrates worldwide is alarming. Several potential causes have been proposed but metal pollutants, while being widespread in the air, soils and water, have so far been largely overlooked. Here, we reviewed the results of 527 observations of the effects of arsenic, cadmium, lead and mercury on terrestrial invertebrates. These four well-studied metals are considered as priorities for public health and for which international regulatory guidelines exist. We found that they all significantly impact the physiology and behavior of invertebrates, even at levels below those recommended as 'safe' for humans. Our results call for a revision of the regulatory thresholds to better protect terrestrial invertebrates, which appear to be more sensitive to metal pollution than vertebrates. More fundamental research on a broader range of compounds and species is needed to improve international guidelines for metal pollutants, and to develop conservation plans to protect invertebrates and ecosystem services.

Chronic exposure to trace lead impairs honey bee learning.

Pollutants can have severe detrimental effects on insects, even at sublethal doses, damaging developmental and cognitive processes involved in crucial behaviours. Agrochemicals have been identified as important causes of pollinator declines, but the impacts of other anthropogenic compounds, such as metallic trace elements in soils and waters, have received considerably less attention. Here, we exposed colonies of the European honey bee Apis mellifera to chronic field-realistic concentrations of lead in food and demonstrated that consumption of this trace element impaired bee cognition and morphological development. Honey bees exposed to the highest of these low concentrations had reduced olfactory learning performances. These honey bees also developed smaller heads, which may have constrained their cognitive functions as we show a general relationship between head size and learning performance. Our results demonstrate that lead pollutants, even at trace levels, can have dramatic effects on honey bee cognitive abilities, potentially altering key colony functions and the pollination service.

Pheromone components affect motivation and induce persistent modulation of associative learning and memory in honey bees.

Since their discovery in insects, pheromones are considered as ubiquitous and stereotyped chemical messengers acting in intraspecific animal communication. Here we studied the effect of pheromones in a different context as we investigated their capacity to induce persistent modulations of associative learning and memory. We used honey bees, Apis mellifera, and combined olfactory conditioning and pheromone preexposure with disruption of neural activity and two-photon imaging of olfactory brain circuits, to characterize the effect of pheromones on olfactory learning and memory. Geraniol, an attractive pheromone component, and 2-heptanone, an aversive pheromone, improved and impaired, respectively, olfactory learning and memory via a durable modulation of appetitive motivation, which left odor processing unaffected. Consistently, interfering with aminergic circuits mediating appetitive motivation rescued or diminished the cognitive effects induced by pheromone components. We thus show that these chemical messengers act as important modulators of motivational processes and influence thereby animal cognition.

Acute thiamethoxam toxicity in honeybees is not enhanced by common fungicide and herbicide and lacks stress-induced changes in mRNA splicing.

Securing food supply for a growing population is a major challenge and heavily relies on the use of agrochemicals to maximize crop yield. It is increasingly recognized, that some neonicotinoid insecticides have a negative impact on non-target organisms, including important pollinators such as the European honeybee Apis mellifera. Toxicity of neonicotinoids may be enhanced through simultaneous exposure with additional pesticides, which could help explain, in part, the global decline of honeybee colonies. Here we examined whether exposure effects of the neonicotinoid thiamethoxam on bee viability are enhanced by the commonly used fungicide carbendazim and the herbicide glyphosate. We also analysed alternative splicing changes upon pesticide exposure in the honeybee. In particular, we examined transcripts of three genes: (i) the stress sensor gene X box binding protein-1 (Xbp1), (ii) the Down Syndrome Cell Adhesion Molecule (Dscam) gene and iii) the embryonic lethal/abnormal visual system (elav) gene, which are important for neuronal function. Our results showed that acute thiamethoxam exposure is not enhanced by carbendazim, nor glyphosate. Toxicity of the compounds did not trigger stress-induced, alternative splicing in the analysed mRNAs, thereby leaving dormant a cellular response pathway to these man-made environmental perturbations.

Honey bees increase their foraging performance and frequency of pollen trips through experience.

Honey bee foragers must supply their colony with a balance of pollen and nectar to sustain optimal colony development. Inter-individual behavioural variability among foragers is observed in terms of activity levels and nectar vs. pollen collection, however the causes of such variation are still open questions. Here we explored the relationship between foraging activity and foraging performance in honey bees (Apis mellifera) by using an automated behaviour monitoring system to record mass on departing the hive, trip duration, presence of pollen on the hind legs and mass upon return to the hive, during the lifelong foraging career of individual bees. In our colonies, only a subset of foragers collected pollen, and no bee exclusively foraged for pollen. A minority of very active bees (19% of the foragers) performed 50% of the colony's total foraging trips, contributing to both pollen and nectar collection. Foraging performance (amount and rate of food collection) depended on bees' individual experience (amount of foraging trips completed). We argue that this reveals an important vulnerability for these social bees since environmental stressors that alter the activity and reduce the lifespan of foragers may prevent bees ever achieving maximal performance, thereby seriously compromising the effectiveness of the colony foraging force.

Changes in responsiveness to allatostatin treatment accompany shifts in stress reactivity in young worker honey bees.

Exposing honey bees to isopentylacetate (IPA) can cause stress-related changes in learning performance. In bees of foraging age, IPA's effects on learning are mimicked by C-type allatostatins (AstCC, AstCCC) injected into the brain. Here we ask whether allatostatins induce a similar response in young (6-day-old) bees and if so, whether their effects on learning performance are modulated by queen mandibular pheromone (QMP). We found that young bees exposed to IPA responded less to the conditioned stimulus during training than controls (Type 1-like stress response). AstCC treatment induced a similar response, but only in bees maintained without QMP. Bees exposed to QMP responded to AstCC with increased odour responsiveness and odour generalisation in the 1-h memory test (Type 2-like response). Type 2-like responses could be induced also by the A-type allatostatin, AstA. However, in bees exposed to QMP, AstA-induced odour generalisation was absent. Effects of AstCCC treatment in young bees were weak, indicating that responsiveness to this peptide changes with age. Our findings are consistent with the hypothesis that honey bee allatostatins play a role in stress reactivity, but suggest in addition that allatostatin signalling is age dependent and susceptible to modulation by pheromone released by the queen bee.

Relationship between brain plasticity, learning and foraging performance in honey bees.

Brain structure and learning capacities both vary with experience, but the mechanistic link between them is unclear. Here, we investigated whether experience-dependent variability in learning performance can be explained by neuroplasticity in foraging honey bees. The mushroom bodies (MBs) are a brain center necessary for ambiguous olfactory learning tasks such as reversal learning. Using radio frequency identification technology, we assessed the effects of natural variation in foraging activity, and the age when first foraging, on both performance in reversal learning and on synaptic connectivity in the MBs. We found that reversal learning performance improved at foraging onset and could decline with greater foraging experience. If bees started foraging before the normal age, as a result of a stress applied to the colony, the decline in learning performance with foraging experience was more severe. Analyses of brain structure in the same bees showed that the total number of synaptic boutons at the MB input decreased when bees started foraging, and then increased with greater foraging intensity. At foraging onset MB structure is therefore optimized for bees to update learned information, but optimization of MB connectivity deteriorates with foraging effort. In a computational model of the MBs sparser coding of information at the MB input improved reversal learning performance. We propose, therefore, a plausible mechanistic relationship between experience, neuroplasticity, and cognitive performance in a natural and ecological context.

Aversive learning of odor-heat associations in ants.

Ants have recently emerged as useful models for the study of olfactory learning. In this framework, the development of a protocol for the appetitive conditioning of the maxilla-labium extension response (MaLER) provided the possibility of studying Pavlovian odor-food learning in a controlled environment. Here we extend these studies by introducing the first Pavlovian aversive learning protocol for harnessed ants in the laboratory. We worked with carpenter ants Camponotus aethiops and first determined the capacity of different temperatures applied to the body surface to elicit the typical aversive mandible opening response (MOR). We determined that 75°C is the optimal temperature to induce MOR and chose the hind legs as the stimulated body region because of their high sensitivity. We then studied the ability of ants to learn and remember odor-heat associations using 75°C as the unconditioned stimulus. We studied learning and short-term retention after absolute (one odor paired with heat) and differential conditioning (a punished odor versus an unpunished odor). Our results show that ants successfully learn the odor-heat association under a differential-conditioning regime and thus exhibit a conditioned MOR to the punished odor. Yet, their performance under an absolute-conditioning regime is poor. These results demonstrate that ants are capable of aversive learning and confirm previous findings about the different attentional resources solicited by differential and absolute conditioning in general.

Experience during early adulthood shapes the learning capacities and the number of synaptic boutons in the mushroom bodies of honey bees (Apis mellifera).

The honey bee mushroom bodies (MBs) are brain centers required for specific learning tasks. Here, we show that environmental conditions experienced as young adults affect the maturation of MB neuropil and performance in a MB-dependent learning task. Specifically, olfactory reversal learning was selectively impaired following early exposure to an impoverished environment lacking some of the sensory and social interactions present in the hive. In parallel, the overall number of synaptic boutons increased within the MB olfactory neuropil, whose volume remained unaffected. This suggests that experience of the rich in-hive environment promotes MB maturation and the development of MB-dependent learning capacities.

Pheromones modulate reward responsiveness and non-associative learning in honey bees.

Pheromones are chemical messengers that trigger stereotyped behaviors and/or physiological processes in individuals of the same species. Recent reports suggest that pheromones can modulate behaviors not directly related to the pheromonal message itself and contribute, in this way, to behavioral plasticity. We tested this hypothesis by studying the effect of pheromones on sucrose responsiveness and habituation in honey bees. We exposed workers to three pheromone components: geraniol, which in nature is used in an appetitive context, and isopentyl acetate (IPA) and 2-heptanone (2H), which signal aversive situations. Pheromones associated with an aversive context induced a significant decrease of sucrose responsiveness as 40% and 60% of bees exposed to IPA and 2H, respectively, did not respond to any sucrose concentration. In bees that responded to sucrose, geraniol enhanced sucrose responsiveness while 2H, but not IPA, had the opposite effect. Geraniol and IPA had no effect on habituation while 2H induced faster habituation than controls. Overall, our results demonstrate that pheromones modulate reward responsiveness and to a lower degree habituation. Through their effect on sucrose responsiveness they could also affect appetitive associative learning. Thus, besides conveying stereotyped messages, pheromones may contribute to individual and colony-level plasticity by modulating motivational state and learning performances.

Inter-individual variability in the foraging behaviour of traplining bumblebees.

Workers of social insects, such as bees, ants and wasps, show some degree of inter-individual variability in decision-making, learning and memory. Whether these natural cognitive differences translate into distinct adaptive behavioural strategies is virtually unknown. Here we examined variability in the movement patterns of bumblebee foragers establishing routes between artificial flowers. We recorded all flower visitation sequences performed by 29 bees tested for 20 consecutive foraging bouts in three experimental arrays, each characterised by a unique spatial configuration of artificial flowers and three-dimensional landmarks. All bees started to develop efficient routes as they accumulated foraging experience in each array, and showed consistent inter-individual differences in their levels of route fidelity and foraging performance, as measured by travel speed and the frequency of revisits to flowers. While the tendency of bees to repeat the same route was influenced by their colony origin, foraging performance was correlated to body size. The largest foragers travelled faster and made less revisits to empty flowers. We discuss the possible adaptive value of such inter-individual variability within the forager caste for optimisation of colony-level foraging performances in social pollinators.

C-type allatostatins mimic stress-related effects of alarm pheromone on honey bee learning and memory recall.

As honey bee populations worldwide are declining there is an urgent need for a deeper understanding of stress reactivity in these important insects. Our data indicate that stress responses in bees (Apis mellifera L.) may be mediated by neuropeptides identified, on the basis of sequence similarities, as allatostatins (ASTA, ASTC and ASTCC). Effects of allatostatin injection are compared with stress-related changes in learning performance induced by the honeybee alarm pheromone, isopentylacetate (IPA). We find that bees can exhibit two markedly different responses to IPA, with opposing effects on learning behaviour and memory generalisation, and that strikingly similar responses can be elicited by allatostatins, in particular ASTCC. These findings lend support to the hypothesis that allatostatins mediate stress reactivity in honey bees and suggest responses to stress in these insects are state dependent.