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.

Interspecific variation of antennal lobe composition among four hornet species.

Olfaction is a crucial sensory modality underlying foraging, social and mating behaviors in many insects. Since the olfactory system is at the interface between the animal and its environment, it receives strong evolutionary pressures that promote neuronal adaptations and phenotypic variations across species. Hornets are large eusocial predatory wasps with a highly developed olfactory system, critical for foraging and intra-specific communication. In their natural range, hornet species display contrasting ecologies and olfactory-based behaviors, which might match to adaptive shifts in their olfactory system. The first olfactory processing center of the insect brain, the antennal lobe, is made of morphological and functional units called glomeruli. Using fluorescent staining, confocal microscopy and 3D reconstructions, we compared antennal lobe structure, glomerular numbers and volumes in four hornet species (Vespa crabro, Vespa velutina, Vespa mandarinia and Vespa orientalis) with marked differences in nesting site preferences and predatory behaviors. Despite a conserved organization of their antennal lobe compartments, glomeruli numbers varied strongly between species, including in a subsystem thought to process intraspecific cuticular signals. Moreover, specific adaptations involving enlarged glomeruli appeared in two species, V. crabro and V. mandarinia, but not in the others. We discuss the possible function of these adaptations based on species-specific behavioral differences.

A systems-based approach to the environmental risk assessment of multiple stressors in honey bees.

The European Parliament requested EFSA to develop a holistic risk assessment of multiple stressors in honey bees. To this end, a systems-based approach that is composed of two core components: a monitoring system and a modelling system are put forward with honey bees taken as a showcase. Key developments in the current scientific opinion (including systematic data collection from sentinel beehives and an agent-based simulation) have the potential to substantially contribute to future development of environmental risk assessments of multiple stressors at larger spatial and temporal scales. For the monitoring, sentinel hives would be placed across representative climatic zones and landscapes in the EU and connected to a platform for data storage and analysis. Data on bee health status, chemical residues and the immediate or broader landscape around the hives would be collected in a harmonised and standardised manner, and would be used to inform stakeholders, and the modelling system, ApisRAM, which simulates as accurately as possible a honey bee colony. ApisRAM would be calibrated and continuously updated with incoming monitoring data and emerging scientific knowledge from research. It will be a supportive tool for beekeeping, farming, research, risk assessment and risk management, and it will benefit the wider society. A societal outlook on the proposed approach is included and this was conducted with targeted social science research with 64 beekeepers from eight EU Member States and with members of the EU Bee Partnership. Gaps and opportunities are identified to further implement the approach. Conclusions and recommendations are made on a way forward, both for the application of the approach and its use in a broader context.

Conflicts of interest and improvement through peer review: the case of IPBES report on pollinators.

To understand the real causes of disorders and abnormal mortalities of bees (honeybees and wild bees) in the world, the scientific method requires that each category of potential stressors be analyzed in a balanced manner. In this article, I show that the first version of the section dealing with the evaluation of the toxic effects of pesticides on bees, in the IPBES assessment report on pollinators and pollination, revealed an incomplete and biased literature review in many places, especially downplaying the risks that pesticides in general, and neonicotinoids in particular, pose for pollinating insects. Then, according to the rules of IPBES, an independent peer review by external experts of this first version allowed the published report to be more in line with the reality of scientific knowledge, which shows, for example, that sublethal effects of pesticide exposure can impair the ability of bees to provide pollination. However, some other key points remain unchanged in the published version.

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.

Risk assessment of pesticides and other stressors in bees: Principles, data gaps and perspectives from the European Food Safety Authority.

Current approaches to risk assessment in bees do not take into account co-exposures from multiple stressors. The European Food Safety Authority (EFSA) is deploying resources and efforts to move towards a holistic risk assessment approach of multiple stressors in bees. This paper describes the general principles of pesticide risk assessment in bees, including recent developments at EFSA dealing with risk assessment of single and multiple pesticide residues and biological hazards. The EFSA Guidance Document on the risk assessment of plant protection products in bees highlights the need for the inclusion of an uncertainty analysis, other routes of exposures and multiple stressors such as chemical mixtures and biological agents. The EFSA risk assessment on the survival, spread and establishment of the small hive beetle, Aethina tumida, an invasive alien species, is provided with potential insights for other bee pests such as the Asian hornet, Vespa velutina. Furthermore, data gaps are identified at each step of the risk assessment, and recommendations are made for future research that could be supported under the framework of Horizon 2020. Finally, the recent work conducted at EFSA is presented, under the overarching MUST-B project ("EU efforts towards the development of a holistic approach for the risk assessment on MUltiple STressors in Bees") comprising a toolbox for harmonised data collection under field conditions and a mechanistic model to assess effects from pesticides and other stressors such as biological agents and beekeeping management practices, at the colony level and in a spatially complex landscape. Future perspectives at EFSA include the development of a data model to collate high quality data to calibrate and validate the model to be used as a regulatory tool. Finally, the evidence collected within the framework of MUST-B will support EFSA's activities on the development of a holistic approach to the risk assessment of multiple stressors in bees. In conclusion, EFSA calls for collaborative action at the EU level to establish a common and open access database to serve multiple purposes and different stakeholders.

Defensive behaviour of Apis mellifera against Vespa velutina in France: testing whether European honeybees can develop an effective collective defence against a new predator.

We investigated the prey-predator interactions between the European honeybee, Apis mellifera, and the invasive yellow-legged hornet, Vespa velutina, which first invaded France in 2004 and thereafter spread to neighbouring European countries (Spain, Portugal and Italy). Our goal was to determine how successfully honeybees are able to defend their colonies against their new predator in Europe. Experiments were conducted in the southwest of France-the point of entry of the hornet in Europe-under natural and semi-controlled field conditions. We investigated a total of eight apiaries and 95 colonies subjected to either low or high levels of predation. We analyzed hornet predatory behaviour and collective response of colonies under attack. The results showed that A. mellifera in France exhibit an inefficient and unorganized defence against V. velutina, unlike in other regions of Europe and other areas around the globe where honeybees have co-evolved with their natural Vespa predators.

Native Prey and Invasive Predator Patterns of Foraging Activity: The Case of the Yellow-Legged Hornet Predation at European Honeybee Hives.

Contrary to native predators, which have co-evolved with their prey, alien predators often benefit from native prey naïveté. Vespa velutina, a honeybee predator originating from Eastern China, was introduced into France just before 2004. The present study, based on video recordings of two beehives at an early stage of the invasion process, intends to analyse the alien hornet hunting behaviour on the native prey, Apis mellifera, and to understand the interaction between the activity of the predator and the prey during the day and the season. Chasing hornets spent most of their time hovering facing the hive, to catch flying honeybees returning to the hive. The predation pressure increased during the season confirming previous study based on predator trapping. The number of honeybee captures showed a maximum peak for an intermediate number of V. velutina, unrelated to honeybee activity, suggesting the occurrence of competition between hornets. The number of honeybees caught increased during midday hours while the number of hornets did not vary, suggesting an increase in their efficacy. These results suggest that the impact of V. velutina on honeybees is limited by its own biology and behaviour and did not match the pattern of activity of its prey. Also, it could have been advantageous during the invasion, limiting resource depletion and thus favouring colonisation. This lack of synchronization may also be beneficial for honeybee colonies by giving them an opportunity to increase their activity when the hornets are less effective.

The bite of the honeybee: 2-heptanone secreted from honeybee mandibles during a bite acts as a local anaesthetic in insects and mammals.

Honeybees secrete 2-heptanone (2-H) from their mandibular glands when they bite. Researchers have identified several possible functions: 2-H could act as an alarm pheromone to recruit guards and soldiers, it could act as a chemical marker, or it could have some other function. The actual role of 2-H in honeybee behaviour remains unresolved. In this study, we show that 2-H acts as an anaesthetic in small arthropods, such as wax moth larva (WML) and Varroa mites, which are paralysed after a honeybee bite. We demonstrated that honeybee mandibles can penetrate the cuticle of WML, introducing less than one nanolitre of 2-H into the WML open circulatory system and causing instantaneous anaesthetization that lasts for a few minutes. The first indication that 2-H acts as a local anaesthetic was that its effect on larval response, inhibition and recovery is very similar to that of lidocaine. We compared the inhibitory effects of 2-H and lidocaine on voltage-gated sodium channels. Although both compounds blocked the hNav1.6 and hNav1.2 channels, lidocaine was slightly more effective, 2.82 times, on hNav.6. In contrast, when the two compounds were tested using an ex vivo preparation-the isolated rat sciatic nerve-the function of the two compounds was so similar that we were able to definitively classify 2-H as a local anaesthetic. Using the same method, we showed that 2-H has the fastest inhibitory effect of all alkyl-ketones tested, including the isomers 3- and 4-heptanone. This suggests that natural selection may have favoured 2-H over other, similar compounds because of the associated fitness advantages it confers. Our results reveal a previously unknown role of 2-H in honeybee defensive behaviour and due to its minor neurotoxicity show potential for developing a new local anaesthetic from a natural product, which could be used in human and veterinary medicine.

Does Patriline Composition Change over a Honey Bee Queen's Lifetime?

A honey bee queen mates with a number of drones a few days after she emerges as an adult. Spermatozoa of different drones are stored in her spermatheca and used for the rest of the queen's life to fertilize eggs. Sperm usage is thought to be random, so that the patriline distribution within a honey bee colony would remain constant over time. In this study we assigned the progeny of a naturally mated honey bee queen to patrilines using microsatellite markers at the queen's age of two, three and four years. No significant changes in patriline distribution occurred within each of two foraging seasons, with samples taken one and five months apart, respectively. Overall and pair-wise comparisons between the three analyzed years reached significant levels. Over the three-year period we found a trend for patrilines to become more equally represented with time. It is important to note that this study was performed with a single queen, and thus individual and population variation in sperm usage patterns must be assessed. We discuss long-term changes in patriline composition due to mixing processes in the queen's spermatheca, following incomplete mixing of different drones' sperm after mating.

Attack or retreat: contrasted defensive tactics used by Cyprian honeybee colonies under attack from hornets.

This study describes the tactics used by Cyprian honeybees (Apis mellifera cypria) to defend their colonies against hornet (Vespa orientalis orientalis) attacks. We use simulated hornet attacks and a combination of video recordings and image analysis to reveal, for the first time, contrasted intra-subspecies defensive tactics that operate at the colony level during predation. In some colonies, when attacked, the numbers of guards at the hive entrance increases rapidly to attack, engulf, and kill invading hornets. In other colonies, guards avoid conflicts with hornets by retreating gradually and by forming a defensive line of honeybees at the hive entrance. Retreater colonies have propolis walls at the hive entrances with small apertures that are too narrow to allow the hornet to access the hive and that therefore reinforces entrance protection. On the contrary, attacker colonies have propolis walls with large openings through which the hornet can pass; these bees block the hornet's access by intensively guarding the hive entrance. We experimentally destroy propolis walls to test whether colonies consistently rebuild walls with the same intrinsic characteristics and we also monitor the survival rate of each anti-predator tactic after massive natural predation by hornets.

Deformed wing virus is not related to honey bees' aggressiveness.

Guards of Cyprian honey bee colonies, Apis mellifera cypria, display a great defensive behaviour against hornets' attacks. The deformed wing virus (DWV) and the kakugo virus (KV) genomes are very similar, but unlike KV, the presence of DWV is not related to honey bees' aggressiveness. This discrepancy is further discussed.