Food wanting is mediated by transient activation of dopaminergic signaling in the honey bee brain.

The biological bases of wanting have been characterized in mammals, but whether an equivalent wanting system exists in insects remains unknown. In this study, we focused on honey bees, which perform intensive foraging activities to satisfy colony needs, and sought to determine whether foragers leave the hive driven by specific expectations about reward and whether they recollect these expectations during their waggle dances. We monitored foraging and dance behavior and simultaneously quantified and interfered with biogenic amine signaling in the bee brain. We show that a dopamine-dependent wanting system is activated transiently in the bee brain by increased appetite and individual recollection of profitable food sources, both en route to the goal and during waggle dances. Our results show that insects share with mammals common neural mechanisms for encoding wanting of stimuli with positive hedonic value.

The short neuropeptide F (sNPF) promotes the formation of appetitive visual memories in honey bees.

Motivation can critically influence learning and memory. Multiple neural mechanisms regulate motivational states, among which signalling via specific neuropeptides, such as NPY in vertebrates and NPF and its short variant sNPF in invertebrates, plays an essential role. The honey bee (Apis mellifera) is a privileged model for the study of appetitive learning and memory. Bees learn and memorize sensory cues associated with nectar reward while foraging, and their learning is affected by their feeding state. However, the neural underpinnings of their motivational states remain poorly known. Here we focused on the short neuropeptide F (sNPF) and studied if it modulates the acquisition and formation of colour memories. Artificially increasing sNPF levels in partially fed foragers with a reduced motivation to learn colours resulted in significant colour learning and memory above the levels exhibited by starved foragers. Our results thus identify sNPF as a critical component of motivational processes involved in foraging and in the cognitive processes associated with this activity in honey bees.

The short neuropeptide F regulates appetitive but not aversive responsiveness in a social insect.

The neuropeptide F (NPF) and its short version (sNPF) mediate food- and stress-related responses in solitary insects. In the honeybee, a social insect where food collection and defensive responses are socially regulated, only sNPF has an identified receptor. Here we increased artificially sNPF levels in honeybee foragers and studied the consequences of this manipulation in various forms of appetitive and aversive responsiveness. Increasing sNPF in partially fed bees turned them into the equivalent of starved animals, enhancing both their food consumption and responsiveness to appetitive gustatory and olfactory stimuli. Neural activity in the olfactory circuits of fed animals was reduced and could be rescued by sNPF treatment to the level of starved bees. In contrast, sNPF had no effect on responsiveness to nociceptive stimuli. Our results thus identify sNPF as a key modulator of hunger and food-related responses in bees, which are at the core of their foraging activities.

Charles Henry Turner and the cognitive behavior of bees.

Social movements in several countries are stimulating a reconsideration of academic structures and historic figures and promoting reparation and recognition of marginalized and forgotten black scientists. A paradigmatic case in that sense is Charles Henry Turner (1867-1923) who was the first African American to receive a graduate degree at the University of Cincinnati and one of the first in earning a PhD degree of the University of Chicago. He performed numerous experiments on sensory perception, orientation, and mating of solitary and social bees, most of which have been unjustly forgotten despite the fact that they anticipated fundamental concepts of animal cognition. We review these studies and highlight the importance of his ideas for modern views of animal cognition and the study of bee behavior. We conclude that besides his scientific contributions, Turner is an inspiration for scientists fighting against social adversity and prejudices.

Peripheral taste detection in honey bees: What do taste receptors respond to?

Understanding the neural principles governing taste perception in species that bear economic importance or serve as research models for other sensory modalities constitutes a strategic goal. Such is the case of the honey bee (Apis mellifera), which is environmentally and socioeconomically important, given its crucial role as pollinator agent in agricultural landscapes and which has served as a traditional model for visual and olfactory neurosciences and for research on communication, navigation, and learning and memory. Here we review the current knowledge on honey bee gustatory receptors to provide an integrative view of peripheral taste detection in this insect, highlighting specificities and commonalities with other insect species. We describe behavioral and electrophysiological responses to several tastant categories and relate these responses, whenever possible, to known molecular receptor mechanisms. Overall, we adopted an evolutionary and comparative perspective to understand the neural principles of honey bee taste and define key questions that should be answered in future gustatory research centered on this insect.

Degradation of an appetitive olfactory memory via devaluation of sugar reward is mediated by 5-HT signaling in the honey bee.

Conditioned taste aversion (CTA) learning induces the devaluation of a preferred food through its pairing with a stimulus inducing internal illness. In invertebrates, it is still unclear how this aversive learning impairs the memories of stimuli that had been associated with the appetitive food prior to its devaluation. Here we studied this phenomenon in the honey bee and characterized its neural underpinnings. We first trained bees to associate an odorant (conditioned stimulus, CS) with appetitive fructose solution (unconditioned stimulus, US) using a Pavlovian olfactory conditioning. We then subjected the bees that learned the association to a CTA training during which the antennal taste of fructose solution was contingent or not to the ingestion of quinine solution, which induces malaise a few hours after ingestion. Only the group experiencing contingent fructose stimulation and quinine-based malaise exhibited a decrease in responses to the fructose and a concomitant decrease in odor-specific retention in tests performed 23 h after the original odor conditioning. Furthermore, injection of dopamine- and serotonin-receptor antagonists after CTA learning revealed that this long-term decrease was mediated by serotonergic signaling as its blockade rescued both the responses to fructose and the odor-specific memory 23 h after conditioning. The impairment of a prior CS memory by subsequent CTA conditioning confirms that bees retrieve a devaluated US representation when presented with the CS. Our findings further highlight the importance of serotonergic signaling in aversive learning in the bee and uncover mechanisms underlying aversive memories induced by internal illness in invertebrates.

Aversive gustatory learning and perception in honey bees.

Taste perception allows discriminating edible from non-edible items and is crucial for survival. In the honey bee, the gustatory sense has remained largely unexplored, as tastants have been traditionally used as reinforcements rather than as stimuli to be learned and discriminated. Here we provide the first characterization of antennal gustatory perception in this insect using a novel conditioning protocol in which tastants are dissociated from their traditional food-reinforcement role to be learned as predictors of punishment. We found that bees have a limited gustatory repertoire via their antennae: they discriminate between broad gustatory modalities but not within modalities, and are unable to differentiate bitter substances from water. Coupling gustatory conditioning with blockade of aminergic pathways in the bee brain revealed that these pathways are not restricted to encode reinforcements but may also encode conditioned stimuli. Our results reveal unknown aspects of honey bee gustation, and bring new elements for comparative analyses of gustatory perception in animals.

Absence of food alternatives promotes risk-prone feeding of unpalatable substances in honey bees.

The question of why animals sometimes ingest noxious substances is crucial to understand unknown determinants of feeding behaviour. Research on risk-prone feeding behaviour has largely focused on energy budgets as animals with low energy budgets tend to ingest more aversive substances. A less explored possibility is that risk-prone feeding arises from the absence of alternative feeding options, irrespectively of energy budgets. Here we contrasted these two hypotheses in late-fall and winter honey bees. We determined the toxicity of various feeding treatments and showed that when bees can choose between sucrose solution and a mixture of this sucrose solution and a noxious/unpalatable substance, they prefer the pure sucrose solution and reject the mixtures, irrespective of their energy budget. Yet, when bees were presented with a single feeding option and their escape possibilities were reduced, they consumed unexpectedly some of the previously rejected mixtures, independently of their energy budget. These findings are interpreted as a case of feeding helplessness, in which bees behave as if it were utterly helpless to avoid the potentially noxious food and consume it. They suggest that depriving bees of variable natural food sources may have the undesired consequence of increasing their acceptance of food that would be otherwise rejected.

Insulin effects on honeybee appetitive behaviour.

Worker honeybees (Apis mellifera) carry out multiple tasks throughout their adult lifespan. It has been suggested that the insulin/insulin-like signalling pathway participates in regulating behavioural maturation in eusocial insects. Insulin signalling increases as the honeybee worker transitions from nurse to food processor to forager. As behavioural shifts require differential usage of sensory modalities, our aim was to assess insulin effects on olfactory and gustatory responsiveness as well as on olfactory learning in preforaging honeybee workers of different ages. Adults were reared in the laboratory or in the hive. Immediately after being injected with insulin or vehicle (control), and focusing on the proboscis extension response, bees were tested for their spontaneous response to odours, sucrose responsiveness and ability to discriminate odours through olfactory conditioning. Bees injected with insulin have higher spontaneous odour responses. Sucrose responsiveness and odour discrimination are differentially affected by treatment according to age: whereas insulin increases gustatory responsiveness and diminishes learning abilities of younger workers, it has the opposite effect on older bees. In summary, insulin can improve chemosensory responsiveness in young workers, but also worsens their learning abilities to discriminate odours. The insulin signalling pathway is responsive in young workers, although they are not yet initiating outdoor activities. Our results show strong age-dependent effects of insulin on appetitive behaviour, which uncover differences in insulin signalling regulation throughout the honeybee worker's adulthood.

Learning context modulates aversive taste strength in honey bees.

The capacity of honey bees (Apis mellifera) to detect bitter substances is controversial because they ingest without reluctance different kinds of bitter solutions in the laboratory, whereas free-flying bees avoid them in visual discrimination tasks. Here, we asked whether the gustatory perception of bees changes with the behavioral context so that tastes that are less effective as negative reinforcements in a given context become more effective in a different context. We trained bees to discriminate an odorant paired with 1 mol l(-1) sucrose solution from another odorant paired with either distilled water, 3 mol l(-1) NaCl or 60 mmol l(-1) quinine. Training was either Pavlovian [olfactory conditioning of the proboscis extension reflex (PER) in harnessed bees], or mainly operant (olfactory conditioning of free-walking bees in a Y-maze). PER-trained and maze-trained bees were subsequently tested both in their original context and in the alternative context. Whereas PER-trained bees transferred their choice to the Y-maze situation, Y-maze-trained bees did not respond with a PER to odors when subsequently harnessed. In both conditioning protocols, NaCl and distilled water were the strongest and the weakest aversive reinforcement, respectively. A significant variation was found for quinine, which had an intermediate aversive effect in PER conditioning but a more powerful effect in the Y-maze, similar to that of NaCl. These results thus show that the aversive strength of quinine varies with the learning context, and reveal the plasticity of the bee's gustatory system. We discuss the experimental constraints of both learning contexts and focus on stress as a key modulator of taste in the honey bee. Further explorations of bee taste are proposed to understand the physiology of taste modulation in bees.

Bitter stimuli modulate the feeding decision of a blood-sucking insect via two sensory inputs.

The gustatory system of animals is involved in food quality assessment and controls the feeding decision of an individual confronted with a potential alimentary source. Triatomines are haematophagous insects that feed on vertebrate blood. Once they reach a potential host, they walk over the host skin searching for an adequate site to pierce. Then, they insert their stylets and take a first sampling gorge to decide whether food is acceptable. Our work reveals that the presence of bitter compounds inhibits the feeding behavior of these bugs. Firstly, triatomines decreased their feeding behavior if substrates spread with quinine or caffeine were detected by external receptors localized exclusively in the antennae. Morphological inspections along with electrophysiological recordings revealed the existence of four gustatory sensilla located in the tip of the antenna that respond to both bitter tastants. The absence of these bitter detectors by antennal ablation reversed the observed feeding inhibition evoked by bitter compounds. Secondly, once triatomines pumped the first volume of food with bitter compounds (quinine, caffeine, berberine, salicin), a decrease in their feeding behavior was observed. Morphological inspections revealed the existence of eight gustatory sensilla located in the pharynx that might be responsible for the internal bitter detection. Finally, we found that a brief pre-exposure to bitter compounds negatively modulates the motivation of bugs to feed on an appetitive solution. Results presented here highlight the relevance of bitter taste perception in the modulation of the feeding behavior of a blood-sucking insect.

The tarsal taste of honey bees: behavioral and electrophysiological analyses.

Taste plays a crucial role in the life of honey bees as their survival depends on the collection and intake of nectar and pollen, and other natural products. Here we studied the tarsal taste of honey bees through a series of behavioral and electrophysiological analyses. We characterized responsiveness to various sweet, salty and bitter tastants delivered to gustatory sensilla of the fore tarsi. Behavioral experiments showed that stimulation of opposite fore tarsi with sucrose and bitter substances or water yielded different outcomes depending on the stimulation sequence. When sucrose was applied first, thereby eliciting proboscis extension, no bitter substance could induce proboscis retraction, thus suggesting that the primacy of sucrose stimulation induced a central excitatory state. When bitter substances or water were applied first, sucrose stimulation could still elicit proboscis extension but to a lower level, thus suggesting central inhibition based on contradictory gustatory input on opposite tarsi. Electrophysiological experiments showed that receptor cells in the gustatory sensilla of the tarsomeres are highly sensitive to saline solutions at low concentrations. No evidence for receptors responding specifically to sucrose or to bitter substances was found in these sensilla. Receptor cells in the gustatory sensilla of the claws are highly sensitive to sucrose. Although bees do not possess dedicated bitter-taste receptors in the tarsi, indirect bitter detection is possible because bitter tastes inhibit sucrose receptor cells of the claws when mixed with sucrose solution. By combining behavioral and electrophysiological approaches, these results provide the first integrative study on tarsal taste detection in the honey bee.

Taste perception in honey bees.

Taste is crucial for honeybees for choosing profitable food sources, resins, water sources, and for nestmate recognition. Peripheral taste detection occurs within cuticular hairs, the chaetic and basiconic sensilla, which host gustatory receptor cells and, usually a mechanoreceptor cell. Gustatory sensilla are mostly located on the distal segment of the antennae, on the mouthparts, and on the tarsi of the forelegs. These sensilla respond with varying sensitivity to sugars, salts, and possibly amino acids, proteins, and water. So far, no responses of receptor cells to bitter substances were found although inhibitory effects of these substances on sucrose receptor cells could be recorded. When bees are free to express avoidance behaviors, they reject highly concentrated bitter and saline solutions. However, such avoidance disappears when bees are immobilized in the laboratory. In this case, they ingest these solutions, even if they suffer afterward a malaise-like state or even die from such ingestion. Central processing of taste occurs mainly in the subesophageal ganglion, but the nature of this processing remains unknown. We suggest that coding tastants in terms of their hedonic value, thus classifying them in terms of their palatability, is a basic strategy that a central processing of taste should achieve for survival.

Toxic but drank: gustatory aversive compounds induce post-ingestional malaise in harnessed honeybees.

BACKGROUND: Deterrent substances produced by plants are relevant due to their potential toxicity. The fact that most of these substances have an unpalatable taste for humans and other mammals contrasts with the fact that honeybees do not reject them in the range of concentrations in which these compounds are present in flower nectars. Here we asked whether honeybees detect and ingest deterrent substances and whether these substances are really toxic to them. RESULTS: We show that pairing aversive substances with an odor retards learning of this odor when it is subsequently paired with sucrose. Harnessed honeybees in the laboratory ingest without reluctance a considerable volume (20 µl) of various aversive substances, even if some of them induce significant post-ingestional mortality. These substances do not seem, therefore, to be unpalatable to harnessed bees but induce a malaise-like state that in some cases results in death. Consistently with this finding, bees learning that one odor is associated with sugar, and experiencing in a subsequent phase that the sugar was paired with 20 µl of an aversive substance (devaluation phase), respond less than control bees to the odor and the sugar. Such stimulus devaluation can be accounted for by the malaise-like state induced by the aversive substances. CONCLUSION: Our results indicate that substances that taste bitter to humans as well as concentrated saline solutions base their aversive effect on the physiological consequences that their ingestion generates in harnessed bees rather than on an unpalatable taste. This conclusion is only valid for harnessed bees in the laboratory as freely-moving bees might react differently to aversive compounds could actively reject aversive substances. Our results open a new possibility to study conditioned taste aversion based on post-ingestional malaise and thus broaden the spectrum of aversive learning protocols available in honeybees.

Behavioral studies on tarsal gustation in honeybees: sucrose responsiveness and sucrose-mediated olfactory conditioning.

Although the forelegs of honeybees are one of their main gustatory appendages, tarsal gustation in bees has never been systematically studied. To provide a more extensive account on honeybee tarsal gustation, we performed a series of behavioral experiments aimed at characterizing (1) tarsal sucrose sensitivity under different experimental conditions and (2) the capacity of tarsal sucrose stimulation to support olfactory conditioning. We quantified the proboscis extension reflex to tarsal sucrose stimulation and to odors paired with tarsal sucrose stimulation, respectively. Our experiments show that tarsal sucrose sensitivity is lower than antennal sucrose sensitivity and can be increased by starvation time. In contrast, antennae amputation decreases tarsal sucrose sensitivity. Furthermore, we show that tarsal sucrose stimulation can support olfactory learning and memory even if the acquisition level reached is relatively low (40%).

Understanding the logics of pheromone processing in the honeybee brain: from labeled-lines to across-fiber patterns.

Honeybees employ a very rich repertoire of pheromones to ensure intraspecific communication in a wide range of behavioral contexts. This communication can be complex, since the same compounds can have a variety of physiological and behavioral effects depending on the receiver. Honeybees constitute an ideal model to study the neurobiological basis of pheromonal processing, as they are already one of the most influential animal models for the study of general odor processing and learning at behavioral, cellular and molecular levels. Accordingly, the anatomy of the bee brain is well characterized and electro- and opto-physiological recording techniques at different stages of the olfactory circuit are possible in the laboratory. Here we review pheromone communication in honeybees and analyze the different stages of olfactory processing in the honeybee brain, focusing on available data on pheromone detection, processing and representation at these different stages. In particular, we argue that the traditional distinction between labeled-line and across-fiber pattern processing, attributed to pheromone and general odors respectively, may not be so clear in the case of honeybees, especially for social-pheromones. We propose new research avenues for stimulating future work in this area.

Electrophysiological and behavioural characterization of gustatory responses to antennal 'bitter' taste in honeybees.

We combined behavioural and electrophysiological experiments to study whether bitter taste is perceived at the antennal level in honeybees, Apis mellifera. Our behavioural studies showed that neither quinine nor salicin delivered at one antenna at different concentrations induced a retraction of the proboscis once it was extended in response to 1 M sucrose solution delivered to the opposite antenna. Bees that extended massively their proboscis to 1 M sucrose responded only partially when stimulated with a mixture of 1 M sucrose and 100 mM quinine. The mixture of 1 m sucrose and 100 mM salicin had no such suppressive effect. No behavioural suppression was found for mixtures of salt solution and either bitter substance. Electrophysiological recordings of taste sensillae at the antennal tip revealed sensillae that responded specifically either to sucrose or salt solutions, but none responded to the bitter substances quinine and salicin at the different concentrations tested. The electrophysiological responses of sensillae to 15 mM sucrose solution were inhibited by a mixture of 15 mM sucrose and 0.1 mM quinine, but not by a mixture of 15 mM sucrose and 0.1 mM salicin. The responses of sensillae to 50 mM NaCl were reduced by a mixture of 50 mm NaCl and 1 mM quinine but not by a mixture of 50 mM NaCl and 1 mM salicin. We concluded that no receptor cells for the bitter substances tested, exist at the level of the antennal tip of the honeybee and that antennal bitter taste is not represented as a separate perceptual quality.

Inhibitory and excitatory effects of iodobenzene on the antennal benzoic acid receptor cells of the female silk moth Bombyx mori L.

As shown in single-sensillum recordings, iodobenzene has a bimodal effect on the receptor cell tuned to benzoic acid (BA) of the female silk moth Bombyx mori. Exposure to iodobenzene causes an inhibition of the response to BA. With stimulation by iodobenzene alone, a reduction of basic nerve impulse firing during exposure is followed by a transient post-stimulus excitation (rebound). We suggest that inhibition suppresses excitation during exposure but fades afterwards more rapidly than excitation. Due to the spatial equivalence of the iodine and the acid residue, these effects might indicate opposing interactions of iodobenzene with the specific site for the key compound BA. This is supported by the fact that substitutions by smaller halogens are less effective in both inhibition and rebound. The inhibitory effect but not the rebound with iodobenzene alone was also observed in receptor cells tuned to key compounds other than benzoic acid, e.g. in the cell most sensitive to 2,6-dimethyl-5-heptene-2-ol (DMH-cell) occurring in the same sensillum as the BA-cell, or in the bombykol- and bombykal-cells of the male. At least in these cells the inhibitory effect might reflect the action of iodobenzene on a general site, e.g. the lipid matrix of the plasma membrane of the receptor cells.

The antennal benzoic acid receptor cell of the female silk moth Bombyx mori L.: structure-activity relationship studies with halogen substitutes.

Studies on structure-activity relationships were carried out to characterize the response specificity of the benzoic acid cell of the female of the moth Bombyx mori by means of single sensillum electrophysiological recordings. We demonstrated that this cell type responds best to a natural key substance (benzoic acid) and has similar response profiles for less effective compounds, including various halogen substitutes of benzoic acid, benzaldehyde and other derivates of the key compound. Using different halogen substitutes (F, Cl, Br, I), we showed that the cellular response decreases with increasing atomic size of the substitute and that halogen substitutes were most effective in the meta-position. Thus, m-fluor benzoic acid was even more effective than benzoic acid. These results indicate that a critical feature of the stimulus molecule is the inductive effect generated by the halogen substitutes. Increasing the atomic size of the halogen substitute impairs the recognition of the molecule by the receptor cell, possibly due to steric effects. Decreasing the electron density in the aromatic ring improves the receptor response. The benzoic acid receptor cell can be considered as specialist despite not being involved in pheromone detection as it responds maximally to a key substance and has similar response profiles for less effective compounds.