Multisite imaging of neural activity using a genetically encoded calcium sensor in the honey bee.

Understanding of the neural bases for complex behaviors in Hymenoptera insect species has been limited by a lack of tools that allow measuring neuronal activity simultaneously in different brain regions. Here, we developed the first pan-neuronal genetic driver in a Hymenopteran model organism, the honey bee, and expressed the calcium indicator GCaMP6f under the control of the honey bee synapsin promoter. We show that GCaMP6f is widely expressed in the honey bee brain, allowing to record neural activity from multiple brain regions. To assess the power of this tool, we focused on the olfactory system, recording simultaneous responses from the antennal lobe, and from the more poorly investigated lateral horn (LH) and mushroom body (MB) calyces. Neural responses to 16 distinct odorants demonstrate that odorant quality (chemical structure) and quantity are faithfully encoded in the honey bee antennal lobe. In contrast, odor coding in the LH departs from this simple physico-chemical coding, supporting the role of this structure in coding the biological value of odorants. We further demonstrate robust neural responses to several bee pheromone odorants, key drivers of social behavior, in the LH. Combined, these brain recordings represent the first use of a neurogenetic tool for recording large-scale neural activity in a eusocial insect and will be of utility in assessing the neural underpinnings of olfactory and other sensory modalities and of social behaviors and cognitive abilities.

Ants act as olfactory bio-detectors of tumours in patient-derived xenograft mice.

Early detection of cancer is critical in medical sciences, as the sooner a cancer is diagnosed, the higher are the chances of recovery. Tumour cells are characterized by specific volatile organic compounds (VOCs) that can be used as cancer biomarkers. Through olfactory associative learning, animals can be trained to detect these VOCs. Insects such as ants have a refined sense of smell, and can be easily and rapidly trained with olfactory conditioning. Using urine from patient-derived xenograft mice as stimulus, we demonstrate that individual ants can learn to discriminate the odour of healthy mice from that of tumour-bearing mice and do so after only three conditioning trials. After training, they spend approximately 20% more time in the vicinity of the learned odour than beside the other stimulus. Chemical analyses confirmed that the presence of the tumour changed the urine odour, supporting the behavioural results. Our study demonstrates that ants reliably detect tumour cues in mice urine and have the potential to act as efficient and inexpensive cancer bio-detectors.

Coevolution of the olfactory organ and its receptor repertoire in ray-finned fishes.

BACKGROUND: Ray-finned fishes (Actinopterygii) perceive their environment through a range of sensory modalities, including olfaction. Anatomical diversity of the olfactory organ suggests that olfaction is differentially important among species. To explore this topic, we studied the evolutionary dynamics of the four main gene families (OR, TAAR, ORA/VR1 and OlfC/VR2) coding for olfactory receptors in 185 species of ray-finned fishes. RESULTS: The large variation in the number of functional genes, between 28 in the ocean sunfish Mola mola and 1317 in the reedfish Erpetoichthys calabaricus, is the result of parallel expansions and contractions of the four main gene families. Several ancient and independent simplifications of the olfactory organ are associated with massive gene losses. In contrast, Polypteriformes, which have a unique and complex olfactory organ, have almost twice as many olfactory receptor genes as any other ray-finned fish. CONCLUSIONS: We document a functional link between morphology of the olfactory organ and richness of the olfactory receptor repertoire. Further, our results demonstrate that the genomic underpinning of olfaction in ray-finned fishes is heterogeneous and presents a dynamic pattern of evolutionary expansions, simplifications, and reacquisitions.

Evolutionary Dynamics of the OR Gene Repertoire in Teleost Fishes: Evidence of an Association with Changes in Olfactory Epithelium Shape.

Teleost fishes perceive their environment through a range of sensory modalities, among which olfaction often plays an important role. Richness of the olfactory repertoire depends on the diversity of receptors coded by homologous genes classified into four families: OR, TAAR, VR1, and VR2. Herein, we focus on the OR gene repertoire. While independent large contractions of the OR gene repertoire associated with ecological transitions have been found in mammals, little is known about the diversity of the OR gene repertoire and its evolution in teleost fishes, a group that includes more than 34,000 living species. We analyzed genomes of 163 species representing diversity in this large group. We found a large range of variation in the number of functional OR genes, from 15 in the Broad-nose Pipefish Syngnathus typhle and the Ocean Sunfish Mola mola, to 429 in the Zig-zag Eel Mastacembelus armatus. The number of OR genes was higher in species when a multilamellar olfactory rosette was present. Moreover, the number of lamellae was correlated with the richness of the OR gene repertoire. While a slow and balanced birth-and-death process generally drives the evolution of the OR gene repertoire, we inferred several episodes of high rates of gene loss, sometimes followed by large gains in the number of OR genes. These gains coincide with morphological changes of the olfactory organ and suggest a strong functional association between changes in the morphology and the evolution of the OR gene repertoire.

Biological constraints on configural odour mixture perception.

Animals, including humans, detect odours and use this information to behave efficiently in the environment. Frequently, odours consist of complex mixtures of odorants rather than single odorants, and mixtures are often perceived as configural wholes, i.e. as odour objects (e.g. food, partners). The biological rules governing this 'configural perception' (as opposed to the elemental perception of mixtures through their components) remain weakly understood. Here, we first review examples of configural mixture processing in diverse species involving species-specific biological signals. Then, we present the original hypothesis that at least certain mixtures can be processed configurally across species. Indeed, experiments conducted in human adults, newborn rabbits and, more recently, in rodents and honeybees show that these species process some mixtures in a remarkably similar fashion. Strikingly, a mixture AB (A, ethyl isobutyrate; B, ethyl maltol) induces configural processing in humans, who perceive a mixture odour quality (pineapple) distinct from the component qualities (A, strawberry; B, caramel). The same mixture is weakly configurally processed in rabbit neonates, which perceive a particular odour for the mixture in addition to the component odours. Mice and honeybees also perceive the AB mixture configurally, as they respond differently to the mixture compared with its components. Based on these results and others, including neurophysiological approaches, we propose that certain mixtures are convergently perceived across various species of vertebrates/invertebrates, possibly as a result of a similar anatomical organization of their olfactory systems and the common necessity to simplify the environment's chemical complexity in order to display adaptive behaviours.

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.

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.

The neuroethology of olfactory sex communication in the honeybee Apis mellifera L.

The honeybee Apis mellifera L. is a crucial pollinator as well as a prominent scientific model organism, in particular for the neurobiological study of olfactory perception, learning, and memory. A wealth of information is indeed available about how the worker bee brain detects, processes, and learns about odorants. Comparatively, olfaction in males (the drones) and queens has received less attention, although they engage in a fascinating mating behavior that strongly relies on olfaction. Here, we present our current understanding of the molecules, cells, and circuits underlying bees' sexual communication. Mating in honeybees takes place at so-called drone congregation areas and places high in the air where thousands of drones gather and mate in dozens with virgin queens. One major queen-produced olfactory signal-9-ODA, the major component of the queen pheromone-has been known for decades to attract the drones. Since then, some of the neural pathways responsible for the processing of this pheromone have been unraveled. However, olfactory receptor expression as well as brain neuroanatomical data point to the existence of three additional major pathways in the drone brain, hinting at the existence of 4 major odorant cues involved in honeybee mating. We discuss current evidence about additional not only queen- but also drone-produced pheromonal signals possibly involved in bees' sexual behavior. We also examine data revealing recent evolutionary changes in drone's olfactory system in the Apis genus. Lastly, we present promising research avenues for progressing in our understanding of the neural basis of bees mating behavior.

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.

Configural perception of a binary olfactory mixture in honey bees, as in humans, rodents and newborn rabbits.

How animals perceive and learn complex stimuli, such as mixtures of odorants, is a difficult problem, for which the definition of general rules across the animal kingdom remains elusive. Recent experiments conducted in human and rodent adults as well as newborn rabbits suggested that these species process particular odor mixtures in a similar, configural manner. Thus, the binary mixture of ethyl isobutyrate (EI) and ethyl maltol (EM) induces configural processing in humans, who perceive a mixture odor quality (pineapple) that is distinct from the quality of each component (strawberry and caramel). Similarly, rabbit neonates treat the mixture differently, at least in part, from its components. In the present study, we asked whether the properties of the EI.EM mixture extend to an influential invertebrate model, the honey bee Apis mellifera. We used appetitive conditioning of the proboscis extension response to evaluate how bees perceive the EI.EM mixture. In a first experiment, we measured perceptual similarity between this mixture and its components in a generalization protocol. In a second experiment, we measured the ability of bees to differentiate between the mixture and both of its components in a negative patterning protocol. In each experimental series, the performance of bees with this mixture was compared with that obtained with four other mixtures, chosen from previous work in humans, newborn rabbits and bees. Our results suggest that when having to differentiate mixture and components, bees treat the EI.EM in a robust configural manner, similarly to mammals, suggesting the existence of common perceptual rules across the animal kindgdom.

Aminergic neuromodulation of associative visual learning in harnessed honey bees.

The honey bee Apis mellifera is a major insect model for studying visual cognition. Free-flying honey bees learn to associate different visual cues with a sucrose reward and may deploy sophisticated cognitive strategies to this end. Yet, the neural bases of these capacities cannot be studied in flying insects. Conversely, immobilized bees are accessible to neurobiological investigation but training them to respond appetitively to visual stimuli paired with sucrose reward is difficult. Here we succeeded in coupling visual conditioning in harnessed bees with pharmacological analyses on the role of octopamine (OA), dopamine (DA) and serotonin (5-HT) in visual learning. We also studied if and how these biogenic amines modulate sucrose responsiveness and phototaxis behaviour as intact reward and visual perception are essential prerequisites for appetitive visual learning. Our results suggest that both octopaminergic and dopaminergic signaling mediate either the appetitive sucrose signaling or the association between color and sucrose reward in the bee brain. Enhancing and inhibiting serotonergic signaling both compromised learning performances, probably via an impairment of visual perception. We thus provide a first analysis of the role of aminergic signaling in visual learning and retention in the honey bee and discuss further research trends necessary to understand the neural bases of visual cognition in this insect.

Neural substrate for higher-order learning in an insect: Mushroom bodies are necessary for configural discriminations.

Learning theories distinguish elemental from configural learning based on their different complexity. Although the former relies on simple and unambiguous links between the learned events, the latter deals with ambiguous discriminations in which conjunctive representations of events are learned as being different from their elements. In mammals, configural learning is mediated by brain areas that are either dispensable or partially involved in elemental learning. We studied whether the insect brain follows the same principles and addressed this question in the honey bee, the only insect in which configural learning has been demonstrated. We used a combination of conditioning protocols, disruption of neural activity, and optophysiological recording of olfactory circuits in the bee brain to determine whether mushroom bodies (MBs), brain structures that are essential for memory storage and retrieval, are equally necessary for configural and elemental olfactory learning. We show that bees with anesthetized MBs distinguish odors and learn elemental olfactory discriminations but not configural ones, such as positive and negative patterning. Inhibition of GABAergic signaling in the MB calyces, but not in the lobes, impairs patterning discrimination, thus suggesting a requirement of GABAergic feedback neurons from the lobes to the calyces for nonelemental learning. These results uncover a previously unidentified role for MBs besides memory storage and retrieval: namely, their implication in the acquisition of ambiguous discrimination problems. Thus, in insects as in mammals, specific brain regions are recruited when the ambiguity of learning tasks increases, a fact that reveals similarities in the neural processes underlying the elucidation of ambiguous tasks across species.

Differential combinatorial coding of pheromones in two olfactory subsystems of the honey bee brain.

Neural coding of pheromones has been intensively studied in insects with a particular focus on sex pheromones. These studies favored the view that pheromone compounds are processed within specific antennal lobe glomeruli following a specialized labeled-line system. However, pheromones play crucial roles in an insect's life beyond sexual attraction, and some species use many different pheromones making such a labeled-line organization unrealistic. A combinatorial coding scheme, in which each component activates a set of broadly tuned units, appears more adapted in this case. However, this idea has not been tested thoroughly. We focused here on the honey bee Apis mellifera, a social insect that relies on a wide range of pheromones to ensure colony cohesion. Interestingly, the honey bee olfactory system harbors two central parallel pathways, whose functions remain largely unknown. Using optophysiological recordings of projection neurons, we compared the responses of these two pathways to 27 known honey bee pheromonal compounds emitted by the brood, the workers, and the queen. We show that while queen mandibular pheromone is processed by l-ALT (lateral antennal lobe tract) neurons and brood pheromone is mainly processed by m-ALT (median antennal lobe tract) neurons, worker pheromones induce redundant activity in both pathways. Moreover, all tested pheromonal compounds induce combinatorial activity from several AL glomeruli. These findings support the combinatorial coding scheme and suggest that higher-order brain centers reading out these combinatorial activity patterns may eventually classify olfactory signals according to their biological meaning.

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.

Odourant dominance in olfactory mixture processing: what makes a strong odourant?

The question of how animals process stimulus mixtures remains controversial as opposing views propose that mixtures are processed analytically, as the sum of their elements, or holistically, as unique entities different from their elements. Overshadowing is a widespread phenomenon that can help decide between these alternatives. In overshadowing, an individual trained with a binary mixture learns one element better at the expense of the other. Although element salience (learning success) has been suggested as a main explanation for overshadowing, the mechanisms underlying this phenomenon remain unclear. We studied olfactory overshadowing in honeybees to uncover the mechanisms underlying olfactory-mixture processing. We provide, to our knowledge, the most comprehensive dataset on overshadowing to date based on 90 experimental groups involving more than 2700 bees trained either with six odourants or with their resulting 15 binary mixtures. We found that bees process olfactory mixtures analytically and that salience alone cannot predict overshadowing. After normalizing learning success, we found that an unexpected feature, the generalization profile of an odourant, was determinant for overshadowing. Odourants that induced less generalization enhanced their distinctiveness and became dominant in the mixture. Our study thus uncovers features that determine odourant dominance within olfactory mixtures and allows the referring of this phenomenon to differences in neural activity both at the receptor and the central level in the insect nervous system.

Cyclic nucleotide-gated channels, calmodulin, adenylyl cyclase, and calcium/calmodulin-dependent protein kinase II are required for late, but not early, long-term memory formation in the honeybee.

Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee Apis mellifera, olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM) are formed upon multiple-trial conditioning: an early phase (e-LTM) which depends on translation from already available mRNA, and a late phase (l-LTM) which requires de novo transcription and translation. Here we combined olfactory PER conditioning and neuropharmacological inhibition and studied the involvement of the NO-cGMP pathway, and of specific molecules, such as cyclic nucleotide-gated channels (CNG), calmodulin (CaM), adenylyl cyclase (AC), and Ca(2+)/calmodulin-dependent protein kinase (CaMKII), in the formation of olfactory LTM in bees. We show that in addition to NO-cGMP and cAMP-PKA, CNG channels, CaM, AC, and CaMKII also participate in the formation of a l-LTM (72-h post-conditioning) that is specific for the learned odor. Importantly, the same molecules are dispensable for olfactory learning and for the formation of both MTM (in the minute and hour range) and e-LTM (24-h post-conditioning), thus suggesting that the signaling pathways leading to l-LTM or e-LTM involve different molecular actors.

Chromatic processing in the anterior optic tubercle of the honey bee brain.

Color vision in honey bees (Apis mellifera) has been extensively studied at the behavioral level and, to a lesser degree, at the physiological level by means of electrophysiological intracellular recordings of single neurons. Few visual neurons have been so far characterized in the lateral protocerebrum of bees. Therefore, the possible implication of this region in chromatic processing remains unknown. We performed in vivo calcium imaging of interneurons in the anterior optic tubercle (AOTu) of honey bees upon visual stimulation of the compound eye to analyze chromatic response properties. Stimulation with distinct monochromatic lights (ultraviolet [UV], blue, and green) matching the sensitivity of the three photoreceptor types of the bee retina induced different signal amplitudes, temporal dynamics, and spatial activity patterns in the AOTu intertubercle network, thus revealing intricate chromatic processing properties. Green light strongly activated both the dorsal and ventral lobes of the AOTu's major unit; blue light activated the dorsal lobe more while UV light activated the ventral lobe more. Eye stimulation with mixtures of blue and green light induced suppression phenomena in which responses to the mixture were lower than those to the color components, thus concurring with color-opponent processing. These data provide evidence for a spatial segregation of color processing in the AOTu, which may serve for navigation purposes.

Honeybee drones are attracted by groups of consexuals in a walking simulator.

During the mating season, honeybee males, the drones, gather in congregation areas 10-40 m above ground. When a receptive female, a queen, enters the congregation, drones are attracted to her by queen-produced pheromones and visual cues and attempt to mate with the queen in mid-air. It is still unclear how drones and queens find the congregations. Visual cues on the horizon are most probably used for long-range orientation. For shorter-range orientation, however, attraction by a drone-produced aggregation pheromone has been proposed, yet so far its existence has not been confirmed conclusively. The low accessibility of congregation areas high up in the air is a major hurdle and precise control of experimental conditions often remains unsatisfactory in field studies. Here, we used a locomotion compensator-based walking simulator to investigate drones' innate odor preferences under controlled laboratory conditions. We tested behavioral responses of drones to 9-oxo-2-decenoic acid (9-ODA), the major queen-produced sexual attractant, and to queen mandibular pheromone (QMP), an artificial blend of 9-ODA and several other queen-derived components. While 9-ODA strongly dominates the odor bouquet of virgin queens, QMP rather resembles the bouquet of mated queens. In our assay, drones were attracted by 9-ODA, but not by QMP. We also investigated the potential attractiveness of male-derived odors by testing drones' orientation responses to the odor bouquet of groups of 10 living drones or workers. Our results demonstrate that honeybee drones are attracted by groups of other drones (but not by workers), which may indicate a role of drone-emitted cues for the formation of congregations.

The genome of the blind bee louse fly reveals deep convergences with its social host and illuminates Drosophila origins.

Social insects' nests harbor intruders known as inquilines,1 which are usually related to their hosts.2,3 However, distant non-social inquilines may also show convergences with their hosts,4,5 although the underlying genomic changes remain unclear. We analyzed the genome of the wingless and blind bee louse fly Braula coeca, an inquiline kleptoparasite of the western honey bee, Apis mellifera.6,7 Using large phylogenomic data, we confirmed recent accounts that the bee louse fly is a drosophilid8,9 and showed that it had likely evolved from a sap-breeder ancestor associated with honeydew and scale insects' wax. Unlike many parasites, the bee louse fly genome did not show significant erosion or strict reliance on an endosymbiont, likely due to a relatively recent age of inquilinism. However, we observed a horizontal transfer of a transposon and a striking parallel evolution in a set of gene families between the honey bee and the bee louse fly. Convergences included genes potentially involved in metabolism and immunity and the loss of nearly all bitter-tasting gustatory receptors, in agreement with life in a protective nest and a diet of honey, pollen, and beeswax. Vision and odorant receptor genes also exhibited rapid losses. Only genes whose orthologs in the closely related Drosophila melanogaster respond to honey bee pheromone components or floral aroma were retained, whereas the losses included orthologous receptors responsive to the anti-ovarian honey bee queen pheromones. Hence, deep genomic convergences can underlie major phenotypic transitions during the evolution of inquilinism between non-social parasites and their social hosts.