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.

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.

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.

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.

Olfactory coding in the antennal lobe of the bumble bee Bombus terrestris.

Sociality is classified as one of the major transitions in evolution, with the largest number of eusocial species found in the insect order Hymenoptera, including the Apini (honey bees) and the Bombini (bumble bees). Bumble bees and honey bees not only differ in their social organization and foraging strategies, but comparative analyses of their genomes demonstrated that bumble bees have a slightly less diverse family of olfactory receptors than honey bees, suggesting that their olfactory abilities have adapted to different social and/or ecological conditions. However, unfortunately, no precise comparison of olfactory coding has been performed so far between honey bees and bumble bees, and little is known about the rules underlying olfactory coding in the bumble bee brain. In this study, we used in vivo calcium imaging to study olfactory coding of a panel of floral odorants in the antennal lobe of the bumble bee Bombus terrestris. Our results show that odorants induce reproducible neuronal activity in the bumble bee antennal lobe. Each odorant evokes a different glomerular activity pattern revealing this molecule's chemical structure, i.e. its carbon chain length and functional group. In addition, pairwise similarity among odor representations are conserved in bumble bees and honey bees. This study thus suggests that bumble bees, like honey bees, are equipped to respond to odorants according to their chemical features.

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.

Morphological and functional correlates of vestibular synaptic deafferentation and repair in a mouse model of acute-onset vertigo.

Damage to cochlear primary afferent synapses has been shown to be a key factor in various auditory pathologies. Similarly, the selective lesioning of primary vestibular synapses might be an underlying cause of peripheral vestibulopathies that cause vertigo and dizziness, for which the pathophysiology is currently unknown. To thoroughly address this possibility, we selectively damaged the synaptic contacts between hair cells and primary vestibular neurons in mice through the transtympanic administration of a glutamate receptor agonist. Using a combination of histological and functional approaches, we demonstrated four key findings: (1) selective synaptic deafferentation is sufficient to generate acute vestibular syndrome with characteristics similar to those reported in patients; (2) the reduction of the vestibulo-ocular reflex and posturo-locomotor deficits mainly depends on spared synapses; (3) damaged primary vestibular synapses can be repaired over the days and weeks following deafferentation; and (4) the synaptic repair process occurs through the re-expression and re-pairing of synaptic proteins such as CtBP2 and SHANK-1. Primary synapse repair might contribute to re-establishing the initial sensory network. Deciphering the molecular mechanism that supports synaptic repair could offer a therapeutic opportunity to rescue full vestibular input and restore gait and balance in patients.

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.

Long-term Sensory Conflict in Freely Behaving Mice.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

Differential Processing by Two Olfactory Subsystems in the Honeybee Brain.

Among insects, Hymenoptera present a striking olfactory system with a clear neural dichotomy from the periphery to higher order centers, based on two main tracts of second-order (projection) neurons: the medial and lateral antennal lobe tracts (m-ALT and l-ALT). Despite substantial work on this dual pathway, its exact function is yet unclear. Here, we ask how attributes of odor quality and odor quantity are represented in the projection neurons (PNs) of the two pathways. Using in vivo calcium imaging, we compared the responses of m-ALT and l-ALT PNs of the honey bee Apis mellifera to a panel of 16 aliphatic odorants, and to three chosen odorants at eight concentrations. The results show that each pathway conveys differential information about odorants' chemical features or concentration to higher order centers. While the l-ALT primarily conveys information about odorants' chain length, the m-ALT informs about odorants' functional group. Furthermore, each tract can only predict chemical distances or bees' behavioral responses for odorants that differ according to its main feature, chain length or functional group. Generally l-ALT neurons displayed more graded dose-response relationships than m-ALT neurons, with a correspondingly smoother progression of inter-odor distances with increasing concentration. Comparison of these results with previous data recorded at AL input reveals differential processing by local networks within the two pathways. These results support the existence of parallel processing of odorant features in the insect brain.

AhR-deficiency as a cause of demyelinating disease and inflammation.

The Aryl hydrocarbon Receptor(AhR) is among the most important receptors which bind pollutants; however it also regulates signaling pathways independently of such exposure. We previously demonstrated that AhR is expressed during development of the central nervous system(CNS) and that its deletion leads to the occurrence of a congenital nystagmus. Objectives of the present study are to decipher the origin of these deficits, and to identify the role of the AhR in the development of the CNS. We show that the AhR-knockout phenotype develops during early infancy together with deficits in visual-information-processing which are associated with an altered optic nerve myelin sheath, which exhibits modifications in its lipid composition and in the expression of myelin-associated-glycoprotein(MAG), a cell adhesion molecule involved in myelin-maintenance and glia-axon interaction. In addition, we show that the expression of pro-inflammatory cytokines is increased in the impaired optic nerve and confirm that inflammation is causally related with an AhR-dependent decreased expression of MAG. Overall, our findings demonstrate the role of the AhR as a physiological regulator of myelination and inflammatory processes in the developing CNS. It identifies a mechanism by which environmental pollutants might influence CNS myelination and suggest AhR as a relevant drug target for demyelinating diseases.

Long-Lasting Visuo-Vestibular Mismatch in Freely-Behaving Mice Reduces the Vestibulo-Ocular Reflex and Leads to Neural Changes in the Direct Vestibular Pathway.

Calibration of the vestibulo-ocular reflex (VOR) depends on the presence of visual feedback. However, the cellular mechanisms associated with VOR modifications at the level of the brainstem remain largely unknown. A new protocol was designed to expose freely behaving mice to a visuo-vestibular mismatch during a 2-week period. This protocol induced a 50% reduction of the VOR. In vivo pharmacological experiments demonstrated that the VOR reduction depends on changes located outside the flocculus/paraflocculus complex. The cellular mechanisms associated with the VOR reduction were then studied in vitro on brainstem slices through a combination of vestibular afferent stimulation and patch-clamp recordings of central vestibular neurons. The evoked synaptic activity demonstrated that the efficacy of the synapses between vestibular afferents and central vestibular neurons was decreased. In addition, a long-term depression protocol failed to further decrease the synapse efficacy, suggesting that the VOR reduction might have occurred through depression-like mechanisms. Analysis of the intrinsic membrane properties of central vestibular neurons revealed that the synaptic changes were supplemented by a decrease in the spontaneous discharge and excitability of a subpopulation of neurons. Our results provide evidence that a long-lasting visuo-vestibular mismatch leads to changes in synaptic transmission and intrinsic properties of central vestibular neurons in the direct VOR pathway. Overall, these results open new avenues for future studies on visual and vestibular interactions conducted in vivo and in vitro.

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.

Parallel Olfactory Processing in the Honey Bee Brain: Odor Learning and Generalization under Selective Lesion of a Projection Neuron Tract.

The function of parallel neural processing is a fundamental problem in Neuroscience, as it is found across sensory modalities and evolutionary lineages, from insects to humans. Recently, parallel processing has attracted increased attention in the olfactory domain, with the demonstration in both insects and mammals that different populations of second-order neurons encode and/or process odorant information differently. Among insects, Hymenoptera present a striking olfactory system with a clear neural dichotomy from the periphery to higher-order centers, based on two main tracts of second-order (projection) neurons: the medial and lateral antennal lobe tracts (m-ALT and l-ALT). To unravel the functional role of these two pathways, we combined specific lesions of the m-ALT tract with behavioral experiments, using the classical conditioning of the proboscis extension response (PER conditioning). Lesioned and intact bees had to learn to associate an odorant (1-nonanol) with sucrose. Then the bees were subjected to a generalization procedure with a range of odorants differing in terms of their carbon chain length or functional group. We show that m-ALT lesion strongly affects acquisition of an odor-sucrose association. However, lesioned bees that still learned the association showed a normal gradient of decreasing generalization responses to increasingly dissimilar odorants. Generalization responses could be predicted to some extent by in vivo calcium imaging recordings of l-ALT neurons. The m-ALT pathway therefore seems necessary for normal classical olfactory conditioning performance.

Genotypic influence on aversive conditioning in honeybees, using a novel thermal reinforcement procedure.

In Pavlovian conditioning, animals learn to associate initially neutral stimuli with positive or negative outcomes, leading to appetitive and aversive learning respectively. The honeybee (Apis mellifera) is a prominent invertebrate model for studying both versions of olfactory learning and for unraveling the influence of genotype. As a queen bee mates with about 15 males, her worker offspring belong to as many, genetically-different patrilines. While the genetic dependency of appetitive learning is well established in bees, it is not the case for aversive learning, as a robust protocol was only developed recently. In the original conditioning of the sting extension response (SER), bees learn to associate an odor (conditioned stimulus - CS) with an electric shock (unconditioned stimulus - US). This US is however not a natural stimulus for bees, which may represent a potential caveat for dissecting the genetics underlying aversive learning. We thus first tested heat as a potential new US for SER conditioning. We show that thermal stimulation of several sensory structures on the bee's body triggers the SER, in a temperature-dependent manner. Moreover, heat applied to the antennae, mouthparts or legs is an efficient US for SER conditioning. Then, using microsatellite analysis, we analyzed heat sensitivity and aversive learning performances in ten worker patrilines issued from a naturally inseminated queen. We demonstrate a strong influence of genotype on aversive learning, possibly indicating the existence of a genetic determinism of this capacity. Such determinism could be instrumental for efficient task partitioning within the hive.

Olfactory coding in the honeybee lateral horn.

Olfactory systems dynamically encode odor information in the nervous system. Insects constitute a well-established model for the study of the neural processes underlying olfactory perception. In insects, odors are detected by sensory neurons located in the antennae, whose axons project to a primary processing center, the antennal lobe. There, the olfactory message is reshaped and further conveyed to higher-order centers, the mushroom bodies and the lateral horn. Previous work has intensively analyzed the principles of olfactory processing in the antennal lobe and in the mushroom bodies. However, how the lateral horn participates in olfactory coding remains comparatively more enigmatic. We studied odor representation at the input to the lateral horn of the honeybee, a social insect that relies on both floral odors for foraging and pheromones for social communication. Using in vivo calcium imaging, we show consistent neural activity in the honeybee lateral horn upon stimulation with both floral volatiles and social pheromones. Recordings reveal odor-specific maps in this brain region as stimulations with the same odorant elicit more similar spatial activity patterns than stimulations with different odorants. Odor-similarity relationships are mostly conserved between antennal lobe and lateral horn, so that odor maps recorded in the lateral horn allow predicting bees' behavioral responses to floral odorants. In addition, a clear segregation of odorants based on pheromone type is found in both structures. The lateral horn thus contains an odor-specific map with distinct representations for the different bee pheromones, a prerequisite for eliciting specific behaviors.