14th European Symposium for Taste and Olfaction (ESITO)

ABSTRACTS

September 20-25, 2015

Villasimius (Cagliari – Italy)

www.ice.mpg.de/esito

Agustina Falibene, Flavio Roces, Wolfgang Rössler Department of Behavioral Physiology and Sociobiology, Biozentrum, University of Würzburg, Germany, [email protected] Odor learning in ants: synaptic plasticity associated with long-term avoidance memory formation Long-term behavioral changes related to learning and experience have been shown to be associated with structural remodeling in the brain. Leaf-cutting ants learn to avoid previously preferred plants after those are experienced to be harmful for their symbiotic fungus, a process that involves long-term . We studied the dynamics of brain microarchitectural changes after long-term olfactory memory formation following avoidance learning in Acromyrmex ambiguus. Quantification of synaptic complexes (microglomeruli, MG) in olfactory regions of the (MB) at different times after learning revealed a transient change in MG densities. Two days after learning, lip MG density was higher than before learning. At days 4 and 15 after learning – when ants still showed plant avoidance – MG densities had decreased to the initial state. Changes were observed in the lip but not in the visual collar in which MG densities remained unaffected. Furthermore, enriched experience such as the simultaneous collection of several, instead of one, non-harmful plant species resulted in a decrease in MG densities (pruning). The results indicate that learning and sensory experience affect the synaptic architecture of the MB calyces via different processes. While sensory experience leads to MG pruning in the MB olfactory lip, long-term memory formation appears to involve growth of new MG followed by the elimination of others. Supported by DAAD (A/11/76441) and DFG SFB 1047 Insect timing (projects B6 and C1).

Ahmed A. M. Mohamed, Tom Retzke, Markus Knaden, Bill S. Hansson & Silke Sachse Max Planck Institute for Chemical Ecology, Jena, Germany, [email protected] To go or not to go? Olfactory processing of odor features: Good vs. Bad Natural olfactory stimuli are mainly composed of complex blends of volatiles, which might be mixtures of attractive and aversive odors. The identities and ratios of these odor components need to be evaluated by the fly to decide whether the odor mixture is still attractive or has shifted to an aversive stimulus. Despite this importance, the mechanism by which the olfactory system processes and integrates this complex information remains so far unclear. By combining behavioral experiments - using the FlyWalk - with neurophysiological experiments - using functional calcium imaging at different processing levels of the fly brain - we aim at understanding how and where the information about odor valence, i.e. attractive and aversive odors, is encoded and ultimately integrated along the olfactory pathway. The first results of the behavioral assay show that Drosophila adults behave differently when they are faced to different ratios of odor mixtures of attractive and aversive odors. The flies are strongly attracted to a mixture of an attractive food odor (ethyl acetate) and a well- known fly repellent (benzaldehyde) when the attractive odor occurs at high concentration. However, if the concentration of the attractive odor is decreased, the flies get less attracted, i.e. the aversive odor suddenly dominates the mixture. Remarkably, our imaging results show that this integration of attractive and aversive odors seem to take place already at the output level of the , i.e. at the level of the projection neurons. We are planning to silence different populations of LNs to identify those

2 neurons that are evoking mixture interactions and therefore mediating the integration of odors with opposing valences.

Alexander Haverkamp, Julia Bing, Bill S. Hansson, Markus Knaden Max Planck Institute for Chemical Ecology, Department of evolutionary Neuroethology, [email protected] Optimal foraging by smell The intimate relationship between sphingid moths and the flowers they pollinate is a long standing model system for insect-plant coevolution. Pollinator shifts and pollinator preferences have been argued as the main forces driving this coadaptation; however the preference of hawkmoths for flowers with a matching corolla still remains controversial. The hawkmoth Manduca sexta visits a variety of night-blooming flowers. While foraging, it relies mainly on olfactory information and a preference for flowers emitting aromatic compounds and terpenes has been demonstrated. First, we used the close relationship between Manduca and different plant species within the Nicotiana genus to test for a relationship between olfactory preference and the match of proboscis and flower length. 3-d flight tracking of moths entering a well characterized flower odor plume revealed significantly higher upwind speeds and more source contacts when the headspace of matching flowers was presented, indicating that the olfactory preference of Manduca relates to the match of proboscis and flower. We next tested whether the moth gains an advantage from foraging on matching flowers. Hawkmoths feed “on the wing” - making energetic costs an important factor of foraging. We balanced flower-handling costs measured by respirometry against the energy provided by floral nectar. The net energy gain was indeed highest for moths foraging on matching flowers. Hence, olfactory information enables moths to predict foraging outcomes and strengthens the coadaptation of pollinator and flower.

Anthi A. Apostolopoulou, Saskia Köhn, Michael Lutz, Alexander Wüst, Lorena Mazija, Anna Rist, Giovanni Galizia, Alja Lüdke and Andreas S. Thum University of Konstanz, Department of Biology, Konstanz, Germany, andreas.thum@uni- konstanz.de The neuronal and molecular basis of taste signaling in Drosophila larvae Caffeine is a widely consumed substance for humans, which strongly affects behaviour. Yet, for humans and the molecular and neuronal background underlying caffeine perception and processing is not entirely understood. Here, we use Drosophila larva as a simple system to investigate caffeine information processing. In larvae, consumption of caffeine induces avoidance behaviour, suppresses feeding and decreases survival but, does not reinforce aversive odour associations. Only 12 gustatory neuron pairs co-express the bitter receptors Gr33a and Gr66a and these neurons are necessary for avoidance behaviour. We found that individual receptor gene mutants for these two genes show partially reduced caffeine avoidance. Performing a neuronal screen to pinpoint total loss of caffeine avoidance to single gustatory bitter neurons out of the Gr33a/Gr66a set identified a single Gr93a positive pair of dorsal pharyngeal sensilla that responded to caffeine. Likewise, Gr93a receptor gene function is necessary for caffeine avoidance. In conclusion, our data suggests that the detection of caffeine is encoded using a dedicated sensory channel for bitter sensation involving Gr93a. This finding opens new avenues to investigate how caffeine

3 affects behaviour by separating stimulation of the sensory system from internal brain functions.

Andy Sombke, Elisabeth Lipke University of Greifswald; Zoological Institute and Museum; Dept. for Cytology and Evolutionary Biology, Germany, [email protected] Visualizing the antennal lobe using X-Ray microscopy – a new approach in neuroanatomy Traditionally, arthropod neuroanatomy is explored by methods such as classical histology or immunohistochemistry combined with fluorescence- or confocal laser scan microscopy. However, these methods are time consuming, prone to artifacts, and often require extensive sample preparations to obtain desired information. Furthermore, as a consequence of e.g., dissection or sectioning of the nervous system, or parts of it, information on the natural coherence is usually missing. Non-destructive approaches such as micro-computed X-ray tomography (µCT) overcome these limitations and have been shown to be a valuable tool in understanding and visualizing internal anatomy and structural complexity of a variety of studied taxa, including . Nevertheless, only little is known about the usability of this method for analyzing the architecture and organization of the arthropod nervous system. We analyzed the brains in representatives of several arthropod taxa and compare our results to previously obtained histological and immunohistochemical data. Our data show that the resolution obtained by µCT is sufficient to (1) analyze the overall organization as close to their natural state as possible, (2) distinguish specific neuropils, such as olfactory glomeruli of the antennal lobes, and (3) obtain quantitative data. Moreover, we compare and discuss limitations, as well the influence of staining and preliminary sample preparation procedures.

Aniruddha Mitra and Frédéric Marion-Poll Laboratoire Evolution, Genomes, Comportement, Ecologie, Centre National de la Recherche Scientifique, Gif sur Yvette, France, [email protected] Volatile odorants activate taste neurons on the proboscis of Drosophila Taste and smell share the same function: detecting chemicals from the external world. Despite this common functionality, both systems are designed very differently both at the level of the sensory receptors and in the way information is mapped into the brain. While olfaction is tuned towards detecting volatile chemicals from air, the taste system is specialized for detecting non-volatile molecules through contact. Usually the two systems have been studied separately, under the assumption that both systems are tuned to detect divergent and non-overlapping ensemble of molecules. We show through electrophysiology that taste sensilla on the proboscis detect airborne molecules associated with food like acetic acid, acetone and isoamyl propionate (banana odor), as well as compounds known to be aversive to the olfactory system like 1-octen-3-ol (associated with pathogenic microbes) and 1-octanol. Behavioral tests like Y-maze assay and proboscis extension response (response to sucrose in presence/absence of volatiles blowing over proboscis) show that flies ablated of their olfactory organs retain the ability to express behavioral responses to acetic acid vapors. Therefore the taste system has the ability to detect some volatile compounds at close range, raising questions about whether there is a clear separation between the olfactory and gustatory sensory space, at least in . 4

Anna Rist, Andreas Thum University of Konstanz,Department of Biology, Germany, [email protected] Taste Perception in Drosophila Larva: Cellular Architecture of the Terminal Organ Drosophila larvae provide a simple, genetically tractable nervous system to study gustatory coding. A detailed knowledge of the anatomy of their taste system is essential to understand the mechanisms of how taste is perceived and processed. We examine structural and molecular properties of the larva`s major external taste organ, the terminal organ, by means of advanced electron and light microscopy. Taking advantage of focused ion beam scanning electron microscopy, we obtain a precise three dimensional analysis of the terminal organ fine architecture. This allows us to describe the diverse structural organization of its different external sensilla types, which are named: papillum, pit, knob, spot and modified papillum. To further characterize these, members of the gustatory, pickpocket and ionotropic receptor gene family are observed for expression in the sensilla`s sensory neurons by using the GAL4/UAS system and confocal microscopy. Expression is observed in three out of five sensilla types: the papillum, pit and knob. Each houses a different subset of receptor neurons which corroborates functional differences. Considering their ultrastructural properties only the papillum and pit sensilla might be contact chemosensors which perceive tastants. Our findings suggest additional sensory functions for the terminal organ than merely taste perception.

Arthur de Fouchier, William B. Walker, Nicolas Montagne, Muhammad Binyameen, Claudia Steiner, Bill S. Hansson, Thomas Chertemps, Peter Anderson, Fredrik Schlyter, Mattias C. Larsson & Emmanuelle Jacquin-Joly Institute of Ecology and Environmental Sciences of Paris (iEES-Paris), INRA, Versailles, France, [email protected] Functional analysis of olfactory receptors in a phytophagous pest insect indicates a prevalence of narrow tuning Insects use their chemical senses (olfaction and taste) to analyze the information from the environment with a unique efficiency which contributes to their adaptive success. The olfactory detection capacities of an insect depend extensively on its repertoire of (OR) genes, expressed by the Olfactory Sensory Neurons (OSN), and the functional properties of its OR proteins. For now, large OR repertoires have been investigated only in two Diptera: Drosophila melanogaster and the malaria mosquito Anopheles gambiae. These pioneering works notably demonstrated that ORs exhibit various tuning breadths, with individual ORs being usually activated by multiple odorants and individual odorants activating multiple ORs. These studies also showed that OR repertoires are specialized in detecting ecologically relevant odorants. As such, the functional characterization of a large OR repertoire from an herbivorous species is still lacking. Here, we present a systematic functional analysis, using the “Empty Neuron” system, of 35 ORs from the phytophagous moth Spodoptera littoralis, which has been established as a model of crop pest in the fields of chemical ecology and neurobiology of olfaction. Our results provide insights into the odor- coding mechanisms of S. littoralis and highlight a narrow tuning of its OR, especially at low dose of odorant stimulus. Our study thus represents an important step toward improving the understanding of the molecular determinants of olfactory-driven behaviors in S. littoralis and other crop pest moths. 5

Ashiq Hussain, Mo Zhang, Habibe K. Üçpunar, Thomas Svensson, Elsa Quillery, Nicolas Gompel, Rickard Ignell, and Ilona C. Grunwald Kadow Max-Planck Institute of Neurobiology, Munich, Germany, [email protected] Neuropeptides enhance female sensitivity to polyamines upon mating How gravid females identify foods to match their specific dietary needs remains elusive. Among these needs are pungent smelling polyamines, such as putrescine and spermidine, which are vital organic polycations with multiple cellular functions essential for cell proliferation, reproduction, and embryonic development in all animals. Using Drosophila, we have identified a behavioral, neuronal, and genetic mechanism that adapts the senses of smell and taste, the major modalities for food quality, to the physiological needs of a gravid female. We identify the mechanism of polyamine detection and show that female flies use specific olfactory and gustatory ionotropic receptors (IRs) and bitter taste neurons to seek polyamines. Polyamine attraction is enhanced in a sex-specific manner in gravid females through the G-protein coupled receptor SPR (sex peptide receptor) and its neuropeptide ligands, MIPs (myoinhibitory peptides) that act directly in the polyamine-detecting chemosensory neurons. Together, our data shows that neuropeptide-mediated modulation of specific chemosensory neurons increases pregnant females’ preference for important nutrients to ensure optimal conditions for her growing progeny. How gravid females identify foods to match their specific dietary needs remains elusive. Among these needs are pungent smelling polyamines, such as putrescine and spermidine, which are vital organic polycations with multiple cellular functions essential for cell proliferation, reproduction, and embryonic development in all animals. Using Drosophila, we have identified a behavioral, neuronal, and genetic mechanism that adapts the senses of smell and taste, the major modalities for food quality, to the physiological needs of a gravid female. We identify the mechanism of polyamine detection and show that female flies use specific olfactory and gustatory ionotropic receptors (IRs) and bitter taste neurons to seek polyamines. Polyamine attraction is enhanced in a sex-specific manner in gravid females through the G-protein coupled receptor SPR (sex peptide receptor) and its neuropeptide ligands, MIPs (myoinhibitory peptides) that act directly in the polyamine-detecting chemosensory neurons. Together, our data shows that neuropeptide-mediated modulation of specific chemosensory neurons increases pregnant females’ preference for important nutrients to ensure optimal conditions for her growing progeny.

Ashiq Hussain, Mo Zhang, Ilona C. Grunwald Kadow Max-Planck Institute of Neurobiology, Munich, Germany, [email protected] Identification of neural mechanisms underlying the relationship of ageing and olfaction The loss of the is one of the earliest indicators of neurodegenerative diseases such as Alzheimer and Parkinson, but also a frequent symptom of ageing in healthy people. Ageing associated olfactory decline may change the distribution, density, and function of specific receptor proteins, ion channels, and signaling molecules that affect the ability of neural elements throughout the olfactory pathway to signal and process odorant information. The anatomical and molecular causes underlying ageing-associated olfactory sensitivity loss and their relationship to neurodegenerative diseases are unidentified. Using the fruit fly Drosophila melanogaster as a genetic model , we identified gradual regressions in olfactory driven choice behavior with ageing. Remarkably, the visual choice 6 behavior remained largely intact. Although old flies contrary to younger flies, show slightly but significantly declined olfactory neuron responses in single sensillum recordings these results likely do not fully explain the behavioral decline. To begin to address the critical neuronal and molecular mechanisms of this decline we have tested the potential role of previously identified lifespan-altering genes. Among these was superoxide dismutase 2 (SOD2), whose absence causes premature ageing and death in many organisms. We found that ageing-related decline in olfactory choice behavior can be mimicked by knocking-down of SOD2 expression in projection neurons, but not in olfactory receptor neurons suggesting that projection neurons are primarily affected during ageing. To identify additional genetic factors in ageing-associated olfactory decline, we have carried out high throughput deep RNA sequencing of young and old fly olfactory tissues that revealed 181 inheritable factor showing major loss of gene expression in old flies. Furthermore, we will use functional imaging and anatomical analysis to characterize the neural circuit as well as cellular mechanisms that lead to loss of olfactory function in old animals. We anticipate that this comprehensive investigation will identify conserved and potentially causative mechanisms of ageing-associated olfactory functional decline.

Astrid Rohwedder, Nana L. Wenz, Bernhard Stehle, Annina Huser, Nobuhiro Yamagata, Marta Zlatic, Jim Truman, Bertram Gerber, Hiromu Tanimoto, Timo Saumweber and Andreas S. Thum University of Konstanz, Konstanz, Germany, [email protected] Sugar reward is signaled via four dopaminergic neurons Where or when to find energy-rich food is important for all living beings and thus, the ability to learn cues that hint towards these is essential. In various studies Drosophila melanogaster showed to be able to form memories that associate odours with punishing or rewarding stimuli via distinct dopaminergic cell clusters. The DA-associated neuronal circuit of flies and even larva of Drosophila is centered on the mushroom body (MB), a brain center known to be important for the formation and storage of odor memory. The olfactory pathway from sensory neurons to the MB is well described. About perception and processing of the gustatory stimulus, however little is known. In our study we show that a cluster of four dopaminergic neurons in the anterior part of the larval protocerebrum, termed primary protocerebral anteriormedial cluster (pPAM) conveys the rewarding function of sugar to the MB. The pPAM cluster is necessary and sufficient for signaling an acute reinforcing function during odor-reward conditioning for different types of sugar that vary in their nutritional value. We conclude that for Drosophila larvae based on our results on the reward processing DA pPAM cluster, cellular specificity underlying the various roles of DA exists that is reflected by spatially segregated local circuits within the MB.

Brian H Smith School of Life Sciences, Arizona State University, USA, Zukunftskolleg, University of Konstanz, Germany, [email protected] The olfactory ‘scene’ statistics of natural of floral odors We now have a wealth of information about the chemical composition of natural floral odors. They are typically composed of several different chemical compounds, and there is information – e.g. species identity, pollination status, presence of nectar – in the blend amounts and ratios that is important to pollinators. As a generalist pollinator facing 7 significant demands for carbohydrate and protein to support the colony, the honey bee has the capability to learn about many different floral components. It is therefore unlikely that a small subset of odors, or a sub-mixture, can explain recognition of floral odors, as is the case for more specialist insect/flower associations. Instead, the honey bee olfactory system is sensitive to slight changes in blend composition from flower-to-flower. Yet most investigations of olfactory coding and plasticity in the brain use simple odors or uncontrolled floral blends. It may well be that use of more controlled blends that mimic the natural statistics of olfactory scenes will reveal new types of coding complexity in early or later stages of processing. This approach has been used successfully to reveal new coding dimensions in visual and auditory processing, where neural networks from early through deep stages of process extract features based on statistics of physical scenes. Using two species of flowering plants that honey bees visit, we have shown how variance affects the information that honey bees extract in behavioral studies. And we have shown how variance affects plasticity in early processing of the antennal lobe. These analysis point to a new way of looking at olfactory scenes that affect honey bee foraging behavior.

Walker S.J. 1, Leitão-Gonçalves, R. 1, Francisco, A.P. 1, Herbert, S. 1, Corrales-Carvajal, V.M. 1, Carvalho-Santos, Z. 1, Itskov, P. 1, Fioreze G. T. 1, Elias, A. P. 1, Piper, M. D. 2, Carlos Ribeiro 1 1 Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, 1400- 038 Lisbon, Portugal; 2 Institute of Healthy Ageing, Department of Genetics, Evolution and Environment, University College London, London [email protected] The gourmet fly – matching feeding decisions to the internal state of the animal Nutrition is a key determinant of health, wellbeing and aging. To optimize survival and reproduction, animals must match their nutrient intake to their current needs. Many factors, including reproduction, profoundly change nutritional requirements, with many species showing an appetite for specific nutrients during specific periods of their lifecycle. How different internal states modify neuronal information processing to ensure homeostasis is poorly understood. I will discuss different circuit and molecular mechanisms used by the Drosophila brain to read out internal state information allowing the animals to appropriately select among different nutrients such as carbohydrates, amino acids and salt. A general theme emerging from our work is that internal states modify chemosensory processing using a combination of feed-forward and feed-back mechanisms leading to changes in the microstructure of feeding and hence feeding decisions. Importantly, these changes are adaptive as they allow the animal to optimize its reproductive output over its lifetime. Changes in chemosensory processing and feeding microstructure may represent common mechanisms through which internal state-sensitive circuits modify feeding decisions across species.

Carlotta Martelli, Andre Fiala Georg-August-Universität Göttingen, Dept. of Molecular Neurobiology of Behaviour, Julia- Lermontowa-Weg 3, 37077 Göttingen, Germany, [email protected] goettingen.de Single-cell, population-based and network adaptation in the Drosophila olfactory system

8

Insects use olfactory cues to recognize and localize odor sources. Airborne odor landscapes can range from smooth concentration gradients to highly intermittent plumes. Likely, the statistics of natural odor stimuli exceeds the dynamic range of a neuron response. I investigate which strategies allow the insect olfactory system to adapt its sensitivity to relevant stimulus statistics. At a single-cell level, sensory neurons are able to adjust their dynamic range to the current mean stimulus. Using single sensillum recordings in Drosophila, I show that the firing rate of Olfactory Receptor Neurons (ORNs) adapts under sustained stimulation, ultimately causing a shift in sensitivity. This gain adaptation occurs on a time-scale of about one second, but has no effect on the response dynamics at shorter time-scales, therefore allowing a rapid response also in presence of a background stimulus. The combinatorial activation of a large number of contributes to the encoding of odor identity. But do the receptors equally encode stimulus statistics? Using calcium imaging from ORNs terminals in the Antennal Lobe (AL), I show that only a subset of all responding glomeruli precisely follow fluctuations in stimulus concentrations. As the amplitude of these fluctuations increases, the capability to encode the stimulus statistics shifts from one subset of glomeruli to another. This population-based adaptation allows the system to maintain sensitivity beyond the capability of single ORNs. Single-cell and population-based adaptation in the ORNs are not the only forms of adaptation in the Drosophila olfactory system. Odor-evoked calcium influx in Projection Neurons (PNs) terminals decreases over a slow time-scale (10-20 seconds), ultimately inducing a rearrangement of odor representation in PNs. Such rearrangement is not visible in the ORNs, suggesting that is mediated by the AL synaptic network. I will discuss possible mechanisms that might cause such adaptation and its functional roles in odor coding.

ChunSu Xu, Melanie Rast, Jan Pielage Friedrich Miescher Institute, Switzerland, [email protected] The PP1 adaptor protein spinophilin is required for Drosophila olfactory associative memory Drosophila has been a successful model organism for dissecting the molecular basis of behavior, including associative learning. The neuronal circuits of appetitive and aversive olfactory associative learning are already well characterized and genetically tractable. Central to associative learning is the Drosophila mushroom body, a third-order olfactory center. In combination with the versatile molecular tools, mushroom body dependent associative learning represents a great model to explore the molecular machinery controlling synaptic plasticity in the insect’s brain. In the mammalian brain long-term depression (LTD) has been identified as a potential mechanisms contributing to learning and memory. LTD is, at least in part, mediated by posttranslational protein modifications. It has been demonstrated that protein phosphatase 1 (PP1) activity is necessary for LTD. During LTD formation, activated protein phosphatase 1 (PP1) relocates to dendritic compartments and its activation and relocation requires the adaptor protein Neurabin/Spinophilin. To address the role of Drosophila Spinophilin during learning and memory we generated a specific antibody and loss-of-function mutations. Spinophilin is present throughout the brain similar to the presynaptic protein Bruchpilot (Brp), indicating a potential synaptic localization. Spinophilin is particular enriched in the calyx, the dendritic region of the mushroom body neurons. Here, Spinophilin is present in postsynaptic compartments opposite Brp. First behavioral experiments indicate that Spinophilin is not required for learning acquisition but

9 for memory retrieval. We are currently investigating whether Spinophilin may affect LTD as a potential mechanisms controlling associative memory in the insect brain.

Agustina Falibene, Flavio Roces, Wolfgang Rössler, Claudia Groh Department of Behavioral Physiology and Sociobiology, Biozentrum, University of Würzburg, 97074 Würzburg, Germany, [email protected] Daily fluctuations in pupal thermal experience affect synaptic organization in olfactory centers of the adult ant brain Environmental temperature is crucial for development and affects physiology and behavior in insects. Social insects can control brood development by regulating the brood temperature via different mechanisms. In honeybees, slight deviations from a constant brood temperature affect olfactory learning and the architecture of brain areas involved in this process, the mushroom bodies (MB). Camponotus mus ants show a different behavior: nurses translocate the brood inside the nest to two different temperatures showing a circadian rhythm. Brood is located at 30.8°C at the middle of the photophase and 8h later at 27.5°C. Here we investigate whether daily thermal fluctuations experienced by the pupae affect the synaptic organization of the olfactory MB calyx lip in the adult ant brain. Newly spun pupae were reared under daily fluctuating temperatures with different thermal amplitudes, but the same mean temperature. Young worker brains were synapsin- immunolabeled to combine volume measurements of the olfactory lip with the quantification of synapsin-positive presynaptic boutons. Our results show that pupal development time and mortality did not differ between thermal treatments. Temperature mediated effects became evident at the neuronal level. While the lip volume was not affected, the density of presynaptic boutons significantly varied among thermal treatments. The thermal fluctuation experienced by pupae through the nurses’ thermal preferences maximizes the formation of presynaptic boutons in the non-dense lip, a subarea most likely innervated by projection neurons (PNs) from the lateral antennal lobe tract (lALT). In the dense region of the lip – a subarea most likely innervated by PNs from the medial ALT – bouton density was not affected by temperature. We conclude that thermoregulatory control of brood rearing can generate area specific effects on olfactory synaptic neuropils in the adult ant brain. Resulting differences in the synaptic circuitry may affect olfactory communication and olfactory learning abilities in ants. Supported by the DFG SFB 1047 Insect timing (B5 to C.G., B6 to W.R. & C1 to F.R.).

Claudia Steiner, F. Bozzolan, T. Chertemps, M. Maibeche University Pierre et Marie Curie, Institute of Ecology and Environmental Sciences Paris, France, [email protected] Juvenile Hormone Esterase Duplication (JHEdup): Involvement in food odor detection in Drosophila melanogaster? The reception of odor molecules within insect olfactory organs involves several steps, including their transport through the sensillar lymph, their interaction with the corresponding receptor and eventually their inactivation. Despite the detection of olfactory cues in insects has recently gained a lot of attention and became a well studied field, the molecular processes involved in signal termination remain unclear. Odorant degrading enzymes (ODEs) are thought to be responsible, at least in part, for olfactory signal

10 termination in the chemosensory system by rapid degradation of odorants in the vicinity of the receptors. Until now, this hypothesis has been mainly supported by in vitro results. We have recently shown that the esterase “Juvenile hormone esterase duplication (JHEdup)” might be involved in the physiological dynamics of the response of Drosophila melanogaster to its food odors ethyl butyrate and ethyl propionate. Fluorescent microscopy, using the GAL4-UAS-GFP-system, unveiled that JHEdup is strongly expressed in olfactory tissues like the antennae and maxillary palps in comparison to the remaining body. Applying an electrophysiological approach, we performed Single Sensillum Recordings (SSR) on Ab1 sensilla in JHEdup mutants, lacking the enzyme, and a control strain (same genetic background). The flies of the JHEdup mutant showed an increased response in comparison to the control line towards ethyl butyrate and ethyl propionate. Our findings indicate that JHEdup might be involved in the physiological dynamics of Drosophila’s response to these common food odors and suggest it as a potential ODE.

Daniel Münch, Jennifer S. Ignatious Raja, Tom Laudes, Anja Nissler, C. Giovanni Galizia Neurobiology, University of Konstanz, Germany, [email protected] Olfactory signaling on the Drosophila - Response profiles and individual response dynamics Olfactory sensory systems usually consist of multiple classes of olfactory receptor neurons (ORNs) that are tuned to different but overlapping sets of odorants. Thus, when odors are presented, specific activation patterns arise. Tuning profiles of ORNs range from generalists that are sensitive to many odors, to specialists that respond to only a handful or even single substances. Responses vary in strength as well as in dynamics, e.g. polarity or duration. ORN responses towards mixtures are even more complex. Two substances might interfere, generating a mixture response that is distinct in strength or dynamics from the components response. We recorded response profiles of eight classes of Drosophila ORNs using a set of ~100 odorants. We analyzed the individual response dynamics in detail. For a smaller set of ORNs and odorants we studied responses towards binary mixtures. We found both, generalist- and specialist ORNs in our set. For Or56a neurons which are known to be specifically tuned to geosmin we found several new ligands, even though these were much weaker. Dynamical response-features differed across ORN—odorant combinations: Some generated strong and prolonged responses, others were short-lived (phasic), still others were complex over time, including excitatory and inhibitory bouts. Most binary mixtures tested produced weak or no interactions, though a few combinations lead to suppressive or synergistic mixture responses. Across ORNs, about half of the mixture responses followed one component, while the rest had distinct activity patterns. This is relevant for understanding how odor information is coded in combinatorial activity patterns.

Elena Ian and Bente G. Berg Norwegian University of Science and Technology, Norway, [email protected] New findings on the anatomical arrangement of antennal-lobe tracts in the moth brain Like in other insects, second order olfactory neurons of moths form three main antennal- lobe tracts, the medial, the medio-lateral, and the lateral tract. Previous anatomical studies have shown that these paths terminate mainly in two higher order centers in the ipsilateral protocerebrum, the calyces of the mushroom bodies and the lateral horn (Homberg et al.

11

1988, Cell Tiss Res 254; Rø et al. 2007, J Comp Neurol 500). By performing mass staining experiments including retrograde and anterograde labeling techniques, we here present new anatomical data on the antennal-lobe tracts in the moth brain. The small size of the brain allowed us to achieve whole mount scans that visualize the second order level of the olfactory pathway in its entirety. In addition, the quality of the stainings provided high resolution confocal images uncovering distinct details of the tracts, both in terms of origin in the antennal lobe and projection pattern in the protocerebrum. The findings demonstrate that only a few confined to the lateral tract make a direct link between the antennal lobe and the calyces, meaning that this kind of connection is maintained primarily by the medial tract in the moth. Also, the retrograde staining of the last mentioned tract provided new data concerning arrangement of cell clusters and their connections to specific roots in the antennal lobe. Moreover, in addition to the classical tracts previously described, a new transverse antennal-lobe tract was discovered. Finally, the results revealed a specific organization of antennal lobe axon terminals in the lateral horn, not previously described.

Fotini A. Koutroumpa, Christelle Monsempes, Marie-Christine Francois, Royer Corinne, De Cian Anne, Concordet Jean-Paul, Emmanuelle Jacquin-Joly INRA, UMR iEES-Paris, France, [email protected] Trying ORco KO in Noctuids via the development of CRISPR-Cas9 targeted transgenesis Olfaction in insects requires two classes of olfactory receptors: 1) the odorant receptors (ORs) that function via heterodimerization with a common co-receptor (ORco) and that are involved in /plant odorant detection, and 2) the ionotropic receptors (IRs), known in Drosophila to detect acids coming from organic degradation. The combination of OR and IR expression in insect antennae allows detecting a wide range of odorants. Since Drosophila cannot resume insects, a better understanding of the functioning of these olfactory receptors requires functional tools to be developed in non-model species such as crop pest moths. Loss of-function studies using RNAi are generally difficult to achieve in and heritable transgenesis has been developed on a very limited number of species, namely the silkworm moth Bombyx mori and the monarch butterfly Danaus plexippus. Nowedays, new approaches for targeted transgenesis represent promising tools for non-model species. Here, we report our efforts to develop a new genome editing method based on CRISPR-Cas9 to target ORco knock-out in the noctuid crop pest Spodoptera littoralis. Validation of CRISPR/Cas9 as a heritable transgenesis technique for crop pest Lepidoptera will open unprecedented routes in the understanding of insect olfaction and in insect functional genomics in general.

Christine Missbach, Bill S. Hansson, Ewald Grosse-Wilde MPI for Chemical Ecology, Jena, Germany, [email protected] ORs and OBPs in basal insects The olfactory system is responsible for the detection of a plethora of distinct and behaviouraly relevant chemical stimuli. Over the course of evolution, selection pressure has led to the emergence of extraordinarily diverse gene families in different animal phyla. Insects detect volatiles using olfactory (OR) or ionotropic receptors (IR). While IRs are expressed in chemosensory organs in many Protostomia, the insect specific ORs are speculated to be an adaptation to a terrestrial lifestyle. Connected to OR function is the coreceptor ORCo, which acts as both chaperone and partner in signal transduction. In 12 addition, odorant binding proteins (OBP) are hypothesized to ferry hydrophobic odorants across the sensillum lymph to the receptors. Both ORCo and OBPs are speculated to have coevolved with ORs. We have analysed basal, primarily flightless insects using a variety of methods, including electrophysiology, immunohistochemistry and molecular biology to test these hypotheses and unravel the evolutionary history of the olfactory sense in insects.

Fabio Miazzi, Sabine Kaltofen, Bill S. Hansson, Dieter Wicher Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Jena, Germany, [email protected] Ex vivo functional imaging of olfactory sensory neurons in Drosophila melanogaster Insects smell using two pairs of main olfactory organs, the antennae and the maxillary palps, located in the anterior part of their head. They possess three types of olfactory receptors: Ionotropic Receptors (IRs), Gustatory Receptors (GRs) and Odorant Receptors (ORs). ORs, in particular, are involved in the perception of a plethora of behaviourally relevant organic compounds, like food odours, danger odours (e.g. Geosmin) and . Each olfactory neuron houses a specific odorant receptor (OrX) and a ubiquitous coreceptor (Orco), which is necessary in order to target the OrX to the dendritic membrane and to transduce the olfactory signal. Despite significant progress in the knowledge of the structure of ORs, the signal cascade that leads to olfactory perception and its regulation is still controversial. Studies involving functional imaging and patch-clamp techniques have been so far conducted expressing insect ORs in heterologous systems, due to the difficulty to access native olfactory neurons inside the antennae or to establish cell cultures of these neurons. Here, we present a new preparation that allows exposing olfactory neurons in Drosophila antennae. We show how this technique makes possible to stimulate these cells and to monitor the response of individual olfactory neurons expressing genetically encoded fluorescence indicators using functional imaging techniques. Moreover, coupling imaging analysis with pharmacological experiments it is possible to study olfactory transduction in ex vivo conditions. We tested this technique using both a Ca2+ indicator (GCamP3.0) and a cAMP indicator (Epac1-camps). Using flies expressing GCaMP3.0 in neurons carrying the Or22a receptor, we investigated the role of Calmodulin on the sensitization of Drosophila olfactory sensory neurons, while using Epac1-camps we could show that the stimulation of olfactory receptors induces a cAMP production inside olfactory neurons. These results suggest the effectiveness of our new technique and indicate its potentialities to advance in the study of olfactory transduction mechanisms in insects.

Felipe Borrero-Echeverry, Federica Trona, Sebastien Lebreton, Florian Bilz, Veit Grabe, Paul G Becher, Bill S Hansson, Peter Witzgall, Silke Sachse Swedish University of Agricultural Sciences, Sweden, [email protected]; Max Planck Institute for Chemical Ecology, Jena, Germany Modulation of pheromone attraction and mating in Drosophila Behavioural response to olfactory stimuli change, following mating or feeding, to match physiological internal states. Our behavioural tests with Drosophila melanogaster show that upwind flight attraction to food odour and sex pheromone is modulated according to nutritional state and sexual receptivity.

13

Results: Starvation increases, and feeding reduces the response to food odour (vinegar), in both sexes. Adding sex pheromone cis-vaccenyl acetate (cVA) to vinegar, however, maintains attraction in fed females, while it has no effect in males. This strong synergistic effect of food odour and cVA in fed females is paralleled by the increased receptivity of fed females to male courtship. Functional imaging of the antennal lobe shows that cVA activates the DA1 , and the sexually isomorphic DM2 and VM2 glomeruli, which also respond to vinegar. We show that cVA and food odour interact in these glomeruli, in a gender-specific fashion and that olfactory response in the DA1 and VM2 glomeruli is also modulated by starvation. Conclusion: In Drosophila, gender-specific behavioural responses to male-produced sex pheromone cVA rely on sexually dimorphic, third-order neural circuits. We show that nutritional state in female flies modulates cVA perception in first-order olfactory neurons in Drosophila females. The strong synergism between cVA and vinegar demonstrates that investigations of physiological and behavioural responses to cVA should take habitat or food odours into account. In nature, the flies perceive social and environmental signals together.

Florencia Campetella, Rolf Beutel, Bill S. Hansson and Silke Sachse Max Planck Institute for Chemical Ecology, Jena, Germany, [email protected] Odor-guided behavior in kissing bugs Haematophagous relay on multimodal sensory cues in order to identify and infect their vertebrate host. In the blood sucking bug Rhodnius prolixus, vector of Chagas disease, olfactory information has been proven sufficient for these insects to orientate. However, to date, a limited number of olfactory cues have been tested and their hedonic value (i.e. whether attractive or aversive) is still unknown. Furthermore, how this information is coded at the periphery and higher brain areas remains to be elucidated. Here we intended to unveil how olfactory cues are used in the context of odor-guided behavior. We designed a comprehensive odor panel that was first tested by means electrophysiology. These potential odor cues were later assed in a two-choice Y-maze, where we were able to determine an index of attractiveness or aversion for each of the studied odorants. Given the current lack of specific control methods for these insect vectors, we believe that our findings might contribute to the design of more effective chemical control strategies.

Georg Raiser, C. Giovanni Galizia, Paul Szyszka University of Konstanz, Germany, [email protected] Drosophila Kenyon cell responses to temporally complex odor mixtures Natural odor plumes distribute in spatially complex filaments, and thereby contain much temporal structure when they reach a nose. This temporal structure contains information about the number of different odor sources, as odorants from the same sources fluctuate more synchronously, whereas odorants from spatially separated sources fluctuate more asynchronously. Behavioral experiments have shown that insects can use rapid temporal cues for odor-background segregation. So far, it is not known how insect brains process and extract information from temporal stimulus cues. To address this question we developed an olfactory stimulus delivery device that is able to generate temporally complex odor mixtures with both high temporal precision and reproducibility. We characterize the device with a photoionization-detector and 14 demonstrate that it is capable of generating high bandwidth fluctuations of natural odors. We used this odor delivery device to measure how synchronous and asynchronous mixtures are represented in the olfactory system of Drosophila. Employing calcium imaging of Kenyon cell bodies with single-cell resolution, we found mixture interactions in the responses of Kenyon cells with responses either weaker or stronger than predicted from the individual components alone. In more than 40% of the Kenyon cells, the interactions differed between synchronous and asynchronous binary mixtures. These time sensitive mixture effects could serve as a neural basis for the analytic extraction of individual mixture components, and suggest that Drosophila should also be capable to perform odor-background segregation based on fast temporal odor stimulus structures, as observed in other insects.

Geraldine Wright and Sebastien Kessler Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE1 7RU, GB, [email protected] Sipping from a poisoned chalice: Mechanisms for the detection of plant toxins and pesticides in nectar in bees Insect pollinators like bees have a long-standing mutualism with plants in which they visit flowers to obtain nectar and pollen. This mutualism is complicated by the fact that nectar and pollen are often laced with toxic compounds that plants produce as a means of defence against herbivores. In contrast to insect herbivores, bees have few genes for gustatory receptors, implying that they have poor acuity for the detection of toxins. Here, I will describe the pre-ingestive mechanisms that honeybees and possess for detecting toxins such as pesticides. Using tip recordings from the mouthparts’ sensilla, we have identified neurons that respond with tonic activation during stimulation with toxic substances such as nicotine, but only when the toxins are present at high concentrations (mM). These neurons did not respond to the range of concentrations of pesticides encountered by bees in the field. By examining how the responses of the mouthparts sensilla correlate with the behaviour of bees, we have found that tonic activation of putative ‘bitter’ neurons is not sufficient to enable rejection of food solutions. Thus, we present evidence that the detection and rejection of toxins in food solutions is complex and is likely to involve specific activation of the entire population of gustatory sensilla present on the mouthparts.

Gina Pontes, Marcos Horacio Pereira, Romina B. Barrozo Group of Neuroethology of Insect Vectors, Lab, IBBEA, CONICET-UBA, DBBE, FCEyN, Universidad de Buenos Aires, Buenos Aires, Argentina, email: [email protected]; Laboratório de Insetos Hematófagos, Instituto de Ciencias Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil, email: [email protected] Salts control feeding decisions in a blood-sucking insect Salts are necessary for maintaining homeostatic conditions within the body of all living organisms. Like with all essential nutrients, deficient or excessive ingestion of salts can result in adverse health effects. The taste system is a primary sensory modality that helps animals with adequate feeding decisions in terms of salt consumption. In this work we show that salt concentration controls the feeding behavior in Rhodnius prolixus. Notably, feeding was triggered only by a solution with an optimal salt concentration of NaCl or LiCl or KCl, i.e. 0.1- 15

0.15M and ATP. Not surprisingly, this concentration is isosmotic with the blood plasma of vertebrates. Below or above this narrow range of salinity, the feeding response of bugs was significantly reduced. Additionally, we recorded the electrical activity of the cibarial pump while insects were feeding. Solutions of low (0.6M) salt concentrations elicited different activity patterns of the pump, even if in both cases the insects did not ingested a significant volume of solution. Moreover, at low salt concentrations insects bite a few times and maintained their mouthparts inside the feeder for long periods. On the other hand, at high salt concentrations insects performed numerous bites for short periods. Finally, we found that administration of amiloride (an epithelial sodium channel blocker) reduced the feeding behavior of insects confronted to optimal salt solutions (NaCl or LiCl). Not only they did not feed over appetitive solutions, but they also exhibited a similar pumping pattern to what was observed when low salt concentration solutions were offered. Our results confirm the importance of the taste system for a blood-sucking insect, showing that the salt concentration plays a relevant role on their feeding decisions. Moreover, we described here for the first time the existence of an amiloride-sensitive salt (Na+/Li+) detector involved in salt recognition in these insects. Salts are necessary for maintaining homeostatic conditions within the body of all living organisms. Like with all essential nutrients, deficient or excessive ingestion of salts can result in adverse health effects. The taste system is a primary sensory modality that helps animals with adequate feeding decisions in terms of salt consumption. In this work we show that salt concentration controls the feeding behavior in Rhodnius prolixus. Notably, feeding was triggered only by a solution with an optimal salt concentration of NaCl or LiCl or KCl, i.e. 0.1- 0.15M and ATP. Not surprisingly, this concentration is isosmotic with the blood plasma of vertebrates. Below or above this narrow range of salinity, the feeding response of bugs was significantly reduced. Additionally, we recorded the electrical activity of the cibarial pump while insects were feeding. Solutions of low (0.6M) salt concentrations elicited different activity patterns of the pump, even if in both cases the insects did not ingested a significant volume of solution. Moreover, at low salt concentrations insects bite a few times and maintained their mouthparts inside the feeder for long periods. On the other hand, at high salt concentrations insects performed numerous bites for short periods. Finally, we found that administration of amiloride (an epithelial sodium channel blocker) reduced the feeding behavior of insects confronted to optimal salt solutions (NaCl or LiCl). Not only they did not feed over appetitive solutions, but they also exhibited a similar pumping pattern to what was observed when low salt concentration solutions were offered. Our results confirm the importance of the taste system for a blood-sucking insect, showing that the salt concentration plays a relevant role on their feeding decisions. Moreover, we described here for the first time the existence of an amiloride-sensitive salt (Na+/Li+) detector involved in salt recognition in these insects.

Giorgia Sollai, Maurizio Biolchini, Paolo Solari, Roberto Crnjar University of Cagliari, Dept. Biomedical Sciences, Cagliari, Italy, [email protected] Chemosensory basis for feeding acceptance of two host plants in the larvae of Papilio hospiton Papilio hospiton Géné, endemic of the Sardinian and Corsican islands, is an oligophagous species, using various plants in the Apiaceae and Rutaceae families as host, and larvae do not feed on plants outside of these two families. In Sardinia it uses Ferula communis as an almost exclusive host plant: when this species is unavailable, two other plants are used, 16

Ferula arrigonii and Ruta lamarmorae. Besides, in the Tavolara island, just off the coast of Sardinia, we found that the adult females lay the eggs on Seseli tortuosum, an endemic plant of the island. However, when we tried to raise the larvae on the same plant in captivity they hardly ever grew beyond the first-second instar. In fact, host specificity of lepidopteran insects is determined not only by female oviposition preferences, but also by larval food acceptance. Functional characterization of each GRN housed in both styloconic sensilla showed that the lateral sensillum has two deterrent GRNs (L-lat and M2-lat neurons), one phagostimulant (M1-lat neuron) and one salt neuron (S-lat neuron), while the medial sensillum has two phagostimulant GRNs (L-med and M1-med neurons), one deterrent (M2- med neuron) and one salt neuron (S-med neuron) (Sollai et al., 2014). In addition, we found that the L-lat GRN may act as a “labeled-line” which indicates the presence of toxic compounds (Sollai et al., 2015). In this respect we considered that the larval peripheral taste sensitivity plays an important role in feeding acceptance. Aim of this work was to study the sensitivity of each GRN to saps of F. communis and S. tortuosum and evaluate the discriminating capability between the two saps and which neural code/s is/are used in the peripheral taste system of P. hospiton larvae. The spike activity of the medial and lateral maxillary styloconic taste sensilla of larvae was recorded after stimulation with plant saps, with the aim of comparing the GRN response patterns in the light of the different feeding acceptance. The results show that the spike responses of the GRNs are statistically different. The neurons activated by both plant saps are the same, but L-lat GRN shows a higher activity in response to S. tortuosum with respect to F. communis, while the opposite was observed for the M1-lat and M1-med neurons. Besides, the results suggest that the larvae of P. hospiton may be able to discriminate between the saps of F. communis and S. tortuosum by means of ensemble code.

C. Giovanni Galizia, Georg Raiser, Paul Szyszka, Alja Lüdke University of Konstanz, Universitätsstr. 10, D - 78457 Konstanz, Germany, [email protected] Post-stimulus activity in the olfactory pathway of Drosophila Animals are able to associate immediate positive or negative experiences with stimuli that lie in the past. This time-bridging learning ability requires the existence of prolonged stimulus information (a so called stimulus trace). In trace conditioning experiments, we train animals to associate a conditioned stimulus with a reinforcing stimulus that are separated by a temporal gap. Olfactory trace conditioning has shown that Drosophila and other insects have an odor trace in the olfactory system that lasts for several seconds. However, the neural substrate of this odor trace is still unknown. Searching for this substrate, we investigated whether olfactory information is kept after odor offset in the different stages of the olfactory pathway of Drosophila. We performed in vivo calcium imaging in receptor neuron axons (driver: Orco), projection neurons dendrites and somata (GH146) and Kenyon cell somata (OK107) and measured spatio-temporal odor response patterns in these three consecutive processing stages. Receptor neurons and projection neurons responded to odors with combinatorial response patterns of activated and inhibited glomeruli, as previously described (see http://neuro.uni.kn/DoOR). After odor offset, the activity patterns turned into prolonged post-odor response patterns, which were dissimilar to the initial odor response, but odor specific. These post-odor activity patterns were invariant to changes in stimulus length. Kenyon cell somata also showed odor specific activation patterns during the odor stimulus and prolonged calcium activity after odor offset. However, in contrast to receptor neurons and projection neurons, post-odor responses in several Kenyon cells 17 remained similar to the initial odor response patterns for several seconds. These results show that both the antennal lobe and mushroom body exhibit ongoing odor specific calcium activity, but whether they serve as neural substrate for an odor trace, or how they contribute to it, still remains elusive.

Melanie Marquardt, Kim Heuel, C. Giovanni Galizia, Manfred Ayasse, Stefan Dötterl, Hannah Burger University of Konstanz, Germany, [email protected] Odour responses to host-plant odorants in specialized Andrena bees Specialized (oligolectic) bees rely on a small range of host plants for their pollen collection, in strong contrast to bees like honeybees which forage on a broad and generalized flower spectrum. As compared to generalists, the specialists might have evolved neural adaptations allowing them to effectively locate their specific host flowers. We performed bioassays with specialized Andrena vaga bees to identify the relative importance of visual versus olfactory cues to locate Salix (willows) host plants. To investigate how host odours are processed in their brains, we used calcium imaging experiments and recorded odour-evoked activity patterns in the antennal lobe for the specialist A. vaga and, for comparison, in the generalist honeybee Apis mellifera. We recorded responses to synthetic compounds in serial solutions as well as to volatile compounds of the odour bouquet of Salix flowers that were separated by a gas chromatograph. Our behavioural experiments showed that A. vaga bees orientate mainly on olfactory cues to locate host flowers. In our physiological experiments, A. vaga bees, but not the honeybee A. mellifera, had a particularly high sensitivity for 4- oxoisophorone and 1,4-dimethoxybenzene, two characteristic components of the host- flower odour. Our experiments suggest that A. vaga females show correlates between neural organization and host-plant finding behaviour. Burger H, Ayasse M, Dötterl S, Kreissl S, Galizia CG (2013) Perception of floral volatiles involved in host-plant finding behaviour – comparison of a bee specialist and generalist. J Comp Physiol A 199:751-761

Kaouther Rabhi, Sylvia Anton, Emmanuelle Jacquin-Joly, Christophe Gadenne, Helene Tricoire-Leignel University of Angers-INRA, France, [email protected] Effects of a on nicotinic acetylcholine receptors involved in olfaction In male moths, the behavioural attraction to female-produced sex pheromones relies on a highly sensitive olfactory system, which detects and processes this vital olfactory cue. The neural circuit of olfaction involves cholinergic neurons and therefore cholinergic receptors in postsynaptic areas. These receptors mediating fast neurotransmission are mainly nicotinic acetylcholine receptors (nAChRs), cell-surface complexes of five proteins, i.e. subunits. Because 10 to 12 subunit specific genes are detected in the genome of various insect species, different complexes arise leading to distinct receptor subtypes with different pharmacological and functional properties. Indeed, a lack of a specific subunit can abolish a whole nAChR population, which in turn abolishes a binding site for acetylcholine and/or for an agonist. On the contrary, overexpression of a subunit involved in high affinity binding sites for a specific agonist, can increase the effect of this ligand. Among agonists,

18 as are widely used in pest insect control for crop protection and therefore accumulate at low concentrations in the environment. Recent work showed that previous intoxication with sublethal concentrations of a neonicotinoid insecticide, clothianidin, can modulate nAChR subunit expression in the pea aphid, changing the impact of each nAChR population in the agonist effect. In males of the moth , treatment with a specific low dose of clothianidin increases pheromone detection in males, suggesting a higher sensitivity of the olfactory system, including the antennal lobe, where olfactory input is firstly processed. Here, we investigated the effect of this dose in males of A. ipsilon on the nAChR subunit expression in the antennal lobe, using qPCR. Because the genome of this insect is not yet available, partial sequences of 8 subunits were previously identified from a cDNA bank (E. Jacquin-Joly, personal communication), or through cloning experiments using degenerated primers.

Hiromu Tanimoto Tohoku University Graduate School of Life Sciences, [email protected] circuits and memory formation A biologically relevant event such as finding a food source or being poisoned can drive memory for life with a single encounter. Neuronal mechanisms by which such a strong reward or punishment induces stable memory are poorly understood. A single presentation of a sugar reward to Drosophila activates distinct subsets of dopamine neurons that independently induce short- and long-term olfactory memories. We show that distinct subsets of dopamine neurons signal reward for short- and long-term appetitive memories in Drosophila. The temporal dynamics of memory components triggered by these distinct reward signals are complementary, and together contribute to a temporally stable memory retention. Two subsets of dopamine neurons could signal different reward properties, sweet taste and nutritional value of sugar. Furthermore, we found that a recurrent reward circuit underlies the formation and consolidation of LTM. Sugar reward is thus intricately encoded in the fly brain, given the importance of long-lasting food-related memory in survival. Our results suggest that a reward signal transforms a nascent memory trace into stable LTM using a feedback circuit.

Ian Keesey, Markus Knaden, Nicolas Buchon, Bill S.Hansson Max Planck Institute for Chemical Ecology, Jena, Germany, [email protected] The Sweet Smell of Sickness Flies that have been infected with bacteria have been well studied in terms of their immune system and metabolism, but only recently in regard to the olfactory cues that might indicate infection or disease. Here we show that flies are able to distinguish between healthy and infected adults as well as between the frass or feces from these individuals. Moreover, because the bacteria can survive ingestion, attraction of healthy flies to infected frass results in additional sick adults that can carry the bacteria to the next host plant.

Jakob Krieger, Bill S. Hansson, Steffen Harzsch University of Greifswald; Zoological Institute and Museum; Dept. for Cytology and Evolutionary Biology, Germany, [email protected]

19

Evolution of terrestrial olfaction in representatives of hermit crabs (Anomala) with notes on true crabs (Brachyura) The conquest of land occurred at least seven times convergently within Crustacea. We are interested in terrestrial adaptations of the sensory organs and the central nervous system of different crustacean taxa in comparison to their nearest marine relatives. Our focus is on the behavior, the morphology of central olfactory processing areas as well as on their development. We examined the brains of different species of the true crabs (Brachyura) and hermit crabs (Anomala) by the use of x-ray microscopy and immunohistochemical labelings. Morphological data including basic morphometric analyses are accompanied by behavioral data based on bioassays of selected species, both in the field as well as in the laboratory. Our aim is to improve the knowledge of the brain architecture and the central olfactory pathway as well as a better understanding of different adaptation strategies for conquering land within crustaceans. Our results indicate that terrestrial Brachyura have smaller olfactory processing areas compared to their marine cousins, suggesting that olfaction may play a minor role in the sensory ecology of these animals. Contrary, terrestrial Anomala have inflated their neuronal substrate dedicated to primary olfactory processing which coincides with behavioral observations that suggest proper sense of aerial olfaction in this taxon.

Jan Armida, J.R. Arguello, L. Abuin, R.Benton Center for Integrative Genomics, University of Lausanne, Switzerland, [email protected] Evolution of novel glomerular targeting specificities in Drosophila olfactory sensory neurons How novel neural circuits emerge during evolution is an important but poorly understood process. To gain insights into this question we are studying the Drosophila olfactory circuits that express members of a tandem cluster of olfactory receptor genes – IR75a, IR75b and IR75c – which are relatively recently evolved neural pathways. IR75a, IR75b and IR75c have segregated their expression into three distinct olfactory sensory neuron (OSN) populations that project their axons to different, but adjacent, glomeruli. Here we aim at unraveling the molecular mechanisms underlying these subtle targeting differences to give rise to these three distinct olfactory circuits. To identify molecules involved in IR75a, IR75b and IR75c neuron axon targeting, we have pursued three complementary approaches: high-throughput gene expression analysis of developing and mature antennal tissue by RNASeq, targeted DamID (TaDa) to identify transcriptionally active genes in each OSN population, and manual isolation and transcriptomic analysis of GFP-labeled OSNs expressing different receptor genes. Here we present preliminary results from these methods and show that we can identify putative axon guidance molecules responsible for the differential wiring of IR75a, IR75b and IR75c expressing neurons. We are currently analyzing the most promising candidates by RNAi and classical loss-of-function genetic analysis to confirm their role in circuit wiring. Our results will shed light into the molecular mechanisms underlying the assembly and evolution of neural circuits.

Jan E. Bello, Gabriel Hughes, Matthew D. Ginzel, and Jocelyn G. Millar MPI for Chemical Ecology, Jena, Germany, [email protected] Removing the Mystery from Chiral Methyl-Branched Hydrocarbons as Contact Pheromones

20

Methyl-branched hydrocarbons (MBCHs) are ubiquitous components of insect cuticular lipids. Several have been shown to function as contact pheromones, and it is likely that many more remain to be discovered. The majority of insect produced MBCHs are chiral, but there have been no studies to determine whether they are biosynthesized enantiospecifically. In fact, there have been only a handful of studies on the effects of chirality on the biological activities of MBCH contact pheromones. This is primarily a result of the small to vanishingly small specific rotations of MBCHs (~3º to a tiny fraction of a degree), which in the past made enantiomeric analysis through polarimetry impractical, particularly in light of the small amounts of hydrocarbons (ng to µg) that can be obtained from many insects. The problem was compounded by difficulties in isolation of individual MBCHs from the crude mixture, and the time-consuming synthesis of chiral MBCH standards, both of which have hindered research on MBCH chirality. We will describe the isolation of insect MBCHs from crude extracts, using a combination of simple fractionating techniques and reverse phase HPLC with nonaqueous solvent systems, detecting all compounds with an evaporative light scattering detector. Stereochemical analysis of the isolated MBCHs with a digital polarimeter revealed that the absolute stereochemistry of these insect natural products is conserved through at least nine orders of Insecta. We also present an efficient asymmetric synthesis of chiral MBCH standards. The resulting enantiopure compounds were used to test the effects of chirality of the bioactivity of the contact pheromones of the red- headed ash borer, Neoclytus acuminatus (Cerambycidae).

Jen Beshel and Yi Zhong Cold Spring Harbor laboratory, USA, [email protected] Alterations in Brain-derived Leptin-homolog Unpaired 1 Lead to Obesity Phenotypes in Drosophila through Regulation of Food Odor Value Signaling Motivated feeding results from the interplay of homeostatic and hedonic drives and dysregulation of either may contribute adversely to conditions of overweight and obesity. Recent work demonstrates domeless receptors in Drosophila can be activated by human Leptin, a typically adipose-derived “satiety hormone” with a long-established role in weight regulation. Interestingly, knockdown of endogenous domeless-ligand upd2 in the fat body of flies leads to smaller body size and has no effect on feeding, the opposite behavior of leptin- deficient mammals. While we replicate the lower weight and unchanged food intake in upd2-manipulated flies, we further show manipulations to another endogenous ligand for the domeless receptor, brain-based unpaired 1 (upd1), recapitulate mammalian obesity phenotypes in flies. Flies with reductions in upd1 restricted to neural tissue show increased weights, increased food intake, and increased attraction to food odors. We additionally report domeless receptors likely mediate observed phenotypes. We show behavior-relevant domeless receptors are located on neurons expressing Drosophila Neuropeptide F (dNPF), the Neuropeptide Y (NPY) homolog, in the central brain with targeted receptor knockdown specifically to these cells replicating increased weight, attraction and intake phenotypes. In vivo 2-photon imaging demonstrates upd1 acts as the homeostatic regulator of our previously reported dNPF food odor value signal, up- or down-regulating this hedonic signal as a function of satiety state. Our findings suggest Leptin-NPY and upd1-dNPF represent functionally homologous circuits across diverse species and imply in mammals adipose- and less understood brain-derived Leptin may play different roles in feeding and weight regulation.

21

Kang Ko, Cory Root, Scott Lindsay, Orel Zaninovich, Andrew Shepherd, Steven Wasserman, Susy Kim, Jing Wang University of California - San Diego, USA, [email protected] Starvation Promotes Concerted Modulation of Appetitive Olfactory Behavior via Parallel Neuromodulatory Circuits The internal state of an organism influences its perception of attractive or aversive stimuli and thus promotes adaptive behaviors that increase its likelihood of survival. The mechanisms underlying these perceptual shifts are critical to our understanding of how neural circuits support animal cognition and behavior. Starved flies exhibit enhanced sensitivity to attractive odors and reduced sensitivity to aversive odors. Here, we show that a functional remodeling of the olfactory map is mediated by two parallel neuromodulatory systems that act in opposing directions on olfactory attraction and aversion at the level of the first synapse. Short neuropeptide F (sNPF) sensitizes an antennal lobe glomerulus wired for attraction, while tachykinin (DTK) suppresses activity of a glomerulus wired for aversion. Thus we show parallel neuromodulatory systems functionally reconfigure early olfactory processing to optimize detection of nutrients at the risk of ignoring potentially toxic food resources.

Jocelyn Millar Department of Entomology, University of California, Riverside, USA, [email protected] Chemical conundrums: Insects responding when they shouldn’t There are now numerous examples of insects responding to long-chain and seemingly nonvolatile hydrocarbons and related compounds as volatile pheromone components. The first part of my presentation will discuss recent advances in the analysis, bioassay, and synthesis of these types of compounds, and from there, some unexplained and currently unexplainable results from behavioral bioassays with these types of compounds. In the second part of the presentation, I will focus on another group of seemingly inexplicable results from our work with volatile pheromones of cerambycid beetles. Here, we have several recent cases of:  a species X responding strongly to the pheromones of another species Y, when species X does not produce those compounds,and  a species Z producing large amounts of sex-specific volatiles, for which we have not been able to determine a function. These will be discussed in the context of some very recent identifications of likely cerambycid pheromones, which has considerably expanded the known pheromone repertoire within this large insect family.

Jürgen Rybak1 Giovanni Talarico1,4 Santiago Ruiz2 Christopher Arnold1 Rafael Cantera2,3 Bill S. Hansson1 1 Max Planck Institute for Chemical Ecology, Dept. Evolutionary Neuroethology, Jena, Germany; 2 Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay; 3 Zoology Department, Stockholm University, Stockholm, Sweden; 4 Institute of Legal Medicine, Dept. of Forensic Toxicology, University Medicine Greifswald, Germany Synaptic circuitry of identified neurons in the antennal lobe of Drosophila melanogaster 22

In the antennal lobe (AL), a first relay station of the Drosophila melanogaster brain, olfactory cues are processed in multiple microcircuits comprising olfactory sensory cells, projection neurons, and local interneurones. We have dissected this circuitry by combining confocal microscopy with serial section transmission electron microscopy. Projection neurons (PNs) were labelled with a genetically encoded membrane-bound electron dense marker in order to trace the PN synaptic sites in the VA7, DM2 and DL5 glomerulus. Here, we would like to emphasize on three aspects of sensory and its structural correlates: the spatial segregation of PN input-output sites into different zones of the glomerulus. PNs were predominantly postsynaptic to putative olfactory sensory neurons (OSN), thus sensory convergence, important for high signal-to-noise ratio, and reliable transfer of olfactory signals at the AL output, is mediated by the OSN-to-PN synapse. OSN terminals form conspicuous, enlarged T-bars, with several pedestals, opposed to multiple postsynaptic processes, including PN profiles. PN feedback synapses exhibited T-shaped presynaptic densities, mostly in tetradic constellation, consisting of pedestal and platform and surrounded by clusters of clear vesicles, are distributed non-randomly over the PN dendrites. PNs showed an invariant ratio for the number of input to-output synapses of approximately three-to-one. A second non-labeled cell type (presumably local interneurons, LNs) had a light cytoplasm and two types of vesicles. The PNs thus formed reciprocal microcircuits, i.e. feed-forward- and feedback-synapses with putative OSNs and LNs. The results are discussed with regard to current models of olfactory glomerular microcircuits in the AL of insects and the of vertebrates.

Kathrin Steck, Célia Baltazar, Ana Paula Elias, Gabriela Fioreze, Pavel Itskov, Ricardo Leitão- Gonçalves, Carlos Ribeiro Behavior and Metabolism Laboratory, Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Doca de Pedrouços, 1400-038 Lisboa, Portugal, [email protected] Identification of sensory neurons and receptors that mediate yeast and amino acid feeding in Drosophila melanogaster In most animals olfaction and taste play a major role in the selection of the appropriate food sources needed to satisfy their metabolic requirements. These requirements vary a lot depending on the current needs of the animal. The molecular and neuronal mechanisms the brain uses to detect these specific metabolic requirements and modify the chemosensory input to prioritize among the different available feeding possibilities are currently largely unknown. Despite being key nutrients the identity of the chemosensory channels insects use to decide to ingest amino acid rich food remains to be identified. This is also the case in Drosophila melanogaster where the chemosensory basis for the ingestion of yeast, its main source of amino acids, or pure amino acids remains elusive. We used a combination of two choice feeding assays, internal state manipulations, neuronal silencing approaches and neuroanatomy to identify a subset of chemosensory neurons that is required for ingestion of yeast and amino acid rich food. The identified neurons are part of the gustatory system and label the taste pegs of the labellum and the internal sense organs of the cibarium. We are currently aiming at characterizing the function of the identified neurons using calcium imaging and identifying the receptor genes mediating amino acid feeding. This work thus provides important novel insights into the chemosensory basis of amino acid feeding, one of the elementary gustatory modalities promoting the fitness of the animal. 23

Ke Dong, Peng Xu, Yuzhe Du, Elizabeth Bandason and Kamal Chauhan Michigan State University, USA, [email protected] Pyrethrum and pyrethroid insecticides activate specific olfactory receptors in Drosophila melanogaster Pyrethrum is a botanical insecticide extracted from dry flowers of certain species of Chrysanthemum. Pyrethroid insecticides, synthetic analogues of pyrethrum, are used extensively in controlling arthropod pests and disease vectors due to their low mammalian toxicity and favorable environmental properties. These compounds exert insecticidal activity by modifying the gating of voltage-gated sodium channels. Besides the insecticidal activities, these compounds have been observed to induce behavioral avoidance. In particular, excito- repellency has been documented for mosquitoes in response to pyrethroid-treated bednets for malaria control. Furthermore, volatile pyrethroids are used extensively in mosquito coils, vaporizer mats and emanators to repel mosquitoes. However, the molecular mechanism underlying pyrethroid repellency remains elusive. To understand the mechanism of spatial repellency, we examined olfactory responses of adults of Drosophila melanogaster to these compounds using electroantennogram (EAG) and single sensillum recordings (SSR). Our results show that fly antennae could detect these compounds and specific olfactory neurons/receptors are activated by these compounds in a dose-dependent manner. Our identification of pyrethroid-responsive ORs provides a new paradigm for the study of the role of spatial repellency in pyrethroid-treated bednets and a new platform for screening effective mosquito repellents.

Kevin Cury and Richard Axel Columbia University, USA, [email protected] Chemical Sensing in the Drosophila Uterus Chemical sensing occurs not only outside an organism but inside as well. Internal sensory circuits such as those in the gut for example must detect and induce appropriate response to the presence of nutrients or toxins. The uterus offers another example, whereupon mating, a female is exposed to a number of chemicals contained within the seminal fluid of males. In Drosophila melanogaster, a handful of these chemicals have been identified and their detection by the female has been shown to play key roles in reproduction, including the facilitation of sperm storage and release, as well as the induction of post-mating behavioral programs that favor egg production and deposition over continued receptivity. The sensors or neurons responsible for detecting these signals are largely unknown. Sensory neurons that innervate the uterus have also been shown to express the gustatory receptor Gr43a and to be responsive to fructose. The functional relevance of these cells is also not known. Here we describe experiments aimed at identifying those cells receptive to these various signals through functional imaging experiments. As a follow up, we trace their projections into the abdominal ganglion of the ventral nerve cord where we further attempt to identify second order neurons. Identification of such a neural circuit provides key insight into this specialized communication system that has evolved between mating pairs.

Kristina V. Dylla, C. Giovanni Galizia, Paul Szyszka University of Konstanz, Germany, [email protected] 24

Dynamics and heterogeneity of associative plasticity across different types of dopaminergic neurons during odor-shock conditioning in Drosophila In Drosophila the cellular and molecular correlates of memory formation after odor- punishment conditioning are well understood. Dopaminergic neurons mediate the reinforcement in odor-punishment learning, and odor-punishment memories are encoded in Kenyon cell output synapses. However, the dynamics of neural plasticity during memory acquisition remain elusive. Here, we investigated the dynamics of neural plasticity in the Drosophila mushroom body during multiple-trial odor-shock conditioning. Using confocal laser scanning microscopy we imaged calcium responses in different populations of dopaminergic neurons and Kenyon cells. We monitored the experienced electric shock strength for each individual, allowing correlating shock strength with neural responses and neural plasticity. We took advantage of the trace conditioning paradigm in which the odor and the shock application are separated by a few seconds, allowing analyzing the neural responses to odor and shock separately. We found that odor- and shock-induced calcium responses in dopaminergic neurons and Kenyon cells differed across mushroom body compartments. The experienced shock strength correlated with the calcium response strength in dopaminergic neurons. Repeated stimulation induced a non-associative decrease in the calcium response to odors in both Kenyon cells and dopaminergic neurons, and to shock in dopaminergic neurons. Pairing odor and shock stimuli during conditioning counteracted the non-associative decrease in response strength to the odor stimulus in dopaminergic neurons, but not in Kenyon cells, suggesting an associative plasticity effect in dopaminergic neurons. The strength and the dynamics of associative plasticity differed across different types of dopaminergic neurons. Understanding the dynamics and heterogeneity of associative and non-associative plasticity in dopaminergic neurons and Kenyon cells in the mushroom body may provide critical insight into the neural mechanisms of stimulus-punishment learning in insects.

Larry J. Zwiebel Vanderbilt University, USA, [email protected] The Evolution, Genomics and Molecular Biology of Olfaction in Anopheles gambiae and other Anopheline Mosquitoes The ability to sense and discriminate an almost infinite range of chemical cues is central for several behaviors of mosquito vectors. In particular, olfaction plays a major role in host seeking /preference and oviposition behaviors of blood feeding and non-blood feeding female mosquitoes. This includes non-vector species as well as the principal Afrotropical malaria vector species Anopheles gambiae whose strong preference for human hosts (anthropophily) is largely responsible for its high vectorial capacity. Genomic, RNAseq and functional data will be discussed concerning the identification and characterization of several large super-families of receptors that together make up essential elements of the peripheral chemosensory signal transduction cascades across vector and non-vector mosquitoes. These include conserved and highly divergent odorant receptors (AgORs), variant ionotropic glutamate-like ionotropic receptors (AgIRs) and gustatory receptors (AgGRs) associated with olfactory signaling pathways in An. gambiae and other mosquitoes. We will describe recent studies of the chemosensory apparatus in An. gambiae and other Anopheline mosquitoes relative to host-selection/location, oviposition preference and reproduction as well as detailed functional studies on AgORs that have key implications for the mechanistic elements of signaling as well as provide a role of peripheral signaling in this 25 system. Understanding of the olfactory system of An. gambiae, is informing efforts to develop novel approaches to modulate oviposition and human host (blood meal) preferences that are critical mosquito behaviors that drive vectorial capacity. In addition, progress will also be presented on ongoing efforts that utilize recently developed AgOR agonist/antagonists to design novel repellents and attractants for anti-malarial programs that target chemosensory receptors and the behaviors/processes they control in vector mosquitoes. This work has received generous support from the Bill and Melinda Gates Foundation, the FNIH through the Grand Challenges in Global Health Initiative, NIAID and Vanderbilt University.

Lena van Giesen, Simon G. Sprecher Universite de Fribourg, Switzerland, [email protected] Multimodal stimulus coding in sensory neurons of the Drosophila larvae Chemical signals in the environment are perceived by specialized sensory neurons and their accurate perception is crucial for survival. The Drosophila larvae possesses only a small number of chemosensory neurons to sample the environment but displays a wide repertoire of behaviors related to chemosensation, spanning from simple innate repulsive/attractive responses to complex computations such as learning and memory. Using calcium based activity recording we show, that the larval chemosensory neurons apply a multimodal coding scheme and chemical stimuli might already be integrated context-dependent on the sensory level. Ablation of individual sensory neurons affects behavioral decisions towards individual chemicals and blends. Molecular analysis of this phenomena suggests, that the larva enlarges its chemical perception by using multiple receptors per cell and by applying a combinatorial coding in order to broaden the spectrum of chemical signals that can be perceived with a small number of neurons.

Lothar Baltruschat, P. Ranft, A. Fiala, G. Tavosanis German Center for Neurodegenerative Disease (DZNE), Bonn, Germany, [email protected] Addressing structural and functional plasticity of microglomeruli in the mushroom body calyx in olfactory learning The fly mushroom body (MB) is essential for olfactory associative memory formation and retrieval. In the MB calyx the Kenyon cells (KCs) receive presynaptic input from second-order cholinergic projection neurons (PNs) delivering olfactory information. Together they form distinct synaptic complexes called calycal microglomeruli (MG). Those consist of a single presynaptic PN bouton, enclosed by claw-like postsynaptic sites of several, random KCs. However, whether changes of synaptic strength or connectivity between PNs and KCs play a role in olfactory learning is not understood. Thus, we designed an experimental approach that allows us investigating synaptic plasticity within MGs on a structural as well as a functional level. First, we modified the classic two-odor appetitive learning paradigm and used the pheromone 11-cis-vaccenyl acetate (cVA) to train the flies with a single odor. We found that females and males display long-term memory of this appetitive conditioning at 24 hours after training. Then, to observe potential structural changes correlated with long-term associative memory formation in a subset of MGs, we labeled the presynaptic site of only

26 those PNs responsive to cVA with a specific driver line expressing the fluorescently-tagged presynaptic marker Brp-short. We additionally visualized the postsynaptic compartment of MGs using a GFP-tagged Dα7 subunit of the acetylcholine receptor expressed in most KCs. A high throughput, automated 3D reconstruction method developed in our lab allows analyzing morphological changes precisely in those MGs that receive input from odor- specific PNs during appetitive conditioning. To further investigate functional alterations of the input/output relation between PNs and KCs following learning, we established an in vivo Two-Photon-Microscopy set up. We are thus able to reveal stimulus-evoked neurotransmission in the calyx using calcium indicators during olfactory appetitive conditioning with cVA.

Makoto Hiroi, Tetsuya Tabtata Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan, [email protected] Functional imaging of the third-order olfactory neurons Kenyon cells in the mushroom body of Drosophila Olfactory information in Drosophila is conveyed by projection neurons from olfactory sensory neurons to Kenyon cells (KCs) in the mushroom body (MB). A subset of KCs responds to a given odor molecule, and the combination of these KCs represents a part of the neuronal olfactory code. KCs are also thought to function as coincidence detectors for memory formation, associating odor information with a coincident punishment or reward stimulus. Associative conditioning has been shown to modify KC output in the vertical lobes of MBs containing α/α' branches of KCs, which is revealed by measuring the average Ca2+ levels in the branch of each lobe. In order to asses neuronal dynamics of those neurons, we established an in vivo Ca2+ imaging preparation to monitor plasticity of molecular and physiological properties in the brains, induced by pairing odor and shock under the microscope. Employing two-photon microscopy, we could monitor specific patterns of MB activities to different odors at cellular-resolution. After pairing an odor with electric shock, a part of MB cells showed increased Ca2+ signal to the conditioned odor. To quantitatively describe the population activity patterns recorded from axons of >1000 KCs, we did principal component analysis of the population activity patterns that clearly differentiated the responses to distinct odors. Currently we are analyzing neural plasticity in one of the MB output neurons to reveal the functional connectivity with MB neurons upon conditioning. The functional imaging allows us to analyze temporal and spatial dynamics of many neurons simultaneously. This is an important feature because populations of MB cells, which consist of ~2000 cells, will be almost heterogeneous in function and fate after conditioning. In addition, available fluorescent probes for several molecules (Ca2+, cAMP, PKA etc.) and recent tremendous progress of developing new probes will cover more comprehensive trace of cells physiology.

Mamiko Ozaki, Masaru K. Hojo, Hitomi Mizutani, Yusuke Takeichi, Akino Miyamae Department of Biology, Graduate School of Science, Kobe University, Japan, [email protected] Self-grooming of antennae manipulates nestmate and non-nestmate discrimination in Camponotus japonicus

27

Grooming is a common behavior in animals. It serves function of removing foreign materials and excess amounts of self-secreted materials from the body surfaces. Especially in ants that have developed a sophisticated social communication style using colony-specific mixture of cuticular hydrocarbons (CHCs), it is expected that the self-grooming of antennae have a particular role concerned with the nestmate recognition. When the self-grooming of antennae was limited in Camponotus japonicus, unstructured materials were accumulated on such a self-glooming-limited antennae, unlikely on the normally self-groomed antennae. We hypothesized that the accumulated material includes a large amount of self-secreted CHCs, blend of which were qualitatively the same but quantitatively different from those on the self-groomed antennae. In comparison of the aggressive behavior expression between self-grooming-limited and normally self-groomed workers, we found that the self-grooming- limited workers frequently failed to discriminate conspecific non-nestmates from nestmates, but still exhibited aggressive behavior towards hetero-specifics. For the self-grooming- limited workers, the compatibility-incompatibility boundary, which should be set between nestmates and nonnestmates within conspecifics for the normally self-groomed ants, was moved between conspecifics and hetero-specifics. The CHC sensilla on the self-grooming- limited antennae hardly responded not only to the conspecific nestmate CHCs but also to the non-nestmate CHCs, but all responded to the hetero-specific CHCs. On the other hand, the CHC sensilla on the self-groomed antennae responded to the CHCs of the non-nestmates CHCs. Based on these results, we will discuss how the ants discriminate opponents with their sensory organs.

Maren Reuter, Martin Brill, Martin Strube-Bloss, Wolfgang Rössler University of Würzburg, Biozentrum, Behavioral Physiology and Sociobiology (Zoology II), Am Hubland, 97074 Würzburg, Germany, [email protected] Concentration Coding in the Honeybee Dual Olfactory System The brains of insects provide excellent model systems to investigate the principles of olfactory coding. Their neural components for odor perception are remarkably similar to those in the vertebrate brain. In Honeybees, communication in the hive as well as orientation behavior critically depends on olfactory cues and signals. Many different odors need to be perceived and memorized over a bee’s lifetime. In the honeybee brain, olfactory information is sent via parallel pathways from the antennal lobe (AL) to higher order processing centers in the mushroom body (MB) and the lateral horn (LH). The unique configuration of a dual olfactory pathway in Hymenoptera comprises the medial and lateral antennal lobe tracts (m- and l-ALT). The m-ALT projects to the MB first and then to the LH, whereas the l-ALT runs in the opposite direction. Recent electrophysiological and calcium imaging studies have focused on functional differences between the two pathways leading to partially contradictory conclusions. Thus, the function as well as evolutionary benefits of odor processing via a dual olfactory pathway are still not fully clarified. In the present study we tested whether odor identities and concentrations are coded differently by projection neurons of both AL tracts. Using an established multi-unit electrophysiological recording technique, olfactory processing was monitored simultaneously and with high temporal resolution from multiple projection neurons (PNs) of the two tracts over long time periods (Brill et al. 2014 J Vis Exp). The results show that while different odors evoke different activity profiles, PNs of both tracts seem to code for odor concentrations in a similar way. Supported by the DFG SPP 1392 “Integrative Analysis of Olfaction” (RO 1177/5-1/2).

28

Marianna I. Zhukovskaya, E.S. Novikova Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Russia, [email protected] Threshold and suprathreshold pheromone stimuli in isolated Periplaneta americana males Periplanone B, the main component of American cockroach sex pheromone, when presented in natural or laboratory colony settings elicit noticeable searching and courtship behaviors, such as antennal waving, upwind flight and run toward the odor source, as well as wing-raising. In our experiments a cockroach supplied with threshold dose (10-13 g on the dispenser) of Periplanone B, slightly increased its locomotion, but no other sign of orientation towards pheromone source was noticed. Increasing pheromone load by two orders of magnitude did not change the behavior of a male. Bioassay performed with group of cockroaches shows increased antennation and attraction with 10-13 g of PB and strong attraction with wing-raising behavior at 10-11 g (Seelinger, 1985 and our data). Thus, the luck of any cues from conspecifics and environmental odors suppresses most of pheromone- elicited behaviors. Antennal grooming, significantly enhanced by plant odorants, hexanol and eucalyptol, was not affected by both pheromone doses. Frequency of genital grooming was raised during threshold dose exposure, showing some degree of male arousal. Supported by RFBR grant 1304-00610

Markus Knaden, Roman Huber, Cornelia Bühlmann MPI for Chemical Ecology, Jena, Germany, [email protected] Desert ant navigation Desert ants, Cataglyphis fortis, individually forage for dead arthropods in the Tunisian salt pans. During foraging runs of more than 1.5 km the ants use path integration, and visual as well as nest-derived olfactory cues to return to their nest. Here we describe additional olfactory strategies used by the ants to increase their foraging efficiency.

Marta Scalzotto, Michael Saina, Richard Benton Universite de Lausanne, Switzerland, [email protected] Identification of genes that functionally distinguish the OR and IR olfactory subsystems Many of the major sensory modalities – olfaction, taste, vision, touch, audition – have several submodalities that respond to a specific set of stimuli. The olfactory system of Drosophila melanogaster (and most other insects) is also comprised of two subsystems characterised by a common organisational logic, but distinct anatomical, functional and evolutionary properties. These subsystems are defined by expression of different olfactory receptor families, the Odorant Receptors (ORs) and Ionotropic Receptors (IRs). Beyond these genes, remarkably little is known about the molecular underpinnings of the developmental and functional divergence of these subsystems. To identify novel factors distinguishing these subsystems, we performed a microarray screen for genes differentially expressed in antennae of animals mutant for the proneural genes amos and atonal, which selectively eliminate the OR and IR subsystems, respectively. Analysis of expected expression differences of OR and IR genes confirmed the specificity and sensitivity of the microarray screen of picking up olfactory subsystem-related genes. We are currently performing RNAi screens of the other differentially expressed genes to determine their developmental, 29 structural or functional role in the olfactory subsystems. First, we are performing a morphological screen to identify defects in the different types of sensory structure in the antennae (basiconic and trichoid sensilla for the OR subsystem; coeloconic sensilla, sacculus and arista for the IR subsystem). Subsequently, we plan to perform a functional using single sensilla recordings and/or calcium imaging.

Martin F. Brill & Glenn C. Turner Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, 11724 New York, USA, [email protected] Glomerular latency coding in the olfactory system of the fly Animals need to perceive odors in an efficient and reliable manner. How nervous systems have implemented odor detection and recognition in higher brain areas is still not fully understood. In flies, odors are detected by an array of receptors along the antenna and maxillary palp, which project in a receptor-specific manner to glomeruli in the first processing stage, the antennal lobe (AL). Different odors have different binding affinities and glomeruli are differently innervated by local interneurons. Hence several glomeruli are typically recruited during an odor response, with characteristic spike rates as well as response latencies. These signals are then sent to deeper layers of the circuit via projection neurons (PNs). PNs project to Kenyon Cells (KCs) the principal neurons of the Mushroom Body (MB), an association center involved in learning and memory. The connectivity of PNs to KCs is divergent and allows sparse odor representation in the MB. To shed light on the physiological connectivity of PNs to KCs, we ask the following questions: Are KCs able to read out a latency code from PNs and do KCs have a specific response window for inputs with different response onsets as has been found in the vertebrate olfactory cortex (Haddad et al. 2013 Nat Neurosci)? And is the order of glomerulus activation important to elicit a particular response pattern in the MB, since the order of glomerular activation is likely to be concentration-invariant? To answer these questions we use different manipulations to artificially manipulate odor representations in the AL and examine the impact on odor representations in the KCs via two-photon-imaging. The results will shed light on an alternative coding mechanism that could be a widespread feature of different olfactory systems. This work is funded by the National Institutes of Health (Grant #R01 DC010403-01A1 to G.C.T.).

Martin Paul Nawrot, Rinaldo Betkiewics Computational Systems Neuroscience, Institute for Zoology, University of Cologne, Germany, [email protected] Sparse coding in a spiking neural network model of the insect olfactory pathway In their natural environment, insects sense and evaluate olfactory cues of time-varying composition and concentration. Their olfactory pathways are adapted to the natural stimulus statistics, thus it is not surprising that odor processing is fast. Honeybees, for example, learn to discriminate odors presented for as short as 200 ms (Wright, Carlton & Smith, 2009). The neural odor code emerges within 50ms after stimulus onset and neural representation changes dynamically during and after an odorant is present (Krofczik, Menzel & Nawrot, 2008). We present a comprehensive spiking neural network model of the

30 olfactory pathway that reproduces the spatial and temporal patterns of the odor code in the antennal lobe (AL) and the mushroom body (MB) observed in neurophysiological experiments. The model makes new predictions on the reliability of the odor code and on the subthreshold representation of a prolonged odor trace. Our model relies on two simple and experimentally confirmed mechanisms: (1) uniform lateral inhibition within the AL, and (2) cell intrinsic spike-frequency adaptation. The latter is particularly relevant and prominent in the Kenyon cells (KCs) of the mushroom body (Demmer & Kloppenburg, 2009). Together, both mechanisms robustly regulate the spatial and temporal sparseness in the KC population and they reduce the trial-to-trial variability of the neural ensemble code. Moreover, they provide an explanation for latency differences between projection neurons (PNs) and interneurons in the AL (Meyer, Galizia & Nawrot, 2013) and the broad latency distribution in PNs (Strube-Bloss, Herrera-Valdez & Smith, 2012). Finally, we show how across-odor correlation in the spatial response pattern is reduced from AL to MB. Demmer, Kloppenburg (2009). J Neurophysiol, 102, 1538-1550; Krofczik, Menzel, Nawrot (2008). Frontiers Comp Neurosci 2; Meyer, Galizia, Nawrot (2013). J Neurophysiol 110, 2465- 2474; Strube-Bloss, Herrera-Valdez, Smith (2012). PLoS ONE, e50322; Wright, Carlton, Smith (2009). Behavioral Neuroscience 123, 36

Martin Strube-Bloss Department of Behavioral Physiology & Sociobiology/Theodor-Boveri-Institute of Bioscience/Biocenter University of Würzburg, Germany, martin.strube-bloss@uni- wuerzburg.de Prolonged Computation of Behaviorally Relevant Olfactory Stimuli in the Antennal Lobe and Mushroom Body There are mainly two categories of animal behaviors - one is innate, meaning if confronted with a specific stimulus, animals respond with a stereotypical behavior. The other one is learned, and manifestation of a behavior depends on experience. The latter needs to be flexible, whereas there is broad consent that innate behaviors are hard wired and, therefore, inflexible. However, in both cases the initiating stimulus needs to be separated from irrelevant environmental information by neural networks acting at different processing levels. We focus on the olfactory system of Hymenoptera to analyze and compare odor representation at two processing stages, the antennal lobe (AL), the first order olfactory neuropil receiving input from olfactory sensory neurons located on the antennae, and the mushroom body (MB), a higher order multimodal integration center. Olfactory information into the MB is provided by projection neurons (PN) of the AL. MB output is represented by MB-output neurons (MBON). Comparing the ensemble response of MBONs before and after classical differential conditioning revealed that computation of the reward associated stimulus at the MB level was prolonged and drastically increased (Strube-Bloss et al., 2011). In a specific case, we detected strong and prolonged ensemble responses associated with the separation of a behaviorally relevant stimulus already at the level of the AL. We tested different sesquiterpenoids, one of them was farnesol, and recorded the PN activity in bumblebees (Bombus terrestris). Farnesol has been identified as a key component of the recruitment pheromone in bumblebees. It is released by successful foragers and activates unemployed nest mates by triggering an innate behavior (recruitment). The neural representation of farnesol was unique compared to all other tested odors. Most importantly, the separation of farnesol was prolonged in time reaching half maximum separation more than ~90 ms after the tested “non-relevant” odors. Our results suggest,

31 that neural computations at the level of the MB (learned) as well as at the level of AL (innate) separate behaviorally “relevant” from “non-relevant” information. At both processing levels separation of behaviorally relevant information is prolonged in time suggesting that the computation of behaviorally important information requires more time for neuronal computation. Acknowledgements: Martin Strube-Bloss was supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg; Reference: Strube- Bloss, M.F., Nawrot, M.P., & Menzel, R. (2011) Mushroom body output neurons encode odor-reward associations. J. Neurosci., 31, 3129-3140.

Giorgia Sollai(1), Maurizio Biolchini(1), Roberto Romani(3), Valerio Rossi(2), Roberto Crnjar(1), Gianfranco Anfora(2) (1) University of Cagliari, Dept. of Biomedical Sciences, Cagliari, Italy, [email protected] (2) Fondazione Edmund Mach, Research and Innovation Center, S. Michele a/A (TN), Italy (3) University of Perugia, Dip. di Scienze Agrarie Alimentari e Ambientali, Perugia, Italy Morpho-functional study of the ovipositor sensilla in Drosophila suzukii Drosophila suzukii, an invasive and destructive crop pest that originated in South East Asia, is extremely fond of otherwise undamaged, ripening fruits and lays eggs on fresh fruits, unlike most other Drosophila species which attack only decaying or rotten fruits. It uses a serrated ovipositor to pierce the relatively hard-skin of fruits and lay eggs in them. This saw-like ovipositor represents one key adaptation, but other traits, such as fruit recognition mediated by the olfactory and/or gustatory systems, are also implicated. D. suzukii presents several sensilla housed on the surface of its ovipositor, the functional role of which is still unknown. Aim of this study was to examine the functional significance of these sensilla, by means of a morphological, electrophysiological and behavioural approach. The scanning electron microscopy (SEM) analysis showed that the ovipositor surface in D. suzukii presents three different types of sensilla, among which 10 uniporous sensilla (5 per each valve). The electrophysiological recordings from these sensilla displayed that they are sensitive to different sugars (sucrose, glucose, fructose), bitter compounds (caffeine, nicotine) and ascorbic acid. Finally, behavioural trials indicate that the number of eggs laid on the sugar substrates was significantly different from those on bitter or acid substrates. In conclusion, morphological, electrophysiological and behavioral results suggest for the first time, in a Drosophila species, a chemosensory role for the ovipositor sensilla and their possible involvement in the choice of the oviposition sites.

Michiyo Kinoshita, Mina Yoshida, Yuki Itoh, Hisashi Omura, Basil el Jundi, Uwe Homberg, Kentaro Arikawa SOKENDAI (The Graduate University for Advanced Studies), Shonan Village, Hayama 240- 0193, Japan, [email protected] Plant scents modify innate colour preference in foraging swallowtail butterflies Flower visiting insects exhibit innate preferences to particular colours and scents. Here we demonstrate that the innate colour preference of the Japanese yellow swallowtail butterfly 32

(Papilio xuthus) is modified by olfactory stimuli in a sexually dimorphic manner. When naive P. xuthus were presented with four coloured discs (blue, green, yellow, and red), the majority of individuals first landed on and attempted to feed from the blue one. When the scents of either orange flower or lily essential oils were introduced to the room, females’ tendency to select the red disc increased. The odours of lavender and flowering potted Hibiscus rosa-sinensis, however, were less effective. Interestingly, the odour of the non- flowering larval host plant shifted the preference to green in females. In males however, all plant scents were less effective, such that blue was always the most favoured colour. These observations indicate that interactions between visual and olfactory cues play a more prominent role in females. To understand the neural basis of this behavioural phenomenon, we first initiated a neuroanatomical study focusing on the first olfactory ganglion, the antennal lobe (AL). The Papilio AL is composed of about 60 glomeruli. Three glomeruli in the ventrolateral AL are enlarged in females; this sexual dimorphism may be related to the sexual difference in behavior. We also investigated where in the brain visual and olfactory information may be integrated. Local dye injection into the optic lobe (OL) and the AL revealed that outputs of both the OL and the AL innervate the calyces of the mushroom bodies, suggesting that the mushroom body might integrate visual and olfactory signals in Papilio.

Monika Zielonka and Jürgen Krieger University of Hohenheim, Institute of Physiology, 70599 Stuttgart, Germany, [email protected] Sex pheromone detection in larvae of Heliothis virescens In many moth species mate finding depends on female-released sex pheromone blends and the sensitive and accurate detection of the components by the males. In the noctuid moth H. virescens females release a blend containing (Z)-11-hexadecenal (Z11-16:Ald) as the major and (Z)-9-tetradecenal (Z9-14:Ald) as principle minor sex pheromone components. Previously, we showed that the detection of the major component involves three molecular elements: (i) the pheromone receptor HR13 that is expressed by olfactory sensory neurons (OSNs) projecting their dendrites into hair-like sensilla trichodea (type A); (ii), the pheromone binding protein type 2 (PBP2) supposed to capture pheromone molecules from the air and deliver them to the pheromone receptor in the dendritic membrane; and (iii), the so-called “sensory neuron membrane protein” (SNMP1) possibly operating as docking site for PBP2/pheromone complexes and/or contributing to the release of pheromones to the pheromone receptor. Similarly, HR6 was found coexpressed with SNMP1 and identified as the receptor for Z9-14:Ald that is detected by OSNs in male type B trichoid sensilla. Interestingly, a recent study has suggested an unexpected role of female main sex pheromone component in food search of Spodoptera littoralis larvae. It was found that larvae are preferentially attracted to a food source containing female sex pheromone and can detect pheromone components by OSNs on the larval antenna. However, expression of the respective pheromone receptor was not analyzed. Here we investigated whether larvae of H. virescens may detect the major and minor components of the female sex pheromone and whether this involves the pheromone receptors, binding proteins and SNMP that are used in adult moths. Toward this goal we assessed the expression of HR6, HR13, SNMP1 as well as for PBP1 and PBP2 in RT-PCR experiments on larval antenna. Furthermore, whole mount fluorescence in situ hybridization (WM-FISH) and whole mount fluorescent immunohistochemistry (WM-FIHC) were performed to visualize the cells expressing the olfactory detection elements in sensilla on the larval antenna. 33

This work was supported by the Deutsche Forschungsgemeinschaft, SPP1392.

Nicola K. Simcock, Luisa A. Wakeling, Dianne Ford & Geraldine A. Wright Newcastle University, GB, [email protected] Honeybee gustatory receptors genes: what they lack in number they make up in coverage and plasticity. Locating nutrients, in addition to detecting and avoiding toxins in food is essential for all animals. For the foraging honeybee (Apis mellifera) this ability is not only vital to the individual, but to the colony as a whole. Gustation, mediated via gustatory receptors (Grs), is the chemical sense that aids this process, but interestingly, the honeybee possesses one of the smallest gustatory gene repertoires of any insect annotated to date. Additionally, as bees age their role within the hive shifts and the transition from newly emerged to foraging adults is accompanied by a number of metabolic, physiological and behavioural changes. This task-differentiation links strongly with nutrition and gustatory sensitivity and unsurprisingly, demands upon the bee gustatory system vary throughout their lifetime. In the present work we used quantitative reverse transcription (qRT)-PCR to analyze the expression of the 10 honeybee Gr genes across the bodies of newly emerged and forager bees. We find that Gr gene expression is widespread, with all detectable genes discovered in all gustatory tissues, including the brain and guts. This is the first example of internal Gr gene expression in the honeybee and may suggest a role in nutrient regulation and feeding regulation. Additionally, expressions of some Gr genes were found to differ between the two age groups, notably, a greater expression of all genes in the forager brains compared to the newly emerged brains. This result may demonstrate plasticity within the gustatory system that aids the roles of individual bees within the hive.

M Karageorgi, L Bräcker, B Prudhomme, and Nicolas Gompel Ludwig-Maximilians-Universität, München, Germany, [email protected] The evolution of sensory preferences between Drosophila suzukii and close relative The environment chosen by a female insect for the development of her progeny is a key determinant of reproductive success. Likely driven by interspecies competition, shifts in breeding site selection is not uncommon among closely related insect species. How this translates at the genetic and neuronal levels is generally not well understood. In an endeavor to tackle this problem, we are characterizing the behavioral and sensory basis of such a shift, by looking at the oviposition site selection of different Drosophila species. The recent pest Drosophila suzukii is known to target ripening fruits still attached to their plant, while the two closely related species D. biarmipes and D. melanogaster are mostly targeting rotting fruits for egg-laying. Focussing on this behavior in a comparative framework, we have characterized the preferences of each species in relationship to fruit maturity. In particular, we have determined which sensory modalities mattered to this choice, by reducing the complexity of the substrates we offered to the flies. The evolution of this behavior entails changes in the perception of substrate taste and odor, as well as substrate hardness.

Nicolas Montagne, Arthur de Fouchier, William B. Walker, Xiao Sun, Stefania Robakiewicz, Christelle Monsempes, Mattias C. Larsson, Emmanuelle Jacquin-Joly

34

Institute of Ecology and Environmental Sciences (iEES-Paris), Université Pierre et Marie Curie & INRA, France, [email protected] Receptors for sex pheromone components in the pest moth Spodoptera littoralis Moth sex pheromone communication is recognized as a long-standing model in insect biology and a widespread knowledge has been accumulated on this subject. In those nocturnal insects, mate finding strongly rely on the detection by the male of several components of the pheromone blend emitted by calling females. This detection is ensured by a sub-family of olfactory receptors called pheromone receptors (PRs). In the noctuid moth Spodoptera littoralis, the sex pheromone is composed of a large number of 14-carbon acetates but for now, the receptor for only one of these compounds could be identified. Here, we will present recent advances in the identification and functional characterization of PRs in this species. In the course of characterizing the function of previously and newly identified candidate PRs, we expressed them in a specific population of Drosophila olfactory sensory neurons and stimulated them with a panel of 26 closely related pheromone compounds, in order to investigate in depth their response specificity. We notably found that two PRs detect the same minor component of the female pheromone blend, albeit with different specificity and sensitivity. Interestingly, the expression of one of these two PRs is restricted to the distal part of male antennae, where we could identify a novel functional class of pheromone-sensitive neurons whose response spectrum matches that of the heterologously expressed PR.

Olivia Schwarz, Xinyu Liu, Jan Pielage FMI, Friedrich Miescher Institute for Biomedical Research, Switzerland, [email protected] Identification of the Neuronal Circuitry Controlling Drosophila Taste Behavior The sense of taste is essential for the survival of most animals as it enables the discrimination between nutritious and harmful substances prior to ingestion. Here, we are using the gustatory system as a model to study the principles underlying the development and function of a simple sensory-motor circuit. In adult Drosophila, attractive and aversive substances are detected by gustatory sensory neurons that relay taste information to the primary gustatory center, the subesophageal ganglion (SEG). Sweet stimuli evoke a robust and highly stereotypic motor behavior, the extension of the proboscis towards the food source. This behavior can be assessed in a non-invasive manner to test for the integrity of the circuit. So far, the neuronal ensembles forming the sensory-motor circuitry within the SEG are largely unknown. We performed a combined behavioral and morphological screen to identify genetic control elements allowing the selective manipulation of individual neurons within the taste circuit. Here, we identified and characterized motoneurons and upstream regulatory interneurons that are necessary and sufficient for the execution of the motor program. The combination of opto- and thermogenetic tools with classical genetic approaches will allow us to unravel the principles underlying information processing, integration and computation of the taste circuitry.

Paromita Saha, Aniruddha Mitra, Maximilian Choulideer, Anindita Bhadra and Raghavendra Gadagkar Centre for Ecological Sciences, Indian Institute of Science, Bangalore, India, [email protected] 35

Chemical communication in a social wasp: difference in composition does not guarantee recognition A typical colony of Indian social wasp Ropalidia marginata has a single queen who maintains reproductive monopoly through a pheromone produced by her Dufour’s gland. Upon experimental removal of this queen, one of the workers (Potential queen, PQ) becomes extremely aggressive and takes over as the next queen of the colony. However, this PQ immediately drops her aggression if the queen is returned. Our previous study has shown PQ drops her aggression also upon exposure to queen’s Dufour’s gland macerate, suggesting that the queen’s Dufour’s gland content mimicked the queen herself. Dufour’s gland of hymenopteran insects are known to synthesize hydrocarbons which are primarily used to prevent desiccation, and their role in communication is thought to be secondarily evolved. Interestingly, Dufour’s gland chemical profiles are adequate to statistically classify queens and workers as well as individuals from different colonies with 100% accuracy. Therefore, we argued that the Dufour’s gland macerate of a queen would signal both her fertility and colony identity. We tested this prediction by exposing PQ to Dufour’s gland of a foreign queen. As we already know that queens are attacked and not accepted when introduced to foreign colonies, PQs are not expected to drop aggression in presence of a foreign queen. Contrary to our expectation, PQs dropped their aggression in response to both own and foreign queens’ Dufour’s gland macerate. This suggests that only the queen signal is perceived universally across colonies and the Dufour’s gland profiles are not used to infer nestmateship. Hence, this study concludes that the ability to statistically discriminate organisms using their chemical profiles does not necessarily imply that the organisms themselves are evolved to make such discrimination.

Patrick Lhomme, David Carrasco, Mattias Larsson Mattias, Bill S. Hansson and Peter Anderson Swedish University of Agricultural Sciences, Sweden, [email protected] Effect of larval experience to plant olfactory cues on host plant choice in a polyphagous moth In the polyphagous moth Spodoptera littoralis, host plant choice is based on a stable plant preference hierarchy that can be modulated by larval host experience. This plant preference plasticity could facilitate fast adaptation to changing environments. However, larval experience of a suboptimal host plant does not affect subsequent female host plant choice. This plasticity thus appears to be selective and associated with an ability of larvae to evaluate host plant quality. In this context, the aim of this work was to explore the mechanisms underlying this experienced-based behavioral plasticity. We first tested if experience to plant olfactory cues during larval stage mediates behavioral decision-making in subsequent adults. Our results showed that female plant preference was affected by preimaginal olfactory experience. We then tested if food quality could mitigate the transfer of larval olfactory experience. For that we exposed groups of larvae to different plant odors and fed them simultaneously with different qualities of artificial diet (normal diet, low protein diet or aversive diet). Our results show that larvae are able to associate plant odors with the diet quality. They are attracted to plant odors associated with good quality diet and avoid the same odors when associated with poor quality diet. As expected, the positive larval experiences affect female plant preference but interestingly the larval negative experiences do not. Females reared as larvae on poor quality diets do not avoid the associated plants but instead seem to rely on their innate plant preference.

36

These results shed new light on the mechanisms associated with the phenotypic plasticity involved in oviposition decisions.

Peter Anderson Chemical Ecology, Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Box 102, Alnarp, Sweden, [email protected] Experience changes host plant preferences in a polyphagous moth Selection of a suitable host plant is crucial for herbivorous insects and may require the insect to handle and integrate information from many potential host plants. Earlier experiences to plant cues could facilitate host plant choice decisions in complex environments by focusing search behaviour and reducing effects of neural limitations. We have studied experience- driven phenotypic plasticity in host plant choice in the polyphagous moth Spodoptera littoralis. Effects of experience on host plant were studied in female oviposition experiments and male attraction to sex pheromone with a background of plant odour. We found that the adult moth host plant choice was guided by a stable plant preference hierarchy, which can be modified by larval experience. The larval rearing host plant was in general elevated to the most preferred plant for the adult. Exposure to plant volatiles during the larval feeding on artificial diet was sufficient to change adult preferences and an association between food quality and the response to experienced odours was found. Furthermore we have demonstrated that mating experience on a plant modulates plant preference in subsequent reproductive behaviours, whereas exposure to the plant alone or plant together with sex pheromone does not affect this preference. When exposing individuals to both larval feeding and mating experience experiences, we found that both experiences modulate host plant preference. Our results show that S. littoralis change their host plant preferences based on experience to plant cues during specific behavioural events. They also show that different experiences may interact to modulate host plant choice behaviours.

Peter Christ, Anna Reifenrath, Sharon Hill, Frank Hauser, Joachim Schachtner, Rickard Ignell Chemical Ecology, Swedish University of Agricultural Sciences, 23053 Alnarp, Sweden, [email protected] Neuropeptide Signature of Blood-feeding Behavior in Aedes aegypti Mosquitoes transmit a wide range of viral and parasitic diseases. Two significant threats for human health are the viral-borne diseases Yellow fever and Dengue fever that are transmitted by Aedes aegypti. Female Ae. aegypti display a repertoire of stereotypic odor- mediated behaviours, each dependent on the physiological state of the insect. The aim of the research in our group is to understand the mechanisms underlying these behavioral changes. Neuropeptides are the largest and most diverse class of , with 22 known genes in Ae. aegypti, which thereby provide a huge toolbox for the regulation of physiology and behavior. Due to the nature of neuropeptides, which undergo significant post-translational modifications, transcript-based methods cannot be used to analyze changes in neuropeptide concentration in a specific tissue and correlate this to a switch in behavior. To overcome this problem, we established a semi-quantitative mass spectrometry method. In the study presented here we used this method to analyze changes in the concentration of neuropeptides belonging to five distinct neuropeptide families (tachykinin, short neuropeptide F, allatostatin-A, neuropeptide-like precursor and SIFamide) expressed in the antennal lobe in response to feeding. Female mosquitoes were blood-fed six days after 37 adult emergence and the concentration of neuropeptides was compared between blood-fed and sugar-fed animals 1h, 24h, 48h and 72h after a blood meal, as well as 24h after oviposition. We show a significant decrease in the concentration of short neuropeptide F, allatostatin-A and neuropeptide-like precursor in the antennal lobes of blood-fed mosquitoes at 24h and 48h post blood meal. This corresponds to a period of inhibition of the host-seeking behavior of Ae. aegypti. Ongoing behavioral studies are aimed at elucidating the significance of these neuropeptides in regulating odor-mediated behavior.

Peter Witzgall, Francisco Gonzalez, William Walker, Maria Sousa, Marie Bengtsson, Alan Knight SLU, Alnarp, Sweden, [email protected] New host-finding paradigm in insect herbivores Male moths use highly sensitive olfactory neurons, expressing compound-specific receptors, for sexual communication. Our knowledge of sex pheromones is derived from chemical analysis of female pheromone glands, followed by electrophysiological and behavioural screenings, followed by more recent molecular work. Chemical analysis is facilitated by production of only a few, characteristic volatile chemicals in female glands. Electrophysiological recordings from the entire antenna largely reflect responses of pheromone-sensitive neurons, and single-sensillum from sensilla trichodea housing these neurones are technically feasible. Last but not least, pheromones immediately trigger a characteristic behavioural reaction in males, upwind flight behaviour can readily be investigated in the lab. In contrast, the female response to host plant kairomones - thought to stimulate host orientation and oviposition - is slower and behavioural work is therefore very time- consuming. Absence and presence of background volatiles in the lab and field is a serious technical difficulty. Therefore, adult food and oviposition cues have been confounded, and attractants have been described which obviously lack behavioural or ecological relevance. Single sensillum recordings from kairomone-sensitive neurons are technically intricate. Plants produce many volatiles and there is obviously no correlation between the amounts produced and behavioural relevance, especially since current insect-plant associations may have been established only recently, in agricultural environments. The most abundant compounds in plant headspace, which are often found in many host and non-host plants, will invariably produce electrophyisological responses from entire moth antennae, which do not necessarily reflect behaviorally activity. Probably only few kairomones have been identified thus far. What if the female antennae is as sensitive as the male antenna, capable of detecting key compounds in subnanogram amounts against the odour background, and if the compounds, which reliably signal host plants to the ovipositing female, are plant species-specific and produced in very small amounts?

Philippe Lucas, Vincent Jacob, Christelle Monsempes, Jean-Pierre Rospars, Jean-Baptiste Masson Institute of Ecology and Environmental Sciences - Paris; INRA, route de Saint Cyr, 78026 Versailles Cedex, France, [email protected] Olfactory coding in the turbulent realm

38

Insects, such as male moths attracted to the female-emitted pheromone, perform efficient olfactory searches in their environments. Successful searches can start at distances as far as several hundred meters. At these scales olfactory cue transport is dominated by turbulence. The most direct consequence of turbulent transport is the intermittency of the olfactory signal. The time course of the pheromone signal matters, in addition to its quality and intensity, as insects perform better with discontinuous odor stimuli than in steady, uniform plume. Moreover, olfactory searching moths face the problem of long periods without detection and long detection times, especially at large distances from the odor source. The signal of odor plume is known to be intermittent, yet quantitative analysis of olfactory coding in turbulent stream and at large distances from the source is lacking. To investigate the coding of turbulent plumes, we built a stimulator able to deliver any sequence of time- varying olfactory stimuli. We recorded the responses of olfactory receptor neurons (ORNs) and antennal lobe (AL, the primary olfactory centre) neurons to sequences of pheromone puffs with white-noise statistics and with turbulent statistics calculated at different distances from the odor source. We fitted a Linear Non-Linear model to the responses to white-noise stimuli to characterize the dynamic coding properties of ORNs and AL neurons. We then used the model to predict responses to turbulent stimulations at various concentrations and compared it with experimental responses. We show that the performance of olfactory coding varies with the stimulus properties (concentration and temporal dynamic) so that the neuronal coding of the stimulus dynamics decreases with the distance to the odor source. The better characterization of onset and offset of odor puffs at the AL level likely contributes to the efficient insect odor-search behavior at long distances.

Qike Wang, Jason Q.D. Goodger, Ian E. Woodrow, Mark A. Elgar University of Melbourne, Australia, [email protected] Ants use antennae to both convey and receive social signals It is widely accepted that the antennae are the key sensory organs that receive chemical signals in ants and other insects. Social insects use their antennae to distinguish between nestmates and non-nestmates by detecting differences among individuals in the composition of cuticular hydrocarbons (CHCs). Despite their crucial significance in maintaining the social integrity of the colony, the exact source and identity of CHCs that act as nest-specific identification signals remain largely unknown. Typically, studies that identify CHC signals in ants and other insects use organic solvents to extract a single sample from the entire animal. We take a novel approach by first identifying CHC profiles from different body parts of ants (Iridomyrmex purpureus), then use behavioural bioassays to reveal which body parts receive the most attention from workers, and finally establish the location of these recognition signals. CHC profiles varied between different body parts, and CHCs located on the antennae reveal nestmate identity. Workers paid more attention to the antennae of non-nestmates and to the legs of nestmates. Workers responded less aggressively to non- nestmates if the CHCs on the antennae of their opponents were removed with a solvent. This remarkable finding shows that antennae both convey and receive social signals. Our approach and findings could be valuably applied to chemical signalling in other behavioural contexts, and provide insights that were otherwise obscured by including chemicals that either have no signal function or may be used in other contexts.

Raquel Teixeira-Sousa and Geraldine Wright

39

Institute of Neuroscience, Medical School, Newcastle University, UK; Centre for Behaviour and Evolution, School of Biology, Newcastle University, UK, Graduate Program in Basic and Applied Biology (GABBA), Universidade do Porto, Portugal, [email protected] A preference for salt? Studying the regulation of the intake of micronutrients by adult worker honey bees (Apis mellifera). Nutrition is the selection process that enables animals to obtain macronutrients and micronutrients required for survival and optimal performance. Unlike macronutrients, micronutrients, such as mineral salts, are required in tiny amounts to make up most of body functions. These nutrients are known to be limiting for phytophagous insects and, thus ingesting the mineral optimal requirements may be challenging. The mechanisms involved in the regulation of micronutrients have been rarely studied to date in insects, mainly for social insects. Here, we studied how adult worker honey bees regulate their intake of several mineral salts and dietary metals (Na, Cu, Fe, Ca, K, Mg and Zn). To assess salt preferences and regulation, we used choice assays (no salt vs. salt diets) in which different micronutrient concentrations were added individually to basal diets: 1M sucrose solutions or to 1M sucrose solutions containing the 10 essential amino acids. Cohorts of 30 newly emerged bees were randomly assigned to acrylic ventilated cages with different salt treatment diets and kept at 34°C. We recorded daily food consumption and survival over 6 consecutive days. We found clear evidence for the pre-ingestive regulation of most tested salts except for Na and Cu salts. We predict that regulation is mainly by taste (pre-ingestively) and secondarily by post-ingestive regulation of intake of food. Dietary context also plays a role in the ability of honey bees to regulate their intake of micronutrients. Adult honey bees maintained in lab conditions showed different sensitivities and preferences across tested dietary metals. Furthermore, the average amount of mineral salts present in pollen is likely to be associated with less sensitivity and total food ingestion. Further behaviour and physiological experiments will be conducted in order to assess feeding responses to different salts and whether taste neurons are tuned to specific dietary metals in a nectar-like diet.

Ricarda Scheiner, Anne Steinbach, Henrike Scholz University of Wuerzburg, Germany, [email protected] The role of in insect sweet taste and taste-related learning behaviors In insects like honey bees and fruit flies, appetitive learning performance strongly depends on individual taste. In different appetitive learning assays relying on sucrose as a reward, honey bees with high responsiveness to sucrose perform better than those with lower responsiveness. Habituation to gustatory stimuli also correlates with responsiveness to these stimuli both in fruit flies and bees. Octopamine is an important insect and has long been assumed to mediate the rewarding function in appetitive associative learning in honey bees. We here investigated the role of octopamine signaling in regulating sweet taste and taste-related appetitive learning performance in honey bees and fruit flies. Our data provide strong evidence for octopaminergic modulation of sweet taste in both insect species. They suggest that octopamine receptor gene expression and octopamine titers affect appetitive learning performance through modulation of sweet taste. In honey bees, mRNA expression of the Ca2+-coupled octopamine receptor AmOCT-R1 correlates with age-dependent differences in sweet taste and appetitive learning performance. In fruit flies, octopamine is required for normal sweet taste and habituation. Loss of octopamine results in lower responsiveness to sucrose and faster habituation, while restoring octopamine levels 40 by pharmacological treatment or by using the UAS/GAL4 system restores sweet taste and learning performance. Our results suggest an important and conserved role of specific octopamine receptors and octopamine signaling in the vicinity of the subesophageal ganglion in the regulation of sweet taste and taste-related learning performance of insects.

Adria Le Boeuf, Colin Brent, Laurent Keller, Richard Benton Center for Integrative Genomics, University of Lausanne, Switzerland, [email protected] Chemical communication by trophallaxis in ants Trophallaxis is an unusual behaviour of social insect species that permits mouth-to-mouth liquid transfer between members of a colony. Although it is widely considered to be a simple food-sharing mechanism, we hypothesised that trophallaxis also offers insects a means of private communication. To test this idea, we have analysed the chemical components of trophallaxis fluid in the Florida carpenter ant, Camponotus floridanus. Using nano-liquid and gas-chromatography mass spectrometry, we have identified a number of endogenous ant proteins implicated in regulation of juvenile hormone (JH) levels, as well as JH itself. With a radioactive JH tracer, we have detected the passage of JH between individuals and from adults to larvae. We are currently testing the effect of JH on larval fate by providing colonies food supplemented with JH. Our initial results suggest that trophallaxis-based JH transfer from adults to larvae may contribute to brood development and caste determination.

Romina B. Barrozo, Gina Pontes, Agustina Cano, Sebastian Minoli, Santiago Masagué, Laura Gutierrez CONICET and University of Buenos Aires, Argentina, [email protected] Tasting the world by a blood-sucking insect The ability to discriminate between nutritive and harmful food is essential for animals’ survival. Though olfaction contributes to find a potential food source from a distance, the taste sense works as a final control system driving food acceptance or rejection. Triatomines are blood-sucking insects, vectors of Chagas Disease in Latin America. As soon as they reach a potential vertebrate host, they walk over their skin searching for an adequate site to pierce. Then, they take a first sampling gorge to decide if food is acceptable or not. Our work shows that in triatomines, feeding can be inhibited during the gustatory assessment of both, the surface to bite or the first gorge of food. Insects can detect bitter compounds and high salt concentration and both processes can prevent them from feeding. Morphological inspections along with electrophysiological recordings confirm that the sensory organs involved in taste detection are located in the antenna and in the pharynx. These two independent sensory stages work with different thresholds of response: internal sensors are by far more sensitive to detect aversive compounds than external ones. Additionally, applying a multiapproach strategy, we study whether bugs are able to discriminate among aversive stimuli, or alternatively if they are simply indistinguishable negative input signals that induce feeding inhibition. On the one hand we target on the putative salt receptors studying the transduction pathway possibly involved in the aversive detection. On the other hand, we use a cognitive approach to gain more insights on the ability of insects to behaviorally discriminate among aversive taste modalities. We also study the effects of previous gustatory experiences on feeding. Our results highlight the relevance

41 of taste perception of aversive compounds in modulating the feeding behavior in a blood- sucking insect and the importance of previous experience in the final feeding decision.

Ryosuke Yagi, Nobuaki Tanaka Hokkaido University, Japan, [email protected] The brain regions connected with multiple primary sensory centers in Drosophila Integration of multimodal sensory information such as olfactory, gustatory, and visual information is essential for animal life. However, its processing mechanism still remains unclear. Drosophila melanogaster is a suitable model to study this problem due to abundant genetic approaches combined with physiological methods. To first identify the brain regions, which are connected with multiple primary sensory centers in Drosophila, we injected tracers into the primary sensory centers: olfactory antennal lobe, gustatory subesophageal ganglion, or visual optic lobe. We found that the fibers from these centers converged onto the same region within the accessory calyx of the mushroom body and posteriorlateral protocerebrum. In some insects, such as the honeybee, the mushroom body calyx received putative olfactory, visual, and gustatory inputs. Our results indicate that the mushroom body in Drosophila receives the multimodal sensory inputs as well as the other insects.

David Owald, Johannes Felsenberg, Clifford Talbot, Gaurav Das, Emmanuel Perisse, Wolf Huetteroth, Scott Waddell Centre for Neural Circuits & Behaviour, University of Oxford, GB, [email protected] Activity of defined mushroom body output neurons underlies learned olfactory behavior in Drosophila During olfactory learning in fruit flies dopaminergic neurons assign value to odor representations in the mushroom body Kenyon cells. Here we identify a class of downstream glutamatergic mushroom body output neurons (MBON) called M4/6, or MBON-β2 β´2a, MBON-β´2mp and MBON-γ5β´2a, whose dendritic fields overlap with dopaminergic neuron projections in the tips of the β, β´ and γ lobes. This anatomy and their odor-tuning suggests that M4/6 neurons pool odor-driven Kenyon cell synaptic outputs. Like that of mushroom body neurons, M4/6 output is required for expression of appetitive and aversive memory performance. Moreover, appetitive and aversive olfactory conditioning bi-directionally alters the relative odor-drive of M4β´ neurons (MBON-β´2mp). Direct block of M4/6 neurons in naïve flies mimics appetitive conditioning, being sufficient to convert odor-driven avoidance into approach, while optogenetically activating these neurons induces avoidance behavior. We therefore propose that drive to the M4/6 neurons reflects odor-directed behavioral choice.

Veit Grabe, Amelie Baschwitz, Bill S. Hansson, and Silke Sachse Max Planck Institute for Chemical Ecology, Hans-Knoell-Strasse 8,D-07745 Jena, Germany, [email protected] Elucidating the neuronal architecture of olfactory glomeruli in the Drosophila antennal lobe 42

Species of quite diverging animal phyla with an advanced olfactory system share an important similarity, which is the presence of olfactory glomeruli. During the last decades the wiring properties of these spherical compartments has been elucidated in great detail while only little is known about the numerical neuronal composition of individual glomeruli. The lack of exact numbers leads to a common basic assumption of glomerular uniformity, although different glomeruli do not accomplish a uniform function. In order to scrutinize whether each glomerulus possesses a unique neuronal architecture or whether glomeruli are uniform structural units, we characterized the detailed neuronal architecture of individual glomeruli and correlated these anatomical features with their functional properties in the model organism Drosophila melanogaster. We report a complete quantitative mapping of all receptor-specific sensory neurons that innervate a certain glomerulus, including sexually dimorphic distributions and glomerular volumes. Our data disprove the so far assumed universal 30:1 convergence and demonstrates for the first time the impact of OSN number on glomerular dimensions. Moreover, we show sex-specific differences in neuron number and glomerular volume also for fruitless negative glomeruli. In addition, we demonstrate a glomerulus-specific projection neuron innervation. Finally, we correlate these morphological features with functional properties and provide evidence for a unique neuronal architecture of glomeruli encoding behavioral relevant odors. Supported by DFG SPP 1392.

Sonja Bisch-Knaden, Silke Sachse, Bill S. Hansson Max Planck Institute for Chemical Ecology, Hans-Knoell-Strasse 8, D-07745 Jena, Germany, [email protected] Olfactory coding in the moth antennal lobe Female hawk moths, Manduca sexta, use olfactory cues to locate nectar sources and oviposition sites. These environmental odorants are represented in the insect’s brain by spatial activation patterns across the antennal lobe, the first olfactory processing centre. With the help of in vivo calcium imaging we aimed at establishing a functional atlas of the antennal lobe. Therefore, we stimulated the insects with a panel of more than 100 compounds belonging to different chemical classes (terpenes, aromatics, aldehydes, alcohols, esters, ketones, and acids). A subset of these compounds was used as diagnostic odorants that enabled us to identify 23 glomeruli, the functional units of the antennal lobe, based on their response pattern. We analysed the molecular receptive range of these individual olfactory glomeruli, and found that most of them were broadly tuned but had distinct response spectra, allowing a combinatorial olfactory coding strategy. More specialised glomeruli were preferentially activated by acids and were located at the medial part of the antennal lobe opposite to the entrance of the antennal nerve. The proposed functional atlas will help to understand the olfactory coding strategy of Manduca sexta.

Sudeshna Das, Silke Sachse and Bill Hansson Max Planck Institute for Chemical Ecology, Hans-Knoell-Strasse 8, D-07745 Jena, Germany, [email protected] Lateral Horn neurons: from molecules to function

43

In Drosophila melanogaster, the first olfactory processing center is the antennal lobe where individual olfactory stimuli are encoded by the interplay and combined activity of different chemical and electrical synapses between multiple olfactory sensory neurons, local interneurons and projection neurons. How the higher center processes this odorant information, coming from the antennal lobe and helps the animal to elicit an appropriate odor-guided behavior is poorly understood. How the animal spontaneously decides whether a smell is good or bad? Who are the players behind this crucial decision-making? Lateral horn, cluster of the third order neurons receives the majority of glomerular projections from the antennal lobe in a stereotyped manner and is thought to play an important role in mediating innate odor-guided responses (Strutz et al., 2014). In this current study, using photoactivatable GFP and immunohistochemistry, we aim at characterizing the molecular architecture of different lateral horn neurons (LHNs) and generating a comparative map regarding different neurotransmitters and neuromodulators. By performing calcium imaging of these neurons to a set of ecologically relevant attractive and repulsive odors, we will also characterize their functional properties. These results will help us to investigate the mechanisms underlying coding and processing of odorant valence by these higher-order neurons.

Sylvia Anton, Kaouther Rabhi, Helene Tricoire-Leignel, Elodie Demondion, Philippe Lucas, Nina Deisig, Christophe Gadenne Neuroethology-RCIM, INRA-Angers University, 42 rue Georges Morel, 49071 Beaucouzé, France, [email protected] Low doses of an insecticide modify pheromone responses in a pest insect Sex pheromones are essential for reproduction in many pest insects, such as noctuid moths. Male moths are highly sensitive to the species-specific female-emitted pheromone. Nevertheless, many factors, such as the physiological state or previous experience can modify their sensitivity at the behavioural and neurophysiological level. Actual pest management strategies rely on treatments with neurotoxic molecules, including neonicotinoid insecticides. The wide spread use of these insecticides results in residual accumulation of low concentrations in the environment. Neonicotinoids are known to disrupt neurotransmission through nicotinic acetylcholine receptors and therefore kill insects at high doses. At low doses, neonicotinoids affect sensory systems and could therefore potentially disrupt pheromone communication. On the other hand adaptation processes to environmental pollution have been shown to occur in some insects and lead to an increase in reproduction after exposure to low insecticide doses. We investigated the impact of low doses of the neonicotinoid clothianidin on the olfactory system of the noctuid moth, Agrotis ipsilon. We determined the toxicity of oral clothianidin treatments and subsequently tested behavioural responses to the sex pheromone 24 h after intoxication. Surprisingly, we observed an increase in pheromone responses after intoxication with the lethal dose 20 (LD20) and a decrease in pheromone responses after intoxication with a very low dose.

Teun Dekker Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden, [email protected] Olfactory Correlates of a Taste for Fresh Fruit 44

The search for baits for monitoring and control of the spotted wing Drosophila (SWD, Drosophila suzukii) should benefit from the extensive knowledge of Drosophila melanogaster‘s olfactory circuitry and the difference in ecology between these species. Whereas D. melanogaster requires damaged, fermenting or rotting fruit for oviposition, SWD prefers to oviposit on ripe fruit. We investigated olfactory correlates of this, for Drosophila rather unique, shift and analyzed side-by-side the olfactory circuitry of SWD and D. melanogaster. Some of the shifts will be highlighted and discussed in the context of demands and opportunities of its ecological niche.

Thomas C. Baker, Q. Zhou, and T. Tighe Penn State University, USA, [email protected] Specializations for Odorant Capture Revealed by Atomic Force Microscopy of Moth Trichoid Sensilla Across Species The functional olfactory unit on the antennae of male moths for adsorbing and reporting the abundance of sex pheromone molecules is the trichoid sensillum. The surface of each of these sensilla is festooned with hundreds of pores that facilitate entry of pheromone molecules into the sensillar lumen. If the pores do not allow pheromone molecules to enter the lumen, then the dendrites of olfactory sensory neurons cannot receive molecules and nothing else neurophysiologically can happen. Thus, understanding how these pores and their associated ridges work is important to understanding pheromone olfaction. We used two new kinds of Atomic Force Microscopy, PeakForce Tapping mode and Kelvin Probe Force Microscopy (PF-KPFM) to characterize the topographic and chemical features of the trichoid sensilla of males of Manduca sexta, Lymantria dispar, Helicoverpa zea, and the E and Z pheromonal strains of Ostrinia nubilalis. PeakForce Tapping mode allowed us to collect information on the topography as well as changes in adhesion, deformation, and modulus (“sponginess”) across the sensillar surfaces of these species. Our recordings showed details regarding the adhesion and deformation related to these features for all four species, giving us information on the surface-lipid adhesive forces and hence their relative hydrophilic vs. lipophilic chemistries. The pores and the ridge structures were always intimately associated in a stereotypical way across species, and thus the ridges must play an important role in odorant capture and transport to the pores. The pores of all four species were found to be more hydrophilic than the surrounding surfaces. We used PF-KPFM to collect information on topography and surface potential, and found how surface potential changes significantly across pores compared to the rest of the sensillar surfaces. This change in potential may somehow be related to the transport of pheromone molecules into the sensillum lumen.

Toshihide Hige, Yoshinori Aso, Gerald M. Rubin and Glenn C. Turner Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, [email protected] Plasticity-driven individualization of olfactory coding in mushroom body output neurons Although all sensory circuits ascend to higher brain areas where stimuli are represented in sparse, stimulus-specific activity patterns, relatively little is known about sensory coding on the descending side of neural circuits, as a network converges. In insects, mushroom bodies (MBs) have been an important model system for studying sparse coding in the olfactory system, where this format is important for accurate memory formation. In Drosophila, the 2000 Kenyon cells (KCs) of the MB converge onto a population of only 35 MB output neurons (MBONs). Here we provide the first comprehensive view of olfactory representations at the 45 fourth layer of the circuit, where we find a clear transition in the principles of sensory coding, and demonstrate their flexibility. We show that MBON tuning curves are highly correlated with one another. This is in sharp contrast to the process of progressive decorrelation of tuning in the earlier layers of the circuit. Instead, at the population level, odor representations are reformatted so that positive and negative correlations arise between representations of different odors. At the single-cell level, we show that uniquely identifiable MBONs display profoundly different tuning across different animals. Interestingly, tuning of the same neuron across the two hemispheres of each fly was nearly identical. These results show that individual-specific coordination of tuning arises at this level of the circuit. Furthermore, we find that this individualization is an active process that requires a learning-related gene, rutabaga. Ultimately, neural circuits have to map highly stimulus-specific information in sparse layers onto a limited number of different motor outputs, in a way that is flexible. The reformatting of sensory representations we observe in the MBON layer may mark the beginning of this sensory-motor transition in the olfactory system.

U. Benjamin Kaupp Center of Advanced European Studies and Research (caesar), Department Molecular Sensory Systems, Germany, [email protected] How to detect single molecules by a Membrane-spanning guanylyl cyclases (GC) serve as versatile sensory receptors, in particular chemoreceptors, from C. elegans to mammals. GCs can be stimulated by semio-chemicals as diverse as carbondioxid, Ca2+, and peptides. In sperm of marine invertebrates, GCs regulate a cGMP-signaling pathway involved in chemotactic swimming of sperm in a gradient of chemoattractant peptide. The signaling pathway endows sperm with single-molecule sensitivity. I will discuss the sequence of signaling events from the chemoreceptor to the Ca2+ signal that controls the beat of the flagellum and, thereby, swimming behavior.

Vanessa Ruta The Rockefeller University, USA, [email protected] Adaptive Olfactory Processing in Drosophila In a complex and dynamic environment, animals must constantly vary their behavior to accommodate changing circumstances and contingencies. Yet, how associative brain centers flexibly couple a single sensory input to alternative behavioral outputs remains unclear. We are exploiting the relative simplicity of the Drosophila olfactory system to gain insight into the synaptic and circuit mechanisms through which context and experience can modify odor processing. In Drosophila, the mushroom body is a higher brain center that integrates olfactory and contextual signals to generate flexible and adaptive behaviors. Using functional synaptic imaging with electrophysiology, we show that the mushroom body functions like a switchboard in which dopaminergic neuromodulation can reroute the same odor signals to different behavioral circuits depending on the state and experience of the fly. Our data suggest a circuit mechanism for behavioral flexibility in which neuromodulatory networks act with exquisite spatial precision to transform a single sensory input into different patterns of output activity.

46

William Walker, Arthur de Fouchier, Nicolas Montagne, Claudia Steiner, Muhammad Binyameen, Peter Anderson, Fredrik Schlyter, Bill S. Hansson, Emmanuelle Jacquin-Joly, Mattias Larsson. Swedish University of Agricultural Sciences, Sweden, [email protected] Perspectives on insect odorant receptor ligand responses in an evolutionary context Numerous insect odorant receptors (ORs) have been functionally characterized, with respect to odorant response profiles, in Dipteran and Lepidopteran species, including Drosophila melanogaster, Anopheles gambiae, Spodoptera littoralis, and Bombyx mori. We have constructed a phylogenetic analysis of Lepidopteran ORs with key activating ligands mapped to receptors, where possible. In general, we observe a conservation of function across OR subfamily clades, such that there is a tendency for more similar ORs to respond to more similar classes of volatile odorant compounds. This principal has potential to facilitate predictive power regarding orphan receptor orthologues within and across species. Here, we expand phylogenetic analyses of OR response profiles to Dipterans and discuss potential implications for diverse insect taxa.

Wolf Huetteroth, E Perisse, S Lin, M Klappenbach, C Burke, and S Waddell University of Konstanz, Department of Biology, Neurobiology - M1124a, 78457 Konstanz, Germany, [email protected] Representation of short-acting sweet reward and long-acting nutrient reward in mushroom body dopamine neurons Recent studies in Drosophila unveiled a cluster of dopaminergic neurons that provide reward-learning signals like in mammals. All neurons of this cluster innervate distinct regions on the mushroom body, filling the gaps of previously identified dopaminergic neurons that convey aversive value. It appears as if quality and value of reinforcing stimuli are encoded in these different subregions of the mushroom body. But despite the fact that all neuronal cell types of the mushroom body have been identified by now, a complete assignment of reinforcing signals has not emerged yet. Previous work demonstrated that sugars can have two separable reinforcing properties: hedonic sweetness and nutritional value. Experimentally this can be addressed by using sweet sugars without any caloric value like arabinose, sweet and nutritious sucrose, or nutritious but tasteless sorbitol. Sweet reinforcement leads to short-lasting memory only, whereas long-lasting food memory requires a nutritional component. Subsequently we have shown that octopaminergic neurons convey exclusively hedonic sweetness, and act upon a subset of rewarding dopamine neurons through the alpha-adrenergic receptor OAMB. Here we were able to narrow down the short-term reinforcing effects of sweet taste to its responsible dopaminergic neurons innervating the b’2 and g4 regions of the mushroom body lobes. The nutritional reward signal requires separable dopamine signaling in the g5b region, while artificial activation of dopamine neurons projecting to the b lobe and adjacent a1 region alone are sufficient to implant a long-lasting memory. Interestingly, this implanted long-term memory responds differently to the caloric state of the animal; while artificial implantation and expression of short-term memory is independent of satiety state, acquisition and retrieval of long-lasting food memory requires the flies to be hungry. Taken together, a more complete picture emerges how different reinforcing stimuli are represented, integrated and modulated in the fly on the cellular level.

47

Wolfgang Rössler University of Würrzburg, Biozentrum, Behavioral Physiology & Sociobiology, Am Hubland, D- 97074 Würzburg, Germany, [email protected] Developmental plasticity and maturation of the honeybee olfactory pathway Eusocial insects express a high degree of developmental plasticity. The resulting behavioral plasticity represents a major prerequisite for division of labor and task allocation in social insect colonies. The honeybee is an excellent social insect model species to investigate mechanisms of plasticity in the olfactory pathway and their importance for behavior. The talk will highlight recent progress in our understanding of mechanisms underlying developmental plasticity between the female (queen, worker) and the male (drone) castes - from differences in olfactory sensory input, to antennal lobe (AL) circuits, all the way up to higher integration centers in the mushroom bodies and lateral horn. We have recently focused on mechanisms of developmental plasticity and adult maturation in MB olfactory microcircuits, in particular at the level of MB input synapses (microglomeruli MG) that form highly divergent synaptic complexes between olfactory projection neurons and MB Kenyon cells. I will highlight recent advances in our understanding of cellular and molecular processes that shape olfactory microcircuits in the MB throughout life, from postembryonic development to adult maturation and the formation of long-term memory. The results suggest that the different levels of structural plasticity in olfactory circuits play important roles in regulation of social organization in honeybee colonies. Supported by DFG SPP 1392.

Yusuke Takeichi, Masaru K. Hojo, Kouji Yasuyama, Naoyuki Miyazaki, Kazuyoshi Murata, Mamiko Ozaki Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan, [email protected] Ultrastructure of the sensilla basiconica and the putative mechanism for nestmate- nonnestmate discrimination in the Japanese carpenter ant, Camponotus japonicus It has been reported that in the Japanese carpenter ant, Camponotus japonicus, workers discriminate between nestmates and non-nestmates by the sensilla basiconica on the antennae. Previous studies indicated that this type of sensilla house 130 olfactory receptor neurons (ORNs) projecting 130 glomeruli of T6 region in the antennal lobe. We observed ultrastructure of the sensilla basiconica using serial block face scanning electron microscope (SBF-SEM) and constructed its 3D model showing the particular shape like a twisted thick rope of ORNs. Dendritic processes of those over hundred ORNs have no branches but characteristic swellings (1-7 swellings/dendritic process). In this swelling region, cell membranes of ORNs are closely adjacent with complicated borders. We supposed to exist some interaction among these ORNs at such a swelling region. If it happens, it may affect the functional mechanism of the sensilla basiconica as a sensory unit to detect difference between CHC patterns of self and others. Now we are investigating involvement of gap junction in such a mechanism, searching localization of innexin in the antennae of C. japonicus.

Béla Péter Molnár, Zoltán Tóth, Zsolt Kárpáti Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary, [email protected]

48

Larval frass volatiles deter oviposition in the invasive box tree moth (Cydalima perspectalis) Insects find their oviposition site using olfactory cues. Volatile stimuli emitted by an intact as well as herbivore occupied host plant or the herbivore itself influence females in the final decision. Box tree moth, Cydalima perspectalis (Lepidopera, ) as an invasive, strictly monophagous species recently introduced to Europe causing enormous damage on European box tree (Buxus sempervirens). Frass produced by the voracious larvae cover the plants and have an unpleasant scent, which could be detectable from long distance. Therefore we investigated whether the females use this odor bouquet in oviposition site location. Volatile substances surrounding larval frass of box tree moth were collected and physiological activity was investigated by coupled gas chromatographic electro- antennographic detection (GC-EAD). Based on the structure elucidation two aromatic derivate and one terpene alcohol have been identified as antennaly active compounds emitted by larval fecal pellets on male and female antennae. Behavioral bioassays indicated that natural frass has only short-term oviposition deterrent effect due to the altered volatile profile caused by desiccation. However synthetic mixture of the active compounds shows persistent effect of oviposition deterrence on conspecific females. Aerial entrainments (SPME) revealed altered volatile profile of larval frass exposed to room temperature after a day as the potential explanation of the lost of deterrent activity. To identify the response characteristics of the olfactory sensory neurons responding to the frass volatiles we obtained single sensillum recordings on female antenna. Assessment revealed that sensilla trichodea responded strongly to the identified frass compounds. In conclusion the headspace volatile of frass could be act as oviposition deterrent pheromone therefore should be sufficient to prevent multiple egg-laying on a host which cannot support high population density and thus reduce intraspecific competition.

49