15th European Symposium

for Insect Taste and Olfaction (ESITO)

ABSTRACTS

September 17-22, 2017

Villasimius (Cagliari – Italy)

www.ice.mpg.de/esito

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Abstracts are ordered alphabetically by the last name of the presenter (in bold)

Álvarez-Ocaña R., Auer T.O., Arguello J.R. & Benton R. Center for Integrative Genomics, University of Lausanne, Switzerland Evolution of olfactory pathways in Drosophilids

Animals’ brains are continuously evolving, as can be observed through the enormous variety in brain morphology found among closely-related animals. However, not all brain structures change at the same time or in the same way. Brain regions that are involved in processing environmental information, such as chemosensory-related regions, have been of particular interest because it appears they evolve rapidly. The reasons for this rapid evolution remains unclear, but it likely is related to constant changes in environmental conditions, and other driving forces. Among the evolutionary differences that have been observed in chemosensoy-related systems, two intriguing examples include (1) changes in the size of sensory neuron populations and (2) the expansion and contraction of chemosensory receptor families. We have employed Drosophila melanogaster, and its closely-related species, as a model system to examine these two modes of sensory system evolution. Arican C., Pinger K. & Nawrot M.P. University of Cologne, Institute of Zoology, Germany Operant and classical conditioning of the cockroach Periplaneta americana in a forced choice task

The forced choice paradigm is regularly employed in learning experiments with vertebrates. However, only few successful attempts have been made in insects, most prominently in honeybees. The aim of the present study is to establish a novel forced choice paradigm in Periplaneta americana and advertising the cockroach as a model organism for learning and memory. Cockroaches have several advantages and complementary properties in comparison with other well-established insect models for learning and memory. We developed a novel experimental setup for cockroaches based on a T-maze. The first task uses a paradigm of operant aversive conditioning. Animals had to choose the walking direction (left or right) and we punished their first choice with an aversive light stimulus. The results show a significant learning success and indicate stable individual learning behavior (Pamir et al. 2011). The second task employs classical olfactory conditioning (Watanabe et al. 2003). For the retention test we transferred the animals to the T-maze (de Brito Sanchez et al. 2015). We find that cockroaches accomplished the transfer task with olfactory stimuli. For the third task an operant olfactory conditioning paradigm was used. The cockroaches had to choose between benzaldehyde and citral that were placed in the two arms of the T-maze. One odor was presented together with sucrose solution, the other odor was presented with saline solution. The cockroaches showed a significant learning success after few trials. References: de Brito Sanchez, M. G., Serre, M., Avarguès-Weber, A., Dyer, A. G. & Giurfa,M. (2015). Learning context modulates aversive taste strength in honey bees. The Journal of Experimental Biology, 218: 949-959. Pamir, E., Chakroborty, N. K., Stollhoff, N., Gehring, K. B., Antemann, V., Morgenstern, L., Felsenberg, J., Eisenhardt, D., Menzel, R. & Nawrot, M. P. (2011). Average group behavior

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does not represent individual behavior in classical conditioning of the honeybee. Learning & Memory, 18(11), 733-741. Watanabe, H., Kobayashi, Y., Sakura, M., Matsumoto, Y. & Mizunami, M. (2003). Classical Olfactory Conditioning in the Cockroach Periplaneta Americana. Zoological Science, 20(12): 1447-1454.

Baker T.C., Andreadis S.S., Cloonan K.R. & Myrick A.J. Penn State University, USA Coupled GC/EAD and GC/behavior assays used to isolate and identify the sex pheromone of the fungus gnat Lycoriella ingenua

Lycoriella ingenua Dufour (Diptera: Sciaridae) is acknowledged as the major pest species of the white button mushroom, Agaricus bisporus, throughout the world. Previous reports concerning the identification of a sex pheromone comprised of n-C15-n-C18 saturated straight-chain hydrocarbons useful for trapping L. mali, with the major component declared to be heptadecane, have proven to be questionable. The purpose of our present study was therefore to reinvestigate the sex pheromone of this species, beginning with the collection of extract from virgin females and thereafter isolating the behaviorally active fractions of these extracts that would evoke wing-fanning and copulatory abdomen-curling in males. Our investigations using coupled gas chromatography electro-antennographic detection (GC/EAD), plus a rarely used gas chromatography-coupled behavioral bioassay (GC/BB), resulted in a behaviorally active pheromone component being isolated and partially characterized via gas chromatography-mass spectrometry (GC/MS). This component was found definitively to not be n-heptadecane or any of the other n-C15-n-C19 hydrocarbons previously erroneously identified, but rather appears to be one of the isomers of a sesquiterpene alcohol, germacradienol. Bardos V. & Kloppenburg, P. Biocenter, Institute for Zoology, CECAD, University of Cologne, Germany Octopamine modulation of local interneurons type I in the antennal lobe of the cockroach Periplaneta americana

In insects, processing of peripheral olfactory input is achieved by a strongly interconnected network of interneurons. The interaction of antennal lobe neurons shapes the olfactory representations, such as regulating the tuning profile of output projection neurons. The olfactory system is highly adaptable to changes in their environment by modulating the sensitivity and performance. Characterizing the modulation of antennal lobe neurons is crucial for understanding odor response processing in this network. Our focus is to investigate the modulatory effect of octopamine on identified, synaptically isolated local interneurons type I in the antennal lobe of the American cockroach Periplaneta americana. Octopamine and its precursor tyramine are often considered as the invertebrate homologues to the vertebrate noradrenalin and adrenalin, respectively. Based on previous work in a variety of insect preparations octopamine has been shown to act as neurotransmitter, neurohormone and/or neuromodulator by that regulating a wide spectrum of physiological processes. Immunohistochemical and biochemical experiments were able to detect octopamine in the insect antennal lobe. Additionally, Ca2+ imaging coupled with odor experiments showed a network effect upon octopamine application in the honeybee (Rein et al. J.

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Comp. Physiol. Vol. 11:947-62). In the cockroach our perforated patch clamp recordings revealed that the general octopamine effect on local interneurons type I is a concentration dependent increase in excitability. Currently, we are analyzing the octopamine mediated effect on intrinsic electrophysiological properties. Preliminary results suggest an increase in input resistance and firing frequency, a decrease in first spike latency and threshold as well as a modulatory effect on the rebound property. Bastin-Héline L., Caballero-Vidal G., Meslin C., Wang G., Jacquin-Joly E. & Montagné N. Université Pierre et Marie Curie, France Characterization of a putative novel subfamily of pheromone receptors in Spodoptera species

Sex pheromone receptors (PRs) are key players in chemical communication between mating partners in insects, and thus participate in reproductive isolation between species. In Lepidoptera, these PRs are supposed to form a dedicated subfamily of olfactory receptors (ORs). Hence, all the PRs functionally characterized to date belong to the same clade of the lepidopteran OR phylogeny. Unexpectedly, we recently identified an OR that responds to the major sex pheromone component of the cotton leafworm Spodoptera littoralis, but does not belong to this clade. We identified orthologues and paralogues of this new PR in different lepidopteran genomes and rebuilt its evolutionary history. Interestingly, in the S. littoralis genome, these OR genes are organized in a cluster, suggesting recent gene duplication events. Currently, we are conducting functional studies on several paralogues and orthologues of the new S. littoralis PR, using heterologous expression in Xenopus oocytes. Together with evolutionary analyses, our data will help assessing whether PRs evolved twice in Lepidoptera and if the evolution of this subfamily participated in speciation events among Spodoptera species. Benton R. University of Lausanne, Switzerland Olfactory receptor evolution

Olfactory receptor repertoires have evolved remarkable functional diversity, but the underlying molecular basis is poorly understood. Through analysis of variant ionotropic glutamate receptor (IR) olfactory receptors in ecologically-diverse drosophilids, we have delineated a molecular path that reveals how the specificity of an IR has shifted from one salient odor to another. We identify a “hot-spot” in the receptor ligand-binding pocket, where mutations both in different IRs, and within the same IR at different evolutionary times, have driven alterations in specificity. Surprisingly, this site does not affect receptor affinity, but rather receptor efficacy, counter to widespread models of odor/receptor interactions. Finally, we show that mutations outside the ligand-binding pocket can also contribute to evolution of receptor specificity, revealing an epistatic network of residues that tunes olfactory receptor responses.

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Caballero-Vidal G., Audic G., Heligon C., Meslin C., Walker W.B., Montagné N. & Jacquin-Joly E. INRA, Institute of Ecology and Environmental Sciences of Paris (iEES-Paris), Versailles, France Sorbonne Universités, Université Pierre et Marie Curie, iEES-Paris, Paris, France Development of a high-throughput screening platform for the study of Spodoptera littoralis olfactory receptors

Insect olfactory receptors (ORs) are at the core of the olfactory detection process. However, only a few ORs have been functionally characterized and, outside the two dipteran models Drosophila melanogaster and Anopheles gambiae, we lack a complete view of how a given species uses its OR repertoire to efficiently detect its environment. Recently, we have functionally characterized the first large OR repertoire in a Lepidoptera, the cotton leafworm Spodoptera littoralis, using the “empty neuron” system, that is heterologous expression in Drosophila olfactory neurons coupled to single-sensillum recordings. However, thanks to the recent annotation of ORs in the genome of S. littoralis, we estimate that the deorphanized ORs only represent one third of the total OR repertoire of this species. We thus aim at developing high-throughput deorphanization of ORs on a HiClamp platform that uses Xenopus oocytes coupled to two-voltage clamp electrophysiology. We will present our first results on previously deorphanized ORs used to calibrate the Xenopus system. This system has the potential to screen a large panel of ligands on a wide range of ORs. Cardé R.T., Hughes G., Lacey E. & Zhang J. University of California, Riverside, USA Pheromone antagonists of Heliocoverpa

The pheromones of both Heliocoverpa zea and H. zea are comprised of a 97:3 mixture of (Z)-11- and (Z)-9-hexadecenals. Several related compounds antagonize attraction to pheromone in both species, including (Z)-11-hexadecenol. It has been proposed that this antagonist regulates optimal mating in H. armigera by delaying male attraction until females are mature. To explore this hypothesis, we have used wind-tunnel studies to establish male sensitivity to antagonists, particularly (Z)-11-hexadecenol. Documentation of possible emission of this compound included behavioral observations of calling periodicities in immature and mature females, pheromone gland extractions and SPME wipes, and airborne collections. Chertemps T., Zbinden M., Berthod C., Montagné N., Machon J., Léger N., Rabet N., Shillito B. & Ravaux J. Institute of ecology and environmental sciences (iEES-Paris), France Comparative study of chemosensory organs of shrimp from hydrothermal vent and coastal environments

The detection of chemical signals is involved in a variety of crustacean behaviors, such as social interactions, search and evaluation of food and navigation in the environment. At hydrothermal vents, endemic shrimp may use the chemical signature of vent fluids to locate active edifices, however little is known on their sensory perception in these remote deep-sea habitats. Here, we present the first comparative description of the sensilla on the antennules and antennae of 4 hydrothermal vent shrimp (Rimicaris exoculata, Mirocaris fortunata, Chorocaris chacei, and Alvinocaris markensis) and of a closely related coastal shrimp (Palaemon elegans). These observations revealed no

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specific adaptation regarding the size or number of aesthetascs (specialized unimodal olfactory sensilla) between hydrothermal and coastal species. We also identified partial sequences of the ionotropic receptor IR25a, a co-receptor putatively involved in olfaction, in 3 coastal and 4 hydrothermal shrimp species, and showed that it is mainly expressed in the lateral flagella of the antennules that bear the unimodal chemosensilla aesthetascs. Chu X., Hodson R.M., Juell M.S. & Berg B.G. Department of Psychology, Norwegian University of Science and Technology, Norway Morphological and physiological properties of pheromone-sensitive projection neurons confined to parallel tracts in the male moth brain

In the noctuid moth, Helicoverpa armigera, three identified female produced molecules activate the male-specific olfactory system including the second-order projection neurons (PNs) passing from the macroglomerular complex (MGC) to secondary olfactory centers, i.e. the calyces of the mushroom body and the lateral horn. This male-specific arrangement is associated with two stereotyped behaviors: 1) attraction and mating behavior in response to two pheromone compounds released by the conspecific females and 2) avoidance during exposure to one substance emitted by heterospecifics. Previous data from mass staining experiments have demonstrated that MGC PNs project along all three classical antennal-lobe tracts (ALTs), the medial, mediolateral, and lateral ALT (Homberg et al. 1988). However, except for medial-tract neurons, little is known about individual PNs originating in the MGC. In this study, we identified single male-specific PNs confined to all three ALTs by performing intracellular recording/staining combined with confocal microscopy. Several new types of MGC PNs were identified, including neurons passing in the lateral and the mediolateral ALT, respectively. Additional double mass-staining experiments demonstrated that a substantial amount of the MGC PNs are lateral-tract neurons terminating in the column. In addition to the electrophysiological experiments, we performed calcium imaging experiments for measuring odor-evoked responses from populations of MGC output neurons confined to different ALTs. Two types of staining procedures were carried out. One implied application of the calcium-sensitive dye to the calyces, leading to staining of the medial-tract PNs exclusively, whereas the other included dye application to the lateral horn, resulting in labeling of PNs in all tracts. Data from the two categories of stained preparations showed similar response patterns to the stimuli. However, medial-tract PNs displayed a longer response latency (about 200 ms) than the group of PNs confined to all ALTs.

Conchou L., Anderson P.*, Birgersson G.*, Lucas P. & Renou M. Institute of Ecology and Environmental Sciences – Paris, Institut National de la Recherche Agronomique, Versailles, France *Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden Olfactory perception in complex and changing odour landscapes

Phytophagous insects use olfaction in order to navigate their environment. Plant odours constitute odour landscapes, which convey information about the presence and location of potential host plants, but also constitute a complex and changing background against which insects need to detect cues of interest (such as sex pheromones).

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At SLU university, we investigated whether 1) the polyphagous moth S. littoralis has the sensory ability to perceive alternative host plant species as different (a pre-requisite to the ability to choose among them) 2) whether variable environmental conditions would change host plant odour profiles enough to impair this ability. We grew 3 host plant species under 3 levels of soil fertility, and compared their odour profiles, as detected by S. littoralis (identity and proportion of GC-EAD active volatiles). The 3 plant species are characterised by different combinations of GC-EAD active volatiles, such that olfaction alone is enough for S. littoralis to perceive alternative host plant species as different. On the contrary, soil fertility only had a minor effect on GC-EAD active volatile ratios, therefore did not affect S. littoralis’ ability to differentiate plant species. Many abiotic factors have been shown to impact volatile emissions by plants, such that global change might modify the odour landscapes insects will face in the future. As part of the ODORSCAPE project at INRA Versailles, I investigate how plant volatile backgrounds (composition, intensity, temporal structure and variation) influence sex pheromone perception and neural coding in the moth Agrotis ipsilon.

Cook B., Haverkamp A., Roulston T., Lerdau M. & Knaden M. University of Virginia, USA; Collaborators Max Planck Chemical Ecology, Jena, Germany Pollination in the Anthropocene: Manduca sexta can learn ozone-altered floral plumes

Insect pollinators rely on floral scents, often in synergy with visual cues, to locate their host flowers. Emerging research, however, indicates that the integrity of olfactory cues may be jeopardized in polluted atmospheres. Anthropogenically elevated pollutants, like the ubiquitous oxidant ozone, can react with floral plumes, decreasing the overall concentration or altering the composition of a plume, potentially eroding pollinators’ ability to recognize floral scents. However, current work has not considered whether insect learning could mitigate complications in host-finding induced by pollution. Here we demonstrate that the pollinating moth Manduca sexta, while not innately attracted to ozone-altered plumes without visual cues, can learn to associate ozone-altered plumes with rewards. Moths that were visually attracted to a flower emitting an ozone-altered plume, later followed this plume also in the absence of visual cues. Our results, while describing a potential mechanism for pollinators to use floral plumes in polluted atmospheres, stress the complicated nature of pollination and the need to include insect perception and behavior in future studies concerning the intersection of atmospheric chemistry and entomological ecology. Biolchini M., Sollai G. & Crnjar R. Dept. Biomedical Sciences, University of Cagliari, Italy Antennal and palpal sensitivity in Ceratitis capitata to sex pheromone components depends on mating condition and time of day

The Mediterranean fruit fly, Ceratitis capitata Wied., is a worldwide pest for horticulture given its high biological potential, the difficulty of its control and the broad polyphagy, mainly addressed, in Southern Europe, to pomaceous and citrus cultures. Recent studies have characterized the chemical composition of the sex pheromone and the Pheromone Binding Proteins (PBPs) involved in the detection of these compounds. Siciliano et al. (2014) identified in C. capitata 17 unique Odour Binding Protein genes, 5 of which seem to be putative pheromone binding proteins. The transcript of one of them

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(CcapObp69a) results highly enriched both in the antennae and maxillary palps and the expression of its gene appears to be modulated both by mating and time of the day. Based on these considerations, we measured by EAG and EPG recordings, the olfactory sensitivity of antennae and palps of C. capitata in both sexes, in different physiological states (virgin and mated), and at different times of the day (morning and afternoon) following stimulation with α-farnesene, β-farnesene and β-myrcene (Jang et al., 1989). The results show that the olfactory sensitivity was higher in the antennae than in palps. EAG amplitude values in response to all stimuli were higher in the morning than in the afternoon for both sexes and in both virgin and mated insects. Besides, in both sexes, the olfactory sensitivity of virgin insects was higher than in mated ones. The EPG amplitude in response to all stimuli was higher in the morning in mated females than in virgin females and higher in the morning than in the afternoon in both mated sexes. A possible relationship between the olfactory sensitivity of C. capitata and the gene expression of its PBP CcapObp69a based on sex, physiological state and time of day is discussed. De Obaldia M.E. & Vosshall L.B. Rockefeller University, USA Dissecting the role of the human skin microbiota in mosquito attraction

Aedes aegypti mosquitoes spread viral diseases, including yellow fever, dengue, chikungunya, and Zika, among human populations, sickening hundreds of millions of people per year. Ae. aegypti are able to transmit disease so effectively because they prefer to bite humans over other mammals, and they blood-feed on multiple humans over their lifetime to support egg development. In addition to their strong human preference, Ae. aegypti prefer to bite some humans over others. The mechanistic basis of these phenomena remains unknown. Cues such as body odor, carbon dioxide exhaled in the breath, and body heat alert mosquitoes to the location of nearby humans. Prior work suggests that human body odor mediates mosquito attraction to humans, and is important for discrimination among potential hosts. We hypothesize that bacteria contribute to the uniquely attractive odor blend emanating from human skin, and that variation in the human skin microbiome governs how attractive a given person is to mosquitoes. We are developing porcine skin explants as a model in which to dissect the role of the human skin microbiome in mosquito attraction to humans. We anticipate that these studies could lead to novel strategies to curb mosquito biting behavior and disease transmission, such as skin microbiome manipulation. Hanoune J.L., Murmu M.S. & Deisig N. iEES Paris, UMR 1392, Département d’ Ecologie Sensorielle, INRA Versailles, Route de Saint-Cyr, 78026 Versailles cedex, France Aversive olfactory learning and the effect of pheromone exposition in male Agrotis ipsilon moth

Pheromones are chemical messengers eliciting stereotyped behavioural or physiological responses in another organism of the same species. They are responsible for triggering immediate or delayed innate responses to relevant stimuli, and are thus crucial for the regulation of social or sexual interactions. In addition to their well-documented function as communication signals, some pheromones have been recently shown to play a role as “modulators” of cognitive phenomena, facilitating or inhibiting associative learning and memory both in vertebrates and invertebrates. This modulation corresponds to a previously unknown effect of pheromones as it targets behaviours that

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are different from the innate responses typically controlled by a given pheromone. Pheromones could thus provide contextual information rendering subsequent learning and memory more or less relevant for the context signalled. To test the potential influence of pheromones on aversive learning in moths, we first developed an aversive olfactory learning and memory testing paradigm, which does not exist for moths so far. For this, a mild electric shock was paired with an odour during conditioning and the moths’ responses were tested in an olfactometer for olfactory memory after 1h and 24h. Our results show that exposition to a ‘potential aversive pheromone’ before conditioning increased aversive olfactory learning, while exposition to the ‘appetitive’ sexual pheromone or solvent control did not have an effect. Further, moths avoided the previously aversively learned odour during memory tests. However, exposition of neither an aversive, nor an appetitive pheromone modulated memory performance 1h or 24h later. Thus, pheromones seem to have an ‘immediate’ modulatory effect (on learning), but not a ‘long-lasting’ effect (on memory). Since biogenic amines (dopamine and octopamine) seem candidate molecules for mediating appetitive and aversive learning, we currently investigate their role in aversive learning and memory in moths combining pharmacological and behavioural approaches. Devaud J.-M., Cabirol A. & Barron A. Research Center on Animal Cognition, CNRS/Paul Sabatier University, Toulouse, France Experience-dependent plasticity of brain and learning performance in the honey bee

During foraging, honey bees are likely to experience more complex learning situations than those usually tested in laboratory conditions. For example, they may need to update information about cue associations that are no longer relevant while learning new ones, such as in a reversal learning protocol. We have previously shown that that this task involves specific brain circuits involving the mushroom bodies (MBs), a major brain center that undergoes structural changes over the lifetime of honey bees. How does experience acquired by the animal influences the maturation of MB circuits, and how does this modulate its capacity to perform MB-dependent learning tasks? To address these questions, we have performed several studies to compare learning capacities and MB structural characteristics of bees with different experiences. So far our results indicate that experience of environmental factors, both early in life within the hive and later during foraging, is a key factor of both behavioural and structural brain plasticity. Droujinine I.A. Department of Genetics, Harvard Medical School, USA Long non-coding RNA-derived small open reading frame peptide is a Drosophila pheromone

Animal communications are mediated by metabolite and protein pheromones. However, pheromones, neurons, and receptors governing interaction from adults to developing individuals are poorly characterized. Furthermore, long non-coding RNA (lncRNA)-derived small open-reading frame (sORF) peptides of <100amino-acids are a novel class of signaling-factors, yet their roles as putative pheromones are unknown. We serendipitously observed that Drosophila melanogaster larvae are retained on medium preincubated with males versus no-adults. Also, larvae of both sexes choose parchments preincubated with males over females or no adults. Moreover, biochemical fractionation-activity experiments and mass-spectrometry reveal male excretion of a

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69amino-acid lncRNA-derived sORF, which we term “Male Peptide” (MP). MP is expressed significantly higher in males than females/larvae, conserved in Drosophila species, and has a putative uncharacterized human ortholog. Further, MP is necessary and sufficient for male-larvae interactions, as larvae prefer synthesized-MP over scramble-peptide, and wt males over MP CRISPR-knockout males (rescued by synthesized-MP). Moreover, MP is produced by testis, as larvae do not respond to males with superglued penis (but not anus/headless), and to males with MP-RNAi in testis somatic cyst-cells (but not germline, gut, muscle RNAi). MP-Gal4 is expressed in testis cyst-cells. Also, live-imaging shows MP-GFP excretion from penis. Interestingly, masculinized females interact with larvae and express MP. Using larval calcium-imaging, we demonstrate that Or35a olfactory receptor-neurons (ORNs) are activated by MP. Further, Or35a-ORN-inactivated or mutant larvae do not respond to MP. Also, a single-functional Or35a-ORN in background of inactive ORNs is sufficient for larvae to respond to MP. Moreover, synthesized-MP or wt but not MP-mutant males promote larval residence in food, regulating developmental-progression. During starvation, larvae leave the food early and die above it; however, MP decreases egress from food and lethality, dependent on Or35a. Thus, we reveal a novel developmental paradigm in which MP accumulation from adult males’ presence transmits environmental information to regulate food retention, development, and survival. Fandino R.A., Haverkamp A., Bisch-Knaden S., Zhang J., Bucks S., Knaden M., Hansson B.S. & Große-Wilde E. Max Planck Institute for Chemical Ecology, Jena, Germany Novel approaches in understanding the role of olfaction in host-seeking behaviors in Manduca sexta

Due to the Tabacco Hawkmoth’s (M. sexta) ability to travel long distances seeking a host or a mate it has been a major model in the in the field of insect olfaction for many years. Not only has this organism been useful for the understanding of odor seeking behavior it also presents an excellent model for the olfactory basis of co-evolved plant and pollinator interactions. However, the lack of tools for genetic manipulation has prevented application of more modern analytical methods in the species. Our group has established efficient germ-line cell manipulation mediated by CRISPR-Cas9 in M. sexta. We have established a M. sexta line with a loss-of-function frameshift mutation in the OR co-receptor (Orco) gene. The Msexta Orco -/- has allowed us to analyze the contribution of the gene to the olfactory function in a variety of tissues and behaviors. In addition to using modern gene editing techniques we have demonstrated the applicability of a novel method of OR deorphanization in M. sexta using the recently published DREAM method, allowing association of ecologically relevant odors with a corresponding set of detecting ORs. Together, these two tools will facilitate future studies from a novel view point and enhance our understanding of the ecological significance of olfactory mediated behaviors.

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Braccini C.L., Barrozo R., Davila C., Coll Aráoz M.V. , Gershenzon J., Reichelt M., Zavala J. & Fernandez P.C. INTA - UBA - CONICET, Argentina The role of phenolic glycosides during host location behavior of the willow swfly Nematus oligospilus

Once an insect has located and reached a plant, contact cues from the leaf surface determine final host acceptance. The outermost leaf surface possesses an epicuticular wax layer which consists mostly of very long chain aliphatic wax components. Previous studies show that the epicuticular wax composition of Salix spp. plays a role in the selection of preferred and non-preferred willow genotypes by the willow sawfly Nematus oligospilus (Hymenoptera: Tenthredinidae). However, in several insect species, epicuticular wax compounds act in synergism with secondary metabolites as oviposition stimulants. Phenolic glycosides are the most important secondary metabolites present in Salicaceae. We investigated if these compounds might have a role in host recognition of preferred Salix genotypes by N. oligospilus females. More specifically, we first determined the sequence of behaviors of the ovipositing females after contacting the leaf surface of preferred (S. nigra) and non-preferred (S. viminalis) species. Second, we evaluated differences in the chemistry of phenolic glycosides present on the cuticular surface of preferred and non-preferred willow genotypes. Then we studied the morphology and electrophysiological response of chemosensory structures present on female’s antennae and ovipositor with which females could detect host plant cues. Finally, we performed single sensillum recordings from chemosensory sensilla to phenolic glycosides differentially present in both experimental genotypes. Our results suggest the presence of contact cues on the leaf surface of preferred genotypes, which increase ovipositor probing and trigger egg laying in sawflies. Phenolic glycosides present on the leaf surface seem to be part of a chemical signature that this highly specialized insect seeks on host plants. Chaetic chemosensory sensilla present in antennae and cerci of the ovipositor are sensitive to salicin and may play a role in the detection of phenolic glycosides. Bilz F. & Fiala, A. University of Göttingen, Molecular Neurobiology of Behavior Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Julia-Lermontowa-Weg 3, 37077 Göttingen, Germany Monitoring learning-induced plasticity in single Kenyon Cells in Drosophila melanogaster

Drosophila melanogaster is a key model organism to study neuronal circuits underlying olfactory learning and memory formation. Flies can associate conditioned stimuli (e.g., odors) and unconditioned stimuli (e.g., electric shocks as punishment or sugar stimuli as reward) during classical conditioning procedures. In a typical differential training paradigm the flies learn to associate one odor (CS+) with a simultaneously presented punishment. A second odor (CS-) is presented without punishment. In a subsequent choice situation in which both odors are presented the animals avoid the CS+. The mushroom body of the Drosophila central brain represents a key structure underlying associative odor learning. The intrinsic neurons of the mushroom body (Kenyon cells) are sparsely activated by a given odor stimulus, and dopaminergic neurons projecting to the main output regions of the mushroom bodies, the lobes, mediate the reinforcing effects of the punishment. The coincidence of odor-evoked activity of Kenyon cells and dopaminergic input is hypothesized to cause synaptic plasticity of Kenyon cell

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presynapses, ultimately changing the animals’ behavior in response to the learned odor stimulus. The mushroom body lobes can be subdivided in lobe-specific compartments by the innervation of extrinsic neurons (e.g., dopaminergic neurons or mushroom body output neurons). These compartments were shown to be differentially involved in distinct aspects of learning (e.g., appetitive and aversive olfactory learning). We ask how odor-evoked, neuronal activity within single Kenyon cell axons is represented across these compartments, how it is modulated in the course of aversive associative olfactory learning, and whether neuromodulation is restricted to specific sub-regions of the axon. Using optical imaging we monitor calcium activity in single Kenyon cell axons and its modulation in the course of associative training. Results will be presented and discussed. Fleischer J.1, Pregitzer P.2, Jiang X.2, Grosse-Wilde E.3, Hansson B.S.3, Lemke R.S.1, Breer H.2 & Krieger J.1 1. Martin Luther University Halle-Wittenberg, Institute of Biology/Zoology, Department of Animal Physiology, Halle (Saale), Germany 2. University of Hohenheim, Institute of Physiology, Stuttgart, Germany 3. Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Jena, Germany In search for pheromone receptors in the desert locust Schistocerca gregaria

The desert locust (Schistocera gregaria) can exist in two anatomically and behaviorally distinct phases, in a solitary or in a gregarious phase. The latter can form swarms of billions of animals, leading to massive destruction of crop yields. In both phases, the behavior of desert locusts is largely directed by olfactory cues and is supposed to rely on pheromonal substances. However, little is known about the molecular processes underlying odorant detection in this species. Due to the key role of odorant receptors (ORs) in odor and pheromone recognition, we set out to investigate the OR repertoire of the desert locust in the gregarious phase. By sequencing the antennal transcriptome and subsequent bioinformatical analyses, we identified 119 Schistocerca gregaria ORs (SgreORs). In situ-hybridization approaches with selected SgreORs demonstrated that they are expressed in distinct subsets of antennal olfactory sensory neurons (OSNs). Pheromone-sensitive OSNs in insects are characterized by the expression of the “sensory neuron membrane protein 1” (SNMP1). Therefore, to identify SgreOR types that might be implicated in pheromone detection, we analyzed the co-expression of SgreORs and SNMP1 in antennal OSNs. Among the SgreORs tested, a subset of them were found to be expressed in SNMP1-positive OSNs, indicating that these receptors might detect pheromones. In contrast to moth species in which pheromone receptors form a distinct branch in the OR repertoire in phylogenetical analyses, the putative pheromone receptors from the desert locust were found to belong to phylogenetically different OR groups. Currently, experimental approaches are conducted to assess whether the SgreOR types co-expressed with SNMP1 are indeed activated by known pheromones from Schistocera gregaria.

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Fusca D., Pippow A. & Kloppenburg P. Biocenter, Institute for Zoology, CECAD, University of Cologne, Germany Two tasks for local interneurons: inter- and intraglomerular signaling

In the insect antennal lobe local interneurons (LNs) mediate complex excitatory and inhibitory interactions between the glomerular pathways to structure the olfactory representation, which ultimately determines the tuning profiles of the (output) projection neurons. In the antennal lobe of the cockroach Periplaneta americana, we can discriminate two main LN types with distinctive physiological properties and morphological features, which imply important consequences for their computational properties and the olfactory processing that they perform. To get an understanding of the character of the glomerular interactions involved in olfactory processing we combined whole-cell patch clamp recordings and odor stimulation with simultaneous calcium imaging of single LNs. Spiking type I LNs express different branching patterns in different glomeruli suggesting a polar organization with defined input and output regions. This suggests that synaptic input from a defined ‘receptive field’ (e.g., one or a few glomeruli) is integrated into action potential firing. In turn the action potentials can spread to other glomeruli and provide a defined array of glomeruli with synaptic input. In optophysiological recordings this is reflected in identical glomerular tuning curves of every imaged glomerulus of a type I LN. In this model, glomeruli can interact independently of their distance: not only nearest-neighbor glomeruli can interact, but also glomeruli that are distributed throughout the entire antennal lobe. Nonspiking type II LNs have very similar branching patterns in all glomeruli, suggesting that they can receive synaptic input from all innervated glomeruli. During odor stimulation synaptic input is typically restricted to a few glomeruli, in which graded postsynaptic potentials are generated. These potentials can spread only within the same glomerulus or to glomeruli that are electrotonically close to the stimulated glomerulus. Accordingly, glomeruli of type II LNs show individual odor specific tuning curves, suggesting spatially restricted, intraglomerular signaling. Galizia C.G. Universität Konstanz, Germany Olfaction: a review across the ages

The ESITO 2017 marks my personal 20 year anniversary, since I attended ESITO for the first time in 1997. What did we discuss then, what are the advances since? I will focus on three big questions: what is the role of combinatorial coding? how plastic is the code? and how fast is the code? These questions were relevant 20 years ago, and controversially discussed. They are relevant still – though many details have been solved. Most data will be from honeybee research, but other insects will also be featured. Paoli M., Münch D., Haase A., Skoulakis E., Turin L., Galizia C.G. Universität Konstanz, Germany Minute impurities contribute significantly to olfactory receptor ligand studies: tales from testing the vibration theory

Several studies have attempted to test the vibrational hypothesis of odorant receptor activation in behavioral and physiological studies using deuterated compounds as odorants. The results have been mixed. Here we attempted to test how deuterated compounds activate odorant receptors using calcium imaging of the fruit fly antennal

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lobe. We found specific activation of one area of the AL corresponding to inputs from a specific receptor. However, upon more detailed analysis, we discovered that an impurity of 0.0006% ethyl acetate in a chemical sample of benzaldehyde-d5 was entirely responsible for a sizable odorant-evoked response in Drosophila melanogaster olfactory receptor cells expressing dOr42b. Without gas chromatographic purification within the experimental setup, this impurity would have created a difference in the responses of deuterated and non-deuterated benzaldehyde, suggesting that dOr42b be a vibration sensitive receptor, which we show here not to be the case. Our results point to a broad problem in the literature on use of non GC-pure compounds to test receptor selectivity, and we suggest how the limitations can be overcome in future studies. Kraft N., Spaethe J., Rössler W. & Groh C. University of Würzburg, Biozentrum, Behavioral Physiology and Sociobiology (Zooloogy II), 97074 Würzburg, Germany Plasticity of synaptic organization in olfactory integration centers of the adult bumblebee brain

In many social insects factors such as age, task or body size are associated with structural changes within the brain. Bumblebees represent ideal model organisms for investigating structural neuronal plasticity in the brain in relation to these factors as the worker caste does not exhibit a strictly age-related task allocation. This suggests that possible structural neuronal changes might be primarily correlated with general maturation rather than specific changes in task performance. Furthermore, bumblebee workers exhibit a distinct size polymorphism, which allows for the investigation of a possible correlation between body size and the synaptic organization of the olfactory MB calyx lip region in the adult brain. Bumblebee colonies were reared in constant darkness (DD 24 h, 25° C, 50 % rH), and freshly emerged workers were age-marked every day. At defined ages, brains of workers were dissected for whole-mount synapsin immunolabeling. Using confocal laser scanning microscopy and the 3D reconstruction software AMIRA, we combined volume measurements of the olfactory MB calyx lip with the quantification of synapsin-positive presynaptic boutons. Our results show that the volume of the MB-calyx lip did not differ between different age groups, but age-related effects became evident at the synaptic level. Age had a significant effect on the density of presynaptic boutons in the lip, in particular a significant pruning of MG already within the first two days of adult life. The lip volume and the total number of olfactory synaptic boutons positively correlated with body size, while their density remained constant, supporting the idea that size differences between bumblebee workers are in fact isometric, even at the synaptic level. Exposure to light did not affect volume or synaptic organization in olfactory and visual calyx MB subcompartments. We conclude that in contrast to honeybees and ants investigated so far, neuroanatomical maturation in the bumblebee proceeds faster suggesting that these structural changes might be correlated with a fast maturation high flexibility in behavior. Supported by the DFG, SFB 1047 “Insect timing” (projects B3, B5 and B6) to JS, CG, and WR.

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Halty-deLeon L. & Wicher D. Max Planck Institute for Chemical Ecology (Jena), Germany Investigating the role of the Drosophila Na+/Ca2+ exchanger CALX in the odor response

Flying insects can detect faint odor traces thanks to an especially sensitive class of olfactory receptors called odorant receptors (ORs). These receptors are heteromeric complexes formed by a specific ligand-binding subunit (OrX) and a highly conserved co-receptor protein (Orco). The ORs are expressed in the plasma membrane of olfactory sensory neurons (OSNs) and function as non-selective cation channels which are tuned by intracellular signalling systems. In the OSNs, Ca2+ plays a major role in regulatory processes such as activation and termination of the odor response. OR stimulation leads to an influx of Ca2+ ions which act as a second messenger in signalling cascades. However, too high intracellular Ca2+ levels are potentially dangerous for a cell. The Na+/Ca2+ exchanger (NCX) is one of the most important Ca2+ extrusion mechanisms. In mammals, NCX acts as a bidirectional transporter. In its efflux mode (physiological conditions) it exports one Ca2+ ion for the uptake of 3 Na+. During pathological conditions or after agonist receptor stimulation, however, the reversed mode is triggered. This results in the uptake of Ca2+ and extrusion of Na+. Interestingly CALX, the NCX homolog in Drosophila, exhibits an anomalous regulation being inhibited by increasing intracellular calcium concentration [Ca2+]i. Disruption of Ca2+ extrusion might have an important effect on the response profile of OSNs. Using calcium imaging we are studying the influence of CALX in the response of ORs after agonist stimulation of the co-receptor Orco both in ex vivo preparations and heterologous systems. This study was supported by the Max Planck Society. Harzsch S. University of Greifswald, Zoological Institute and Museum, Department of Cytology and Evolutionary Biology, Soldmannstrasse 23, D-17498 Greifswald, Germany Evolution of olfaction in Crustacea and a crustacean perspective on insect olfactory systems

Malacostracan crustaceans display a large diversity of sizes, morphs and life styles. However, only a few representatives of the decapod taxa have served as models for analyzing crustacean olfaction, such as for example crayfish and spiny lobsters. Crustaceans bear multiple parallel chemosensory pathways represented by different populations of unimodal chemosensory and bimodal chemo- and mechanosensory sensilla on the mouthparts, the walking limbs and primarily on their two pairs of antennae. Here, I will focus on the olfactory pathway associated with the unimodal chemosensory sensilla on the first antennal pair, the aesthetascs. I will explore the diverse arrangement of these sensilla across malacostracan taxa and point out evolutionary transformations which occurred in the central olfactory pathway. The evolution of chemoreceptor proteins, comparative aspects of active chemoreception and the temporal resolution of crustacean olfactory system will be discussed. Viewing the evolution of crustacean brains in the light of energetic constraints can help us understand their functional morphology and suggests that in various crustacean lineages, the brains were simplified convergently because of metabolic limitations. Comparing the wiring of afferents, interneurons and output neurons within the olfactory glomeruli suggests a deep homology of insect and crustacean olfactory systems. However, both taxa followed

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distinct trajectories during the evolutionary elaboration of their olfactory systems. A comparison with insects suggests their olfactory systems - especially that of the vinegar fly - to be superb examples for “economy of design”. Such a comparison also inspires new thoughts about olfactory coding and the functioning of malacostracan olfactory systems in general. Supported by DFG grants Ha 2540/14-1 and 16-1. Harzsch S., Sombke A., Beltz B.S. & Hansson B.S. Zoological Institute and Museum, Department for Cytology and Evolutionary Biology, Ernst-Moritz-Arndt-University of Greifswald, Soldmannstraße 23, D-17487 Germany Persistent neurogenesis in the adult crustacean brain: the neurogenic niche in Coenobita clypeatus and Birgus latro (Anomala, Coenobitidae)

For almost 20 years it is known that neurogenesis persists in the central olfactory pathways of adult decapod crustaceans. In the brains of e.g. crayfish, clawed lobsters, and spiny lobsters it has been shown that distinct proliferation zones generate new olfactory interneurons associated with the primary olfactory centers and that the newly born neurons in fact survive and are integrated into the existing circuitry of the central olfactory pathway. Furthermore, in crayfish adult neurogenesis is driven by a neurogenic niche associated superficially with the brain. The niche is a spherical cluster of cells with glial characteristics and a core of extracellular matrix (cadherins), and dextran injections into the crayfish circulatory system showed that the niche is associated with the vascular system. In these animals, fibrous migratory streams from the neurogenic niche serve as pathways from which neuronal precursors from the niche travel towards the two proliferation zones associated with the clusters of olfactory interneurons, collectively called the “deutocerebral proliferative system”. We wanted to know if persistent neurogenesis also characterizes the central olfactory pathway of terrestrial crustaceans. To that end, we analyzed terrestrial hermit crabs of the Coenobitidae with S-phase specific proliferation markers and immunohistochemistry against tubulin, synaptic proteins and actin. Our results suggest that in these animals all components of the deutocerebral proliferative system are present so that persistent neurogenesis characterizes not only the olfactory systems of crustaceans living in aquatic but also in terrestrial environments. Supported by DFG grant Ha 2540/16-1 and the Max Planck Society. Hillier N.K., Rizzato A.R., MacKay C., McGuire M. & Rose S. Biology, Acadia University, 33 Westwood Avenue, Wolfville, Nova Scotia B4P 2R6, Canada Pheromone blend complexity in heliothine moths

Heliothine moths are ubiquitous, representing some of the most serious agricultural pests on the planet, causing massive annual crop losses particularly in the developing world. We are developing a global initiative to investigate comparative evolution of olfactory systems in this important group of agricultural pests to develop this species complex as a model system for evolution of complex pheromone communication. Using comparative physiology, chemistry and molecular tools to characterize odorant receptor shifts associated with communication, we are developing a comprehensive comparative database of olfaction for this group. The long term goal of this study will be the establishment of a global network of collaborators using heliothines as a model system to correlate shifts in communication manifested by differential olfactory receptor (OR) expression with larger trends in speciation (both sympatric and allopatric) and

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evolution in insects. Overall, this initiative will determine genetic factors mediating shifts in female pheromone production, male behavioral pheromone preference and physiological processing of these odorants as these species diverge. Ichinose T., Kanno M., Aso Y. & Tanimoto H. Niigata University, Tohoku University, Japan Neural circuits that distinguish memory processes in the fly brain

Memory acquired from salient experiences guides future behavior. Fruit flies Drosophila melanogaster bidirectionally adapt the response to an odor based on appetitive and aversive memories of sugar reward and electric shock punishment, respectively. Accumulating evidence suggest that the output from a brain structure called the mushroom body (MB) is necessary for acquisition, consolidation, and retrieval of appetitive and aversive olfactory memories. However, it is largely unclear how the MB circuit operates these distinct memory processes. To comprehensively characterize the MB circuit usage in different processes, we measured the behavioral significance of each MB output neurons for all of the documented cell types. Thereby we found that different sets of the MB output neurons are recruited in appetitive and aversive memories. Our comprehensive behavioral analysis revealed circuit motifs for processing these two different memories. KC P., Reitstøen Arnesen M. & Berg B.G. Norwegian University of Science and Technology, Norway Complete mapping of central projections from the labial pit organ of the noctuid moth, Helicoverpa armigera

Moths detect carbon dioxide (CO2) via sensory neurons housed inside the labial pit organ (LPO), localized on the outermost segment of the labial palp. Previous studies have reported that these neurons project to three main parts of the central nervous system: 1) the labial pit organ glomerulus (LPOG) in both antennal lobes, 2) the gnathal ganglion, and 3) the ventral cord (Kent et al. 1984; Zhao et al. 2013). Whereas the main target area, the LPOG, has been thoroughly described previously, less is known about the terminals ending in the two remaining regions. In the study presented here, we performed mass staining from the LPO combined with confocal scanning for analyzing more thoroughly the CO2 projections. The detailed projection pattern in the gnathal ganglion, described for the first time, showed a dense network of thin terminals targeting the ipsilateral region, close to the midline of the ganglion, plus a few processes in the antennal mechanosensory and motor center (AMMC). The projection pattern in the ventral cord, described for the first time as well, demonstrated relatively extensive processes in the ipsilateral part of the first thoracic ganglion. In addition, a few axons innervated the second thoracic ganglion. According to previous data reporting about two morphological types of sensilla in the LPO, one located more peripherally than the other (Zhao et al. 2013), we also performed distinct mass labeling experiments by applying dye to the tip and the middle segment of LPO, respectively. Staining from the tip showed axonal bundles projecting to the gnathal ganglion and the ventral cord exclusively whereas dye application to the middle part of the LPO gave rise to the well-known projection pattern including the LPOG as well. Finally, application of two dyes, one in the antenna and one in the LPO, showed no overlapping terminals.

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Khallaf M.A., Dweck H., Knaden M. & Hansson B.S. MaxPlanck institute for chemical ecology, Jena, Germany A male specific pheromone mediate the sexual and social behavior in D. mojavensis

Mating bahaviors in Drosophila are driven by olfaction. Once flies land a food patch colonized by conspecifics, males almost immediately court females. Males of the melanogaster-species clade generically produce male-produced cis-vaccenyl acetate (cVA) which induce female receptivity and functions as an aggregation pheromone. The males transfer it to females during mating as an anti-aphrodisiac signal for further males. However, little is known what regulates the social and sexual behaviors in the non cVA-producing species, like cactiphilic Drosophila mojavensis. In this study we identify previously anonymous male-specific compounds which D. mojavensis males transfer to females during copulation. From single molecules and genes, to neurons, to behavioral responses, we could dissect the sex pheromones evolution in closely related species living under different ecological conditions. Our results increase the understanding of the evolution of Drosophila pheromones and how reproductive isolation barriers between species are created mainly by species-specific signals. Kleineidam C.J. & Neupert S. University of Konstanz, Germany Aggressive ants in social context

Recognizing nestmates (members of the same colony) and discriminating against conspecific individuals from foreign colonies is a fundamental ability of social insects. It is crucial for the emergence of colony coherence and ultimately benefits colony fitness. Colony coherence is based on individual decisions of the colony members in encounters with other ants. At the individual level, recognition and discrimination is based on colony specific profiles of cuticular hydrocarbons (CHC label) that are represented in the olfactory centers of the ants’ brain. I will present our latest results on the neuronal mechanisms underlying nestmate recognition and the amazingly fast classification of complex and changing colony odor cues. In behavioral experiments, we investigated how modulation of the nestmate profile (containing an additional or removing one component) affects recognition. Our results support the idea that the CHC label is perceived as an entity where both, added or subtracted components render the nestmate label as non-nestmate specific. In further behavioral experiments we address the importance of nestmate recognition cues for division of labor. I will demonstrate how interactions with nestmates provide the social context for task allocation by modulating the individual propensity to react aggressively to non-nestmates. I will discuss our current view on nestmate recognition by combining conclusions from behavioral experiments, modeling approaches and neurophysiological data. Radermacher J., Klussmann J., Bardos V., Nawrot M. & Kloppenburg P. Biocenter, Institute for Zoology, CECAD, University of Cologne, Germany Functional analysis of interneurons and projection neurons in the antennal lobe network of the cockroach Periplaneta americana

In the insect antennal lobe (AL) processing of olfactory information is accomplished by a highly interconnected recurrent network of interneurons. This interaction in the AL shapes olfactory representations, e.g. regulating the tuning profile of projection neurons. In the cockroach Periplaneta americana we distinguish two broad

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classes of spiking (Type I) and non-spiking (Type II) interneurons. A relative small number of projection neurons represent the AL output and project to higher brain centers. This small projection neuron population encodes olfactory information with a dense spatiotemporal odor code. The processing in the AL network not only reduces the spatial dimension - from a high dimensional input of many thousands of input neurons to a small population of output neurons - but also processes fine temporal structure of the olfactory input within few milliseconds to compute and construct the spatiotemporal population code of projection neurons. Characterizing the functional properties of AL neurons is crucial for understanding odor response processing in this network. Here we investigate the intrinsic cell properties of projection neurons, type I and type II local interneurons using current clamp recordings. To this end we employ two complementary approaches. The traditional approach derives active and passive cell parameters from responses to constant current injections, through a wide array of hyperpolarizing and depolarizing protocols. In a model-driven approach we perform noise current injections in order to fit a generalized integrate-and-fire neuron model and add on to the characterization of AL neurons. Such inputs allow us to take into account the fluctuating nature of synaptic inputs. Our results indicate functionally relevant mechanistic differences for the AL neuron types - such as different spike frequency adaptation behaviors - and we anticipate this work to ground realistic models of AL neurons in P. americana. Knaden M. & Huber R. Max Planck Institute for Chemical Ecology, Jena, Germany The capacity and storage duration of the olfactory memory in the desert ant Cataglyphis fortis The desert ant Cataglyphis fortis inhabits the open saltpans of Tunisia. The individually foraging Cataglyphis workers cover long distances and use path integration as well as visual and olfactory cues to pinpoint their nest entrance during homing. The ants localize their food, mainly dead arthropods, by following odor plumes. Here we show, that the ants can associate an otherwise neutral odor with food. After a single experience, an odor, that does not attract naïve ants provokes, plume following in trained ants. Furthermore, the ants are able to memorize multiple odor-food associations and do so for several weeks. Obviously individual ants store food-odor related information that they gained during their foraging trips for their entire lifetime.

Koutroumpa F.A., Unbehend M., Groot A.T., Dekker T. & Heckel D.G. University of Amsterdam, IBED, Netherlands Characterization of candidate genes underlying variation in male sex pheromone response in Ostrinia nubilalis

Moths possess a unique sexual communication system with great interest for the evolution of reproductive isolation: females attract conspecific males by using a species-specific sex pheromone blend. Although the male response is governed by the pheromone receptor genes, little is known about how other genes enable evolution of male response. We study the European Corn Borer, Ostrinia nubilalis that consists of two sex pheromone strains, the males of which respond to a 99:1 vs. a 3:97 ratio of the E and Z isomers of the female pheromone. The male response is governed by a single, sex-linked locus. Using a QTL mapping approach, we previously found (Koutroumpa et al. 2016 PNAS) that the

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chromosomal region most tightly linked to this locus contains genes involved in neurogenesis but, in accordance with an earlier study (Lassance et al. 2011 Evolution), does not contain the odorant receptors expressed in the male antenna that detect the pheromone. The aim of our current study is to compare the expression of the candidate genes and the pheromone receptor genes at different levels of the development of the moth’s olfactory system. Three of these candidate genes are expressed almost exclusively at the antenna level and in the olfactory sensilla. One of these has been implicated in differentiation of olfactory sensory neurons in Drosophila; the other two candidates have no known role in the olfactory system. A diversity of mechanisms appears to be responsible for the behavioral response differences among the males in this system. Blankenburg S.1, Zielonka M.2, Pregitzer P.2 & Krieger J.1 1. Martin Luther University Halle-Wittenberg, Institute of Biology/Zoology, Department of Animal Physiology, 06120 Halle, Germany 2. University of Hohenheim, Institute of Physiology, 70599 Stuttgart, Germany Molecular equipment of sex pheromone-responsive sensilla in adult and larval stages of the moth Heliothis virescens

Female moths release species-specific sex pheromone blends to attract potential mating partners over long distances. Data we have accumulated for the tobacco budworm, Heliothis virescens, indicate that on the male antenna the recognition of distinct sex pheromone components is based on an interplay of specific pheromone receptors (PRs) in the dendrites of olfactory sensory neurons (OSNs) and specialized pheromone binding proteins (PBPs) in the lymph of hair-like sensilla housing the OSNs. In the detection process, the “sensory neuron membrane protein 1”(SNMP1) appears to play a crucial role by operating as docking site for PBP/pheromone complexes and/or by contributing to the transfer of pheromones to PRs. Previously, we identified a second SNMP type (SNMP2) in male antennae that is expressed in support cells which are associated with SNMP1-expressing OSNs in the same sensillum. The differential expression of the two SNMP subtypes suggests distinct functions of the proteins; while SNMP1 is indicated to operate in pheromone detection, SNMP2 might be involved in pheromone clearance of the sensillum lymph. Electrophysiological and behavioural studies have shown that also the females themselves and moth larvae have the ability to recognize the female-released sex pheromone components. By comparative analyses of the molecular equipment of pheromone-responsive sensilla on the antenna, we revealed evidence that the recognition of distinct pheromone components in the two sexes and different developmental stages is based on the same PRs, PBPs and SNMP1. To assess whether a differential expression of SNMP1 and SNMP2 is also realized across sexes and developmental stages, we have investigated the cells expressing the two SNMP-types in the antennae of adult females and of moth larvae. This work was supported by the Deutsche Forschungsgemeinschaft SPP1392

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Frei J.1, Kröber T.1, Troccaz M.2*, Starkenmann C.2 & Guerin P.M.1 1 Institute of Biology, University of Neuchâtel, Rue Emile-Argand 11, 2000 Neuchâtel, Switzerland. 2 Firmenich S.A., Corporate Research and Development Division, PO Box 239, CH-1211 Geneva 8, Switzerland * present address: Vifor Pharma, 22, rue du Bois-du-Lan, P.O. Box 368, CH-1217 Meyrin 1 / Geneva, Switzerland Behavioural response of the malaria mosquito, Anopheles gambiae, to human sweat inoculated with axilla bacteria and to two volatiles specific to human axillary odour

A dual-choice olfactometer was used to record the responses of Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) to odours from human axillary sweat incubated with human axilla bacteria. Staphylococcus epidermidis was selected for its low odour-producing pattern, Corynebacterium jeikeium for its strong Nα-acylglutamine aminoacylase activity liberating carboxylic acids like (R)/(S)-3-hydroxy-3-methylhexanoic acid (HMHA) and S. haemolyticus for its capacity to liberate sulphur-containing compounds including (R/S)-3-methyl-3-sulfanylhexan-1-ol (MSH). Experiments were carried out in presence of regular and synchronized CO2 pulses in both olfactometer arms as a behavioural sensitizer. The behaviour of An. gambiae was analysed under following criteria: (1) take off and sustained upwind flight and (2) discrimination between the two olfactometer arms bearing a test odour in either one or both arms. An. gambiae clearly discriminated for the olfactometer arm conveying odor generated by incubating sweat with any of the three bacteria species. When presented on its own, the two molecules HMHA and MSH suspected to be unique to human sweat did not elicit a dose-dependent response for one side of the olfactometer but did affect mosquito responsiveness. This suggests that they may not to be involved in the anthropophilic behaviour of An. gambiae. These findings may serve as a basis for further research on the odour-mediated anthropophilic host-seeking behaviour of An. gambiae. Reference: J Frei, T Kröber, M Troccaz, C Starkenmann, PM Guerin (2017) Behavioral response of the malaria mosquito, Anopheles gambiae, to human sweat inoculated with axilla bacteria and to volatiles composing human axillar odor. Chemical Senses, 42 (2); 121–131. Locatelli F.1,2 , Gascue F.1 & Pírez N.1,2 1. Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina 2. Instituto de Fisiología Biología Molecular y Neurociencias, UBA-CONICET, Argentina Sensory adaptation and its effect on detection of minor components in odor mixtures

Animals process and perceive environmental information in flexible ways. Some changes in perception are sustained and involve learning and memory processes while others occur quickly and are only transient. In this framework, sensory adaptation is defined as the phenomenon by which the sensitivity to a stimulus decreases after a sustained exposure to it. This phenomenon is characterized by a rapid loss of sensitivity and full recovery within a few seconds after the stimulus disappears. Curiously, this phenomenon has been mostly described and studied by focusing on what the animal fails to perceive, but not on the consequences that it has on the perception of stimuli for which the animal has not experienced adaptation. In this project we study the enriching effect

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that sensory adaptation has on the ability of animals to detect stimuli to which it has not been adapted and that would remain overshadowed by dominant stimuli under normal conditions. We use honey bees Apis mellífera that have a high capacity to learn and recognize odors. By doing behavioral experiments we show that this phenomenon reduces appetitive learning of adapted stimuli while it favors learning of minor components that would normally stay occluded. By doing calcium imaging experiments, we determined that activation patterns that encode mixtures in the antennal lobe are drastically altered after sensory adaptation, in a way that favors the representation of stimuli that are present at sub-threshold concentrations. The results obtained so far emphasize that sensory adaptation is a fundamental mechanism to increase the sensitivity of the animal and not to reduce it. Mansourian S. & Stensmyr M. Department of Biology, Lund University, Sweden Identification of natural odor ligands for Drosophila from the ancestral range

Flies do not detect odors randomly but respond to odors associated with their ecological needs (Hansson & Stensmyr 2011). From an ecological perspective, the general odor world of the animal’s ancestral habitat have to be sought, and the relevant odor ligands identified. In the case of Drosophila melanogaster, the original habitat is found in Southern Africa, and more precisely in Miombo and Mopane forests (Pool et al., 2012). I will here present results from our ongoing efforts to elucidate the chemical ecology of the vinegar fly. Meslin C., de Fouchier A., Caballero-Vidal G., Bastin-Héline L., Monsempès C., François M.-C., Walker W.B., Bretaudeau A., Legeai F., Robin S., Montagné N. & Jacquin-Joly E. INRA, France Functional and evolutionary dynamics of odorant receptor gene family in Noctuidae species

By combining transcriptomics and genomics, we identified the complete repertoire of odorant receptors of the noctuid Spodoptera littoralis, a polyphagous pest found in Africa, South-West Asia and the Mediterranean Basin. In a previous work, heterologous expression in Drosophila antennae coupled to electrophysiology was used to functionally characterize numerous of these receptors, which appeared to be narrowly tuned to ubiquitous plant volatiles at low, relevant odorant titres. By using data from newly available genomes of noctuid moths, we reconstructed a phylogeny of the whole odorant receptor family in the Noctuidae. This phylogeny highlighted the dynamics of evolution of the family, exhibiting gains and losses of genes, duplication events, rapid evolution as well as potential adaptive evolution and functional radiation of the different clades. This work contributes to understand how insect species evolved an OR repertoire that fits their ecology. Miazzi F., Hansson B.S. & Wicher D. Max Planck Institute for Chemical Ecology, Jena, Germany Functional properties of Drosophila melanogaster olfactory sensory neurons

Insect olfactory organs house olfactory sensory neurons (OSNs), the first site of odor detection and subsequent information processing. The OSNs are polarized cells; they possess a single dendritic process dividing in multiple olfactory cilia equipped with

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olfactory receptors. Insect olfactory organs are everted, but protected by a cuticle layer that reduces the ability to access the neurons they house. The antennae of the vinegar fly Drosophila melanogaster make no exception. Although their cuticle is thin enough to enable researches to record extracellular activity via single-sensillum recordings or to perform functional imaging from the antennal surface, other approaches such as intracellular recordings and sub-cellular resolution functional imaging remain difficult. There is currently no established protocol to isolate the fly OSNs and cultivate them in vitro. In order to better access these cells and to study their functional properties, we recently developed an ex vivo preparation to perform functional imaging from the fly OSNs with subcellular spatial resolution. We successfully used it to investigate the role of calmodulin in modulating the response of odorant receptors following weak repeated odor stimulations. We found that calmodulin inhibition was affecting the sensitization only within the olfactory cilia but not in other cellular compartments, suggesting that there are various mechanisms that regulate the function of olfactory receptors and the processing of the odor information in insect OSNs. As a next step in order to gain access to these neurons, we are currently developing methods to gently dissociate the antennal tissue and to embed it on a support. We show that the dissociated neurons retain their complete morphology including their olfactory cilia, together with their functionality. This opens the possibility to use new approaches to study how olfactory signal transduction is accomplished in the olfactory cilia and to investigate the fly OSNs with higher detail. Montagné N., Koutroumpa F., Meslin C., Bretaudeau A., Legeai F., Robin S. & Jacquin-Joly E. Institute of Ecology and Environmental Sciences of Paris, France Large expansion of gustatory receptors in the genome of the noctuid moth Spodoptera littoralis

The evolution of chemosensory receptors is expected to play an important role in the adaptation of insects to a diversity of ecological niches. Whereas the function and the evolution of olfactory receptors have been the focus of intense research in the last years, the gustatory receptors (GRs) have received less attention. GRs are expressed in taste organs and are believed to detect non-volatile molecules such as sugars and bitter compounds found on food sources and oviposition sites. We annotated the repertoire of candidate GRs in the genome of Spodoptera littoralis, a polyphagous noctuid moth. We found a massive expansion of GRs, with more than 200 genes annotated. Most of these genes were found in large gene clusters, suggesting recent and repeated tandem duplications. Phylogenetic analyses revealed that this expansion occurred in candidate bitter receptor clades, and is not restricted to S. littoralis as a large number of candidate bitter receptor genes arranged in clusters could also be identified in closely related species. GR expansion could thus constitute a hallmark of Noctuidae, the most diversified family of Lepidoptera. In order to investigate expression patterns of GR genes, we also carried out a transcriptomic study on different taste organs of S. littoralis, and compared expression patterns with those observed in other lepidopteran species. This work lays the foundation for future studies on the link between the evolution of GRs and the adaptation to polyphagy in Noctuidae.

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Münz T.1, Bauer V.1&2, Stigloher C.³ & Rössler W.1 1 University of Würzburg, Biozentrum, Behavioral Physiology and Sociobiology, Am Hubland, 97074 Würzburg, Germany ² Heinrich Heine University Düsseldorf, Institute of Evolutionary Genetics, Universitätsstraße 1, 40225 Düsseldorf, Germany 3 University of Würzburg, Biozentrum, Division of Electron Microscopy, Am Hubland, 97074 Würzburg, Germany CLEM - Approaching the next level of olfactory synaptic plasticity in the mushroom body calyx

Colonies of social insects consist of thousands of individuals and have often been described as “superorganisms”. The workers, as the main individual ‘unit’ for the functioning of the superorganism, show a highly adaptive behavioral repertoire enabling the colony to express emergent responses to varying environmental conditions. Variations in behavior as well as learning and memory processes on an individual level have been shown to be associated with plastic neuronal changes in the brain (e.g. olfactory stimuli processing brain centers). Such changes are not only triggered by sensory stimuli but are also linked to age- and task-related maturation processes. Therefore, a detailed understanding of the underlying neuronal circuits and the localization of key proteins playing a role in synaptic plasticity and memory formation represent fundamental prerequisites to gain insights in the neuronal and molecular processes mediating behavioral plasticity. Thus far, high-resolution information on neuronal structures exhibiting synaptic plasticity such as the prominent microglomerulus complex synapse in the mushroom body calyx (a higher sensory integration as well as learning and memory center) mainly derives from electron microscopy (EM) and confocal laser-scanning microscopy. Both methods, however, have certain disadvantages: while EM in combination with immunogold labeling allows for precise protein detection in specific cell compartments, it clearly lacks possibilities for advanced colocalization analyses. In contrast, conventional confocal fluorescence microscopy offers the tools for multi-protein labeling, but lacks the high resolution of EM for subcellular detection levels. In the present study we applied and adapted, for the first time, array tomography (Micheva and Smith, Neuron, 2007) for use in the brain of the honeybee and ants (Markert et al., Correlative Light and Electron Microscopy III, 2017). This technique combines the advantages of two worlds by correlation of images taken from the same ultra-thin resin sections with high-resolution EM and super-resolution fluorescence microscopy (structured illumination microscopy – SIM). We established a protocol using high-pressure freezing and LR-White embedding that preserves the tissue in a close to in-vivo status allowing for ultrastructural analyzes while leaving antigens intact. The obtained overlay of EM and fluorescent images of ultra-thin serial sectioned tissues provided high-resolution 3D morphological models of synapses with the additional super-resolution 3D information of synaptic protein localization. This approach allowed us to push the limits of the so far achieved resolution in all three dimensions and the analysis of pre- and postsynaptic structures with respect to the distribution of synaptic proteins (e.g. synapsin, pCaMKII) in the mushroom body neuropil. With the additional possibility to elute and re-label sections several times before EM imaging, array tomography offers the chance to create reference models for the 3D localization of a bibliography of synaptic proteins. In the future array tomography can easily be adapted for the characterization of other neuropils and for other insect species. It seems likely that this combination of ultrastructural functional synapse morphology in combination with high-resolution

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protein localization will take neuroanatomical analyses to new levels. Supported by DFG SFB 1047 (B6) to WR. Nawrot M.P.1,2, Haenicke J.1,2, D’Albis T.2, Yamagate N.3, Menzel R.3 & Strube-Bloss M.4 1) Computational Systems Neuroscience, Institute of Zoology, University of Cologne 2) Bernstein Center for Computational Neuroscience Berlin 3) Institute for Biology – Neurobiology, Freie Universität Berlin 4) Department of Behavioral Physiology & Sociobiology, Biocenter, University of Würzburg Multi-site plasticity underlying olfactory learning in the honeybee – physiological evidence and computational model

We studied plasticity at two sites within the honeybee mushroom body using an appetitive olfactory conditioning paradigm. In a first set of experiments we investigated plasticity in a cluster of mushroom body output neurons (MBONs) using extracellular single unit recordings. Following classical conditioning MBONs encode the odor value in the honeybee [1,2] and the fruit fly [3,4]. MBON plasticity does not take effect immediately but is evident during memory retention 3h after training. We show here that the quantitative change of single MBONs correlates strongly and significantly with the behavioral performance across individual honeybees. We conclude that the value coding at the MB output is required for behavioral decision making during memory retention. A second set of experiments studied plasticity in the projection neuron boutons that form the core of the microglomerular structures in the MB calyx using Ca-imaging. We find that individual boutons show learning-induced plasticity during retention tests 15min after conditioning. Quantitative analysis shows that the learning-induced response change to the rewarded odor correlates strongly and significantly with the behavioral performance across individual animals. The temporal profile of average response changes fits the time course of Ca activation in inhibitory MBONs that provide feedback to the calyx. We conclude that functional plasticity in the calyx takes effect rapidly after or during conditioning. Both experimental approaches demonstrate inter-individual differences in the behavioral memory retention performance that reflect the plasticity level in the same individuals. This might underlie the general observation of individuality of learning success [5,6]. Based on these and earlier experimental observations we established a computational neural network model for short-term memory. Plasticity in the interneurons of the AL allows for a de-correlation of odor response. Local plasticity of the inhibitory feedback in the calyx supports associative memory formation. Our model can reproduce the rapid learning success [6] in a range of behavioral data from elemental and non-elemental learning paradigms. [1] Strube-Bloss MF, Nawrot MP, Menzel R (2011) J Neurosci 31 [2] Strube-Bloss MF, Nawrot MP, Menzel R (2016) Proc. R. Soc. B 283 Novikova E.S. & Zhukovskaya M.I. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Russia Complete mating behavior in males Periplaneta americana in response to sex pheromone requires additional unknown volatile

It seems the mating behavior of American cockroach is well-studied. Olfactory perception of female sex pheromone by male releases alertness, antennal waving, oriented locomotion, wing-raising, backing and attempt to copulate, including in relation

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to another male (Barth, 1969). The main component of sex pheromone produced by females, Periplanone B, acts as a long distance attractant and elicit the complete sequence of the male mating behavior (Seelinger, 1985; Okada et al., 1990). Contacts with other individuals play a modulatory role, prolonging wing-raising and stimulating backing (Barth, 1964, 1969). In our studies of the effect of Periplanone B on grooming and locomotion of isolated males, we noticed the absence of the complete sequence of the male mating display, as was presented at the ESITO meeting in 2015. Our present studies showed that a small group of males (2-5 individuals) placed in a clean air blowing chamber stimulated with 10-11 g of Periplanone B rarely showed full sequences of mating behavior, although there were regular contacts with other cockroaches and males exhibited antennal waving and erect body posture. When the chamber was connected with shelter containing about 10 resting males, the individuals in the chamber and especially the newcomers from the shelter became more active and showed complete mating behavior. This effect did not depend on the number of cockroaches. The frequency of following behaviors were evaluated: erect body posture as an indicator of the exploratory behavior, wing-raising, backing, as well as body jerks. Wing-raising and backing significantly rose after connecting the chambers. Obtained data suggest that there is a volatile substance in the shelter that triggers the complete mating behavior. Chan H.K., Hersperger F., Szyszka P. & Nowotny T. School of Engineering and Informatics University of Sussex, Falmer, Brighton BN1 9QJ, UK Biophysical model of the olfactory system of honey bees predicts qualitatively different responses to mixtures

In their natural environment, insects typically encounter complex mixtures of odorants. It is an important open question whether and how responses to odorant mixtures differ from those to single components. To approach this question, we built a statistical model of the full receptor repertoire and antennal lobe of honey bees. Our model was developed to reproduce a variety of statistics of olfactory response patterns observed experimentally (Galizia, et al. (1999) Nat Neurosci), taking into account biophysical processes, such as receptor binding and activation, and spike generation and transmission in neurons. Our model can predict responses to both, single components and mixtures, and reproduces temporal and spatial features of neuronal odor representations that were not used when building it (Ditzen (2005) FU Berlin; Szyszka, et al. (2014) PNAS; Deisig, et al. (2010) J Neurophysiol). In particular, it reproduces the strong correlations among receptor neuron responses that weaken among projection neurons (Galizia, et al. (1999) Nat Neurosci), and suggests that this decorrelation is predominantly due to inhibition. Using simulations and mathematical analysis, we found that the receptor dynamics alone already lead to significant differences between the responses to mixtures and to single components, long before any neural processing takes place. The model predicts that a) the response latency of olfactory receptor neurons decreases and b) response patterns become less variable across concentrations with increasing number of chemical components in the mixture. These effects are preserved in the projection neurons. We confirm our prediction for response latencies by single sensillum recordings in Drosophila. Our results suggest that the insect olfactory system encodes mixtures more efficiently than single odorants, which resonates well with the observation that chemical signaling in nature predominantly utilizes mixtures. This work was supported by HFSP, RGP0053/2015 and EPSRC, EP/J019690/1.

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Obiero G.F., Pauli T., Hansson B.S., Niehius O. & Große-Wilde E. Max Planck Institute for Chemical Ecology, Jena, Germany Chemoreceptor gene evolution in Apoidea

Hymenoptera play important ecological roles: they are pollinators, scavengers, parasitoids, predators and invasive pests of food crops and forests. Importantly, the order exhibits transitions in lifestyles, for example solitary to eusocial, and entomophagous to phytophagous. Hymenoptera rely heavily on chemosensory detection of food sources, nests, mates and oviposition sites. Eusocial hymenoptera underwent unusual expansion of their chemosensory receptor families; there is evidence that this expansion is connected to their eusocial lifestyle. However, to test this hypothesis we need to analyse non-eusocial hymenopteran species, and only very few species that mostly are distantly related to eusocial lineages have been scrutinized. Can an expansion of chemoreceptor repertoires be explained by the emergence of eusociality? We aim to explain evolution of eusociality in Apoidea using comparative genomics and transcriptomics of chemoreceptor genes generated from a select number of non-eusocial sister species, Psenulus fuscipennis, Ampulex compressa and Cerceris arenaria. The focus of our analysis will be the chemoreceptor gene families, odorant receptors (ORs), gustatory receptors (GRs), and ionotropic receptors (IRs). Preliminary results indicate that C. arenaria possesses >300 ORs, a larger number than any analysed Apoidea. It also has clearly distinct species-specific gene family expansions. The OR coding genes are mostly organized in clusters containing 2-18 OR genes. The 9-exon OR clade, which is supposedly connected to eusociality, is present but with fewer genes in comparison to Apoidea. Overall, our preliminary results indicate that the expansion of the OR receptor family predates emergence of eusociality in Apoidea, and was likely driven by alternative selection pressures. Olsson S.B., Nordström K., Tait C. & Kaushik P. National Centre for Biological Sciences, India From insect dreams to virtual reality

One of the most important tasks for any organism is to identify objects in the world around them. All organisms must, for example, discriminate what to eat from what might eat them. Identifying complex objects in an even more complex world is a difficult task. Most insects are solitary, which means they must initially identify some objects, such as food or enemies, innately. Our group is interested in how insects identify objects across different environments, and how they can detect new introduced objects in an environment, such as invasive species. Using field assays, multivariate analysis, and physiological analyses, we have found that cosmopolitan species of hoverflies use unique combinations of visual and olfactory cues to identify flowers in tropical South India, the alpine Himalayas, or cold temperate Sweden. For the latter question, we have compared olfactory processing of host odors for different fruit-specific populations of Tephritid flies that have recently diverged in preference for various fruit within the past 300 years. Finally, we are quantitatively characterizing object identification itself using a novel chemo-visual virtual reality arena. We hope to offer a comparative approach to understand how insects parse the environment to identify objects in nature.

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Lyutova R., Eichler K., Thum A. & Pauls D. University of Würzburg, Neurobiology and Genetics, Biocenter University of Würzburg, Am Hubland, 97074 Würzburg, Germany Neuronal networks underlying associative odor memories in the Drosophila larva

During the last years, the Drosophila larva has become an attractive model organism in neuroscientific research based on the elementary organization of the central larval nervous system that consists of only about 10.000 neurons; the availability and robustness of behavioral assays, and the existence of transgenic techniques, which allow one to interfere with neuronal networks, small sets of neurons or even individually identified neurons. These advantages together with the ongoing efforts toward the reconstruction of the complete larval connectome brings a full-brain, single-cell, and single-synapse understanding of learning and memory for the first time into reach. In previous work, we could dissect neuronal networks underlying associative learning and memory in the Drosophila larva. Here, we could demonstrate that only embryonic-born Kenyon cells of the mushroom body are required for associative odor-sugar learning. Furthermore, dopaminergic and octopaminergic neurons are necessary and sufficient to mediate reward and punishment signals within the brain during learning. Now, in a new set of experiments, we paired odor exposure with optogenetical activation of mushroom body Kenyon cells to gain new insights into the connectivity of mushroom body extrinsic and intrinsic neurons important for associative learning and memory in the Drosophila larva. Jiang X., Krieger J., Breer H. & Pregitzer P. University of Hohenheim, Institute of Physiology, Stuttgart, Germany Locust odorant binding proteins: phylogenetic relationship, structural polymorphism and sensilla specific expression

Insect olfactory sensory neurons (OSNs) are enveloped by auxiliary cells and some of them secrete odorant binding proteins (OBPs), which are supposed to facilitate transportation of odorous molecules in the sensillum lymph. Currently the knowledge about locust OBPs and the OBP-expressing cells is limited. In this study we set out to investigate OBPs of the desert locust Schistocerca gregaria, a representative Orthoptera species. From an antennal transcriptome we have identified 12 classical SgreOBPs with 6 conserved cysteine residues and two plus-C OBPs. Using the newly identified sequences of SgreOBPs together with OBPs from three other locust species, we have generated a phylogenetic tree which led to the identification of 4 OBP families (I-IV). Subsequently, the families I-III were subdivided at interior branch nodes resulting in three additional subfamilies (I-A to III-A); the subfamilies I-A and II-A were investigated in greater detail. By determining the nonsynonymous substitution rates (dN), the synonymous substitution rates (dS) and the ω rate, we found a negative selection pressure was acting on OBPs of subfamilies I-A and II-A. Modeling of the tertiary structure for OBPs of subfamilies I-A and II-A resulted in subfamily-specific differences in the surface topology and interior cavity. To unravel the topographic expression pattern of OBPs from the two subfamilies in the various sensilla types, we performed RNA in situ hybridizations using riboprobes for different SgreOBPs. The results indicate that subfamily I-A OBPs are expressed in both sensilla trichodea and sensilla basiconica; in contrast, OBPs of subfamily II-A are expressed in sensilla coeloconica. These findings give first insight into the repertoire, the relatedness and the sensilla specific expression of locust OBPs, which is considered as a step to unravel the specific functional roles of different OBP-types in locust olfaction.

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Riabinina O., Potter C. & Baines R. University of Manchester, UK Live imaging of single-cell olfactory responses in Anopheles gambiae larvae

Anopheles gambiae mosquitoes are the major vector of malaria, a disease that affects more than a half of the world&#39;s population and results in more than 400.000 deaths every year (WHO report, 2016). Human body smell and CO2 from breath are the essential cues that adult mosquitoes use to find humans. Larval mosquitoes also use olfactory cues to identify sources of food and harmful elements in their habitat. Interfering with olfactory processing is thus a promising approach for limiting mosquito populations and rates of disease transmission. In order to manipulate olfactory processing in live mosquitoes, we need to have a way to specifically target olfactory neurons, and knowledge about what neurons to target. Genetic tools served both of these purposes well in Drosophila, providing us with detailed information about olfactory perception in this insect. Inspired by Drosophila research, we have very recently used the Q-system for transgene expression to develop the first transgenic line of A. gambiae that allows us to express effectors, such as GFP or a fluorescent calcium sensor GCamp, in olfactory ORCO-dependent receptor neurons. Consistent with previous reports, we found that A. gambiae larvae possess approx. 10 ORCO neurons per antenna, and 5 ORCO neurons per maxillary palp. We are now using Orco-QF2/QUAS-GCamp6 larvae as a simple aquatic genetic olfactory model. For the first time, we can record live responses of ORCO neurons to behaviourally relevant odorants. The responses of single neurons will be used to establish the neuronal basis of olfactory attraction and repulsion in the mosquito larvae. This knowledge is essential for subsequent genetic manipulations of olfactory behaviours, and for development of novel mosquito attractants and repellents. Roces F. & Arenas A. Behavioural Physiology and Sociobiology, University of Würzburg, Am Hubland, 97074 Würzburg, Germany Learning through the waste: olfactory cues from the colony refuse determine plant preferences in foraging leaf-cutting ants

Leaf-cutting ants are polyphagous herbivores that show distinct preferences in the choice of plants as substrate for their fungus. Foragers are known to reject previously accepted plants if they prove to be unsuitable for their symbiotic fungus once incorporated into the nest, a phenomenon that involves olfactory avoidance learning. Inside the nest, waste particles removed from the garden likely contain cues originating from both the unsuitable plant and the damaged fungus. We investigated whether leaf-cutting ant foragers learn to avoid unsuitable plants solely through olfactory cues available at the colony dump. We fed subcolonies of Acromyrmex ambiguus privet leaves treated with a fungicide undetectable for the ants, collected later the produced waste, and placed it into the fungus chamber of naïve subcolonies. In individual choice tests, naïve foragers preferred privet leaves before, but avoided them after waste was given into the fungus chamber. Evidence on the influence of olfactory cues from the waste on decision making by foragers was obtained by scenting and transferring waste particles from subcolonies that had been fed either fungicide-treated or untreated leaves. In choice experiments, foragers from subcolonies given scented waste originating from fungicide-treated leaves collected less sugared paper disks smelling to it, as compared to foragers from subcolonies given scented waste from untreated leaves. Results indicate that foragers learn to avoid plants unsuitable for the fungus by associating plant odours and

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cues from the damaged fungus that are contingent in waste particles. It is argued that olfactory cues at the dump enable foragers to predict the unsuitable effects of plants even if they had never been experienced in the fungus garden, and that waste particles may contribute to spread information about noxious plants for the fungus within the colony. Ruedenauer F.1,2, Strube-Bloss M.2, Spaethe J.2 & Leonhardt S.1

1 Department of Zoology III, University of Würzburg, Würzburg, Germany 2 Department of Zoology II, University of Würzburg, Würzburg, Germany Bees rely on their sense of taste to assess pollen nutritional composition

Numerous studies have investigated visitation patterns and floral preferences of bees, whereas the proximate mechanisms underlying bee foraging choices are still poorly understood. For example, it is still unknown whether and how bees assess the nutritional quality of pollen, or whether information on pollen quality is used to adjust individual foraging patterns. Assessing pollen nutritional quality would be highly beneficial for bees, because it would facilitate composing an ideal diet for their colonies. To better understand whether and how social bees assesses pollen quality, we used chemotactile conditioning of the proboscis extension reflex (PER), developed a new technique to measure electroantennogram (EAG) activity with chemotactile stimulation, and performed feeding choice assays to test whether bumblebees (Bombus terrestris) and honeybees (Apis mellifera) are able to differentiate between pollen of different nutrient concentrations. Our results show that both individual Bombus terrestris and Apis mellifera workers use their sense of taste to discriminate pollen differing in nutrient content. We also show that bumblebees are able to differentiate between pollen types and receive as well as perceive particular amino acids. However, the bees failed to discriminate between pollen types, when we manipulated the amino acid profile of pollen, but were sensitive to changes in fatty acid content. Khallaf M., Das S., Baschwitz A., Hansson B.S. & Sachse S. Max Planck Institute for Chemical Ecology, Jena, Germany Odor coding mechanism within and beyond the Drosophila antennal lobe

Animals use sensory systems to navigate the environment in a way that optimizes their survival and reproduction. The olfactory system plays here a major role in encoding chemical information and translating the outside world into a neuronal representation that enables an animal to take odor-guided decisions. The vinegar fly represents a premier model system for studying olfactory processing mechanisms since it exhibits a stereotyped architecture which is similar to its mammalian counterpart, but is less complex and highly tractable as well as susceptible to genetic manipulations. By exploiting these genetic techniques and linking them to neurophysiological, molecular and behavioral methods, we are dissecting the neural circuits underlying olfactory coding and processing in the context of odor-guided behavior in Drosophila. In order to understand higher odor processing we are scrutinizing the lateral horn (LH) of the protocerebrum, a brain region that is assumed to be involved in innate behavior. Two populations of projection neurons – excitatory (ePNs) and inhibitory (iPNs) – convey the odor information from the antennal lobe to the LH. We analyzed how different odor features such as hedonic valence and intensity are functionally integrated in this brain area. We could previously demonstrate that the LH can be classified into three functional odor response domains that decode opposing hedonic valences and odor intensity that

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derive from iPNs and third-order neurons that further innervate the ventro-lateral protocerebum. To investigate whether ePNs accomplish a comparable categorization of odor features and whether iPNs and ePNs interact, we have elucidated the neuronal circuitry of the two PN populations at a morphological and functional level. The talk will summarize our recent insights into the processing strategies of the two parallel output pathways to the higher brain and their contribution to odor perception. Scheiner R., Pölloth L. & Thamm M. University of Würzburg, Germany Modulation of taste in honeybees

Taste is an essential sensory modality, because it allows to discriminate between edible and non-edible items which may cause harm or even death. In honeybees (Apis mellifera), taste additionally appears to be involved in the regulation of social organization, because bees performing different tasks in the hive reliably differ in their taste. Furthermore, taste is an excellent physiological indicator in honeybees, because it correlates with responses to other stimulus modalities and with behaviors such as learning. Modulation of taste is a fascinating topic in insect physiology, because multiple behaviors can be affected simply by modulating the taste of an animal. We are particularly interested in the modulation of taste through biogenic amines such as tyramine. This neurotransmitter can bind to two specific receptors in honeybees, both of which have been characterized. We investigate the distribution of the tyramine receptor AmTAR1 in the brain with a specific antibody and investigate the role of tyramine in modulating sweet taste in different social roles of honeybees. Schmitt T. University of Würzburg, Germany Cuticular hydrocarbons as chemical cues for recognition processes in antagonistic interactions

All insects investigated so far exhibit a complex blend of hydrocarbons (CHC) on their cuticle. These CHC profiles are often species and sex specific and play a crucial role in intra- and interspecific recognition processes and communication. Although CHC composition and function is studied intensively, our knowledge on the evolution of this ubiquitous trait is rather scarce. In order to understand the factors shaping these complex chemical profiles, we studied antagonistic interaction networks of solitary wasps (Crabronidae, Vespidae, Chrysididae). These interaction networks involve chemical mimicry since CHCs of cuckoo wasps are suggested to be important cues for recognizing parasitization processes. In my talk I will show how the interaction between predatory wasps and their antagonists impacts the composition of complex CHC profiles and thus, potential recognition processes. Our research sheds light on the co-evolutionary processes selecting for ubiquitous chemical cues in Hymenoptera. Schuh E., Bisch-Knaden S., Hansson B.S. & Sachse S. Max Planck Institute for Chemical Ecology, Jena, Germany The ecological significance of trichoid sensilla in female silkmoths

Sensory hairs on the antenna of male silkmoths (Bombyx mori) house two neurons that are involved in long-range attraction to conspecific females via female-produced sex

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pheromones. Female silkmoths also possess these so-called trichoid sensilla, which were described to have all the same response characteristics, and to house neurons exclusively tuned to lüalool and benzoic acid, respectively. However, the specific ecological significance of those volatiles remains unknown. We therefore asked which ecological role the trichoid sensilla on the antenna of female silkmoths might play. In order to address this question, we collected volatiles of mulberry leaves, the only host plant of B. mori larvae. Furthermore, we collected the headspace of female and male moths to identify potential pheromonal compounds in addition to the female-produced sex pheromone. We analyzed these odor collections using gas chromatography, and tested the physiological activity of the most prominent compounds by single sensillum recordings from female trichoid sensilla. In total we tested a panel of 79 monomolecular odorants with a potential ecological significance for female silkmoths. In mulberry extracts we found several aromatic compounds, terpenes and a few green leaf volatiles, whereas volatile collections of female and male moths contained predominantly terpenes. Our single sensillum recordings revealed the existence of a second physiological type of trichoid sensilla, and also a broader response profile of trichoid sensilla than reported in earlier studies. The first physiological type of trichoid sensilla corresponds to the already described one, containing the “linaool cell” and the “benzoic acid cell”. However, the ‘linalool cell’ was in addition tuned to α-terpineol, cis-jasmone, and some aromatic compounds, and the ‘benzoic acid cell’ showed equally high spike frequencies to benzoic acid, 3-methyl butanoic acid and pentanoic acid as well as to benzaldehyde. Interestingly, there was one mulberry compound (indole) that elicited an inhibitory response lasting 4-5 seconds. The second type of trichoid sensilla housed two olfactory sensory neurons responding to none of the acids, but mostly to methyl salicylate, 2-phenylethanol, α-terpineol, linalool and cis-jasmone. In this second sensillum type none of the tested odorants ecilited an inhibitory response. We furthermore established a behavioural assay for female silkmoths, the y-maze, in order to investigate the significance of the identified ligands. In summary, our data show that neurons housed in trichoid sensilla of female silkmoths are more broadly tuned than previously thought, and detect mainly host plant volatiles. However, the ecological significance of the neuron that is narrowly tuned to acids is still unknown. Our behavioural results will help us to draw conclusions about the potential relevance of the identified ligands. This work is supported by the Deutsche Forschungsgemeinschaft (SPP1392). Steiner C., Bozzolan F., Montagné N., Maïbèche M & Chertemps T. Sorbonne Universités, UPMC Univ Paris 06, UPEC, INRA, CNRS, IRD, Institute of Ecology and Environmental Sciences of Paris, Paris, France Neofunctionalization of “juvenile hormone esterase duplication” in Drosophila as an odorant-degrading enzyme towards food odorants

Odorant degrading enzymes (ODEs) are thought to be responsible, at least in part, for olfactory signal termination in the chemosensory system by rapid degradation of odorants in the vicinity of the receptors. A carboxylesterase, specifically expressed in Drosophila antennae, called “juvenile hormone esterase duplication (JHEdup)” has been previously reported to hydrolyse different fruit esters in vitro. Here we functionally characterize JHEdup in vivo. We show that the jhedup gene is highly expressed in large basiconic sensilla that have been reported to detect several food esters. An electrophysiological analysis demonstrates that ab1A olfactory neurons of jhedup mutant flies exhibit an increased response to certain food acetates. Furthermore, mutant flies

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show a higher sensitivity towards the same odorants in behavioural assays. A phylogenetic analysis reveals that jhedup arose as a duplication of the juvenile hormone esterase gene during the evolution of Diptera, most likely in the ancestor of Schizophora, and has been conserved in all the 12 sequenced Drosophila species. Jhedup exhibits also an olfactory-predominant expression pattern in other Drosophila species. Our results support the implication of JHEdup in the degradation of food odorants in D. melanogaster and propose a neofunctionalization of this enzyme as a bona fide ODE in Drosophilids. Nolte A., Gawalek P., Schumann R., Werckenthin A. & Stengl M. University of Kassel, Germany Pheromone transduction in the hawkmoth Manduca sexta is not based upon an Orco-dependent ionotropic transduction mechanism but upon G-protein-coupled cascades

Manduca sexta males detect pheromones with olfactory receptor neurons (ORNs) innervating long trichoid sensilla on their antennae. Binding of Bombykal (BAL), the main sex pheromone component, to a specific olfactory receptor (OR) generates the sensillum potential which elicits phasic-tonic action potentials (APs) followed by a late, long-lasting pheromone response. Insect ORs are 7-transmebrane receptors with inverse topology compared to the family of G-protein coupled receptors (GPCRs). They occur together with a conserved olfactory receptor co-receptor (Orco). Orco locates and maintains ORs in the ciliary membrane of ORNs, termed “chaperon-function”. Additionally, Orco forms a spontaneously opening unspecific cation channel controlling spontaneous activity with its “pacemaker channel-function”. It is still under debate, whether Orco underlies an ionotropic odor transduction mechanism in all insect species, forming an OR-Orco odor-gated receptor ion channel complex. Here, we examined the function of M. sexta Orco with Ca2+ imaging and patch clamp recordings in heterologous expression in HEK293 cells, in primary cell cultures of mature ORNs, and with tip recordings in vivo of BAL-sensitive ORNs of intact hawkmoths. With the Orco agonist VUAA1, the Orco antagonists OLC15, the amilorides MIA, and HMA, as well as with antagonists of G-proteins we examined BAL transduction in hawkmoths. We found that Orco is not activated within the first 100 ms of pheromone transduction. Orco does not underlie the first, transient BAL-dependent action potential response, but activates slower during the late, long-lasting pheromone response that is maintained for seconds to minutes after pheromone exposure. In contrast, interference with G-protein- and with phospholipase C functions interferes with the primary events of the pheromone response. Thus, M. sexta does not employ ionotropic but metabotropic pheromone transduction mechanisms. Supported by DFG grants STE531/20-1,2 to MS, by SPP 1392. Strube-Bloss M. & Rössler W. Department of Behavioural Physiology & Sociobiology (Zoology II), Biozentrum, University of Würzburg, Am Hubland, 97074 Würzburg, Germany Multimodal Integration and Stimulus Categorization in Mushroom Body Output Neurons of the Honeybee

Flowers attract pollinating insects like honeybees by sophisticated compositions of olfactory and visual cues. Using honeybees as a model to study olfactory-visual integration at the neuronal level, we focused on mushroom body (MB) output neurons (MBON). From a neuronal circuit perspective MBONs represent a prominent level of sensory-modality convergence in the insect brain. We established an experimental design

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allowing electrophysiological characterization of olfactory, visual, as well as olfactory-visual induced activation of individual MBONs. Despite the obvious convergence of olfactory and visual pathways in the MB, we found numerous unimodal MBONs, conserving the layered organization of the MB input side (Calyx) However, a substantial proportion of MBONs (32%) responded to both modalities and thus integrated olfactory-visual information across MB input layers. In these neurons representation of the olfactory-visual compound was significantly increased compared to single components suggesting an additive, but non-linear integration. Population analyses of olfactory-visual MBONs revealed three output categories - (i) olfactory, (ii) visual, and (iii) olfactory-visual compound stimuli. Interestingly, no significant differentiation was apparent regarding different stimulus qualities within these categories. We conclude that encoding of intramodal stimulus properties is largely completed at the level of MB input, and information at the MB output is integrated across modalities to efficiently categorize sensory information for downstream behavioural decision processing. Tang Q., Hong Z., Wang Y., Hou W., Zhao X. & Dang J. Department of Entomology, Henan Agricultural University, Zhengzhou, China L-arabinose, a phagostimulant or a deterrent for herbivorous insects?

L-arabinose, a natural sweetener for mammals. It was reported that monomeric arabinose could be a natural pharmaceutical that could selectively inhibit intestinal maltase or sucrase activities in mammals and therefore is considered one of the weight-reducing aid in humans. Our previous works proved that other sugars, the sucrose, glucose, fructose and maltose functioned as phagostimulants, whereas L-arabinose deterred the feeding preference in caterpillar of Helicoverpa armgiera. Then we found the developmental duration of larvae and pupal duration were prolonged after caterpillars were exposed to artificial diet containing L-arabinose. It also demonstrates that the exposure of caterpillars to L-arabinose-containing artificial diet could inhibit the sucrase activities and the glucose level in midgut of H. armigera. Finally, no single L-arabinose-sensitive taste neuron was found in the taste sensillum of H. armigera catepilalrs, while taste sensilla were simulated by the mixtures of phagostimulant and L-arabinose, the sensitivity of neurons to phagostimulants decreased significantly. Therefore, our studies indicate the mammal-sensitive-phagostimulant, L-arabinose, is not a phagostimulant, but function as a non-direct deterrent for the caterpillars of H. armigera. Waddell S. University of Oxford, UK Re-evaluation of learned information reveals a role for opponency in the Drosophila dopaminergic reinforcement system

Animals constantly reassess the reliability of learned information to optimize their behavior. On retrieval, memory can be neutralized by extinction if the learned prediction was inaccurate. I will discuss our mechanistic understanding of this memory updating process. Extinction of sugar-reinforced memory requires output neurons with dendrites in the vertical lobes of the mushroom body, which drive negatively reinforcing dopaminergic neurons that innervate neighbouring zones. The aversive valence of these new extinction memories neutralizes previously learned odour preference. In contrast, extinction of aversive electric shock-reinforced memory requires output neurons with dendrites in the horizontal lobes of the mushroom body, which drive positively reinforcing dopaminergic neurons that innervate the same zones. These data suggest that

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learning establishes a valence specific imbalance in the mushroom body output neuron network, which when reactivated during memory retrieval allows the flies to re-evaluate what they have previously learned, and adjust their behaviour accordingly. Wang C.-Z., Yang K., Xu M., Wu H., Jiang X.-J., Guo H., Hou C. & Huang L.-Q. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Beijing 100101, China The cellular and molecular bases of the peripheral pheromone olfactory systems in two sister species, Helicoverpa armigera and H. assulta

Sex pheromones released by female moths are detected by the olfactory sensilla with high specificity and sensitivity in antennae of conspecific males. The two sympatric sister species, Helicoverpa armigera and Helicoverpa assulta share two major sex pheromone components, cis-11-hexadecenal and cis-9-hexadecenal, but in reversed relative concentrations, 97:3 and 7:93, respectively. We combined molecular biology, electrophysiology, optical imaging and behavior to investigate the peripheral coding mechanisms in males to their sex pheromone blends. Five pheromone receptors from the two species were identified functionally in the Xenopus laevis oocyte expression system. Specific olfactory sensory neurons (OSNs) in three types (including some subtypes) of antennal sensilla were proved to be involved in the pheromone coding. The projection of the axons of OSNs to the male specific macroglomerular complex in the antennal lobe was also compared between the two species. I will report how we assign different PRs to OSNs and types of sensilla responding to the pheromone components and the related compounds. We find that the PR orthologs in the two species do not necessarily have the same selectivity, and their species-specific changes in the tuning selectivity in male moths could be achieved with just a few amino acid mutations. We conclude that evolution of the peripheral olfactory coding mechanisms for pheromones of the two species satisfies the need of specific-species pheromone detection, which shed new light onto the reproductive isolation between these species. Witzgall P. SLU, Sweden A Drosophila female pheromone elicits species-specific long-range attraction via an olfactory channel with dual specificity for sex and food

Mate finding and recognition in animals involves social signals and habitat cues. We have discovered an olfactory channel in Drosophila melanogaster with a dual affinity to sex and food odorants. Female flies release a pheromone (Z)-4-undecenal (Z4-11Al), that elicits flight attraction in both sexes. Its biosynthetic precursor is the cuticular hydrocarbon (Z,Z)-7,11-heptacosadiene (7,11-HD), which is known to afford reproductive isolation between the sibling species D. melanogaster and D. simulans during courtship. Twin olfactory receptors, Or69aB and Or69aA, are tuned to Z4-11Al and food odorants, respectively. They are co-expressed in the same olfactory sensory neurons, and feed into a neural circuit mediating species-specific, long-range communication: the close relative D. simulans, which shares food resources with D. melanogaster, does not respond to Z4-11Al. The Or69aA and Or69aB isoforms have adopted dual olfactory traits. The underlying gene yields a collaboration between natural and sexual selection, which has the potential to drive speciation.

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Wright G., Miriyala A. & Kessler S. Newcastle University, UK The sweet tooth of the bumblebee: the temporal pattern of firing of 2 sugar-sensing neurons encodes concentration

Bees and other insect pollinators have mouthparts are specialized consuming sugary solutions such as floral nectar. Bees prefer to drink solutions high in sugar concentrations but the composition of nectar varies in concentration over a range from 8-70% (wt/vol). We predict, therefore, that the gustatory neurons in the sensilla of bees should be sensitive to changes in sugar concentration and capable of detecting sugars over a large range. Here, we report electrophysiological recordings of the population of neurons housed in the galeal sensilla of the buff-tailed bumblebee, Bombus terrestris. Surprisingly, stimulation with sucrose solutions of greater than 50mM produced a distinctive bursting pattern of firing in two neurons, in which a single action potential fired by one neuron is followed by a ~20 ms period of inhibition in a cycles of 40 Hz. This pattern of firing was also observed when galeal sensilla were stimulated with > 50 mM fructose solutions, maltose and melezitose, but only rarely with glucose or other sugars. Interestingly, this response was completely ablated by stimulation with a sucrose solution containing 1 mM quinine. Thus we show that the identity and concentration of sugars and the presence of toxins is represented by a temporal pattern of firing of the gustatory neurons from the mouthparts and propose that the brain uses this information to guide feeding behaviour. Yilmaz A.1, Dyer A.G.2,3, Rössler W.1 & Spaethe J.1 1 Department of Behavioural Physiology & Sociobiology (Zoology II), Biozentrum, University of Würzburg, Am Hubland, 97074 Würzburg, Germany 2 Department of Physiology, Monash University, Clayton, Victoria, Australia 3 School of Media and Communication, Royal Melbourne Institute of Technology, Melbourne, Victoria 3000, Australia Innate colour preference, individual learning and memory retention in the ant Camponotus blandus

Despite large interest in their olfactory-based navigational skills and various aspects of olfactory cognition, no attempt, so far, has been made to analyze the visual memory dynamics of ants after associative colour learning. In this study we investigated the colour preference, learning and memory of Camponotus blandus foragers under controlled laboratory conditions. Our results show that C. blandus foragers exhibit a strong innate preference for ultraviolet (UV, 365 nm) over blue (450 nm) and green (528 nm) wavelengths. Ants can learn to discriminate 365 nm from either 528 nm or 450 nm, independent of intensity changes. However, they fail to discriminate between 450 nm and 528 nm. Foragers trained to discriminate blue or green from UV light are able to retain the learned colour information in an early mid-term (e-MTM), late mid-term (l-MTM), early long-term (e-LTM) and late long-term (l-LTM) memory from where it can be retrieved after 1h, 12h, 24h, 72h and 7 days, respectively, after training, indicating that colour learning may induce different memory phases in ants. Overall, our results show that ants can use chromatic information in a way that should promote efficient foraging and orientation in complex natural environments. Future experiments are aimed to explore multimodal sensory influences on ant orientation, learning and memory. Supported by the Bavarian Gender Equality Grant (BGF) and the DFG SFB 1047 (B3, B6).

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Younger M.A., Ehrlich A. & Vosshall L.B. The Rockefeller University, USA Processing human cues in the mosquito brain

Female mosquitoes require a blood-meal for reproduction, and show intense attraction to human hosts. They rely on host sensory cues, including carbon dioxide (CO2), and components of human body odor, such as lactic acid. These stimuli alone elicit little or no attraction, but in combination they synergize to trigger host-seeking behavior. After obtaining a blood-meal, female host-seeking behavior is switched off for several days. It is unknown where and how any human host cues are represented in the mosquito brain. It is also unknown how human host cues synergize to drive host attraction and ultimately trigger biting behavior, or how attraction is suppressed after a blood-meal. We are examining these questions in the Zika vector mosquito Aedes aegypti. To examine activity in the mosquito brain we are generating a suite of transgenic mosquitoes that express the calcium indicator GCaMP6s in subsets of neurons. To characterize the expression of GCaMP6s in these lines, we are generating a digital 3D standard atlas of the mosquito brain on which we can register new transgenic strains. We have also established a system for imaging activity in the antennal lobe of live mosquitoes using two-photon excitation microscopy, and are mapping responses in the antennal lobe to chemosensory cues. This data will be used to generate the first odor map of the mosquito antennal lobe, and we will explore how this map is modulated after blood-feeding and in the presence of the CO2. These experiments will provide the first insights into how human cues are processed in the brain of Aedes aegypti. Klinner C.F.1, Zhang J.1, Missbach C.1, Daly K.C.2, Bisch-Knaden S.1, Hansson B.S.1† & Große-Wilde E.1*† 1. Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany 2. Department of Biology, West Virginia University, Morgantown, WV, USA Chemosensation in the ovipositor of Manduca sexta

In many moth species, females locate oviposition sites and mating partners over great distances by means of olfactory cues mainly detected by olfactory and gustatory sensilla on antenna and other organs. However, these behaviors may be also guided by sensory systems located on ovipositor in short range. We present evidence of chemosensory sensilla on the ovipositor of the hawkmoth Manduca sexta. Scanning electron microscopy identified eight to twelve porous sensilla on each of the anal papillae. HRP immunostaining indicated that these sensilla are innervated by dendrite-like structures from multiple neurons. Single sensillum recordings with a panel of 142 monomolecular odorants verified the olfaction function of some of these sensilla. Furthermore, in tip recordings we found neurons inside these sensilla also responding to both sugar and bitter tastants, indicating multi-modality.

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Zhao X.-C.1, Ma B.-W.1, Xie G.-Y.1, Tang Q.1, Berg B.G.2 & Wang G.R.3 1. College of Plant Protection, Henan Agricultural University, Zhengzhou, 450002, China. 2. Department of Psychology, Norwegian University of Science and Technology, Trondheim 7489, Norway. 3. State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China Revisiting sexual dimorphism of antennal lobe glomeruli in three species of heliothine moths

It is well demonstrated that the glomerular organization of the antennal lobe (AL) in many insect species, including moths, exhibits sexual dimorphism corresponding to the sex-specific roles in odor-evoked behaviors. For instance, the male-specific macroglomerular complex (MGC) of the AL is responsible for encoding information about pheromones emitted by the conspecific female. Female-specific glomeruli, on the other hand, located in the corresponding position of the MGC, are responsible for encoding input about odorants mediating female-related behaviors. In the heliothine moths, such sex-specific glomeruli have been identified. In the present study, three species of heliothine moths were investigated with respect to sex-specific glomeruli. By using immunostaining and three-dimensional reconstruction, complete glomerular maps of male and female in each species were reconstructed and compared. Both in males and females, about 78-80 glomeruli were identified in each AL, a number considerably larger than that previously reported in these species. In addition, we found that the sexual dimorphism includes not only the glomeruli previously identified as sex-specific, but also some of the other ordinary glomeruli. The findings presented here may lead to studies examining the AL more in detail and to identification of new glomeruli in other moth species. In addition, the complete identification and global-wide comparison of the two sexes provide an important basis for determining the function of distinct glomeruli and for understanding chemosensory mechanisms underlying reproductive behavior. Zhukovskaya M.I., Schenikova A.V., Selitskaya O.G., Grushevaya I.V., Malysh Y.M., Berim M.N., Kononchuk A.G., Kononchuk P.Y., Ryabchinskaya T.A. & Frolov A.N. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences; All-Russia Institute for Plant Protection, Russian Academy of Sciences; Agrophysical Institute, Russian Academy of Sciences, Russia Intra- and interspecific relatioships between Ostrinia moths in changing environment

The European corn borer, Ostrinia nubilalis, became a main insect pest of maize after this crop spread into Europe from America more than 500 years ago. Two pheromone races in O. nubilalis using 99:1 and 3:97 mixtures of Z- and E- stereoisomers of 11-14 tetradecenyl acetate as main pheromone components are intensively studied. However, according to Frolov et al. (2007) at least two sibling species inhabit Europe, namely O. scapulalis and O. nubilalis which prefer to feed on dicotyledonous, such as hemp and hop, and monocotyledonous, such as maize, plants accordingly. Harsh weather condition with dry and wet periods, together with temperature variations created a preadaptation to divergence of borers also. Now identified as E-race (Vinal, 1917), and Z-race (Felt, 1919) populations of O. nubilalis penetrated into North America about 100 years ago. It is interesting, that in Russia, maize also was practically free from the pest until the middle of 19th century (Frolov, 1982). Local population of sibling O. scapulalis, which share the same pheromone compounds with O. nubilalis interact with newcomers mutually shifting pheromone compositions. Eastern populations of O. nubilalis experience

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similar influences from populations of O. furnacalis. Cornfields in newly established northern area of Russia suffer from the moths which are not attracted to commercially available pheromone traps. Our field studies together with chemical analysis of female pheromone gland content and EAG testing of male antenna to pheromone compounds and blends suggest that some natural population of borer experience high level of hybridization between intruders and aborigines due to low population densities and prolonged breeding season in lower ambient temperatures. The study was partly supported by RFBR grant #16-54-00144.