ESITO IX European Symposium for Insect Taste and Olfaction

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9TH EUROPEAN SYMPOSIUM FOR INSECT TASTE AND OLFACTION (9th ESITO)

September 24-30, 2005 - Sardinia, Italy

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Abstracts 

1

Dmitry N. Akhaev¹ and Khanh D. Nguyen<sup>1

1</sup> Moscow State University, Faculty of Biology, Department of Entomology, Moscow, 119992, Russia. ahaeff@mail.ru, ndkhanmgu@mail.ru

Electrophysiological response of sensillae styloconica of Galleria mellonella L. (Lepidoptera, Pyralidae) larvae to plant sugars: sucrose, D(+)-glucose, D(-)-fructose.

A plant’s chemical composition is in many cases the most important source of information which herbivorous insects use to discriminate between host and non-host plants. Plants generally contain sucrose and its constituent monosaccharides glucose and fructose as primary metabolites resulting from their photosynthetic activity. These compounds function as strong phagostimulants to most herbivorous insects, equipped with specialized receptors to detect sugars  (Schoonhoven, van Loon, 2002). The object of our research was fifth instars larvae of the greater wax moth Galleria mellonella L. The larvae of this moth are an international pest in beehives, tunneling through the combs feeding on pollen, wax and honey which has a high concentrations of plant sugars.

Electrophysiological research of medial and lateral sensillae styloconica was carried out using a tip-recording method (Gothilf, Hanson, 1994) with mechanical immobilization on live fifth-instars larvae. The test solutions were three plant sugars (sucrose, D(+)-glucose and D(-)-fructose) of the following concentrations: 5, 10, 20, 50, 100, 200 mmol l^-1. Each of them was dissolved in distilled water containing 50 mmol l^-1 KCl to ensure adequate electrical conductance. The results show that, plant sugars didn’t produce any response in the lateral sensilla styloconica. In contrast, the medial sensillum has one phagostimulatory cell which had identified as a sugar-sensitive cell and responded to all tested sugars. The threshold of response of this cell for tested sugars was 30 mmol l^-1. The order of stimulating effectiveness for sugar cell was glucose>fructose>sucrose. The study showed  dose/response effect in concentrations of  tested plant sugars ranging from 50 to 200 mmol l^-1. The work was supported by RFBR (grant # 04-04-48779).

Schoonhoven L.M., van Loon J.J.A. (2002). An inventory of taste in caterpillars: each species its own key / Acta Zool. Acad. Sci. Hung. Vol. 48. Suppl. 1. P. 215-263

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2

Institute of Molecular Biotechnology (IMBA) Austrian Academy of Sciences, Dr. Bohr-gasse 3-5, A-1030 Vienna, Austria

^* These authors contributed equally to this work

With the aim to further understanding the logic of olfactory coding in Drosophila, we have constructed near-complete maps of odorant receptor (Or) expression in olfactory receptor neurons (ORNs) of the antenna and maxillary palp, and of ORN axon targeting to individual glomeruli of the antennal lobe. These receptor-to-neuron and receptor-to-glomerulus maps, the first for any organism, establish the general validity of the two key principles of olfactory organization: one neuron – one receptor and one receptor – one glomerulus. Our molecular maps also reveal novel features of olfactory organization in drosophila. Inputs from the antenna segregate in a topographic fashion in the antennal lobe creating a regionalized projection pattern. However within these regions the specific location of each glomerulus is stereotype but not dependant on the cell bodies location in the antenna. Combined with previous physiological data, our molecular maps also allow us to construct odor maps of the antennal lobe. Central representations of aliphatic and aromatic odors are spatially segregated, with those for aliphatic odorants arranged topographically according to carbon number. Besides giving important evidence for the principles behind the olfactory circuit this study also provide a basis for developmental and functional studies of both the antenna and the antennal lobe.

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3

Hubert Amrein

Department of Molecular Genetics and Microbiology, Duke University Medical Center

252 CARL Bldg/Research Drive, Durham, NC 27710, USA. hoal@duke.edu

Taste and Pheromone Coding in Drosophila

Drosophila melanogaster deploy chemosensory systems to evaluate chemical cues present in their environment.  Volatiles are detected by olfactory receptors (ORs) expressed in olfactory sensory neurons present in the antenna and maxillary palps whereas non-volatiles activate taste (gustatory) sensory neurons found in the labial palps, legs and wings through interaction with gustatory receptors (GRs). Unlike many vertebrates, Drosophila has no specialized pheromone sensory system, but utilizes sets of olfactory and/or gustatory sensory neurons for the recognition of pheromones. The behaviors triggered by these two sensory systems are multifaceted and include flight to and from chemicals, feeding and courtship.

We and other have performed extensive expression studies of the Drosophila Gr genes encoding G-protein coupled receptors that are thought to detect non-volatile chemicals in the environment and potential food sources.  Based on these investigations we divided the sixty Gr genes into at least four groups. Members of the first and largest group (I), represented by Gr22e and Gr66a, are expressed in 6 to 44 gustatory receptor neurons (GRN) of large (L) and intermediate (I) taste bristles in the labial palps. Interestingly, different Gr genes are expressed in partially overlapping sets of GRNs, such that some might express only one or two Gr genes, whereas others might express most group I Gr genes. Moreover, only one of the several neurons associated with a single bristle express group I genes. Finally, some group I genes are also expressed in GRNs of secondary taste organs (legs, wings, pharynx). The second group of Gr genes (II), represented by a single Gr gene (Gr5a) encoding a receptor for the sugar trehalose, is expressed in many more GRNs (up to 140) of all three bristle types and does not overlap with the expression of group I genes. Most bristles contain more than one Gr5a expressing neuron. The third set of Gr genes is not expressed in labial GRNs, but only found in neurons of secondary taste organs, such as the legs. And finally, a group of genes (IV) appears to have acquired functions other than (or in addition to that of) classical taste receptors, as these genes are expressed mainly in neurons in the brain and peripheral sensory neurons unrelated to taste.

Functional analyses of Gr genes have mostly relied on behavioral analysis of flies lacking specific sets of functional GRNs (i.e. expressing a given Gr gene). However, these investigations cannot address the specific function of specific Gr genes, because individual GRNs express often multiple Gr genes. Here, we report the generation of flies that lack individual Gr genes, using gene targeting by homologous recombination and PiggyBac-mediated gene deletion strategies. We are currently focusing on a group III gene, Gr68a, a prime candidate gene for a pheromone receptor, and the Gr39a gene cluster, which consists of four, alternatively spliced Gr genes expressed in non-identical sets of GRNs in the labial palps. Behavioral analyses of flies lacking these Gr genes indicate a role for these receptors in male courtship. We also will discuss the possibility and wisdom of a comprehensive genetic analysis of the entire Gr gene repertoire using the reported gene knock-out strategies.

4

Sergio Angeli and Stefan Schütz

University of Göttingen, Institute for Forest Zoology and Forest Conservation, Büsgenweg 3, D-37077 Göttingen.angeli@sssup.it

Purification and first characterization of soluble proteins involved in odorant recognition of Colorado potato beetle

Odorant Binding Proteins (OBP) and Chemo-Sensory Proteins (CSP) are two classes of soluble globular proteins secreted in a very high concentration in the neuron-bathing fluid of insect chemosensory sensilla. In the last ten years, members of both classes were discovered and fully characterized in several insects mainly belonging to the Orders of Lepidoptera, Hymenoptera and Diptera. Despite some members of both classes are recently fully characterized in terms of three-dimensional  structure, binding activity and histological localization, a full explanation of their specific role in the sensillar lymph is still missing. However, several evidence addresses to an important function in the early coding process of odorant and taste compounds during the olfactory perception of insects.

These proteins may also be a useful tool in developing artificial biomimetic chemosensors, since all members characterized so far have a high stability, no post-translational modification and can be easily expressed as recombinant proteins in a full functional form.

Therefore, we decided to investigate the chemosensory perception of Colorado potato beetle (Leptionotarsa decemlineata (Say)), where this type of protein had not been discovered. Extracts of body parts were obtained from males and females of our lab population and analyzed by SDS and NATIVE-PAGE.  Protein profile of chemosensory organs, like mouthpart, antennae and tarsi, did not show any specific band in SDS condition, but a peculiar weak band specific for these extract became visible when the extracts were run in their native state.  Therefore, a mass-rearing of males and females were performed in order to obtain about 2000 antennae for each sex and to reach a full purification of the target proteins.  A combination of gel-filtration and ion-exchange chromatography allowed the purification of a protein of an apparent molecular weight of 12 kDa and a low isoelectric point. A Western blot experiment with the polyclonal antibody raised against the CSP-Sg4 was performed in order to check if this protein belongs to the CSP or OBP family. No reaction of the polyclonal antibody was observed against our purified protein.  A further purification was achieved by HPLC, and the isolated peak was subjected to Edman N-terminal degradation. The aminoacid sequence will be used to design a specific degenerated primer in order to clone the full sequence with RT-PCR and later to characterize the structure and the possible function of this new protein.

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5

Sylvia Anton and Christophe Gadenne

INRA, UMR Santé Végétale, Centre de Recherche de Bordeaux, 33860 Villenave d'Ornon, France

INRA, UMR Physiologie de l'Insecte, Route de St Cyr, 78026 Versailles Cedex, France. santon@bordeaux.inra.fr

To smell or not to smell: plasticity in the adult insect brain

The sense of smell plays an important role in guiding behaviour of many animals including insects.  The attractiveness of a volatile is not only dependent on the nature of the chemical, but might change with the physiological status or environmental conditions of the individual. We study plasticity of olfactory-guided behaviour and its neurobiological basis in Lepidoptera.

The age and the mating status of male moths have an important influence on their behaviour in response to sex pheromones. In the male moth, Agrotis ipsilon, only sexually mature individuals with a high juvenile hormone (JH) titre are attracted by the female-produced sex pheromone. Shortly after mating, males in this species are not attracted any longer by the pheromone. Mating changes also the attractiveness of plant odours for female moths (Lobesia botrana, Tortricidae): only mated females respond to host plant odours.

In all studied cases of behavioural plasticity, we found changes in the sensitivity of olfactory interneurons in the antennal lobe, whereas the peripheral system does not seem to show any plasticity in that context. The changes in the central nervous system are slow under the influence of JH (days) or fast after mating (minutes). The olfactory system seems thus to adapt to the physiological or environmental situation of an animal to avoid a waste of energy. We hypothesize that biogenic amines might play a role in the plasticity of antennal lobe neuron characteristics.

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6

Kiyoshi Asaoka

National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan. asaoka@affrc.go.jp

Involvement of Ca²⁺ cascade in the taste transduction of the caterpillar, Bombyx mori

Several molecules functioning in the process of insect taste transduction have been proposed. Most of these findings are based on studies using flies. In this study, I use a Lepidopteran caterpillar, Bombyx mori  and investigate the comparative involvement of the proposed second messengers and some of the related molecules in the taste transduction process. Using pharmacological agents, the tip recording method was employed to record spike responses from the three different identified taste neurons - the sugar, the inositol and the deterrent cell present in either of the two styloconic sensilla on the maxillary galea. Gustatory stimuli used to distinguish the response of the three taste neurons included sucrose, myo-inositol and strychnine nitrate, respectively. In contrast to results reported using a blowfly, Phormia regina (Murata et al., 2004, Amakawa et al., 1990), NOC-7, a donor of nitric oxide, did not elicit spike responses in any of the taste neurons of B. mori; in addition, membrane-permeable cyclic nucleotides did not have any effect on the responses.

Involvement of IP₃ and release of endoplasmic Ca²⁺ stores have been suggested whereby xestospongin C, an inhibitor of IP₃ receptor, decreases spike responses of some taste neurons. W-7, a Ca²⁺-calmodulin inhibitor suppressed the responses in a dose-dependent manner in all the three cells. Diltiazem, amiloride and SKF-96365 clearly suppress the responses of the sugar and the inositol cell and probably the deterrent cell; these agents possibly block voltage-sensitive Ca²⁺ channels on the receptor membrane. The effect of another Ca²⁺ channel inhibitor, nifedipine is however intriguing and might be different between the sugar and the inositol cell. All inhibitors mentioned above do not affect the initial phasic part of the responses but suppress the following tonic response. These results are in contrast to those reported by Liscia et al. (2002), wherein W-7 and SKF-96365 decrease the overall response in the sugar cell of the blowfly, Protophormia terraenovae.

Results obtained in the present study indicate the possible involvement of both endoplasmic and extracellular Ca²⁺ in the taste transduction process of B. mori, particularly in the tonic phase of the response whereby adaptation occurs. The initial part of responses being independent on Ca²⁺ cascades suggests the presence of ionotrophic receptors as revealed in the sugar receptor of the fleshfly, Boettcherisca peregrina (Murakami and Kijima, 2000). Since the divalent cations, including Ca²⁺ and Mg²⁺ suppress the overall responses as observed in an earlier study (Asaoka, ISOT/JASTS 2004), it is suggested that one of the possible targets of the blockade is an ionotrophic receptor.

Amakawa T., Ozaki M, Kawata K. (1990) J. Insect Physiol. 36: 281-286.
Liscia A., Crnjar R., Masala C., Sollai G., Solari P. (2002) J. Insect Physiol. 48: 693-699.
Murakami M., Kijima H. (2000) J. Gen. Physiol. 115: 455-466.
Murata Y., Mashiko M., Ozaki M., Amakawa T., Nakamura T. (2004) Chem. Senses 29: 75-81.

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7

Manfred Ayasse

Scent variation, hybridization and speciation in sexually deceptive orchids

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8

Sergi Bermúdez i Badia, Pawel Pyk, Philipp Knuesel, Paul Rogister and Paul F.M.J.  Verschure.

Institute of Neuroinformatics, ETH/University Zurich. Winterthurerstr. 190, CH-8057 Zurich, Switzerland. sergi@ini.phys.ethz.ch

A model of moth optomotor-anemotactic chemical search applied to a flying robot.

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9

Thomas C. Baker¹ and Neil J. Vickers²

¹ Penn State University, Department of Entomology, Chemical Ecology Laboratory, University Park, PA, 16802, USA  tcb10@psu.edu

² Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA vickers@biology.utah.edu

Behavior and ORN responses of hybrid heliothine male moths explained by co-expression of two sex pheromone  receptors on a single type of ORN

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10

Richard Benton, Silke Sachse and Leslie B. Vosshall

Laboratory of Neurogenetics and Behavior, The Rockefeller University, 1230 York Avenue Box 63, New York, NY10021, USA; bentonr@mail.rockefeller.edu, leslie@mail.rockefeller.edu

Odorant receptor trafficking in Drosophila

The localization of odorant receptors (ORs) to the ciliated endings of olfactory sensory neuron (OSN) dendrites is essential for their function in translating odor stimuli in the environment into spatial and temporal patterns of neuronal activity in the brain. How ORs navigate from their site of synthesis in the endoplasmic reticulum in the OSN cell bodies to these specialized sensory compartments is largely unknown. In Drosophila, mutations that disrupt the broadly expressed and highly conserved receptor, OR83b, result in the complete absence of ORs from sensory dendrites, and these receptors are detected only at low levels in the cell body (Larsson et al., 2004). Using cell biological and transgenic techniques, we have explored in detail the in vivo function of OR83b in promoting correct OR localization. ORs show a continuous requirement for OR83b to maintain localization, but there is no essential developmental role for Or83b in OR trafficking. The localization of OR83b to sensory cilia is, however, independent of other ORs. OR83b is sufficient to promote OR trafficking and function in ciliated sensory neurons that normally mediate responses to tastants, sounds, and carbon dioxide. Unlike all other known chemosensory receptors, Drosophila ORs adopt an inside-out membrane topology, placing the most conserved loops of these proteins in the cytoplasm. OR83b physically interacts with ORs via these conserved C-terminal domains in vitro and OR83b and ORs form homomeric and heteromeric complexes in vivo. Together these results suggest a model in which association of OR83b with ORs is critical to couple these receptors to the sensory cilia transport machinery to permit their correct localization. Funded by NIH/NIDCD, NSF, EMBO, Helen Hay Whitney Foundation

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11

Neuroscience Unit/Dept. of Psych., Norwegian University of Science and Technnology, 7489 Trondheim, Norway. Bente.Berg@svt.ntnu.no

Distinctive characteristics in the male-specific olfactory pathway of the Oriental Tobacco Budworm Moth, Helicoverpa assulta, as compared to related heliothine species

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12

Stacey L. Brown, Joby Joseph, and Mark Stopfer

NIH-NICHD, Bethesda, MD, USA

Temporal Response Patterns in Locust Antennal Lobe Neurons Evolve over Short-Interval Odor Pulse Trains

Projection neurons (PNs) in the locust antennal lobe (AL) respond to odor puffs with odor identity- and concentration-specific sequences of spiking, inhibition, and quiescence. How does this spatiotemporal mechanism encode odors in rapid trains of nearly overlapping brief pulses, as could occur in a natural odor plume?

In adult locusts, we made intracellular and extracellular “tetrode” recordings from PNs, intracellular recordings from local neurons (LNs), local field potential (LFP) recordings from the mushroom bodies, and simultaneous electroantennogram (EAG) records.  We delivered 100 ms odor pulses in trains of 3 or 10 pulses, with inter-pulse intervals ranging from 0.5 sec to 2 sec. For each pulse pattern, blocks of 10 trials (15 or 20 sec inter-trial interval) were delivered in random order. We found that odor responses of AL neurons changed reliably and significantly with the position of the odor pulse within the train. This change was caused, at least in part, because PN- and odor-specific periods of inhibition from earlier odor pulses affected responses to later pulses.

For most PN-odor combinations, numbers of odor pulse elicited spikes changed reliably and often greatly with pulse position.  In some cases, the numbers of spikes increased during the train; in others, spikes decreased.  Often, these effects were observed with 750ms or longer inter-pulse intervals, times greatly exceeding the duration of pulse-elicited EAG deflections. A PN’s response to pulse position could change with odor or concentration, making unlikely that certain PNs serve as specialized “channels” for temporal information. Over trains of 10 odor pulses, response amplitudes of EAGs, LNs, and LFPs decreased dramatically (~30%), likely reflecting odor receptor adaptation; however, on average, numbers of spikes in PN responses decreased much less (~10%), (and spikes for some PN-odor combinations increased over the pulse trains), perhaps reflecting decreased inhibition from LNs.

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13

Mikael A. Carlsson¹, Philipp Knüsel², Paul F.M.J. Verschure² and Bill S. Hansson¹

¹ Division of Chemical Ecology, Department of Crop Science, Swedish University of Agricultural Sciences, P.O. Box 44, SE-230 53 Alnarp, Sweden;

² Institute of Neuroinformatics, ETH-Universitat Zürich, Winterthurerstrasse 190, Zürich CH 8057, Switzerland. mikael.carlsson@vv.slu.se

Spatio-temporal Ca²⁺ dynamics of moth olfactory projection neurons

We studied the Ca²⁺ dynamics of odour-evoked glomerular patterns in the antennal lobe (AL) of the moth Spodoptera littoralis using optical imaging. Here we selectively stained a large population of AL output neurons, projection neurons (PN), by retrograde filling with FURA-dextran from the inner antennocerebral tract (IACT) in the protocerebrum. Different plant-associated odorants evoked distributed patterns of activated glomeruli that were odour-dependent and repeatable. These patterns were, however, dynamic during the period of odour exposure. Temporal responses differed across glomeruli and were stimulus dependent. Next we examined how the correlations between patterns evoked by different odorants changed with time. Initially, responses to structurally similar compounds were highly correlated, whereas responses to structurally different compounds differed. Within the period of odour exposure (1 sec) we found a significant reduction in similarity of responses evoked by different odours, irrespective of initial similarity, whereas trial-to-trial correlations remained high. Our results suggest an ability for coarse classification at the initial encounter with an odour source. With time, however, the discrimination ability increases and structurally similar odours can be distinguished.  Grant sponsor: EU-FET AMOTH IST-2001-33066 and the Swedish Research Council (VR).

14

Teun Dekker, Irene Ibba, Marcus Stensmyr, Bill Hansson

Drosophila sechellia is a specialist on Morinda fruit, a smelly fruit toxic to its sibling species. How this has affected its olfactory circuitry is poorly studied. Here we report on shifts at various levels in the olfactory circuitry, which are in part adaptive. Combined gas chromatography and Electro-Antenno Detection (GC-EAD) and GC- MS (mass spectrometry) revealed that both D. melanogaster and D. sechellia  antennae respond strongly to the fruit’s characteristic hexanoates. Acids, which dominate the fruit’s headspace elicited very little antennal responses. Further single sensillum screening of antennal sensory neurons revealed that in D. sechellia large basiconic sensillae type 3 (AB3) were overrepresented (approximately 3.5x times more) on the costs of AB2 (not found) and AB1 sensillae (50-70% fewer). AB3 sensilla responded down to femtogram quanitities of its key ligand methyl hexanoate. Concordantly, we found that neuronal projections of large AB inhabiting neurons had undergone substantial rewiring in the antennal lobe, creating two enlarged glomeruli receiving input from the AB3 type sensillae. The physiological and morphological changes are reflected in shift in D. sechellia ‘s behavior. Behaviorally D. sechellia is attracted to lower concentrations of hexanoates, than its sibling D. melanogaster, whereas no tapering was observed at high concentrations. However, whereas D. sechellia was behaviorally also more sensitive to the fruit’s acids, particularly caproic acid, no evidence for a peripherally mediated shift was found on antennae or palpae. Several classes of olfactory sensillae responded to hexanoic acid, but no obvious changes in either frequency, their distribution or sensitivity were observed. Clearly, the shift accounting for the acid preference is located downstream at a higher level of integration. These findings uniquely indicate how evolution can act at several levels of the olfactory circuitry in mediating the fly’s unique preference for fruit that kills its sibling species.

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15

Charles Derby

Department of Biology, Georgia State University. Atlanta, Georgia  USA. cderby@gsu.edu

Why do crustaceans have two parallel antennular chemosensory pathways?

Decapod crustaceans such as the spiny lobster Panulirus argus have two chemosensory pathways associated with their major chemosensory organ, the first antennae. These are the aesthetasc – olfactory lobe pathway, and non-aesthetasc – lateral antennular neuropil pathway (1). The aesthetasc – OL pathway has input from aesthetasc sensilla, which are the only unimodal chemosensory sensillar type. Aesthetasc sensory neurons project exclusively to the olfactory lobe, which is organized into glomeruli much like the antennal lobe of insects and is thought to have a odotopic organization. This pathway is sometimes referred to as the ‘olfactory pathway’ (1). The non-aesthetasc – LAN pathway has input from many different types of sensilla – 9 types in P. argus (2). Non-aesthetasc sensilla are bimodally (chemo- and mechanoreceptor) innervated, and their sensory neurons project to the LAN. The LAN, which also receives motor innervation, has a stratified organization reminiscent of a topotopic organization.  What is the functional distinction between these two pathways?  Some functional redundancy in these pathways is known – either pathway can mediate learning, discrimination, and distance localization of food odors (3-5). But some functional differences are also known. One type of non-aesthetasc sensillum – asymmetric sensilla – is necessary and sufficient to mediate a motor behavior evoked by the food odor L-glutamate – antennular grooming behavior (6). Aesthetascs appear to be necessary for behavioral responses to pheromones such as social (aggregation) and sexual cues (7,8). Thus, these two antennular chemosensory pathways have some redundancy, but also appear to differ in their responsiveness to pheromones and in their control of sensory-motor behaviors. Supported by NIH DC00312, NSF IBN-0324435, and NSF IBN0077474.

1. Schmidt, M. and B.W. Ache. 1996a,b. J. Comp. Physiol. A 178: 579-604, 605-628.

2. Cate, H.S. and C.D. Derby. 2001. Cell Tissue Res. 304: 439-454.

3. Steullet, P. et al. 2001. J. Exp. Biol. 204: 4259-69

4. Steullet, P. et al. 2002. J. Exp. Biol. 205: 851-867.

5. Horner, A.J, M.J. Weissburg, and C.D. Derby. 2004. J. Exp. Biol. 207: 3785-3796.

6. Schmidt, M. and C.D. Derby. 2005. J. Exp. Biol. 208: 233-248.

7. Horner, A.M. and C.D. Derby. 2005. Abstract from AChemS meeting.

8. Gleeson, R.A. 1982. Biol. Bull. 163: 162-171.

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16

Joseph Dickens and Benedict Hollister

USDA, ARS, Harry A. Wallace Beltsville Agricultural Research Center, Plant Sciences Institute, Chemicals Affecting Insect Behavior Laboratory, Beltsville, MD, USA 20705. dickensj@ba.ars.usda.gov

Interaction Between Olfactory and Gustatory Inputs in Insect Behavior

Behavior of insects is governed by chemical and physical signals impinging on specialized sensory organs.  Volatile chemical signals are detected by the olfactory sense and result in upwind orientation by conspecifics receiving them.  Detection of less volatile chemical signals by the gustatory sense results in behaviors such as copulation, biting and ingestion.  However, seldom are these signals encountered in isolation; more often multiple chemical signals are detected simultaneously and the insect receiving them must process the information and appropriate a response.  We investigated behavior of Colorado potato beetles challenged by simultaneous olfactory and gustatory stimuli.  An open ambulatory Y-track olfactometer was used to measure insect preferences, number of turns made during orientation, and time to make a choice between stimuli.  Olfactory stimuli were delivered to one-side of the device; gustatory stimuli were painted on the other side.  Behavioral conflicts and preferences were discovered between stimulus pairs.  For example, females took more time and made more turns when confronted with a volatile plant attractant and sucrose compared to the plant attractant and a male extract.  We will examine behavioral hierarchies revealed by our studies and discuss how they may relate to reproduction and feeding.

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17

Patrizia D’Ettorre

University of Copenhagen , Institute of Biology , Universtitetsparken 15, 2100 Copenhagen , Denmark . pdettorre@bi.ku.dk

A multi-significant queen signal in the primitive ant, Pachycondyla inversa

Primitive ant societies, with relatively simple social structure, give us the opportunity to explore the evolution of chemical communication, in particular of mechanisms underlying within-colony discrimination. In the same colony, slight differences among individual odours can be the basis to discriminate among different castes, classes of age and social status. Correlative studies have given some evidence that such inter-individual variation is associated with differences in reproductive status, but a direct proof for certain chemical compounds being detected and recognized by ants was lacking.
In the ant Pachycondyla inversa, fertile queens and, in orphaned colonies, dominant, egg-laying workers are characterized by the predominance of a branched hydrocarbon (3,11-dimethylheptacosane) on the cuticle. Using electroanntennography and gas chromatography with electroantennographic detection, we showed that workers detect and react to this key compound. 3,11-diMeC 27 is correlated with ovarian activity and, because it is detected, is likely to assume the role of a fertility signal reflecting the quality of the sender.
P. inversa workers prevent each other from reproducing by killing (policing) worker-laid eggs. 3,11-diMeC 27 is also present in significantly higher amount on queen-laid eggs than on worker-laid eggs. Since ant colonies keep eggs in piles, worker-laid eggs might become more acceptable once placed in the egg pile, by acquiring odour from touching queen-laid eggs. Using behavioural manipulations and chemical analyses, we showed that such “ cue scrambling ” does not occur. Policing, therefore, is stable against this potential cheating mechanism, probably because queen-laid eggs are marked with a queen signal which is not easily transferred by physical contact. This is likely to be an example of the widespread pheromonal parsimony, with a key chemical compound serving as a fertility signal and protecting queen-laid eggs from policing. Supported by EU and DFG.

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18

Hossein Farazmand ^1&2 and Stanislav Yu. Chaika ¹

¹ Dept. of Entomology, Faculty of Biology, Moscow State University, 119899 Moscow, Russia. ² Plant Pests & Diseases Research Institute, P. O. Box 1454 Tehran 19395, Iran.

hfarazmand@yandex.ru, biochaika@mtu-net.ru

Effect of precocene II - juvenile hormone inhibitor on chemoreceptor organs of Colorado potato beetle, Leptinotarsa decemlineata Say. (Col.: Chrysomelidae)

The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say. (Col.: Chrysomelidae), is the most serious insect pest of the cultivated potato and a major pest worldwide. The increasing incidence of resistance to almost very insecticide used against it may lead to serious control problems. Precocene, a juvenile hormone inhibitor, exerts cytotoxic effects on the corpora allata of sensitive insect species, which leads to the necrosis of parenchymal cells, the source of juvenile hormone. Recently it has been observed that precocenes significantly reduce the life of the last instar larvae, induce ecdysis of larval cuticule and formation of abnormal puparia and that these effects can be reversed by juvenile hormone administration.

Chemoreceptor organs of holometabola insect larvae are good models for analysing the effect biological compound, as far as the number of sensilla permanently for all larvae instars. Topical application of precocene II occurred on the dorsal part of 2^nd instar larval abdomen by applying 1 μl (10 ng) solution of precocene II in acetone (1%) with a micropipette. Sensillae on the apex of the third segment and 2 basiconica, 2 trichoid and 1 conical sensillae on the distal part of the second segment. Based on the laboratory studies, with the second instar larval, which were treated 1% Precocene II, after the first molting the considerable changes of antenna cuticle structure were observed. The second and third segments of antenna in many larvae were merged, on second segment have 1-5 sensilla; on top third segment some larvae have only 2-6 sensilla. For some larvae other anomalies were observed also full reduction conical sensilla and preservation of a cuticle of the previous instar.

On the maxillary palp of control larvae have 16 basiconica, 4 trichoid and 1 digitiform sensilla. All basiconica sensilla are placed on the distal apex of third segment. Results of experiments on treated larvae showed, that boundary between two terminal segments of palp often fades, and so the number of sensilla is reduced.

On the labial palp of control larvae have 11 basiconica sensilla. All this sensilla are placed on the distal apex of second segment. Based on experiments, on labial palp often have remainder of cuticle of the previous instar, the number of sensilla is reduced up to 3-11, and in some cases all sensilla are reduced.

Study of section through antenna and palps in treated larvae showed reduction of receptor cells and their dendrites. The structure of cuticle of sense organs differs from the control. Study of imago after emergence from pupa showed, that in some imago were observed reduction in number of sensilla only in maxillary and labial palps and were not observed change in antenna.

Thus, precocene II for CPB larvae cause considerable changes in chemoreceptor organs that expressed in a reduction number of sensilla, and neurons. Most considerable changes in chemoreceptor organs in antenna, maxillary and labial palps are observed after larvae treatment on several series instars. The work was supported by RFBR (grant  04-04-48779).

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19

Cécile Faucher ¹, Monika Hilker ² and Marien de Bruyne ¹

¹ Freie Universitaet Berlin, Neurobiologie, 14195 Berlin, Germany

² Freie Universitaet Berlin, Angewandte Zoologie / Oekologie der Tiere, 12163 Berlin, Germany.

cfaucher@zedat.fu-berlin.de, mdebruyn@zedat.fu-berlin.de

Drosophila melanogaster is repelled by carbon dioxide: behavioural observations in adults and larvae

With their olfactory and gustatory systems insects are able to monitor their chemical environment. They selectively detect and respond to those molecules, which aid their orientation toward feeding sources, oviposition sites and mates. Detection of certain chemicals may also be essential to avoid toxins or other dangerous situations. CO₂ is a rather unspecific cue, constantly present at a relatively high level of 0.035%in the atmosphere. Nevertheless, it is perceived by many insect species and modulates their behaviours.

We are investigating the behavioural responses of Drosophila melanogaster to CO₂. These flies feed on fermenting fruits, which produce large amounts of CO₂. We have characterized a class of CO₂ specific receptor neurons in the antenna and discovered that the G-protein coupled receptor Gr21a is expressed exclusively in these cells. Flies in which we have genetically ablated Gr21a expressing cells do not respond to CO₂.

In a choice situation with four converging airflows, individual flies are repelled by high CO₂ concentrations, above 0.1%. However, from physiological experiments we know that their receptor neurons can detect shifts in CO₂ concentrations of as little as 0.02%. In order to reveal behavioural responses close to sensory thresholds we tested 0.02% CO₂ on a background of an attractive odour mixture and found that females were repelled while males were not. This suggests that Drosophila avoids even minor increases of CO₂ and that this behaviour is sexually dimorphic.

In the same behavioural setting, larvae avoid CO₂ at a high concentration but are less sensitive than adults. The Gr21a receptor is also expressed in a single larval neuron, innervating the terminal organ. We therefore tested larvae that have the cell expressing Gr21a deleted. These results indicate that these larvae do not show repulsion to CO₂ and prove that the Gr21a receptor is expressed in CO₂ detecting cells of larvae as well.

The particular sensitivity of females suggests CO₂ perception could modify oviposition behaviour. Our analysis of CO₂ emission from fruits indicate the levels fall during ripening but may rise again due to the growth of micro-organisms. Low CO₂ concentration could be correlated with late phases of ripening before toxic levels of fermentation are reached.

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20

Kenny A. Fernandez, Richard G. Vogt

University of South Carolina, Dept. of Biological Sciences, Columbia, SC  29208, U.S.A.
fernandk@biol.sc.edu, vogt@biol.sc.edu

Metamorphosis of an olfactory system: hormonal regulation of growth and patterning in the antennal imaginal disc of the moth Manduca sexta.

Peripheral olfactory systems of insects undergo metamorphosis, transforming from a simple larval antenna to the highly complex adult antenna mediating diverse chemosensory behaviors. Adult antennae derive from imaginal discs which grow during the larval stage, and undergo neurogenesis and morphogenesis during the pupal stage. We are characterizing patterns of morphogenic activities in the imaginal disc and early developing antenna to identify events which lead to patterns observed in the adult antenna.

This study focuses on development the antennal disc in M. sexta.  Disc growth occurs throughout most of the fifth larval instar, initiating just prior to the 4th-5th molt.  This final instar is divided into a 4 day feeding period and a 5 day non-feeding wandering period.  At the onset of wandering animals find a suitable site in soil to dig a pupation chamber; after about 2 days, the animals become inactive and prepares for the larval-pupal molt.  The antennal imaginal disc grows inward from an epithelial ring surrounding the base of the larval antenna; developmental committment for this growth occurs immediatly following 4th-5th larval molt (Ohtaki et al., 1986; Kremen & Nijhout, 1989; Obara et al., 2002).  Disc growth continues up until mid-wandering at which time the body epidermis undergoes apolysis (detachment from cuticle) and the imaginal discs evert.   Final pre-pupation morphogenesis continues; secretion of pupal cuticle begins about 24 hr prior to pupation.

We have quantified DNA content during disc growth as an indicator of cell number, observing a sharp decline in DNA content just prior to disc eversion.  We have subsequently identifed apopoptotic activity in a spatial pattern which is reflected in the spatial organization of the adult antenna.  We have characterized the expression of genes such as Notch and Distal-less, known to regulate neurgenic activity and imaginal disc development (Bohbot & Vogt).  We have explored the role of ecdysteroids regulating disc growth.  Shortly after disc growth initiates, we have observed expression of the Broad gene within the peripodial epithelium; Broad is one of only several immediatly downstream genes of the ecdysteroid pathway and is thought to direct tissue development through metamorphosis.  We have demonstrated ecdysteroid sensitivity of disc eversion, and are currently exploring the role of ecdysteroids in regulating the post eversion apoptotic events.  These studies are establishing a foundation for identifying the hormonal regulation of growth and patterning that will give rise to the selection of specific chemosensory phenotypes of adult olfactory sensilla.

Ohtaki, T., Yamanaka, F., Sakurai, S., 1986. Differential timing of pupal commitment in various tissues of the silkworm Bombyx mori. J. Insect Physiol. 32, 635–642.

Kremen, C., Nijhout, H.F., 1989. Juvenile hormone controls the onset of pupal commitment in the imaginal disks and epidermis of Precis coenia (Lepidoptera: Nymphalidae). J. Insect Physiol. 35, 603–612.

Obara, Y., Miyatani, M., Ishiguro, Y., Hirota, K., Koyama, T., Izumi, S., Iwami, M., Sakurai, S., 2002. Pupal commitment and its hormonal control in wing imaginal discs. J. Insect Physiol. 48, 933– 944.

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21

Kenny A. Fernandez, Paul Kobres, Geoffry Fourqurean, Richard G. Vogt

University of South Carolina, Dept. of Biological Sciences, Columbia, SC  29208, U.S.A. fernandk@biol.sc.edu

Characterization of a Drosophila melanogaster sensory specific SNMP.

SNMP is an antennal specific, two transmembrane domain protein, abundantly present in the receptive membrane of olfactory neurons in Lepidopterans (Rogers et al. 1997, 2001 a,b). SNMP is expressed late in adult development and into adult life, after morphogenic events have occurred.  SNMPs are homologous with the vertebrate CD36 family of two-transmembrane domain receptor proteins that can bind lipid-protein complexes and transport lipids across the cell membrane. SNMPs are the only CD36 family member known to express in neurons. These properties of SNMPs suggest they play a central role in odor detection, but their exact function is still unknown.

The genome of Drosophila melanogaster contains at least 13 SNMP/CD36 homologues. Three of the D. melanogaster proteins have been characterized and are neither olfactory nor neuronal, but do have CD36-like functions such as playing a role in cell-cell interactions and transporting lipids. We are now characterizing the expression pattern of D. melanogaster SNMP gene family members focusing on the identification of olfactory specific SNMPs. One Drosophila homologue, CG7000, shares significant sequence similarity with the lepidopteran SNMPs. Our study focuses on characterizing the expression of CG7000 as a first step towards using Drosphila to elucidate the function of SNMPs in sensory chemosensory neurons. We have constructed a transgenic fly containing the promoter for the CG7000 gene that drives the expression of cd8::GFP, labeling cells that express this Drosophila SNMP homologue. Studies of the temporal and spatial patterns of this protein suggests CG7000 expresses in subsets of chemosensory (olfactory and gustatory) and mechanosensory neurons of adults, and chemosensory neurons of larvae, and that the CG7000 promoter is activated at around 40% of adult development in the pupa and continuing well into the adult stage. These results suggest that CG7000 is a suitable candidate for studying SNMP function as it relates to insect olfaction as well as studying the roles of diverse SNMP/CD36 homologues in a single species.

Rogers M, Sun M, Lerner MR, Vogt RG (1997) SNMP-1, a novel membrane protein of olfactory neurons of the silk moth Antheraea polyphemus with homology to the CD36 family of membrane proteins. Journal of Biological Chemistry 272, 14792-14804.

Rogers ME, Steinbrecht RA, Vogt RG  (2001a) Expression of SNMP-1 in olfactory neurons and sensilla of male and female antennae of the silkmoth Antheraea polyphemus.. Cell and Tissue Research, 303, 433-446.

Rogers ME, Krieger J, Vogt RG  (2001b) Antennal SNMPs (Sensory Neuron Membrane Proteins) of Lepidoptera define a unique family of invertebrate CD36-like proteins. Journal of Neurobiology 49, 47-61.

22

André Fiala, Thomas Riemensperger, Thomas Völler, Patrick Stock and Erich Buchner

Julius-Maximilians-Universität Würzburg, Theodor-Boveri-Institut, Lehrstuhl für Genetik und Neurobiologie, Biozentrum, Am Hubland, 97074 Würzburg, Germany.
afiala@biozentrum.uni-wuerzburg.de

Visualization of odour and reinforcer representation in the Drosophila brain: an imaging approach to olfactory memory traces.

How does a brain apply a relevance to a stimulus? In behavioural terms, animals learn to associate a neutral stimulus with a relevant stimulus (carrot or stick) in form of Pavlovian conditioning. Associative learning involves the convergence of the signals from the neutral stimulus (CS) with the reinforcement signal (US). Drosophila provides a prime model system for investigating learning and memory on the genetic and behavioural level. In the most typical learning paradigm, am odour stimulus as the CS is paired with an electric shock as the US, leading to an aversive behavioural response to the odour (1). The neuronal substrate underlying this type of experience-dependent plasticity could be localized to the mushroom body lobes (2). However, due to the lack of physiological methods, hypotheses about cellular mechanisms remain tentative as yet. We use an optical imaging approach to visualize the activity of neurons in the brain in vivo during associative training.  A DNA-encoded calcium sensor is selectively expressed in second and third-order neurons of the olfactory pathway (olfactory projection neurons or Kenyon cells, respectively), or in dopaminergic neurons, which are candidates for mediating an aversive reinforcement signal (3). We describe on the one hand the representation of odours at different levels of processing in the Drosophila brain.  On the other hand, we report that dopaminergic neurons innervating the mushroom body lobes respond strongly to an aversive electric shock stimulus, but only weakly to an odour stimulus.  Moreover, we demonstrate that after pairing a neutral odour stimulus with an electric shock, the response to the odour alone is selectively prolonged as a result of the training. This demonstrates that the activity of dopaminergic neurons in Drosophila displays similar predictive features for an expected reinforcer as has been described for mammals.

2) B. Gerber et al. (2004). Curr. Opin. Neurobiol. 14:737-744.

3) M. Schwaezel et al. (2003). J. Neurosci. 23: 10495-10502.   

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23

Walter Fischler, Sunanda Marella, Priscilla Kong, Sam Asgarian and Kristin Scott

University of California, Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, Berkeley, CA. fischler@berkeley.edu

Taste recognition in Drosophila

The ability to distinguish nutritious and toxic foods is essential for an animal's survival, but how taste quality is encoded in the brain is not understood.  The simple nervous system and behaviors in Drosophila provide a tractable model to study taste perception.  Drosophila sense taste compounds with members of the Gustatory Receptor (GR) family of approximately 70 genes. We used a combination of molecular genetic, functional and behavioral approaches to determine how different tastes are recognized in the periphery and how they are represented in the brain.  We show by functional imaging experiments in the live fly brain that taste cells selectively respond to bitter compounds or sugars. Moreover, cell-specific ablations and inducible activation experiments demonstrate that different taste cells mediate taste acceptance or avoidance behaviors.  These studies demonstrate that taste cells are broadly tuned to recognize different taste categories. 

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24

Kazuyo Fujikawa, Keiji Seno and Mamiko Ozaki

Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan. fujikawa@ipc.kit.ac.jp

Identification and localization of a novel takeout-like protein of the blowfly, Phormia regina.

To understand the mechanisms of the insect chemosensory system, we attempted to isolate the functional proteins from taste sensilla of the blowfly, Phormia regina. The proteome analysis revealed that a soluble 28.5kDa protein shared similarity with takeout homolog of Drosophila melanogaster. We isolated its cDNA from the labellar mRNA of Phormia and sequenced. Molecular phylogenetic analysis revealed that the Phormia takeout-like protein (TOL) belongs to a gene family that includes takeout of Drosophila melanogaster and several takeout-like proteins of other insects. TOL is most similar (65% amino acid identity) to CG14661, a member of the Drosophila takeout protein family. Recently, it has been reported that Drosophila Takeout may be involved in circadian control of feeding behavior (Sarov-Blat et al., 2000). However, the functions of other members of the takeout family, including CG14661, so far remain unclear. Western blot analyses revealed that Phormia TOL was exclusively expressed in antenna and labellum of the adult blowfly in both sexes. In order to estimate the functional localization of the protein, immunohistochemical analysis was carried out. Immunofluorescent signals were observed at the base of the taste and olfactory sensilla in the labellum and antenna, respectively. In addition, immunohistochemical experiments using electron microscopy showed that labeling of TOL was observed at the tip of the lamella of the auxiliary cells and in the sensillar lymph of the labellar taste sensillum. These observation suggest that Phormia TOL is involved in the early event in perception of the chemical signals in both taste and olfactory systems. This work was supported by a grant of ProBRAIN to M. O.

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25

Bertram Gerber

University of Würzburg, Biocenter, Department of Genetics and Neurobiology, Am Hubland, 97074 Würzburg, Germany. bertram.gerber@biozentrum.uni-wuerzburg.de

Olfactory learning in Drosophila

It is a major tasks for behavioural neuroscience to understand how synaptic plasticity relates to associative learning, and further to understand how associative learning relates to cognition-like function. I address these issues using fruit flies (Drosophila), and fruit fly larvae.

The larval neuromuscular junction is one of the best understood preparations for synaptic plasticity. However, the study of learning has been limited to adult flies. Therefore, I have developed two learning paradigms for the larva: one uses association of visual stimuli with food reward, the other of odours and food reward. Both paradigms are used for a functional analysis of presynaptic proteins, in particular of synapsin.

Concerning cognition-like function, the main problem is to get an operational handle on the cognitive process in terms of behaviour. As generating predictions is a central function of brains and a basic building block of cognition, I ask: (I) Can animals predict not only the upcoming presence of an event, e.g. of shock, but also its absence? (II) Can animals combine two predictions to resolve ambiguity?

(I) Flies repeatedly receiving a shock after an odour subsequently avoid the odour because it predicts shock and signals danger. Almost all learning research is concerned with this kind of prediction. What, however, if the sequence of events is reversed? If a shock repeatedly comes before odour? Does then the odour predict cessation/ absence of shock? Does the odour become a safety signal and is prefered? We have shown that this is the case (Tanimoto et al., 2004). However, practically nothing is known about the neuronal underpinnings of this paradoxically “rewarding” effect of shock. First results will be presented.

(II) Four stimuli (A to D) are equally often presented with (+) and without (-) reinforcement. Thus, the individual stimuli are ambiguous; however, the combination of stimuli is arranged to be predictive: AB+ CD+ AC- BD-. Such a task cannot be solved by simple predictions (A predicts reward). It can be solved, however, by conditional predictions: A predicts reward if it occurs with B. Such conditional predictions are a hallmark of cognition because they require a two- rather than one-level network architecture. I have shown that adult flies can solve a conditional prediction task in the visual domain. I currently investigate whether fly larvae can also solve such a discrimination task.

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26

Yongmei Gong, Erika Plettner

Department of Chemistry, Simon Fraser University, Burnaby, BC. V5A 1S6, Canada.ygong@sfu.ca

Studies of the Kinetic Binding Properties of Pheromone-binding Proteins from Gypsy Moth

The gypsy moth, Lymantria dispar, is a serious forest pest in North America. The flightless female moths use pheromone, the major component of which is (7R, 8S)-cis-2-methyl-7, 8-epoxyoctadecane ((+) disparlure) 1, as a powerful attractant to mates. The males have expanded feather-like antennae acting as a very sensitive chemoreceptor that can sense the females several miles away. There are two remarkable properties of the moth olfaction system. 1. It has very high selectivity towards particular pheromone blend components that are structurally related. 2. It has a broad range of 10 orders of magnitude in sensitivity of the pheromone concentration in the plume.

Up to date, little has been known about the mechanism of processing pheromone information. From previous studies, the pheromone-binding proteins (PBPs), which have a high concentration of about 10mM in the sensillum lymph bathing the receptor neuron 2, have been observed to bind pheromone ligands. PBPs are small (~16 kDa), tight and hydrophilic proteins. They are required to transport hydrophobic pheromone molecules from the hair surface to the receptor cell through the aqueous lymph. However, a lot of observations suggest that PBPs play an important role in both pheromone detection and discrimination, more than just passive shuttles⁶.

In thermodynamic studies, the two PBPs, PBP1 and PBP2, found in the antennae of the gypsy moths, selectively bound different ligands with subtle differences in the equilibrium binding constants 4: PBP1 prefers (-) disparlure and PBP2 prefers (+) disparlure 3. However, PBPs take about 30 minutes to reach the equilibrium with a ligand, while the moth spends only 1 second in the odor plume. We suggest that binding kinetics is a better measurement of in vivo PBP selectivity than equilibrium binding constants. Besides, there was an average delay of several hundred milliseconds for the first nerve impulse elicited by a low intensity pheromone stimulus 5. Biophysical, biochemical and electrophysiological studies indicate this latency is determined by the kinetic processes located within the peripheral sensory hairs. This indicates that most probably it is the kinetic binding and/or dissociation of PBPs with pheromones that account for the major part of the response latency.

We have used dansyl chloride to chemically modify the PBPs found in gypsy moth antennae and have developed a valid method to measure binding kinetics. For the first time, we are able to determine multiple off rates of PBPs with various ligands. Our results show that the binding kinetics contributes to the pheromone discrimination.

Reference:

1. Grant et al., Naturwissenschaften, 1996, 83, 328

2. Vogt, R. G., Riddiford, L. M., Nature, 1981, 293, 161

3. Plettner et al., Biochemistry, 2000, 39, 8953

4. Honson et al., Chem. Senses, 2003, 28, 479

5. Kaissling, K.E., and Priesner, E., Naturwissenschaften, 1970, 57, 23

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27

Birgit Greiner¹, Carlos José de Carvalho Pinto^2,3, Christophe Gadenne^3,4, Eve Pomarez³, Sylvia Anton^3,4

² Lund University, Department of Cell and Organism Biology, Helgonav. 3, 22362 Lund, Sweden          

² University of Florianopolis, Depto MIP/CCB/UFSC, 88040-900 Florianopolis, SC, Brasil

³ INRA, UMR Santé Végétale, Centre de Recherche de Bordeaux, 33860 Villenave d'Ornon, France

⁴ INRA, UMR Physiologie de l'Insecte, Route de St Cyr, 78026 Versailles Cedex, France. birgit.greiner@cob.lu.se

Possible role of octopamine in the plasticity of the moth olfactory system

Male moths use female-emitted pheromones to find their mating partners. In the noctuid moth Agrotis ipsilon, it is known, however, that adult males change their behaviour towards sex pheromones during adult life. Freshly hatched males, which have a low juvenile hormone (JH) level in this species are not sexually mature and do not respond to the pheromone produced by conspecific females. After some days, biosynthesis of JH increases, males become sexually mature and are then highly attracted by the female-produced sex pheromone. Maturation can be accelerated by injection of JH in young males and the behavioural response can be inhibited in mature males if they are deprived of JH. In parallel, our studies have shown that central neurons in the male antennal lobe change their sensitivity with age and juvenile hormone level. Both the behavioural and central nervous effects of JH appear relatively slowly, during one to two days. We therefore hypothesized that the hormonal effect might be indirect and one candidate for a neuromodulator, which might serve as an "interface" is octopamine.

To test our hypothesis we studied the effect of octopamine and an octopamine receptor antagonist, mianserine, injected in the head capsule on the behaviour of males in a wind tunnel and on the thresholds of neurons in the antennal lobe. First results indicate, that octopamine can mimic effects observed e.g. under JH treatment, i.e. young males injected with octopamine respond better to the sex pheromone than control males in the windtunnel and the percentage of very sensitive neurons is higher than in control males. Inversely, in sexually mature males injected with mianserine, a large proportion of neurons shows high thresholds compared to control males. We will now investigate if there are interactions between the hormonal and octopamine effects.

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28

Bill S. Hansson, Susanne Erland, Marcus Sjöholm and Marcus Stensmyr

Division of Chemical Ecology, SLU, Box 44, SE-230 534 Alnarp , Sweden, bill.hansson@vv.slu.se

The olfactory sense of robber crabs – structure, function and behaviour

The robber crab, Birgus latro, has during the last 5 million years evolved to cope with a terrestrial life. Several parts of its body have gone through dramatic changes to fit the requirements of a life in air. We investigated how the olfactory system has been affected by the sea-to-air transition and we also initiated a study of the population structure and the mating behaviour of these animals.

Here I report on an already published study on peripheral olfactory structure and function (Stensmyr et al 2005). I also describe preliminary data from our second expedition to Christmas Island, when we studies the central nervous architecture of the robber crab olfactory system, and population movements that form a base for future investigations of olfactory-dependent behaviour.

The robber crab antennule with its aesthetascs displays a remarkable similarity to the insect system. In our investigations of both morphological and physiological characteristics we could show a strong convergent evolution of the crab system towards the insect one (Stensmyr et al 2005). In the continued studies we have so far found very crustacean-like structures in the central olfactory system. When studying population movements, interesting patterns were observed, patterns allowing pheromones as a possible means of sexual communication.

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29

Joerg Hipp¹ and Alex Bäcker^2,3

² Sandia National Laboratories and the California Institute of Technology.

Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000

³ Presenting author joerg@ini.phys.ethz.ch, alex@caltech.edu

An activity-dependent model of the development of the nose to brain connection

The first processing stage in olfactory systems creates a mapping of olfactory sensory neurons (OSNs) to glomeruli. In the adult animal, each glomerulus receives connections from only one OSN species (i.e. all expressing the same olfactory receptor), and each OSN species connects to at least one glomerulus. The topology of glomerular specialization is conserved across individuals. This elegant mapping sorts out the complex arrangement of OSNs on the epithelium into an orderly projection pattern. How the map is established, and maintained throughout life in the face of constant neuronal replacement, remains enigmatic after many years, perhaps the most hotly contested question in olfaction today.

Here, we review the recent experimental literature, which suggests a role for activity-dependent learning in the development of the OSN-glomerular mapping, and then propose and implement a biologically plausible activity dependent model for the development of this mapping.

We implement an initial gradient-guided migration of the OSN axons. The chemoattractant receptors involved in the guidance are the very olfactory receptors (ORs). Therefore, axons from different OSN species with similar odor-induced activity patterns end up spatially close in the olfactory bulb (OB). This explains the chemotopic structure of the OB that is well preserved across individuals. To explain the specialization of glomeruli in single OSN species, we propose a weight-dependent Hebbian learning rule. This rule allows cells with activity highly correlated to their target (OSNs from the same species) to map with approx. equal strength to the target cell, while connections from less correlated cells (OSNs from other species) degenerate. Interglomerular inhibition and inhomogeneity, due to the differential binding of ORs to guidance molecule gradients during initial growth, accounts for diversity across glomeruli.

The proposed model explains the establishment and maintenance of the map based on a gradient–guided initial growth followed by activity-dependent specialization of glomeruli. It explains results of recent experiments and makes testable experimental predictions.

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30

Irene Ibba, Teun Dekker and Bill Hansson

Department of Crop Science, Swedish University of Agricultural Sciences,Alnarp, Sweden. ireneibba@hotmail.com

A behavioural and sensory physiological comparison of Drosophila melanogaster and Morinda fruit specialist D. sechellia to acids

Drosophila melanogaster a Dipteran insect is the species of fruitfly that was chosen as a genetic animal model at the beginning of  20^th Century by Nobel prize winner Thomas Hunt Morgan. Since then it has been a very successful animal model for biological research and it has become a prime insect model for studying olfaction. Nowadays Drosophila melanogaster’s olfactory circuitry is increasingly understood, but many questions remain.

D. melanogaster is cosmopolitan and generalist, whereas D. sechellia, a sibling species of  D. melanogaster, is endemic to the Seychelles archipelago in the Indian Ocean and breeds in a single resource: Morinda citrifolia, a fruit toxic for other Drosophilids.

D. sechellia is resistant to the toxic and prefers to mate and oviposit on Morinda. We addressed the question of how the olfactory system has been affected as a result of specialization and drift in these two relative species:  D. melanogaster and D. sechellia.

First we have collected odours from M. citrifolia, that is commonly known as Noni fruit, from various stages of ripeness in order to identify and quantify the main volatile compounds present in Morinda fruit headspace.

Many different chemicals have been found in M. citrifolia and two of these, present in high content, especially in the ripe stage of the fruit, are hexanoic and octanoic acids. These acids are responsible for the general toxicity of the fruit.

We investigated the behavior of the two species towards these and other acids with a trapping bioassay. D. sechellia seemed very attracted to hexanoic acid to all concentration ranges tested whereas D. melanogaster seemed more variable to all acids.

The sensitivity of D. sechellia and D. melanogaster’s antennae to acids has been screened  also with electro-antennogram (EAG) in order to investigate possible shifts in the peripheral olfactory system.

Preliminary results show significant differences in the behavior of the two Drosophilids species, but relatively small differences in their peripheral olfactory system.

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31

Rickard Ignell, Teun Dekker, Majid Ghaninia and Bill S. Hansson

Div. Chemical Ecology, Agricultural University of Sweden, Alnarp, Sweden. rickard.ignell@vv.slu.se

The antennal lobe of mosquitoes

Mosquito behavior is heavily dependent on olfactory cues, which are detected by receptor neurons on the antennae and maxillary palps. Recent progress in mosquito sensory genomics has highlighted the need for an up-to-date understanding of the neural architecture of the mosquito brain. By applying classical histological techniques we here present the architecture of neural elements residing within the primary olfactory centre, the antennal lobe, of two species of mosquitoes; the African malaria mosquito, Anopheles gambiae, and the yellow fever mosquito, Aedes aegypti. The presented data reveal striking similarities with previous reports from other insects, but also specialisations within the olfactory system of mosquitoes. The most striking differences are efferent neurons that innervate single glomeruli and send axons into the antennal nerve, as well as a novel neuropil within the antennal nerve. The novel morphological features may indicate a vital requirement of rapid fine tuning or modulation of signals acquired by the olfactory system.

Furthermore, we present high-resolution three-dimensional models of the glomerular organisation of the antennal lobes of males and females of the two species. These models indicate low variability between individuals and show difference between sexes and species. These differences may be interpreted as specialisations of the olfactory system in either sexes or species.

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32

Claudio Ioriatti, Gianfranco Anfora, Marco Tasin and Silvia Schmidt

U.O. DIF  and SAFECROP CENTER - Istituto Agrario S.Michele - 38010 S.Michele a/A, Italy . claudio.ioriatti@iasma.it

Use of semiochemicals for the pest control

In the early ’80s most of the experiences aimed to develop a commercial application of mating disruption in Italy failed. Commercial application took off in the early ’90s in Trentino and South Tyrol when codling moth resistance to chitin synthesis inhibitors developed in apple orchards and with the implementation of IPM systems in vineyards to reduce chemical inputs. This pushed researchers, technicians and farmers associations to find alternative strategies. In 15 years the application of pheromone MD expanded to more than 35,000 ha of apple, pear, peach and vines; Trentino and South Tyrol region, despite its small size, still accounts for more than 2/3 of the national mated disrupted area. The experience gathered in these years established that reliable dispenser devices, population density and area wide applications are the key factors for the success.

Dependable release dispenser system having stable performances through the season, high quality standards, pheromone compatibility and low cost are a basic need of MD technology. At present hand-applied dispensers are largely the most widespread in the fruit crops and grape.

The amount of pheromone delivered with these dispensers is between 100-150 g/ha per season. Field scouting at critical timing is an irreplaceable practice for the successful implementation of MD in field. The density of the moths, the target of MD, is estimated on the basis of the larval population. Action thresholds have been developed according to the crops and the pest behavior; differences appear dependent on the female potency and reproductive potential of the pest in relation to the host phenological stage. Monitoring of adult density helps to predict the pest outbreak risk; the use of overloaded pheromone traps is a standard practice to check the pest presence in the MD orchard.

The small acreage of the single farm hampers the efficacy of the MD. In this case area wide application is possible only if promoted by grower associations and strongly supported by the technical advisory service.

When the key factors are under control, MD efficiency is generally high. However, plots located in urban areas and in slope still represent critical situations that require intensive monitoring. Improvements in the MD efficacy are still possible if more field and basic research will be done to reach a fine tuning of pheromone applications. The use of kairomone bisexual attractant has been recently developed to monitor the codling moth presence in the apple orchard. A new semiochemical simple blend able to attract both females and males of grape moth in the wind tunnel has been recently discovered. Pheromones are species specific and their wide application on single pests will increase risks for other similar species of insects. Development of multi-species MD technology and strategy is the new challenge. New methods, like false trail following and autoconfusion, that are based on different mechanisms and that use a less amount of active ingredient are under development. Semiochemicals could be used to induce a change in the choice of the oviposition sites and to modify larval behavior as well. First experiences in that sense will be reported.

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33

Guillaume Isabel¹, Romain Franconville¹, Julie Neveu¹, Hélène Coulom², Serge Birman², Philippe Vernier¹ and Thomas Preat¹

¹ DEPSN, CNRS, 91190 Gif-sur-Yvette, France. ² IBDM, Marseille, France. preat@iaf.cnrs-gif.fr

Dynamic of olfactory memory phases in Drosophila

The major challenge faced by neuroscientists studying memory is to define the links between the various levels of nervous system organization, starting with molecules and cells, and then neuronal circuits, and finally the global cognitive functions of the brain. Despite the small size of the Drosophila brain (about 100,000 cells in total), it can generate various forms of associative memories after presentation of an odor paired with electric shocks. We previously showed that Drosophila mushroom bodies are involved in long-term memory (1), and that the brain is the site of dynamic interactions between consolidated memory phases (2). We have further analyzed olfactory learning and memory systems. In particular, we propose that parallel learning processes lead to cAMP-dependent labile memory and anesthesia resistant memory.

Dopaminergic neurons project onto mushroom body lobes. The enhancer-trap Gal4 system combines with UAS-shibire construct that encodes a thermosensitive mutant toxin (3), allows to reversibly block the activity of particular brain circuits. We show that a transient block of dopaminergic neurons after conditioning leads to a strong memory enhancement. This extra memory  is labile, and depends on Rutabaga adenylate cyclase. Thus dopamine actively controls labile memory level in Drosophila.

(1) Pascual A. and Preat T. (2001). Localization of long-term memory within the Drosophila mushroom body. Science, 294 :1115-1117.

(2) Isabel, I. Pascual, A. and Preat, T. (2004). Exclusive consolidated memory phases in Drosophila. Science 304 : 1024-1027.

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34

Emmanuelle Jacquin-Joly, Christine Merlin, Marie-Christine François, Philippe Lucas and Martine Maïbèche.

UMR « Insect Physiology : Signalisation and Communication », INRA Versailles, Route de Saint-Cyr, F-78026 Versailles Cedex, France. jacquin@versailles.inra.fr

An expressed sequence tag approach for the identification of olfactory genes in a pest moth, the cotton leafworm Spodoptera littoralis (Lepidoptera, Noctuidae)

Our previous studies resulted in the identification of several insect olfactory specific genes by biochemical purification, molecular cloning and expression pattern analyses. However, the logic of olfaction relays on the combinatorial expression and intervention of a multitude of different actors, most of which remain to be discovered, such as olfactory receptors or ion channels. Indeed, only few olfactory receptors are known to date in insects and they appear as new families of extremely divergent genes. Only one S. littoralis olfactory receptor has been cloned in the lab through homology strategies, but it remains atypical since it is quite conserved between insect species.

We then set up a transcriptomic strategy for the discovery of new olfactory genes in our model through the elaboration and sequencing of an antennal EST library. This library was constructed and normalized from 2mg of RNA extracted from 12000 S. littoralis male antennae (InVitrogen, CA, USA) in a Gateway compatible vector (pSPORT6). Normalization efficiency analyses showed a 20-fold decrease of abundant genes, as measured with beta-actin, then enriching the library in rare transcripts. Random sequencing led to the identification of a panel of molecular elements potentially involved in the discrimination and specific recognition of odours, including odorant-binding proteins, olfactory receptors, degrading enzymes, ion channels. Through their diversity in the same species, they are supposed to act in a combinatorial/sequential way to ensure dynamic discrimination of several thousand of possible ligands. S. littoralis antennal sequences will be deposited in the SPODOBASE (http://bioweb.ensam.inra.fr/spodobase/index_uk.html), an integrated database for the genomic of Spodoptera, as well as in international databases.

Since many insect behaviours (sexual recognition, animal/plant host interaction) are mediated by olfactory cues, the discovery of new olfactory molecular targets is a particular challenge in agriculture (crop pests) and human health (insect disease vectors).

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35

Bettina A. Johne, Bernhard Weissbecker and Stefan Schütz

Georg-August-University Goettingen, Institut for Forest Zoology and Forest Conservation, Buesgenweg 3, D-37077 Goettingen, Germany.  bjohne@gwdg.de

Olfactory detection of host odor compounds in different generations of the horse chestnut leaf miner Cameraria ohridella

The small leafminer Cameraria ohridella (DESCHKA & DIMIC) attacks the horse chestnut Aesculus hippocastanum. The horse chestnut leaf miner establishes 3 generations in Central Europe. Host plant volatiles are affected by phenology and stress status of the tree. It was examined if specific volatiles of special importance during a generation time are detected with different sensitivity by individuals of each generation. Utilizing the GC-MS/EAD method, more than 30 olfactory active compounds of the host plant could be identified. The host odor pattern shows compounds appearing during the whole season. Furthermore, substances typical for flowers as well as young and old green leaves were analysed. Moreover, leaf mining activity of C. ohridella larvae and infection by the phytopathogenic fungi Guignardia aesculi and Erysiphe flexuosa affect the host volatile pattern. Moths of the 1^st generation encounter flower scent of host trees and odor of young green leaves. Volatile compounds caused by an increased number of competitors as conspecific larvae and phytopathogenic fungi gain importance during the 2^nd and 3^rd generation.

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36

Walton D. Jones¹, Thuy-Ai T. Nguyen², Brian Kloss², Kevin J. Lee², and Leslie B. Vosshall¹

¹ Laboratory of Neurogenetics and Behavior , The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA ;

² Sentigen Biosciences, Audubon Biomedical Research Building, 3960 Broadway, New York, NY 10032, USA

Functional conservation of an insect odorant receptor across 250 million years of evolution.

A family of 62 odorant receptors (ORs) mediates olfaction in Drosophila. These receptors are highly divergent between even closely related insect species. One of these receptors, Or83b, is remarkably conserved through all insect phyla investigated thus far. Unlike other OR family members, which are expressed in limited sub-populations of olfactory sensory neurons (OSNs), Or83b is co-expressed with other ORs in nearly all OSNs of the fly antenna. The sequence conservation and broad expression pattern of Or83b suggests a general olfactory function working in conjunction with other ORs. Consistent with this hypothesis, mutant flies lacking Or83b fail to traffic other OR family members to the sensory dendrite. The absence of ORs in the OSN dendrites, the site of interaction with airborne odorants, eliminates odor-evoked potentials, and severely attenuates olfactory-associated behaviors in Or83b mutant flies. We asked whether Or83b orthologues from Anopheles gambiae, Ceratitis capitata, and Helicoverpa zea are able to functionally rescue the Or83b mutant defects in OR trafficking and electrophysiological deficits. These results confirm that members of the Or83b family subserve a general and important role in insect olfaction.

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37

Karl-Ernst Kaissling

Max-Planck-Institut für Verhaltensphysiologie/Ornithologie, Seewiesen, 82319 Starnberg, Germany, kaissling@orn.mpg.de

Modeling PBP function in moths antennae

Recent investigations revealed rate constants for the binding of bombykol and pheromone binding protein of Bombyx mori (BmorPBP) and for the pH-induced conformational change of BmorPBP from the A-form to the B-form, and in the reverse direction^1-3. These data were included in a modified quantitative model of PBP function^4,5. It is assumed that the pheromone entering the hair lumen is first loaded on the A-form, transported through the lymph by the B-form, and finally interacts with the receptor while bound to the A-form. In order to simulate the kinetics of the receptor potential it was necessary to increase the number of receptor molecules and the affinity of the pheromone-receptor interaction as compared with the previous model⁴. Furthermore, the previous assumption that the pheromone-PBP complex interacts with the receptor molecules was compared with the assumption that the pheromone dissociates from the PBP before interacting with the receptor. In the latter model the dissociation rate constant had to be increased by 3,000-fold above the experimental value in order to obtain sufficient amounts of free pheromone. This suggests that it is indeed the pheromone-PBP complex rather than the free pheromone which interacts with the receptor molecule. These studies support the idea that - for relatively brief and weak stimuli - the receptor potential reflects the kinetics of the network rather than the one of intracellular transducer processes.

1) Sandler BH, Nikonova L, Leal WS, Clardy J (2000) Chemistry & Biology, 7, 143-151.

2) Horst R, Damberger F, Luginbühl P, Güntert P, Peng G, Nikonova L, Leal WS, Wüthrich K (2001)

PNAS 98, 14374-14379.

3) Leal WS, Chen AM, Ishida Y, Chiang VP, Erickson ML, Morgan TI, Tsuruda JM (2005) PNAS 102, 5386-5391

4) Kaissling KE (2001) Chemical Senses, 26:125-150.

5) Minor AV, Kaissling KE (2003) J Comp Physiol A 189, 221-230.

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38

Blanka Kalinová¹, Rostislav Zemek², Edwin A.P. Bouman³, Helena Zahradníková², Petr Imek² and Frantiek Dusbábek³

¹ Institute of Organic Chemistry and Biochemistry CAS, Prague blanka@uochb.cas.cz

² Institute of Entomology CAS, České Budějovice

³ Institute of Parasitology CAS, České Budějovice

Sexual pheromones in Ixodes ricinus

The sexual attractiveness of females of Ixodes ricinus was recorded using computer assisted video-tracking technique. Experiments were performed during daytime in the absence of any host stimuli. Unengorged, semi-engorged and fully engorged field collected females and males were tested. Experiments showed that females are attractive for males from a distance. The attractiveness of females was dependent on their feeding status. Unengorged females were less attractive than semi- and fully engorged ones. Video-tracking analysis also revealed that females deposit chemical trails on a substrate. These trails, attractive for males, are not persistent and completely diminish within 24 hrs. Males are not attractive to other males and probably do not deposit any compounds modifying behaviour of other males.

In order to identify female-specific semiochemicals, SPME, dynamic headspace, deposit and female washes were analyzed using GC-MS and GCxGC/TOF-MS. Analysis revealed two types of female specific compounds – i) more volatile compounds including already known methyl 3-chloro-4-methoxybenzoate and ii) less volatile unknown steroid-like C27H40 hydrocarbons. Possible behavioural and physiological meaning of identified compounds will be discussed.

Acknowledgement: Grant 206/04/0751 of the Grant Agency of the Czech Republic

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39

Koichi Kawabata and Kiyoshi Asaoka

National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan. cloud3@affrc.go.jp

Molecular cloning and characterization of candidate taste receptor genes in the silk worm, Bombyx mori.

Feeding is a main biological activity in insect larvae.  We have investigated feeding mechanism, focussing on taste perception by using electrophysiological methods in the silk worm, Bombyx mori.  Based on our previous study and recent reports from other groups, two types of taste receptors, G-protein coupled receptors (GPCRs) and channel type receptors seem to be involved in gustatory perception in insects.  As a next step to understand the mechanism of taste perception, we aimed to clarify which type of the gustatory receptors function in taste perception.  Using the genome database timely disclosed from Silk Worm Genome Research Program (NIAS, Japan), some partial sequences of candidate taste receptors were extracted and full-length sequences of both types of GPCRs and channel type receptors were determined by RACE method.  These receptor genes were cloned into appropriate expression vectors and were tried to reconstruct in the heterologous cell systems.  We report structural and expressional characters of these receptor genes.

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40

Andreas Keller and Leslie B. Vosshall

Laboratory of Neurogenetics and Behavior, The Rockefeller University,  New York, NY 10021 USA

Animal species differ enormously in their olfactory preferences, the numbers and types of olfactory sensory neurons, and the repertoire of odorant receptors they express. We designed novel, high-throughput olfactory behavior paradigms to ask how Drosophila responds to odor stimuli. Despite their limited repertoire of 61 odorant receptor genes and small number of olfactory neurons, we find that flies are extraordinarily sensitive to odors.

In a screen of 63 structurally diverse odors, flies show odor-evoked locomotor responses that are characteristic for a given odor and strongly dose-dependent. Odor detection thresholds in the fly are remarkably similar to the far more complex human species. Comparative judgements of odor similarity by fly and human subjects show marked species-specificity. Distortion of the fly olfactory system by selective removal of single odorant receptor genes produces subtle behavioral phenotypes that suggest extensive overlap in the receptive range of olfactory neurons. Taken together, these results show that fruit flies can efficiently solve problems in odor detection despite having a simple olfactory system.

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41

Philipp Knüsel¹, Mikael A. Carlsson², Bill S. Hansson², Tim C. Pearce³, Paul F.M.J. Verschure¹

¹ Institute of Neuroinformatics, University/ETH Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland

² Division of Chemical Ecology, Dept. of Crop Science, SLU, P.O. Box 44, S-230 53 Alnarp, Sweden

³ Dept. of Engineering, University of Leicester, Leicester, LE1 7RH, United Kingdom

pknuesel@ini.phys.ethz.ch, mikael.carlsson@vv.slu.se,

bill.hansson@vv.slu.se, t.c.pearce@leicester.ac.uk, pfmjv@ini.phys.ethz.ch

Independent coding dimensions of the optical antennal lobe output neuron response

In the encoding of information by the nervous system a distinction is usually made between its organization in time, i.e. temporal versus rate codes, and space, single cell versus population codes. This raises the question whether these coding strategies are exclusive or complementary [1].  Here we experimentally address this question by analyzing the optical response of the projection neurons (PNs), the output neurons of the moth antennal lobe (AL), to odor stimuli. In this study we propose a new biophysically motivated model based approach that allows us to parametrize the optical imaging response in terms of its amplitude and duration.

Following this approach we assess how both aspects of the optical imaging response contribute to the encoding of the odor stimuli. Our results show that both response parameters, i.e. the amplitude and the duration, are different between glomeruli and are significantly modulated by odor stimuli. We observe that all possible combinations of amplitude- and duration-modulation can be found. Classification experiments using the data of single glomeruli show that both response parameters contribute to the encoding of odor stimuli. Whereas using either amplitude or duration of a single glomerulus renders 46% and 44% correct classifications (chance level 25%), respectively, their combination significantly increases the percentage of correct classifications to 56%. Hence, the encoding of odor stimuli into amplitude and duration is complementary in the sense that their combination increases the amount of information encoded, suggesting that both response parameters constitute independent coding dimensions. Finally, combining these two coding dimensions in a temporal population code where both the PN response amplitude and duration are considered across a population of glomeruli renders a performance of about 75% compared to 60% when either of these dimensions is excluded (chance level 12.5%).  Moreover, we show that this temporal population code is faster than coding scenarios that rely solely on amplitude or duration.

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42

Jürgen Krieger, Ewald Grosse-Wilde, Thomas Gohl and Heinz Breer

University of Hohenheim, Institute of Physiology, Garbenstrasse 30, 70599 Stuttgart, Germany.  krieger@uni-hohenheim.de

Identification and characterization of pheromone receptors from moths

In many insects mating behavior is initiated and controlled by female released pheromones. The remarkable ability of male moths to recognize very low concentrations of female sex-pheromone components is based on the extremely sensitive and selective reaction of specialized sensory cells in the male antennae. Seven transmembrane domain receptor proteins in the dendritic membrane of these neurons are supposed to recognize the pheromonal compounds and initiate intracellular transduction cascades, generating the electrical response of the cells.

Due to their key role in the initial step of pheromone perception and their potential as targets for novel agents to control pest insects, intensive efforts have been employed over the past decade to identify pheromone receptors of moths. Recently, we have discovered a small family of putatitive pheromone receptors in the tobacco budworm Heliothis virescens and the silkmoth Bombyx mori. RT-PCR-analysis revealed that several subtypes were predominantly or exclusively expressed in the antennae of male moths. More detailed in situ hybridization studies in combination with laser scanning microscopy indicated that expression of these receptor types was confined to antennal cells, which were surrounded by cells expressing pheromone binding protein. In addition, in situ hybridization signals  were located beneath sensillar hair structures (sensilla trichodea) containing pheromone sensitive neurons. Using a receptor-specific antiserum allowed to visualize the receptor protein in the sensory dendrites projecting into these sensilla trichodea. To approach the ligand specificity of candidate pheromone receptors, cDNAs for distinct receptor types were stably integrated into the genome of modified HEK293 cells carrying also the Ga₁₅ gene. The responsiveness of receptor expressing cells to pheromonal compounds and other odorants was assessed by Ca²⁺-imaging. These experiments showed that expression of candidate pheromone receptors rendered HEK cells sensitive to low concentrations of pheromone components; no response was obtained with general odorants. These findings indicate that the identified receptor types may in fact be the receptors for pheromones. This work was supported by the Deutsche Forschungsgemeinschaft.

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43

Lina Kristoffersen¹, Eric Hallberg², Rita Wallén², Olle Anderbrant¹

¹ Lund University, Chemical Ecology and Ecotoxicology, Department of Ecology, Ecology Building, Sölvegatan 37, SE-223 62 Lund, SWEDEN.

² Lund University, Zoology, Department of Cell and Organism Biology, Zoology Building, Helgonavägen 3, SE-223 62 Lund, SWEDEN.

Antennal morphology of the carrot psyllid (Trioza apicalis)

The carrot psyllid, Trioza apicalis (Homoptera: Psylloidea), is a specialist plant-sucking insect and a serious pest on carrots in northern and central Europe. The species is univoltine and hibernate as adults on coniferous trees. In May or June they invade carrot fields to feed and reproduce. The initial aim of the project was to identify volatiles mediating behaviour in carrot psyllids. However, limited success with numerous bioassays, both behavioural and electrophysiological, led to the hypothesis that olfactory cues are of little importance in the natural history of this species. Therefore, to investigate the morphological basis for olfactory reception in the carrot psyllid antennae, we used scanning and transmission electron microscopy (SEM and TEM, respectively).

Our study reveals a very sparse sensillar setup. Based on physiological features, we suggest probable functions for the different types of sensilla. We identify and describe several different types of single walled sensilla likely to have an olfactory function. These sensilla are either hair shaped or located in cavities in the cuticula. There are also mechanosensory hairs and intracuticular sensilla. The low number of sensilla indicate that carrot psyllids require high levels of odour stimuli to respond. It also implies that T. apicalis probably use olfactory cues, at least to some extent, to locate their host plants. No pheromones are known in the Psylloidea at this point, and in concordance, there is no sexual dimorphism in the sensillar setup. This is the first description of psyllid sensilla based on TEM, providing a profound basis for future electrophysiological experiments, e.g. single sensillum recordings (in progress). 

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44

Walter S. Leal

Maeda-Duffey Lab, Department of Entomology, University of California, Davis, CA 95616; wsleal@ucdavis.edu

Quickly come, quickly go: pheromone signaling in moths

To communicate with small-molecule signals, insects live a life in the fast lane. Odorant-oriented navigation requires a dynamic sensory system to detect pheromone pockets and reset the detectors (on a few milliseconds time scale) while flying in the clean air spaces between pockets of chemical signals.

Molecular interactions of the silkworm moth (Bombyx mori) pheromone (bombykol) with both native and mutated pheromone-binding proteins were analyzed by a novel binding assay. A recombinant protein with the native conformation (rBmorPBP) showed high binding affinity at pH 7, but no affinity at pH 5 when tested at both low and high KCl concentrations. A protein with a C-terminal segment deleted (BmorPBPP129-V142) was found to bind bombykol at pH 7 and at pH 5 with the same affinity as the native protein at pH 7.

Transient kinetic studies revealed that the basic to acidic conformational change (BmorPBP^B à BmorPBP^A) is much faster (t_1/2 9.3 ms) than the reverse process. The uptake of bombykol by BmorPBP proceeds with an “on” rate of 0.068±0.01 M^-1.s^-1. With the high concentration of PBP in the sensillar lymph, the half-life for the uptake of pheromone in vivo is only ca. 1 ms.

On the other hand, pheromone inactivation was studied in the wild silkmoth, Antheraea polyphemus. The previously identified pheromone-degrading enzyme (PDE) (Vogt et al., 1985; Klein, 1987) was isolated to purity for the first time. Internal amino acid sequences were obtained by by MS-MS and the full-length cDNA was cloned. The role of PDEs in the fast inactivation of pheromone will be discussed.

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45

H. Lei, T.A. Christensen and J.G. Hildebrand

ARL Division of Neurobiology, University of Arizona, Tucson, AZ 85721, USA
hlei@neurobio.arizona.edu; tc@neurobio.arizona.edu; jgh@neurobio.arizona.edu

Pharmacological dissection of the inhibitory interactions in the glomerular network of the moth, Manduca sexta

There is considerable interest in the function of interneurons containing gamma-aminobutyric acid (GABA) in olfactory information coding, but few studies have characterized the pharmacology of the diverse receptor subtypes that mediate GABAergic inhibition. In view of their significant subunit diversity, it is possible that GABA receptors (GABA-Rs) with distinct subunit composition serve different functions at different synapses in the olfactory pathway. We are studying the relative contributions of intra- and inter-glomerular inhibition in olfactory signal coding in the antennal lobes of the moth Manduca. In this presentation, we examine the hypothesis that intra- and interglomerular inhibition are mediated by pharmacologically distinct GABA-Rs.

Using a juxtacellular recording method, we examined the responses of glomerular output neurons to their known odor inputs. We tested a range of stimulus concentrations and examined olfactory responses before, during and after bath application of agonists and antagonists of mammalian GABA_A and GABA_B -Rs. We found that: (1) muscimol (GABA_A agonist) reduced the rate of spontaneous firing as well as response magnitude; (2) bicuculline methiodide (competitive GABA_A antagonist) induced a change in spontaneous activity from a random to a regular pattern of firing while also potentiating and prolonging intraglomerular excitatory odor responses, while in sharp contrast, this treatment did not affect interglomerular inhibitory odor responses; (3) picrotoxinin (non-competitive GABA_A antagonist) did not alter spontaneous firing but lowered the threshold for intra-glomerular excitatory responses; and (4) 2-hydroxysaclofen (GABA_B antagonist) broadened the molecular receptive range of some neurons, suggesting an action on inter-glomerular synapses.

To study further how neuronal interactions are modulated by these drug treatments, we used neural-ensemble recording, which permitted simultaneous monitoring of firing patterns from multiple neurons innervating the same or different glomeruli. Physiologically identified projection neurons and local interneurons were affected differentially by agonists and antagonists of different GABA-Rs. Consistent with the results obtained with the single-unit recording method, we again found that GABA_A antagonists increased the sensitivity of projection neurons while the GABA_B antagonist seemed to broaden the cell’s molecular receptive range.

In summary, our data support the hypothesis that intra- and interglomerular inhibitory synaptic interactions are mediated by different subtypes of GABA-Rs and that these receptors differentially modulate odor-signal processing in the glomerular network. [Supported by NIH grant R01 DC02751.]

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46

Matthieu Louis¹, Thomas Huber², Thomas P. Sakmar² and Leslie B. Vosshall¹

¹ Laboratory of Neurogenetics and Behavior, ² Laboratory of Molecular Biology and Biochemistry,

The Rockefeller University, 1230 York Avenue, New York NY 10021, U.S.A. louism@mail.rockefeller.edu

Making sense of graded olfactory stimuli

Chemotaxis involves directed navigation toward attractive stimuli and away from aversive stimuli. Chemotaxis is critical for the survival of all motile animals since it allows them to track food and mating partners while avoiding danger (assuming that mating partners are not dangerous). The biochemical principles underlying chemotaxis have been thoroughly investigated in bacteria, such as Escherichia coli, which chemotax using improved random locomotion biased in the direction of the chemical gradient. However, the mechanisms by which higher organisms with complex nervous systems navigate through chemical gradients remain poorly described. We are studying this problem in the simple, genetically tractable system of fruit fly (Drosophila melanogaster) larvae. Here we show that chemotaxis represents a powerful paradigm to investigate the neurobiological principles of odor coding. Drosophila larvae have a pair of bilateral olfactory organs each containing 21 olfactory sensory neurons (OSNs). Each OSN expresses one or two specific odorant receptor (OR) genes in addition to the ubiquitously expressed OR gene Or83b.

To characterize the navigation strategy of wild type larvae, we have developed a novel chemotaxis assay where odorant conditions can be both controlled and measured. The assay comprises multiple odor sources that diffuse with semi-predictable dynamics to generate stable odor gradients. We have used infrared spectroscopy to quantify directly the concentrations of odors in gas phase. In this manner, we can precisely control and describe the shape and concentration of odor gradients detected by our experimental animals. In the presence of a stable odor gradient, larvae chemotax by constantly aligning their direction of motion with that of the local odor gradient (klinotaxis).

Using transgenic techniques, we have interfered with the normal expression of OR genes to program an animal with an altered repertoire of ORs. To determine the minimum components of the peripheral system that are required for chemotaxis, we have genetically silenced 20 out of the 21 OSNs. The ability of animals with only a single functional OSN to navigate toward an odor source was assessed in the chemotaxis assay. Our behavioral results demonstrate that the information transmitted by a single OSN is sufficient to mediate robust chemotaxis responses and that intensity coding does not necessitate the combinatorial activation different OR types. Extending this analysis to different combinations of OR-expressing neurons will allow us to deconstruct odor coding in this model organism at the level of behavior.

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47

Martine Maïbèche-Coisne, Christine Merlin, Marie-Christine François, Julien Pelletier, Françoise Bozzolan and Emmanuelle Jacquin-Joly.

Unité UPMC-INRA-INA.PG, « Insect Physiology : Signalisation and Communication », INRA, Route de Saint-Cyr, Bat A, 78026 Versailles Cedex France. maibeche@versailles.inra.fr

Odorant-degrading enzymes of noctuid moths

Perireceptor events in insect antennae consist of sequential steps from the odorant binding to the activation of the olfactory receptors of the sensory neurons. These interactions take place in the sensillar lymph, where sensory neurons are not only exposed to odorants but also to potential xenobiotics. The olfactory system has thus evolved mechanisms for inactivating odors to minimize signal saturation, as well as mechanisms for detoxifying cytotoxic substances. The ability of various enzymes found in Lepidoptera antennae, such as esterases or aldehyde-oxidases, to degrade pheromone has been demonstrated over the past two decades by biochemical approaches. This finding has led to the hypothesis that these Odorant-Degrading Enzymes (ODEs) could participate in ligand degradation after interaction with receptors, and thus to play an important role in signal termination. More recent studies have shown that other enzymes, such as cytochromes P450 or gluthatione-S-transferases, well known for their role in detoxification processes, are expressed in the antennae, where they could be involved in both odorant and/or xenobiotic metabolism.

Using noctuid moths as models to investigate the molecular mechanisms of olfactory termination, we have previously identified several genes that appear to be involved in odorant/xenobiotic metabolism in the cabbage armyworm Mamestra brassicae. We isolated four cytochrome P450 cDNAs expressed in the olfactory sensilla, tuned to sex pheromone and plant’s volatile detection, suggesting a role in odorant clearance (1, 2). All these P450s present structural features of microsomal P450s and they presumably act on odorants only after their internalization. More recently, preliminary analysis of the antennal EST library from the moth Spodoptera littoralis, led to the discovery of seven putative antennal P450s, confirming the microdiversity of this kind of biotransformation enzymes in the antennae. Taken together, these results strongly suggest the occurrence of a P450-dependant oxidative metabolism involved in odor degradation in insects, as shown in vertebrates.

In addition, we have cloned four esterase cDNAs expressed in the antennae of various moths, which used acetates as major pheromone compounds: M. brassicae (3), Sesamia nonagroïdes and S. littoralis. The comparison of the deduced protein sequences of these putative esterases revealed a diversity that could reflect distinct substrate specificities, in agreement with the different chemistry of the pheromone components. More recently, an aldehyde-oxidase expressed in the antennae of M. brassicae has been isolated, leading to the first molecular characterization of this kind of enzyme in insect antennae (4). For a best understanding of odor termination in insects, further studies are now necessary to precise the functional properties of these enzymes.

(1) Maïbèche-Coisne M. et al. 2002. Insect Mol. Biol. 11, 273-281; (2) Maïbèche-Coisne M et al. 2005. Gene 346, 195-203; (3) Maïbèche-Coisne M et al. 2004. Chem. Senses, 29, 381-390; (4) Merlin C. et al., 2005. Biochem. Biophys. Res. Com., 332, 4-10.

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48

Armenak Margaryan^1,2, Ruin Moaddel¹,Jeffrey R. Aldrich², Jennifer M. Tsuruda³, Angela M. Chen³, Walter S. Leal³, and Irving W. Wainer¹

¹ Gerontology Research Center, National Institutes in Aging, National Institutes of Health, Baltimore, Maryland  21224-6825.

² USDA-ARS Chemicals Affecting Insect Behavior Laboratory, Beltsville, Maryland. 20705. aldrichj@ba.ars.usda.gov

³ Department of Entomology, University of California, Davis 95616 

An Immobilized Bombyx mori Pheromone Binding Protein Liquid Chromatography Stationary Phase for Ligand Fishing: Initial Synthesis and Characterization

The pheromone binding protein from the silkworm moth, Bombyx mori (BmorPBP) has been covalently bonded to a liquid chromatographic stationary phase through either the amino or carboxyl terminal groups. The resulting columns were evaluated using radiolabeled bombykol. The immobilized protein retained its ability to bind this ligand, and the two immobilization techniques produced equivalent columns. The BmorPBP column was able to distinguish between four compounds, and rank them in their relative order of affinity for the protein from highest to lowest: bombykol > bombykal >1-hexadecanol > (Z,E)-5,7-dodecadien-1-ol. Furthermore, the immobilized BmorPBP retained its pH-dependent conformational mobility, and the column was stable over a 10 month period. The results of this study demonstrate that the concept of immobilizing pheromone binding proteins and odorant binding proteins in order to create affinity chromatographic columns is viable approach to the development of online screens for new biologically active compounds.

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49

Christine Merlin, Marie-Christine François, Françoise Bozzolan, Julien Pelletier, Emmanuelle Jacquin-Joly and Martine Maïbèche-Coisne

Unité 1272, UPMC - INRA - INA.PG, Physiologie de l’Insecte: Signalisation et Communication, Route de Saint-Cyr, Bat A, 78026 Versailles Cedex France. merlin@versailles.inra.fr

A new aldehyde oxidase selectively expressed in chemosensory organs of insects

Insect’s olfaction consists of spatial and temporal dynamic interactions occuring in sequential steps: the odorant binding, reception, termination and signal transduction leading to the transformation of the chemical signal into an electrical message. Signal termination is a crucial step in the dynamic of the olfactory process, as it prevents the olfactory organs to a continuous chemical stimulation and minimizes signal saturation. It involves different classes of Odorant-Degrading Enzymes (ODEs). Extracellular ODEs specialized in pheromone degradation, such as esterases and aldehyde oxidases, have been identified by early biochemical studies in Lepidoptera. Antennal esterases have been cloned only recently in two moth species, which used acetates as major sex pheromones, the wild silk moth Antheraea polyphemus and our model, the cabbage armyworm Mamestra brassicae. However, no molecular data were available on aldehyde oxidases (AOXs) until now.

In our work, we characterized an aldehyde oxidase in insect antennae, using M. brassicae as model. Indeed, this species used aldehyde components in its sex pheromone: in particular 11-cis-hexadecenyl aldehyde (Z11-16: Ald), which is detected by males and is a synergist for their attraction behaviour, and benzaldehyde, which is emitted by males during the sexual courtship.

Using a PCR-based strategy, we isolated for the first time a partial cDNA coding for a putative antennal aldehyde oxidase from M. brassicae male antennae. Its expression pattern was studied by RT-PCR, Northern blot and in situ hybridization. The transcript was specifically expressed in chemosensory organs, with the strongest expression in antennae of both sexes. In these organs, expression was restricted to the olfactory sensilla. Our results suggest that the corresponding enzyme could degrade aldehyde odorant compounds, such as pheromones or plant’s volatiles.

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50

Christine Merlin, Marie-Christine François, Martine Maïbèche-Coisne and Emmanuelle Jacquin-Joly

UMR « Insect Physiology : Signalisation and Communication », INRA Versailles, Route de Saint-Cyr, F-78026 Versailles Cedex, France. merlin@versailles.inra.fr

The molecular characterization of two clock genes, period and cryptochrome, in moth antennae suggests the occurrence of an antennal circadian clock.

Circadian rhythms are daily 24h mechanisms occurring in most living organisms, from cyanobacteria to human. These rhythms are generated by endogenous circadian clocks, whose molecular elements are well characterized in the fruit fly Drosophila melanogaster. Nocturnal insects, such as moths, mainly use olfactory cues in many aspects of their life. In particular, their pheromonal communication has been demonstrated to undergo daily rhythms, like the female pheromone emission, used to attract conspecific males for mating, as well as the corresponding male behavioural response. Thus, moths appear as good models for olfactory rhythm studies.

In this work, we investigated the possible occurrence of an antennal peripheral clock in a moth, Mamestra brassicae, which could be implicated in olfactory rhythms. Indeed, in Drosophila, such a peripheral antennal clock has been demonstrated to be necessary and sufficient for the control of olfactory rhythms.

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51

Ruthann Nichols

University of Michigan, Biological Chemistry Department, Ann Arbor, MI, 48109-0606 USA nicholsr@umich.edu

Drosophila melanogaster RFamide peptides: olfactory associative behavior.

Neuropeptides regulate a wide range of behaviors in animals from insect to humans. A focus of our research is to investigate the peptide superfamily related by a common RFamide C terminus. This brain-gut peptide family is conserved across the phylogenetic tree. Typically, multiple RFamide genes are present in a species and each mRNA encodes a polyprotein precursor that can be processed to yield several structurally-related peptides; however, in some cases only a single peptide is present. Thus, this conserved superfamily provides the opportunity to investigate several aspects of biology from DNA and protein processing to the physiological function(s) of peptides including a role(s) in feeding behavior. I will present our studies involved in mapping individual RFamide peptide temporal and spatial expression patterns and bioassay activity data. In particular, our research focuses on three D. melanogaster RFamide genes: dromyosuppressin (Dms), drosulfakinin (Dsk), and FMRFamide. D. c melanogaster sulfakinin peptides (drosulfakinin I and II, DSK I and DSK II) are structural homologs of the vertebrate peptide, cholecystokinin (CCK). Data suggest RFamide peptides play a role(s) in behavior in D. melanogaster. Our research shows RFamide peptides, although, related by structure and similar activities are uniquely expressed and, thus, are not redundant.

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52

Tomoyosi Nisimura¹, Atsushi Seto¹, Miki Shimohigashi², Masayuki Iwasaki², Ryohei Yamaoka¹ and Mamiko Ozaki¹

¹ Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto 606-8585, Japan,

² Division of Biology, Faculty of Science, Fukuoka University, Fukuoka, 814-0180, Japan, tomoyosi@kit.ac.jp

The central projections from the antenna and maxillary palp that share olfactory input regulating the feeding preference in the blowfly, Phormia regina

Dietary experience with an odor influences feeding sensitivity of the blowfly Phormia regina. When the flies fed on sucrose flavored by D-limonene after emergence for 5days, the flies show a subsequent decrease in the feeding sensitivity to plain sucrose. When they fed on sucrose flavored by dithiothreitol (DTT), they show an increased sensitivity. The purpose of our study is to reveal how these olfactory experiences affect the feeding sensitivity. In this study, we focused on the olfactory organs and their projections in the brain. We removed either antennae or maxillary palps and examined whether such influences of the dietary experience on the feeding sensitivity was altered or not. Removal of antennae cancelled the effects of the dietary experience with D-limonene, but did not influence the effects of the dietary experience with DTT. On the other hand, removal of maxillary palps cancelled the effects of the dietary experience with DTT, but did not with D-limonene. Thus, the maxillary palps and the antennae of the flies might share olfactory inputs for memory formation affecting the feeding preference. The maxillary palp had sensilla basiconica, sensilla trichodea and spinules, although the gustatory sensilla were conspicuously absent in our scanning electron microscopy observation. In order to clarify the processes from maxillary palps and antenna into the brain, the sensory projections in the brain were examined. Fluorescence labeling of maxillary afferents revealed a distinct fiber bundles projecting into some area in the suboesophageal ganglion (SOG) and several glomeruli in the ipsilateral and contralateral antennal lobe. The antennal afferents innervated antennal lobe, and some fiber bundle projected into SOG. In maxillary and antennal afferents, the termination areas within the SOG were close to each other, though their interaction remains unknown. In order to analyze the innervating mechanism of those afferents from the maxillary palp and antenna to the second order neurons, 3D digital models of the glomeruli were produced based on these images of confocal laser scanning microscopy . This analysis demonstrated that the afferents of the maxillary palps and the antennae projected into the distinct gromeruli in the antennal lobe, respectively.

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53

Mamiko Ozaki

Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto 606-8585, Japan. mamiko@kit.ac.jp

Behavioral switching by contact chemical cues in flies and ants: Role sharing between the peripheral and the central nervous systems.

Feeding behavior in flies is triggered by palatable taste of foods, and aggressive behavior in ants is triggered by cuticular hydrocarbons (CHCs) of strangers.  Those behaviors were quantitatively controlled by adequate contact chemical cues, which stimulate particular chemosensory neurons.  However, those behavioral thresholds, which are determined in the CNS, were modulated by various factors; feeding threshold is altered by coexistence of toxic taste substances or appetitive and non-appetitive olfactory food flavors, cognitive food preference, postnatal dietary experiences, magnitude of starvation, etc; aggression threshold is altered by presence of nestmates and non-nestmates or even their foot prints, fighting history, basic activity, etc.

In the taste sensillum of the adult blowfly, Phormia regina, we recorded the response to toxic monoterpenes, which were applied with an OBP, and certified existence of deterrent cell.  The deterrent cell and the sugar receptor cell discriminatively categorize edible and non-edible substances.  Recently, we found the particular antennal sensilla in the ant, Camponotus japoicus.  Using a CSP, we recorded selective response to CHC patterns from non-nestmates and other ant species but did not to nestmate CHCs.  This sensillum looked like an olfactory sensillum but functionally contact chemosensory organ for non-volatile CHCs.  Surprisingly, it housed 200 receptor neurons.  This number of cells may be needed to categorize various CHC patterns into signs of nestmates and others.  In both cases of the fly and the ant, regardless of using 2 or 200 receptor neurons, chemical signs were categorized based on their biological meaning at the most peripheral level. 

In the CNS, the electrophysiological signal coming from each sensory neuron would be quantitatively evaluated and integrated with each other.  Neural activity balance among neurons or other secondary factors influenced determination of behavioral threshold via biogenic amines.  We showed that in the fly, increase in tyramine level enhanced feeding motivation, and that in the ant, increase in octopamine level decreased aggressiveness.

This study was supported by the grant from ProBRAIN to M.O.

Pézier A.¹ and Lucas P.¹

¹ INRA, UMR 1272, Physiologie de l’Insecte - Signalisation et Communication, Route de St Cyr, 78026 Versailles Cedex, France. pezier@versailles.inra.fr

A combined in vivo and in vitro electrophysiological study of Ca 2+ involvement in moth olfactory transduction

Responses of olfactory receptor neurones (ORNs) involve a two-step transduction pathway in vertebrates 1,2 and invertebrates 3. Second-messengers activate Ca 2+ permeable channels, leading to an increase in intracellular Ca 2+ concentration [Ca 2+]i. Ca 2+ then gates channels that depolarize the neurone. In insects, the olfactory transduction involves p hospholipase C that mediates the production of inositol 1,4,5-trisphosphate (IP 3) and 1,2-diacylglycerol (DAG). IP 3 was proposed to be the first second messenger of the transduction cascade, IP 3 opening Ca 2+ channels and then Ca 2+ activating cationic channels 4. Using in vivo and in vitro electrophysiological approaches in Spodopteralittoralis (Lepidoptera, Noctuidae), we reinvestigated (1) the involvement of Ca 2+ in insect olfactory transduction and (2) the second messenger cascade leading to an increase in Ca 2+.

In vivo , sensillar potentials (SP) and action potential (AP) were recorded from ORNs in response to the main pheromone component with the extracellular tip recording technique. [Ca 2+] in the sensillum lymph was adjusted to 6.10 -3, 10 -3 or 2.10 -8 M. The kinetics and amplitude of the rising phase of SPs were the same in the three [Ca 2+]. The facts that SPs were recorded in low [Ca 2+] and that there is no Ca 2+ stores in the outer dendrite (transduction zone) indicate that second messenger-gated channels can depolarize ORNs in absence of Ca 2+. In vertebrates Ca 2+ down regulates the opening and conductance of second messenger (cAMP)-gated channels 5 and these channels can alone depolarize ORNs in Ca 2+-free solutions. We thus hypothesized that, as in vertebrate ORNs, the opening and/or conductance of second messenger-gated channels is down regulated by Ca 2+. In vitro experiments confirm this hypothesis. We identified a novel DAG-activated cationic current permeable to Ca 2+ from cultured ORNs with whole-cell patch clamp recordings. Decrease in ext. [Ca 2+] or addition of a calmodulin antagonist strongly increased DAG-gated current amplitude confirming that in insects ORNs Ca 2+ also down regulates this second messenger-activated current.

Decreasing [Ca 2+] in the sensillum lymph significantly lengthened SP responses by increasing the repolarising phase. Thus, Ca 2+ is necessary for a fast ORN repolarisation. Two mechanisms are proposed from the in vitro approach to explain this result. First, a fast activating Ca 2+-dependent K + current is the largest conductance found in Mamestra brassicae 6 and S. littoralis ORNs. This current is most probably implicated in ORN repolarisation. Second, the Ca 2+ modulation of the DAG-activated current cited above could be due to a negative Ca 2+ feedback as already described for IP 3-gated channels 4. The importance of Ca 2+ to get fast responses to odours is consistent with the insect orientation behaviour to pheromone sources since odour plumes are intermittent with fast series of bursts of odour.

1 Schild and Restrepo (1998) Physiol. Rev. 78:429-466.

2 Lucas et al. (2003) Neuron 40:551-561.

3 Stengl et al. (1999) In Insect Olfaction. Ed. Hansson, Springer Verlag, Berlin .

4 Stengl (1994) J. Comp. Physiol. 174:187-194.

5 Zufall and Firestein (1993) J. Neurophysiol. 69:1758-1768.

6 Lucas and Shimahara (2002) Chem. Senses 27:599-610.

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55

Pézier A.¹, Papaefthymiou C.^1,2, and Lucas P.¹

¹ INRA, UMR 1272, Physiologie de l’Insecte - Signalisation et Communication, Route de St Cyr, 78026 Versailles Cedex, France, ²Animal Physiology, Aristotle University, GR-54124 Thessaloniki, Greece pezier@versailles.inra.fr

Involvement of Diacylglycerol in insect olfactory transduction. An electrophysiological study in the moth Spodoptera littoralis

Animals have developed highly specialized sensory organs to detect olfactory cues that are abundantly used in their life. Vertebrates and insects share a common design in their olfactory system 1 that makes insects a good model for basic studies on the sense of smell. Their olfactory system is less complex, specific olfactory ligands are available and in genetically engineered insects altered behaviors can be examined 2. Olfactory receptor neurons (ORNs) convert the information about the quality, quantity and temporal pattern of the odor stimuli they detect into electrical responses, which are propagated as action potentials to the brain where the olfactory information is processed. In insects, the process of chemo-electrical signal transduction has been studied using biochemical, electrophysiological and molecular genetic techniques. However, its molecular mechanisms remain controversial. The transduction cascade is mediated by G-protein-coupled receptors which activate phospholipase C b (PLC- b ) 2,3. PLC mediates the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP 2) in inositol 1,4,5-trisphosphate (IP 3) and 1,2-diacyl glycerol (DAG). IP 3 was proposed to be the first second messenger of the pheromone transduction cascade, opening Ca 2+ channels 4. However, DAG was also found to activate moth ORNs both in vivo 5 and in vitro 6. Moreover, olfactory responses but not adaptation were normal in IP 3-receptor Drosophila mutants 7 suggesting that IP 3 might not be required at the primary step of olfactory transduction in the fruitfly. We reinvestigated the role of DAG in moth olfactory transduction. Whole-cell patch clamp recordings on cultured ORNs of the moth Spodoptera littoralis have revealed a novel DAG-activated cationic current for insects. The DAG-gated channel is permeable to Ca 2+. Its activation does not require Ca 2+ or PKC. Decrease of external Ca 2+ concentration or the presence of a calmodulin antagonist strongly increased the current amplitude, which demonstrates that this channel is modulated by intracellular Ca 2+. The inhibition of the degradation of DAG, using a DAG kinase (DGK) inhibitor, produced a sustained activation of a current that shares the properties of the DAG-gated current. The pheromone-gated current matches the kinetic, ionic and pharmacological profile of the DAG-evoked conductance. On the basis of these results, we propose that DAG is involved in chemo-electrical transduction in the moth S. littoralis and that an active DGK regulates the concentration of endogenously generated DAG. The insect DAG-gated channel shares common properties with TRP channels. Such a DAG-gated TRP channel, TRPC2, has been recently characterized in mouse vomeronasal neurons 8 demonstrating the high similarity of pheromone transduction between vertebrates and invertebrates.

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56

Jean-François Picimbon

Department of Ecology, Lund University, Sölvegatan 37, SE-22362 Lund, Sweden

Organization, evolution and expression of odorant binding proteins in moths

Given the remarkable sensitivity of the insect olfactory system, one may assume that the controlled responses of an olfactory cell are established following the assembly of the supramolecular structure odorant-binding protein-receptor. In moths, different classes of binding proteins may mediate the binding of the different classes of chemicals from most generalist odorants to highly specific pheromone compounds. Expression studies suggest that pheromone binding proteins and general odorant binding proteins are expressed in a distinct sex- and tissue-specific manner.  In this talk I will review our understanding of evolution and function of odorant binding proteins, with a particular focus on studies of the phylogenetic distribution and genomic structures in different species of moths, as revealed by the analysis of the genome of Bombyx mori and cloning of OBPs in noctuids.

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57

Erika Plettner, Nicolette Honson, Yongmei Gong and Ivy Ling

Simon Fraser University, Department of Chemistry, Burnaby B. C. V5A 1S6, Canada. plettner@sfu.ca

Early and late events in insect pheromone olfaction

Early events.  Pheromone-binding proteins (PBPs) are soluble, acidic proteins that bind small molecules (such as pheromones) reversibly.  PBPs, which are the most abundant protein in the lymph of pheromone-sensing hairs, are thought to be the first protein in the insect to come in contact with pheromone.  Since the original discovery of PBPs in 1981, many studies have revealed structure and sequence relationships among the large variety of PBPs and odorant-binding proteins (OBPs).  For example, all PBPs and many OBPs have 6 highly conserved cysteine residues, which form three interlocked disulfide bridges. 

For the past 7 years, we have studied the physical properties of the two PBPs from the gypsy moth, Lymantria dispar.  We are particularly interested in the mechanism of binding and dissociation of small ligands, as well as structure-function relationships in insect OBPs.   New data from N. Honson has shown that the second disulfide bridge in these PBPs (cys 2-cys5) is most easily reduced.  Interestingly this disulfide bridge is missing in the four-cysteine OBPs.  Conformational analysis of all disulfide bridges from all available PBP structures has revealed that the reducible disulfide may act as a conformational guide by accessing conformations that are unusual for disulfide bridges.  Moreover, rotation around the reducible disulfide significantly moves several helices relative to each other.

New data from Y. Gong (poster) has shown that it is possible to attach fluorescent probes to PBPs, without affecting dissociation constants for known ligands.   She has used this approach to study kinetics of ligand association and dissociation. 

Late events.  When a moth is exposed to odorants, these adsorb on various body surfaces.  If the adsorbed odorants accumulate and eventually desorb, then the body surface could become a confounding source.  For this reason, deactivation of odors is thought to be very important.  We have found an unusual pathway of pheromone odorant degradation in the gypsy moth.  This pathway starts with oxidation of the hydrocarbon pheromones and continues with degradation to smaller metabolites.  Unexpectedly, these metabolites are built up again to methyl and ethyl oleate and linoleate, as well as to very polar compounds.  We have detected the enzymatic activity in extracts from antennae, legs and wings.  We have studied the cofactor requirements and some of the intermediate metabolites.

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58

Pawel Pyk¹, Sergi Bermudez i Badia¹, Philipp Knüsel¹, Eric Chanie², Paul F.M.J. Verschure¹

¹ Institute of Neuroinformatics, ETH-UZ, Winterthurerstr. 190, CH-8057 Zürich, Switzerland

² Alpha MOS, Toulouse, France. ppyk@ini.phys.ethz.ch, www.amoth.org

Building an artificial moth: An outdoor flying robot for optomotor anemotactic behavior. 

Nature has performed a great job in optimizing male moths for olfactory search. Indeed the resources needed to carry their large chemical sensor arrays, i.e. antennae, suggest that these must serve a vital purpose. Constructing an artificial moth including its sensors, neuronal structures and real world behavior, can increase our understanding of this biological system and will also lead us to the development of new technologies.

Here we describe the technologies we have developed and integrated to construct an artificial moth. Our approach is to rely on the most advanced technologies available while building on off-the-shelf components to reduce engineering overhead.

A  polyurethane blimp (4m long) filled with helium is propelled by 4 DC motors fixed to a rigid carbon fiber frame. Our system uses lithium-polymer batteries with a very advantageous energy/weight ratio, resolving a key limiting technology for UAVs, i.e. energy autonomy. A vision system based using two miniature wireless (2.4GHz) color cameras with wide angle optics are used for course stabilization and obstacle avoidance. Two olfactory sensors, each containing an array of six broadly tuned chemosensors, each served by their own microcontroller, support chemical search. A third microcontroller is responsible for communication and motor control. Additional microcontrollers are interfacing monitoring sensors including GPS, 3D magnetic compass and barometric altimeter. All of these microcontrollers are connected to a common bus which allows for easy system expansion. Wireless communication with a ground station using 2.4GHz long range radio link allows us to control the device with a conventional autopilot or large-scale simulations of insect neuronal systems that are running on a groundstation.  This solution keeps the onboard computation to a minimum. Flight tests have shown that this is a stable and robust platform for both indoor and outdoor experimentation and the infrastructure can be generalized to other types of vehicles including mobile robots.  We will present the basic properties of our artificial moth and show its performance in a number of flight tests. This project is supported through the EU Future and Emerging Technologies programme (IST-2001-33066 (AMOTH))

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59

Ariane Ramaekers¹, Edwige Magnenat¹, Elizabeth C. Marin², Nanaë Gendre¹, Gregory S.X.E. Jefferis², Liqun Luo² and Reinhard F. Stocker¹

¹ University of Fribourg, Department of Biology, Chemin du Musée 10, CH-1700 Fribourg, Switzerland
² Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA Ariane.Ramaekers@unifr.ch, Reinhard.Stocker@unifr.ch, lluo@stanford.edu

In summary, the basic design of the larval olfactory system is similar to the adult one. However, ORNs and projection neurons lack cellular redundancy and in general do not exhibit convergent and divergent connectivity: 21 ORNs confront essentially similar numbers of antennal lobe glomeruli, projection neurons and calycal glomeruli. Hence, we propose the larval olfactory circuit of Drosophila as an elementary model for olfactory studies, a system that still possesses the basic design of the mammalian system, but in its simplest form.

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60

Carolina E. Reisenman¹, Thomas Christensen and John Hildebrand

¹ ARL Neurobiology, University of Arizona, Tucson, AZ 85721-0077, USA. carolina@neurobio.arizona.edu

Odor selectivity of output neurons innervating an identified, sexually isomorphic glomerulus in the main antennal lobe of the moth Manduca sexta

The antennal lobe (AL) of insects contains an orderly array of glomeruli, primary sites of olfactory information processing. In some insects (e.g. moths and cockroaches) a few glomeruli are sexually dimorphic and constitute labeled lines for processing of sex pheromones. Active debates still exist, however, about whether glomerular output cells (PNs) in the main AL are also narrowly tuned. We thus examined this critical issue in the AL of the moth Manduca sexta using intracellular recording and staining techniques. We consistently found that PNs innervating an identifiable sexually isomorphic glomerulus in the main AL, G35, selectively responded to low concentrations (down to 10^-6 vol/vol or less) of the host-plant volatile cis-3-hexenyl acetate (c3HA). We also found that PNs innervating glomeruli neighboring G35 showed weak or no response to c3HA, suggesting that information processing about this compound is mainly restricted to G35. We found that the dose-response properties, odor-tuning and morphological properties (including their projection sites in the protocerebrum) of G35-PNs were identical in both sexes, but the sensitivity of male G35-PNs was 1-2 orders of magnitude lower than that of females. These results thus indicate that glomeruli in the main olfactory system have, as the well-studied sexually dimorphic glomeruli, characteristic and limited molecular receptive ranges. These findings stress the functional significance of spatial coding in olfactory information processing. To understand the neuronal mechanisms underlying this coding scheme, we are now characterizing interglomerular synaptic interactions in both sexes by testing G35-PNs with the odor stimulus that specifically drives PNs in a neighboring glomerulus (and vice-versa). Supported by the PEW Latin American Program in the Biomedical Sciences from the PEW Charitable Trust to CR and NIH Grant R01-DC-02751 to JGH.

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61

Carlos Ribeiro and Barry Dickson

Institute of Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences, Dr. Bohr-Gasse 3-5, A-1030 Vienna, Austria. Carlos.ribeiro@imba.oeaw.ac.at

Using inducible RNAi to study the genetic basis of taste perception in Drosophila melanogaster

The perception and analysis of the environment is one of the most important and fascinating tasks every living organism has to perform. While our understanding of olfaction in Drosophila has been rapidly growing in the last years less is known about the genetic basis of gustation. In the last years RNA interference (RNAi) has emerged as a novel approach to perform functional genomic analyses of complex biological processes. Combined with the Gal4-UAS system for controlled gene induction in Drosophila it allows the silencing of genes of interest in almost any tissue in a time specific manner. We have started analyzing the involvement of a set of candidate genes in taste discrimination and taste perception by combining RNAi silencing with classic behavioral assays. First results from this approach will be presented.

¹ UMR 1272 "Physiologie de l'insecte", INRA, 78026 Versailles Cedex, France

² Laboratoire de Physique Théorique des Liquides, Université Pierre et Marie Curie (Paris 6), 75252 Paris Cedex 05, France. rospars@versailles.inra.fr

Modelling the early steps of transduction in olfactory receptor neurons

Olfactory transduction is a multistep process whose basic function is to convert a low energy reaction that may involve a single odorant molecule – the odorant-receptor interaction – into a whole cell electrical response – the receptor potential, which triggers the firing of one or several action potentials. The ultimate goal of our study is to account quantitatively for the various reactions involved in the transduction of pheromone signals in moth olfactory receptor neurons (ORNs) specialized in the reception of sexual pheromones using experimental (in vivo and in vitro) and modelling approaches.

The system we investigated includes reactions taking place both in the sensillum lymph and within the membrane of the ORN outer dendritic segment. We took into account the main reactions involving pheromone molecules, i.e. their translocation from air to lymph, their transport by pheromone-binding proteins, their enzymatic deactivation and their interaction with receptors. However, the present study was focused on the membrane events. These reactions involve the sequential interaction of three types of proteins – receptors (R), G-proteins (G) and effectors (E, generating second messengers) –, whose reaction rates are limited by lateral diffusion in the membrane. The network of reactions we considered includes 13 molecular species and both activating (forward) and inactivating (backward and feedback) reactions.

The present study, based on the law of mass action, led us to solve the system of 13 coupled differential equations representing the rates of change of the 13 molecular species based on realistic values of the reaction rate constants. The three activated proteins (R*, G* and E*) were especially examined and their evolutions in time were computed for various numbers of odorant molecules and various kinds of stimulation (single pulse and periodic train of pulses). In particular the height (number of molecules per ORN) and temporal characteristics (durations of the rising and falling phases) of the modelled peak responses of R*, G* and E* were determined.

The results for E* were compared to the experimentally known characteristics of the receptor potential measured in the same conditions. Measurements and calculated results are in good agreement which suggests that the model studied, i.e. both the reactions considered and the order of magnitude of their associated constants, may retain essential features of the real system.

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63

Wolfgang Rössler

University of Würzburg, Biozentrum, Zoology II, Am Hubland, 97074 Würzburg, Germany. roessler@biozentrum.uni-wuerzburg.de

Neuronal plasticity within the olfactory pathway and its importance for polyethism in social hymenoptera

Olfaction plays a major role throughout the life of social insects. Pheromone communication, chemical recognition and olfactory orientation induce a rich diversity of olfactory-guided behaviors. These behaviors can vary significantly depending on caste, group, age or sensory experience. Social hymenoptera are excellent model systems to study the neuronal basis of long-term plasticity in olfactory behavior. I will present results from our recent studies on developmental and adult neuronal plasticity in primary and secondary olfactory centers in the brain of two ant species and the honeybee.

Developmental plasticity mediated via brood care in the honeybee: Queen-worker polymorphism is induced by larval feeding and does not depend on genomic differences. We found that the total number and volume of olfactory glomeruli in the antennal lobe (AL) of queens is smaller than in workers. In addition, the number and density of synaptic complexes (microglomeruli) in the olfactory input region (lip) of the mushroom body (MB) – calyx was significantly lower. Thermoregulatory influences during the pupal period affected the volume of AL glomeruli and the number of MB-calyx microglomeruli in the adult brain. These changes may underlie differences observed in olfactory learning abilities.

Age- and experience dependent plasticity during brain maturation in the ant Camponotus rufipes: Ants go through an extended period of brain maturation during early adulthood. In the C. rufipes we observed remarkable changes in the synaptic organization within the MB-calyx. Both the number and density of microglomeruli in the olfactory lip increased markedly during the first three weeks of adult life. These changes correlate with important transitions in olfactory-guided behavior.

Status-dependent adult plasticity in the ponerine ant Harpegnathos saltator: H. saltator can switch from the non-reproductive to the reproductive caste during the adult stage. This change in status is correlated with profound changes in behavior. Compared to the honeybee, the changes in olfactory centers were more subtle among both female castes, but an extreme sexual dimorphism was observed. This was also the case for the distribution of serotonergic and dopaminergic neurons.

Our results demonstrate that long-term plasticity within olfactory centers of hymenoptera occurs at various stages during development and adulthood. We propose that the associated changes in neuronal and synaptic structures play an important role in the regulation of polyethism and the division of labor.

M. Rützler, H. W. Kwon, Lu T., Knepper J., Pitts, R.J. and L. J. Zwiebel

Department of Biological Sciences and Center for Molecular Neuroscience, Vanderbilt University, Nashville, Tennessee 37205. m.ruetzler@vanderbilt.edu

Expression and functional studies of odorant receptors in the malaria vector mosquito anopheles gambiae

The biting behavior of Anopheles gambiae (An. gambiae) is largely influenced by olfactory cues emanating from host animals and the strong preference of the An. gambiae s.s. species for human hosts contributes significantly to the transmission of human malaria in sub-Saharan Africa.  We have previously identified a family of genes in An. gambiae that encodes candidate G protein-coupled odorant receptors (AgORs). We now present functional studies carried out in Drosophila and Xenopus oocytes that provides evidence in support of their identification as odorant receptors.  One of these genes, GPRor7, is highly conserved with respect to single genes from many insect orders.  Immunocytochemistry demonstrates that its protein product is localized to most sensilla of olfactory organs of An. gambiae.  By RT-PCR analysis, GPRor7 is also expressed in the proboscis of An. gambiae, a known gustatory organ in other mosquitoes.  Immunocytochemistry indicates that GPRor7 is found within each of a small subset of sensilla on the labellar lobes, a pair of bulbous organs at the tip of the proboscis.  Scanning electron micrographic analysis reveals that the GPRor7 expressing sensilla are short, grooved hairs residing in a socket on the cuticle surface and that there are about 25 of these hairs per labellar lobe.  Their morphology is reminiscent of other grooved olfactory sensilla, and the presence of an odorant receptor within them indicates either a potential olfactory function for them, or a potential gustatory function for GPRor7.  This work was supported by grants from the US National Institutes of Health. M. Rützler is currently supported by the Max-Kade Foundation.

Camilla Ryne¹, Mats Ekeberg², Niclas Jonzén³, Cam Oelschlager⁴, Christer Löfstedt¹ and Olle Anderbrant¹

¹ Lund University, Chemical Ecology and Ecotoxicology, Dept of Ecology, Solvegatan 37, SE-223 62 Lund, Sweden

² Cloetta-Fazer AB, Ljungsbro, Sweden.

³ Theoretical Ecology, Dept of Ecology, Lund University, Sweden.

⁴ChemTica Internacional, Apdo. 159-2150, San Jose, Costa Rica. Camilla.Ryne@ekol.lu.se

Reduction in an almond moth Ephestia cautella (Lepidoptera: Pyralidae) population by means of mating disruption

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66

Silke Sachse, Andreas Keller and Leslie B. Vosshall

The Rockefeller University, Laboratory of Neurogenetics and Behavior, 1230 York Avenue, New  York, NY 10021, USA.  sachses@mail.rockefeller.edu

Carbon dioxide mediates specific synaptic and behavioural adaptation in Drosophila

The ability of the brain to adapt structurally and functionally in response to sensory stimuli is a striking property across animal phyla. Several studies have reported that continuous exposure to odors leads to global morphological effects in the first olfactory neuropil, the olfactory bulb of vertebrates or the insect antennal lobe.

To investigate the specific effect of an odorant on the neuronal network it activates, we studied the plasticity in identified olfactory neurons with known odor response profiles in Drosophila melanogaster. Fruit flies are highly sensitive to CO₂, which activates a population of approximately 25 olfactory sensory neurons that project to a single glomerulus in the antennal lobe (De Bruyne et al., 2003; Suh et al., 2004). Using the GAL4/UAS system to visualize the different olfactory neurons innervating this specific glomerulus, anatomical changes on separate processing levels due to long-term CO₂ exposure were investigated. The results showed an enlargement of the CO₂-glomerulus in a concentration-dependent manner. This effect was stimulus- and glomerulus-specific.

In order to analyze physiological consequences of long term exposure, we performed calcium imaging in the antennal lobe by specifically labelling sensory neurons as well as local interneurons. The sensory neurons were not influenced by the CO₂ exposure, while on the level of local interneurons a significant decrease in response to the exposed stimulus was observed.

To determine whether these stimulus-evoked changes in olfactory circuitry produced behavioral consequences, we tested olfactory-evoked locomotor responses to CO₂ and other stimuli. Flies showed a reduced sensitivity to CO₂ after CO₂ pre-exposure, but showed normal responses to all other odors tested. The behavioral changes were stimulus-specific and concentration-dependent. Importantly, both anatomical and behavioral effects of CO₂ exposure were reversible. We are in the process of identifying the cellular and molecular basis of these experience-dependent changes. Support contributed by: NIH/NIDCD (1R01DC005036-03), NSF (IBN-0092693), Beckman Foundation, McKnight Foundation, John Merck Fund.

De Bruyne et al. (2003). XXV. AChemS meeting, .no. 379; Suh et al. (2004). Nature 431:854-9.

Hansjürgen Schuppe, Matthew Cuttle, Philip L Newland

School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom. hs8@soton.ac.uk

Modulation of sodium chloride taste sensitivity by nitric oxide

Locusts require sodium chloride (NaCl) at low concentrations as part of their diet, and detect it with taste receptors (basiconic sensilla) scattered over the body surface, including the legs. When NaCl makes contact with a sensillum a volley of action potentials is elicited in 1-3 of its chemosensory neurons. Previous studies have suggested that one or more of the sensory neurons contain soluble guanylate cyclase, one target for nitric oxide (NO). The aim of our work is to determine the role of NO in taste perception, and in particular to analyse its effect on salt taste sensitivity.

Perfusion of the leg with saline containing drugs that alter NO levels within the preparation shows that NO modulates sensory responses to NaCl. For example, drugs that increase NO levels decrease the spike rate of the responses, whereas drugs that decrease NO levels cause an increase in spike rate. This suggests, that NO is endogenously generated and continuously attenuates the sensitivity of the chemosensory neurons to NaCl. The effect of NO does, however, not involve the NO/cGMP pathway, but is likely to be mediated by direct binding of NO to sulfhydryl groups.

There are two potential sources of endogenous NO that could act on sensory neurons in the periphery: from within the sensory neurons and support cells of the basiconic sensilla themselves, or from nearby cells in the epidermal layer. Close to basiconic sensilla the epidermis contains glandular cells, which synthesise NO, however there is no evidence that NO is generated by chemosensory neurons. Thus glandular cells represent a potential source of NO that can modulate the sensitivity of NaCl taste receptor neurons.This work was supported by a project grant to P.L.N. from the BBSRC.

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Maria Dolores Setzu, Iole Tomassini Barbarossa, and Anna Maria Angioy

Department of Experimental Biology, Section of General Physiology, University of Cagliari, Cittadella Universitaria, 09042 Monserrato-Cagliari, Italy

tomassin@unica.it; mdsetzu@unica.it; amheart@unica.it 

Olfactory sensitivity to pure volatile compounds and their mixture in the adult moth, Spodoptera littoralis

With their highly specialized olfactory system, insects are able to detect and discriminate a number of odorants present in the environment as mixtures of chemical volatiles. Olfaction guides specific behavioural performances, allowing them to orient towards sexual partners, food sources and oviposition sites. Several insect species also display olfactory-induced heart responses which reliably monitor odour perception (Angioy et al., 2004). In the adult moth Spodoptera littoralis, a highly sensitive heart response occurs to stimulation with single components of the sex pheromone and their blend, as well as with pure plant odour volatiles (Angioy et al., 2003).

In the present research study, the heart response to olfactory stimulation has been used as an experimental tool for testing the sensitivity of Spodoptera littoralis moths to plant odours mixtures. 

By performing electrophysiological recordings, the heart activity (ECG) and the antennal olfactory input (EAG) were simultaneously monitored on intact specimens of both sexes. Pure chemicals were diluted in decadic steps and singly tested in order of increasing concentrations until a cardiac response was induced. The EAG amplitudes to stimulus doses above and below the heart response threshold were measured. A binary mixture containing half dose of each odour below the heart response threshold was tested, and heart response occurrence or absence as well as EAG amplitude were measured.

Extremely low doses of single odour stimuli induced a heart response in most specimens. Response occurrence was associated to a much higher EAG amplitude compared to that measured in response to the stimulating odour dose below the heart response threshold.

Insects displayed a heart response when stimulation was performed with lower single-odour doses in binary mixture. In addition, the related EAG amplitude was higher than that measured in response to the single-odour stimulus dose below the heart response threshold, and equivalent to that to the single-odour stimulus dose above the response threshold.

Taken together, these results show a synergic effect of odours in a binary mixture leading to an enhancement of sensitivity and reactivity of the insect to single components of the mixture. This work was supported by the Italian Ministry of University and Scientific Research - 2003

Angioy AM, Muroni P, Setzu MD, Urru I, Cansella G, Banni S and Tomassini Barbarossa I (2004) Cardiac responses to external stimuli: a precious tool for testing insect responsiveness. Trends in Comp Biochem & Physiol, 10: 65-73.

Angioy AM,  Desogus A, Tomassini Barbarossa I,  Anderson P,  Hansson BS (2003) Extreme sensitivity in an olfactory system. Chemical Senses, 28: 279-284.

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Ana Florencia Silbering¹; Ryuichi Okada¹; Elane Fishilevich²; Leslie Vosshall²; Kei Ito³ and Giovanni C. Galizia⁴

¹ Institut für Neurobiologie, Freie Universität Berlin, Königin-Luise-Str. 28/30, D14195, Berlin, Germany;

² Laboratory of Neurogenetics and Behaviour, The Rockefeller University, 1230 York Avenue, Box 63 New York, NY 10021-6399, USA;

³ Institute of Molecular and Cellular Biosciences, Centre of Bioinformatics, University of Tokyo/National Institute for Basic Biology, Myodaiji, Okazaki, 444-8585 Aichi, Japan;

⁴ Department of Entomology, Room 383, University of California, Riverside, CA 92521, USA.

anafs@zedat.fu-berlin.de, okada@neurobiologie.fu-berlin.de, fishily@mail.rockefeller.edu, leslie@mail.rockefeller.edu, itokei@iam.u-tokyo.ac.jp, giovanni.galizia@ucr.edu

Coding of odor mixtures in the antennal lobe of Drosophila

The antennal lobes (ALs) are the first brain structures involved in odor processing in insects, and are the functional and structural analogues of the vertebrate olfactory bulb. In the ALs, olfactory sensory neurons (OSNs) converge in glomerular structures where they make synapses with local inhibitory neurons (LNs) and projection neurons (PNs).

We have used binary odor mixtures to study coding mechanisms in the AL of Drosophila melanogaster. Specifically, we investigated the role that different neuron populations play in odor processing. We imaged odor elicited Ca⁺⁺ responses in three neuron populations of the AL: the OSNs, LNs and PNs. We expressed the genetically encoded calcium sensor GCaMP using the UAS-GAL4 system with the following lines: Or83b, Or22a, Or33b and Or85a (OSNs, Leslie Vosshall), Np2426 and Np1227 (LNs, Kei Ito) and GH146 (PNs, Gertrud Heimbeck). Or83b stains 70-80% of the antennal OSNs, while in the other OSN lines expression is driven only in a few OSNs. The two LN enhancer trap lines drive expression in two non-overlapping subpopulations of local neurons which have different arborization patterns (Okada, unpublished data). GH146 induces expression in the calcium sensor in ~60% of the PNs.

The glomerular responses of OSNs to a mixture equalled the responses to the components alone. In contrast, we found inhibitory mixture effects in the PNs, where the responses to a mixture were reduced in some of the glomeruli of the activity pattern. Consequently, at the level of the PNs the representation of a binary mixture differs from the linear combination of its components. This is an important evidence of transformation of odor representations in the AL of Drosophila. We propose a glomerulus specific mechanism through which the AL network actively shapes the PN odor responses.

¹ Department of Crop Science, Swedish University of Agricultural Sciences, Alnarp, Sweden;

² Arizona Research Laboratories Division of Neurobiology, University of Arizona, Tucson, AZ 85721.

A small but elaborate mushroom body in the moth, Spodoptera littoralis.

The mushroom bodies are paired structures in the insect brain involved in complex functions such as memory formation, sensory integration and context recognition. In many insects they are highly complex structures comprising sometimes several hundred thousand neurons. Spodoptera littoralis is a moth that has been extensively used for olfactory research and in conditioning experiments and the present study describes in detail the gross anatomy of the spodopteran mushroom body, as well as the morphology of the different kinds of Kenyon cells, the intrinsic neurons of mushroom bodies. The mushroom bodies in Spodoptera consist of only about 4000 large-diameter Kenyon cells. However, these neurons are recognizably similar to morphological classes of Kenyon cells identified in other insects. The calyx of the spodopteran mushroom body is clearly divided in discrete zones receiving different input. There are three major divisions in the vertical and medial lobe, one of which, the gamma lobe, is supplied by clawed class II Kenyon cells as in other described taxa. Of special interest is the presence of a discrete tract (the Y tract) of axons leading from the calyx, separate from the pedunculus, that innervates lobelets above and beneath the medial lobe, close to the latter's origin from the pedunculus. The results allow speculations about possible functional roles of the spodopteran mushroom body divisions when comparing with mushroom bodies of other species, and with the background of the olfactory behavior of Spodoptera.

² Stazione Sperimentale del Sughero, Via Limbara 9, 07029 Tempio Pausania (SS), Italy

Electrophysiological and behavioural responses to gypsy moth (Lymantria dispar) sex pheromone analogues.

The female gypsy moth Lymantria dispar, an important defoliator of cork-oak forests in Sardinia, produces a single-component species-specific sex pheromone called (+)disparlure (2-methyl-7,8-epoxy-octadecane) to attract  males for mating. With the aim of identifying stronger or more stable attractants than the natural pheromone several analogues were synthesized and tested both by electrophysiological and behavioural experiments. Functional tests consisted of EAG and single-cell recordings from excised gypsy moth male antennae. Compounds were tested within the 10^-1-10^-3 dilution range in CH₂Cl₂, pure or added to an equal concentration of (+)disparlure, and their responses compared to those of pure pheromone. The results of two of these analogues that displayed significant effects, 2-decyl-1-oxaspiro[2.2]pentane (Epo-014) and 4-(1-oxaspiro[2.2]pent-2-yl)butan-1-ol (Epo-025), are here reported. In fact, Epo-014 strongly decreased the response of male antennal receptors to (+)disparlure in a dose-dependent manner when the two compounds were presented as a mixture at the two lowest concentrations. Conversely, Epo-025, at the 10^-2 dilution, had a stronger stimulatory effect than that of (+)disparlure; besides, at the same concentration the mixture evoked an additive-type response from male antenna. Epo-025 also had a greater attractive effect than (+)disparlure, as estimated in behavioural experiments by field trap counts. In conclusion, both electrophysiological and behavioural data suggest that synthetic analogues may mimicking the natural gypsy moth pheromone effects to various extents.

¹ Department of Entomology, 318 West 12 th Ave. , Ohio State University , Columbus , OH 43210 . smith.210@osu.edu

² Mathematical Biosciences Institute, The Ohio State University , 231 W. 18th Avenue , Columbus , Ohio 43210

Plasticity and interaction among sensory representations of components in odor mixtures: Predictions from a computational model.

Behavioral and physiological analyses of odor mixture processing in several animal species have revealed several ways in which components of odor mixtures interact as they are being associated with reinforcement. Sometimes components are unaffected by their presence in a mixture. More commonly, components suppress each other such that they are less effectively perceived in a mixture than when presented alone, which is called mixture suppression or overshadowing. This can arise because mixtures typically activate unique neural representations that only partially reflect representations of the components. Interaction of this nature can come about through either sensory interaction or associative mechanisms (e.g. associative plasticity among sensory pathways that process odors). Furthermore, prior conditioning of one component can potentiate (enhance) or block (hinder) learning about another component of a mixture when that mixture is subsequently conditioned using the same reinforcement.

Published reports differ markedly in the degree to which any of these mechanisms – overshadowing, potentiation, blocking – are detected or whether they are symmetric (A overshadows or blocks X but not vice versa). These differences can arise from several factors. Odorants can differ in salience as conditioned stimuli and in the degree to which their neural representations interact. In particular, interaction between neural representations can arise from overlap of both excitatory and inhibitory processing in the Antennal Lobe.

In order to provide a more thorough account for mixture processing, and to provide a more complete account of published data, we have developed a computational model of the honeybee AL . The model incorporates known aspects of the AL circuitry in the honeybee and other insects, including a pathway that represents sucrose reinforcement. Differences in manifestation of any of the conditioning phenomena in the model AL are a product of how neural representations for odorants interact. Therefore, it is to be expected that overshadowing, blocking and potentiation will not occur for all pairs of stimuli. Furthermore, the failure of the prediction of an earlier model to account for blocking strictly in terms how inhibition is spread among neural activity patterns may have resulted from a failure to take into account how excitation and inhibition interact in the AL . The presentation will discuss these findings as well as ecological reasons that odorant components may differ in the ways they interact.

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Marit Stranden¹, Tonette Røstelien^1,2, Anna-Karin Borg-Karlson³ and Hanna Mustaparta¹

¹ Department of Biology, Norwegian University of Science and Technology, Trondheim , Norway , Marit.Stranden@bio.ntnu.no

² Department of Nursing, Gjøvik University College , Gjøvik , Norway

Plant odour receptor neurones characterized by GC-SCR and GC-MS in heliothine moths

Moths of the subfamily Heliothinae (Lepidoptera: Noctuidae) are used to compare mechanisms evolved for detecting and processing plant odour information among monophyletic species. In addition, the pest status of the polyphagous Heliothis virescens and Helicoverpa armigera makes it important to identify the odorants the females use to locate the host plants.

These experiments started out trapping headspace samples of various host and non-host plants to stimulate the olfactory receptor neurones on the antennae. Essential oils and chemical standards were also included to increase the number of volatile compounds to be tested. Using electrophysiological recordings from single receptor neurones linked to gas chromatography (GC-SCR) and GC-MS, we have functionally identified olfactory receptor neurones and classified them into 19 types. All neurones were narrowly tuned, responding strongest to one compound (primary odorant) and weaker to a few others with related structures (secondary odorants). The neuron types were named according to the primary odorant, and the molecular receptive ranges were similar within each type. The co-location of the types was also constant among individuals within and across species. Five of the types were identified in all three heliothine species studied: H. virescens, H. armigera and the oligophagous Helicoverpa assulta (Røstelien et al. 2000a, b, Stranden et al. 2002, 2003a, b). Five others were found in H. virescens and H. armigera, seven only in H. virescens, and two only in H. armigera (Røstelien et al. 2005). The number of receptor neurone types identified in the three species (17 in H. virescens, 12 in H. armigera, and 5 in H. assulta) reflects the number of recordings made in each of them. The odorants, mainly being mono- and sesquiterpenes, a few aliphatic green leaf volatiles and aromatic compounds, were often present in minor amounts in the plant samples. The receptor neurones tuned to six of the odorants (E,E-α-farnesene, geraniol, (-)-germacrene D, (+)-linalool, E-β-ocimene, and E-4,8,12-trimethyl-1,3,7,11-tridecatetraene) constituted 88% of the recorded neurones, and the (-)-germacrene D receptor neurones alone constituted 60%. The results suggest that these neurones may detect the most important odorants for the species. The behavioural effect has only been shown for (-)-germacrene D, which was attractive to mated H. virescens females when added to host plants not containing this odorant (Mozuraitis et al. 2002).

Anatomical studies of the antennal lobes of the three moth species have shown consistent numbers and positions of most glomeruli (Berg et al. 2002, Skiri et al. 2005). Interesting questions are i) whether the number of ordinary glomeruli (60-62) reflects the number of plant odour receptor neurone types, ii) whether the same odour quality is represented in corresponding glomeruli, and iii) whether the preferred host plants of the oligophagous H. assulta females are based on odour information mediated by the species specific glomerulus?

Berg et al. 2002 J Comp Neurol 446:123-134, Mozuraitis et al. 2002 Chem Senses 27:505-509, Røstelien et al. 2005 Chem Senses 30:443-461, 2000a Chem Senses 25:141-148, 2000b J Comp Physiol A 186:833-847, Skiri et al. J Comp Neurol 2005 in press, Stranden et al. 2002 Chem Senses 27:143-152, 2003a J Comp Physiol A 189:563-577,

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Nicholas J. Strausfeld

University of Arizona, Tucson, AZ 85721. flybrain@neurobio.arizona.edu

Arthropod olfactory lobes: Their evolution and neuroanatomical ground plans

Glenn P. Svensson¹, Olle Pellmyr² and Robert A. Raguso³

¹ Lund University, Dept. of Ecology, Solvegatan 37, SE-223 62 Lund, Sweden. glenn.svensson@ekol.lu.se

² Univ. of Idaho, Dept. of Biological Sciences, P.O. Box 443051, Moscow ID 83844, USA, ³Univ. of South Carolina, Dept. of Biological Sciences, Columbia SC 29208, USA.

The role of floral odours in the yucca - yucca moth association

The obligate pollination mutualism between yuccas (Agavaceae) and yucca moths (Lepidoptera, Prodoxidae) is a classically cited example of coevolution, yet the sensory signals mediating these insect-plant interactions are poorly understood. The first step in a program to elucidate the role of floral odours in the mutualism is presented. Floral scent has been collected from 12 yucca species, including all three sections, and extracts analysed by GC-MS, and for some species also GC-EAD.

All capsular-fruited yuccas analysed (n=5) produced compounds from two major biosynthetic pathways: homoterpenes derived from nerolidol, and aliphatic hydro-carbons. Two novel compounds with a prominent signal at m/z 66 in the mass spectrum were also found, and one of these elicits strong EAD responses from pollinator antennae. Surprisingly, an analysis of geographic variation in floral scent of Yucca filamentosa revealed no difference in the odour blend among populations that rely on two different yucca moths for pollination. Traps baited with fragrant flowers of Y. filamentosa attracted the local pollinator, Tegeticula cassandra, and the non-mutualistic yucca moth Prodoxus decipiens, that feeds on the inflorescence stalk, and both moths responded to the same floral volatiles in GC-EAD analyses.

In contrast, the fleshy-fruited species (n=6) showed considerable interspecific variation in floral scent. Yucca treculeana only produces the unique m/z 66 compounds, whereas these compounds are absent in other species. The single member of spongy-fruited yuccas (Joshua Tree) produces fungus odours, like 3-octanone, in addition to homoterpenes and hydrocarbons. As yuccas are exclusively pollinated by yucca moths, plants are predicted to produce highly specific floral odors to facilitate the attraction of pollinators, either by emitting unique compounds or by using strongly canalized odor blends. Flight tunnel and field trapping experiments will reveal what compounds in odour extract that are important for attraction of exclusive pollinators to flowers, and thus how specialised the floral fragrance chemistry is in this highly specific pollination system.

Paul Szyszka,¹ Mathias Ditzen,¹ Alexander Galkin,¹ Giovanni Galizia² and Randolf Menzel¹

¹ Institut für Biologie - Neurobiologie, Freie Universität Berlin, Königin-Luise-Strasse 28/30, 14195 Berlin, Germany

² Department of Entomology, University of California, Riverside, Riverside CA, 92521, USA. szyszka@zedat.fu-berlin.de

Sparsening and temporal sharpening of olfactory representations in the honeybee mushroom bodies

We characterized odor-evoked network activity in the honeybee brain at three consecutive neural compartments. Using Ca²⁺ imaging, we recorded activity in the dendrites of the projection neurons that connect the antennal lobe with the mushroom body, a higher-order integration center. Next, we recorded the presynaptic terminals of these projection neurons. Finally, we characterized their postsynaptic partners, the intrinsic neurons of the mushroom body, the Kenyon cells. We found fundamental differences in odor coding between the antennal lobe and the mushroom body. Odors evoked combinatorial activity patterns at all three processing stages, but the spatial patterns became progressively sparser along this path. Projection neuron dendrites and boutons showed similar response profiles, but the boutons were more narrowly tuned to odors. The transmission from projection neuron boutons to Kenyon cells was accompanied by a further sparsening of the population code. Activated Kenyon cells were highly odor specific. Furthermore, Kenyon cells responded to projection neuron activity only within the first 200 ms and transformed complex temporal patterns into brief phasic responses. Thus, two types of transformations occurred within the MB: Sparsening of a combinatorial code, mediated by pre- and postsynaptic processing within the mushroom body microcircuits, and temporal sharpening of postsynaptic Kenyon cell responses, probably involving a broader loop of inhibitory recurrent neurons.

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Nobuaki Tanaka¹ and Kei Ito

Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

¹ Current address; National Institute of Child Health and Human Development, 35 Lincoln Drive, Bethesda, MD 20892, USA.      tanakano@mail.nih.gov

Internal assembly of the Drosophila mushroom body

The mushroom body (MB) is one of the secondary olfactory centers in insect brains. Recent behavioral works have suggested that the olfactory pathway via the MB is essential for odor learning and memory, but the internal assembly of the MB still remains unclear. We here analyzed how the MB of Drosophila is organized by comparing projection patterns between MB intrinsic and extrinsic neurons. The MB is composed of three parts; the calyx, pedunculus, and lobes. We found that the projection patterns of Kenyon cells, the most abundant intrinsic neurons linking all the three parts, formed two separable areas in the calyx and layer structures in lobes, whose architectures were different among cell types. However, those layer structures were topologically identical between the medial and vertical lobes, suggesting that the distribution of terminals of Kenyon cells are similar between them. In the calyx, one of two areas was observed to be connected with the antennal lobe, but the other area was not, implying that there would be different patterns of input in the calyx.

On the other hand, 16 types of MB extrinsic neurons (MBENs) had specific terminal areas, whose terminals showed segmental divisions in each lobe, that divided lobes perpendicularly to the longitudinal axons of Kenyon cells. Axons of each Kenyon cell pass through multiple segments, suggesting that information of each Kenyon cell could be processed differently among segments. One group of MBENs connected among segments of the medial and vertical lobes proximal to the pedunculus, while the other linked the distal segments of either the medial or vertical lobe. The latter group of MBENs had different patterns of connections with protocerebral areas between the medial and vertical lobes. Those neurons would allow the distributed parallel processing between the medial and vertical lobes of the MB. Many types of MBENs linked among multiple lobes, indicating that the information of each lobe of Kenyon cells are reciprocated among lobes.

Richard S. Vetter ¹ and Giovanni C. Galizia ^1,2

¹ University of California, Riverside. Riverside, CA 92521. USA. 2. Universität Konstanz, D-78457 Konstanz, Germany. Giovanni.Galizia@uni-konstanz.de

Temporal integrity of an odor stimulus in common insect olfactometer settings is greatly affected by physical aspects of the flow system

There is currently a great debate about the role that temporal patterns in neural activity play for olfactory coding. An accurate analysis of this question, however, is only possible if temporal properties of a stimulus itself are under good control. So far, no technique was available to accomplish this. Using a photoionization detector (PID) we show that there is great variability in the integrity of the signal profile within an odor delivery apparatus, which is highly influenced by the physical components of the set-up.  Some of these variables are: the location of the PID within the air stream, airspeed of the odor deliverant, exit tube length, exit tube diameter, orientation of the odor delivery device in relation to the exhaust flow, exhaust tube airspeed.   With most variables that we tested, the caliber of the signal deteriorated by 2 cm from the exit of the tubing.  This has significant implications for researchers who are addressing issues of insect olfaction; significant preliminary planning must be incorporated into the design of the experiment to provide an optimized odor delivery system.  Additionally, this demonstrates that highly variant olfactory response cannot be wholly attributed to neurological activity of the experimental animal and may be a reflection of the caliber of the odor plume hitting the sensory receptors.  We present the critical parameters for ameliorating control over the physical properties of olfactory stimuli.

Richard Vogt, Kenny Fernandez, Jonathan Bohbot and Marie-Dominique Franco

University of South Carolina, Dept. of Biological Sciences, Columbia, SC  29208, U.S.A. vogt@biol.sc.edu

Establishing peripheral phenotypes: early events in post-embryonic development leading to the patterned organization of the adult Manduca sexta antenna.

Central olfactory pathways are topographically organized in a manner consistent with and, to a degree, controlled by the phenotypes of primary olfactory neurons.  These ORN phenotypes are determined early during post embryonic development, suggesting that early developmental decisions in the periphery are critical to the ultimate establishment of central the pathways.  Genetic approaches in Drosophila have identified genes that likely serve as central and hierarchical gateposts regarding the phenotypic fates of sensory organs (sensilla/neurons) within specific regions within the antenna.  These studies have not yet, however, revealed much information regarding pathways downstream from these gates.  How are these hierarchical decisions translated into specific sensillar and neuronal phenotypes?  We have undertaken an non-genetic approach in the moth M. sexta, screening early developmental tissue for patterns of morphology, mitosis, apoptosis and gene expression which are reflected in patterns observed in the adult antenna.  We have examined the activities of ecdysteroids in regulating the expression of these early developmental phenotypes.  Our studies are identifying pathways and gene activities, testable in Drosophila, that may contribute to the phenotypes of specific classes of olfactory organs.

The adult M. sexta antenna is a flagellum divided into some 80 segments, or annuli; the ventral surface is sensory (olfactory sensilla), while the dorsal surface is largely non-sensory (scales). In male antennae, the sensory epithelium of each annulus is further divided into a peripheral region containing trichoid sensilla and a medial region containing a mixed population of basiconic, ceoloconic and other sensilla (Lee & Strausfeld, 1990). In females, a higher density of short trichoid sensilla within the peripheral annular regions suggests a similar organization (Shields & Hildebrand 1999). Sanes and Hildebrand (1976) described M. sexta antenna and sensilla development in considerable detail, noting that mitotic events giving rise to sensilla cells (ORNs, support cells) occurred during a 20-60 hour after pupation window of an 18 day developmental period (pupal stage).

We have characterized mitotic patterns in the early pupa,  noting a mitotic "wave" initiating 24-48 hr after pupation along the proximal and distal annular borders and progressing towards the medial regions until about 72 hr.  We've identified several transcription factors expressing within these zones.  Activities are ecdysteroid sensitive in a manner suggesting that their temporal pattern and hormone sensitivity accommodate decisions whether to enter diapause.  The initial proximal-distal activities strongly resemble the distribution of adult trichoid sensilla, these regions are already visible shortly after pupation based on cell density and gene expression.  Antennal development begins with imaginal disc growth which initiates at the 4^th-5^th larval molt.  Characterization of disc development has revealed temporal patterns of gene expression and ecdysteroid sensitive morphogenesis, and a highly patterned apoptotic event that is allowing us to focus on a specific time in disc development when the adult sensilla phenotypes may actually be determined.

Shields, V. D. C. and Hildebrand, J. G. (1999a). Fine structure of antennal sensilla of the female sphinx moth, Manduca sexta (Lepidoptera: Sphingidae). I. Trichoid and basiconic sensilla. Can. J. Zool. 77, 290–301.

Lee, K. and Strausfeld, N. J. (1990). Structure, distribution and number of surface sensilla and their receptor cells on the olfactory appendage of the male moth Manduca sexta. J. Neurocytol. 19, 519–538.

Sanes, J. R. and Hildebrand, J. G. (1976). Origin and morphogenesis of sensory neurons in an insect antenna. Dev. Biol. 51, 300–319.

Kevin W. Wanner, Kimberly K.O. Walden and Hugh M. Robertson

University of Illinois at Urbana-Champaign, Department of Entomology, Urbana, IL 61801 USA. kwanner@life.uiuc.edu

The chemoreceptor superfamily in honey bees: support for the one receptor-one neuron-one glomerulus model for insect olfaction

Annotation of the odorant receptors (Ors) and gustatory receptors (Grs)  encoded in the honey bee (Apis mellifera L) genome sequence reveals an expansion of the Ors and a reduction in the Gr repertoire relative to Drosophila flies and  Anopheles mosquitoes. The 10X reduction in the Gr repertoire to just  seven genes might result from the nurturing hive environment for the  larvae and the symbiotic relationship of bees with plants; however, highly divergent Grs might remain undetected. Expansion of the Or family  involves multiple species-specific subfamilies and a total of 160 genes  and 6 pseudogenes. This expansion might be commensurate with the increased dependence of bees on floral odors for food location, pheromonal signals for social communication, and cuticle hydrocarbons for kin recognition. The concordance of Or number with the ±160  glomeruli identified in the bee antennal lobe provides support for the current model for insect olfaction in which each olfactory receptor neuron expresses one (or occasionally two) Ors and these extend axons to a particular glomerulus.

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Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, B3H 1X5, Canada

¹ Present address: Institute of Molecular Biotechnology (IMBA), Dr. Bohr-Gasse 3-5, 1030 Vienna, Austria. widmer@imp.univie.ac.at

Spider peripheral mechanosensory neurons are directly innervated and modulated by octopaminergic efferents

Peripheral mechanosensory neurons of the spider (Cupiennius salei) receive extensive efferent innervation. Previous work has shown that many of these efferent neurons are immunoreactive to GABA, while ionotropic and metabotropic GABA receptors are found on the mechanosensory neurons themselves. In addition, octopamine immunoreactive neurons have previously been found in the spider CNS, making it possible that the sensory neurons are also modulated by octopamine. Here we investigated the distribution and function of octopamine receptors on mechanosensilla in the spider leg. Immunocytochemistry against octopamine receptors (OARs) indicated that these receptors are present in all mechanosensory neurons, concentrated in the proximal parts of the somata and axon hillocks. Double labeling experiments with an antibody against synapsin suggested that OARs are associated with presynaptic vesicles. Octopamine immunolabeling demonstrated the presence of octopamine immunoreactive efferent fibers in close proximity to the sensory neurons. Using electrophysiology, octopamine and its precursor tyramine were shown to increase the firing rate of mechanically stimulated filiform tactile hairs (trichobothria). This effect was inhibited by the OAR blocker phentolamine. The cAMP analog 8-Br-cAMP mimicked the effect of octopamine, while Rp-cAMPS, a PKA inhibitor, blocked the octopamine response, suggesting that OARs in spiders are positively coupled to adenylyl cyclase and act via a PKA mediated pathway. Frequency response analysis revealed that octopamine increases the overall sensitivity of mechanosensory neurons over a broad frequency range. Therefore, the main effect of octopamine is to enhance neuronal sensitivity to wind-borne signals from sources such as prey or predators. Supported by: The Canadian Institutes of Health Research and the Natural Sciences and Engineering Council of Canada.

PingXi Xu and Dean P. Smith

Department of Pharmacology and Center for Basic Neuroscience, University of Texas Southwestern Medical Center. 5323 Harry Hines Blvd. Dallas, TX 75390-9111

Drosophila odorant binding protein lush is absolutely required for pheromone sensitivity and is a direct activator of pheromone-sensitive neurons

Odorant binding proteins (OBPs) are a diverse family of extracellular proteins localized to the chemosensory systems of most terrestrial species.  OBPs are expressed by non-neuronal cells and secreted into the fluid bathing olfactory neuron dendrites.  Several members have been shown to interact directly with odorants, but the significance of this is not clear.  Here we show that a Drosophila odorant binding protein,  LUSH (OBP76a), is required for responses to the male-produced pheromone 11-cis vaccenyl acetate (VA).  Mutants lacking LUSH are completely devoid of VA-evoked activity in a small subset of olfactory neurons that normally respond to this pheromone, revealing that this binding protein is absolutely required for activation of these chemosensory neurons. lush mutants are also completely defective for pheromone-evoked behavior.  We detected a genetic interaction between lush and spontaneous activity in VA sensitive neurons in the absence of pheromone, suggesting LUSH protein can activate pheromone sensitive neurons, even in the absence of pheromone. The defects in spontaneous activity and VA sensitivity are reversed by germline transformation with a cloned, wildtype copy of lush or by introducing recombinant LUSH protein into mutant sensilla. These studies link expression of an OBP with activity in a specific subset of olfactory neurons and pheromone-induced behavior.

Sarah Young, Hans Schuppe, Guy Poppy, David Shepherd, and Philip Newland.

School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, U.K. syoung@soton.ac.uk

Does Larval Diet Affect Adult Food Choice? Gustatory Learning and Memory in Drosophila melanogaster.

Gustation underlies much insect behaviour, including finding and assessing food and egg laying sites.  Drosophila are capable of learning sensory information about their environment, however, whether they are capable of learning gustatory information and using this information to modify their adult behaviour is as yet unknown.  A behavioural assay was therefore designed to try to test this issue.

Drosophila larvae were raised on food media with carbohydrate concentration and type varied.  Individuals were removed from these diets as pupae and subsequently as adults given the choice to feed and lay their eggs on either the diet upon which they were raised or a standard diet. 

Results show that adult individuals raised during the larval stages on food media lacking sucrose have a preference to feed on this medium over a standard medium containing sucrose.  When raised as larvae on other concentrations of sucrose, however, adult preference is always for the highest carbohydrate content medium.  Nevertheless adults are more likely to accept the reduced carbohydrate content medium if they were exposed to it as larvae.  When raised as larvae on a diet containing no sucrose adults are less likely to lay their eggs on this medium than those raised as larvae on a standard medium.  At all other concentrations of sucrose larval diet had no effect on subsequent egg laying preferences.  Further experiments using the mutants dunce and rutabaga suggest these modifications in behaviour could be due to learning and memory.

Adult individuals raised as larvae on reduced concentrations of trehalose were less accepting of these media as adults compared to those raised as larvae on a standard trehalose diet.  When raised as larvae on a diet containing no trehalose adults were more likely to lay their eggs on this medium than those raised as larvae on a standard trehalose medium.  At all other concentrations of trehalose larval diet had no effect on subsequent egg laying preferences.

In summary, results show that varying larval diet can affect subsequent adult feeding and egg laying preferences, although the effects depend on the constituent of the diet that is being varied.  Possible reasons for these modifications in behaviour are discussed.   This work was funded by a studentship from the Gerald Kerkut Trust.

Satoji Yuji, Tomoyosi Nisimura, Ryohei Yamaoka and Mamiko Ozaki

Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto 606-8585, Japan, yuuchann1013hp@yahoo.co.jp

3D models of the glomeruli in antennal lobe of Camponotus japonicus

Ants get a variety of environmental information through sensory organs on antenna; CO₂ sensors, olfactory, mechanical, and contact chemical sensors. Recently, it was found that the specific chemosensilla on the antenna in the carpenter ant, Camponotus japonicus, responded to cuticular hydrocarbons (CHCs), which are used for nestmate recognition marker in ant species.

The afferent neurons from those antennal sensory organs should project into the antennal lobes  which would play important role for processing of olfactory information. However, the structural composition of the antennal lobes remains unknown in ant species. In order to analyze the innervating mechanism of those afferents from the antenna to the second order neurons, we stained glomeruli of the carpenter ant by retrograde tracing of all axons in the antennal nerves and observed by confocal laser scanning microscopy. Based on these data, 3D digital models of the glomeruli of the antennal lobe were produced. We also tried to stain afferents from individual chemosensillum.

Marianna I. Zhukovskaya

Thorez Ave. 44, 194223 Saint-Petersburg, Russia. mzhukovskaya@yahoo.com

Peripheral modulation of antennal chemoreceptor cells in American cockroach.

A small animal such as an insect is surrounded by a permanently changing environment. An individual also alters its physiological state with age, time of day, season, weather or influence of conspecifics. The octopamine content of the antennal heart of a cockroach rises under stress condition (Mobius, Penzlin, 1993), but the role of octopamine in the antenna is not yet clear.

Octopamine applied to the antennal cuticle gets into the hemocoel causing simultaneous changes in EAG of both cockroach antennae. Our experiments repeatably found a clear drop in EAG amplitude, unlike the data reported for bee and totricid moth (Spivak et al, 2002; Stelinsky et al, 2003). In contyrast, the spike activity of sensilla increased under elevated octopamine level. Similar results for the spike counts were shown for some Lepidoptera species (Pophof, 2000, 2002, Grosmaitre et al. 2001).

Some clues to understand octopamine action in sensilla come from the experiments with anoxia. When all active processes in a cell are suppressed by an energy lack due to oxygen deficit, both spike activity and EAG amplitude decrease. Action potential generation, according to arguments of Dolzer and colleagues (2001), is supposed to be in the base of a receptor cell. Taking into consideration these findings together with our data we can suggest that octopamine decreases membrane resistance of the receptor cell near the axonal hillock and depolarizes it. This would make the membrane less stable, increasing spike rate. The EAG in this case would decrease due to smaller changes in membrane potential in response to adequate stimuli. Transepithelial potential created by auxiliary cells of a sensillum is believed to be a driving force for receptor current (Kaissling, 1987). Anoxia reduces the transepithelial potential due to suppression of the energy depended ionic pump resulting in decrease of spike frequency.

This hypothesis is supported by our data on perfused antennae. The spike quantity is higher under octopamine versus plain ringer. Receptor cell membrane seems to trigger more spikes being depolarized by octopamine both in response to pheromone and without stimulation.

Abstracts and program compiled with support from the University of Arizona's Center for Insect Science.

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