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 i