ESITO VIII European Symposium for Insect Taste and Olfaction

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8TH EUROPEAN SYMPOSIUM FOR INSECT TASTE AND OLFACTION (8th ESITO)

July 2-7, 2003 - Harstad, Norway

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Abstracts of Posters

Abstracts

1

Johan Andersson ^1,2, Anna-Karin Borg Karlson ¹, Christer Wiklund ², and Hanna Mustaparta

1. Royal Institute of Technology, Sweden; 2. Stockholm University, Sweden; 3. Neuroscience unit, NTNU, Norway;
joan@kth.se

The antennal responses to intra- and inter species signals of three Pieris butterflies.

Inter- and intra-species interactions in butterflies are cued by both visual and chemical stimuli. Olfactory stimuli are important at close range and the message contains different kind of information, like female receptiveness, interspecies recognition, male performance and sex recognition. In the genus Pieris we know the message of the odours emitted by three species of butterflies. The male produced methyl salicylate (P. napi and P. rapae), indole (P. rapae) and benzyl cyanide (P. brassicae) are transferred during mating to females and later used as anti-aphrodisiac, displayed by the female to show unreceptivness to male courtship. We have also shown that the male released odor, citral (P. napi), act as an aphrodisiac to make virgin female more receptive to the courtship. By screening the antenna of the three species with these and related compounds, the responses (electroantennogram) showed a species specific sensitivity and selectivity to the insect produced compounds. Thus, the result reflects the impact of these compounds in the intra- and inter species communication in the three Pieris species. We here present the EAGs elicited by the different insect produced odorants in the three Pieris species, and show the correlation of these responses to the behavioural importance of the odor as a signal within and between the three species.

Andersson, J., Borg-Karlson, A.K., and Wiklund, C. 2000. Sexual cooperation and conflict in butterflies: A male-transferred anti-aphrodisiac reduces harassment of recently mated females. Proceedings of the Royal Society of London B 267(July 7):1271.
Andersson J, Borg-Karlson A.K., and Wiklund C., 2003 Anti-aphrodisiacs in Pierid butterflies; a theme with variation! Accepted Feb. 13, 2003, Journal of Chemical Ecology
Bergström, G. & Lundgren, L. 1973 Androconial secretion of three species of butterflies of the genus Pieris (Lep., Pieridae). Zoon Suppl. 1, 67-75.
Blaakmeer, Anton; van der Wall, Dick; Stork, Andre; van Beek, Teris A.; de Groot, Aede; van Loon, Joop J. A. Structure-activity relationship of isolated avenanthramide alkaloids and synthesized related compounds as oviposition deterrents for Pieris brassicae. Journal of Natural Products (1994), 57(8), 1145-51. CODEN: JNPRDF ISSN:0163-3864. CAN 121:175699 AN 1994:575699 CAPLUS
Silberglied, R.E. 1984 Visual communication and sexual selection among butterflies. In TheBiology of Butterflies (ed. R.I.Vane-Wright, & P.R. Ackery) pp. 207-223 London: Academic Press.

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2

Sylvia Anton ^1,2 and Jean-Pierre Rospars ³

1 Department of Chemical Ecology, Swedish Agricultural University, Box 44, 230 53 Alnarp, Sweden
2 INRA, UMR Santé Végétale, Centre de Recherche de Bordeaux, BP 81, 33883 Villenave d’Ornon, France
3 INRA Unité de phytopharmacie et Médiateurs Chimiques (UPMC), Route de Saint-Cyr, 78026 Versailles, France
santon@bordeaux.inra.fr

Central projections of olfactory receptor neurons from single antennal and palpal sensilla in mosquitoes

Female mosquitoes use the odours emitted by their hosts to detect potential sources for a bloodmeal. These odours are detected by specific receptor neurons situated on the antennae and maxillary palps. The receptor neuron axons reach the primary olfactory neuropil, the antennal lobe, via the antennal nerve and arborize in glomeruli.

We developed a method to stain central projections of intact receptor neurons responding to host odour compounds in the malaria mosquito, Anopheles gambiae. Axons from single antennal olfactory receptor neurons arborized in one glomerulus each. Different axons originating from the same sensillum, presumably responding to different host odours, never branched in the same glomerulus. Three-dimensional analysis of the projection patterns of receptor neurons from sensilla which were stimulated with a specific odour revealed more or less limited target areas within the antennal lobe depending on the type of sensilla and the used compound.

Olfactory receptor neurons originating from maxillary palp sensilla projected into one posterior-medial glomerulus in both antennal lobes, which never received input from antennal receptor neurons. Our results support the hypothesis that functionally different receptor neurons send their information to anatomically distinct units within the antennal lobe.

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3

R. Crnjar, A. Liscia, C. Masala, P. Solari and G. Sollai

Dipartimento di Biologia Sperimentale, Sezione di Fisiologia Generale, Università di Cagliari, Cittadella Universitaria di Monserrato, SS. 554 Km 4.500, I-09042 Monserrato (CA) ITALY.
crnjar@unica.it

Possible transport mechanisms sustaining the transepithelial potential (TEP) in labellar chemosensilla of the blowfly Protophormia terraenovae.

The cell complex (sensory + accessory cells) of insect taste sensilla exhibits a transephitelial potential (TEP), for which an important role has been proposed in modulating the receptor responses [1]. The TEP is sustained by an electrogenic potassium transport associated with the accessory cells. It is generally accepted that an apical vacuolar-type H+-ATPase and the related K+/H+ antiport are involved in the generation of the TEP, as described in other insect epithelia, such as Malpighian tubules in Drosophila melanogaster [2] or olfactory sensilla in Manduca sexta [3]. By means of the tip-recording technique [4], in the present work we have investigated the effects of specific inhibitors of the K+/H+ antiport (amiloride, omeprazole and Na-orthovanadate), of an inhibitor of K+/Cl- and Na+/K+/2Cl- cotransport systems (bumetanide) and of a K+ channel blocker (BaCl2) on the labellar chemosensilla in the blowfly Protophormia terraenovae.

Drug solutions were directly injected in the labellum or added to the recording electrode saline, with the aim of reaching, via hemolymph or receptor lymph (through the apical pore), the accessory cells on the basolateral or luminal side, respectively. Results indicate that, in P. terraenovae, all tested K+/H+ antiport inhibitors significantly decreased the TEP up to 46% of the initial value, when injected in the labellum over a 20-min period, and can be thus regarded as having a diminishing effect on the antiport activity. On the contrary, no effect was detected with respect to controls when the same compounds were applied through the apical pore. Conversely, both bumetanide and BaCl2 lowered the TEP value only when added with the latter modality.

The information obtained with the two modes of drug administration suggests the existence of different active and passive potassium transport mechanisms, localized on either pole of the sensillar accessory cells.

[1] U. Thurm and G. Wessel, J. Comp. Physiol. 134: 119-130, 1979
[2] S.M. Linton and M.J. O’Donnell, J. Exp. Biol. 202: 1561-1570, 1999
[3] J. Dolzer et al, J. Exp. Biol. 204: 2781-2794, 2001
[4] E. S. Hodgson et al, Science 122: 417-418, 1955

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4

A.M. Dacks, V.M. Pawlowski, T.A. Christensen & J.G. Hildebrand

ARL-Neurobiology, University of Arizona, Tucson, AZ 85721

Effects of varying stimulus concentration on ensemble representations in the moth antennal lobe

For many animals, the task of identifying and locating the source of an odor depends on the animal’s ability to track the stimulus even if its physical features change in space and time. In moths like Manduca sexta, it has been shown that these insects are well adapted behaviorally to track odor plumes that are both dynamic and unpredictable. We therefore examined the effects of varying stimulus concentration on the neural representations of odors at the first stage of processing in the brain. Neural-ensemble recording using a four-pronged silicon multielectrode array was used to record the odor-evoked population activity across several glomeruli in the antennal lobe (AL). We found that different members of the coding ensemble exhibited differences in both response threshold and dynamic range. Depending on stimulus concentration, some units were excited by odor while others were inhibited. Elevated stimulus concentrations triggered increased ensemble activity, and the ensemble representations evoked by different odorants became less and less discriminable. Furthermore, because participating members of the ensemble change their response characteristics with concentration, both the spatial and temporal characteristics of the ensemble responses to a single odorant can change dramatically at different stimulus intensities. These results suggest that key elements in the odor representation are fixed, but the overall representation is dependent on contextual features. Because it has been shown that serotonin titres are elevated in the AL during the moth’s active period, we also examined the effects of 5-HT on the concentration-dependent modulation of ensemble responses to odor. Supported by NSERC-PGSA-244345-2001 & NIH.

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5

Nina Deisig¹, Jean-Christophe Sandoz², Harald Lachnit³, Martin Giurfa²

1 Neurobiology, Institute of Biology, Free University of Berlin, D-14195 Berlin, Germany;
2 Department of Psychology, Philipps University of Marburg, D-35032 Marburg, Germany;
3 Centre de Recherches sur la Cognition Animale, Université Paul Sabatier - Toulouse III, 118 Route de Narbonne, 31062 Toulouse cedex 4, France
deisig@cict.fr

Olfactory mixture processing in the honey bee: conditioning and optical imaging

We investigated how honeybees perceive odor mixtures using both behavioral and optical imaging methods. At the behavioral level, we used olfactory conditioning of the proboscis extension reflex (PER), a paradigm in which odors can be associated with a reinforcement of sucrose solution. In a series of conditioning experiments, we investigated the capability of honeybees to discriminate between single odorants and binary and ternary olfactory mixtures. Bees were subjected to two different discrimination problems (A/+, B+, and /C+, )AB/AC/BC+ versus ABC-, and A+, BC+ versus ABC-). We investigated whether the course of differentiation between stimuli could be predicted by different theories of “compound” processing (unique cue, modified unique-cue or Pearce’s configural theory). In the first discrimination problem (A/+, B+, and /C+, )AB/AC/BC+ versus ABC-) contradicted the predictions of Pearce’s configural theory, while differentiation in the second discrimination problem (A+, BC+ versus ABC-) was only in line with a modified unique cue theory. This suggests that in honeybees, an olfactory mixture is perceived as the sum of its elements, plus a unique stimulus (the “unique cue”), proper to the mixture. Furthermore, it indicates that the salience of odors is reduced in a mixture, due to interferences between them at the peripheral or central processing levels. To investigate mixture processing at the physiological level, we used calcium imaging in the primary olfactory neuropil of the olfactory pathway, the antennal lobe. We thus compared induced-induced activation patterns in the antennal lobe for the same single odors and binary and ternary mixtures that were used in behavioral experiments. The data show that the glomerular patterns obtained for the mixtures differ from the simple addition of the patterns recorded for the single odors, thus suggesting that the information necessary for the extraction of a “unique cue” may already be found at the level of the antennal lobe.

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6

Ana Domingos^1,2 and Leslie B. Vosshall¹

1The Rockefeller University, New York, USA.
2Instituto Gulbenkian de Ciencia , Oeiras, Portugal.
dominan@rockefeller.edu

Neural Basis of olfaction in Drosophila melanogaster larva

Drosophila melanogaster larva possess a simple chemosensory system capable of perceiving both volatile and non-volatile gustatory stimuli. Chemosensation, before metamorphosis, is thought to be accomplished by several chemosensory sensilla distributed over the body of the larva: the dorsal organ (DO), the terminal organ (TO), the ventral organ (VO), the ventral pits (VP), and other internal sensilla. Anatomical studies have led to the hypothesis that the DO mediates olfaction while the TO and the VO mediates taste. The DO is a multiporous structure, ideal for volatile reception, in contrast with the single pore structure of the VO, TO.

However, no functional studies to date have shown the exclusive requirement of the DO for olfaction. Here we provide functional evidence that the DO neurons are required for olfaction but not for taste.

We used a genetic approach to selectively ablate the neurons that innervate the DO and tested the larvae for olfactory- and taste-driven behavior. The larvae whose DO neurons were silenced had defective olfactory-driven behavior while their wild type siblings and parental lines had normal olfactory-driven behavior. Taste-driven behavior in these manipulated animals was unaffected. Future experiments will examine the molecular basis of odor perception in Drosophila larvae, and how these odors are represented in the larval brain.

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7

Kazuyo Fujikawa and Mamiko Ozaki

Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, Japan
fujikawa@ipc.kit.ac.jp

Bacterial expression and ligand binding of a lipophilic ligand-binding protein, CRLBP, from the blowfly Phormia regina.

Chemical sense-related lipophilic ligand-binding protein (CRLBP) is a unique member of the odorant-binding protein since it is distributed in both taste and olfactory organs in the blowfly, Phormia regina. It is considered that CRLBP carry the lipophilic odorous components to the taste or olfactory receptor membrane in both chemosensory systems.
In the present study, to investigate the binding property of CRLBP, the fusion protein carrying a His-tag at the N-terminal of CRLBP was expressed in E. coli cells. The fusion protein was purified with Ni-NTA agarose resin and was then used for ligand binding experiments. We visualized that CRLBP interacted with fluorescent 7-hydroxycoumarin on the native PAGE gel. We are further examining the ability of the fusion protein to bind other various odorous components and carried out a functional characterization of CRLBP.
Supported by a grant of ProBRAIN to MO

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8

Martin Gabriel and Uwe T. Koch

Abteilung für Tierphysiologie, AG Pheromone,Universität Kaiserslautern, Postfach 3049, 67653 Kaiserslautern, Germany

Computer controlled multiple odour sources for defined antennal stimulation in EAG experiments

Electroantennogram (EAG) setups are in use for a large number of experiments. Recently, an EAG system has been constructed to measure airborne pheromone density in mating disruption experiments [1]. For further insight into receptor mechanisms, it seems worthwhile to study the interaction of odours using controlled superposition of odour stimuli. For such experiments it is essential to use stable and reproducible odour sources. In standard procedures filter papers are used [2], but these sources lack reproducibility [3]. A syringe containing varying ratios of oil and odour material is an odour source with the required stability [4]. If such syringes are to be applied in a superposition experiment, they should be positioned all at the same distance from the antenna (i.e. in one plane) to achieve the same stimulating effect. Thus, only 3 syringes can be permanently connected to a central air channel leading to the antenna. This layout was used in the pheromone measuring system [5]. Since at least 2 syringes are needed for the calibration of the antenna, the old system can measure the superposition of only 2 odours at one time. For more comprehensive measurements of odour interactions it is desirable to apply many different odours in a short sequence, since the antenna’s sensitivity and threshold vary and intervals between calibrations must be kept small. Since 10-15 syringes are desirable, the existing construction had to be changed dramatically. In the new system one syringe is replaced by a barrel carrying 12 syringes. The barrel is rotated by a motor with position feedback. Thus, each of the 12 syringes can be selected and pushed into the injection hole. Air puffs from the syringes in use are generated by moving the piston by a step motor, which gets defined impulses from a microcontroller. The step motor’s feed rate controls the concentration of the odour at the antenna. In order to get interaction between all 3 odour sources in use, the step motor control system permits to activate each motor independently with its own feed rate, with total or partial activity overlap. All functions of the system are remotely controlled by a PC which also records the EAG trace.

[1] U.T. Koch, P. Witzgall (2001), IOBC wprs Bulletin Vol. 24(2): 55-62
[2] K.E. Kaissling (1979), Chem. Ecology (F.J. Ritter ed.), Amsterdam: Elsevier, 43-56
[3] A.E. Sauer, U.T. Koch (1989), Proceedings of the 17th Göttingen Neurobiology Conference (N. Elsner, W. Singer ed.), Georg Thieme Verlag, p.78
[4] U.T. Koch, A.M. Cardé, R.T. Cardé (2002), J. Appl. Ent. 126, 431-435
[5] P. Färbert, U.T. Koch, A. Färbert, R.T. Staten, R.T. Cardé (1997), Environ. Entomol. 26(5): 1105-1116

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9

Birgit Greiner¹, Christophe Gadenne³ and Sylvia Anton^2,3

1 Department of Cell and Organism Biology, Lund University, Helgonavägen 3, 22362 Lund, Sweden
2 Department of Crop Science, Swedish University of Agricultural Science, 23053 Alnarp, Sweden
3 Institut National de la Recherche Agronomique, Unité Mixte de Recherches en Santé Végétale, Centre de Recherches de Bordeaux, BP 81, 33883 Villenave d´Ornon cedex, France
santon@bordeaux.inra.fr

A 3-D antennal lobe atlas of a male moth (Agrotis ipsilon) and glomerular representation of projection neurons responding to plant volatiles

The olfactory glomerulus is the universal anatomical and functional unit for primary olfactory processing. In many male insects a limited number of enlarged glomeruli, known as the macroglomerular complex, process information on the female-produced sex-pheromone compounds. This has been studied in great detail in the last decades for many insect species. The ordinary glomeruli, however, regarded as being important for processing of plant volatiles, only recently gained more interest and were the focus of this anatomical and physiological study.

In the black cutworm moth, Agrotis ipsilon, both sexes locate food sources using flower volatiles. Males seem to use plant volatiles to detect food sources throughout their adult life and could use plant volatiles, beside the female-produced sex-pheromone, as additional cues to detect females on their oviposition sites. Preliminary behavioural studies have shown that heptanal is attracting A. ipsilon males in the wind tunnel.

In our study we have constructed a three-dimensional (3-D) atlas of the entire antennal lobe of the male A. ipsilon using confocal images of formaldehyde-fixated brains. Four macroglomeruli and sixty-two ordinary glomeruli were identified in three different animals. Furthermore, the glomeruli harbouring arborisations of sixteen physiologically characterised projection neurons, responding with different specificity to seven tested plant volatiles, were identified in the antennal lobe. Projection neurons responding to the same compounds were not projecting to the same glomerulus. Moreover, projection neurons targeting the same glomerulus responded to different compounds. These results support the current hypothesis that complex patterns of activated glomeruli are representing specific plant volatiles.

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10

Bernhard T. Hovemann, Denise Pargmann, Sven Fischer, and Klemens F. Störtkuhl

AG Molekulare Zellbiochemie, Ruhr-Universität Bochum, NC 5/170, Universitätsstrasse 150, 44780 Bochum, Germany
bernhard.hovemann@rub.de

EGFP-fusion reveals Or43a expression in ORNs that innervate basiconic sensilla and target glomerulus DA4.

In Drosophila more than 60 putative olfactory receptor genes have been identified. Recently, three of those including or43a have been shown to encode receptor proteins that exhibit olfactory function in vivo by responding to a limited number of volatile compounds. To visualize the sensilla that show Or43a expression either in vivo by fluorescence or in vitro by immunocytochemistry we generated lines with a carboxyterminally EGFP-tagged Or43a receptor. Electrophysiological recordings from the third antennal segment verified that this receptor-EGFP construct retained olfactory function when compared to wild type Or43a receptor. To disclose endogenous Or43a activity, we generated an or43a-enhancer-gal4 line for either Or43a-EGFP or EGFP-tagged Synaptobrevin expression. Double labeling of or43a-egfp RNA and wild type receptor RNA revealed faithful activity of the or43a-enhancer-gal4 construct consistent with neurons of wild type or43a expression. EGFP fluorescence and immunocytochemistry revealed Or43a-EGFP in small basiconic sensilla and identified glomerulus DA4 in the antennal lobe as the projection target of Or43a expressing olfactory receptor neurons. Our data show that DA4 perceives signals from basiconic sensilla through application of the Or43a receptor ligands cyclohexanol, benzaldehyde, cyclohexanone and benzyl alcohol.

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11

Inoue A. Takashi

JAPAN, 305-8634, Ibaraki, Tsukuba, Ohwashi 1 – 2, NIAS;
inoueatp@affrc.go.jp

Papilio butterflies detect sweet taste by their mouth (Video presentation)

“Butterflies detect sweet taste by their legs.” This is one of the most popular facts on insect sensory systems and was established by MINICH (behavior), ANDERSON (behavior), ELTRINGHAM (morphology) and MORITA (electrophysiology). This was, however, doubted by many naturalists of JAPAN in which nation six-legs butterflies are majority. Japanese Rhopalocerist researchers well know that there are many different not only in morphology but also in behavior between four-leg butterflies and six-leg butterflies. So we thought that Papilio butterflies that are one of the major six leg butterflies group would not detect sweet taste by their legs but by their mouth, we carried out morphological, electrophysiological, behavioral and biochemical research and finally, we ensured that Papilio butterflies really detect sweet taste by their mouth. We also notice that some other species of butterflies also might sweet detect by their mouth because we found taste sensilla from the proboscis of some Pieridae and Nymphalidae butterflies. The reason that the previous researcher could not point out this possibility in the behavioral works might be in the conditions of butterflies that were used in their experiments. Those butterflies were almost death of hunger so they already could not show normal response to the stimulation of the taste solutions. All biological researchers using behavioral experiments should remember “Hunger is the best sauce” and we suggest that in behavioral works, we must carry out a questionnaire survey but torture.

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12

Nunzio Isidoro¹, Roberto Romani² and Ferdinando Bin²

1 Department of Agricultural and Environmental Biotechnologies – University of Ancona, via Brecce Bianche, 60131, Ancona, Italy
2 Department of Arboriculture and Plant Protection – Entomology, University of Perugia, Borgo XX Giugno, 06121, Perugia, Italy
isidoro@unian.it

Morpho-functional features of multiporous gustatory sensilla in female parasitic Hymenoptera

Various kind of sensory structures have been described in insect parasitoids, among which olfactory and gustatory sensilla are the most studied because of their involvement in sex and host recognition mechanisms. Numerous studies have been focused on olfactory sensilla, and sometimes these structures have been misinterpreted being in fact release site of glands or involved in gustation. In the latter case, these sensory structures (which have been found only ventrally on female antennae in the most important parasitoid families, i.e. Trichogrammatidae, Mymaridae, Scelionidae, Platygastridae, Eulophidae, Diapriidae) show peculiar morphological features, i.e. a cuticular apparatus consisting of a basiconic shaft with a localised multiporous area; cellular components made up of a number of sensory neurons ranging from 6 up to 400. In some cases (Scelionidae) these multiporous sensilla have tubular accessory glands that release their secretion through the sensillum socket. The number of these gustatory sensilla is variable, and it seems to be correlated with the number of sensory neuron found per sensillum. These morphological characters, combined with behavioural observations, strongly suggest a gustatory function, although electrophysiological studies are still needed to confirm this hypothesis. We propose for these sensory structures the term “Multiporous Gustatory Sensilla” (MGS), based on their morpho-functional features.

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13

Melissa Jordan^{1, 3}, Richard Newcomb¹, Dave Greenwood¹, Kim Plummer³ and Paul Sutherland²

1 Gene Technologies Sector, The Horticulture and Food Research Institute of New Zealand Limited, Private Bag 92169, Auckland, New Zealand
2 Future Horticulture, New Varieties Team, The Horticulture and Food Research Institute of New Zealand , Private Bag 92169, Auckland, New Zealand
3 School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
mjordan@hortresearch.co.nz

Sniffing Out Olfactory Proteins from Light Brown Apple Moth Antennae

The Light Brown Apple Moth, Epiphyas postvittana, is a major horticultural pest in New Zealand and Australia. We are using this tortricid moth as a model to investigate the molecular mechanism of pheromone reception. We are employing a combination of molecular biology and proteomics to isolate and identify proteins specific to the olfactory process, in particular the pheromone receptor and proteins of the signal transduction pathway.

First the ultrastructure of the male and female antenna was compared using Scanning Electron Microscopy revealing some morphological differences, most notably the number and distribution of long sensilla tricodea. Each segment of the male antenna possesses many long sensilla tricodea positioned in two radial rows. These have been shown to be sensitive to the pheromone of this moth. Female moth antenna lack any long sensilla tricodea. We anticipate that this difference may be detectable at the protein level. Proteins have been extracted from male and female antennae and also from the legs of male moths using methods optimised to enrich for membrane proteins in addition to soluble proteins. Two-dimensional gel electrophoresis was used to separate the proteins and the resulting differential profiles compared. Antennal-specific proteins were identified by comparing the two-dimensional profiles of leg proteins with those of the antennal proteins. These antennal specific proteins have also been compared between male and female profiles and proteins of interest are being sequence analysed by Electrospray Mass Spectrometry (LC-ESI-MS). The resulting MS-MS information is being searched against public and the HortResearch proprietary insect EST databases.

Concurrently, a candidate chemosensory receptor with sequence similarity to the Heliothis virescens HR6 was isolated from an Epiphyas postvittana antennal cDNA library. In situ experiments are currently being undertaken to determine the location of expression of this receptor in antennal tissue.

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14

Blanka Kalinová¹, Aleš Svatoš²

1 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic
2 Max-Planc- Institute for Chemical Ecology, Winzerlaer Straße 10; D-07745 Jena, Germany
blanka@uochb.cas.cz

Identification of host semiochemicals for horse chestnut leafminer, Cameraria ohridella, by gas chromatography-electroantennography

The host range of a new invasive species in Europe, the horse chestnut leafminer, Cameraria ohridella (Lepidoptera, Gracillariidae, Lithocolletinae) is restricted to species of Aesculus (Hipocastanaceae). C. ohridella massively colonizes broadly cultivated white blooming common horse chestnut trees, Aesculus hippocastanum (Hipocastanaceae). The red horse chestnut, Aesculus x carnea (a hybrid of the common horse chestnut and the North American Aesculus pavia), is usually far less infested. Infestation on trees of other families was not reported except for rare records on the sycamore and Norway maple, A. platanoides and A. pseudoplatanus (Aceraceae).
Volatile semiochemicals possibly involved in host selection were analysed by high-resolution gas chromatography-electroantennography and gas chromatography-mass spectrometry. Samples were obtained by air entrainment of foliage from host trees. Volatile spectra of the main host Aesculus hippocastanum and the resistant Aesculus x carnea were similar. Experiments showed a similar discriminatory capacity of males and females to plant related compounds. Behavioural experiments are in progress to ascertain behavioural roles of identified compounds.

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15

Erik Kristian Lassa, Tor Jørgen Almaas and Hanna Mustaparta

Norwegian University of Technology and Science, Department of Biology, Neuroscience Unit, MTFS, Trondheim, Norway
lassa@stud.ntnu.no

Detection of temperature and humidity by receptor neurons in the antennal sensilla styloconica of heliothine moth

Because of the high surface/volume ratio in insects, body temperature and water loss is highly affected by the ambient temperature and humidity. In previous studies of insects, receptor neurons that are sensitive to temperature and humidity have been demonstrated (review: Tichy and Loftus 1996). The antenna of the heliothine moth have one sensilla styloconica on each segment, located on the leading edge. The fine structure of these sensilla shows two triads, each containing one lamellated dendrite (Færavaag 1999). This indicate a function of temperature and humidity detection.

In the present study, electrophysiological recordings from single receptor neurons of s. styloconica in Heliothis virescens, was carried out. Stimulation was performed by switching between two airstreams with different temperatures and humidities. The switch was made by an electromagnetic valve. The air was dried through a silica gel column before the temperature was regulated.

On the basis of different spike characteristics the recordings show the presence of three different neurons. The temperature receptor neurons were characterized by a spontaneous firing rate in the range of 10 to 20 imp/s at 24ºC. By stimulation with a higher temperature the neurons immediately stopped firing, and resumed the spontaneous activity after the stimulation period. When stimulated with a lower temperature an immediate increase of firing occurred, followed by a decreasing firing rate. At the end of the stimulation the neuron stopped firing for 2-20s, and then resumed the spontaneous activity. Curves describing the temperature- response strength, and selectively stained neurons for determining the projections in the brain, will be presented.

Tichy H, Loftus R (1996) Hygroreceptors in insects and a spider: Humidity transduction models. Naurwissenschaften 83:255-263
Færavaag AC (1999) En transmisjonselektronmikroskopisk- og immunocytokjemisk studie av sensilletyper på antennen hos to arter nattfly, Heliothis virescens og Helicoverpa zea (Lepidoptera: Noctuidae). Master thesis. NTNU

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16

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

ARL-Neurobiology, University of Arizona, Tucson, AZ 85721
hlei@neurobio.arizona.edu

Temporal reinforcement of spatially distributed ensemble codes for odors in the antennal lobe of Manduca Sexta

The spatial and temporal patterns of activity observed at the initial stages of olfactory information processing in the brain are currently under intense investigation in numerous species. In the ongoing search for “the” odor code, many groups have employed imaging methods that reveal distributed patterns of neural activity with high spatial resolution, while others have used single-unit electrophysiological recordings that can measure the time course of neuron spiking on a millisecond time scale. Neither method, however, can achieve high resolution in both domains. In an attempt to resolve this fundamental problem, we used silicon multi-electrode recording arrays to study the temporal dynamics of odor-evoked population responses across a spatially defined subset of olfactory glomeruli in the AL of the moth Manduca sexta. The stimulus set consisted of selected pheromonal, monoterpinoid, aliphatic and aromatic odorants. We found that whether the stimulus was pheromonal or non-pheromonal in nature, each odorant evoked a pattern of activity that involved a unique subset of output (projection) neurons that were distributed across the recording array. While the pattern evoked by each odorant usually had its focus of activation in one or possibly several contiguous glomeruli, there was nevertheless some degree of overlap between the distributed patterns evoked by different odorant classes. This overlap was likely a consequence of the inhomogeneous nature of the output neurons that are involved in a given odor response. Some neurons responded preferentially to only one category of odorant while others were broadly responsive to all tested odorants. Importantly, this method also allowed us to measure the temporal correlations across the neural populations represented in these spatial patterns. A key finding was that neurons that showed similar response profiles across odorants were also more likely to exhibit synchronized firing. These neurons were also more likely to be recorded from the same or nearby sites. Therefore, in accordance with what we recently found in pheromone-processing networks, these data provide new evidence that intraglomerular synchrony also functions to augment the spatially defined and chemotopic representation of non-pheromonal odorants across populations of olfactory glomeruli.

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17

Anna Luxová ^1,2 and Anna-Karin Borg Karlson ³

1 Institute of Organic Chemistry and Biochemistry, Academy of Science CR, Department of Natural Product, Flemingovo nám.2, 166 28 Prague 6, Czech Republic
2 Institute of Chemical Technology, Technická 5, 166 28 Prague 6, Czech Republic
3 Royal Institute of Technology, Organic Chemistry, Teknikringen 56, 100 44 Stockholm, Sweden
luxova@uochb.cas.cz, akbk@kth.se

Male bumblebee odours as cues in deceit pollinated flowers

Orchis pauciflora and Barlia robertiana are two non-rewarding orchid species pollinated by bumblebees. Bombus terrestris queens are typical pollinators of these flowers. We collected the floral scents on short PorapakÔ columns by entrainment. After rinsing the column with hexane we analyzed the concentrated sample by GC-MS. Major compounds were, (E)-b -farnesene, (E,E)-a-farnesene and also 2,3-dihydrofarnesol (2,3-DHF) of Orchis pauciflora scent. Main constituent in Barlia robertiana scent was citronellol. By our two dimensional GC and a chiral column we were able to separate both the enantiomers and identify the (S)-citronellol and (S)-2,3-DHF in both bumblebee pheromone and orchid scents. (S)-2,3-DHF is used as a marking pheromone of B. terrestris males and has been shown to attract virgin conspecific females.

Borg-Karlson A.-K., Luxova, A., Liblikas, I., Valterová, I., Kunze, J., and Gumbert, A. Male mating pheromones enhance attraction of queens to deceptive flowers. Submitted to Nature 2003.

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18

C. Masala, A. Liscia, R. Crnjar, G. Sollai and P. Solari

Dipartimento di Biologia Sperimentale, Sezione di Fisiologia Generale, Università di Cagliari, Cittadella Universitaria di Monserrato, SS. 554 Km 4.500, I-09042 Monserrato (CA) ITALY.
liscia@unica.it

The “deterrent” cell in the blowfly is activated by Na-saccharin.

In the attempt to gain more information on the mechanisms underlying bitter and/or sweet taste reception, we have investigated the responses of labellar chemosensilla in the blowfly Protophormia terraenovae to Na-saccharin, as compared to sweet (sucrose and fructose) or bitter chemicals (amiloride).
Our electrophysiological data, obtained by means of the tip-recording technique [1], indicate that a dose-response relationship exists in response to Na-saccharin for at least one chemoreceptor cell in the 10-100 mM concentration range.

Cross-adaptation occurred between Na-saccharin and both sugars when the adapting stimulus was Na-saccharin, but it did not when the adapting stimuli were sucrose or fructose. On the other hand, addition of Na-saccharin to both sucrose and fructose significantly depressed the spike firing frequency and a mutual inhibition between the “sugar” and the “deterrent” cells takes place.
In the case of amiloride, cross adaptation with Na-saccharin takes place, regardless of the order of adapting stimuli. An increase of the “deterrent” cell activity was also detected following addition of Na-saccharin.

In free-choice behavioural tests, addition of Na-saccharin completely abolished sucrose or fructose ingestion, while pure saccharin is virtually untouched.
Both electrophysiological and behavioural data demonstrate that in blowflies Na-saccharin inhibits both the electrophysiological and behavioural responses to sugars, analogously to what observed for quinine in blowflies [2, 3, 4]. Conversely, our results are consistent with the hypothesis of a “deterrent” chemoreceptor sensitive to Na-saccharin.

[1] Hodgson, E.S.; Lettvin, J.Y.; Roeder, K.D. Science 122: 417-418; 1955.
[2] Dethier, V.G.; Bowdan E. Physiol. Entomol. 14:127-136; 1989.
[3] Dethier, V.G.; Bowdan E. Physiol. Entomol. 17:325-330; 1992.
[4] Liscia, A.; Solari, P. Physiol. Behav. 70: 61-65; 2000.

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19

Ingwild Masante-Roca, Marie-Cécile Dufour, Lionel Delbac and Christophe Gadenne

Institut National de la Recherche Agronomique, UMR Santé Végétale, Centre de Rercherches de Bordeaux, BP 81, 33883 Villenave d’Ornon cedex, France
gadenne@bordeaux.inra.fr

Olfactory orientation responses of the grapevine moth, Lobesia botrana, to the host plant in a wind tunnel

Lobesia botrana (Lepidoptera : Tortricidae) is one of the most serious pests of vineyards in the paleartic area. Different generations of this species are confronted with different volatiles emitted from the main host plant, grapevine, during different seasons. In particular, females lay their eggs on either flower buds, green berries or ripe berries depending on the generation. As part of a general study on the plasticity of olfaction in L. botrana, we investigated the behavioral responses of laboratory-reared virgin or mated males and females. Various parts of the plant were tested for attraction in wind tunnel experiments.

First results show that the plant parts that elicit a behavioural response in the tested moths were correlated with the egg laying periods in the vineyards (mainly, flower buds, green and ripe berries). Moreover, only mated females responded to plant parts. No response was obtained with virgin males and females or with mated males.

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Tomoyosi Nisimura ¹, Kyoko Nakamura ¹, Takasi Nagao ², Satoshi Tamotsu³, Ryohei Yamaoka ¹ and Mamiko Ozaki ¹

1 Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto 606-8585, Japan
2 Human Information Systems, Kanazawa Institute of Technology, Ishikawa 924-0838, Japan
3 Graduate School of Human Culture, Nara Women's University, Nara 630-8506, Japan.
tomoyosi@ipc.kit.ac.jp

Dietary experience with smell alters the feeding sensitivity of the blowfly : Biogenic amine concerned with the feeding sensitivity regulation.

Associative memories of some odors with taste of diet influence feeding preference of the blow fly Phomia regina. In the flies fed on sucrose solution with the smell of limonene for five days after eclosion, the feeding sensitivity to sucrose was obviously reduced. In order to investigate change in the fly brain when the feeding sensitivity of the fly was reduced by dietary experience with smell of limonene, we quantified biogenic amines in the brain. Of all measured biogenic amines, both octopamine and tyramine significantly were decreased in the fly showing the feeding sensitivity reduction. In such flies, injection of tyramine but not that of octopamine recovered the feeding sensitivity to the normal level. Thus, we concluded that tyramine modulated the feeding sensitivity, but not octopamine. Using immunohistochemical methods, we found the tyramine positive cells in the brain. They were classified into seven groups. It is probable that some of them are concerned with the feeding sensitivity regulation in the fly.
Supported by a grant of ProBRAIN to MO.

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21

Nobuaki Tanaka¹, Takeshi Awasaki², Kei Ito³

1 Grad. Univ. Advanced Studies, Natl. Inst. Basic Biol., Myodaiji, Okazaki, 444-8585, Japan
2 Natl. Inst. Basic Biol., PRESTO (JST), Univ. of Tokyo, Institute of Molecular and Cellular Biosciences,1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
3 Natl. Inst. Basic Biol., BIRD (JST), Univ. of Tokyo, Institute of Molecular and Cellular Biosciences,1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
nobuchan@nibb.ac.jp

Projection patterns of the antennal lobe relay interneurons of the Drosophila brain

The antennal lobe (AL) of Drosophila has about 50 glomeruli, each of which is likely to receive information from a distinct type of olfactory sensory neurons. In order to understand the mechanisms underlying odor recognition and olfactory learning and memory, it is important to reveal how olfactory information from each glomerulus is transmitted to, and integrated in, other brain areas that are responsible for such functions.

To visualize neural circuits for olfactory processing, we used genetical approaches. We screened 3939 GAL4 enhancer-trap strains that label specific subsets of AL projection neurons (PNs). Previously, three antenno-cerebral tracts (ACTs) have been identified to connect the AL with the secondary olfactory centers, the mushroom body (MB) and lateral horn (LH). In addition to these ACTs, we identified ten novel pathways that connect the AL with various regions of the protocerebrum. These PNs arborize in distinct small areas within each part of the protocerebrum.

We next analyzed the projection pattern of each pathway in the LH, where the majority of PNs terminate. In the LH, the PNs of each pathway terminate at the characteristic zone that overlaps only partially with each other. We then screened for the strains that label intrinsic and other extrinsic neurons of the MB and LH, and compared the arborization patterns with those of the PNs. There are spatial correlation of arborization patterns between those neurons in the secondary olfactory centers, suggesting that there are subcompartments each of which is preferentially responsible for the processing of the information deriving from particular subsets of AL glomeruli. We lastly show that such a subcompartmentation in the secondary olfactory centers is eatablished during development and maintained regardless of the deprivation of olfactory input after eclosion.

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22

Marzia Cristiana Rosi¹, Roberto Romani², Nunzio Isidoro³ and Ferdinando Bin²

1 Department of Agricultural Biotechnologies, University of Firenze, Piazzale delle Cascine 18, 50144, Firenze, Italy
2 Department of Arboriculture and Plant Protection – Entomology, University of Perugia, Borgo XX Giugno, 06121, Perugia, Italy
3 Department of Agricultural and Environmental Biotechnologies – University of Ancona, via Brecce Bianche, 60131, Ancona, Italy
mcrosi@unifi.it

Differences in antennal multiporous gustatory sensilla in Trissolcus basalis and their possible involvement in the host selection process

Trissolcus basalis female (Hymenoptera :Scelionidae) carry multiporous sensilla on the antennae, distributed according to the claval formula 1-2-2-2-2. A gustatory function has been suggested for these sensory structures, therefore defined as MGS (multiporous gustatory sensilla).
Morphological investigations carried out on these structures showed differences on the external cuticular features of the apical mutiporous area between the sensillum on the apical antennomere and the other sensilla. This intra-sensilla difference is due to the apical mutiporous area, possibly related to differences on how the sensillum comes into contact with the host egg.

During the host selection antennation process play a foundamental role in detecting non volatile semiochemicals acting on contact. Upon landing the female parasitoid antennate the substrate surface to locate the host. As soon as she reach the host, she antennates the host surface to perceive inter- or intraspecific signals that can affect the host acceptance process. T. basalis host acceptance is induced by host recognition kairomone present in the host egg glue. The same behaviour can be induced by substances not directly related to the attacked host stage, e.g. secretion from the dorsal abdominal glands (DAG) of the Nezara viridula adult which induces acceptance of dummy eggs. While antennating, female wasps can also perceive intraspecific signals such as host marking pheromone to discriminate already parasitized hosts. T. basalis female starts antennation when it comes into contact with the egg (encounter), then it mounts the egg-masses and continues more extensively, standing on the host (examination). Host acceptance or rejection then can follow.

In the case of females exposed to glass beads coated with N. viridula DAG extracts, the encounter time varied significantly. T. basalis female responds to the host marking pheromone by increasing the examination time but not the encounter time. The encounter and examination behaviours are affected by the progressive ablation of the MGS-bearing antennomeres varying the allocation time of the encounter and of the examination behaviour. With amputations the encounter duration increases, while the examination time decreases. Significant time shifts start after the ablation of A11 and A10 antennomeres. These first results suggest that host recognition occurs during the encounter, and host discrimination during the examination. MGS play an important role in host recognition and host discrimination behaviour, mainly in the encounter and the examination phases respectively. Further investigation are needed to study the role of these sensilla on the perception of chemicals acting on contact during host location and a possible function of A11 antennomere in this step.

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23

Oystein Olav Roten¹, Anna-Karin Borg-Karlson², Johan Andersson² and Hanna Mustaparta¹

1 Norwegian University of Science and Technology, Department of Biology, Neuroscience Unit, MTFS, Trondheim, Norway
2 The Royal Institute of Technology, Group of Ecological Chemistry, Stockholm, Sweden
oysteiro@stud.ntnu.no

Behavioural responses by the pine weevil Hylobius abietis to conifer produced odorants detected by receptor neurones

The pine weevil, Hylobius abietis, causes great damage on seedlings of Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) in reforestation areas in Northern and Middle Europe. The damage is caused by adult weevils feeding on the soft bark of the young plants. Attention to olfaction in this insect is based on numerous observations that host plant volatiles are very attractive to the pine weevil. Compounds known to be important attractants are (+)-a-pinene1 and the mixtures of a-pinene and ethanol2. An extensive study of olfactory receptor neurones in the pine weevil has revealed numerous plant compounds that elicit activation3. The neurones could be classified in 30 types according to the compound that elicited the best responses. The present study was carried out in order to test the attractive effect of selected key odorants, including enantiomers of a-pinene and limonene. The weevils were tested in a multiple-choice bioassay, with two test tubes and four-control tubes1. The experiments were carried out in darkness, each with 3-15 weevils, and lasted at least 8 hours.

The results showed that both enantiomers of a-pinene were stronger attractants than the other bicyclic monoterpenes ((-)-b-pinene, (+)-3-carene and camphor) of the host plants and the bicyclic monoterpene E-verbenol, which is a bark beetle pheromone component. A slight difference of attraction to (+)- vs. (-)-a-pinene was found for weevils fed on Scots pine and Norway spruce, respectively. Another interesting finding was that a non-host compound, para-methylanisol, had a stronger attractive effect than (+)-a-pinene. The compounds with lowest effect (below controls) were (-)-limonene and verbenol, which are previously shown to inhibit attraction in field tests4,5. Interestingly, (+)-limonene was found to attract the weevils, and when tested against the mixtures of limonene enantiomers, (+)-limonene attracted more weevils than the mixture. This indicates that the (-)-limonene acted as an inhibitor. In contrast, when testing the (+)-a-pinene against a mixture of (+)- and (-)-a-pinene, this mixture attracted far more weevils than the single compound. Also when testing (+)-a-pinene against a mixture containing (+)-a-pinene and four other attractive components, the mixture was far more attractive than the single compound in all tests. All together these results have elucidated which of the selected compounds attract and inhibit the pine weevil. It is shown that mixtures have stronger attractive effects than single attractive compounds. The aim is to construct the most attractive blend of host plant compounds for the pine weevil.

1Mustaparta H., 1975, Responses of single olfactory cells in the pine weevil Hylobius abietis L. (Col.: Curculionidae). J Comp Physiol A, 97: 271-290
2Tilles D. A., Sjødin K., Nordlander G. and Eidmann H.H., 1986, Syngerism between ethanol and conifer host volatiles as attractants on the pine weevil, Hylobius abietis (L.) (Coleoptera: Curculionidae, J Ecol Entomol, 79: 970-973.
3Wibe A., Borg-Karlson A.K, Norin T. and Mustaparta H., 1997, Identification of plant volatiles activating single receptor neurones in the pine weevil (Hylobius abietis), J Comp Physiol A, 180: 585-595
4Nordlander G., 1989, Limonene inhibits attraction to a-pinene in the pine weevil, Hylobius abietis and H. pinastri, J Appl Ecol, 4: 1307-1320
5Lindgren B.S., Nordlander G. and Birgersson G., 1996, Feeding deterrence of verbenon to the pine weevil, Hylobius abietis (L) (Col, Curculionidae), J Appl Entomol, 120: 397-403

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24

Tonette Røstelien^1,2, Marit Stranden¹, Anna-Karin Borg-Karlson^1,3 and Hanna Mustaparta¹

1 Norwegian University of Science and Technology, Department of Biology, Neuroscience Unit, NO-7489 Trondheim, Norway
2 Gjøvik University College, Faculty of Health Studies, NO-2802 Gjøvik, Norway
3 The Royal Institute of Technology, Department of Chemistry, Ecochemistry, SE-100 44 Stockholm, Sweden
tonette.roestelien@hig.no

Receptor neurone responses to naturally produced plant volatiles identified in two closely related heliothine moth species.

We are studying how plant odour information is encoded in the receptor neurones of heliothine moths. By the use of gas chromatography linked to single cell recordings (GC-SCR) and to mass spectrometry (GC-MS), plant volatiles that are detected by receptor neurones have been identified in two species; Heliothis virescens (America) and Helicoverpa armigera (south Europe, Asia, Australia, Africa, eastern Pacific). According to the plant compounds eliciting responses, 15-20 types of receptor neurones have been classified in these species. The receptor neurones respond to compounds within the chemical groups of acyclic, monocyclic and bicyclic monoterpenes, oxygenated monoterpenes, sesquiterpenes, homo-terpenes, aromatic hydrocarbons and oxygenated aliphatic compounds. The specificity and structure–activity relationships of five of these types have been described in other studies (Røstelien et al. 2000, Chem Senses, Røstelien et al. 2000, J Comp Physiol A, Stranden et al. 2002, Chem Senses, Stranden et al. 2003 J Comp Physiol A, submitted, Stranden et al. 2003 Chemoecology, submitted). We here present other receptor neurone types for which the active components have been identified; one type responding with high sensitivity to the chemical analogs (S)-(+)-linalool and dihydrolinalool. A group of receptor neurones appearing together in the recordings, all responded to oxygenated aliphatic compounds. The activity of these neurones was separated on the basis of different spike amplitudes and waveforms. Typically, the neurones showed narrowly tuning, responding to only a few chemically related compounds out of the hundreds of the constituents present in the various host- and non-host plant materials tested. No overlap of the molecular receptive ranges of the receptor neurone types was found. One exception is for linalool, the key compound for one of the neurone types presented here, and also a secondary active compound for another neurone type tuned to geraniol.

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25

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