ESITO X European Symposium for Insect Taste and Olfaction

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10 TH EUROPEAN SYMPOSIUM FOR INSECT TASTE AND OLFACTION (10th ESITO)

September 15-22, 2007 -Roscoff, Brittany, France

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Abstracts 

Alisha R Anderson ^1,2, Kevin Wanner⁴, Marien deBruyne², Stephen Trowell¹, Coral Warr² and Richard Newcomb³.

1 Food Futures Flagship and CSIRO Entomology, Canberra, Australia

2 School of Biological Sciences, Monash University, Victoria, Australia

3 HortResearch, Mt Albert, Auckland, New Zealand

4 University of Illinois, Urbana-Champagne, Illionis, USA

Elucidating the molecular basis of odour coding requires an understanding of the odorant responses of olfactory receptors. The functional analysis of these receptors was pioneered in Drosophila and has shown that receptor-odorant tuning ranges along a continuum from narrowly- to broadly-tuned. Initial studies in Lepidoptera focused on the very narrowly-tuned male-specific pheromone receptors, which respond to the female produced pheromone components in both Heliothis virescens and Bombyx mori. We have recently identified 41 novel odorant receptors from the recently sequenced genome of the silk moth, B. mori. We have shown that six of these receptors are either female-specific or overexpressed in females compared to males. Functional analysis of these receptors using the Drosophila empty neuron system and calcium imaging in insect lines will be discussed and compared. The results provide an interesting example of how genomic information can lead to insights into the behaviour and ecology of an insect.

Martin Andersson , Mattias C. Larsson, and Fredrik Schlyter

SLU, Dept. Plant Protection Biology, Chemical Ecology, P.O. Box 44, SE-230 53, Alnarp, Sweden

martin.andersson@ltj.slu.se

Single-cell responses to host, non-host, and pheromone compounds in the European spruce bark beetle ( Ips typographus )

Host selection by conifer-inhabiting bark beetles is governed by pheromones from conspecifics and kairomones from host trees. The response to attractive odours is inhibited by non-host volatiles (NHV), originating from angiosperm trees. For the European spruce bark beetle (Ips typographus), the importance of aggregation pheromone and inhibitory NHV has been demonstrated, whereas the role of host kairomones in host location is questionable. Several inhibitory NHV have been identified, some with redundant, others with synergistic effects. By means of electrophysiological recordings from single olfactory receptor neurones (ORNs), we investigate the relative proportions of ORNs that respond to pheromones, host, and non-host volatiles, respectively, and we look for spatial distribution patterns among ORN types. We also study whether the behavioural pattern of redundancy and synergism among NHV is reflected at the antennal level of olfaction. Although we are at an early stage, clear functional patterns have emerged. Most ORNs respond specifically to one or a few compounds. In both sexes, pheromone sensitive ORNs seem to be more common than ORNs tuned to host or non-host volatiles, respectively. For one of the compounds (cis-verbenol), ORNs are mainly present on the distal part of the antennae. Selective ORNs that respond to host compounds exist, suggesting that volatiles released from conifers might be relevant in host location. In addition, at least four ORN types that are sensitive to NHV have been found. The response specificity of these neurones seems to partly explain the behavioural patterns of redundancy and synergism among active NHV. Synergizing compounds appear to be perceived by selective ORNs, while somewhat more broadly tuned ORNs respond to redundant compounds. However, we are careful to draw any definite conclusions until response specificity has been studied at lower stimulus doses. Yet, the preliminary results indicate that this study may provide a basis for how different attractive, and especially inhibitory signals, are integrated in the insects’ olfactory system.

Peter Anderson

Plant Protection Biology, Swedish University of Agricultural Sciences, Box 44, S-230 53 Alnarp, Sweden. 

peter.anderson@ltj .slu.se

Experience modulates male response to female pheromone in a moth.

Living in a variable environment it is adaptive for most animals to change their behaviour depending on the current conditions. In insects, plasticity in the response to stimuli related to food and oviposition cues is well documented. However, very little is known about if experience changes the response to signals related to reproduction.
We have shown that brief pre-exposure to sex pheromones, without ensuing reward, lowers the threshold for behavioural response. The behavioural attraction to sex pheromone increases after pre-exposure in a time-dependent manner: a short-term effect, possibly a form of sensitization, and a long-term effect after more than 24 hours. The behavioural long-term effect is paralleled by an increase in sensitivity of interneurons in the antennal lobe, whereas the peripheral olfactory system does not change its sensitivity.

Romina B. Barrozo^1,2, Louise Couton²; Teresita Insausti1; Nadine Fresquet¹; Sebastian Minoli², Jean-Pierre Rospars²; Sylvia Anton² and Claudio Lazzari¹

1. IRBI, UMR6035 CNRS - Univ. François Rabelais, Tours, France.

2. INRA, UMR Physiologie de l'Insecte, Versailles, France.

claudio.lazzari@univ-tours.fr

Antennal pathways in the central nervous system of Rhodnius prolixus

The haematophagous bug Rhodnius prolixus has been a classical model system in insect physiology for a long time, and more recently sensory aspects are being revealed. Nevertheless, very few data are available on the basic organisation of its nervous system. By means of neuronal fills, histology, confocal microscopy and 3-D reconstruction methods, we established the first three-dimensional map of the antennal lobe (AL) of this species, based on both a qualitative and a quantitative approach. Computer reconstruction of the antennal lobe has revealed at least 22 glomeruli. Several prominent glomeruli are recognizable by their location, size, and shape; others are identifiable only by their positions relative to prominent glomeruli. In general, the ALs of this species are relative diffuse structures, with certain regions receiving more fibres than others. No obvious sexual dimorphism of the glomerular architecture was observed. Interestingly, antennal afferents not only project into the deutocerebrum, but fibres descend through the ventral nervous chain, up to neuropiles belonging to abdominal segments. Our results will be discussed in a comparative frame and as related to the biology of these bugs.

Romina B. Barrozo; Xenia Simeone; Christophe Gadenne; Sylvia Anton

Institut National de la Recherche Agronomique, Unité Mixte de Recherches en Physiologie d'Insectes: Signalisation et communication, Centre de Recherches de Versailles, Route de St Cyr 78026 Versailles cedex, France

rbarrozo@versailles.inra.fr

Olfactory plasticity in insects: the role of biogenic amines on pheromone sensitivity

Odour perception in insects is directly influenced by diverse internal and external factors such as the reproductive status, age, experience, and photoperiod. An identical stimulus may elicit distinct reactions under different environmental or behavioural conditions. Since neural circuits are in finite number in animals, neurons and networks must accomplish multiple functions. Therefore, neuromodulation provides a potent means to reach functional and reversible adjustments of a sensory circuit. The olfactory system is an attractive model for the study of neuronal wiring and information processing. In moths, the sensitivity to the sex pheromone may vary with the physiological state of the insects as well as with experience. Such phenomena have been shown at the behavioural and at the central nervous level. In males of Agrotis ipsilon, the behavioural and central nervous responses to sex pheromone increase with age and juvenile hormone levels (Anton and Gadenne, 1999). On the other hand, a transient post-mating inhibition of behavioural and central nervous responses to sex pheromone was also observed (Gadenne et al 2001). This transient neuronal plasticity serves as an energy-saving strategy by switching off the olfactory system and therefore preventing males from mating unsuccessfully. Thus, nocturnal moths are able to progressively set in motion or disengage their olfactory system in correlation with their physiological state. Neuromodulators like the biogenic amines octopamine (OA) and serotonin (5HT) could be implicated in this task. Both have been shown to be involved in regulating sex-specific behaviour, and to modulate peripheral and central nervous processing of pheromones in insects and 5HT even in vertebrates. We combined behavioural studies, intracellular recording techniques and biochemical methods to investigate the role of OA and 5HT in the plasticity of the olfactory system during mating. We show that both neuromodulators are implicated in regulating sex-specific behaviour and central nervous processing of olfactory signals.

 Bente G, Berg¹ , Sandra Utz² , Joachim Schachtner² , Uwe Homberg²

1 Dept of Psych/Neurosci Unit, Norwegian University of Science and Technology, 7489 Trondheim, Norway

2 Dept. of Biology, Animal Physiology, Philipps University, 35032 Marburg, Germany

Bente.Berg@ntnu.no

Neurochemical organization of the primary olfactory centre of the heliothine moth

The noctuid moth Heliothis virescens is a widely used organism for studying the neural pathways mediating olfactory signal information. In particular, the chemotopic organization of the male‑specific macroglomerular complex has been thoroughly evidenced by tracing of functionally characterized receptor neurons and antennal lobe projection neurons in this species. To gain further insight into the anatomical and neurochemical organization of the central olfactory pathways we have analyzed the distribution of neuropeptides and their possible co‑localization with g -amino-butyric acid (GABA) in the antennal lobe of H. virescens. Immunocytochemical experiments and MALDI-TOF mass spectrometry show that the neuropeptides are present in the antennal lobe - more precisely, in distinct populations of local interneurons, in certain types of projection neurons, and in centrifugal neurons. Interestingly, one antiserum, recognizing members of the tachykinin-related peptides (TKRPs), was uniquely distributed in the antennal lobe glomeruli as compared to the other antisera tested. Besides, a total lack of co‑localization of tachykinin with GABA as well as the other neuropeptides turned up. This indicates that TKRPs are present in a particular subtype of antennal lobe local interneurons. In addition to local neurons a pair of centrifugal neurons with cell bodies in the subesophageal ganglion, arborisations in the antennal lobe, and projections in the inner antennocerebral tract, showed tachykinin immunostaining.

Jonas Bengtsson , Mattias C. Larsson, Ylva Hillbur

SLU (Swedish Agricultural University), Plant protection biology, Vaxtskyddsv. 3, 230 53 Alnarp, Sweden

Jonas.Bengtsson@ltj.slu.se

Response of olfactory receptor neurons in the sorghum chafer, Pachnoda interrupta, to plant and fruit odours

The adult sorghum chafer beetle is a polyphagous herbivore, feeding on a diverse array of plants such as acacia, abutilon and sorghum, as well as fruit, e.g. banana and mango. Different host plants are available to the adults during their lifespan. The emergence of new adults in September-October coincides with the availability of sorghum with seeds in a palatable stage. The beetles feed extensively on cultivated sorghum, a host to which they have recently shifted, becoming a pest during the early 1990s. After this intense feeding period, adults aestivate for six months in the topsoil. The beetles re-emerge to mate and lay eggs in June, feeding on acacia flowers.

The olfactory receptor system of an odour-oriented polyphagous insect herbivore must detect several potential hosts, and possibly also assist in choosing among them. A wide variety of volatile chemicals make up the odour bouquet emitted by host plants. While flowering plants may purposely emit odour cues to enhance pollination or seed dispersal via fruits, plants should otherwise be under selection pressure not to offer any reliable, specific or detectable odour cues for herbivores.

We have characterized morphological and physiological types of olfactory sensilla in P. interrupta by means of scanning electron microscopy and electrophysiological recordings from single sensilla. One purpose of these investigations has been to screen for potential attractants for this economically important pest. Another purpose has been to determine how the receptor neuron assembly of this species is adapted to handle the many different types of potentially relevant olfactory stimuli available. We have investigated sensitivity and specificity of single neurons in various sensillum types to volatile compounds identified from host plants such as sorghum, abutilon and banana. Olfactory receptor neurons identified in these scarab beetles have been functional specialists, rather than generalists, detecting a small number of compounds with a high degree of specificity and sensitivity.

Aurélie Bodin

Université de Tours, Faculté des sciences et techniques, IRBI – Institut de Recherche sur la Biologie de l’Insecte, Parc Grandmont, Avenue Monge, 37200 Tours.

aurelie.bodin@etu.univ-tours.fr

Chronobiology of insect odour perception: adaptive control by endogenous and exogenous mechanisms

Circadian rhythms of chemosensitivity have been well established in insects and a general modulation of the response to odours was postulated. However, some fundamental questions arise concerning the adaptive value of such general variation. Indeed, it would be more adaptive to modulate the sensitivity to each odour separately and maximal responsiveness should occur at the moment when a given odour becomes biologically relevant. We tested both hypotheses in Rhodnius prolixus, which exhibits a bimodal activity, each peak associated to mutually excluding behaviours, at dusk search for food and at dawn for refuges. We studied the daily dynamics of the response to carbon dioxide (host-associated) and to aggregation cues (refuge associated).  We show that the responsiveness to these signals does not remains constant along the day, but is limited to precise temporal windows.  However, whereas CO2 sensitivity is restricted to dusk, that to assembling odours is limited to dawn. The responsiveness to carbon dioxide revealed to be controlled by an endogenous circadian clock, whereas that to assembling signals evinced an exogenous dependence.  Thus, our results support a specific modulation of odour responsiveness, rather than a global modulation. The control of specific sensitivities is adjusted according to the behavioural context associated to each individual odour. Finally, endogenous and exogenous control systems act together to control the response of the insects to different odours. We discuss the adaptive value of this type of control of sensory sensitivity in a complex environment.

Isabelle Brigaud, Sébastien Malpel, Marie-Christine François & Emmanuelle Jacquin-Joly

 UMR Physiologie de l’insecte : signalisation & communication, INRA, Route de Saint-Cyr, F-78026 Versailles cedex, France

ibrigaud@versailles.inra.fr

Identification and molecular characterization of genes encoding candidate pheromone receptors in the cotton leafworm, Spodoptera littoralis.

In Lepidoptera, pheromone receptor (PR) identification became possible only with progress in genome sequencing, notably in Bombyx mori and Heliothis virescens. Indeed, olfactory receptors, including PRs, are very divergent within insects, making homology-based cloning strategies unsuccessful. Here, we bypassed this problem using an Expressed Sequence Tag (EST) approach. An antennal cDNA library was constructed from male antennae of Spodoptera littoralis. About 21000 EST were sequenced and blasted with all the insect olfactory receptors known to date. Four PR candidates were found based on their homology with B. mori and H. virescens PRs. Their entire sequences were determined and the encoded proteins clustered in a phylogenetic group gathering B. mori and H. virescens PRs. In addition, the proteins were predicted to present a 7 transmembrane domain topology (TMHMM2.0), common to all olfactory receptors, including PRs. Using RT-PCR and in situ hybridization, we showed that their expression was restricted to the antennae, in association with the olfactory sensilla. Quantitative PCR experiments revealed that two of them were enriched in male antennae while the two others were expressed equally in the two sexes. The four genes appeared to be expressed late in development (2 days before adult eclosion), in good agreement with the development and functionality of the adult olfactory system. Taking into account these different characteristic features, we then conclude that these genes encode good pheromone receptor candidates in S. littoralis. Functional experiments are now in progress to address their respective ligands.

Daniela Büsser¹ and Patrick M. Guerin¹

1University of Neuchâtel, Animal Physiology, Institute of Biology, Rue Emile-Argand 11, Case Postale 158, 2009 Neuchâtel, Switzerland,

daniela.buesser@unine.ch

Attraction of the European grape berry moth to its pheromone is affected by plant volatiles

Host plant compounds are involved in many aspects of the lives of phytophagous insects, not least the problem of finding a suitable resource to feed on, mate and oviposit. In addition, volatile host plant products have been shown to influence the sexual behaviour of insects as enhancers of pheromone perception and production.

The grape berry moth Eupoecilia ambiguella Hb. (Lepidoptera, Tortricidae) is one of the main pests in European vineyards and some integrated methods for its control are based on sex pheromones. The disruption of pheromone communication is, however, insufficient at high population densities, is costly, and affects only male moths as females do not perceive the pheromone. One way to improve the pheromone-based control of grape moths may be found by considering host plant volatiles that may, in unison with the grape berry moth’ pheromone, act in signalling rendezvous sites for mating.

We have studied the influence of plant compounds on pheromone perception by the male grape berry moth using antennal sensory cell recordings and in a wind tunnel where test compounds were presented in a mixture with a synthetic pheromone blend. In the presence of a high pheromone concentration most males flew upwind to within a certain distance of the source, but then showed in-flight arrestment or flew out of the plume. The addition of host plant volatiles to the pheromone changed male flight behaviour and rendered the overdosed pheromone dose acceptable. Our results are important in the context of currently applied mating disruption strategies based on semiochemicals.

Gerard Carot-Sans¹, Gloria Rosell¹, Fançoise Bozzolan², Angel Guerrero¹ and Martine Maïbèche-Coisne²

1 Department of Biological Organic Chemistry, IIQAB (CSIC), Barcelona, Spain.

2 Unité 1272, UPMC-INRA-INA.PG, Physiologie de l'Insecte: Signalisation et Communication, Université Pierre et Marie Curie, France.

gcsqob@iiqab.csic.es

Peptide identification and cDNA cloning of a putative odorant-degrading esterase from the corn borer S. nonagrioides

 During pheromone reception process, a rapid degradation of pheromone compounds is required so that pheromone receptors may be activated by new incomming pheromone molecules. In the case of S. nonagrioides, esterases are mainly the enzymes responsible for the catabolism of the pheromone, particulary for (Z)-11-hexadecenyl acetate, the major component of the pheromone blend.

Preliminary studies in non-denaturing PAGE revealed the presence of at least seven different bands with esterase activity in male antennae. Although most of these bands were also present in non-olfactory tissues, one of them appeared to be antennae-specific, suggesting a more specific role in pheromone degradation. In this work we present an initial approach to understand the significance of this tissue specificity.

In order to identify the antennae-specific esterase, we have developed a bidimensional gel electrophoresis procedure to separate proteins with esterase activity. Mass spectrometry analysis of these proteins allowed us to find peptide sequences corresponding to the carboxylesterase family. A degenerated primer was designed from one peptide sequence and allowed us to amplify a partial cDNA by PCR. After gene analysis and data base comparison of the resulting sequences, one clone was identified as a putative new carboxylesterase. The characterization of the corresponding cDNA is running: full length cDNA isolation by RACE-PCR, study of the tissue distribution of the transcript by RT-PCR approach, as well as alignment and phylogenetic analysis to compare the sequence with other insect carboxylesterases.

Antoine Chaffiol and Christophe Pouzat

Brain Physiology Laboratory, CNRS UMR 8118, Paris-Descartes University, 45, rue des Saints-Pè res, 75006, Paris, France

antoine.chaffiol@univ-paris5.fr
christophe.pouzat@univ-paris5.fr

Quantitative analysis of odor responses in the cockroach Periplaneta americana.(POSTER PRESENTATION)

The American cockroach, Periplaneta americana is one of the few insects that has been extensively studied in the context of physiology, thanks to its robustness for in vivo experiments, its rich behavioral repertoire and visual or olfactory conditioning possibilities. Using Multi-Electrode Array (MEA) and Local Field Potential (LFP) techniques we record routinely neuronal populations activities mainly in the Antennal Lobe (AL) of the insect. We will illustrate the spike-sorting method, and describe our results about odor representations in the AL.

Bastien Chouquet^1,2, Françoise Bozzolan¹, Marthe Solvar¹,  Line Duportets¹,  Emmanuelle Jacquin-Joly², Philippe Lucas², Stéphane Debernard¹.

UMR 1272, UPMC-INRA-INA.PG, Physiologie de l’Insecte : Signalisation et Communication,

1Centre INRA, Bât. A, route de Saint-Cyr, F-78026 Versailles Cedex.

2Université Paris VI, 12 rue Cuvier, 75005 Paris, France.

stephane.debernard@snv.jussieu.fr

Molecular cloning and expression patterns of olfactory diacylglycerol kinase from Spodoptera littoralis

Insects especially use pheromones as cues for conspecific relationships such as sexual and social behaviours. Among insects, male noctuid moths are known for the ability to respond to low concentrations of the sex pheromone delivered by females. The sex pheromone components are detected via specialized olfactory receptor neurons (ORNs) innervating trichoid sensilla located on antennae. The chemical signal is converted into electrical activities which are conveyed as potential actions to antennal lobes where the olfactory information is processed to elicit behavioural and physiological responses. To this date, the different steps of chemo-electrical cascade are not fully elucidated.
The interaction of pheromone compound with theirs receptor proteins evokes the stimulation of a G-protein and phospholipase C which catalyses the cleavage of phosphatidylinositol 4,5-biphosphate  (PIP2) in  inositol 1,4,5- triphosphate (IP3) and 1,2-diacylglycerol (DAG). Numerous data support the current concept that pheromone perception of moths is mediated by the IP3 transduction pathway. Nevertheless, biochemical and electrophysiological evidence also indicated the involvement of DAG as a second messenger in insect pheromone transduction. Extracellular recordings from moth pheromone-sensitive sensilla demonstrated that DAG is able to stimulate ORN firing activity and a DAG-gated cationic current has been recently revealed from cultured neurons of the moth Spodoptera littoralis.
 The pivotal role of DAG in different cellular events has arisen great importance to a specific of lipid kinases, the diacylglycerol kinases (DGKs). These enzymes act as regulators of signal transduction by attenuating the endogenous level of generated DAG in a phosphorylation reaction that yields phosphatidic acid. In Spodoptera littoralis cultured ORNs, it has recently been demonstrated that the inhibition of DAG degradation, using a DGK pharmacological blocker, produced a sustained stimulation of a current that shared the properties of the DAG-activated current. A such result provided also evidence for the contribution of a putative DGK in insect olfactory transduction cascade.

The aim of our work was to pursue the study of this olfactory DKG by undertaking its molecular identification. We succeeded to clone the full cDNA of Spodoptera littoralis DGK (SlDGK) and its deduced amino acid sequence shared domains and biochemical features essential to DGK protein function. In male adult, the SlDGK transcript was detected predominantly in the brain and antennae where its presence was restricted to the olfactory trichoid sensilla. On the other hand, in male antennae, SIDGK gene expression started at the end of the pupal stage and reached a maximum during the adult life. This expression profile was consistent with an olfactory function limited to adults. Taken together, these findings brought evidence the molecular characterization of first DGK potentially involved in insect olfactory transduction.

Sylwester Chyb

CSIRO Entomology, Canberra ACT 2601, Australia

sylwester.chyb@csiro.au

Drosophila norpA encodes a phospholipase C-ß (PLC-ß),  an effector enzyme of the phosphopinositide (PI) signaling pathway. While its role in phototransduction is well documented we have only limited evidence of its involvement in the chemoreception. Here, we will show that norpA is expressed in large subset of gustatory receptor neurons (GRNs) in Drosophila labellum and tarsi. The potential role of norpA-encoded PLC in  taste transduction will be discussed.

Violaine Colson, Michael King, Frédéric Marion-Poll

INRA, UMR Physiologie de l'Insecte: Signalisation et Communication, Route de St Cyr, 78026 Versailles Cedex, France.

vcolson@versailles.inra.fr

Appetitive and aversive learning of odors in Spodoptera littoralis larvae : methodological adjustments

Secondary plant products can have insecticide, toxic or aversive actions on phytophagous insects. It was observed that caterpillars were able to avoid food containing phytoecdysteroids, as compared to food free of these compounds. This implies that they are able to prevent intoxication and that they could learn to be attracted by or to avoid odors associated with a reward or a punishment. Recent studies have demonstrated appetitive and aversive learning in Drosophila larvae ( Scherer et al 2003; Gerber et al 2007 ). We examined whether the classical associative learning used in Drosophila is applicable to larvae of a polyphagous species, the Egyptian cotton leafworm, Spodoptera littoralis. We developed a conditioning paradigm in which an odorant (hexanol) was paired with positive (fructose) or negative (chloride sodium or quinine) gustatory reinforcers. After expositions to an odorant (with the positive or the negative reinforcer), larvae (first to third instar) were given a choice between the odorant and paraffin oil, applied on 2 filter papers dropped on the opposite sides of a Petri dish. Attractiveness or repellence of the test odor to larvae was also assessed in a locomotor compensator. Larvae were slightly attracted by hexanol when previously associated with fructose. It appeared that LiCl provided a lack of mobility of the insects, probably due to the illness induced. Hexanol exposed with quinine induced an aversion that has to be confirmed with repetitions.

Louise Couton^1,2, Sylvia Anton¹, Kiên Kiêu², Jean-Pierre Rospars^1,2.

1- UMR 1272  PISC, INRA, Route de St Cyr, 78026 Versailles, France.
2- UR 341 MIA, INRA, Domaine de Vilvert 78350 Jouy-en-Josas, France.

lcouton@versailles.inra.fr

Mapping the antennal lobe of male Spodoptera littoralis

The antennal lobe (AL) is the primary olfactory centre in insects. The AL is divided into spherical sub-compartments called glomeruli, which house synaptic connections between olfactory receptor neurons (ORNs), local interneurons and projection neurons. Each glomerulus receives afferents from ORNs that all express the same olfactory receptor and thus has a functional identity. In addition, each glomerulus is similarly located in all individuals of a given species, sex and developmental stage (Rospars, 1983).
. The pattern of activated glomeruli constitutes a combinatorial code of odours which confers a functional significance to the AL glomerular organization.
The noctuid moth Spodoptera littoralis, with its well studied pheromone communication system, has become a model species for several research groups interested in olfaction. However, only a partial map of the female AL is currently available (Sadek et al., 2002). In order to prepare functional investigations and comparisons with related species, we have begun analyzing the AL organization in this species, focusing on the anatomical identification of individual glomeruli.
In the present work we first developed a complete image processing chain, using the Matlab language. This tool covers all steps, from confocal image segmentation to quantitative analysis of the 3D reconstruction. Using the newly developed tool, we established standardized 3D “maps” of ALs in three male individuals of S. littoralis based on the size of glomeruli and their spatial coordinates in a standard, carefully oriented, coordinate system. We analyzed the invariance and variability of the glomerular array using several criteria and methods.
On average, 62 glomeruli per AL were distinguished. Three landmark glomeruli - easily recognised in all six lobes - could be found so far. The other homologous glomeruli, in the right and left ALs of the same individual and between individuals, were determined by visual comparison of confocal images and 3D reconstructions, by progressing around landmark glomeruli. The pairs were validated by comparing their sizes and by a computer algorithm using only the spatial coordinates of the glomeruli. We found that the variability is significantly less within a single brain than between ALs of different individuals. The level of both intra- and inter-individual variability in the ALs of male S. littoralis seems higher than that observed in other moth species.
Finally, we propose a map of the male AL in this species, where the identity of glomeruli has been confirmed through different approaches. These data will serve to identify glomeruli in future studies, e.g. for identifying target glomeruli of stained neurons.

Rospars JP. 1983. Invariance and sex-specific variations of the glomerular organization in the antennal lobes of a moth, Mamestra brassicae, and a butterfly, Pieris brassicae. J Comp Neurol 220(1):80-96.
Sadek MM, Hansson BS, Rospars JP, Anton S. 2002. Glomerular representation of plant volatiles and sex pheromone components in the antennal lobe of the female Spodoptera littoralis. J Exp Biol 205(Pt 10):1363-1376.

Andrew Dacks, Thomas Christensen, John Hildebrand, Alan Nighorn

Arizona Research Laboratories Division of Neurobiology, University of Arizona, Tucson, AZ, 85721, U.S.A.

adacks@email.arizona.edu

Serotonergic modulation of olfactory processing in the antennal lobe of Manduca sexta.

Insects have evolved to be masters of miniaturization partly by the efficient use of their nervous systems. For instance, neuromodulators are often used to fine tune information processing capabilities of specific neural circuits to suit the current physiological state of the individual insect. In the antennal lobes (ALs) of the tobacco hawkmoth, Manduca sexta, serotonin is thought to function as a circadian modulator of responsiveness and sensitivity. Previous studies have observed that serotonin application caused a reduction in the membrane resistance of individual AL neurons that resulted in enhanced excitability of individual AL neurons in response to either antennal nerve shock or sex pheromone blend. However, these previous studies tested a limited stimulus set and only examined individual neurons. In this study, we used multi-channel extracellular arrays to observe the effects of serotonin on the responses of multiple AL units to olfactory stimuli that varied in concentration and chemical structure.

Serotonin only affected a subset of units within each AL ensemble recorded. For the AL units affected, serotonin enhanced the odor-evoked response magnitudes, but had no effect on the slow temporal patterning of responses. Serotonin also increased the coincident firing, although not the synchrony, between AL unit pairs. The slopes of the dose-response curves of AL units were increased by serotonin and in some instances serotonin caused AL units to respond to concentrations of stimuli that were previously sub-threshold. Principal components analysis determined that the differences between responses of AL ensembles to different concentrations of a single odor stimulus were enhanced by serotonin as were the responses of AL ensembles to structurally dissimilar odors (although not structurally similar odors). These data suggest that while serotonin has little effect on the actual coding of odors, it can act as a modulator of sensitivity and contrast between responses depending on the physiological state of the moth.

Gabriela de Brito, Joao Ramalho Ortigao Farias , Isabelle Massou, Monique Gauthier and Martin Giurfa

Research Centre on Animal Cognition, CNRS – University Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9, France.

debrito@cict.fr

New explorations of honeybee taste

One of the natural contexts in which honeybees have been most studied is that of their foraging behaviour. Although the processing of sensory information of flowers (e.g. colors, odors, shapes) has been intensively studied in the last decades, less is known about the processing of gustatory stimuli that are perceived when bees contact different natural products such as nectar and pollen. Honeybee taste, contrarily to other sensory modalities, remains a mainly unexplored research field.
In the honeybee, the antennae, mouth pieces and tarsi of the forelegs constitute the main chemosensory organs. They include gustatory but also hygro, thermo, mechanosensory and olfactory receptors. Gustatory receptor cells on these structures are located within specialized sensillae (trichoid or basiconic). We have studied honeybee taste using three main approaches: 1) behavioural studies, 2) electrophysiological recordings of gustatory receptor cells and 3) molecular characterization of gustatory receptor genes.
By means of behavioural and electrophysiological experiments we studied whether a sense for bitter taste exists in honeybees. We tested the effect of substances like quinine, salicine and caffeine which have proved to elicit consistent aversive responding in Drosophila melanogaster. In parallel, we recorded the responses to sucrose and saline solutions, and mixtures of these substances with quinine or salicine. We analyzed the responses of bees when these tastants were applied at the level of the antennae, mouth parts and tarsi of the forelegs. Behavioral responses showed no effect or a slight inhibitory effect of bitter substances on appetitive responses (proboscis extension reflex after sucrose stimulation). No effect was found when stimulations were performed at the level of the antennae and slight inhibition was found at the level of mouth parts and tarsi. Our electrophysiological recordings showed that while dedicated sugar and salt receptors could be identified on the antennae, mouth parts and forelegs, no dedicated bitter receptor could be found in these structures. Nevertheless, some bitter substances like quinine inhibited cellular responses to sucrose when presented together. In other words, bitter substances could be detected indirectly via their action on sucrose receptors.
In order to characterize gustatory receptors we performed bioinformatics analysis of the bee genome taking the gustatory receptor sequences from Drosophila melanogaster as reference. We identified the presence of 10 G-protein coupled gustatory receptor genes (AmGr), two of which (AmGr1 and AmGr2) showed a strong similarity to the DmGr5a, a sweet receptor gene associated with trehalose responding in flies. We used the RT-PCR technique to analyze the transcription of 9 of these AmGrs in different parts of the honeybee body. Besides the antennae, mouth parts and the three leg types, we found AmGr transcription in unsuspected body parts like the abdomen carcass and the crop. A transversal analysis across various developmental stages showed that some genes like AmGr2 and AmGr6 are transcribed in the egg and the pupa in equal or superior quantities compared to the antennal levels.
Our results yield new light on the unexplored world of honeybee taste perception and open the door for new functional studies on the role and specificity of gustatory receptors in honeybees.

Marien de Bruyne¹, Cecile Faucher

Freie Universit ä t Berlin, Neurobiologie, K ö nigin-Luise-St. 28-30, 14195 Berlin, Germany,

1. presently: Monash University, Wellington Rd, Clayton VIC 3800, Australia.

Marien.DeBruijne@sci.monash.edu.au

Behavioural and physiological responses to carbon dioxide and vinegar odours

With their olfactory system, insects detect and respond to complex chemical stimuli which can lead them to food, oviposition sites or mates. Odours may also help them avoid toxins or other dangerous situations. Drosophila melanogaster is attracted to the odour of apple cider vinegar but generally avoids CO 2 . Are these responses hard-wired?

We have studied a sex-specific interaction between CO 2 and attractive odours in two different assays. In a four-field olfactometer, individual walking flies avoided high concentrations of CO 2 but didn’t respond to a small rise of 0.02%. However, in a background of vinegar, females avoid concentrations as low as 0.02% whereas males do not.

In a more natural situation, groups of free flying Drosophila were also attracted to vinegar and still avoided CO 2 . However, adding CO 2 to vinegar made it more attractive than vinegar alone. The interaction was still sex-specific but the nature of the response to the combination was different in this assay.

To study the neuronal basis of these behaviours we recorded from olfactory receptor neurons on the antenna of male and female flies. CO 2 is detected by only one class of receptor neurons but components of vinegar odour are detected by several classes. We discuss some of the physiological properties that make CO 2 -detecting neurons unique.

Heike Demmer, Simon Heß, Andreas Husch, Moritz Paehler and Peter Kloppenburg

University of Cologne, Institute for Zoology and Physiology, Krieler Str. 119, D-50935 Cologne, Germany

peter.kloppenburg@uni-koeln.de

Physiological and morphological characterization of interneurons in the insect olfactory pathway

Olfactory information is processed in multiple stages. In insects, the first step of synaptic processing occurs in the primary olfactory centres, or antennal lobes. Inside the antennal lobe each olfactory receptor cell projects into one glomerulus and many receptor axons converge at each glomerulus, where they form synapses with local interneurons and projection (output) neurons. The arborizations of the local interneurons are confined to the antennal lobe. In contrast, the projection neurons extend their axons to higher-order neuropiles of the protocerebrum. The projection neurons process and integrate olfactory information in the antennal lobe, and convey and distribute processed olfactory information inside the protocerebrum as well; e.g. to the mushroom bodies, where further processing, such as multi-modal integration and olfactory learning, occurs.

To better understand the specific functional roles of the different neuron types that form the central olfactory pathway, we have begun to study their physiological and morphological characteristics. To analyze the intrinsic firing properties, odor induced responses and their underlying voltage activated currents we are using primary cell culture and an intact brain preparation, which allows patch-clamp recordings during physiological relevant stimulations and a subsequent morphological characterization of identified neurons.

Jean-Marc Devaud , Benoît Hourcade, Emmanuel Perisse, Jean-Christophe Sandoz

Research Center on Animal Cognition (CNRS UMR 5169), Paul Sabatier University

118 Route de Narbonne 31062 Toulouse cedex 4, France

devaud@cict.fr

Glomerular plasticity associated to olfactory long-term memory in the honeybee

Consolidation of memory traces can rely on cellular modifications within neural networks and especially in those involved in the processing of the learned stimuli. The antennal lobssociations, and (2) it is organised in clearly-identified structural and functional units, the glomeruli. The glomeruli house synaptic contacts between several neuron types involved in odour processing, but also receive convergent input signalling reinforcement, and are thus a place for forming odour-sugar associations. Besides, they are highly plastic, as shown by previously observed changes in volume and activity related respectively to foraging experience and short/medium term associative memory. Still, the nature of changes specifically related to the formation and maintenance of associative LTM remain unknown.

We have thus used volumetric measurements and in vivo calcium imaging recordings respectively, to evaluate possible structural and functional changes due to LTM in the honeybee antennal lobe. Anatomical and functional data were collected in separate animals that had either (i) formed an odour-specific memory after paired odour-sucrose presentations or (ii) control bees that received only unpaired stimuli. Measurements were carried out on 20 clearly identified glomeruli, 3 days after conditioning, a time corresponding to the late phase of LTM. Our results show for the first time that associative olfactory late-LTM is associated with volume changes and modulations of odour-induced activity in a subset of glomeruli. Besides, these structural and functional changes were odour-specific, such that different glomeruli were affected when learning different odours. This suggests that such glomerular plasticity may represent a part of the memory trace contributing to a stable and specific olfactory long term memory.

Joseph C. Dickens

USDA, ARS, Henry A. Wallace Beltsville Agricultural Research Center, Plant Sciences Institute, Chemicals Affecting Insect Behavior Laboratory, Beltsville, MD 20705

Specialized receptor neurons for aggregation pheromone and host plant volatiles in Colorado potato beetle

The Colorado potato beetle is a major pest of potatoes and other solanaceous crops world-wide.  Previous studies demonstrated orientation of adult beetles to volatiles emanating from host plants (Visser 1976; Bolter et al. 1997).  Using coupled gas chromatography and electroantennograms, volatiles detected by the insect were shown and blends of these volatiles were demonstrated to be attractive in laboratory behavioural studies and field tests (Dickens 1999; 2000).  More recently, a male-produced aggregation pheromone was discovered (Dickens et al. 2002); behavioral activity of the pheromone was enhanced by host plant volatiles (Dickens 2006).  Here we studied the sensitivity and specificity of receptor neurons housed within sensilla on the antennae of male and female CPB when challenged by 25 odorants including host plant volatiles, the aggregation pheromone, and other chemical signals emitted by plants.  Recordings from more than 120 sensilla revealed receptor neurons specialized for the aggregation pheromone and five plant volatiles including constitutive and induced signals.  Receptor neurons for two of the plant volatiles were often co-localized within the same sensilla; possibly indicative of their role in detection of ratios of the two compounds.  The results demonstrate that the Colorado potato beetle has narrowly-tuned specific receptor neurons for its chiral aggregation pheromone and plant volatiles important in locating suitable host plants and enhancement of the pheromone.

Bolter CJ, Dicke M, Van Loon JJA, Visser JH, Posthumus MA (1997) J. Chem Ecol., 23, 1003-1023.

Dickens JC (1999) Agric. For. Entomol., 1, 47-54.

Dickens JC (2000) Agric. For. Entomol., 2, 167-172.

Dickens JC (2006) J. Appl. Entomol., 130, 26-31.

Dickens JC, Oliver JE, Hollister B, Davis JC, Klun JA (2002) J. Exp. Biol., 205, 1925-1933.

Michael J. Domingue¹, Callie J. Musto², Charles E. Linn Jr.² and Wendell L. Roelofs ² , Thomas C. Baker¹

1 Center for Chemical Ecology, Penn State University, University Park, PA.16802, USA.

2 Department of Entomology, Barton Lab, New York State Agricultural Experiment Station, 630 W. North St., Cornell University, Geneva, New York 14456, USA.

mjd29@psu.edu

Altered olfactory receptor neuron responsiveness in rare European and Asian corn borer males that exhibit cross-specific behavioral responses

Olfactory receptor neuron (ORN) response was measured to assess why some males of the Asian corn borer (ACB), Ostrinia furnacalis, and the E strain of the European corn borer (ECB), Ostrinia nubilalis, fly upwind to both ACB and ECB pheromone blends. For the three ORNs housed within each sensillum of both species, we tested responsiveness to the ACB pheromone components Z12- and E12-14:OAc, the ECB pheromone components Z11- and E11-14:OAc, and the common behavioral antagonist Z9-14:OAc. For both species, behaviorally rare and normal males responded to their own pheromone components and the common antagonist with similar ORN specificity and spike frequency. For ACB males that flew upwind only to the ACB blend, the ECB components stimulated the agonistic-pathway ORNs, but Z11-14:OAc also elicited responses in the antagonistic-pathway ORN. For the rare-type ACB males that flew to ACB and ECB pheromone blends, Z11-14:OAc did not stimulate the antagonistic-pathway ORN. This behaviorally significant alteration in ORN responsiveness is described as “olfactory antagonistic release”. In contrast, for both the normal and rare ECB E-strain males, the ACB components stimulated the agonistic-pathway ORNs, but not the antagonistic-pathway ORN. For these rare ECB males, responsiveness to the ACB components increased on the ORN associated with the major pheromone component, E11-14:OAc, while decreasing on the ORN associated with the minor pheromone component, Z11-14:OAc. The olfactory alterations in rare ECB E-strain males appear to allow the ACB pheromone blend to stimulate the two agonistic-pathway ORNs with the same relative strength as the ECB E-strain blend. Thus, while peripheral olfactory alterations are correlated with cross-specific behavioral responses in both species, they differ with respect to whether the changes occur on the agonistic or antagonistic-pathway ORNs.

Maike Forstner, Heinz Breer and Jürgen Krieger

University of Hohenheim, Institute of Physiology, Garbenstr. 30, 70599 Stuttgart, Germany

krieger@.uni-hohenheim.de, maikeforstner@web.de

Candidate pheromone receptors of the two silkmoth species Antheraea pernyi and Antheraea polyphemus

Females of the silkmoth Antheraea pernyi and the sibling species Antheraea polyphemus release a sex-pheromone blend containing the same components albeit in inversed ratios. Accordingly, in the sensilla hairs on the antenna of the males the same types of sensory neurons have been found each tuned to one of three sex-pheromone components. Distinct pheromone components are supposed to be transferred by special pheromone binding proteins (PBPs) through the sensillum lymph towards the dendrites of the sensory neurons, where they interact with specific receptors in the membrane. Three types of PBPs have been identified in both species, however, pheromone receptors are still elusive. As a prerequisite to explore the antennal expression pattern of receptor types in the two Antheraea species which use the same pheromone components in an inversed ratio and to elucidate a possible interplay between PBP- and receptor types we set out to identify genes encoding putative pheromone receptors. As a first approach we have screened cDNA libraries from male antennae. Using probes according to receptor sequences of Heliothis virescens we have identified a cDNA from A. pernyi encoding a protein (AperOR-1) with moderate identity to the screening probes; screening an antennal cDNA library of A. polyphemus with an AperOR-1 probe led to the identification of a highly related sequence (ApolOR-1). Comparison of AperOR-1 and ApolOR-1 with other moth olfactory receptors assigned them to the relatively conserved group of candidate pheromone receptors from Bombyx mori and Heliothis virescens. RT-PCR experiments revealed that ApolOR-1 was predominantly expressed in male antennae and in situ hybridization experiments have demonstrated that ApolOR-1 expressing cells were located beneath long sensilla trichodea. Moreover, double in situ hybridization studies elucidated that these cells were closely associated with cells expressing pheromone binding proteins. Together the results support the notion that the newly identified genes may encode pheromone receptors of the Antheraea species.

This work was supported by the Deutsche Forschungsgemeinschaft.

Majid Ghaninia¹, Mattias Larsson¹, Bill Hansson^1,2, Rickard Ignell¹

1 SLU, Dept. of Plant Protection Biology, 230 53 Alnarp, Sweden

2 Max Plank Institute for Chemical Ecology, Dept. of Evolutionary Neuroethology, DE-07745 Jena, Germany

majid.ghaninia@vv.slu.se

 Gas chromatography coupled single sensillum recordings from female yellow fever mosquitoes, Aedes aegypti.

 Female Aedes aegypti are vectors of dengue and yellow fever. The predominant cues driving the host-seeking behavior are odor volatiles. Olfactory stimuli are detected and discriminated by olfactory receptor neurons (ORNs) housed in sensory hairs, sensilla, which are located on the antennae as well as the maxillary palps. In a previous study we have shown that the antennal ORNs of female Ae. aegypti are divided into functionally different classes based on their response to a set of compounds that previously have shown to be either behaviorally and/or electrophysiologically relevant for Ae. aegypti and other mosquito species. The axons of these ORN classes project and terminate stereotypically into distinct antennal lobe glomeruli. Based on the results obtained in that study we believe that additional ligands remain to be discovered for some of the ORNs.

Through gas chromatographic-single sensillum recordings (GC-SSR) from antennal trichoid and intermediate sensilla (the most abundant sensillum types) of female Ae. aegypti we have been able to elucidate novel olfactory compounds to which the mosquitoes respond. We used headspace collections and solvent washes from biologically relevant sources such as different human body parts including e.g. foot and armpit. Through GC-SSRs we found several types of ORNs that responded strongly to individual compounds within the extracts. Through the GC-MS (mass spectrometry) technique we were able to identify the compounds that elicited response in the ORNs, e.g. heptanal, octanal, nonanal and decanal. These compounds have previously been reported to be present in human emanations. Sensitivity of the ORNs was then examined using different concentrations (ranging from 0.001 to 10%) of the synthetic compounds. At least in the case of heptanal and octanal receptor neurons displayed considerably lower response thresholds to these compounds than to the synthetic compounds previously used for their characterization. Dose-response experiment of the ORNs to other ligands is currently ongoing.

C. Giovanni Galizia, Ana F. Silbering

University of Konstanz , 78457 Konstanz , Germany

giovanni.galizia@uni-konstanz.de

Transforming olfactory information across neurons in the Drosophila antennal lobe

Odor information is encoded in the ensemble of activated olfactory sensory neurons (OSN). In insects OSNs project into the antennal lobe (AL) where axons of OSNs which express the same receptor protein are sorted to the same glomeruli. Within the glomeruli OSNs make synapses with local neurons (LN) and with projection neurons (PN). While most PNs arborize in single glomeruli and thus receive input from a single OSN class, LNs innervate many glomeruli. How the interglomerular LN network affects the activity transfer from OSNs to PNs is still not fully understood.

To investigate this network we performed in vivo recordings of odor-evoked activity patterns in different neuron populations in the AL of the fruit fly Drosophila melanogaster and pharmacologically blocked GABAergic transmission. Using the genetically engineered calcium sensor G-CaMP we recorded odor responses of OSNs, two types of LNs and PNs. We found that (1) the spatio-temporal patterns of odor responses were odor and concentration dependent and stereotyped across animals in all neuron populations. (2) While OSN and PN responses were clearly glomerular, LN responses were spatially structured but distributed over broad areas of the AL. (3) Response patterns and dynamics differed between the two types of LNs, suggesting a functional diversity of these neuron populations. (4) The gain of each glomerulus (relationship between OSN and PN responses) was concentration dependent, and the concentration-gain relationship was different for different odors in some glomeruli. (5) PN responses to odor mixtures could not be predicted from the OSN response alone, suggesting that interglomerular interactions shape PN responses. (6) Odor evoked PN responses are constitutively suppressed by fast GABAergic inhibitory input.

These results show that the transmission of olfactory information from OSNs to PNs is not a simple feed-forward process. Instead, odor processing in the AL involves the interplay of several LN networks, which shape the input-output relationship in an odor dependent manner.

Birgit Greiner¹, Nancy Butcher¹, Nicole Delaney¹, Robert M. Marshall², Yoshitaka Hamanaka¹, Ian A. Meinertzhagen¹

1Life Sciences Centre, Dalhousie University, Halifax, NS, Canada B3H4J1,

2Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305, USA;

birgit.greiner@dal.ca

Electron tomography of Drosophila T-bar ribbon synapses and vesicle fusion sites

All animal behaviour, from insect olfaction to human thought, is generated within circuits of neurons. The unique ability of neurons to send signals within these networks occurs at specialized contacts, or synapses. Neurotransmitter release at synaptic sites is thought to critically depend on the distinct protein architecture of synaptic organelles. The high depth resolution of electron microscope tomography provides first insights into the three-dimensional, macromolecular assembly of synapses. We examined the T-bar ribbon synapse of Drosophila from dual-axis tilt series and found anchoring sites between the T-bar base and presynaptic membrane, release sites with docked and fused vesicles, and a filamentous network interconnecting vesicles and tethering them to the synapse.  Our results provide new structural evidence of a direct linkage between vesicles and T-bar ribbons in Drosophila synapses, supporting the current idea of presynaptic densities being actively involved in efficient neurotransmitter release. As a way to identify the component proteins biochemically, we additionally analysed tomography datasets of silver-enhanced immunogold labelled tissue marking sites of antibody attachment to these organelles.

Support: Schrödinger Fellowship of the Austrian Science Fund (to B.G.); NIH grant EY-03592 (to I.A.M.).

Joaquín Goyret, Poppy Markwell and Robert Raguso

Cornell University, Dept. of Neurobiology and Behavior, Ithaca, NY 14853, U.S.A.

jg549@cornell.edu

Interaction of olfactory and visual cues in the foraging behavior of Manduca sexta

Claudia Groh & Wolfgang Rössler

University of Würzburg, Biocenter, Zoology II, Am Hubland, 97074 Würzburg, Germany

claudia.groh@biozentrum.uni-wuerzburg.de

Caste-specific development of the olfactory pathway in the female honeybee brain

The goal of this study was to investigate cellular components of neuronal plasticity during metamorphic adult development of olfactory neuropils in the brain of the honeybee Apis mellifera. We focused on the two female castes, queens and workers and investigated caste-specific differences in the formation of olfactory glomeruli in the primary olfactory centers (antennal lobe, AL) and synaptic complexes (microglomeruli, MG) in higher olfactory centers in the mushroom-body (MB) calyx.
The differences between workers and queens are not based on genetic differences, but rather reflect phenotypic plasticity caused by differential gene expression initiated via larval feeding and endocrine mechanisms. During postembryonic pupal metamorphosis, queens develop much faster than workers, are larger, live much longer, and are devoted to reproductive behavior. The developmental trajectory for female larvae is determined by the third day of larval development. From there on the developmental pathway is fixed for the queen or the worker phenotype.
To study ontogenetic plasticity at the level of the brain and to begin to unravel possible neuronal correlates for the obvious differences in behavior, we raised synchronized worker and queen brood under controlled conditions in incubators at temperatures within the natural, thermoregulated range (~35°C). Brains of worker and queen pupae were dissected at various stages for both immunofluorescent staining (anti synapsin-immunocytochemistry and f-actin labeling with phalloidin) and prepared for electron microscopy.
The results of immunolabeling experiments revealed substantial differences in the timetable of synaptic development at the level of AL glomeruli as well as MB-calyx MG within olfactory and visual input regions showing that ontogenetic plasticity among the female castes is reflected in the development of the brain. These findings were substantiated by analyses at the ultrastructural level. The results show that differences found in the synaptic organization of adult workers and queens (Groh et al. 2006, Brain Behavior & Evolution 68:1-14) are mainly caused by developmental plasticity during postembryonic pupal metamorphosis. Our findings on MB-calyx MG further indicate that structural plasticity affects both pre- and postsynaptic components. The underlying mechanisms and regulation of this remarkable developmental synaptic plasticity will be the focus future studies.

Yuqiao Gu and Jean-Pierre Rospars

UMR1272, Physiologie de l’Insecte : Signalisation et Communication, INRA, route de Saint-Cyr, F-78026 Versailles Cedex, France,

ygu@versailles.inra.fr

A dynamical model of the receptor potential and its electrode-recorded counterpart in moth pheromone-sensitive sensilla

In male moth, pheromone chemo-electrical transduction takes place in the outer dendrite of olfactory receptor neurons (ORNs) located in the long sensilla trichodea. Because of experimental constraints, intracellular recording of the receptor potential (RP) from the outer dendrite of ORNs in vivo is not feasible at the present time. The most common technique to record from an ORN is the tip-recording technique in which a capillary glass electrode is slipped over the cut end of the hair shaft. This electrode records the extracellular potential at the ORN tip, the so called sensillar potential (SP). It raises the question of knowing to which extent the SP reflects the RP. Furthermore, the ORN soma and the three auxiliary cells that wrapped the ORNs have intrinsic electrical properties (battery, resistance and capacitance) which influence both RP and SP. In this work, using a compartmental model of the sensillum we addressed several related questions: What are the dynamics of RP and SP? What is the relationship between them? How are they affected by the morphological and electrical parameters of the ORN dendrite and the electrical parameters of the ORN soma and ACs? Have these various parameters the same influence on RP and SP? What is the functional significance of the auxiliary cells? These questions are important not only for understanding the sensillum as a whole but also for analyzing the ORN properties from the experimental tip recordings.

In a previous work (Vermeulen and Rospars, 2001), a sensillum model was studied and analytical steady-state solutions given for the SP and RP, i.e. after sufficiently long constant pheromone stimulation. Now, we extend these results to take time into account. First, we showed that the RP and SP amplitudes increase with time and finally reach the values given by the steady-state equations over a wide range of stimulus-dependent conductance. The goodness of fit depends on the number of compartments used for modelling the outer dendrite (at least 10 are needed). Second, we studied the rising and falling times of RP and SP in response to conductance changes in the outer dendrite of same duration and different amplitudes, mimicking pheromone stimulation. We showed that, for realistic parameter values, the influence of auxiliary cells and the ORN soma on the dynamical properties are negligibly small. All response properties (relative amplitudes, half-rise time and half-fall time) of the RP at the base of the outer dendrite and the SP were found to be nearly the same. Third, we utilized our complete model of ORN transduction (see our communication on this topic) to investigate the effect of stimulation with pulses of pheromone. The realistic conductance changes obtained, with a slowly rising and falling “wave” of conductance involving several ionic channels, confirmed the results above.

In conclusion, an integrated description of the sensillum was obtained which is an indispensable tool for analyzing pheromone reception and transduction in vivo.

Harraca, V. & G uerin, P.M.

Institute of Biology, University of Neuchâtel, Rue Emile‑Argand 11, Case Postale 158, CH-2009 Neuchâtel  

Sensing resources in tsetse flies

The tsetse fly and the disease it transmits, African trypanosomiasis, cause a problem for human and agricultural systems affecting both man and his livestock. Because tsetse flies are ovoviparous, their populations can be controlled using visual traps. Attraction of these traps is improved by the addition of odours. Research to date on the sensory ecology of tsetse flies has been fragmented, concentrating on just a few products affecting some savannah species.

By coupling the tsetse fly antennae as a biological detector to the effluent of a high resolution gas chromatographic column, we could obtain a very precise quantification of the electrophysiological response thresholds of the tsetse antennae from different species to stimulation with test products.

Our data indicate that the tsetse spp. studied from different African habitats show a strong similarity in their olfactory sensitivities to different volatile compounds from different resources they exploit. Tsetse flies perceive and are able to respond to a much wider range of products compared to the few compounds used today for trapping.

The comparative approach used in this work with different tsetse species permits to extend our knowledge of this group of disease vectors, and allows us to understand how this group of ectoparasites is able to use certain host animal and plant metabolites to find a host and/or a refuge. The next aim of the work is to incorporate the products in visual traps used to control these important vectors of disease.

Sharon R. Hill1, Rickard Ignell1 and Bill S. Hansson^1,2.

1Division of Chemical Ecology, Plant Protection Biology, Swedish Agricultural University, Alnarp, Sweden. 2Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany.

Odour coding in antennal sensilla of the southern house mosquito, Culex quinquefasciatus.

The Culex pipiens species complex is comprised of important vectors of human pathogens. The southern house mosquito, Culex quinquefasciatus, is a vector for the causative agents of lymphatic filariasis and arbovirus encephalitis. Culex quinquefasciatus originated in Africa, but its populations have spread throughout the tropical and temperate regions of the globe. As the major vector of Wuchereria bancrofti, Cx. quinquefasciatus is responsible for the more than 100 million people infected, and the 45 million people seriously disabled, by this form of lymphatic filariasis worldwide.
As arguably the most important insect vectors of human disease, mosquitoes have come under the auspices of many control programs using a multitude of techniques, such as insecticides, repellents, insect growth regulators, biological agents and environmental manipulation. Each of these techniques has its merits, but also its disadvantages, and currently no single control method yields consistent and global success. It is chemicals that have proven, historically, to be the most effective control agents in use. The development, however, of resistant vector populations due to the indiscriminate use of insecticides remains a major constraint to the continued use of chemicals in this manner. This does not, of course, exclude all chemicals as useful control agents.
Mosquitoes use various volatile chemicals as behavioural cues, particularly in seeking for hosts and oviposition sites (e.g. Puri et al., 2006; Allan et al., 2006; McCall and Cameron, 1995; Mukabana et al., 2004; Meijerlink et al., 2001; Gieir et al., 1999). Human skin emanates with between 300 and 400 chemicals (Bernier et al., 2000) but only a fraction of these volatiles have proven attractive to mosquitoes (e.g. Puri et al., 2006; Allan et al., 2006; Meijerlink et al., 2001).

Irene Ibba¹, Bill Hansson² , AnnaMaria Angioy¹, Teun Dekker³

¹Department of Experimental Biology, Section of General Physiology,
University of Cagliari, Italy.
² Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Jena,Germany.
³Division of Chemical Ecology, Swedish University of Agricultural Sciences, Alnarp, Sweden.

ibba@unica.it

Superspecialism of the olfactory system of Drosophila sechellia

The melanogaster subgroup comprises several closely related species, most of which appear generalistic in their food choice. Within the simulans-clade, however, there are two specialist species, Drosophila sechellia and D. mauritiana. Whereas D. simulans is a cosmopolitan human commensal, D. sechellia is endemic to the Seychelles archipelago and strictly specialized on the fruit of the rubiaceous scrub Morinda citrifolia. The peculiarity of this fruit is that is toxic for other drosophilids. The main volatile compounds present in ripe M. citrifolia fruit headspace are medium chain aliphatic acids and esters thereof. In a previous study we showed that there are shifts at various levels in the olfactory circuitry of D. sechellia related to its sole food source and oviposition site (Dekker et al., 2006). In the antennae the number of ab3 sensilla, which house methyl hexanoate (MeHex) sensitive neurons, are 3 times overexpressed, whereas ab1 and ab2 sensilla are numerically reduced by 65% and 95-100%, respectively. Concordantly, there is a 2.9 times voluminar increase of the glomeruli receiving axonal projections of ab3 inhabiting neurons, DM2 and the VM5d. These observed changes are clearly reflected in D. sechellia behaviour.

In the current study we tested if other minor of Morinda fruit volatiles could be also relevant for the specialist fruit fly to find its food source. In a careful screen we identified another compound, 2-heptanone, which elicited strong antennal responses in D. sechellia. Using gas chromatographic-single receptor neuron recording technique (GC-SS) we found that this compound triggered a response predominantly on the ab3B neuron, which coinhabits the same sensillum as the neuron responding to MeHex. 2-Heptanone elicited similarly strong attraction in D. sechellia as MeHex.

D. sechellia was attracted to all concentrations of 2-heptanone tested from pure to 10 - 8 , whereas the generalist D. melanogaster showed attraction only for intermediate concentrations. Thus, D. sechellia, through overexpression of one sensillum type, has evolved a set of macroglomeruli tuned to its sole host, Morinda fruit. We are currently investigating in detail through backfills how the ab3 overexpression has affected axonal targeting of ORNs in D. sechellia compared to D. melanogaster.

Rickard Ignell¹, Dick Nässel², Åsa Winther²

1 Division of Chemical Ecology, Swedish University of Agricultural Sciences, Box 44, Alnarp, Sweden.

2 Department of Zoology, Stockholm University, Stockholm, Sweden

Rickard.ignell@ltj.slu.se

Peptidergic signalling in the antennal lobe modulates olfaction in Drosophila

Tachykinin-related peptides (TKRPs) have been isolated from several insect species. Based on immunocytochemical data these peptides are predominantly expressed in interneurons within the CNS suggesting a role in neuromodulation. In the primary olfactory system, the antennal lobe ( AL), TKRPs are expressed in a subset of local interneurons (LNs). Through neuron-specific interference with TK signalling in the AL of Drosophila we have studied how these peptides modify the behavioural response to specific odorants.

We have created Drosophila TK (DTK) deficiency in two different partly overlapping populations of LNs. GAD1 is the biosynthetic enzyme for GABA, and when using the GAD1-Gal4 we were able to drive expression in a large population of LNs, since many LNs are GABAergic. GAD1-Gal4 drives expression in several LNs that also express DTK. There is no co-expression of GAD1-Gal4 and other DTK expressing neurons in the brain. We have also tested elav-Gal4;UAS-DTK-RNAi flies, these flies are virtually DTK null flies. When driving the DTK-RNAi construct with GAD1-Gal4 and elav-Gal4 flies were less attracted to one of three tested odors.

To study the role of the DTK receptor (DTKR) we used Gal4-lines that drive expression in the ORNs, LNs and in the PNs to over-express or down-regulate the receptor. DTKR over-expression in the ORNs, using Or83b-Gal4, resulted in flies more repelled (or less attracted) to two of three tested odors. Over-expression of DTKR in a subpopulation of the LNs, using GH298-Gal4, also produced flies more repelled or less attracted to two of three tested odors. Flies with DTKR deficiency in the ORNs and LNs were more attracted to two of three odors tested. Over-expression of DTKR in projection neurons, using the GH146-Gal4 line, did not produce an effect.

These results suggest that DTKs and the DTKR are involved in the modulation of the response to specific odors at the levels of ORNs and LNs, possibly by modulating the inhibitory activity of GABA.

David Jarriault, Jean-Pierre Rospars and Sylvia Anton

INRA Versailles UMR1272 Physiologie de l’Insecte : Signalisation et Communication F-78026 Versailles, France

djarriault@versailles.inra.fr

Sex pheromone coding in projection neurons in the antennal lobes of Agrotis ipsilon

For finding a mating partner, moths rely on pheromonal communication. Released in very low amounts, female pheromones are used by males to identify and localize the females. Interestingly, in the noctuid moth, Agrotis ipsilon, males, depending on their age, respond differently to the pheromonal blend emitted by the females. Electrophysiological studies showed that the sensitivity of central olfactory neurons was higher in 5-days-old than in 1-day-old males (Anton and Gadenne, 1999). Here we investigated how the qualitative, quantitative and temporal aspects of the pheromonal signal are encoded in the central olfactory neurons.
Pheromones are detected by olfactory receptor neurons in the antennae and information is transmitted to a specialized structure of the antennal lobes: the macro glomerular complex. In antennal lobes, integration of the pheromone signal is performed by interactions between local and projection neurons. The output signal is transmitted to the protocerebrum by projection neurons.

Castelli Jérémy, Colson Violaine, Michel Renou et Rochat Didier

UMR1272 : Physiologie de l’insecte : Signalisation et communication, INRA, F-78026 Versailles, France ;

rochatd@versailles.inra.fr

Mating modifies responsiveness to aggregation pheromone in Red Palm Weevil (Coleoptera Curculionidae)

Responses to pheromones can change with physiological status in insects. In short lived species (a few days), particularly moths, responsiveness to sex pheromone reaches maximum soon after emergence and decreases just after mating. The male can recover a good responsiveness afterwards and be able to mate again. In long lived insects (several weeks or months) and for beetles responding to male aggregation pheromone very little is known about the effect of mating on the responsiveness to pheromones. This is particularly the case for the Red Palm Weevil, a major invasive palm pest in Middle-East and Europe.

Mass trapping using synthetic aggregation pheromone + host plant odours capture 2/3 females, mostly mated, and 1/3 males. The individual responsiveness in natural populations is unknown. In order to determine whether mating could change the response to the male aggregation pheromone we measured the response of male and female RPW maintained singly from emergence and provided three times with a sex partner for 1 day (every 2 weeks) under laboratory conditions. Control insects were provided with a conspecific of the same sex. The response to 100 ng of pheromone with or without 1µg of ethyl acetate (EtAc; a known host plant synergist) by each individual were recorded in a 4-armed olfactometer, 10 times successively at day -1, 1, 8, 14, 16, 23, 30, 32, from 39 and 46 days from the first pairing. Weevils were 10-day old at first test. Whatever the age, mating and pairing status, males did not respond to 100 ng pheromone either alone or plus EtAc. On the contrary, virgin 10-days old females (day -1) responded intensely to pheromone alone but not to pheromone + EtAc. The response maintained till the age of 56 days in the virgin control females paired with other females. After the first mating (day 1) established ly responded to this mixture on an average after the first mating (day 1) till the age of 56 days. Responses to EtAc alone are currently measured. Results are discussed in ecological and applied perspectives.

Blanka Kalinová¹, Jan Šobotník¹, Vladimír Ptáček⁴, Irena Valterová¹

1Institute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech Republic, Prague,
4Department of Animal Physiology and Immunology, Faculty of Science, Masaryk University, Brno

blanka@uochb.cas.cz

Age-dependent structural and physiological changes of male labial gland in Bombus terrestris

Bombus terrestris is a common European bumblebee. Like other bumbelbee species, males scent-mark various substrates on their flight routes in order to attract conspecific queens to mate with. The male marking pheromone is synthesized in cephalic part of paired labial gland and consists of complex blend of numerous compounds (esters, alcohols, terpenes, and hydrocarbons) mixed in a species-specific mixture. In spite of accumulated information about the pheromone composition, it is still not clear how the pheromone blend is formed and which compounds are essential to carry the pheromone function. Neither is known whether all compounds of the pheromone are synthesised in the gland or whether some of them are formed from the lipid precursors transported from the fat body. To examine some of these possibilities, we studied age dependent ultastructural and physiological changes associated with labial gland ontogeny in Bombus terrestris. Specifically: i) using optical and transmission electron microscopy, we studied gland ultrastructure and morphology, ii) using gas chromatography we studied age-dependent changes in pheromone composition, iii) using EAG and GC-EAD we determined which compounds of the blend elicit queen antennal responses and how EAG activity correlates with age-dependent changes in gland ultrastructure and pheromone composition.
Our data show that cephalic part of labial gland in B. terrestris consists of numerous acini and connecting ducts. The acini are formed by secretory cells, site of secretion production, and a lumen, where the secretion is stored. The pheromone production starts immediately after eclosion, reaches its maximum at the age of 7 days and lasts up to ten days at longest. The termination of pheromone production is associated with the appearance of apoptic cells in the gland. The metabolism of secretion cells and male primary metabolism are interconnected shortly after eclosion. Prominent structures of active secretory cells are smoth endoplasmic reticulum (SER) and Golgi apparatus. The production of Golgi secretion peaks in 1 day old glands, while the maximal rate of SER production occurs about 1-2 days later. Age-dependent gland structural changes are associated with quantitative and qualitative changes in pheromone composition. The volume of secretion stored in acinar lumen reaches its maximum in 5-7 days old males then decreases and reached zero in older individuals. 2,3-dihydrofarnesol, the main pheromonal component of the male marking pheromone, appears in 1-day old glands and reached maximum at the age of 5-7 days, then its content in the gland gradually decreases. Queen antennae respond to extracts of labial glands of all ages. The maximum sensitivity was observed in response to gland extracts of 4 days old males. Extracts of older glands gradually lose their effectiveness. GC-EAD experiments show that several components of the gland extract elicited queen antennal responses. This suggests that the pheromone function is carried out by specific multicomponent blend.

Blanka Kalinová, Robert Hanus, Jan Šobotník, Anna Luxová, Pavel Jiroš, Irena Valterová¹

1Institute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech Republic, Prague,

blanka@uochb.cas.cz

Sex pheromone in Prorhinotermes simplex (Isoptera: Rhinotermitidae) identified using GC-EAD

Termites generally reproduce during/and or after dispersal flights of alates that leave the mother colony and initiate new colonies from bisexual pairs. Depending on the species, pairing is mediated by females or males and different glands are involved in sex pheromone secretion. With the exception of sporadic behavioral data describing some aspects of premating behavior, nothing was known about sexual communication in Prorhinotermes simplex. This is why we performed behavioral and analytical experiments using our swarming laboratory colony of P. simplex to figure out 1) how the premating behavior is mediated, 2) what glands are involved in sexual communication and 3) what is the chemical nature of compounds involved. Laboratory experiments were performed in Petri dishes (15 cm dia), bottom of which were covered with moisturized filter paper. Behavioral observations were performed under ambient laboratory temperature with unspecified time period. Alates of both sexes were allowed to respond to a) conspecifics (glued in the centre), b) to excised male or female tergal glands and c) to extracts of male or female tergal glands. Chemical analysis was performed using GC-EAD with isolated male or female antennae as biological detectors and GCxGC-MS.
Behavioural experiments confirmed that pairing in P. simplex is initiated by females and determined that tergal glands (located on posterior abdominal tergites) are involved. The secretion contains volatile activity inflicting male behavior from a distance. Males were activated shortly after introduction into the Petri dish and upon activation exhibited typical searching behavior with their antennae rapidly moving. However, the dispersal of tergal gland secretion can be facilitated when necessary. In several experiments we observed female calling in a posture characterized by lifted and protruded abdominal tip with tergal glands fully exposed. Not only females but also excised tergal glands and/or gland extracts activated males. EAG experiments with isolated antennae confirmed that extracts of female tergal glands possess high EAG activity against male antennae. GC-EAD analysis showed consistently the presence of one EAD active compound characterized by Kovats retention indice RI = 1530 (e.g. eluting after synthetic standard saturated aliphatic hydrocarbon with 15 carbon atoms). Two dimensional GCxGC-MS analysis of the area of interest provided evidence of many sexually isomorphic sesquiterpenoids (contaminants from frontal glands) but no female specific compound. However, the retention behavior similar to our EAD active compound, possess dodecatrienol and dodecadienol, compounds identified as sex pheromones in related termite species (P. spiniger, R. santonensis, R. lucifugus grassei). Careful comparison of GC-EAD retention behavior and EAD activity of (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol and (3Z,6Z)-dodeca-3,6-dien-1-ol (obtained thanks to courtesy of Dr. Bordereau) confirmed the identity of the compound from tergal gland secretion as (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol. Further behavioral analysis show that (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol elicited similar pre-mating behavior as females or tergal gland extracts.

Karl-Ernst Kaissling

Max-Planck-Institut fuer Verhaltensphysiologie, Seewiesen, 82319 Starnberg, Germany

Kaissling@orn.mpg.de

Do receptor molecules function as odorant deactivating enzymes?

Odorant deactivation was postulated for odor sensors working as flux detectors (Kaissling 1998a). Modeling revealed that this still hypothetical process must be responsible for the response kinetics of the olfactory receptor cell. The well-known enzymatic odorant degradation proceeds too slow as observed in vivo. Two mechanisms of odorant deactivation were discussed previously, with deactivation catalysed either by the receptor molecules (model R; Kaissling 1998a, b) or by the hypothetical enzyme N (model N, Kaissling 1998a, 2001). The model R was suggested based on the redox shift of the PBP observed in homogenates of olfactory hairs (Ziegelberger 1995). Model R is supported by recent studies showing that properties of the receptor potential kinetices were transfered together with olfactory receptor genes expressed in an “empty” olfactory neuron (Dobritsa et al., 2003; Hallem et al., 2004, Syed et al. 2006). The model N was prefered by Kaissling (2001) since the putative receptor blocker DTFP ( decyl-thio-trifluoro-propanone, Pophof 1998; Pophof et al. 2000) did not seem to interfere with the odorant deactivation. A quantitative comparison of both models reveals that the measured rise of the receptor is simulated well by model R whereas model N produces a slower rise (with 30-80% larger half times). Therefore it seems more likely that the receptor molecules catalyse the odorant deactivation. - The model was computerized by J. Thorson, Oxford.

Ziegelberger (1995) Eur. J. Biochem., 232, 706-711. Kaissling KE (1998a) Chemical Senses, 23, 99-111

Kaissling KE (1998b) Chemical Senses, 23, 385-395. Kaissling KE (2001) Chemical Senses, 26, 125-150

Pophof (1998) J. Comp. Physiol. A, 183, 153-164. Pophof et al. (2000) J. Comp. Physiol. A,186, 315-323. Dobritsa et al., (2003)Neuron37, 827-841. Hallem et al., (2004) Cell117, 965-979. Syed et al. (2006) PNAS 103, 16538-16543

Zsolt Kárpáti^1,2*, Teun Dekker^1*, Bill S. Hansson^1,3

1Division of Chemical Ecology, Swedish University of Agricultural Sciences, PO Box 44, SE-230 53, Sweden teun_dekker@hotmail.com

2Plant Protection Institute of Hungarian Academy of Sciences, PO Box 102, H-1525, Budapest, Hungary zkarpati@hotmail.com

3Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology
Hans-Knoell-Strasse 8, D-07745 Jena, Germany

bill.hansson@vv.slu.se

* Both authors contributed equally to the work.

Putative swap of pheromone receptors along sensory neurons underlies existence of reversed pheromone blend preference in Ostrinia nubilalis males

The European corn borer (Ostrinia nubilalis Hbn) (Lepidoptera: Pyralidae) has two pheromone strains which is a good oportunity to study the evolution of sexual communication in insects. Two strains, the E and the Z strain, do not interbreed freely in sympatry, as they produce and prefer a reversed ratio of the pheromone components E11-14:OAc and Z11-14:OAc. The female pheromone production is oligogenic, whereas the male behavioral preference is controlled by a single autosomal gene. Here we studied how the single gene can swich the preference of males.
Intracellular recording and activity dependent anterograde backfills were used to identify the structure and function of E- and Z-strain males olfactory receptor neurons (ORNs) and the projection neurons (PN) in the primary olfactory neuropil, the antennal lobe (AL). Physiologically well-characterized neurons were filled with neurobiotin tracer and their dendritic arborization patterns were reconstructed using confocal microscopy in alfa-synapsin overview stained ALs. The fine structure and position of the glomeruli was analysed with AMIRA reconstruction.
The males and females ALs have ~66 glomeruli in both strains The male specific macroglomerular complex (MGC) situated at the entrance of the antennal nerve, is morphologically similar in the two strains, but differs substantially from earlier reports. It consists three glomeruli, two large, highly convoluted, variable in shape and dimension, and a smaller posterior disc-shaped glomerulus. In females two enlarged glomeruli (LFG) were found at the same position as in males. ORNs show uniglomerular innervation pattern in the MGC. The two large MGC glomeruli receive input from E11-14OAc and Z11-14OAc responding olfactory receptor neurons, whereas the smaller glomerulus receives input from antagonist responding neurons (Z9-14OAc). The intracellulary well-characterized PNs show the same characteristic arborization as the ORNs in the AL. These demonstrate that the two large MGC glomeruli tuned to the two major pheromone components, and the small, disk-shaped answers to the antagonist. In the Z-strain Z11-14:OAc-specific PNs arborize in the larger, medial MGC glomerulus, whereas E11-14:OAc-specific neurons innervate the medial, smaller one. In the E strain this topology is reversed.

We postulate that the E and Z responding ORNs have exchanged the expression of their olfactory receptors, whereas the ORN as well as the PN innervation patterns in the AL has been conserved between the two strains.

Markus Knaden, Kathrin Steck, Bill Hansson

Max-Planck-Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Hans-Knöll-Strasse 8, 07745 Jena, Germany

mknaden@ice.mpg.de

Do ants use olfactory landmarks?

Korbu O.V., Barabanova A.A., Zhukovskaya M.I

Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences
44 Thorez Ave, Lab. 25, 194223, Saint-Petersburg, Russia

plecotus@inbox.ru

Context dependent hexan-1-ol responses in Periplaneta americana cockroaches

Hexane-1-ol is a general odorant for Periplaneta americana cockroaches perceived by individuals of both sexes. Despite this volatile is a common component of green leaves and fresh fruits consumed by cockroaches; it is shown to be a repellent at least for adults. Electrontennogramm (EAG) recorded in response to hexanol depends on mating status of males: freshly mated males had decreased EAG in compare with virgin cockroaches exposed to calling females and exhibited wing-raising behavior but not allowed to mate. Antennal responses are presumably altered with octopamine, because the drug application was shown to decrease EAG of virgin and freshly mated males but not one of virgin males exposed to calling females. EAG responses of females were decreased by octopamine as well, but mating status did not have any influence on them.

Jürgen Krieger, Ewald Gosse-Wilde, Thomas Gohl, Elisabeth Bouché and Heinz Breer

University of Hohenheim, Institute of Physiology, Garbenstr. 30, 70599 Stuttgart, Germany

krieger@uni-hohenheim.de

Molecular basis for pheromone reception by antennal neurons of Heliothis virescens

The remarkable ability of male moths to detect female-released sex-pheromone with high sensitivity and selectivity is mediated by specific sensory neurons housed in long sensilla trichodea on the antenna. Females of the tobacco budworm Heliothis virescens use a multicomponent blend to attract males and in males electrophysiological studies have assigned identified pheromonal compounds to three different types of sensilla trichodea. This specific responsiveness implies that sensory neurons in the sensilla types express distinct receptors. We have identified candidate pheromone receptors of Heliothis virescens, which form a relatively conserved group of moth olfactory receptors. By in situ hybridisation the receptor types could be allocated to sensory neurons housed in long trichoid sensilla surrounded by cells expressing pheromone binding proteins (PBPs). Immunohistochemical approaches visualized the receptor protein in the dendritic processes of the antennal neurons. Functional analysis of heterologously expressed receptors stimulated with pheromonal compounds solubilized by means of DMSO revealed that distinct receptor types responded to several compounds. Substituting the organic solvent with pheromone binding proteins to solubilize the hydrophobic pheromone compounds revealed an increase in sensitivity and specificity; it was found that cells expressing HR13 responded in the presence of HvirPBP2 specifically to the main component of the sex pheromone blend. These data provide further evidence that the combination of a distinct receptor type and binding protein forms the basis for the specific responsiveness of moth antennae to distinct pheromone components.

This work was supported by the Deutsche Forschungsgemeinschaft.

Thomas Kröber¹, Patrick Guerin¹

1 University of Neuchâtel, Dept. of Animal Physiology, CH – 2007 Neuchâtel, Switzerland

thomas.kroeber@unine.ch

A ligand assay for studies on Anopheles gambiae olfaction

Jean-Marc Lassance¹ and Christer Löfstedt¹

1Department of Ecology, Lund University, Sölvegatan 37, SE-22362 Lund, Sweden.

jean-marc.lassance@ekol.lu.se, christer.lofstedt@ekol.lu.se

Male pheromone mediates female preference and contributes to female pheromone evolution in a moth.

One possible explanation for the evolutionary success of Lepidoptera, the second most species-rich insect order, may be the efficiency of their mate-signalling system. In moths species-specific sex pheromones emitted by females mediate long-range mate-attraction and serve as a primary isolating factor. In some species, males also possess pheromones that are released during courtship. The evolution of male pheromones has been suggested to constitute an adaptative response in species in which the probability of mating mistakes is high. Female mate discrimination via chemical cues emitted by males would then act as a second reproductive barrier. Furthermore, male pheromones have been proposed as a possible trait used by females to assess male quality. We report on the role of scents released during courtship by males of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae). We find that the composition of the male pheromone in O. nubilalis varies in an age-dependent manner and in choice experiments females show preference for older males. Whereas a vast majority of male pheromones identified so far are derived from plant compounds, our data implies that the male pheromone components of corn borersarebiosynthesized de novo by an enzymatic machinery similar to that found in female pheromone glands. Our results reveal that the evolution of specific male and female pheromones in corn borers is an intricate case of adaptive sex-specific transcription/translation of essential pheromone production genes.

Walter S. Leal

Maeda-Duffey Laboratory, Department of Entomology, University of California-Davis
1 Shields Ave., Davis CA 95616 USA

wsleal@ucdavis.edu

Reception of Semiochemicals in Lepidotera and Diptera

Insects utilize semiochemicals and species-specific pheromones to communicate in a noisy environment. In intraspecific chemical communication, emitters produce low amounts of semiochemicals in order to minimize cost of production and eavesdrop. Receivers, on the other hand, have evolved detectors with remarkable sensitivity in order to deal with the low amounts of signal. These detectors excel not only in sensitivity but also in selectivity and dynamic properties. The large number of semiochemical-detecting sensilla distributed over the surface of the insect antennae and palps most likely contribute to the sensitivity of the insect olfactory system. While odorant receptors (ORs) seems to play a pivotal role in the selectivity of the insect olfactory system, odorant-binding proteins (OBPs) may contribute to sensitivity and dynamics (and perhaps even selectivity) of the olfactory system by rapidly capturing molecules reaching the port of entry of sensilla, the pore tubules. By pH-mediated conformational changes, OBPs deliver odorants to membrane-bound ORs. At low pH - as on the surface of dendrites - C-terminal alpha-helices are formed in Lepidoptera OBPs, whereas in Diptera OBPs hydrogen bonding and salt bridges are disrupted. The ligands are delivered by intramolecular occupation of the binding pocket in the former and “deprotection” in the latter. Ongoing research in our laboratory suggests that odorant-degrading enzymes (ODEs) are a key component of signal inactivation. We are now exploiting our current understanding of the molecular basis of insect olfaction in order to expedite the discovery of insect semiochemicals by using a reverse chemical ecology approach.

Supported by the NIH-National Institute of Allergy and Infectious Diseases (1U01AI058267-01), the National Science Foundation (NSF) (0234769), and the USDA – National Initiative Competitive Grants Program (2003-35302).

Philippe Lucas and Adeline Pézier

UMR Physiologie de l’Insecte : Signalisation & Communication, INRA, Route de Saint-Cyr,
F-78026 Versailles cedex, France

plucas@versailles.inra.fr

Calcium activates a chloride current in moth olfactory receptor neurons

The response of moth olfactory receptor neurons (ORNs) involves the production of inositol trisphosphate (IP3) and diacylglycerol (DAG) followed by an increase in intracellular Ca2+ concentration. Consequences of the Ca2+ rise remain elusive. We therefore studied Ca2+-activated currents with the whole-cell patch-clamp technique on cultured ORNs of the Noctuid moth Spodoptera littoralis. The Ca2+-activated current was not modified when intracellular or extracellular cations were replaced with choline, ruling out that it is cationic. By contrast, the Ca2+-activated current was inwardly rectified in low external Cl- and outwardly rectified in low internal Cl-. Moreover, applications of Cl- channel blockers, NPPB and flufenamic acid, reversibly blocked Ca2+-activated currents. These findings support the hypothesis of the expression of Ca2+-gated Cl- channels in insect ORNs. Our results allow us to predict that a Ca2+ increase via its influx through DAG-gated channels and/or an IP3-dependent Ca2+ release from intracellular stores activates a Cl- conductance. Since the equilibrium potential for Cl- remains to be determined, single sensillum recordings were undertaken to determine whether the Cl- flux is involved in ORN depolarization or repolarization.

Marjorie Liénard¹, Tomas Johansson¹, Jure Piškur² and Christer Löfstedt¹

1 Department of Ecology, Lund University, Sölvegatan 37, 22362 Lund, Sweden
2 Department of Cell and Organism Biology, Lund University, Sölvegatan 35, 22362 Lund, Sweden

marjorie.lienard@ekol.lu.se

Functional analysis of ∆11-desaturase genes involved in the biosynthesis of conjugated pheromone components in primitive and advanced moth species.

Gabriel Manrique¹, Ana Cristina Renna de Vitta² , Gina Barcelos Pontes², Claudia Andrea Zacharias¹, Marcelo Gustavo Lorenzo²

1 Contributed equally to this work

1 Insect Physiology Laboratory. Biodiversity and Experimental Biology Departament, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II, Ciudad Universitaria, C1428EHA, Buenos Aires, Argentina.

2 IRR-FIOCRUZ Laboratory of Triatomine and Epidemiology of Chagas Disease, Av. Augusto de Lima 1715, 30.190-002, Barro Preto, Belo Horizonte, Minas Gerais, Brazil.

marcelo@cpqrr.fiocruz.br

Chemical Communication in Triatomine Bugs’ Sexual Context

Triatomine bugs act as vectors of Trypanosoma cruzi, a flagelate parasite that is the etiological agent of Chagas disease. This disease affects more than 16 million people in the Americas. Our work intends to clarify several aspects of the sexual behaviour of three species of relevant bugs, in order to allow the potential development of new tools for their control.

For this, we have developed assays to characterize several phases of the sequence of behaviours that mediate encounters between individuals of both sexes. Furthermore, we have identified diverse chemical compounds produced by the metasternal glands (MG) of Triatoma infestans, Triatoma brasiliensis and Rhodnius prolixus and evaluated their potential role as sexual communication signals. We have demonstrated that males of R. prolixus show a directional take-off response against female odour ladden airstreams. Moreover, olfactometer experiments with the related species T. brasiliensis have shown that males are attracted by female odour ladden airstreams and that metasternal gland (MG) odours mediate this behaviour. In addition, we have performed SPME experiments that demonstrate that MG odours are emitted preferentially by R. prolixus females mostly during the dark phase. Mating pairs of T. infestans and R. prolixus, known to promote the aggregation of other males around them, also emitted some of these odours. Moreover, the occlusion of female MGs abolished male aggregation in T. infestans. Finally, experiments with T. infestans and R. prolixus showed that mating occurrence decreases drastically after the occlusion of MGs openings.

For the first time, the volatile products produced by the MGs of triatomine vectors were identified. We also showed for the first time in bugs that male flight can be triggered by female odours and that MG odours mediate the attraction of males to females. We expect to perform GC-EAD studies in order to determine the capability of the different compounds to promote responses in the antennae of males and EAG experiments to detect thresholds and dose-response relationships. The active compounds produced by each species will also be analysed behaviourally to understand their role in the subsequent phases of the sexual behaviour of bugs. Finally, we intend to evaluate their potential use as baits for the detection or capture of these vector insects, to help avoiding the transmission of Chagas disease.

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

UMR Physiologie de l’insecte : signalisation & communication, INRA, Route de Saint-Cyr, F-78026 Versailles cedex, France

jacquin@versailles.inra.fr

Evidence for a circadian clock in moth antennae

Circadian rhythms are observed in pheromonal-related behaviors in moth: females emit sex pheromones and males are attracted by these pheromones in a rhythmic fashion. In the antennae of the noctuid moths Mamestra brassicae and Spodoptera littoralis , we identified the clock genes, period (per),cryptochrome1 (cry1) and cryptochrome2 (cry2). Their expression patterns were established using in situ hybridization, revealing that the transcripts were expressed in the olfactory sensilla. Using quantitative real-time PCR ( qPCR), per, cry1 and cry2 transcripts were found to cycle in antennae as well as in brain, in light-dark cycle conditions and in constant darkness. Additionally, temporal expression patterns of genes encoding antennal proteins putatively involved in pheromone reception in S. littoralis were analyzed in qPCR assays: one Pheromone-Binding Protein , one Olfactory Receptor and one Odorant-Degrading Enzyme from the esterase family.

Together, these results suggest the presence of a peripheral circadian clock in moth antennae. However, its involvement in the circadian regulation of pheromonal communication remains to be established.

Natalie E. Miller

University of South Carolina, Department of Biological Sciences, Columbia, South Carolina, USA

millern@biol.sc.edu

Investigation of the mechanism of sensillar development

It is important for all organisms to be able to sense their environment in some way.  In olfaction, odor molecules bind to specific receptors on neurons and a signal is sent to the central nervous system, which interprets the signal and decides the correct response to the odorant.  The ability of an organism to respond to different odorants depends in part on the specificity of the receptor molecules that detect odorants.  In insect antennae have evolved specifically for detecting sensory information, including odorants.  Antennae detect odorants using sensilla, sensory structures located on the surface of the antenna.  Drosophila adults have three types ofolfactory sensilla that differ in morphology and function – sensilla coeloconica, basiconica, and trichoidea.  

A group of basic helix-loop-helix (bHLH) transcription factors termed proneural genes is required for specification of the neural precursor cells that give rise to each of these sensillar types.  In this study, a proneural gene was cloned from Manduca sexta antennal cDNA.  The expression pattern of this gene was analyzed using PCR and compared to previous studies in Drosophila.  The goal of this study is to learn more about how sensilla are formed in order to determine whether it is via mechanism conserved within insects.

Sarah E. Miller, Kate-Lyn Best and Linda M. Kennedy

Lasry Bioscience Center, Clark University, Worcester MA 01610, USA

lkennedy@clarku.edu

Blocking the IP3 pathway permits typical sugar receptor cell firing, but not proboscis extension, in response to Na-saccharin in the blowfly, Phormia regina

Since blowflies give no behavioral (PER) and little ‘sugar cell’ responses to artificial sweeteners (Higgins & Kennedy, 2002), it has been thought that they lack receptor mechanisms for these stimuli.  Yet the ‘fifth cell’ responds to Na-saccharin (Na-S), and there is reciprocal inhibition for Na-S and sugar (Liscia et al., 2004).  We studied sugar cell and PER responses to Na-S during inhibition of IP3 mediated adaptation with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and Na-deoxycholate (H7). 
Action potential responses to a concentration series of Na-S with (+) and without (-) H7, and of sucrose, were tip-recorded from single sensilla in isolated proboscises.  Sugar cell firing to sucrose and to Na-S +H7 gave typical concentration response curves, but firing rates to Na-S –H7 were low and atypical.  At most concentrations, firing to Na-S +H7 was greater than firing to Na-S –H7 (p< 0.05, Mann-Whitney).  Sugar cell adaptation rates decreased as sucrose concentration increased, but increased as Na-S +H7 concentrations increased, and did not change with Na-S –H7 concentration. While the highest firing rates to Na-S +H7 were to Na-S 7.4 mM +H7, they remained lower than those to sucrose 50 mM (p=0.01, Kramer).  Fifth cell firing gave similar response curves for Na-S + and Na-S –H7.  Flies that gave a PER to sucrose 50 mM did not to Na-S 7.4 mM +various H7 concentrations. 
The results indicate an excitatory receptor mechanism for Na-S in the sugar cell and suggest that sugar cell firing to Na-S is suppressed by a rapid onset of IP3 mediated adaptation.  It seems unlikely that IP3 mediates either transduction or adaptation in the fifth cell.  Further behavioral tests are in progress to determine whether the lack of PER resulted from inadequate sugar cell firing, inhibition via fifth cell firing, or both.            

Minoli SA, Jaubert S, Tagu D, Anton S

INRA-UMR Physiologie d'Insectes: Signalisation et communication - Route de St Cyr, 78026 Versailles cedex France.
INRA Rennes, ULR 1099 BiO3P INRA/Agrocampus, BP35327, 35657 Le Rheu cedex France

minoli@bg.fcen.uba.ar

Morphology of an aphid brain: comparison between asexual and sexual morphs

Aphids are important crop pests, inducing major losses in crop yields by sucking plant sap and transmitting virus diseases. Part of their reproductive success lies in their capacity to change from fast viviparous clonal reproduction during suitable environmental conditions (spring and summer) to oviparous sexual reproduction in winter. This switch has been shown to be triggered mainly by the photoperiod, particularly by the day-length. However, although in the aphid Megourae viciae the sexual phenotypic plasticity (or polyphenism) was found to be partially controlled by a group of neurosecretory cells located within the pars intercerebralis of the protocerebrum that are directly modulated by the light, not much information is available about the general morphology of the brain of these insects. The pea aphid, Acyrthosiphon pisum, serves as a model insect to understand the mechanisms of polyphenism. Its genome has recently been sequenced and the role of different genes in morph changes will be examined using microarray and gene-expression studies. To be able to interpret data from in situ hybridisation experiments, a profound knowledge of the brain anatomy is essential. The objective of this work is to describe and compare the brain structure of the different asexual and sexual phenotypes of the pea aphid A. pisum: parthenogenetic females, sexual females and males. Different histological approaches, including classical overview staining, antennal backfills and immunocytochemical methods, are being applied to describe brain structures and possible differences between phenotypes. Here, we present our first results that for the first time provide a detailed description of the neuroanatomy of an aphid brain. Structures such as the protocerebrum, antennal lobes and nerves, mushroom bodies, pars intercerebralis, central complex as well as several serotoninergic neurons are revealed by the different staining methods.

Newcomb, R.D.¹, Kiely, A.^1,2, Jordan, M.D.^1,2, Begum, D.^1,2, Anderson, A.R.⁴, Law, A.^1,2, Carraher, C.J.¹, Authier, A.¹, Christie, D.², Warr C.³, Trowell, S.C. ⁴, Kralicek, A.V.¹,

1. Molecular Olfaction Team, Health and Food Group, HortResearch, Auckland, New Zealand
2. School of Biological Sciences, University of Auckland, Auckland, New Zealand
3. School of Biological Sciences, Monash University, Melbourne, Australia
4. Food Futures National Research Flagship and CSIRO Entomology, Canberra, ACT, Australia

rnewcomb@hortresearch.co.nz

Characterisation of odorant receptors from moths and flies

Olfaction is a critical sense for insects, which use it to find mates and to locate host plants.  Central to insect olfaction is a family of odorant receptors that transduce the binding of volatile odorants into changes in the membrane potential of the olfactory sensory neurons.  Here we describe progress towards characterising both the structure and function of odorant receptors from insects.

We have used a calcium imaging system to characterise receptors expressed in Sf9 cells.  Dose response curves were generated for a panel of odorants interacting with a number of Drosophila receptors and used to estimate EC50 values for each odorant-receptor combination.  We selected a small number of orthologous receptors from four different Drosophila species and compared their responses to compounds for which in vivo electrophysiological differences were observed between species.  We have also expressed a Drosophila odorant receptor in tandem with an epitope tag in order to characterise the membrane topology of the receptor.  Preliminary data indicate that the N-terminus of the receptor is intracellular, confirming the results of Vosshall’s group.

Ruthann Nichols

University of Michigan Medical School, Biological Chemistry Department, Ann Arbor, MI 48109-006 USA

nicholsr@umich.edu

Members of the sulfakinin peptide family affect Drosophila melanogaster larval odor preference.

Alan Nighorn and Aaron Ostrovsky

ARL Division of Neurobiology, University of Arizona, Tucson, AZ, USA

nighorn@neurobio.arizona.edu

The gaseous messenger nitric oxide (NO) is thought to play a role in the processing of olfactory information in both vertebrate and invertebrate species. The expression patterns of both nitric oxide synthase (NOS) and a target of NO, soluble guanylyl cyclase (sGC), vary between species but generally suggest a neuromodulatory role. Using molecular biology, electrophysiology, and optical imaging methods, we are investigating the role of this signaling system in the olfactory system of Manduca sexta. We have previously shown that NOS is found in the axons of the olfactory receptor neurons (ORNs) and sGC expressed in a subset of antennal lobe ( AL) neurons that includes GABAergic interneurons (LNs), serotonergic interneurons, and projection neurons. Using optical imaging, we have shown that NO is produced in the antennal lobe in response to stimulation of the antenna.

The function of NO in the olfactory system was examined in individual olfactory neurons with intracellular recording techniques while manipulating levels of NO signaling with pharmacological agents. Blocking NOS with either L-NAME or 7-NI resulted in changes in the behavior of both local interneurons (LNs) and projection neurons (PNs). Both PNs and LNs showed changes in baseline activity, including both increases and decreases in spike firing rate in LNs and the presence of bursts in many PNs. The odor-evoked activity in both neuron types was either missing or altered. The effects were mimicked in several neurons when sGC signaling was blocked using ODQ. However, some of the neurons that were affected by NO blockade did not contain detectable levels of sGC as measured by immunohistochemistry of the recorded and dye-filled neurons. These results indicate that NO has a variety of effects on olfactory neurons and that these effects are mediated by both sGC-dependent and sGC-independent mechanisms. We are currently testing these ideas using electrophysiology and dual imaging of nitric oxide and calcium. In addition, we are using RNAi to knockdown the expression of NOS and other genes important for the function and development of the olfactory system and using both dye labeling and optical imaging to examine the effects of those manipulations. We will discuss our current progress in these attempts to analyze olfactory function and development using RNAi. This work is supported by NIH-NIDCD DC04292.

Lynne A. Oland, Nicholas J. Gibson, Leslie P. Tolbert

University of Arizona, Arizona Research Laboratories Division of Neurobiology, PO Box 210077, Tucson, AZ, USA.

lao@neurobio.arizona.edu

Regulation of olfactory receptor axon sorting and targeting by signaling between axons and glial cells in the sorting zone of the developing moth olfactory pathway

The olfactory pathway of the moth Manduca sexta has long been used as a model system for studying developmentally critical neuron-glia interactions involved in axon sorting and targeting. As axons of olfactory receptor neurons (ORNs) extend toward the olfactory lobe, they grow through a glia-rich “sorting zone” (SZ) where, in a manner dependent on the glial cells, the axons separate from their neighbors, dramatically change directions, and fasciculate according to their target glomerulus. In the SZ, fibroblast growth factor receptors are activated on the glia, epidermal growth factor receptors (EGFRs) in lipid rafts are activated on ORN axons, and the cell adhesion molecule neuroglian (an L1 homolog) becomes tightly anchored in axonal membranes.

Our data support a model of signaling interactions in which (1) SZ glia alter axonal behavior by regulating interactions between IgCAMs that then affect activation of the GFRs, and (2) control of this signaling pathway is exerted by inclusion of these molecules in lipid rafts. Downstream effects of GFR activation include anchoring of neuroglian in the membrane, thus stabilizing fasciculation between axons. Since neuroglian is found on ORN axons along their length, but only becomes stably anchored within the glia-rich SZ, we are testing the specific hypothesis that interaction of axons with glial cells in the SZ elicits a glia-derived signal that changes the competence of the neuroglian molecules to interact homophilically. Because metalloproteases have been shown to regulate L1 interactions in vertebrates (Mechtersheimer et al., 2001; Kalus et al., 2003), we injected GM6001, a general metalloprotease inhibitor, just before axons grow into the olfactory lobe. This treatment resulted in loss of axonal EGFR activation, loss of neuroglian anchoring in the SZ, axon stalling in the SZ, and abnormal glomerular distribution of a known set of axons, similar to the phenotype seen after blocking EGFR activation or after raft disruption. These observations are consistent with a model in which axonal neuroglian interactions are regulated, directly or indirectly, by release of a metalloprotease from SZ glia and in which the IgCAM-EGFR interactions that are subsequently induced are mediated by rafts. Funded by NIH NS28495 and DC004598.     

Kalus et al. (2003) J Biol Chem 278:10381-10388.
Mechtersheimer et al. (2001) JCB 155:661-673

Julien Pelletier¹, Françoise Bozzolan², Marthe Solvar², Emmanuelle Jacquin-Joly¹ & Martine Maïbèche-Coisne²

UMR 1272, « Physiologie de l’Insecte : Signalisation & Communication», 1 INRA Versailles - Route de Saint Cyr, 78026 Versailles, France ; 2 Université Paris VI - 12 rue Cuvier, 75005 Paris, France.

Julien.Pelletier@versailles.inra.fr
martine.maibeche@snv.jussieu.fr

Antennal enzymes of moths

The olfactory system has evolved mechanisms for inactivation of odours to minimize signal saturation, as well as mechanisms for detoxifying a variety of substances. Various enzymes, such as esterases, aldehyde-oxidases and alcohol-dehydrogenases have been shown to degrade pheromone components in moth antennae (acetates, aldehydes and alcohol components, respectively), suggesting that they could participate in odor degradation after its interaction with receptors. Other enzymes, already known for their role in detoxification, such as cytochrome P450, have been also found in moth antennae, where they could be involved in both odorant and/or xenobiotic metabolism.

Using noctuid moths and the silk moth as models, we have investigated the molecular mechanisms of odorant deactivation. In this regard, we have identified potential genes involved in odorant/xenobiotic metabolism in different species, among them esterases, aldehyde-oxidases and cytochrome P450. The diversity of these antennal enzymes, from their catalytic property to their cellular/extracellular localization, reflects complex antennal catabolism.

Christophe Pouzat and Antoine Chaffiol

Brain Physiology Laboratory, CNRS UMR 8118, Paris-Descartes University, 45, rue des Saints-Pères, 75006, Paris, France

christophe.pouzat@univ-paris5.fr, roessler@biozentrum.uni-wuerzburg.de , rantoine.chaffiol@univ-paris5.fr

Residuals and statistical tests for spike train models: Applications to odour responses of antennal lobe neurons recorded from Periplaneta americana.

Multi-Electrode Arrays (MEA) allow neurophysiologists to record many neurons simultaneously with a limited tissue damage at a moderate cost; they are therefore enjoying a growing popularity. Once the raw data of these recordings have been processed the experimentalists is left with sequences of spike occurrence times, or spike trains, from many neurons. The problem of what to do with these spike trains becomes then crucial as witnessed by the already large literature on the subject. Proposed approaches range from descriptive ones based on peri-stimulus time histograms (PSTHs; Gerstein and Kiang, 1960, Biophys J, 1:15) to quantitative model based methods (eg, Brillinger, 1988, Biol Cybern, 59:189). As always in neurophysiology, freely available software implementing anything beyond the most elementary approaches are lacking. The important issue of the adequacy of a particular spike train model seems moreover to have been addressed quantitatively only once (Brown et al, 2002, Neural Comput, 14:325) by an incomplete adaptation of methods developed for earthquakes occurrence time analysis (Ogata, 1988, JASA, 83:9). Faced with a large quantity of data generated by our MEA recordings from the cockroach (Periplaneta americana) antennal lobe we have initiated the development of a spike train analysis software. This software implements, beyond the elementary PSTH analysis, model based approached which are common in the statistical literature on failure time data (Kalbfleisch and Prentice, 2002, The Statistical Analysis of Failure Time Data, Wiley Interscience). Both accelerated failure time models (Fitzhugh, 1958, J Gen Physiol, 41:675) and Cox models (Borisyuk et al, 1985, Biol Cybern, 52:301) are implemented. The complete set of Ogata's test is available (1988). We have moreover gone beyond those tests by exploiting the link between the counting process formalism used for spike trains (Brillinger, 1988) and martingale theory (Fleming and Harrington, 2005, Counting Processes and Survival Analysis, Wiley). We can thereby define rigorously a residual (actual spike train – predicted spike train) behaving like a standard random walk if the model is adequate. When the model is not adequate the shape of the residual suggests directions into which the model could be changed. These tests and their interpretation will be illustrated on our cockroach's MEA data.

Yu Tong Qiu, Joop J. A. van Loon and W. Takken

Laboratory of Entomology, Wageningen University, 6700EH, P.O. Box. 8031, Wageningen, The Netherlands.

yu.tongqiu@wur.nl

Functional studies of ORN populations on the antennae and maxillary palps of Anopheles gambiae

Andreas Reinecke¹, Meredith Schuman², Samir Anssour², Ian T Baldwin², Bill S Hansson¹

Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena, Germany
1 Dept. of Evolutionary Neuroethology
2 Dept. of Molecular Ecology

Perception of host plant volatiles by Manduca sexta females: Do they guide oviposition behaviour?

Peripheral detection of plant volatiles and central nervous processing of this information has been studied in Manduca sexta females. M. sexta oviposition preferences are, however, known to be strongly influenced by host plant species as well as geno- or eco-types. Also manipulation of the plants gene expression as well as damage inflicted by feeding conspecifics or other herbivores is known to have a strong effect on oviposition behavior. The olfactory basis of these choices is presently not established. We will investigate the missing  links between female sensory physiology, behaviour, and decision making.

Our data from comparative electrophysiological investigations using plant volatile samples from point at compounds that modulate female oviposition behaviour. The results are discussed in perspective of forthcoming bioassays to elucidate host location and host acceptance in ovipositing Manduca females. 

Jean-Pierre Rospars, Yuqiao Gu and Philippe Lucas

UMR1272, Physiologie de l’Insecte : Signalisation et Communication, INRA, route de Saint-Cyr, F-78026 Versailles Cedex, France

rospars@versailles.inra.fr

An integrated view of sexual pheromone transduction in moths

Olfactory receptor neurons (ORNs) housed in antennal sensilla trichodea of male moths can detect female-released sexual pheromone with exquisite sensitivity. Moreover the dynamic range of these ORNs is extremely broad, extending on about 8 decades of pheromone concentrations from threshold to saturation. Using electrophysiology, calcium imaging and modelling methods we are investigating the various biochemical and electrical processes involved in the transduction of the pheromone signal. We are giving special attention to the generation of the receptor potential, which is the first integrated response of the ORN and the most precisely measured.

Although much effort has been devoted to the experimental analysis of pheromone transduction, its modelling is less advanced. In a previous work we took into account the translocation of pheromone molecules from air to extracellular space, their deactivation and their interaction with receptors, the subsequent activation of G proteins and phospo-lipase C (PLC). This model describes the number of activated PLC molecules as a function of time for pheromone “square” pulses of various intensities (Biosystems, 89:101-109, 2007). Building on these results we have now studied the next steps of the process, i.e. the production by PLC of second-messenger molecules, IP 3 and DAG, the opening of IP 3-dependent calcium permeable channels, of DAG-dependent cationic channels and of calcium-dependent chloride channels, as well as the various inactivating and regulating feedback mechanisms that are needed to return the whole system to its resting state.

Using many known facts, both qualitative and quantitative, in this and other ORNs, we could infer the likely existence of some processes and put severe constraints on the range of many parameter values. Finally, we developed an integrated model of the whole reaction network accounting quantitatively for the kinetics and dynamic range of the receptor potential. This model suggests a specific role for the various channels at different stimulus intensities and gives insight on calcium regulation for example. Thus it helps to conceive new experimental tests and open a virtuous circle of combined experimental and modelling investigations.

Wolfgang Rössler

University of Würzburg, Biozentrum, Zoology II, Am Hubland, 97074 Würzburg, Germany

roessler@biozentrum.uni-wuerzburg.de

Smell and society: olfactory plasticity in social Hymenoptera

Olfaction is an essential sensory modality for social insects. The organisation of large colonies with thousands of individuals heavily depends on sensory processing of various pheromones and olfactory cues. Social Hymenoptera express a high degree of behavioral plasticity, an important prerequisite promoting division of labor. The underlying developmental and adult changes in the neuronal substrate, however, are largely unknown. Social Hymenoptera, therefore, are ideal models to investigate both evolutionary adaptations of olfactory processing and neuronal changes underlying plasticity of olfactory behavior. I will present comparative data from the honeybee (Apis mellifera) and the carpenter ant (Camponotus floridanus) highlighting two levels of olfactory plasticity: evolutionary adaptations of the olfactory pathway and adult synaptic plasticity in higher olfactory centers. Comparison of the input to the antennal lobe ( AL) revealed a substantially higher number of antennal sensory tracts and olfactory glomeruli in C.floridanus (7 input tracts, ~460 glomeruli) compared to the honeybee (4 input tracts, ~165 glomeruli). Despite these profound differences in sensory input, the antennal lobe output shows a very similar division into two AL hemispheres containing glomeruli that supply one of two major projection-neuron (PN) output tracts - the medial and lateral ACT. In contrast to the honeybee, the two pathways in the ant appear to be under differential neuromodulatory control by serotonin and nitric oxide. To study adult plasticity within the olfactory pathway, we quantified changes in large PN output synapses in olfactory and visual input regions of the mushroom-body (MB) calyx in the honeybee. Pre- and postsynaptic elements of individual synaptic complexes (microglomeruli, MG) were analysed using markers for synaptic proteins, cytoskeletal elements and GABA. The results revealed substantial changes in the structural organisation of MG along with the behavioural transition from nursing to foraging. We studied the ability to express synaptic plasticity over an extended lifespan in winterbees that remain in the hive during fall/ winter and resume foraging in early spring. The results suggest that a high degree of dendritic plasticity in intrinsic MB neurons (Kenyon cells) may be important in promoting adult synaptic reorganisation possibly underlying long-term changes in olfactory guided behavior.

Silke Sachse, Marco Schubert and Bill S. Hansson

Max Planck Institute Chemical Ecology, Department of Evolutionary Neuroethology, Hans-Knöll-Str. 8, 07745 Jena, Germany

ssachse@ice.mpg.de

Role of neural circuits in odor coding and processing in Drosophila

Odors are recognized by primary olfactory sensory neurons (OSNs), which are located on the antennae and the maxillary palps. OSNs express odorant receptor genes (62 defined members) which encode the odorant receptors (ORs). The OSNs send their axons to the antennal lobe, which consists of olfactory glomeruli. Each OSN expresses a single OR gene, and all OSNs expressing the same OR converge onto a common glomerulus.

A glomerulus receives not only the input from OSNs, but contains a highly ordered synaptic organization including synaptic microcircuits between OSNs, local interneurons (LNs) and projection neurons (PNs).

It has been shown that inhibitory interactions play an important role in odor coding on all these processing levels. To analyze inhibitory neural circuits underlying olfactory coding, odor evoked inhibitory responses of entire neuron populations must be directly measured. Using a fluorescent protein that functions as an indicator for chloride ions, odor-evoked inhibitory responses can be visualized. Using the GAL4/UAS system we genetically expressed the chloride-sensitive fluorescent protein Clomeleon in subpopulations of olfactory neurons to measure neuronal inhibitions at different processing levels in the Drosophila olfactory system.

Marie-Jeanne Sellier, Megha Makam, Frédéric Marion-Poll

INRA, UMR Physiologie de l'Insecte: Signalisation et Communication, Route de St Cyr, 78026 Versailles Cedex, France.

mjsellier @versailles.inra.fr

Effect of quinine and 20-hydroxyecdysone on the bitter receptors of Drosophila melanogaster

A number of phytophagous insects detect and avoid phytoecdysteroids using taste receptors detecting bitter substances (Marion-Poll and Descoins, 2002). 20-hydroxyecdysone (20E) is both the molting hormone of insects and the most commonly found phytoecdysteroid in plants. In this work, we asked if 20E is detected as a bitter compound by Drosophila melanogaster adults and compared its effects on behavior and on taste receptors to those of quinine. In order to evaluate the behavioral effects of 20E, we adapted a test proposed by Marella et al. (2006). Flies were allowed to walk freely during 30 minutes over 4x4 squares of agar, alternating plain agar with agar containing 100 mM sucrose and the test substance. We assessed a preference ratio by computing the number of pixels occupied by flies on each surface from images sampled at regular intervals. Using extracellular recordings from the tip of taste sensilla, we also recorded the responses of taste sensilla located on the proboscis and on the legs, to quinine and to 20E.

References:

Marion-Poll F. and Descoins C. 2002. Taste detection of phytoecdysteroids in larvae of Bombyx mori, Spodoptera littoralis and Ostrinia nubilalis. J Insect Physiol 48: 467-476.

Marella S., Fischler W., Kong P., Aasgarian S., Rueckert E. and Scott K. 2006. Imaging taste responses in the fly brain reveals a functional map of taste category and behavior. Neuron 49: 285-295.

Joachim Schachtner

Neuropeptides in the developing antennal lobes: Identification, localization, developmental regulation and possible functions

The paired antennal lobes (ALs) are the first integration centers for odor information in the insect brain and compare to the olfactory bulb of vertebrates ( OB). One characteristic of the ALs is the expression of a variety of neuropeptides (reviewed in Schachtner et al. 2005, Arthropod Struct Dev 34:257). It is however not known how many different neuropeptides are expressed in AL or OB, nor is much known about the function of this largest class of signaling molecules during odor processing or during development. A prerequisite to understand the function of neuropeptides is the knowledge of the identity of all neuropeptides involved (the neuropeptidome) and their cellular localization, and as we are particularly interested in AL development, also the developmental occurrence.

To identify neuropeptides we performed direct peptide profiling of ALs of different insects including Manduca sexta, Heliothis virescens, Apis mellifera and Tribolium castaneum by MALDI-TOF mass spectrometry (Berg et al. 2007 Cell Tiss Res 327:385; Utz et al. 2007 Dev Neurobiol 67:764 and unpublished). In these species we found a similar number of over 40 ion signals representing over 40 putative neuropeptides. Currently we are examining the chemical identity of the measured ion signals.

Immunocytochemical studies in a variety of insects revealed localization of neuropeptides mainly in local neurons, but also in subpopulations of projection and centrifugal neurons. To study localization, developmental occurrence and hormonal regulation we predominantly use the sphinx moth M. sexta as a model but are expanding our data to other holometabolous species including e.g. Apis and Tribolium. In holometabolous insects the adult antennal lobes form during metamorphosis, the transition from the larva to the adult. Immunolabeling in M. sexta revealed for each of the studied peptide families (e.g. a-type allatostatins, RFamides, allatotropins) a unique developmental acquisition pattern in defined sets of AL neurons. Pharmacological manipulation experiments revealed that the occurrence of the neuropeptides is developmentally regulated by 20-hydroxyecdysone. For each of the examined neuropeptide families the temporal pattern of their occurrence correlates with defined developmental phases, suggesting defined roles during AL development, including formation of olfactory glomeruli (Schachtner et al. 2004 J Exp Biol 207:2389; Utz and Schachtner. 2005 Cell Tiss Res 320:149). Mass spectrometric analysis throughout development of the lateral cell group which houses the somata of most of the peptidergic cells revealed the expression pattern of several identified neuropeptides and even suggests the expression of different peptide isoforms at different times during development (Utz et al. 2007 Dev Neurobiol 67:764).

The chemical identification of single neuropeptides at defined phases of development together with the immunocytochemical data allows us now to specifically investigate the possible roles and developmental significance of these neuropeptides during the formation of the AL neuronal network.

supported by DFG Scha 678/3-3

Jackson Sparks, Kazushige Touhara¹, Richard Vogt

University of South Carolina, Department Of Biological Sciences, Columbia, South Carolina 29208

1. University of Tokyo, School of Frontier Sciences, Kashiwa, Chiba, Japan 277-8562

sparksj@biol.sc.edu

Characterization of Bombyx mori Sensory Neuron Membrane Protein (SNMP)  

SNMPs are transmembrane proteins of olfactory neurons thought to play a critical role in the detection of chemical signals in Lepidoptera. It is one of several antennal specific protein factors in Lepidoptera whose function is poorly understood. Proposed functions of SNMP range from direct interactions with odorant binding proteins resulting in efficient shuttling or offloading of odorants to functional associations with traditional odor receptors. SNMP mRNA expression patterns have already been demonstrated in two other species of Lepidoptera, but little is known about what the protein does in a cellular context. The silkmoth Bombyx mori is a convenient model insect to probe for SNMP function because of a well-characterized response to a specific pheromone bombykol. There is evidence that a particular male specific pheromone receptor Bm-OR1 is a G-protein coupled pheromone receptor for the female released chemical bombykol. (Sakurai et al 2004).

Planned research includes coexpression of SNMP and Bm-OR1 in Xenopus oocyte expression system (Touhara). The effect of SNMP expression on previously observed (Touhara) cell depolarization in response to Bombykol will be described. Results may imply a direct or indirect association of SNMP with one pheromone receptor in Bombyx mori.

Sakurai T, Nakagawa T, Mitsuno H, Mori H, Endo H, Tanoue S, Yasukochi, Touhara K, Nishioka T. (2004) Identification and functional characterization of a sex pheromone receptor in the silkmoth. PNAS 101;16653-16658.

Klemens Störtkuhl

University of Bochum, Faculty of Biology Building ND 4/39,4470 Bochum Germany

klemens.stoertkuhl@rub.de

Olfactory behavior of Drosophila larvae induced by photoactivated neurons

Olfactory information is processed in central structures of the insect brain. i.e. the antennal lobes. In order to understand the underlying mechanism of signal processing we used the Gal 4 driver system in Drosophila melanogaster and expressed two proteins, channelrhodopsin-2 (ChoR-2) and a photo activated cyclase (Paca) in individual receptor neurons (ORNs) of the olfactory system in the larvae that allow optical activation of neurons. Both proteins are activated by blue light (485nm). Using this technique we were able to stimulate single ORNs odorant independently. In contrast to other techniques in which a mutant background or a deletion of olfactory neurons is needed, this technique allows the stimulation of individual ORNs without changing the morphology or ablating ORNs in the larval olfactory system.
Wild type larvae perform a phototactic negative response. They try to avoid illuminated areas in the response assay. However expressing ChoR-2 and Paca in all ORNs of the larvae olfactory result in a phototactic positive behavior response. These larvae are attracted by blue light. In contrast expressing CHoR-2 and Paca in ORNS that detect repellant odorants stimulation with blue light leads to an avoidance behavior. These larvae gather in not illuminated sections during the test.

Marit Stranden1, Brian Andersen¹, Anna-Karin Borg-Karlson², Hanna Mustaparta¹

1Norwegian university of Science and Technology, Department of Biology, Trondheim, Norway

2Royal Institute of Technology, Department of Chemistry, Stockholm , Sweden

marit.stranden@bio.ntnu.no

The same functional types of plant odorant receptor neurones in male and female heliothine moths

The specificity of the pheromone receptor neurone types are well described in heliothine males (e.g. Berg et al. 1998, 1995), whereas neurones detecting plant odorants have only been studied in the females. Nineteen plant odorant receptor neurone types have been functionally characterised in female heliothine moths using gas chromatography linked to single cell recordings and to mass spectrometry (Røstelien et al. 2005, Stranden et al. 2003). The presence of the same number of ordinary glomeruli in both sexes (Skiri et al. 2005, Berg et al. 2002) indicates that they have the same number of plant odorant receptor neurone types.

To identify as many odorants as possible, blends of volatiles produced by hosts and non-host plants as well as chemical standards were used as test samples. The protocols used for screening volatiles on female receptor neurones were also used for studying the males. In addition, insect produced volatiles and new plant volatiles were included in the test protocol. Screening the neurones with the GC-separated volatiles of the complex blends excluded a large number of molecules as odorants.  All neurones responded strongest to one compound (primary odorant) and weaker to a few structurally related compounds (secondary odorants). The neurone types were named according to the primary odorant. Eight plant odorant receptor neurone types have so far been identified in the Heliothis virescens males. They are similar to the types identified in females. One example is the (-)-germacrene D neurones. They have identical molecular receptive ranges with the female neurons and seem to be the most common neurone type also in the males. Including new plant samples in the protocol also gave new information about the molecular receptive range of some of the neurone types. For instance, the neurone type identified as tuned to 2-phenyl ethanol in the females was also recorded from males, but being even more sensitive to phenyl acetaldehyde. The new information concerning the pheromone receptor neurones was inhibition elicited by one or a few plant odorants. Although more information in males is needed, the overall results suggest that males and females possess the same type of plant odorant receptor neurones.

Nicholas Strausfeld

Division of Neurobiology and Center for Insect Science, Arizona Research Laboratories, University of Arizona, Tucson. AZ 85721.

flybrain@neurobio.arizona.edu

Homology versus Convergent Evolution of Insects and Malacostracan Olfactory Neuropils.

Recent accounts that claim homology between insect and crustacean olfactory neuropils base their conclusions mainly on one measurable commonalty, that of “glomerular” subdivisions and, associated with this, patterns of immunolabeling. However, the olfactory bulb of vertebrates is also divided into glomerular partitions as are assumed first order olfactory neuropils in annelid, onychophoran, chelicerate, and “myriapod’ brains. If the glomerular olfactory neuropils of insects and crustaceans are proposed to be homologous rather than convergent, does this imply that comparable organization in these other taxa is also homologous?

Studies by several laboratories have shown that genetic and second messenger attributes of deuterostome olfactory receptors are distinct from those of insects. This suggests that olfactory systems are fundamentally distinct in these groups and that in these groups olfactory glomeruli have evolved convergently. Might differences between second messenger systems of insect and malacostracan olfactory receptors similarly suggest convergent evolution of olfactory neuropils?

Neuroanatomical evidence shows that insect and malacostracan olfactory pathways share less in common than is commonly supposed. The organization of receptor terminals in targeting subdivisions of the antennular (crustacean)/antennal (insect) olfactory lobes are distinct between these two groups. Likewise, any morphological similarities between interneurons (local and projection neurons) of crustaceans and insects are, at best, restricted to local interneurons. Axonal projections from the olfactory lobes are entirely different between insects and crustaceans and, thus far, the target neuropils of projection neurons in the two groups cannot be reconciled.

Studies of basal Insecta and Entomostraca have thus far failed to identify common olfactory neuropils and apart from dubious claims by early workers there is no solid evidence that either possess glomerular neuropils associated with odorant receptor axons.

In short, evidence that insect and crustacean glomerular olfactory lobes are homologous is tenuous at best. A parsimonious explanation for such superficial similarities is that both systems have evolved convergently in response to similar evolutionary constraints.

Keshava Subramanya M.R.¹, Subramanian K.A.², K. VijayRaghavan.¹, Veronica Rodrigues.^{1, 3}

1National Centre for Biological Sciences, GKVK Campus, Bangalore 560 065, INDIA.
2Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560 014, INDIA.
3Department of Biological Sciences, TIFR, Homi Bhabha Road, Colaba, Mumbai 400 005, INDIA.

veronica@ncbs.res.in

Antibodies against the synaptic molecules Synapsin and Bruchpilot reveal distinct organization of the antennal lobes of polymorphic ants of Camponotus sericeus and Camponotus compressus.

Olfaction plays a major role in communication and organization in social insects mainly the hymenopterans. There is a caste specific difference in behavior which is necessary for proper division of labor in these colonies.  Ants communicate largely by olfactory stimuli with pheromones being a major stimulus which induces changes in patterns of behavior.  As a basis for investigating the mechanisms of structural and behavioral plasticity, we have begun to characterize the anatomy of the olfactory lobe in the common Indian ants Camponotus sericeus and Camponotus compressus. Colonies of C. sericeus and C. compressus exhibit a pronounced polymorphism and polyethism among the queens, male and worker castes. This provides the advantage of correlating morphological differences in the primary olfactory neuropil, the antennal lobe with caste and age specific differences in behavior. Thus, individuals of C. sericeus and C. compressus colony (mainly the worker castes) will be compared using different techniques to visualize common features and possible caste specific differences in odor processing. 

Antibodies raised against the Drosophila presynaptic molecules Synapsin (mAb 3C11) and Bruchpilot (mAb nc82) which cross-react with the ant brain were used. A three dimensional map has been constructed and we propose a nomenclature for a set of landmark glomeruli. The projection pattern of the olfactory receptor neurons from the peripheral sensilla is being characterized by neurobiotin backfilling. In C. sericeus minor workers posses more number of glomerulei than the major workers, however this is not seen in C. compressus. Preliminary behavioral observations in C. sericeus show a dimorphism in the patrolling and foraging behaviors in the worker castes. The possible correlation between behavioral properties and glomerular organization is being tested by examining a number of different ant species which show distinct polymorphisms.

Isabella Urru, Marcus Stensmyr and Bill S Hansson

Max Planck Institute Chemical Ecology, Department of Evolutionary Neuroethology, Hans-Knöll-Str. 8, 07745 Jena, Germany

iurru@ice.mpg.de

Evolution of complex pollination systems

Pollinator attraction by chemical mimicry, where no reward is received by the pollinator, has evolved in several pollinator/flower systems. In previous studies we showed that the Mediterranean species, H. muscivorus (Araceae), with hairy and flesh-coloured inflorescences, exhibits a highly accurate thermo-olfactory mimicry of a dead animal, and thereby traps potential pollinators.

Belonging to the same Araceae family, 28 species of Arums are known to have evolved similar complex, pollinator-trapping systems. However, earlier studies of the reproductive biology over the genus have shown a high degree of variability at different levels (morphology, flower odour, thermogeny, type and species of attracted insects etc.). These differences within and between species are very likely a consequence of adaptations to different habitats and thereby different pollinators.

We are now investigating morphological characters, chemical mimicry, thermogeny and pollinating fauna of the complete Arum genus and correlating these characters with a molecular phylogeny of the group presently under construction in our laboratory. How are the Arum species related to each other? Which pollination system features correlate with phylogenetic lineages? At the moment, we are focusing on inter-intraspecific floral odour variation and identification of the main pollinator. We are identifying key active compounds in floral-emitted blends by combining GC analysis with peripheral electrophysiology and MS. Here we present our first results. Future work will in addition include comparisons with the unrelated genus Aristolochia, since similar morphological-olfactory deceptive traits are present as in the genus Arum. Possible convergent adaptations in these two genera will help us to understand the evolutionary mechanisms underlying these complex pollination systems.

Joop J.A.van Loon¹, H.-L. Wang², Q.-B. Tang², C.-Z. Wang² & H.M. Smid¹

1 - Laboratory of Entomology, Wageningen University, P.O. Box 8031, 6700 EH Wageningen, The Netherlands; 2 - Institute of Zoology, The Chinese Academy of Sciences, 25 Beisihuanxi Rd., Beijing 100080, P. R. China.

joop.vanloon@wur.nl

Deriving coding principles from multineural responses to leaf saps in caterpillars of two species of Helicoverpa

Electrophysiological studies of contact-chemosensory neurons in insects generally employ single compounds. When binary mixtures are applied, the limitation of single-compound approaches becomes apparent when peripheral interactions are observed: a binary mixture of a deterrent and sucrose can excite a deterrent neuron and concomitantly the response of the sugar-best neuron is suppressed, a result not predicted from the results of single-compound stimulations. Natural food and oviposition substrates exploited by insects are chemically extremely complex. Starting from the behavioural observation that a plant-feeding insect consistently discriminates between two plant species, the chemically undefined plant saps or extracts can straightaway be applied as stimuli. We studied electrophysiological activity of two types of gustatory sensilla styloconica on the maxillary galea of the caterpillars of the related species Helicoverpa armigera and H. assulta (Lepidoptera: Noctuidae) in response to saps of cotton (Gossypium hirsutum L.), pepper (Capsicum frutescens L.) and tobacco (Nicotiana tabacum L.). In dual-choice bioassays, H. armigera, although a polyphagous species, exhibits a strong preference for cotton over pepper and tobacco, whereas H. assulta is oligophagous on Solanaceae. We used this behavioural contrast to assess whether the behavioural preference could be correlated with the response profiles in response to each sap. Multineural responses were obtained for which the response intensity of each neuron separately was quantified. Response profiles were constructed and statistically compared. In addition, we applied cluster analysis to test to what extent response profiles to each stimulus in each sensillum and species differed.

Nélia Varela¹, Louise Couton ^2,3, Sylvia Anton², César Gemeno¹, Jesús Avilla¹, Jean-Pierre Rospars^2,3

1 - Department of Protecció de Conreus, Centre UdL-IRTA, Av. Alcalde Rovira Roure, 191, 25198 Lleida, Spain.

2 – UMR 1272 PISC, INRA, Route de St Cyr, 78026 Versailles, France.

3 – UR 341 MIA, INRA, Domaine de Vilvert 78350 Jouy-en-Josas, France.

nelia.antunes@pvcf.udl.es

Three-dimensional antennal lobe map of the oriental fruit moth, Cydia molesta (Busck) (Lepidoptera: Tortricidae)

In the insect brain, the antennal lobe (AL) is the first structure to process olfactory information. Thousands of receptor neuron axons converge and make connections with higher order neurons in spheroidal areas of dense synaptic neuropil called glomeruli, which are species- and sex-specific. In most insects the number of glomeruli is less than 100 permitting for their individual identification. It is now widely accepted that odours are represented within the AL in a cross-glomerular manner and that olfactory receptor neurons expressing the same odorant receptor converge onto the same glomerulus, thus linking AL structure and function ultimately with odour-mediated behaviour.
In the oriental fruit moth Cydia molesta, previously Grapholita molesta, data are available on the effect of pheromone and specific host-plant compounds on the behaviour of males and females, providing an excellent basis for functional studies. In order to provide the basis for a better understanding of coding mechanisms of odours we constructed the first three-dimensional (3D) map of the AL glomeruli in this pest insect based on confocal microscopy. We compared three male and three female brains by using qualitative and quantitative neuroanatomical and computational approaches.
In both males and females, all glomeruli were visible throughout the AL. Therefore homologous glomeruli were easily found within ALs in the two lobes of the same individual and between individuals for their constant position, shape and size. We identified 48 to 49 "ordinary" glomeruli and one large glomerulus situated at the entrance of the antennal nerve in males and 50 to 52 "ordinary" glomeruli and one large glomerulus in the ventro-medial part of the AL in females. The radii of most “ordinary” glomeruli are between 10 and 14 µm, both in males and females and the large glomerulus has on average 24 µm in males and 21 µm in females.
This map will allow the identification of target glomeruli of physiologically-characterized olfactory receptor neurons (ORN’s) and/or AL neurons and will serve as a tool for future functional studies.

Richard Vogt, Natalie Miller, Rachel Litvak, Jackson Sparks

University of South Carolina, Department of Biological Sciences, Columbia SC 29208, USA

vogt@biol.sc.edu

The Olfactory SNMP / CD36 Gene Family of Insects.

SNMPs (Sensory Neuron Membrane Proteins) are abundantly located in the receptor membranes of lepidopteran olfactory neurons. In Lepidoptera, they are antennal specific, associate differentially with specific classes of sensilla, and express late in development. Two SNMPs have been identified in Manduca sexta; both are antennal specific but are only 40% identical in sequence (Rogers et al., 1997, 2001a,b). These observations suggested that SNMPs play a central role in odor detection. SNMPs are members of a gene family characterized by human CD36 which has diverse functions including transporting cholesterol and interacting with other proteins in cell-cell interactions; CD36 may function in part through interaction with lipid rfts. The Drosophila melanogaster genome contains at least 13 SNMP/CD36 homologues; we are characterizing the most likely SNMP orthologue, CG7000. Several of these genes have characterized function, including Nina D (caratinoid transport) Croquemort (cell-cell recognition of apoptotic cells by macrophage) and Peste (cell-cell recogntion of bacteria by blood cells). Expression analysis suggests CG7000 associates with a broad array of chemosensory sensilla both olfactory and non-olfactory, associates with a subset of olfactory sensilla, and expresses in both neurons and support cells. We are currently expanding this study to other insects, taking advantage of available genomic sequence data bases to examine the evolution of this gene family.

Supported by National Science Foundation (IBN-9731005)

Rogers M, Sun M, Lerner MR, Vogt RG (1997) Journal of Biological Chemistry 272, 14792.

Rogers ME, Steinbrecht RA, Vogt RG (2001a) Cell and Tissue Research, 303, 433.

Rogers ME, Krieger J, Vogt RG (2001b) Journal of Neurobiology 49, 47.

Martin von Arx and Patrick M. Guerin

Institute of Biology, University of Neuchâtel, Rue Emile-Argand 11, Case Postale 158, CH-2009 Neuchâtel

martin.vonarx@unine.ch

Host plant volatiles affect the behavioral responses of male grapevine moths, Lobesia botrana, to their sex pheromone

The grapevine moth Lobesia botrana (Lepidoptera: Tortricidae) is the most important insect pest of the grapevine, Vitis vinifera, in Europe. The identification of host plant volatiles that increase the attraction of pheromone blends may be of practical importance for the enhancement of currently used pheromone-based pest management systems.

Plant volatiles that elicit responses from L. botrana antennal receptor cells have been admixed to the three grapevine moth pheromone components (E,Z)-7,9-dodecadienyl acetate, (E,Z)-7,9-dodecadien-1-ol and (Z)-9-dodecenyl acetate that are currently used for this species. In a wind tunnel, upwind oriented flights of male grapevine moths have been studied to investigate the behavioral effect of plant volatile compounds on the attraction of males to their pheromone blend.

We could show effects of plant volatiles on the attraction of male grapevine moths to the ternary blend of the sex pheromone components in that mixtures including plant compounds attracted significantly more males to the source than the ternary blend alone.

Dongsheng Zhou^1,2, Joop J.A.van Loon¹ & C.-Z. Wang²

1 Laboratory of Entomology, Wageningen University, P.O. Box 8031, 6700 EH Wageningen, The Netherlands;

2 Institute of Zoology, The Chinese Academy of Sciences, 5 Da-Tun-Lu Yi, Chaoyang. Beijing 100101, P. R.

China dozhouds@gmail.com

Mechanism of acquired insensitivity to deterrent plant chemicals in the specialist Pieris rapae and the generalist Helicoverpa armigera

The taste system of insects plays a crucial role in mediating responses to food . In lepidopteran larvae, taste depends primarily on the maxillary palps and galea. The lateral and medial sensilla styloconica on the maxillary galea play a decisive role in gustatory perception of compounds in food. However, the sensitivity of taste cells is not fixed and can change with dietary experience. Plasticity was found in behavioural and/or gustatory responses to deterrents in Pieris brassicae, Pieris rapae and Manduca sexta. Furthermore, dietary exposure of larvae to certain deterrents can profoundly affect taste sensitivity to other, chemically unrelated deterrents. In P.rapae, feeding on an artifical diet resulted in suppression of sensitivity to a range of deterrents such as the phenolic chlorogenic acid and cardenolides. This phenomenon has been termed “ cross-habituation ” . However, the mechanism of the diet-induced taste plasticity are poorly understood.

To elucidate the mechanism of the acquired insensitivity to deterrents, we study the specialist Pieris rapae (Lepidoptera: Pieridae) and the generalist Helicoverpa armigera (Lepidoptera: Noctuidae) as experimental models. We focus on the changes in sensitivity of maxillary gustatory neurons induced by exposure to flavonoids. We report here on changes at the behavioural and electrophysiological levels in response to chlorogenic acid and the flavonoid naringin activity of P. rapae reared on an artificial diet or two host-plants, nasturtium, Tropaeolum majus and cabbage, Brassica oleracea. Chlorogenic acid occurs in relatively high concentrations in nasturtium. Naringin is a flavonoid glycoside structurally similar to apigenin-based flavonoids occurring in the artifical diet. In dual-choice leaf disc arena assays, caterpillars reared on cabbage were strongly deterred by chlorogenic acid and naringin. However, caterpillars reared on nasturtium or artificial diet were insensitive to chlorogenic acid. Caterpillars reared on the artificial diet were also less sensitive to naringin. Results of electrophysiological tests showed that the deterrent neuron in the medial sensilla styloconica of caterpillars reared on cabbage is more sensitive than in caterpillars reared on nasturtium or artificial diet. Therefore, the desensitization of the chemicals can be partly explained by reduced sensitivity of taste receptor cells responding to the deterrents. In future experiments, molecular techniques gene expression analysis in sensitive and insensitive individuals will be used to investigate the molecular basis of acquired taste insensitivity.

M.I. Zhukovskaya

Perception of pheromone versus general odours in American cockroach is modulated differently

Behavioural and antennal responses to different odours as well as central olfactory processing in insects are under neurohumoral control. Neurohormone octopamine is the most known substance which alters the receptor cell functioning (Pophof, 2000, Grosmaitre et al, 2001, Kapitsky, Zhukovskaya 2001; Zhukovskaya, Kapitsky, 2006). Since octopamine is supplied into an antenna with hemolymph flow, all the sensilla on its surface get the same modulatory signal. In fact, octopamine causes similar changes in electroantennogramm (EAG) recorded in response to semantically distinct odours such as sex pheromone components, a repellent 1,8-cineole, and a plant volatile hexan-1-ol. Males and females were affected similarly. Cockroaches consumed octopamine with water and sugar syrup behaved more active, than control ones, often displaying agonistic reactions like stilt-walking, kicking, lateral oscilations and biting (Simon, Barth, 1977), especially in response to occasional contacts of another individual. The behavioural changes found correspond to phenomenon reported before (Adamo et al, 1995; Stevenson et al, 2005) indirectly supporting a rise in hemolymph octopamine level. In case of pheromone responses, the drug enhanced behavioral sensitivity by decreasing the threshold dose of wing-raising behavior, the most obvious sign of sexual arousal. Highly significant avoidance of hexane-1-ol shown in control experiments completely disappeared following octopamine treatment. Vanished responses to hexanol, however, cannot be explained by toxic influence of octopamine because the identical treatment of cockroaches in our experiments with pheromone (Periplanone B) caused increased responses. Similar influence on hexanol responses is exerted by presence of sex pheromone emitting virgin female in close proximity of experimental setup. Taking into consideration the data on the rise in octopamine level under sexual excitation found in crickets (Adamo et al, 1995), the neurohormone’s role in orchestration of sexual behavior becomes more or less clear: perception of the pheromone causes a rise in octopamine level, which is, in turn, leads to increased sensitivity to pheromone and decreased responses to general odors.

Research is supported by RFBR grant № 06-04-49029

C. Zube, C.J. Kleineidam, S. Kirschner, J. Neef, W. Rössler

University of Würzburg, Biozentrum, Zoology II, Am Hubland, 97074 Würzburg, Germany

zube@biozentrum.uni-wuerzburg.de

Organization of glomeruli and pheromone processing in the ant antennal lobe.

Ants use complex olfactory communication systems for social organization within their colonies. Pheromones play a key role and induce specific behavioral tasks such as trail following, alertness or defensiveness and control reproductive behavior. Efficient and accurate recognition of pheromonal and non-pheromonal odors is essential for the survival of the ant colony and requires a precise sensory machinery and neuronal network in the brain of each individual. Using neuroanatomical and neurophysiological techniques we investigated the olfactory pathway of the carpenter ant, Camponotus floridanus. We characterized the glomerular substructure of the antennal lobe ( AL) and analysed its afferent and efferent supply. 3D-analyses revealed a total of ~ 460 olfactory glomeruli. We did not find any significantly enlarged glomeruli (macroglomeruli) that might indicate a specific function in pheromone processing. The afferent supply via the antennal receptor neurons was visualized by a ntennal-nerve (AN) backfills and revealed a division of the AN in seven tracts innervating seven distinct clusters of glomeruli. To analyse the efferent innervation of the AL via olfactory projection neurons (PNs) we selectively stained two prominent output tracts (medial, m- and lateral, l–ACT). We show that PNs leaving the AL via the m- and l-ACT innervate two separate subsets of glomeruli in the anterior and posterior part of the AL.

We used calcium imaging techniques to analyse the spatial and temporal representation of odors in AL glomeruli. PNs were retrogradely filled by injection of fura-2 dextran into the m- and l-ACT. We tested whether responses to pheromonal odors differ from responses to general odors regarding sensitivity and/or spatio-temporal representation. Changes in calcium levels were measured during odor stimulation with a releaser component of the trail pheromone (nerolic acid), an alarm pheromone (n-undecane) and two general odors (heptanal, octanol), all applied at dilutions ranging from 10 -1 to 10 -12. Each odor elicited an odor specific glomerular activation pattern, which was reproducible and comparable across individuals. We did not observe any obvious spatial separation of responses to pheromones vs. general odors since activation patterns were partly overlapping. Response thresholds for pheromonal and general odors showed an equally high sensitivity and ranged between dilutions of 10 -11and 10 -10. Glomerular response patterns to the trail-pheromone component nerolic acid showed a remarkable stability over a large concentration range of ~ 8 log units. Response durations increased with increasing concentrations, and signal intensity increased only in some glomeruli, preferentially at threshold level and at very high concentrations. The results suggest that odor quality of the trail pheromone is maintained over a wide concentration range by a stable spatial response pattern and, at the same time, odor intensity is mainly coded by changing response durations. Supported by DFG: SFB 554 (TP A6, A8) and Evangelisches Studienwerk e.V. Villigst

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

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