ESITO XI European Symposium for Insect Taste and Olfaction

11th ESITO

September 19-24, 2009
Residence Hotel Cormoran, Villasimius, Italy
http://www.hotel-cormoran.com

Abstracts 

1

Richard Benton
Center for Integrative Genomics, University of Lausanne, CH-1015, Lausanne, Switzerland

Richard.Benton@unil.ch

Odour detection by Ionotropic Receptors

We recently discovered a novel family of chemosensory receptors in Drosophila melanogaster, named the Ionotropic Receptors (IRs). IRs are structurally related to ionotropic glutamate receptors - a conserved class of ligand-gated ion channel best studied for their roles in synaptic transmission - but have highly divergent ligand binding domains. IR genes are expressed in specific combinatorials in neurons in the antenna that are distinct from those that express Odorant Receptors. IRs localise to the ciliated endings of olfactory sensory dendrites and expression of an IR in an ectopic neuron is sufficient to confer novel odour responses, providing evidence for a direct role in odour recognition. The IRs are therefore likely to define a largely unexplored “second” olfactory system in Drosophila and other insects. I will present my group’s recent progress in understanding the structural, functional and evolutionary properties of the IRs and their neural circuits in mediating odour detection.

2

Thomas Niewalda¹, Thomas Völler^1#, Julia Ehmer^1*, André Fiala^1,2 & Bertram Gerber¹

1 Julius-Maximilians-Universität Würzburg, Theodor-Boveri-Institut, Lehrstuhl für Genetik und Neurobiologie, Biozentrum, Am Hubland, 97074 Würzburg, Germany.
2 Georg-August-Universität Göttingen, Johann-Friedrich-Blumenbach-Institut für Zoologie und Anthropologie, Abteilung Molekulare Neurobiologie des Verhaltens, c/o ENI-G, Grisebachstr. 5, 37077 Göttingen, Germany.
# Present address: Visitron Systems GmbH, Gutenbergstr. 9, 82178 Puchheim, Germany.
* Present address: Dr. Schwab Inspection Technology GmbH, Industriestr. 9, 86551 Aichach, Germany.

bertram.gerber@biozentrum.uni-wuerzburg.de

Odour perception matches physiological activity patterns in Drosophila second-order olfactory neurons

How do physiology and perception relate? Given that sensory processing typically is multi-layered and parallel, the question more specifically is where along these various processing streams physiological activity patterns and perception correspond. We study the relation between olfactory physiology and perception in Drosophila. ‘Perceived-distance’ between odours is determined by a series of odour recognition experiments. Then, using optical imaging of genetically encoded calcium sensors, odour-induced activity patterns in first- and second-order olfactory neurons are measured to derive, for either site of measurement, ‘physiological-distance’ scores between odours. We find that physiological distances match perceived distances at the second- rather than the first-order olfactory processing stage.

3

Shouwen Ma, Martin Strauch, Daniel Münch, C. Giovanni Galizia

Neuroscience, Universität Konstanz, D-78457 Konstanz, Germany

giovanni.galizia@uni-konstanz.de

A DoOR to the complete olfactome: a common response model for the integration of heterogeneous odor-response data

Odors are coded by combinatorial activity patterns across olfactory receptor neurons, a coding logic that is maintained into the first processing network, the vertebrate olfactory bulb or the insect antennal lobe. Therefore, in order to understand the physiological representation of an odor in the brain, it is not sufficient to know the odor-response profile of individual receptors, but rather odor-response profiles of all receptors need to be measured. This is a formidable task even in the numerically simple system of the adult fruit fly Drosophila melanogaster, where approx. 45 receptors are expressed.
Nevertheless, many studies report odor-response patterns for individual or groups of receptors. Here, we developed a computational approach that allows pooling odor-response profiles from different studies, even if the measured magnitudes differ (e.g. action potentials vs. calcium influx, or in situ measurements vs. heterologous expression). Applying this approach to all receptors of the fruit fly, and pooling all available published data, as well as additional measurements from our lab, we generated a new open access database in which all odor-response profiles are accessible: DoOR (Database of Odorant Receptors).

We present a consensus odor response matrix across odorants and olfactory receptors. Users can query the database for the information of a particular odorant receptor, yielding a consensus odor-response profile, including error estimates and flagging of inconsistent datasets. Users can also query the database for an odor, yielding complete combinatorial odor-response patterns. DoOR handles the construction of odorant response matrices and visualizes the calculated olfactory responses in the antennal lobe. The reconstructed odorant response matrix allows for the application of linear algebra, and can therefore be combined with other matrices, for example with an odorant distance matrix to estimate hitherto unknown odorant responses.
Both the database and the software will be open source and freely available on the internet. Our framework allows to easily integrate future datasets in order to constantly improve our knowledge about combinatorial odor coding. Due to its flexible design, this DoOR can also be opened to odor responses in other species.

4

Leslie B. Vosshall

HHMI-The Rockefeller University
1230 York Avenue, Box 63
New York, NY 10025 USA

leslie@mail.rockefeller.edu

Structure-Function Analysis of the Insect Odorant Receptors

Insects are both beneficial and deleterious to human health and happiness. While honeybees are the principal pollinators of our crops, mosquitoes are major vectors of human infectious diseases and a wide range of insect pests damage human agricultural products. Olfaction - the sense of smell - is an important sensory modality used by insects to find plants and animals to feed on. Recent findings suggest that insects have adopted an evolutionarily distinct family of membrane proteins to detect odor cues. These seven transmembrane domain proteins adopt a topology distinct from the more conventional seven transmembrane domain G protein-coupled receptors that vertebrates and nematodes use to sense odors. Moreover, the insect chemosensory receptors are heteromultimeric protein complexes composed of a variable ligand binding subunit and a constant subunit called Or83b that is critical for targeting the receptor to the ciliated dendrites of olfactory sensory neurons. There is emerging evidence that these atypical receptors function as odor-gated ion channels, but the extent to which these receptors depend on G protein signaling for their function remains a topic of intense debate in the field. I will present new data analyzing the role that discrete protein motifs and domains in Or83b and the ligand binding odorant receptors play in multiple aspects of odorant receptor function, including receptor trafficking, odor selectivity, desensitization, and susceptibility to inhibition by DEET, the active ingredient in many insect repellent formulations.
Supported by the NIH, the Grand Challenges in Global Health Initiative of the Bill and Melinda Gates Foundation, administered by the Foundation for the National Institutes of Health, and the Howard Hughes Medical Institute.

5

Alison R Mercer

Dept of Zoology, University of Otago, Dunedin, New Zealand

alison.mercer@stonebow.otago.ac.nz

QMP: the mother of all pheromones

Queen mandibular pheromone (QMP) is a complex blend of chemicals that not only entices young honey bee workers to feed and groom the queen, but also primes bees to perform colony-related tasks. QMP’s ability to inhibit the rearing of new queens, prevent the development of worker ovaries, alter the biosynthesis of juvenile hormone, and influence the behavioural ontogeny of workers is well documented [reviewed by Slessor et al. 2005]. Our studies suggest that QMP-induced shifts in dopamine signalling underlie many of the queen bee’s strategies for survival. The primary component responsible for targeting dopamine pathways is homovanillyl alcohol (HVA), an aromatic compound that is structurally very similar to dopamine. HVA alters brain dopamine levels and has selective effects on dopamine receptor function in the bee [Beggs et al. 2007]. The behavioural and physiological consequences of these effects are profound. For example, in young workers exposed to HVA aversive learning is blocked [Vergoz et al. 2007]. Our recent studies suggest in addition that not only learning performance, but also perception is altered by QMP [McQuillan et al., unpublished data]. QMP’s effects on perception may be the most critical of the queen’s strategies for survival.

References:

Slessor KN, Winston ML, Le Conte Y (2005) Pheromone communication in the honeybee (Apis mellifera L). J Chem Ecol 31:2731-2745
Beggs KT, Glendining KA, Marechal NM, Vergoz V, Nakamura I, Slessor KN, Mercer AR (2007) Queen pheromone modulates brain dopamine function in worker honey bees. Proc Natl Acad Sci USA 104:2460-2464
Vergoz V, Schreurs HA, Mercer AR (2007) Queen pheromone blocks aversive learning in young worker bees. Science 314:384-386

6

M. Herzog^1,2, K. Jørgensen², H. Mustaparta²

1. Department of Neuroscience, Norwegian University of Science and Technology, Olav Kyrresgate 3, 7489 Trondheim, Norway
2. Neuroscience unit, Department of Biology, Norwegian University of Science and Technology, Trondheim, Norway

herzog@stud.ntnu.no

The tarsal gustatory system in Heliothis virescens: Functional tracing of peripheral pathways

Insects use their tarsal gustatory system both for determining if a food source is palatable or deterrent, and if a plant is suitable for oviposition or not. In order to investigate the functionality of the peripheral taste pathways in the moth Heliothis virescens, extracellular recordings (tip recordings) of different taste substances were measured and the gustatory neurons of single sensilla were stained. In addition, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to study the structure and ultra-structure of sensilla chaetica, the taste sensilla at the tarsi. Finally, the proboscis extension reflex (PER) was used to deteremine the actual behavioural response to the different taste stimuli. The results showed that 3-4 dendrites are located in one s.chaetium, in accordance with the different types of spikes that can be seen in the tip recordings. The electrophysiological data indicate that specific neurons are specialized for specific substances, e.g. a “sweet” neuron responds in a dose response manner to sucrose. The response to quinine shows another type of spikes and is characterized by a bursting response. When applying sunflower leaf extract, several different neurons respond. The response strength to sucrose and sunflower leaf extract are comparable. However, there are only a few mothes that show a PER in response to the sunflower leaf extract, whereas the most mothes show a PER after stimulating the fore tarsi with sucrose. The staining of the receptor neurons suggested a primarily innervation of the thoracic ganglion, with some single dendritic branches extending into the connectives, possibly to the suboesophageal ganglion.

7

PD Henrike Scholz

University of Würzburg
Lehrstuhl für Genetik und Neurobiologie
97074 Würzburg, Germany
Phone: 0049-931 8884479
henrike.scholz@biozentrum.uni-wuerzburg.de

Neuronal bases of ethanol preference in Drosophila melanogaster

Speculations of the evolutionary origin of alcohol consumption are based on the observation that primates as well as humans are attracted by the ethanol containing vapor of ripe or rotten fruits. On the one hand eating rotten fruits causes intoxicating effects and on the other hand these fruits have higher caloric values than unripe fruits. This means eating rotten fruits provide a reward in form of nourishment. With this respect the use of ethanol is different from other drugs like for example cocaine. The development of ethanol tolerance would enable primates to increase their consumption of more fermenting fruits and therefore more calories. Times of excess of food and appetite enforcing stimuli like ethanol vapor might lead to an uncontrollable intake of ethanol and therefore to addiction. This phenomenon would be similar to an eating disorder that causes overweight (Dudley, 2002).

To understand the transition between natural to excessive behavior we are using Drosophila melanogaster as a genetic model system to study the cellular and neuronal basis of ethanol induced behaviors in particular ethanol preference. Currently we are investigating the influence of olfaction on ethanol preference.

Towards this end we are using behavioral analyses to dissect neuronal networks underlying ethanol preference. Our goal is to understand the connection between ethanol preference, ethanol tolerance and chronic ethanol consumption.

8

Jürgen Krieger¹, Maike Forstner¹, Ewald Grosse-Wilde², Thomas Gohl¹, Elisabeth Bouché¹, Inga Gondesen¹ and Heinz Breer¹

1. University of Hohenheim, Institute of Physiology, Stuttgart, Germany
2. Max-Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Jena, Germany

krieger@.uni-hohenheim.de

The molecular equipment of pheromone-responsive sensilla in moths: Pheromone receptors, Binding proteins, SNMPs

In moths finding of mating partners highly depends on female-released sex pheromones and their sensitive perception by the males. The cellular and molecular equipment, which enables males to detect pheromones with exquisite sensitivity and selectivity is found in long sensilla trichodea on their antenna. These hairlike structures typically contain two chemosensory neurons surrounded by several supporting cells, the latter secreting pheromone binding proteins (PBP) into the sensillum lymph, which bathes the dendrites of the sensory cells. When pheromones enter a sensillum through cuticle pores, PBPs are supposed to solubilize the molecules and to transfer them through the sensillum lymph towards pheromone receptor proteins in the dendritic membrane. In addition to binding proteins and receptors a possible role of ‘‘sensory neuron membrane proteins (SNMPs)’’ in pheromone reception has been suggested. We have identified several pheromone receptor types, PBPs and SNMPs in various moth species, including Heliothis virescens, Bombyx mori and Antheraea polyphemus and analyzed their expression in cells of pheromone-responsive sensilla on the male antenna by in situ hybridization and immunohistochemical approaches. Our results from different moth species indicate that certain pheromone-responsive neurons are characterized by the expression of a distinct pheromone receptor and SNMP-1, whereas the surrounding supporting cells express several PBP-types and SNMP-2. Furthermore, functional analysis of heterologously expressed receptors stimulated with combinations of PBPs and components of the female sex pheromone blend revealed that distinct receptor types responded best to certain PBP/pheromone combinations. These data provide evidence that the interplay of a distinct pheromone receptor type and binding protein forms the basis for the specific responsiveness of moth antennae to defined pheromone components.

This work was supported by the Deutsche Forschungsgemeinschaft.

9

Claire Eschbach, Bertram Gerber

University of Würzburg, Department of Genetics and Neurobiology, Biocenter Am Hubland
D97074Würzburg, Germany

claire.eschbach@stud-mail.uni-wuerzburg.de

Odour-mixture perception in Drosophila

Using olfactory learning experiments, we investigate mixture perception in fruit flies using 4 odours and their respective binary mixtures: benzadehyde (BA), n-amylacetate (AM), 4-metylcyclohexanol (MCH) and 3-octanol (OCT). The odours dilutions have been adjusted so that all odours support equal conditioned avoidance after odour-shock associative learning. Two experiments are performed: 1) flies are trained with a pure odour A and tested with a mixture AB; 2) flies are trained with a mixture AB and tested with one of its components, e.g. A. In both cases, the rational is that the more similar the flies regard element and mixture, the stronger conditioned avoidance should be.
Given that the element-to-element similarities have already been determined (see contribution by Niewalda et al), the current data will allow seeing whether the similarities between element and mixture can be predicted based on element-element similarities.

10

Martin F. Brill, Christoph J. Kleineidam and Wolfgang Rössler

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

martin.brill@biozentrum.uni-wuerzburg.de

Multi-unit recordings in the honeybee's dual olfactory pathway

Odor information is encoded in neuronal signals and transferred by olfactory receptor neurons (ORNs) to the primary olfactory centers in the brain. The first relay station in insects is the antennal lobe (AL), the analogue of the vertebrate olfactory bulb, where olfactory glomeruli are interconnected. In the honeybee neuronal information is conveyed from the AL glomeruli via two separate uniglomerular projection-neuron (PN) output tracts, the medial and lateral antennoprotocerebral tracts (m- and l-APT), to higher olfactory integration centers in the mushroom bodies (MBs) and lateral horn (LH). This dual uniglomerular PN olfactory pathway to the MBs is a unique character of Hymenoptera (Kirschner et al., 2006; Zube et al., 2008). It is believed that the dual tracts serves the assessment of quality, quantity and temporal structure of an odor stimulus at the level of the MBs, higher brains centers that are known to be involved in higher-order sensory processing as well as learning and memory.
To investigate the significance of the dual APTs their potential role in temporal coding and coincidence detection at level of the intrinsic MB neurons, the Kenyon cells, electrophysiological properties of both APTs are analyzed simultaneously using multiple wire electrodes (adapted from Okada et al., 2007) together with a custom-designed highly accurate multi-unit recording setup. Besides synchronous recordings of the APTs in response to odors, electrical stimulation of these tracts is used to deliver temporally controlled stimuli to the olfactory pathway.
Supported by DFG, SFB 554 A8

References:
Kirschner S, Kleineidam CJ, Zube C, Rybak J, Grünewald B, Rössler W (2006) J Comp Neurol 499:933-952
Okada R, Rybak J, Manz G, Menzel R (2007) J Neurosci 27:11736-11747
Zube C, Kleineidam CJ, Kirschner S, Neef J, Rössler W (2008) J Comp Neurol 506:425-441

11

Nélia Varela ¹, Jesús Avilla ¹, César Gemeno ², Sylvia Anton ³

1. Department of Protecció de Conreus, Centre UdL-IRTA, Av. Alcalde Rovira Roure, 191, 25198 Lleida, Spain.
2. Department of Crop and Forest Science, University of Lleida, Av. Alcalde Rovira Roure 191, 25198 Lleida, Spain.
3. UMR 1272 PISC, INRA, Route de St Cyr, 78026 Versailles, France.

neliavarela@gmail.com

Sex pheromone processing neurons in the antennal lobes of both sexes of Cydia molesta (Busck) (Lepidoptera: Tortricidae)

Olfactory information plays an important role for insects in different behavioural contexts. Pheromone communication is of utmost importance for male insects in locating females, and in some species, females are also able to detect their own pheromone. It has been speculated that this female “autodetection” might play a role in adjusting calling time, and inducing dispersal or aggregation. The tortricid moth Cydia molesta (Busck) (Lepidoptera: Tortricidae) attacks peaches and apples and its activity is mainly controlled by using mating disruption with pheromone. Also female “autodetection” has been shown at the antennal and behavioural level. How pheromone information is processed in the central nervous system is, however, still unknown for both sexes. We characterized pheromone-responding neurons within the antennal lobe (AL) of C. molesta, by using intracellular recording and staining techniques. The three behaviourally relevant single components of the C. molesta sexual pheromone - (Z)-8 dodecenyl acetate (Z8-12:Ac), (E)-8 dodecenyl acetate (E8-12:Ac) and (Z)-8 dodecen-1-ol (Z8-12:OH) - at different doses were used as stimuli. For both sexes the recorded neurons showed different degrees of response specificity. In males more neurons responded specifically only to the main pheromone component (Z8-12:Ac) than in females, where more than half of the recorded neurons responded to all three components. Three antennal lobe (AL) projection neurons were identified neuroanatomically in male stainings. The three projection neurons arborised in three different "ordinary glomeruli" located within the posterior area of the AL, i.e. far from the macroglomerular complex (MGC) of the AL, and no arborisations were observed in the biggest glomerulus of the MGC, the cumulus in any of these pheromone responding neurons. These results suggest that pheromone information is not exclusively processed within the MGC in C. molesta males. In females two neurons were stained and were classified as PNs. One was located in the ventral-posterior area of the antennal lobe (AL) and the other close to the entrance of the antennal nerve (AN), a glomerulus identified as being homologous to the cumulus in males. These first descriptive data on central pheromone processing in virgin, un-experienced males and females will serve as a basis for studies of plasticity of central olfactory processing in this species.

12

Zsolt Kárpáti^1,2, Bill S. Hansson^1,3,Teun Dekker¹

1. Div. of Chem. Ecol., Swedish Univ. of Agric. Sci., PO Box 44, SE-230 53, Sweden.
2. Plant Protection Institute of Hung. Acad. of Sci., PO Box 102, H-1525, Budapest, Hungary
3. Max Planck Institute for Chem. Ecol., Dep. of Evol. Neuroethol. Hans-Knoell-Strasse 8, D-07745 Jena, Germany

zkarpati@hotmail.com

Pheromone neuroanatomy and physiology in the European corn borer is sex linked

The European corn borer, Ostrinia nubilalis, is a schoolbook example of pheromone polymorphism. Two strains exist, which produce and prefer opposite ratios of their pheromone blend. We demonstrated in a previous study that the preference is mediated by an antennal factor, which reverses the functional topology in the antennal lobe. The preference trait has been reported to be sex-linked, whereas the spike amplitude of sensory neurons was autosomal. The question thus is if the functional topology we previously found, is inherited by a sex-linked or autosomal factor. Extensive recordings, neuronal fills, and overview stainings of the antennal lobe demonstrate that EZ and ZE hybrids have an E-type functional topology. However, the size of E and Z glomeruli is intermediate between the two strains. Paternal backcrosses show follow a pattern predicted under sex-linkage. No differences were found in frequency and types of projection neurons encountered between the parental strains, their hybrid, and backcrosses

13

Agnieszka Ruebenbauer

Lund University, Department of Ecology, Chemical Ecology-Ecotoxicology, Solvegatan 37, SE-223 62 Lund, Sweden

Agnieszka.Ruebenbauer@ekol.lu.se

Odor Selection Mechanism- The Drosophila model

The aim of this study was answering the following questions: is single odorant compound an attractant for Drosophila melanogaster, is Drosophila equally attracted to fruits at different stages of rottening, are there any differences in olfactory behavior among wild type Drosophila melanogaster strains (1), how does a neuronal activity change after odorant stimulation (2)? To answer these questions, behavioral assays, electrophysiology technique and mathematical modeling were used. For the experiments wild type strains such as: Berlin K, Canton S, Oregon-R-C, Oregon-R-S and Wind Type Berlin were taken, followed by populations recently obtained from the filed: Dalby-HL, Helsingborg-E, Helsingborg-F. In parallel with experiments the control tests were performed– EAGs, locomotory assays, survival experiments, to proof no significant differences in the overall health of the investigated strains. By monitoring olfactory behavior in time I observed different olfactory preferences depending on origin of tested Drosophila strain, which might be fenotype dependent- different response to optimal and/or suboptimal stimuli. I have shown that single synthetic olfactory compounds, especially those present during fruits fermentation, are strong attractants for all tested Drosophila lines, however synthetic single compounds are never as good attractants as natural ones (blends such as fruits). Some synthetic compounds e.g. benzaldehyde, (E)-2-hexenal (green leaf volatile) are not attractive for fruit flies. Low attractiveness for single synthetic odorants is not a result of olfactory defects but adaptive selectivity of tested Drosophila strains. Rottening fruits are ideal attractants for all tested wild type Drosophila strains (1). Finally, I have shown that, based on mathematical model, it is possible to characterize different olfactory neuron types in different sensilla after stimulation with different olfactory stimuli (2).

1) Ruebenbauer A., Schlyter F., Hansson B.S., Lofstedt C., Larsson, M.C. (2008) Curr Biol 18(18):1438–1443. 2) Ruebenbauer A. (2007) J Theor Biol 248(2):311–316.

14

Andreas Husch, Debora Fusca, Moritz Paehler, and Peter Kloppenburg

Institute of Zoology and Physiology, Center for Molecular Medicine Cologne (CMMC), and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Weyertal 119, 50923 Cologne, Germany

peter.kloppenburg@uni-koeln.de

Distinct Subtypes of Local Interneurons in the olfactory system of Periplaneta americana

A diverse population of local interneurons (LNs) helps to process, structure and spatially represent olfactory information in the insect antennal lobe (AL). In Periplaneta americana we recently identified two LN types with distinctive physiological properties: type I LNs that generated sodium driven action potentials upon odor stimulation and exhibited GABA-like immunoreactivity and type II LNs, in which odor stimulation evoked depolarizations, but no sodium driven action potentials. Here, we identified two sub-types of LNs from the group of non-spiking local interneurons (type II LNs) by their distinct morphological and intrinsic electrophysiological properties. As an important step towards a detailed understanding of the cellular mechanisms that mediate odor information processing we present a detailed analysis of their distinct voltage activated calcium currents, which clearly correlate with their distinct intrinsic electrophysiological properties. Both type II LNs innervate apparently all glomeruli including the macroglomerulus. Type IIa LNs innervated each glomerulus entirely and homogeneously, while type IIb LNs innervated only parts of each glomerulus. All type II LNs were broadly tuned and responded to odorants of many chemical classes with graded changes in the membrane potential. Type IIa LNs responded with odor specific elaborate patterns of excitation that could also include 'spikelets' riding on the depolarizations and periods of inhibition. In contrast, type IIb neurons responded mostly with sustained, relatively smooth depolarizations. Consistent with the strong active membrane properties of type IIa LNs versus type IIb LNs, the voltage activated calcium current of type IIa LNs had a large transient component and were activated in a significantly more hyperpolarized voltage range. While the type I LNs resemble the ‘classical’ GABAergic inhibitory LNs as described in many insect species, the neurotransmitters of type II LNs are unknown. Using antibodies against choline acetyltransferase we have first evidence that type IIa, but not type IIb LNs are cholinergic.
Supported by grants from the Deutsche Forschungsgemeinschaft (DFG).

15

Markus Knaden, Silke Sachse, Bill Hansson

Department of Evolutionary Neuroethology, Max-Planck Institute for Chemical Ecology, Jena, Germany

mknaden@ice.mpg.de

What makes a good odour, what makes a good blend? Attractiveness and repellence in Drosophila olfaction

Different bioassays exist (e.g. y-tube, four-way olfactometer etc.) that test the behavioural significance of odours in Drosophila. However, as the attractiveness of an odour can be affected e.g. by odour concentration and application, as well as by the motivational state of the flies, results from different studies are difficult to compare. In order to create a functional database of odour attractiveness we conduct standardized experiments with a set of more than 100 odours. Optical imaging of the most attractive and most repellent odours shall reveal, whether the attractiveness of an odour is already coded in the antennal lobe. We furthermore raise the question whether the attractiveness of a blend can be predicted by its single components.

16

Poddighe S ¹, Dekker T ², Scala A ³, and Angioy AM ¹

1. Department of Experimental Biology, University of Cagliari, Italy;
2. Department of Crop Science, Division of Chemical Ecology, Swedish University of Agricultural Science, Alnarp, Sweden;
3. Department of Animal Biology, Section of Parasitology and Parasitic Disease, University of Sassari, Italy

s.poddighe@unica.it

Olfactory function in the bot fly Oestrus ovis L.

The larvae of the insect species Oestrus ovis L. (Diptera: Oestridae) are obligatory parasites in sheep and goats. The myasis can severely affect animal production and can represents a high risk factor for the human health. The gravid female insect is viviparous and strikes first stage larvae into the nasal cavities of the host. At the end of their development in the animal’s frontal sinuses, third stage larvae are sneezed from the animal’s nostril and pupate in the soil. Results of a field study indicated that the larvipositional behaviour of the bot fly was mainly dependent on climatic conditions and on visual information.
We conducted the present investigation to analyze the fly’s ability to detect odours that may guide insect attraction to the specific host and larvipositional site.
Third stage larvae collected from heads of freshly-slaughtered Sardinian sheep were maintained in a sterile substrate to pupate at controlled conditions of photoperiod, temperature and relative humidity. Pupal stage duration and fly emergence were recorded. The adult insect is lacking in the alimentary apparatus and is equipped with small-sized spheroid antennae. Scanning electron microscopy showed that the antennal flagellum carries trichoid and basiconic olfactory sensilla, as well as grooved peg formations. These structures are distributed all over the anterior flagellar surface, while only basiconic and grooved peg sensilla are located on the posterior side. Confocal microscopy on the Central Nervous System showed the presence of well defined antennal lobes with a glomerular organization. Electroantennogram recordings were obtained to stimulation with several classes of pure chemical coumpounds. By performing gas chromatography with simultaneous electroantennographic odour detection, experiments are in progress to test the stimulating effectiveness of host volatile compounds collected with the head space- and the solid phase microextraction techniques.
Our findings give the first evidence on an olfactory function in O. ovis flies. The biological importance of olfactory perception in guiding attraction and larvipositional behaviour of the insect will be the object of future studies.

17

Marie-Jeanne Sellier, Frédéric Marion-Poll

INRA, UMR 1272 Physiologie de l'Insecte, F-78000 Versailles, France

mjsellier@versailles.inra.fr

The MultiCAFE, a new multiple – choice feeding test in Drosophila melanogaster

Drosophila melanogaster reacts to taste molecules in a way which is quite similar to humans and within the detection range of mammals (Amrein and Thorne 2005; Gerber and Stocker 2007). In brief, fruit flies are attracted to sugars, avoid bitter and toxic molecules and adapt acids and salts consumption to their internal needs.
Different tests exist to determine feeding preferences in Drosophila melanogaster. One relies on the hypothesis that the number of flies on two different media reflects their feeding preferences (Marella and others 2006). A preference index is built by counting the number of flies on the two media at different time intervals. This kind of test works well when the taste contrast between the media is high. The most popular test consists in wells filled with two kinds of agar solutions, each one colored with either a red or a blue dye (Tanimura and others 1982). A preference index is calculated, based on the number of flies with a blue, red or purple abdomen. This test has a good sensitivity and relies on the actual consumption of the flies and not only their presence. Nevertheless, the major drawbacks of this test are its inability to perform more than two-choice assays and the relative difficulty in assessing the color of the abdomen. None of the above tests allows us to study precisely the absolute amount of food consumed.
We have adapted a test proposed by Ja et al. (2007), who described a new way of studying feeding, using 5 µl micro-capillary tubes filled with solutions and monitoring the level of liquid inside the tubes. With this system, called Capillary Feeder (CAFE), they analyzed the prandial behavior of their flies. Instead of using it as a no-choice or two-choice assay, we use 6 capillary tubes filled with solutions containing different concentrations of an antifeedant in order to build dose-response curves. We determined the effect of different parameters on the sensitivity of the setup and we show results on fructose and some alkaloids.

References

Amrein H, Thorne N. 2005. Gustatory perception and behavior in Drosophila melanogaster. Current Biology 15(17):R673-84.
Gerber B, Stocker RF. 2007. The Drosophila larva as a model for studying chemosensation and chemosensory learning: a review. Chemical Senses 32(1):65-89.
Ja WW, Carvalho GB, Mak EM, de la Rosa NN, Fang AY, Liong JC, Brummel T, Benzer S. 2007. Prandiology of Drosophila and the CAFE assay. Proceedings of the National Academy of Sciences of the United States of America 104(20):8253-8256.
Marella S, Fischler W, Kong P, Asgarian S, Rueckert E, Scott K. 2006. Imaging taste responses in the fly brain reveals a functional map of taste category and behavior. Neuron 49(2):285-295.
Tanimura T, Isono K, Takamura T, Shimada I. 1982. Genetic dimorphism in the taste sensitivity to trehalose in Drosophila melanogaster. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology 147(4):433-437.

18

Zainulabeuddin Syed, Julien Pelletier, and Walter S. Leal

Department of Entomology, University of California, Davis, CA 95616
wsleal@ucdavis.edu

Reception and perception of attractants and repellents by Culex mosquitoes

In mosquitoes, odorants (aka semiochemicals) are detected by specialized sensory structures present on different chemosensory tissues such as antennae, maxillary palps and proboscis. Odorant-binding proteins (OBPs) assist hydrophobic odorant molecules to pass through an aqueous medium, the sensillar lymph, separating the port of entry on the sensilla (the pore tubules) and receptors neurons. We have employed a combination of bioinformatics and molecular approaches to identify and characterize members of the “classic” OBP family in the Southern House mosquito Culex pipiens quinquefasciatus (=Cx. quinquefasciatus), a vector of pathogens causing several human diseases, including West Nile Virus (WNV). By taking advantage of the recently released genome sequences, we have identified fifty-three putative Cx. quinquefasciatus OBP genes by Blast searches. Tissue-specificity studies suggest the existence of different functional classes within the mosquito OBP family. Most genes were detected in chemosensory as well as non chemosensory tissues indicating that they might be encapsulins, but not necessarily olfactory proteins. On the other hand, thirteen “true” OBP genes were detected exclusively in olfactory tissues and might be involved specifically in the detection of “key” semiochemicals. With a chemical prospecting approach, we have identified natural olfactory ligands from major hosts of Culex spp., humans and birds, which are detected with extreme sensitivity by olfactory receptor neurons (ORNs) in Cx. quinquefasciatus. Previously, we have identified ORNs detecting plant/floral compounds on palps as well as antennal ORNs detecting a known mosquito oviposition pheromone and the insect repellent DEET. We have now identified very sensitive ORNs specialized in the detection of host-derived semiochemicals. These findings set the stage for a better understanding of the sensory ecology of Culex mosquitoes, a key step towards development of attractants and repellents for Cx. quinquefasciatus.

19

Kevin Wanner¹, Andrew Nichols², Jean Allen¹, Peggy Bunger¹, Stephen Garczynski³, Charles Linn Jr⁴, Hugh Robertson⁵ and Charles Luetje²

1. Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59715 USA
2. Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, FL 33136, USA
3. USDA-ARS, Yakima Agricultural Research Laboratory, Wapato, WA
4. Department of Entomology, Barton Lab, New York State Agricultural Experiment Station, Cornell University, Geneva, New York 14456, USA
5. Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

kwanner@montana.edu

Sex pheromone receptor specificity in the Z and E isomer races of the European corn borer

Moth species in the genus Ostrinia have been used as a model to study the evolution of sex pheromone signaling in closely related species. Most of the 20 species in this genus use varying ratios of (Z)- and (E)-11-tetradecenyl acetate, (Z)-11- and (E)-11-14:OAc, as the two main components of their pheromone blend. The European corn borer (ECB), Ostrinia nubilalis, was introduced to North America in 1914 and exists as two different pheromone races. Males of the Z race are attracted to a 97:3 blend of (Z)-11- and (E)-11-14:OAc while males of the E race are attracted to a 1:99 blend of the Z and E isomers. Physiological and morphological studies of the pheromone sensitive olfactory neurons and their projection patterns to the antennal lobe suggested that changes in periphery may account for the differences in behavioral attraction to the two different blends (Kárpáti et al., 2008). To test this hypothesis we identified five candidate ECB sex pheromone receptors by high throughput pyrophosphate sequencing of antennal cDNAs and by PCR with degenerate primers designed to conserved amino acid motifs. Full length cDNA sequences representing OnubOrs 1 and 3-6 were cloned from both the Z and E races of the European corn borer and their specificity for the different sex pheromone ligands assayed in Xenopus oocytes. Insights into the evolution of lepidopteran sex pheromone receptors will be discussed.

Kárpáti Z, Dekker T and BS Hansson (2008). Reversed functional topology in the antennal lobe of the male European corn borer. J Exp Biol. 211:2841-8.

20

Geraldine A. Wright

Centre for Behaviour and Evolution, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne NE1 7RU, UK

jeri.wright@ncl.ac.uk

Do the perceptual qualities of odours remain invariant?

The brain is provided with the highest amount of information about sensory stimuli when the physical features if stimuli are encoded via independent channels. Coding mechanisms which adapt to local variation in stimulus intensity are an important means encoding stimulus features independently of their intensities, but the extent to which these mechanisms function in the olfactory system remains unclear. To the human nose, odour quality varies when experienced over a range of odour intensities suggesting that the olfactory system is limited with respect to its ability to encode odour molecular identity independently of odour intensity. Using a peak-shift paradigm for examining generalization gradients, we present evidence which indicates that honeybees also perceive qualitative changes in odour stimuli which vary as a function of odour intensity. Furthermore, we also show that while GABA-b mediated inhibition within the antennal lobe contributes to the formation of an invariant olfactory percept, it can only do so over a range of concentrations of less than 2 orders of magnitude. Our data suggest that the general olfactory system cannot be both ‘general’ and ‘invariant’ and that it is subject to greater constraints than those placed on other sensory systems.

21

Nina Deisig, Sylvia Anton and Christophe Gadenne

INRA, UMR Physiologie de l'insecte: signalisation et communication, Route de Saint-Cyr, 78000 Versailles, France

ndeisig@versailles.inra.fr

Pheromone-plant odour interactions and mating effects in the antennal lobe of Agrotis ipsilon males

In Lepidoptera, olfaction is essential for searching sexual partners (males), host-plants for egg-laying (females) and food (both sexes). Males are thus able to detect and code sensory information pertaining to plant odours and sexual pheromones, by means of two relatively distinct olfactory pathways. In nature, females emit a blend of sexual pheromone when situated on plants (host or non-host) and it is thus vital for males to extract the information on the females’ pheromone from their olfactory environment. Plant odours could either facilitate the location of females (synergy) or mask the female pheromone (suppression). We found, by means of in vivo optical calcium imaging in Agrotis ipsilon males, that plant odours evoke high activation in ordinary glomeruli, while the pheromone blend strongly activates the macroglomerular complex (MGC). When adding the plant odour to the pheromone blend, this strongly reduced (suppressed) pheromone-evoked activity in the MGC, whereas plant odour-evoked activity in ordinary glomeruli was not altered.
Previous studies have shown that the physiological state of a male moth can change the pheromone-guided behaviour. E.g. male A. ipsilon do not respond to the female pheromone after mating any more and in parallel, central neurons within the antennal lobe have been shown to decrease strongly in sensitivity. We now compared the activation of antennal lobe glomeruli by plant odour, pheromone and their mixture in virgin and mated 5-day old males using in vivo calcium imaging. Our results show that mating did not have any effect on pheromone-evoked activity in the MGC and that it did not change the strong suppression of plant odour on pheromone-evoked responses in the MGC. However, mating induced stronger plant odour-evoked responses in ordinary glomeruli when applied at high concentrations. In conclusion, we find strong interactions of pheromone and plant-odours within the male-specific part of the antennal lobe, independently of the mating status, and further an effect of mating on plant-odour activation within the sexually isomorphic glomeruli of the antennal lobe.

22

Fang Yu^1,2, Shangan Zhang¹, Long Zhang¹, Paolo Pelosi²

1. Dept. of Entomology, China Agricultural University, Beijing, China
2. Dept. of Agricultural Chemistry and Biotechnologies, University of Pisa, Pisa, Italy

shuihewochongming@163.com
ppelosi@agr.unipi.it

Intriguing similarities between two novel odorant-binding proteins of locusts

Despite the great economical interest of locusts, major agricultural pests, particularly in Asia and Africa, their genomes have not yet been sequenced. In particular, information on proteins of chemoreception is very limited. Only one OBP (odorant-binding protein) has been reported and the sequences of several related CSPs (chemosensory proteins) are available, while olfactory receptors have not been discovered.
Searching the EST database, we found two novel sequences encoding proteins that could be assigned to the OBP family on the basis of the six-cysteine conserved motif. These proteins, OBP2 and OBP3, are very different in thei amino acid sequences from each other and from the previously reported OBP1. Moreover, OBP3 contains three additional cysteines, a fact not previously recorded in standard length OBPs. However, OBP2 and OBP3 exhibit remarkably similar binding affinities to a set of organic compounds. Such behaviour is supported by three-dimensional models, showing very similar folding for the two new OBPs, but clearly different for OBP1. Also several amino acid residues lining the binding pockets of the three proteins appear conserved in OBP2 and OBP3, but not in OBP1. Western blot experiments revealed the presence of LmigOBP2 in antennae, mouth parts and cerci, but could not detected OBP3 in any of these tissues. Experiments of immunocytochemistry, using antibodies against OBP2 revealed the presence of this protein in the outer lymph of sensilla chaetica of the antennae, in contrast with OBP1, that was previously reported in sensilla basiconica.

23

Majid Ghaninia¹, Shannon B. Olsson¹, Andreas Reinecke¹, Bill S. Hansson¹

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

Functional classification and topographic mapping of olfactory sensory neurons housed in antennal trichoid and basiconic sensilla of the female moth Manduca sexta

In Manduca sexta vital behaviors such as host location, mating and oviposition rely predominantly on olfactory cues released from host plants, mating partners and oviposition sites. The female antennae bear numerous hair-shaped structures, sensilla, in which olfactory sensory neurons (OSN) reside. The OSNs are responsible for detection and discrimination of olfactory cues occurring in the insect environment. Significant scientific effort has been invested towards unraveling odor processing at the central level in this species. Comparably little is, however, known regarding coding of behaviorally relevant odors by OSNs. Electrophysiological attempts towards defining the function of specific OSNs have often failed due to e.g. the sometimes ambiguous spike amplitudes of the OSNs residing in single sensilla. However elucidating exactly how these neurons react when confronted with behaviorally relevant odorants is indispensable for future investigations, e.g. for using modern molecular techniques to deorphanize receptors and to manipulate receptor expression.
With the aim to functionally characterize single OSNs and determine their detailed topographical location on the surface of the antennae, and as a starting point for molecular manipulations, we performed systematic single sensillum recordings (SSR) on four morphological types of olfactory sensilla: trichoid-A and -B, and basiconic-A and -B. The number of OSNs residing in these sensilla ranged from one to three. We were able to unambiguously differentiate the co-localized cells associated with single sensilla based on their spike amplitudes. Using a panel of 30 biologically relevant compounds established in behavioral and coupled gas chromatography-electrophysiology experiments we made over 100 recordings from the four morphological types of sensilla. Based on the response spectra of individual OSNs the sensilla fell into 10 distinct functional classes. Selectivity of the OSNs varied from narrowly tuned, responding to only a subset of compounds, to very broadly tuned, responding to most, if not all, of the compounds tested. We also performed dose-response investigations on some of the functional classes. Topographic mapping of the sensilla revealed that some physiological sensillum types are confined to particular locations on the antennal surface while other classes are more or less irregularly scattered all over the antennal annuli. In conclusion we could establish the response characteristics and topographical location of a number of sensillum types. This detailed knowledge will form an important background to future receptor deorphanization, in situ hybridization and molecular manipulation experiments.

24

E. Grosse-Wilde and B.S. Hansson

Department Evolutionary Neuroethology, MPI for Chemical Ecology, Jena, Germany

grosse-wilde@ice.mpg.de

Olfactory receptors of arthropods

The odorant specificity of olfactory receptor neurons is mainly determined by the specificity of the expressed receptor protein (OR). In insects, there seem to be between 40 to 270 genes encoding ORs per species. Pronounced intra- as well as inter-specific sequence diversity means that OR-encoding genes are usually identified using complete or partial genomic databases. Using the tobacco hornworm Manduca sexta as model species, we established the use of transcriptomic sequencing for the identification of OR coding genes. Currently we are employing this technology to analyze evolutionary changes in the OR gene repertoir of athropods caused by adaptation to different ecological niches.

25

Marianna I. Zhukovskaya

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

mzhukovskaya@yahoo.com

Sexual state affects pheromone and non-pheromone odour responses in the cockroach.

Sensitivity to chemical signals changes according to the phase of an insect sexual status. Virgin male cockroaches were tested in two states, non-aroused and aroused by calling female. Behavioral responses to the plant-derived volatiles were significantly altered in the aroused state compared with the non-aroused control. Repellency of a hexanol odour in binary choice tests disappeared, while no-preference responses to eucaliptol (1,8-cineole) changed into attraction to the odour. The hemolymph octopamine level was shown to rise during sexual arousal in male insects (Adamo et al, 1995). Supplementation with octopamine before the tests had the same effect as sexual excitation, namely, repellency of hexanol decreased while eucaliptol became attractive. Firing activity of olfactory receptor neurons was recorded from the three sensillar types: swB (housing pheromone-sensitive and eucaliptol-sensitive cells), short swA and long swC, with the latter two responding to hexanol. Octopamine application did not alter the responses of swB sensilla to eucaliptol contrary to the enhanced responses of the same sensilla to pheromone components, periplanones A and B. Thus, the receptor cells within the same sensillum are modulated independently of each other. Sensillar responses to hexanol were altered by octopamine in swA but not swC sensilla. The data suggest that a change in an insect sexual status matches the certain tuning of the sensory periphery.
The study was supported by RFBR grant # 09-04-01042-?.

26

Irene Ibba^1,3, Teun Dekker³, Francesca Cadeddu¹, Bill Hansson², Anna Maria Angioy¹

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

ibba@unica.it

Identification of new natural ligands for OSNs in the coeloconic sensilla of Drosophila

In the Drosophila antenna, OSNs are housed in three major morphological types of sensilla: basiconica, tricoidea and coeloconica. Most of the OSNs express two evolutionarily related insect-specific chemosensory receptor families, the odorant receptors (Ors) and gustatory receptors (Grs). Recently it has been found that some OSNs housed in coeloconic sensilla express a novel family of chemosensory receptors, called ionotropic receptors (Irs). Yao and colleagues demonstrate that there are 4 coeloconic sensilla types: ac1, ac2, ac3, ac4 . Among these, only ac3B neurons, which express coreceptor Or83b, respond to the 80% of the odours, specifically to alcohols. The other sensilla showed only few and weak responses to a set of 45 odours. Here we investigated which natural odour ligands present in fruit elicits response in coeloconic sensilla. We used headspace collections from ecologically relevant sources such as different fruits at different stage of ripeness, yeast and vinegar. We performed GC-EAD and GC-SS to identify which natural compounds elicit a response in which coeloconic neuron in Or83b- as well as wild type Drosophila. Through GC-MS (mass spectrometry) we identified compounds that elicited response in the OSNs, e.g. 2methyl propanoic acid; 2methyl butanoic acid; 3-(methylthio)propanoic acid ethyl ester. We found some coeloconic OSNs that responded strongly to individual compounds within the extracts. Synthetic compounds will be used to establish dose response-curves as well as their effect on Drosophila melanogaster behaviour.

Acknowledgments: MIUR: International Inter-University Cooperation

27

Claudia Groh¹, Ian A. Meinertzhagen² and Wolfgang Rössler¹

1. University of Würzburg, Biozentrum, Zoologie II, Am Hubland, D-97074 Würzburg
2. Dalhousie University, Life Sciences Centre, Halifax, NS, Canada

claudia.groh@biozentrum.uni-wuerzburg.de

Synaptic plasticity in olfactory microglomeruli in the mushroom body calyx of the honeybee and the fruit fly

Synaptic plasticity refers to changes in structure and function of synapses in response to various stimuli. These dynamic changes are likely to be important to accommodate adaptations in olfactory behavior. Dendritic spines are suggested as a primary site for synaptic plasticity. In vertebrate neurons, these tiny dendritic specializations are rich in filamentous (f)-actin and exhibit activity-driven changes in shape. Our research in the honeybee and in the fruit fly has revealed that Kenyon-cell (KC) dendritic spines in the mushroom-body (MB) calyx, labeled with f-actin probes, express developmental and adult structural changes. We comparatively investigated the basis of these changes within MB-calyx microglomeruli (MG) at the cellular and subcellular levels. The olfactory MB-lip region represents a higher-order olfactory association center involved in learning and memory processes. By combining immunolabeling and electron microscopy, we quantified changes in pre- and postsynaptic components of MG. Currently we focus on three major questions: 1) what are the cellular changes underlying age- and task-related volume changes in the MB calyx of the honeybee and the fruit fly; 2) are ultractructrual differences present in the medial and lateral tract projection-neuron input regions of the MB-calyx lip in the honeybee; and 3) does juvenile hormone play a role in modulating synaptic plasticity in the MB calyx of adult honeybees?
Calycal MG can be visualized with f-actin phalloidin labeling in the honeybee and in Drosophila. In flies, phalloidinergic MG rings are less distinct and more densely packed compared to the honeybee. In the honeybee, the number of anti-synapsin labeled presynaptic projection neuron (PN) boutons declines with age while at the same time the associated calyx volume increases. Our results suggest that this mainly results from an increase in KC dendritic processes. The degree to which these plastic processes are associated with structural modifications at the pre- and postsynaptic sites of these spine synapses is further quantified at the ultra-structural level.
Supported by DAAD, DFG, NSERC and HFSP

28

Wolfgang Rössler and Christina Zube

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

roessler@biozentrum.uni-wuerzburg.de

Dual olfactory pathway in Hymenoptera: functional indications from species- and sex-specific adaptations

In most insects, olfactory information from antennal-lobe (AL) glomeruli is transferred by uniglomerular projection neurons (uPNs) to the mushroom bodies (MBs) and lateral horn (LH) via the medial antenno-protocerebral tract (mAPT). Our comparative studies on the input-output connectivity of AL glomeruli in the honeybee (Apis mellifera) and the carpenter ant (Camponotus floridanus) revealed specific features of a dual olfactory pathway. In addition to the mAPT, an equivalent number glomeruli of one AL hemilobe are connected by uPNs that project in a reverse order via the lateral APT, first to the LH and then to the MBs. Antennal sensory input tracts are connected in a stereotypic fashion with the two output tracts, the m- and l-APT. We address the following questions aiming to understand the function and origin of a dual olfactory pathway in Hymenoptera:
- Is a dual reverse uPN pathway to the MBs and LH a specific feature of Hymenoptera?
- If this is the case, where within the Hymenoptera did a dual olfactory pathway arise?
- Does the dual pathway exhibit any sex-specific differences?
- How are odors mapped and processed in glomeruli of the two AL hemilobes?
Comparison with other insect orders shows that a prominent l-APT formed by uPNs and a symmetrical hemilobe division into an m- and l-APT uPN pathway is a specific hymenopteran trait. Tracing studies in solitary Hymenoptera and in selected species of the paraphyletic group of sawflies indicate that this feature may have evolved in the basal Hymenoptera. Our studies in C. floridanus revealed a substantial sex-specific difference in the composition of the dual pathway. In females, 436 glomeruli are symmetrically divided into l- and m-APT innervated AL hemilobes. Males possess a reduced number of 258 glomeruli. Whereas in males the number and organization of glomeruli innervated by the l-APT is similar to the situation in the female AL (around 200 glomeruli), the number of glomeruli innervated by the m-APT is dramatically reduced compared to females (58 compared to 218). As males do not engage in social tasks, this may indicate that the m-APT pathway is associated with preferentially processing social odors. In addition to the numerical differences, serotonergic innervation in the l- and m-ACT hemilobes differs between males and females indicating that the two pathways are under different neuromodulatory control. Calcium imaging of odor-evoked activity of uPNs in the AL of C.flordianus demonstrates that, similar as in the honeybee, glomeruli in the l-APT hemilobe process pheromonal as well as non-pheromonal odors in overlapping patterns. The odor specificity of glomeruli in the m-APT hemilobe and potential differences in temporal aspects of odor coding remain to be investigated.
Supported DFG, SFB 554 A6, A8 and HFSP

29

Sylvia Anton

INRA, UMR Physiologie de l'Insecte, Route de St Cyr, 78000 Versailles Cedex, France

santon@versailles.inra.fr

Experience-induced plasticity of the chemosensory system in moths

Male moths innately have a high sensitivity to female-produced sex pheromones. In the noctuid moth Spodoptera littoralis, however, behavioural responses to the pheromone can be further increased by brief pre-exposure to the sex pheromone. In parallel, an increase in sensitivity of neurons within the primary olfactory centre, the antennal lobe, was observed one day after pre-exposure with pheromone. As a first step to test if the observed effect is a form of general sensitization or rather a phenomenon of selective attention, we tested effects of pre-exposure with stimuli of other modalities on the behavioural and central nervous sensitivity to sex pheromones.
Predator-emitted signals such as bat sounds and attractive, as well as repellent gustatory stimuli used during brief pre-exposure, all elicited an increase in sensitivity to the sex pheromone in S. littoralis males one day after exposure. Also certain plant-emitted volatiles such as geraniol had a similar effect. The exposure to a behaviourally not meaningful stimulus, such as a tone with the same frequency as the bat sound but not pulsed, however, did not change male behaviour to sex pheromone. The observed behavioural changes were in several cases correlated with an increase in the sensitivity of central olfactory neurons to the sex pheromone. Our results indicate that pre-exposure to a variety of behaviourally relevant stimuli might lead to a general sensitization of different sensory systems rather than eliciting selective attention

30

Berna, A.Z.¹ Anderson, A.R¹. & Trowell, S.C¹.

1. Quality Biosensors theme – Food Futures Flagship, CSIRO, Canberra, Australia

alisha.anderson@csiro.au

Characteristics of insect olfactory receptors compared with a set of metal oxide sensors.

Animal noses and antennae are deployed in situations where odours must be detected or discriminated rapidly.  Noses and antennae are also capable of performing discriminations among complex odorant mixtures with great sensitivity and speed. Electronic noses, E-noses, are instruments designed to reproduce the pertinent features of animal noses using arrays of chemical sensors. However, E-noses have not been very widely adopted, in large part because they perform poorly in many real-world discrimination tasks. Replicating the performance of a biological system deepens our understanding of the physiological mechanisms involved and may also lead to technological improvements in instruments, such as E-noses, designed to perform similar tasks. Following recent detailed characterisation of the molecular receptive range of a subset of Drosophila olfactory receptors (ORs) (Hallem et al, 2004) it is now possible to compare the responses of a set of technical sensors with those derived by evolution. Here we compare the sensitivity, tuning and independence of 12 metal oxide (MOx) sensors with those of 24 Drosophila ORs using the same panel of 110 test odorants. We find little difference in levels of sensitivity between the MOx sensors and ORs with the Drosophila ORs being more broadly tuned. Despite this apparent lack of selectivity the Drosophila ORs are highly independent of each other, ie each Drosophila OR is non-selective in its own unique way. We propose that an array of independent sensors, each of which display odorant promiscuity is important for the discriminating power of the insect olfactory system. The array and component MOx sensors we examined lack these features and accordingly have relatively poor ability to discriminate odorants.

31

Cécile Faucher and Sylwester Chyb

CSIRO Entomology, Canberra, Australia

cecile.faucher@csiro.au

Functional study of gustatory receptors in Drosophila

In Drosophila, the detection of taste compounds is thought to be mediated by a family of about 60 putative gustatory receptors (GRs) expressed in chemosensory neurons located in the mouth parts, legs, and ovipositors. For the majority of these receptors, their role in taste discrimination remains to be elucidated. Here, we describe the functional analysis of two GRs heterologously expressed in an insect cell line using calcium imaging. Cell expression vectors containing Gr5a or Gr64f have been generated and transiently expressed in the Sf9 cell line. Their sensitivity to tastants has been assessed by monitoring the rise of cytosolic calcium using fluo-4 as a calcium indicator. For homogeneity of the data, values were related to the maximal fluorescence emitted by the cells by addition of ionomycin, a calcium ionophore that affects the permeability of the plasma membrane and thus leads to maximal entry of external calcium into the cell. The results of this study will be discussed in light of the present knowledge of GR specificity in vivo.

32

David Jarriault, Philippe Lucas, Jean-Pierre Rospars, Christophe Gadenne and Sylvia Anton

INRA UMR 1272 Physiologie de l’Insecte : Signalisation et Communication
Route de Saint-Cyr 78000 Versailles, France

djarriault@versailles.inra.fr

Sex pheromone coding and its plasticity in the moth Agrotis ipsilon

Although much progress has been made recently, many questions are still open in the understanding of olfactory processing in antennal lobes and the transformation they operate on the antennal input into the output to higher brain centres. We are currently investigating these questions by focusing on how sex pheromone signals are processed within the male-specific macroglomerular complex (MGC). Sex pheromone communication is crucial for mate encounter and therefore species survival. Interestingly, in our model species, the noctuid moth Agrotis ipsilon, the attraction to the female sex pheromone is decreased following mating (Gadenne et al., 2001). This plasticity is not only seen at the behavioural level, but is accompanied by a change in the sensitivity of central olfactory neurons. The loss of sensitivity after mating is restored during the next day. This transient neuronal plasticity serves as an energy-saving strategy by switching off the olfactory system and therefore preventing males from mating unsuccessfully.
By means of extra- and intracellular recordings and stainings, we described the physiology of receptor neurons (RNs) and projection neurons (PNs) and compared their coding properties in males of two different reproductive states: sexually mature, virgin males and newly mated males.
Single sensillum recordings showed a long lasting response of the pheromone-sensitive RNs, whereas a large proportion of the intracellularly recorded and stained PNs arborising in the MGC responded with a shorter biphasic excitation / inhibition pattern to pheromone stimulation. These results suggest that the message transmitted by RNs is temporally shaped by interactions between AL neurons. According to studies in other insects, the inhibition period we observe in most of our PN responses would be due to an inhibitory input from local neurons (LNs). Detailed analysis of the recorded activity of the neurons after mating revealed changes in the PN response characteristics but not at the RN level. This suggests that the impairment of the male behavioural response observed after mating could originate from a neuromodulation at the AL level.

Ref: Gadenne, C., Dufour, M. C. and Anton, S. (2001). Transient post-mating inhibition of behavioural and central nervous responses to sex pheromone in an insect. Proc. Royal Soc. London B 268, 1631-1635.

33

Anja Fiedler, Bill S. Hansson, Silke Sachse

Max Planck Institute for Chemical Ecology, Dep. of Evolutionary Neuroethology, Jena, Germany
anfiedler@ice.mpg.de

Inhibitory odour responses in Drosophila ORNs: the transgenic chloride indicator Clomeleon

We study the inhibitory odour responses in olfactory receptor neurons (ORNs) in Drosophila melanogaster using the chloride sensitive reporter Clomeleon. Using the GAL4-UAS system we can express Clomeleon in Or83b-neurons and visualize the inhibition of odorants in the antennal lobe (AL), the first olfactory neuropil, by optical recording of the brain of the fixed fly.
The purified Clomeleon protein is known to be pH sensitive in solution. Therefore, we did control experiments in vivo that prove that the Clomeleon detected odour responses in the AL are unaffected by the pH of the Ringer solution used as brain hemolymph substitute.
Further, we studied the effect of chloride and calcium free Ringer solution, respectively. Chloride free Ringer applied as bath solution results in a significant reduction of the inhibitory odour responses to approximately 50%. In contrast, calcium free Ringer solution had no effect on the inhibitory responses and shows Clomeleon to be chloride specific. This might even be a hind for an inhibitory pathway mediated independently from a calcium induced excitation which needs to be investigated in more detail.
We are in the process of investigating which chloride channels are expressed in ORNs and how they mediate the inhibitory pathway. Analysis of these basic principles of olfactory transduction, coding and processing of odours is critical for an in depth understanding of the olfactory pathway.

34

Virginie Party¹, Christophe Hanot¹, Imene Said², Didier Rochat¹ and Michel Renou¹

1. UMR1272 PISC, INRA UPMC Route de Saint-Cyr 78026 Versailles cedex, France
2. Unité de Biochimie Macromoléculaire et Génétique. Faculté des Sciences de Gafsa, cité Zarroug, 2112 Gafsa, Tunisia

vparty@versailles.inra.fr; renou@versailles.inra.fr

Plant volatile compounds: Cues or noise for mating?

Pheromone communication in moths serves for mate recognition and mate finding. Each component of the pheromone blend is detected by olfactory receptor neurons (ORNs) expressing narrowly tuned olfactory receptors. In natural conditions, pheromone is detected in a complex background of plant volatile compounds (PVCs). It has been showed that PVCs can have an effect on pheromone behaviour, but the mechanisms are unknown. PVCs could affect the detection and/or the integration of the pheromone signal. For detection, there are contradictory results: Ochieng et al showed that linalool synergises the response to pheromone, but Kaissling and Van der Pers showed that linalool inhibits the pheromone response in other species. So our work aimed to understand if PVCs are cues or noise for mating and first to characterise the effect of PVCs on the pheromone response of Spodoptera littoralis.

We analyzed quantitatively ORN firing responses to the main pheromone component, Z9E11-14:Ac, in the presence of four monoterpenes: linalool, geraniol, geranyl acetate, linalyl acetate from S. littoralis host plants and isoprene which is not a host plant compound. To mimic natural contexts in which plant compound and pheromone emanate from different sources, the two stimuli were presented with different temporal patterns and from independent sources and we used a ratio of 1000 between PVCs and pheromone concentrations.

Linalool reversibly reduced the firing response to Z9E11-14:Ac and produced an off effect. These results are consistent with Pophof’s in Silkmoth. Geraniol, geranyl and linalyl acetates reduced the response with a longer time course and isoprene had no effect. Applications of pulses of linalool over prolonged stimulation Z9E11-14:Ac resulted in a discontinuous firing activity. In the reverse experiment, pulses of pheromone were better separated over a background of linalool, compared to odourless air.

We demonstrate that PVCs may reduce the sensitivity, but increase the temporal resolution of pheromone detection in S. littoralis. This suggests that the effects of terpenes on pheromone communication are complex. An inhibitor background might positively affect mate location at high pheromone density especially nearby a pheromone source. Furthermore, the effects differ from one species to the other. To better characterize the effects of PVCs we shall analyse the detection of PVCs by plant specific neurons. Behavioural experiments will be necessary to further determine whether PVCs favours (“cues”) or reduce (“noise”) male orientation.

References
Kaissling et al, 1989, J. Comp. Physiol. A 165:147-154
Ochieng et al, 2002, J. Comp. Physiol. A 188:325-333
Pophof et al, 2002, Chem. Senses 27:435-443
Van der Pers et al, 1980, Chem. Senses 5:365-371

35

Thomas S. Muenz and Wolfgang Rössler

University of Würzburg, Biocenter, Zoology II, Am Hubland, 97074 Würzburg, Germany
thomas.muenz@biozentrum.uni-wuerzburg.de

Pheromonal regulation of behavioral plasticity in the honeybee

In honeybee colonies the female worker caste expresses a pronounced polyethism. Summer bees live for ~6 six weeks and perform a rich behavioural repertoire ranging from various indoor duties (e.g. feeding, building) to foraging nectar, pollen and water outside the hive. Switching between these behaviours depends on age, and our current studies show that this goes along with plastic changes in the synaptic organization in the mushroom-body (MB) calyces. However, division of labour in a honeybee colony is not purely age-dependant and as rigid as described above. In fact, the transition from nurse bees to foragers may additionally be modulated by chemical communication signals enabling the colony to respond in a flexible manner to environmental changes by shifting the work force between indoor and outdoor duties.
One of these chemical cues is ethyloleate (EO), which has earlier been described to delay the onset age of foraging (Leoncini et al., PNAS, 2004). EO is found in high concentrations in the honey crop of foraging bees. As honeybees continuously feed each other via trophallaxis EO is passed throughout the entire colony, thus making it an easy accessible signal for work force distribution. Therefore, EO may be regarded as a primer pheromone that accelerates or delays the onset of foraging in a concentration dependant manner. Using synaptic markers in combination with quantitative confocal analyses, we found evidence that EO may delay maturation of input synapses (microglomeruli) in the mushroom-body calyx. This effect goes along with a delayed onset of foraging behaviour.
Whether EO is received as an olfactory and/or gustatory cue or whether it is internalised is still unclear. Here we investigated whether EO is received as an olfactory cue. Since EO has low volatility at room temperature, heated-air or heated-dummy stimulation was used to evoke behavioural and/or physiological responses. Electroantennography (EAG) recordings of whole antennae gave a first indication that EO is at least partly received via the olfactory system. Furthermore, Ca²⁺-Imaging using selective loading of antennal lobe (AL) projection neurons with calcium sensitive dyes was performed to investigate neuronal representation and processing of EO in AL glomeruli. The results indicate that EO is processed in glomeruli of the T1 cluster, and potentially the T3 cluster of AL glomeruli. In summary, the present results demonstrate that EO is at least partly received as an olfactory stimulus suggesting that it may modulate plastic changes in MB microglomeruli during the transition of nurse bees to foragers via the olfactory pathway.
Supported by Human Frontier Science Program

36

T. Saumweber¹, B. Michels¹, D. Bucher¹, N. Funk¹, D. Reisch², G. Krohne², S. Wegener¹, S. Liedel¹, E. Buchner¹, B. Gerber^1*

1 Julius-Maximilians-Universität Würzburg, Theodor-Boveri-Institut, Lehrstuhl für Genetik und Neurobiologie, Biozentrum am Hubland, 97074 Würzburg, Germany.
2 Universität Würzburg, Abteilung für Elektronenmikroskopie, Biozentrum am Hubland, 97074, Würzburg, Germany

timo.saumweber@biozentrum.uni-wuerzburg.de

Associative learning is impaired upon lack of the presynaptic protein SAP47

The synapse associated protein of 47 kDa (SAP47) is a member of a phylogenetically conserved, yet functionally diverse gene family. In Drosophila, SAP47 is encoded by a single gene and is expressed throughout all neuropil regions of the wild-type larval brain; specifically, electron microscopy reveals anti-SAP47 immuno-gold labeling within 30 nm of presynaptic vesicles. To analyze SAP47 function, we use the viable and fertile deletion mutant Sap47¹⁵⁶, which suffers from a 1.7 kb deletion in the regulatory region and first exon of the gene. SAP47 cannot be detected by either immunoblotting or immunohistochemistry in Sap47¹⁵⁶ mutants. Sap47¹⁵⁶ mutant larvae exhibit normal sensory detection of odors and tastants as well as motor performance and basic neurotransmission at the neuromuscular junction. However, Sap47¹⁵⁶ mutant larvae show an approximately 50 % reduction in learning ability when compared to wild-type animals. A similar learning impairment is observed upon reduction of SAP47 expression using RNA-interference. These data underscore the importance of presynaptic, “vesicular” mechanisms for associative plasticity and provide the first hint to the function of SAP47 in flies and its homologues in other species.

37

Luke Holman¹, Stephanie Dreier¹, Charlotte G. Jørgensen^2,3, John Nielsen² & Patrizia d'Ettorre¹

1. Centre for Social evolution, Section for Ecology and Evolution, University of Copenhagen, 2200 Copenhagen, Denmark.
2. Department of Natural Sciences, Bioorganic Chemistry, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark.
3. Present address: Department of Medicinal Chemistry, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark.

Ant queen pheromones: chemical control or signal of quality?

Social insect societies have been described both as paradigms of cooperation and as uneasy alliances in which conflict is rife. The simultaneous action of cooperation and conflict is especially apparent in the evolution of worker sterility, which may represent either voluntary altruism and/or coercion by other colony members. In many species, queens are hypothesised to produce pheromones that maintain worker sterility, and there has been debate about whether such pheromones are a signal to workers or a form of control. Using the ant Lasius niger, we show that a blend of queen cuticular hydrocarbons reliably indicates reproductive maturation and productivity. A synthetic version of the major hydrocarbon inhibited worker ovarian development and aggression in a bioassay, making this the first sterility-inducing pheromone to be identified in any social insect apart from the honeybee. Several lines of evidence indicate that the workers’ response to the pheromone is mutually beneficial. The pheromone is also apparently used by workers to identify and eliminate the least fertile queens in multi-queen colonies; the honesty of the signal may facilitate punishment of queens that selfishly under-invest in worker production and thereby select for greater cooperation.

38

Rickard Ignell¹, Majid Ghaninia^1,2, Sharon Hill¹, Siju K. Purayil¹, Shahid Majeed¹, Bill S. Hansson²

1. Division of Chemical Ecology, Swedish University of Agricultural Sciences, Alnarp, Sweden
2. Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany
rickard.ignell@ltj.slu.se

Neural correlates of host choice behaviour of mosquitoes

Anthropophilic, opportunistic and zoophilic mosquito species are believed to use distinct odour signatures to locate and identify their blood hosts. We hypothesise that the observed differences in mosquito host preference behaviour are correlated with differential tuning of their peripheral olfactory systems. To test this, we have performed a functional analysis of the near-complete repertoire of olfactory sensilla of Aedes aegypti, Culex quinquefasciatus, Anopheles gambiae, An. arabiensis, An. quadriannulatus and An. stephensi. Using the single sensillum recording (SSR) technique we have identified up to 53 olfactory receptor neuron (ORN) classes in a single species, each with a unique response spectrum to a panel of behaviourally relevant odours. These ORN classes are combined in stereotyped configurations within up to 23 functional types of sensilla. We find that the number of ORN classes as well as the tuning and sensitivity of these neurons are highly correlated with the host preference behaviour of the different species. Ongoing studies focus on the identification of novel ligands for individual ORN classes using gas chromatography linked SSR analysis, to further correlate neural responses to the host preference behaviour of anthropophilic, zoophilic and opportunistic mosquitoes.

39

Jeff Riffell¹, Hong Lei1 and John G. Hildebrand¹

1ARL Div. of Neurobiology, University of Arizona, Tucson AZ
jeffr@neurobio.arizona.edu

The characterization and coding of complex odor mixtures in the moth Manduca sexta

Relatively few studies of neural processing of olfactory information in the brain have used natural stimuli, which typically are mixtures of odorants. We asked how natural odor mixtures that best correlate with the flight and foraging behaviors of the moth Manduca sexta are encoded in its primary olfactory center, the antennal lobe. We focused on floral attractants from two important resources for nectar-feeding, the desert plants Datura wrightii and Agave palmeri. We used gas chromatography coupled with multiunit neural-ensemble recording to identify key odorants from these floral mixtures that elicited responses from individual antennal-lobe neurons. Neural-ensemble responses to the complete A. palmeri scent, comprising >60 odorants, could be reproduced by stimulation with a mixture of only six of its constituents that had behavioral effectiveness equivalent to that of the natural floral scent. Likewise, a mixture of three odorants emitted by D. wrightii flowers elicited normal odor-modulated upwind flight behavior. Recording responses of neural ensembles to floral mixtures of varying behavioral effectiveness enabled us to explore the coding of behaviorally significant odors. Similar to behavioral results, neural ensemble responses to the two floral mixtures containing >100 individual odorants can be reproduced by a much smaller subset of odorants (<5). Finally, spatiotemporal encoding by the AL neural ensemble provides a means by which the olfactory system can generalize between related floral mixtures while maintaining sufficient contrast for discrimination of distinct scents.

40

Violaine Olivier, Christelle Monsempes, Marie-Christine François and Emmanuelle Jacquin-Joly

UMR INRA-UPMC PISC 1272, INRA, Route de Saint-Cyr, 78026 Versailles, France.
violaine.Olivier@versailles.inra.fr
Emmanuelle.Jacquin@versailles.inra.fr

Candidate Chemosensory Ionotropic Receptors in Lepidoptera

Recently, Benton et al. (2009) discovered in Drosophila a new family of candidate chemosensory receptors: the ionotropic receptor (IR) family. Members are structurally related to ionotropic glutamate receptors (iGluRs) and are expressed in Drosophila antennae.
Blast analyses of an EST library made from the noctuid moth Spodoptera littoralis male antennae led to the identification of height sequences related to Drosophila IRs. We thus sequenced the corresponding full length cDNAs and deduced the encoded proteins. Phylogenetic analysis showed that these protein sequences are more closely related to Drosophila IRs than to iGluRs of diverse invertebrates. Sequence analyses revealed considerable variations in the predicted ligand-binding domains and none retained the glutamate-binding residues R, T and D/E, suggesting different specificities. Five of the eight transcripts appeared to be exclusively expressed in male and female chemosensory organs (antennae and proboscis), four of which being restricted to antennae. Thus, they appear as good candidates for new members of the IR chemosensory receptor family, extending their discovery to Lepidoptera. In situ hybridization experiments are in progress to precisely establish their expression pattern within S. littoralis antennae.

Reference:

Benton R., Vannice K.S., Gomez-Diaz C. and Vosshall L.B. (2009) Cell. 136: 149-162.

41

Irene Ibba^1,2, Anna-Maria Angioy², Bill S. Hansson³, Teun Dekker¹

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

A macroglomerular complex for fruit odors radically reverses blend interpretation in specialist Drosophila sechellia.

Whereas the basic layout of the insect olfactory circuitry is increasingly clear, how olfactory processing translates into appropriate behavioral responses is still poorly understood. Through a comparative approach we research the basic coding rules for attraction and repulsion. Here we demonstrate that specialist fruit fly Drosophila sechellia, a closely related sibling of Drosophila melanogaster, has formed a macroglomerular complex (MGC) tuned to host odors, methyl hexanoate and 2-heptanone, of its sole host Morinda citrifolia. The voluminar increase of both glomeruli matches the numerical overrepresentation of AB3 sensillae in the antennae, and radical reversed the fly's behavior. The increase in input into both glomeruli has resulted in a reversal of the fly's behavior from repulsion to attraction as compared to its sibling species D. melanogaster. This is true for single odorants, but much more distinctly so for mixtures. These results show how relatively simple changes in the olfactory circuitry can have large effects on odor coding. The findings elegantly illustrate how olfactory preference is wired, and how evolutionary change may be acccomodated by the olfactory circuitry.

42

Manuela Rebora¹, Gianandrea Salerno², Silvana Piersanti¹, Elda Gaino¹

1. Dipartimento di Biologia Cellulare e Ambientale, Via Elce di Sotto, 06121 Perugia, Italy
2. Dipartimento di Scienze Agrarie e Ambientali, Borgo XX Giugno, 06123 Perugia, Italy

rebora@unipg.it

Olfaction in Paleoptera insects

Traditionally, Ephemeroptera (mayflies) and Odonata (damselflies and dragonflies) have been classified as Paleoptera (old wings), based on their inability to fold the wing over the abdomen. In consideration of their short antennae and very developed eyes, Paleoptera insects have been considered visual dependent insects and investigations on other sensory structures are very limited. Neuroanatomical studies support the hypothesis of anosmia since paleopteran insects lack glomerular antennal lobes. Our recent ultrastructural investigations on the antenna of the mayfly Rhithrogena semicolorata and of the dragonfly Libellula depressa revealed the presence of sensilla coeloconica located in pits on the lateral-ventral side of the flagellum (Rebora et al., 2008, 2009). The structure of these coeloconic sensilla is coherent with that reported for single-walled insect olfactory receptors. In fact, they are innervated by unbranched dendrites, which enter the peg and show a dendrite sheath ending at the base of the peg. The peg has no socket and shows a porous cuticle with pore tubules located between pores and dendrites (Rebora et al., 2008; 2009). An overview on the antennal flagella of mayflies and dragonflies belonging to different families reveals that these putative olfactory sensilla are widespread in these insect orders.
Preliminary electrophysiological investigations on adults of the dragonfly Libellula depressa reveal an olfactory response to cis-3-hexen-1-ol, a leaf alcohol produced in small amounts by most plants and acting as an attractant to many predatory insects.
These data raise new questions regarding the presence of olfaction in basal pterygotes. Further electrophysiological investigations and molecular studies on expressed receptor proteins (OR) in Paleoptera are in progress in order to shed light into the supposed ability of these insects to perceive odors. Also, these studies can contribute to trace evolutionary trends in insect odor perception and OR origin.

References:
Rebora M., Piersanti S., Gaino E. (2008). The antennal sensilla of the adult of Libellula depressa (Odonata: Libellulidae). Arthropod Structure & Development, 37: 504-510.
Rebora M., Piersanti S., Gaino E. (2009) The antennal sensilla of adult mayflies: Rhithrogena semicolorata as a case study. Micron (in press)

43

Elodie Urlacher, Bernard Francés, Martin Giurfa and Jean-Marc Devaud

Centre de Recherches sur la Cognition Animale (UMR 5169) – Université Paul Sabatier, Toulouse
devaud@cict.fr

Modulation of learning by a social signal in an insect brain : a new role for allatostatin, acting through an opioid-like pathway ?

Cognitive processes can be modulated by many factors, including social signals from conspecifics. In social insects, pheromones are such signals that contribute to organize the life of the colony by modulating the behaviour and physiology of individuals. Since the honeybee is a model organism to study both the functions of social signals on behaviour and the neural bases of learning, it offers a unique opportunity to study how pheromones can modulate learning. However, except a recent study (Vergoz et al., 2007), this question has seldom been addressed. Here, we show that olfactory learning is modulated by a particular social experience, i.e. exposure to the sting alarm pheromone. After exposure to this signal (released by other bees to signal life-threatening situations), bees perform less well in a pavlovian conditioning assay, compared to unexposed bees. The same results can be obtained after exposure to the main component of the pheromone, isopentyl acetate (IPA). Thus, exposure to IPA may trigger some neuromodulator(s) that modify the function of the brain centers known to be involved in this learning task.
Pharmacological treatments combined with the exposure to IPA prior to conditioning show that agonists (fentanyl) and antagonists (naloxone) of mammalian opioid receptors can mimick or reverse, respectively, the modulation of learning. This suggests the existence of an opioid-like pathway in bees, in accordance with previous work (Núñez et al., 1998). We searched the honeybee genome for possible homologues of mammalian opioid receptors, and found a predicted receptor. It is also shares homologies with the human somatostatin receptor SSTR2 and the Drosophila allatostatin C receptor 2. Allatostatins are neuropeptides mostly known for hormonal control of crucial steps of development. Our current results indicate that allatostatin C is an endogenous ligand for this receptor, and that it modulates learning in the same way as exposure to IPA. Thus, our results suggest a new role of this neurohormone: that of a neuromodulator acting on the the brain circuits underlying appetitive learning, whose release would be triggered by exposure to the alarm pheromone.

Núñez J., Almeida L., Balderrama N., Giurfa M. (1998). Alarm pheromone induces stress analgesia via an opioid system in the honeybee. Physiology and behavior, 63(1):75-80
Vergoz V., Shreurs H. A., Mercer A. (2007). Queen pheromone blocks aversive learning in young worker bees. Science, 317:384-86

44

Martin N. Andersson, Mattias C. Larsson, and Fredrik Schlyter

SLU, Dept. Plant Protection Biology, Chemical Ecology, P.O. Box 102, SE-230 53, Alnarp, Sweden
martin.andersson@ltj.slu.se

Peripheral coding of ecologically relevant odours by the European spruce bark beetle, Ips typographus

We used single-sensillum recordings to characterize response profiles of olfactory receptor neurons (ORNs) in Ips typographus. Using a large selection of host, non-host, and pheromone compounds, we identified 17 strongly responding and highly selective ORN classes. Strikingly, and for the first time shown for any insect, almost 25 % of the neurons (four ORN classes) responded to anti-attractant compounds typical of non-host angiosperm plants. We also found good correlates between the response specificity of these neurons and behavioural responses. Behavioural synergists were perceived by different ORNs, whereas non-synergistic GLV alcohols activated the same ORN class, even at very low doses. Furthermore, 35 % of the ORNs (six classes) were tuned to spruce-related monoterpenes; the remaining ones (seven classes) responded to con- and heterospecific bark beetle pheromones. The most abundant ORN class responded to the aggregation pheromone component cis-verbenol (cV). Oftentimes, the cV neuron was co-compartmentalized with a small spiking B neuron that responded to 1,8-cineole (Ci). Other cV neurons were paired with a non-responsive B cell. Interestingly, upon stimulation with Ci, the cV cell was inhibited, but only in sensilla in which the cV and Ci neurons were paired. Using binary mixtures of the two odours, the responses to low doses of cV were reduced when the Ci cell simultaneously responded to high doses of Ci. We suggest that the response of the co-localized neuron, rather than receptor-ligand interactions, causes the inhibition. Thus, ORNs within a sensillum may not respond independently of each other. Interestingly, Ci also greatly reduces the attraction to the pheromone in the field.
Our study has improved our understanding about bark beetle olfaction, but also concerning peripheral odour coding mechanisms in general. The high selectivity of the characterized neurons questions the existence of ‘generalist’ ORN types.

45

D. Siaussat, F. Bozzolan, L. Duportets, S. Debernard

UMR 1272A Physiology of insect: Signalling and Communication
Université Pierre and Marie Curie (Paris 6)
7 Quai Saint Bernard
Bâtiment A 4^ème étage
75252 Paris Cedex 05
David.siaussat@snv.jussieu.fr

Possible modulation of olfactory processes by steroid hormones in Lepidoptera

In vertebrates and invertebrates, the hormones are known to be involved in the control of sensory systems. In mammals, it is admitted that steroids exert some modulatory effects on the functioning of olfactory system in correlation with the physiological status and environmental factors (photoperiod, temperature…). In adult insects, few results also suggested an action of ecdysteroids, especially 20-hydroxyecdysone (20E), on the central and peripheral olfactory pathways. Nevertheless, most studies are focused on the identification of 20E regulatory mechanisms underlying in the orchestration of insect post-embryonic development. 20E is known to act via proteins, called nuclear receptors, which are transcriptional factors able to activate genetic cascade leading to the control of expression of different genes.The temporal organization of 20E cascade begins by the induction of EcR (the ecdysone receptor) and its partner USP (ultraspiracle) followed by the activation of E75 early gene, a regulator of the signaling pathway. The 20E action in a developmental context is well deciphered whereas its contribution in olfactory plasticity remains largely unexplored.

The aim of this study is to demonstrate that the insect olfactory processes could be modulated by the ecdysteroids. This project is based on Spodoptera littoralis, a lepidopteran model, usually used because of the high sensitivity of its olfactory system and the availability of EST database established from adult males antennae. Titer analysis of ecdysteroid in hemolymph of S. littoralis revealed the presence of these hormones at detectable levels in male adults. The bioinformatics analysis of EST library coupled to an amplification strategy by RACE PCR led to the characterization of EcR, USP and E75. The antennal expression of these receptors occurred during the whole adult life and was localized more precisely in the olfactory sensilla by in situ hybridization. The injection of 20E exogenous into adult males induced an increase in expression amount of EcR, USP, E75 and changes in transcriptional level of peripheral olfactory genes. Taken together, these results provided molecular and biochemical evidences that the detection system of olfactory signal is probably the target of ecdysteroids in insects. Functional approaches are in progress to better dissect the ecdysteroid-mediated signaling in the regulation of olfactory processes.

46

Geir Kjølberg Knudsen¹ and Anders Aak²

1Bioforsk, Plant Health and Plant Protection, Høyskoleveien 7, N-1432 Ås, Norway, geir.knudsen@bioforsk.no
2Norwegian Institute of Public Health, Pb 4404 Nydalen, N-0403 Oslo, Norway, anders.aak@fhi.no

Lure development and mass trapping of Calliphora vicina (Diptera; Calliphoridae) in Norwegian stockfish production

Blowflies of the species Calliphora vicina are causing severe economic losses in dried fish production in northern Norway. Female flies are attracted to dead animals for both oviposition and foraging. As natural decomposing carcasses are scarce in its distribution and only suitable for the larvae a short time, the odour mediated resource location is expected to be very important. A wind tunnel was used to optimize attraction of female C. vicina and chemicals known to attract blowflies were tested both as single compounds and in blends. A three-component blend of dimethyltrisulphide, mercaptoethanol and o-cresol was found to significantly increase upwind orientation compared to the single attractant dimethyltrisulpide. More than 60% of female flies showed upwind oriented flight to the synthetic blend and this attraction was similar to decomposing liver which is commonly used to trap blowflies. Female attraction to odours from dead animals reached 90%, indicating a large potential for lure improvement. The three-component blend significantly increased field catches compared to dimethyltrisulphide, and the catch consisted of 98.2% females. A large scale mass trapping experiment in Lofoten, northern Norway, has now been ongoing for four years. Damage to stockfish in treated areas has significantly decreased compared to control areas.

47

Esther Lorenzo and María Gabriela de Brito Sanchez

Centre de Recherches sur la Cognition Animale, CNRS - Université Paul Sabatier Toulouse, France
debrito@cict.fr

Can bees detect the bitter with their legs?

The antennae, mouth parts and tarsi of the forelegs constitute the main chemosensory appendages of honeybees. Olfactory, gustatory but also hygro-, thermo- and mechanosensory sensilla are located on them. Although gustatory sensilla of honeybees have been characterized electrophysiologically at the level of the antennae and mouthparts, tarsal gustation has never been systematically studied in this insect. The recent decoding of the Honeybee genome allowed identifying the presence of only 10 gustatory receptor genes apprently encoding for 10 gustatory recepors. These numbers contrast with those of drosophila where 68 gustatory receptors encoded by 60 gustatory receptor genes have been identified. These differences suggested that the gustatory world of a bee is relatively poor despite the large variety of natural gustatory compounds to which the bee is exposed throughout its life. With this in mind, we asked whether honeybees have the the sense of bitter. By means of behavioral and electrophysiological experiments, we first showed that bitter substances (quinine, salicin, cafeine, berberine, etc) are not perceived per se at level of the antennae and mouth parts. Here we present results concerning the possibility of bitter taste perception at the level of the tarsi. We performed a series of behavioral and electrophysiological experiments aimed at determining whether bees exhibit aversive responses when stimulated with bitter substances on the tarsi, and whether sensilla dedicated to bitter substances can be located on the tarsi. Our results constitute the first electrophysiological recordings of gustatory neurons of the honeybee tarsi and, together with our behavioral data, will make possible to better understand gustatory processing in this insect.

48

Pål Kvello, Kari Jørgensen and Hanna Mustaparta

Norwegian University of Science and Technology, Dep. Biolog-Neuroscience Unit, Trondheim, Norway. pal.kvello@bio.ntnu.no

Physiological and morphological characterisations of central gustatory neurons receiving information from four appendages in the moth Heliothis virescens.

The importance of gustation in nectar feeding and selection of host plants by herbivorous insects is well known, and detection of different tastants by the gustatory receptor neurons (GRNs) have been studied in many species. Whereas separate GRNs responding to various phagostimulants and deterrents have been well characterised, there is only scarce knowledge about how gustatory information is integrated/mediated in the central nervous system of insects. Neurons responding to both phagostimulants and deterrents have been shown in the thoracic ganglion of the locust Locusta migratoria (Rogers and Newland 2003) and neurons responding to phagostimulants have been shown in the suboesophageal ganglion (SOG) of the fly Sarchophaga bullata (Mitschell and Itagaki 1992). The particular organization of the gustatory receptor neurons on several appendages in insects, like the antennae, the proboscis and tarsi of the moth Heliothis virescens, has raised the question whether the information about tastants from different appendages are integrated or mediated separately in the central neurons of the primary gustatory areas in the SOG/ tritocerebrum. From behavioural studies it is evident that the gustatory receptor neurons (GRNs) mediate appendage specific behaviours; antennal stimulation with phagostimulants and deterrents eliciting orientation toward versus away from the tastant, as well as extension versus recoiling of the proboscis (Jørgensen et al 2007a). Proboscis stimulation maintains proboscis extension and initiates consumption versus inhibit consumption, and tarsal stimulation elicits proboscis extension and probably behaviours involved in egglaying.
We have recorded intracellularly from neurons in the SOG of H.virescens during application of phagostimulants, a deterrent and a mechanical touch on to the contact chemo- sensilla on four appendages (the two antennae, the proboscis and one tarsus). The stimuli are previously shown to activate separate receptor neurons of the contact chemosensilla containing 2-3 GRNs and one mechanosensory neuron in this moth (Jørgensen et al 2007b; Kvello et al unpublished). We here present the response characteristics/ profiles of the CGNs and patterns of activity in an ensemble of CGNs, elicited by each of the stimuli within and across appendages. Staining and three-dimensional reconstructions revealed the morphology of the CGNs, showing extensive dendrite arborisations in the SOG; many with axon projections within the SOG and others projecting out of the SOG into different brain regions or ganglia. We show movies of CGNs registered into the standard brain atlas, showing spatial relationship of the CGNs as well as overlap of their dendrites with the previously described axon projections of the GRNs (Jørgensen et al 2006; Kvello et al 2006). We discuss the involvement of CGNs in specific appetitive or aversive behaviours as well as in multifuctional roles, i.e. belonging to different networks executing specific functions.

References
Jørgensen K, Stranden M,Sandoz JC, Menzel R, Mustaparta H 2007a.J Exp Biol 210:2563-73
Jørgensen K, Kvello P, Almaas TJ, Mustaparta H. 2006. J Comp Neurol 496:121-134
Jørgensen K, Kvello P, Almaas, TJ Mustaparta H. 2007b. Chem Senses 32:863-879
Kvello P, Almaas TJ, Mustaparta H 2006. Arthropod Struct Dev 35:35-45
Mitchel BK, Itagaki H. 1992. J Comp Physiol A 171:213-230
Rogers SM, Newland PL 2002. J. Neuroscience 22:8324-8333

49

Mamiko Ozaki

Department of Biology, Graduate School of Science, Kobe University, Kobe, 657-8501, Japan

Inter- and intraspecific chemical communication in ants: difference between colonialities.

Animal societies have evolved, adapting to environmental changes. Ants have highly developed various social styles and chemical communications. Nestmate recognition by cuticular hydrocarbons (CHCs) used as the main chemical cues underlies social behaviors of ants, e.g. foraging, colony construction, etc.
We have been studying chemical communication and social behavior especially in a monogynous ant, C. japonicus. In this species, worker and queen exhibit aggressive behavior toward nonnestmate, whereas male does not. SEM images illustrated that the worker and queen possessed CHC sensilla housing ~130 olfactory receptor neurons, which can discriminate colony specific blends of 18 kinds of CHCs. The ventro-medial (VM) region of ~130 glomeruli in antennal lobe was also observed in worker and queen but not in male, suggesting that the VM region might provide a relay for conveying the information from the CHC sensilla.
Formica yessensis is a polygynous species constructing supercolony by fusing neighboring nests. In Hokkaido, Japan, they have a huge supercolony along Ishikari shore, where the workers can freely be accepted in any nests within the supercolony. Discrimination analyses of the CHC components in workers indicated that the CHC profiles of workers in the Ishikari supercolony were similar to each other, regardless of nests, but rather different from those in other outside colonies at Oshoro and Hakkenzan. Actually, the workers in the Ishikari supercolony hardly showed aggressive behavior within the supercolony but occasionally toward workers from Oshoro and Hakkenzan. Nevertheless, they got highly aggressive toward different ant species, Camponotus japonicus, for example. The CHC sensilla of this ant consistently responded in a CHC difference dependent manner and their responses seemed to correspond to aggressiveness. Thus, the supercolonies have various levels of CHC profile variations, responsiveness in the CHC sensillum and aggression-acceptance balance among nest. The variations in those levels might have some correlation within a supercolony, although further studies are necessary to conform this and to elucidate neural and molecular mechanisms.
Now, Argentine ant, Linepithema humile, has invaded many countries and brought economical and ecological damages. In 1999, they were first detected in our research field, Kobe, where L. humile has settled down and species have been disappearing. However, it is not known how L. humile can reject other ant species. Hypothesizing some semiochemical effects were involved in, we started the study about chemical communication between the invaded polygynous ant and the indigenous monogynous ant.

This study was supported by Hyogo Science and Technology Association, Brain Science Foundation

50

Silke Sachse, Veit Grabe, Marco Schubert, Sonja Bisch-Knaden and Bill S. Hansson

Max Planck Institute Chemical Ecology, Department of Evolutionary Neuroethology, Hans-Knöll-Str. 8, 07745 Jena, Germany
ssachse@ice.mpg.de

Neural circuits underlying olfactory processing in Drosophila

Most organisms rely on their olfactory system to detect and analyze chemical cues in the environment, cues which are subsequently utilized in the context of behavior. The basic layout of the first olfactory processing centers, the olfactory bulb in vertebrates and the antennal lobe in insects, is remarkably similar. Odors are encoded by specific ensembles of activated glomeruli in a combinatorial manner. However, a comparison of the transformation of odor representations between input to the antennal lobe and output to higher brain centers yields a complex and contradictory picture. The question of how odors are processed is accordingly open. A central problem regarding our present understanding of olfactory processing is that virtually nothing is known regarding the inhibitory components. The inhibitory processes are assumed to be as important as the well-studied excitatory pathways, however, the necessary tools to study the former processes in imaging studies have so far been lacking. In order to visualize inhibitory responses, we use a newly described fluorescent protein, named Clomeleon, which functions as an indicator for chloride ions — the main mediator of synaptic inhibitions in mature neurons. Using the standard GAL4-UAS system in Drosophila melanogaster, we ectopically expressed Clomeleon in specific subpopulations of olfactory neurons. We have successfully imaged chloride signals following stimulation by odor ligands at the different processing levels of the fly antennal lobe. Odors induced specific patterns of glomerular inhibitions at the input as well as the output level of the antennal lobe. In addition, by using the genetically encoded calcium reporter Cameleon, we measured excitatory odor responses and compared these with the inhibitory patterns. These odor responses have been obtained for the different processing levels and mapped to olfactory glomeruli in order to create an odor-specific map of glomerular inhibitions and excitations. These results will help to decipher inhibitory interactions within the antennal lobe network and will add a new dimension to the olfactory code.

Supported by the BMBF

51

Antonia Strutz¹, Johannes Stökl¹, Bill S. Hansson¹, Silke Sachse¹

1. Max Planck Institute Chemical Ecology, Department of Evolutionary Neuroethology, Hans-Knöll-Str. 8, 07745 Jena, Germany
astrutz@ice.mpg.de

Multiglomerular activation patterns of Arum palaestinum odor components in the Drosophila melanogaster antennal lobe

The Arum palaestinum plant is native at the Mediterranean coast of the Middle East. It has a deceptive flower, which exhibits a striking feature: within only twelve hours of flowering, it is capable to attract more than 500 Drosophila. To investigate this remarkable natural fly-trap, we analyzed the headspace of the flower and identified the active components by performing GC-EAD. Two components, acetoin acetate and 2,3-butandione acetate, display new odor compounds, that have not been analyzed before.
The aim of this study is to decipher which odor components are responsible for the strong attraction of this flower. By using calcium imaging of the Drosophila antennal lobe, we measured the activation patterns of the single components of the Arum headspace, as well as the synthetic flower mixture at different concentration levels. High and median concentrations of all single components evoked a broad response pattern in the antennal lobe of the fly. Interestingly the synthetic flower mixture shows an additive response pattern, by activating all glomeruli, activated by each single component. This multiglomerular activation pattern is exceptional compared to ‘non-natural’ fly-traps like the commercially available Vector 960. In addition, several of the receptors targeted by the flower components are highly conserved between the Drosophila group species and also within the 12 sequenced genomes, which indicates an evolutionary very old attraction strategy of the Arum flower.

52

Caroline Joris¹, M. Susantha B. Mohottalage ^1,2, Raphaë^l Tabacchi ², Patrick Guerin ¹

1. Institute of Biology, University of Neuchâtel, CH-2009 Neuchâtel, Switzerland
2. Institute of Chemistry, University of Neuchâtel, CH-2009 Neuchâtel, Switzerland
caroline.joris@unine.ch

Tsetse fly responses to volatile plant compounds

Although tsetse flies are obligate blood feeders, these sleeping sickness vectors seek cover from the unfavourable hot conditions of Africa under vegetation to survive between blood meals. In recent decades man-made vegetation zones such as conifer, coffee and banana plantations and thickets of the invasive bush Lantana camara have been largely colonized by riverine tsetse as new habitats. Flies from such refugia are implicated in sleeping sickness epidemics due to their association with human settlements. Foliage and plant extracts from Lantana camara have been shown in this laboratory to be attractive to three tsetse spp. from different habitats. Plant products provide important resource signals for tsetse flies, signalling suitable habitats and during host finding as byproducts of the metabolism of animals, mainly ruminants. Gas chromatography analysis of essential oils coupled to electroantennogramme recordings show how tsetse flies respond to terpenes, aromatics and other volatiles derived from all the major biosynthetic pathways of plants. Some 35 chemostimuli from 16 plant spp. could be identified. The behavioural responses of tsetse fly spp. of different physiological status and from different habitats to plant compounds and their mixtures are being quantified in a windtunnel in 3D.

53

Xin-Cheng Zhao and Bente G. Berg

Dept of Psych/Neurosci Unit, Norwegian University of Science and Technology, 7489 Trondheim, Norway
xin-cheng.zhao@samfunn.ntnu.no

Modulation via two spike amplitudes? Physiological properties of the serotonin-immunoreactive antennal-lobe neuron in Helicoverpa assulta

Insects detect odor molecules by a large number of olfactory sensory neurons located on the antennae. The signal information is carried directly to the primary olfactory center of the brain, the antennal lobe, via the sensory axons. Here the axon terminals target spherical structures termed glomeruli. The glomeruli contain, in addition to the receptor neuron terminals, arborizations of three central interneuron categories, namely local interneurons that communicate within the antennal lobe, projection neurons that mediate the signal information from the antennal lobe to the protocerebrum, and centrifugal neurons that send axonal projections from other parts of the central nervous system into the antennal lobe. One centrifugal neuron morphologically identified in several insect species is the so-called serotonin-immunoreactive (SI) antennal-lobe neuron, originally described in the sphinx moth Manduca sexta (Kent et al. 1987, J Neurobiol). We have recently characterized, by intracellular recording and staining combined with immunocytochemistry, this particular neuron in the central olfactory pathway of the male moth Helicoverpa assulta. The SI neuron had branching profiles similar to those found in the sphinx moth. The physiology of the neuron revealed two distinctly different categories of spiking amplitudes — one small showing increased frequencies when odors were blown over the antenna and the other with a large, long-duration amplitude showing no observable responses. We speculate whether the two categories of spikes are initiated in different regions and whether their presence is significant for the modulatory capacity of this widespread neuron. This work was supported by the Norwegian Research Council, project number 178860/V40.

54

Julia Riedl¹, Mariana López-Matas¹, Vivek Jayaraman² & Matthieu Louis¹.

1. Center for Genomic Regulation, EMBL-CRG Systems Biology Unit, 08003, Barcelona, Spain
2. HHMI-Janelia Farm Research Campus, Ashburn, VA 20147, USA

matthieu.louis@crg.es

Odor coding with single fly odorant receptors

We are interested in understanding how ethologically relevant odorant stimuli are encoded in the peripheral olfactory system of Drosophila melanogaster larvae. The larval ‘nose’ is composed of 21 olfactory sensory neurons (OSNs) expressing one, or occasionally two, specific odorant receptors along with the Or83b co-receptor. Individual odorant receptors have overlapping but distinct ligand tuning properties. Accordingly, each OSN can be viewed as distinct information channel to the olfactory system. Using a combination of behavioral analysis and in vivo electrophysiology, we have undertaken a long-term project to disentangle the contribution of single OSNs to the representation of static and dynamic odor stimuli. In past works, we have shown that the information transmitted by a single functional OSN is sufficient to detect minute changes in odor intensity. Whether one type of odorant receptors alone is capable of encoding the quality of an odor remains an open question in Drosophila larvae. Here, we present evidence that a single functional OSN is sufficient to mediate odor quality discrimination

55

Marco Schubert, Silke Sachse & Bill Hansson

Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology
Hans-Knöll-Straße 8, D-07745 Jena, Germany
mschubert@ice.mpg.de

Dissecting the Banana: A Dynamic Gas-Chromatography - Optical Imaging Study

For an insect the natural environment holds a myriad of vital information coded in complex odor blends which are composed of many different single compounds. In order to identify these compounds and to characterize their activity patterns in the olfactory system, we established a new tool of odor coding investigation by combining gas chromatography (GC) and calcium imaging techniques. Using the model organism Drosophila melanogaster, we measured physiological responses of receptor neurons in the olfactory organ, the antenna, and of receptor neurons and projection neurons in the first olfactory neuropil, the antennal lobes. As stimulus we used a banana headspace extract which was either presented as a whole or fractured into its single components. To identify those odor components in the banana extract which evoke neuronal activity in receptor and projection neurons the extract was injected into the GC and from there directed to the fly’s antennae via a transfer line. During the stimulations we measured the intracellular calcium concentration changes in these neurons of the olfactory pathway. In a second step we identified the active components of the banana extract by coupled gas-chromatograph/mass spectrography (GC/MS) analysis. By disassembling the banana odor and identifying neuronal activation patterns of its single components we were able to compare an artificially reassembled pattern (simple additive pattern without any signal processing by the olfactory network) with the activation pattern elicited by the whole banana extract (putatively processed by the olfactory network). This puts us in a position to identify not only active key components in a natural odor blend, responding glomeruli and corresponding olfactory receptors, but also to investigate network properties leading to activation pattern processing in the antennal lobe. In that way we hope to elucidate the processes behind perception of complex natural blends and the role of the peripheral olfactory network.

56

Jonathan D. Bohbot and Joseph C. Dickens

U.S. Department of Agriculture, Henry A. Wallace Beltsville Agricultural Research Center, Plant Sciences Institute, Invasive Insect Biocontrol & Behavior Laboratory, Beltsville, MD 20705

Action of DEET on Mosquito Odorant Receptors

The mosquito Aedes aegypti is an important human pest as it vectors Yellow Fever, Dengue and other important diseases. Among control measures used to curtail the spread of mosquito-borne diseases are repellents as they decrease interactions between mosquitoes and their human hosts. The mechanism of action of the known repellent DEET is unclear and remains a subject of debate. We will present evidence for the action of DEET at the molecular level and propose a model for the observed effects of similar repellents on mosquito behavior.

57

Jonathan D. Bohbot and Joseph C. Dickens

U.S. Department of Agriculture, Henry A. Wallace Beltsville Agricultural Research Center, Plant Sciences Institute, Invasive Insect Biocontrol & Behavior Laboratory, Beltsville, MD 20705

Functional Analysis of a Mosquito Odorant Receptor

Odorant receptors (ORs) are classified as broadly or narrowly tuned depending on selectivity towards odor molecules. Enantioselectivity is a special case of molecular recognition, whereby a receptor protein discriminates between mirror image molecules; such examples abound in receptor-ligand interactions outside olfactory processes. While insects and vertebrates alike display such capabilities at the behavioral and physiological levels, the molecular basis for such selectivity has been scarce. We will provide evidence for an enantioselective OR in the Yellow Fever mosquito, Aedes aegypti, and identify structural features of the ligand required for receptor activation.

58

Chen, Yi-chun

University of Würzburg, Department of Genetics and Neurobiology, Biozentrum am Hubland, 97074 Würzburg, Germany
Chen.Yi-chun@biozentrum.uni-wuerzburg.de

A Role of Synapsin PKA-dependent phosphorylation in olfactory learning of larval Drosophila

Understanding the synaptic plasticity underlying learning, we focus on the molecular function of the evolutionary conserved presynaptic phosphoprotein Synapsin. Synapsin knock-out flies (syn97) are viable and show no obvious structural defects in brain anatomy, but are impaired in olfactory associative learning by approximately 50% (Michels et al., 2005). As Drosophila Synapsin includes phosphorylation consensus sites for PKA, Synapsin may function as downstream target of the cAMP-PKA cascade during memory formation. We currently investigate whether transgenic expression of Synapsin with non-phosphorylatable PKA consensus site(s) can rescue the syn97 learning defect. Furthermore, as one of the PKA consensus motives undergoes ADAR-mediated RNA editing (converting the protein motif from R-R-F-S into R-G-F-S) (Diegelmann et al., 2006), we ask whether transgenic expression of Synapsin carrying either of these motives can rescue the syn97 learning deficit.

In a further project, we test for the degree of odour-specificity of larval olfactory learning. Animals are trained to associate one odour (either 1-octanol, n-amyl acetate, 3-octanol, benzaldehyde, or hexyl acetate) with a reward, and tested for their preferences towards either the trained odour or a previously non-trained odour. It turns out that, when odour concentrations are used which are equal in their learnabilty, in most cases learned responses can hardly be retrieved by novel odours, reflecting preceived dis-similarity between trained and tested odours. An exceptional case seems to be n-amyl acetate and hexyl acetate. We interpret such general responses as reflecting perceived similarity between these two odours. Thus, olfactory learning in larval Drosophila seems to be rather specific with respect to odour
identity.

References:
Michels B, Diegelmann S, Tanimoto H, Schwenkert I, Buchner E, Gerber B (2005) A role for Synapsin in associative learning: the Drosophila larva as a study case. Learn Mem 12:224-231.
Diegelmann S, Nieratschker V, Werner U, Hoppe J, Zars T, Buchner E (2006) The conserved protein kinase-A target motif in synapsin of Drosophila is effectively modified by pre-mRNA editing. BMC Neurosci 14;7:76.

59

Philippe Lucas, Marta Grauso, Adeline Pézier and Jean-Pierre Rospars

INRA, UMR 1272 Physiologie de l’Insecte : Signalisation et Communication
Route de Saint-Cyr, 78000 Versailles, France
plucas@versailles.inra.fr

Properties of the Ca²⁺-dependent chloride current in olfactory receptor neurons of the moth Spodoptera littoralis

The response of moth olfactory receptor neurons (ORNs) is accompanied by an increase in intracellular Ca²⁺ concentration ([Ca²⁺]i). The molecular target of the Ca²⁺ rise remains elusive. We therefore studied Ca²⁺-activated currents with the whole-cell patch-clamp technique on cultured ORNs of the Noctuid moth Spodoptera littoralis.
Our experiments revealed that Ca²⁺ activates a chloride current in a dose-dependent manner with a K_½ of ca. 3 µM. The chloride current had a linear I-V relationship in symmetrical intra- vs extracellular chloride concentrations, it was voltage independent, it showed partial inactivation with time and it had an anion permeability ratio of I : NO3 : Br : Cl : MeS = 1.8 : 1.7 : 1.1 : 1 : 0.6. Pharmacological studies showed that this chloride current was reversibly inhibited by general chloride current blockers; it is insensitive to PKC inhibitors and activators and to CaMKII inhibitors.
This chloride current is cell-volume sensitive because its dependence to calcium was reduced in extracellular hyperosmotic solution and dramatically increased in extracellular hyposmotic solution with currents reaching 56.8 ± 4.5 pA/pF at 100 µM [Ca²⁺]i. Thus, the chloride current in S. littoralis ORNs doesn’t share all the properties of classical neuronal Ca²⁺-activated chloride channels (CaCC) but seems to behave as a Ca²⁺-gated chloride channel and a volume-regulated anion channel (VRAC). This current is most likely involved in the receptor potential generation because in vivo responses to pheromone stimuli were dependent on the chloride concentration in the sensillar lymph bathing ORN outer dendrites.
With the aim of identifying the molecular basis of the S. littoralis ORN chloride currents we screened by BLAST analysis a S. littoralis male EST antennal library including 20,000 clones. We used members of all known families of chloride channels and we found three whole cDNAs belonging to the sub-family of bestrophins. Their sequences share high similarity with the Drosophila Best1 and Best2 genes. RT-PCR demonstrated that they are expressed mainly in peripheral sensory tissues (antennae and proboscis) and in the ORN primary culture that we used for patch clamp experiments.

This work was supported by ANR-BBSRC Pherosys and FP7 Neurochem.

60

Lisa M. Dennison and Kristin Scott

Molecular and Cell Biology Department
University of California, Berkeley
16 Barker Hall #3204, Berkeley, CA 94720-3204, USA
lisadee@berkeley.edu

Dissecting neuronal specification in the Drosophila taste system

Understanding how neurons acquire their identities and how these identities are translated into functional differences is a long-term goal of developmental neuroscience. The Drosophila taste system is well suited to address this question because there are relatively few functionally distinct taste neuron classes. While the functional and molecular differences between the taste neurons occupying a single chemosensory bristle are apparent, the underlying mechanisms that lead to this neuronal diversity remain unclear. We hypothesize that taste neuron classes are specified via a stereotyped lineage and that Notch signaling is required for this process. Notch signaling plays a vital role in neuronal specification in many organisms and has been shown to be important for the specification of different classes of Drosophila olfactory neurons. We propose that Notch may also be necessary to diversify the taste lineage by asymmetrically activating downstream signals that lead to the expression of distinct taste receptors in neighboring neurons. Furthermore, based on differences between the olfactory and gustatory cell lineages, we propose that additional factors are also likely to be involved in determining taste neuron cell fates.

61

Koji Sato and Kazushige Touhara

Department of Integrated Biosciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
E-mail: satou-kouji@k.u-tokyo.ac.jp

Insect chemosensation via ligand-gated cation channels

The G-protein-signaling cascade is a conserved molecular basis for the chemical senses among various organisms from nematode to primates. In vertebrates, the binding of chemosignals to olfactory or gustatory receptors leads to activation of G-protein-mediated second messenger pathways, resulting in the opening of second messenger-gated cation channels that convey sodium and calcium currents, and thereby transform chemical signals into electrical neural activity. In contrast, although insect olfactory receptors (ORs) are classified into the seven-transmembrane receptor family, they lack homology to the G-protein-coupled receptor family and possess a distinct membrane topology with the intracellular N-terminus. Moreover, the functional insect OR consists of a heteromeric complex together with the Or83b family co-receptor, implicating that insects utilize a unique signal transduction mechanism different from that in vertebrates. Indeed, we found that stimulation of Xenopus oocytes expressing insect OR complex with the cognate ligand elicited unknown odor-induced inward cation currents at the resting potential, suggesting that insect OR complex conferred a novel cation channel activity. To characterize the channel properties, we next expressed various combinations of insect OR complex in mammalian cell lines. HeLa and HEK293T cells expressing fruit fly, silkmoth, and mosquito OR complex exhibited extracellular calcium influx and nonselective cation conductance upon odorant stimulation. Inhibitors for known G-protein-coupled second messenger pathways had no effect on the odorant response. The odorant response kinetics was completely different from that in vertebrate olfactory receptor neurons. Insect ORs expressed in HeLa cells showed no desensitization with faster response latency than that of the odor response of vertebrate olfactory sensory neurons. Ion permeability and the degree of response inhibition by a calcium channel blocker were different, depending on the OR subunit composition. These results suggested that OR complexes directly elicited G-protein independent responses. The evidence for the current production by OR complex itself was obtained from the outside-out single-channel recording of HEK293T cell membrane containing the OR complex. The pA order of spontaneous cation conductance was recorded, and its open probability increased upon stimulation with the cognate ligand. Our study demonstrates a novel molecular aspect of seven-transmembrane receptors in that insect OR complex are spontaneously-active odor-gated ion channels that likely regulate the receptor potential of olfactory receptor neurons.

62

Veit Grabe, Bill S. Hansson, Silke Sachse

Max Planck Institute Chemical Ecology, Department of Evolutionary
Neuroethology, Hans-Knöll-Str. 8, 07745 Jena, Germany
vgrabe@ice.mpg.de

In vivo imaging of GABA-related effects on odor-evoked chloride responses in the Drosophila brain

The life of an insect is determined by a complex network of attractive as well as repellent olfactory cues in addition to mechanosensory and optical information. To percept and process the high variety of volatile components, a highly diversified system is required to obtain reliable information of the surrounding, potential food sources or predators. The sensory level of this system is well examined from the antennal sensilla, gathering the odorants, over the olfactory receptors (ORs), interacting with the odor molecules, to the olfactory sensory neurons (OSNs) which project the information from the antenna to the antennal lobes (AL). The functionality of the neuronal components like local interneurons (LNs) and projection neurons (PNs) have been studied intensively, but the precise function of these neurons and their specific subsets is still under research.
We aim to investigate the inhibitory pathway throughout the AL in detail. It is known that the AL consists of GABAergic LNs as well as GABA-mediated neural connections, which are evenly distributed in the lobe. To understand the network of diversely interacting in particular inhibitory neuronal circuits in Drosophila, we analyzed the effect of several GABA-antagonists onto the coding of odors within the AL. Using Clomeleon, a genetically-encoded chloride sensor, we visualized inhibitory odor-evoked responses in OSNs and PNs by functional chloride imaging. We observed odor-specific chloride responses on the antennae as well as within the AL. By applying specific antagonists for GABAA and GABAB-type receptors, the source of odor-evoked chloride signals and the lateral inhibition could be analyzed for the different neuronal processing levels within the AL. The results will help to decipher the inhibitory interactions underlying olfactory coding and processing in Drosophila.

63

Tatsuro Nakagawa, Koji Sato, Kazushige Touhara

Department of Integrated Biosciences, Rm201
Graduate School of Frontier Sciences, The University of Tokyo 5-1-5 Kashiwanoha, Kashiwa, Chiba, Japan
e-mail: 87315@ib.k.u-tokyo.ac.jp

Putative Amino Acids Involved in Ion Channel Activities of Insect Olfactory Receptors

Insects sense odor and pheromone molecules with olfactory sensory neurons (OSNs) housed in olfactory organs. Most OSNs co-express two types of seven-transmembrane olfactory receptors (ORs); one is a canonical ligand-binding receptor, and another is a highly conserved Or83b family receptor. Canonical ORs and Or83b family receptors comprise heteromultimeric complex of unknown stoichiometry. We have demonstrated that the OR-Or83b complex are novel type of ligand-gated nonselective cation channels. However, a molecular basis underlying the ion channel activities of the OR-Or83b complex has remained to be elucidated. In the present study, we performed site-directed mutational analysis to identify amino acids involved in the ion pore formation in the BmOr-1 (bombykol receptor) + BmOr-2 (Or83b family receptor of Bombyx mori) complex. It has been reported that Glu, Asp, and Tyr residues play critical roles in ion permeations of various cation channels in pore domains. Therefore we examined effects of Glu, Asp, or Tyr mutations in BmOr-1 and BmOr-2, on ion permeability by using the Xenopus laevis oocyte expression system. Some mutations of amino acid residues in BmOr-1 and BmOr-2 resulted in altered current-voltage relationships. Furthermore, mutation of the corresponding amino acid residues in BmOr-3 (bombykal receptor) and Drosophila Or83b had similar effects, indicating that these amino acid residues may be involved in the ion permeation of the insect OR complex and functionally conserved. Our results provide structural insights into how activated OR-Or83b complex permeates cations.

64

Marit Stranden and Hanna Mustaparta

Norwegian University of Science and Technology, Dep. Biology, Trondheim, Norway marit.stranden@bio.ntnu.no

Physiologically and anatomically characterized olfactory antennal lobe neurons of the moth Heliothis virescens.

We are studying chemosensory coding in Heliothis virescens, a devastating agricultural pest insect. The goal in the present study is to understand how the primary olfactory center, the antennal lobe, process plant odor information and how the information is further mediated to higher olfactory centers in the brain. The neural network of the antennal lobe is formed by synaptic connections in the glomeruli between the receptor neuron terminals, local interneurons and projection neurons. Also terminals of modulatory neurons, GABAergic as well as peptidergic innervate the glomeruli in H. virescens (Berg et al. 2007 Cell Tiss Res 327:385-398, 2009 Cell Tiss Res 335:593-605). By intracellular recordings we characterize antennal lobe interneurons physiologically by testing for sensitivity to antennal stimulation with biologically relevant plant odorants. Receptor neurons, classified by the use of gas chromatography linked to single cell recordings, are narrowly tuned to one primary odorant and respond weaker to a few structurally related odorants (Røstelien et al. 2005 Chem Senses 30:443-461). The minimal overlap of the molecular receptive ranges of the different receptor neuron types indicates that the plant odor information is mediated to the antennal lobe by a labeled-line mechanism. In the present study the test protocol includes primary plant odorants and mixtures as well as pheromone components. The antennal lobe neurons are stained with fluorescent dyes for visualization in confocal laser scanning microscope followed by 3-dimensional reconstruction. The neurons are morphologically classified according to their innervations of glomeruli (uni- or multi-glomerular) and in which of the four antenno-cerebral tracts the axons project to the mushroom bodies (involved in olfactory learning) and the premotoric area in lateral protocerebrum (Rø et al. 2007 J Comp Neurol 500:658-675). The neurons identified in individual brains are integrated into the standard H. virescens brain atlas (Kvello et al. submitted). We here present physiologically and morphologically classified antennal lobe neurons integrated in the standard brain atlas. Most neurons described by this method belong to uniglomerular projection neurons which responded with excitation or inhibition to several of the primary odorants and blends. The results suggest interglomerular excitation as well as inhibition as input to the projection neurons in this moth species.
The project was supported by the Norwegian Research Council (nr. 170510).

65

Antoine Chaffiol¹, Sylvia Anton¹, Jean-Pierre Rospars¹ and Dominique Martinez²

1. INRA, UMR Physiologie de l'Insecte, Route de St Cyr, 78000 Versailles Cedex, France
2. LORIA Campus Scientifique BP239, F54506 Vandoeuvre-les-Nancy, France

achaffiol@versailles.inra.fr
santon@versailles.inra.fr
rospars@versailles.inra.fr
dominique.martinez@loria.fr

Spike timing precision of pheromone sensitive neurons in the antennal lobe of the moth Agrotis ipsilon

In order to locate their mates, male moths have to detect and interpret specific blends of odours -pheromones- released by the females and dispersed by the wind. In male moths, sex pheromones are detected by specialized olfactory receptor neurons situated in cuticular sensilla on the antennae and processed within the Macro-Glomerular Complex (MGC) of the antennal lobe.
In this study, we show a detailed analysis of the spike timing precision in pheromone sensitive neurons in the antennal lobe of the moth Agrotis ipsilon. We performed simultaneous extracellular recordings within the MGC from several neurons in vivo and identified individual spike trains using spike sorting algorithms. To analyse spike timing precision, a statistical tool was developed to segment and characterize individual spike trains. Our analysis reveals that MGC neurons have a stereotyped and synchronized response in the presence of pheromones. From repeated measurements of single odor stimulations, we show that the response is both precise (temporal jitter of spikes over trials < 4ms) and robust (probability of loosing spikes over trials < 0.1). Moreover, experiments conducted using different stimulation protocols with pheromone pulses reveal how the temporal pattern of a response to previous puffs could affect the responses to the following ones. Based on the stereotyped response and its extreme precision we draw conclusions on intrinsic properties of MGC neurons.

66

Andreas Reinecke¹, Anna Henning¹, Subaharan Kesavan², Shannon Olsson¹, Bill S. Hansson¹

1. Max Planck Institute for Chemical Ecology, Hans Knöll Str. 8, D-07745 Jena, Germany
2. Central Plantations Crops Research Institute, 671124 Kasaragpt, Kerala, India

areinecke@ice.mpg.de, ahenning@ice.mpg.de, subaharan70@yahoo.com solsson@ice.mpg.de hansson@ice.mpg.de

Oviposition in Manduca sexta: An emerging picture of behavioural responses, chemical stimuli, and their sensory perception

The choice of host plants is a critical task for female herbivorous insects. In the field, Manduca sexta hawkmoths are known to prefer to oviposit on plants from the Solanaceae and Martyniaceae families. Furthermore, gravid females avoid previously feeding-damaged plants since the latter recruit parasitoids and predators through the release of volatile secondary metabolites. However, a systematic analysis of female egg deposition behaviour and the underlying neuronal processing of volatile stimuli mediating attraction to the plants is lacking.
We report on female preferences with respect to (a) plant species, (b) caterpillar feeding-damage, and (c) chemically induced plant defence compounds. Odour profiles were specific both at the species and plant state level. By using combined gas chromatographic - electrophysiological techniques we identified a number of compounds from the plant headspace that were previously uncharacterised as volatile stimuli for the M. sexta female.
At the level of single sensillum recordings, the odorant bouquet from host plants elicited responses in a plant species-specific array of olfactory sensilla. However, olfactory sensory neurons from the same array of sensilla responded whether headspace volatiles were collected from feeding-damaged or undamaged individuals of the same plant species. The results suggest that patterns of activated olfactory receptor neurons may allow for plant species discrimination, while the assessment of plant state may require higher processing of olfactory information.

67

Yu-Tong Qiu

Laboratory of Entomology, Binnenhaven 7, 6700 EH, P.O. Box 8031, Wageningen, The Netherlands

Yu.tongqiu@wur.nl

Olfactory function of large coeloconic sensilla of the malaria mosquito Anopheles gambiae

The major African malaria vector Anopheles gambiae is nocturnal and uses predominantly olfactory cues to locate sugar sources, blood hosts and reproduction sites. Coeloconic sensilla are peg-in-pit structures and are thought to contain olfactory neurons. Because of their special morphology, extracellular recordings from these sensilla are technically difficult, possibly explaining why no functional studies on these sensilla have been published thus far.

The response pattern of neurons innervating the large coeloconic sensilla of female An. gambiae to a panel of 138 single compounds and headspace samples of human foot odours was studied using single sensillum recordings. Based on differences in spike amplitude, four neurons could be identified. The neuron with the largest spike amplitude only showed excitatory responses to two short-chain carboxylic acids. A second neuron displayed excitatory responses to about 30 compounds including carboxylic acids, aldehydes, amines and aromatics; furthermore, this neuron was excited by odours from a human-worn sock. Most of the active compounds were reported as human odours and some are known attractants for female An. gambiae. Active compounds were also applied in binary blends to study the occurrence of addition, synergism or antagonism. The results suggest that large coeloconic sensilla house at least two olfactory neurons and the responsiveness of these neurons to chemicals is well correlated with the behavioural attractiveness of these chemicals to the female mosquitoes.

68

Sonja Bisch-Knaden ¹, Silke Sachse ¹, Mikael Carlsson ², Marco Schubert ¹, Yuki Sugimoto ¹, Bill Hansson ¹

1. Dept of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany
2. Dept of Zoology, Stockholm University, Stockholm, Sweden

sbisch-knaden@ice.mpg.de

Comparative study of olfactory coding in the moth antennal lobe

Odor information in insects is coded in the primary olfactory processing centre, the antennal lobe, as a pattern of activated glomeruli. Among different moth species there are similar numbers of glomeruli, and thus presumably similar numbers of olfactory receptor types. We asked whether this concordance is also reflected in a similar coding pattern within the antennal lobe. Similarities or differences in these coding patterns might reflect the phylogenetic relationship of moth species and/or might depend on varying life histories, e.g. the diet of larvae and imagines.
Using calcium imaging we measured the spatial activity pattern evoked by a set of 14 common plant volatiles and floral odors of five chemical classes in the antennal lobes of female moths. We investigated moth species belonging to the phylogenetically remote superfamilies Bombycoidea (Manduca sexta, Acherontia atropos, Smerinthus ocellata) and Noctuoidea (Spodoptera littoralis, Spodoptera exigua).
Within the superfamilies the olfactory code was found to be very similar and to depend not on life history. For example, the coding pattern in M. sexta (nectar-feeding, oviposits on Solanaceae) matches the one of S. ocellata (non-feeding, oviposits on Salicaceae) better than the pattern of A. atropos (honey-feeding, oviposits on Solanaceae).
Between the two superfamilies significant differences were found for some of the odorants (C6-, C8-, C9-aldehydes, methyl salicylate) whereas the other tested compounds (C6-, C8-, C9-alcohols and -ketones, ß-caryophyllene, geraniol, linalool, phenyl acetaldehyde) were similarly coded. The main difference being that these odors evoked more distinct coding patterns in noctuoid moths compared to bombycoid moths.

69

Yoichi Seki, Jürgen Rybak, Dieter Wicher, Silke Sachse and Bill S. Hansson

Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, 07745 Jena, Germany

yseki@ice.mpg.de

Electrophysiology and morphometric analysis of local interneurons in the Drosophila antennal lobe

The Drosophila antennal lobe is an excellent model for investigating the neural mechanism of olfactory information processing. Inhibitory and excitatory populations of local interneurons (LNs) connect a large number of glomeruli and are postulated to play a substantial role in shaping odor information. However, single cell properties of the local interneurons have not yet been investigated in detail. We used whole-cell patch-clamp recordings combining functional characterization with 3D reconstruction morphological analysis and identified four classes of LNs with class-specific electrophysiological and morphological properties. Each class of LNs has unique characteristics in intrinsic electrophysiological properties such as the degree of spike adaptation and the amplitude of spike afterhyperpolarization. For example, one class of LNs shows burst activity while the others are tonically active. Neurons from three classes defined according to their morphological properties innervate almost all glomeruli, while LNs from one class innervate a distinct subpopulation of glomeruli. Furthermore, 3D reconstruction analysis revealed the detailed branching pattern of LNs in relation to the glomerular map and class-specific differences in dendritic density and distribution within a glomerulus. The current study gives a first characterization of LNs and provides essential information to reconstruct the antennal lobe neural circuits. We discuss functional implications of the different classes of LNs based on the physiological and morphological properties.

70

Keshava Mysore^{1, 2}, Philbert Ong Yijie³, Baragur V. Shyamala², K Vijay Raghavan¹, Veronica Rodrigues^{1, 4}

1. National Centre for Biological Sciences, Tata Institute for Fundamental Research, Bellary Road, Bangalore-560 065, INDIA.
2. Department of Zoology, University of Mysore, Manasagangothri, Mysore-570 006, INDIA.
3. Ngee Ann Polytechnic, Molecular Biotechnology, SINGAPORE.
4. Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai-400 005, India.
veronica@ncbs.res.in

Post-embryonic development of the olfactory system in the minor workers of the carpenter ant Camponotus compressus. (Fabricius, 1798)

We have traced the development of the antennal lobe of the carpenter ant- Camponotus compressus. In order to track the development of the olfactory sensory neurons (OSNs), we carried out retograde filling of the pupal antenna with micro-ruby. The neuropile of the antennal lobe was immunostained with a synapse-specific monoclonal antibody 7B1 which we obtained by screening a set of mAbs raised against ant brain homogenate.
The OSNs first enter the brain by stage 2 (20% of pupal life) and invade the antennal lobe neuropile by stage 3 (30% of pupal life) to form protoglomeruli. Glomerular formation is complete by stage 4 (~45% of pupal life). The role of the OSNs in patterning glomerular formation is being investigated. We will describe the details of the development and consolidation of glomeruli and follow the entry of a population of serotonergic and gabergic neurons. The close resemblance of development events during glomerular formation to that of Drosophila is intriguing and will be discussed.

71

Nick Bos¹, Annette Bruun Jensen² Thierry Lefèvre³, David Hughes⁴ and Patrizia d’Ettorre¹

1. Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark
2. Department of Agriculture and Ecology/Section of Zoology, Thorvaldsensvej 40, 1871 Frederiksberg C , Denmark
3. Biology Department, Emory University, 1510 Clifton Road, Atlanta GA 30322, US
4. Museum of Comparative Zoology, Harvard University , 26 Oxford Street, Cambridge MA 02138, US

Nbos@bio.ku.dk

The smell of infection in ant societies

Chemical communication is one of the most widespread forms of communication among living organisms, from bacteria, to plants, animals and also humans. Most animals are therefore able to discriminate among different odours. Chemical messengers, perceived via olfaction or taste, can be important for a range of behaviours, such as mating, feeding and recognizing friends and enemies. In social insects, chemical communication is especially important in the context of nestmate recognition. This is based on the between-colony variation of cuticular hydrocarbons, which, despite being long-chain molecules covering the insect body, appear to be detected via olfaction. Social insects are typically characterized by large communities of related individuals, thus only if group members (nestmates) are well discriminated from non-nestmates, individuals will be successful in protecting their colonies against intruders and parasites and in directing altruistic acts to related individuals. Large societies are also highly attractive targets for pathogens. The honeybee genome revealed that this species has less genes associated with the innate immune response than solitary insects. This surprising finding implies that different strategies may be employed by social insect communities in order to protect themselves against pathogens. Behavioural responses, for example, can contribute to the social immune system when infected individuals are detected and either cared for or excluded from the society. This raises the question of how infected individuals are recognized as being infected. In this study we investigated whether ants infected with a pathogenic fungus change their cuticular hydrocarbon profile. We infected individual Formica rufa ants with a pathogenic fungus (Metarhizium anisopliae or Beauveria bassiana) and analysed their cuticular hydrocarbon profile during the course of infection using gas-chromatography and mass spectrometry. Also, behavioural experiments were done, in order to elucidate the effect of infection on behaviour of nestmates. Our results shed new light on how the immune response of social insects can be so effective, even though the innate immune system appears to be less complex than in solitary insects.

72

Isabella Urru, Johannes Stökl, 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

Inter-and intraspecific variation within the genus Arum

The genus Arum is known to display a non-rewarding-brood-site mimicking pollination systems. A study conducted on five different species in the Island of Crete, shows that they vary in smell, pollinator species as well as in habitats. At least two different pollination types are represented on the island: “dung/urine”, pollinated by flies and beetles; “sweet”, pollinated by bees and bugs. Accordingly, the emitted smell during the one-day flowering period differs considerably between species. Odour volatiles are the typical “dung compounds” as well as more “flower-like” fruity-sweet ones. A broad spectrum of the emitted chemicals can be detected by three ecologically different groups of insects. The pronounced differences found among the species represent a clear example of interspecific isolation between related species.
The genus also contains a good example of intra-specific variation. So called “ecotypes” can be distinguished within the same species. Field experiments performed on four different A. discoridis populations suggest an ongoing process of adaptive speciation in this species.

73

Johannes Stökl¹, Amots Dafni², Markus Knaden¹, Antonia Strutz¹, Silke Sachse¹, Aleš Svatoš¹, Isabella Urru¹, Marcus C. Stensmyr¹, and Bill S. Hansson¹

1. Max-Planck-Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany
2. Institute of Evolution, Haifa University, Haifa 31905, Israel

Lambrusco mimicry? - The chemical ecology of the Drosophila-pollinated Arum palaestinum.

Drosophila melanogaster and D. simulans are widespread and common around the world. Despite this high availability they rarely act as pollinators of flowers. Arum palaestinum (Araceae) occurs in the Middle East and its flowers produce a wine or rotten fruit like odor to attract mainly D. melanogaster and D. simulans for pollination. The floral odor is believed to mimic the breeding substrate of the flies. The flowers are extremely effective in trapping flies. During the short anthesis of 24hours a single flower can attract and catch more than 500 flies.
By electrophysiological experiments (GC-EAD) we could identify seven compounds in the head-space of A. palaestinum that can be perceived by D. melanogaster and D. simulans. Behavioral assays showed the synthetic mixture to be highly attractive for D. melanogaster. Attractiveness rises with concentration, reaching the same level of attractiveness as a banana or the best commercial Drosophila trap. Further experiments with subtractive mixes and singe compounds could not identify a single key compound. The complete mixture seems to be the key to the high attractiveness. This is also supported by calcium imaging data.
To identify the model in this mimicry system we are comparing the floral odor of A. palaestinum with the odor of different potential mimicry models, including various (rotten) fruits, vinegar, and wine.

74

Sofia Lavista Llanos, Marcus C. Stensmyr and Bill S. Hansson

Max Planck Institute for Chemical Ecology, Jena, Germany
slavista-llanos@ice.mpg.de

Gene expression in olfactory organs of Drosophila siblings

Species host-specialization entails divergent developmental programs that allow reconciliation of unique physiological, anatomical and behavioural features with the new preference. Drosophila sechellia presents several appropriate characteristics for its particular niche, Morinda citrifolia fruit, which is toxic and aversive to other Drosophila siblings (simulans, mauritiana, melanogaster): resistance to morinda toxic-compounds and a shifted odorant preference to specific fruit components.

To assess the molecular basis of sechellia olfactory-behavioral specialization we compared the gene expression in the olfactory organs (antennae and maxillary palps) and in the brain of four different Drosophila siblings: sechellia, simulans, mauritiana and melanogaster; assayed by RNA micro-array studies on melanogaster-genome designed microchips. Consistent with our expectations, the expression levels of most known olfactory genes in the antennae samples were >2-fold higher than in brains, for the four siblings assayed; albeit a general lower expression in non-melanogaster samples. To account for possible mismatches between non-homologous genes we performed a separate analysis with those genes showing a perfect match between sechellia, simulans and melanogaster. A Gene Ontology Enrichment Analysis identified several biological processes (fatty-acid biosynthesis, response to toxin/chemical stimulus and defense response) as well as various molecular functions (odorant binding, choline and glutamate metabolism, potassium channel regulatory activity and glucose transmembrane transport) to be differentially expressed in sechellia vs generalist -antennae.

To narrow down our differential gene-expression study to olfactory-receptor neurons (ORN), we have applied an mRNA tagging method for profiling the gene expression of the ORN population in the antennae and maxillary palps of Drosophila melanogaster. The purified ORN-transcriptome will be analyzed by direct sequencing; with the aim of pinpointing genes involved in the olfactory-signal transduction cascade, in Drosophila melanogaster olfactory neurons.

75

Sophie Kromann¹, Jan Bert Gramsbergen², Bill S. Hansson³ and Rickard Ignell¹

1. Institute of Chemical Ecology, Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden.
2. Institute of Medical Biology, Department of Anatomy and Neurobiology, University of Southern Denmark, Odense, Denmark.
3. Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Jena, Germany.
Sophie.Kromann@ltj.slu.se

Moths that a garment fret – neuromodulation of olfaction in mated Spodoptera littoralis.

The behaviour of moths is to a large extent regulated by olfactory stimuli. Moths depend on volatile cues to locate mates, food sources and oviposition sites. Most research on olfaction in moths has until recently focused on male responses to pheromone cues emitted by conspecific females. In contrast, we know little about the olfactory system of female moths and how this system is modulated by neuroactive substances in response to changes in their physiological status, e.g. mating.
Recently, wind tunnel experiments have shown that mated female Spodoptera littoralis are activated stronger, will fly for longer time periods and have a higher landing percentage on cotton plants than unmated females (S. Ahmed, pers. comm.). The observed behavioural difference has been shown to be reflected at the antennal level, where electoantennogram (EAG) studies show that mated females are more sensitive to cotton-related odours than unmated females (V. Martell, pers.comm.). It still remains to be examined whether these differences also are reflected in neuronal populations or single neurons at the central nervous system level. We hypothesise that neurohormones or neuromodulators, e.g. biogenic amines, could act as the effectors in this system. Serotonin, a biogenic amine known to alter sensitivity in olfactory neurons, is a prime candidate along with the other biogenic amines, dopamine, tyramine and octopamine.

Using an array of ecologically relevant odours, we are studying the effect of mating status on olfactory processing in the moth S. littoralis. Using electrophysiology, pharmacology, immunohistochemistry and chromatography, we are correlating the mating status of both male and female S. littoralis to the identification and quantification of biogenic amines in the antennal lobes. This will help us understand the significance of mating status in relation to biogenic amine modulation of the olfactory system in moths.

76

Richard A Fandino¹, Jackson Sparks¹, Jon Staples¹, Joseph C Dickens², Richard G Vogt¹

1. Dept of Biological Sciences, University of South Carolina, Columbia SC 29208, USA.
2. USDA, ARS, BARC, 10300 Baltimore Avenue, Beltsville, MD 20705, USA.

FANDINO@BIOL.SC.EDU

Transcriptional regulation of chemosensory SNMP2 in Drosophila melanogaster

Sensory neuron membrane proteins (SNMPs), are a two-transmembrane protein associated with chemoreception a variety of insects. Analysis of the genomes from species throughout the Holometabola reveal two distinct and conserved groups of SNMPs: SNMP1 and SNMP2 (Vogt et al., 2009). Drosophila melanogaster SNMP2 promoter-GAL4 UAS-cd8GFP expression lines suggest that SNMP2-Dmel is expressed in neurons of coeloconic olfactory sensilla the antennae, as well as in chemosensory neurons of the palps, proboscis, legs and wings. We have initiated a dissection of the D. melanogaster snmp2 promoter has been initiated to identify the transcriptional regulatory elements responsible for this observed pattern of expression. Promoter-GAL4 D. melanogaster transgenic lines were constructed around conserved regions shared between D. melanogaster and D. pseudoobscura, establishing a driver series ranging from ~0.2-5 kb upstream of the transcriptional start site. These animals were crossed with UAS-cd8GFP flies to estimate the influence of these regions on SNMP2Dmel expression. We observed that a region between ~0.5 kb and ~0.9 kb upstream of the SNMP2Dmel transcriptional start site is necessary and sufficient for expression of the "normal" SNMP2Dmel pattern. Future studies, will further refine this region, in order to find what transcriptional elements are necessary to provide the phenotypic distinctions in D. melanogaster sensilla, and allow us to compare the regulatory mechanisms of other genes which co-express in the same or sensilla as SNMP2, such as SNMP1, ORs, GRs and OBPs.

Vogt et al., (2009) Insect Biochem Molec. Biol. (doi:10.1016/j.ibmb.2009.03.007)

Support: NSF (RV), USDA (RGV) and the Deployed War-Fighter Protection Research Program (US Dept. of Defense) through the Armed Forces Pest Management Board (JCD).

77

Richard G Vogt¹, Jackson Sparks¹, Richard Fandino¹, Jon Staples¹, Joseph C Dickens²

1. Dept of Biological Sciences, University of South Carolina, Columbia SC 29208, USA.
2. USDA, ARS, BARC, 10300 Baltimore Avenue, Beltsville, MD 20705, USA.

VOGT@BIOL.SC.EDU

SNMP Gene Family: Evolution, Expression and Function

Chemical cues seem detected by biochemical networks of chemosensory proteins; and while the functional role Odor and Gustatory Receptors are relatively easy to define within these networks, the exact function of other proteins such as Odorant Binding Proteins and Odor Degrading Enzymes remains less clear. These presumably non-receptor proteins might thus be viewed as Chemosensory Orphans, as we continue to search for their specific functions. SNMPs are certainly in this category. Initially identified in Lepidoptera (Rogers et al. 1997, 2001a,b; 2001, Forstner et al. 2008) where they were proposed to play some key role in odor detection (based largely on spatial-temporal expression patterns), we have identified SNMP orthologues throughout the Holometabola (incl. Lepidoptera, Diptera, Hymenoptera, Coleoptera) (Vogt et al., 2009). Two reports (Benton et al., 2007; Jin et al., 2008) have demonstrated that the Drosophila melanogaster SNMP1 orthologue is expressed in trichoid sensilla (along with OR67D and the OBP LUSH), and is required for the detection of the OR67D pheromone ligand 11-cis vaccenyl acetate (cVA). We have been characterizing the spatial patterns of SNMP expression in D. melanogaster; these studies suggest a much broader role for SNMPs. These findings will be presented along with a discussion of alternative functional roles the SNMPs may be playing in chemoreception.

Benton et al. (2007) Nature 450, 289; Jin et al. (2008) Proc Natl Acad Sci U S A. 105, 10996; Forstner et al. (2008) Chem. Senses 33: 291. Rogers et al. (1997) Journal of Biological Chemistry. 272, 14792. Rogers et al. (2001a) Cell and Tissue Research. 303, 433. Rogers et al. (2001b) Journal of Neurobiology. 49, 47. Vogt et al., (2009) Insect Biochem Molec. Biol. (doi:10.1016/j.ibmb.2009.03.007)

Support: NSF (RV), USDA (RGV) and the Deployed War-Fighter Protection Research Program (US Dept. of Defense) through the Armed Forces Pest Management Board (JCD).

78

Jackson Sparks¹ , Richard A Fandino¹, Jon Staples¹, Joseph C Dickens², Richard G Vogt¹

1. Dept of Biological Sciences, University of South Carolina, Columbia SC 29208, USA.
2. USDA, ARS, BARC, 10300 Baltimore Avenue, Beltsville, MD 20705, USA.

SPARKSJ@BIOL.SC.EDU

Expression patterns of the SNMP1 and SNMP2 in Drosophila melanogaster.

SNMPs (Sensory Neuron Membrane Proteins) are membrane proteins observed to associate with chemosensory sensilla in insects. SNMPs belong to a gene family characterized by human CD36 which has diverse functions including transport of cholesterol and other fatty acids, cell-cell interaction (presumably through protein-protein interaction), and taste transduction of fats. CD36 family members have been characterized in species throughout the Holometabola, and include a distinct SNMP group, divided into two sub-groups: SNMP1 and SNMP2 (Vogt et al., 2009). In lepidoptera, SNMP1 and SNMP2 are about 25%-30% identical (amino acid sequence) and appeared to specifically associate with antennal pheromone sensilla (Rogers et al., 1997, 2001a,b); the two genes were more recently observed to differentially express, SNMP1 expressing in neurons and SNMP2 expressing in support cells (Forstner et al., 2008). In Drosophila melanogaster, SNMP1 and SNMP2 are also about 25%-30% identical. SNMP1-Dmel was recently shown to be essential for the detection of the pheromone cis-vaccenyl acetate (Benton et al., 2007; Jin et al., 2008). We have characterized the expression of SNMP1-Dmel and SNMP2-Dmel, using a promoter-GAL4, UAS-cd8GFP system, supported by immunohistochemistry and tissue specific PCR. SNMP1-Dmel and SNMP2-Dmel associate with a broad array of chemosensory sensilla both olfactory and non-olfactory, in both neurons and support cells; expression is typically non-overlapping. The specific expression patterns and possible functional associations will be discussed.
Benton et al. (2007) Nature 450, 289; Jin et al. (2008) Proc Natl Acad Sci U S A. 105, 10996; Forstner et al. (2008) Chem. Senses 33: 291. Rogers et al. (1997) Journal of Biological Chemistry. 272, 14792. Rogers et al. (2001a) Cell and Tissue Research. 303, 433. Rogers et al. (2001b) Journal of Neurobiology. 49, 47. Vogt et al., (2009) Insect Biochem Molec. Biol. (doi:10.1016/j.ibmb.2009.03.007)

Support: NSF (RV), USDA (RGV) and the Deployed War-Fighter Protection Research Program (US Dept. of Defense) through the Armed Forces Pest Management Board (JCD).

79

Mattias C. Larsson¹, Agnieszka Ruebenbauer², Lina Bryngelsson¹, Alexandra Schmidt¹, Paul Becher¹, Fredrik Schlyter¹, Peter Witzgall¹, Bill S. Hansson^1,3, Christer Löfstedt²

1. Dept of Plant Protection Biology, Chemical Ecology, Swedish University of Agricultural Sciences, Alnarp, Sweden
2. Dept of Ecology, Chemical Ecology, Lund University, Lund, Sweden
3. Dept of Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany

Mattias.Larsson@ltj.slu.se

Genetics and context influence hierarchical use of olfactory information in Drosophila

The fruit fly Drosophila melanogaster, like most other insects, is very selective in its use of olfactory information. This species is usually referred to as a host generalist, feeding and ovipositing on a wide range of different fruits and decaying organic material. Nevertheless, its exploitation of different food resources is likely governed by common chemosensory signals that are to a great extent shared by all these resources. We have used complex natural odours and single synthetic odours to determine response characteristics to olfactory stimuli in different contexts, ranging from attraction to oviposition. We demonstrated that truly wild type D. melanogaster is very selective in its attraction towards olfactory stimuli, with natural odours from optimal hosts being far more attractive than single synthetic stimuli. There is great variation among laboratory strains of D. melanogaster, however. Some of these strains retain a conservative wild-type phenotype whereas others are more non-selective and respond in a more generalist fashion to a wider range of stimuli almost as readily as to optimal olfactory stimuli. This broad degree of generalism in non-selective strains is limited to olfactory attraction, however. In their oviposition behaviour, both selective and non-selective strains were much more conservative than in attraction assays. Oviposition appears to be mediated by an even more narrow range of olfactory stimuli than attraction from a distance.

80

Mattias Alenius¹ and Barry Dickson²

1. IKE, Linkoping university, SE-581 85 Linkoping, Sweden
2. IMP, Dr Bohr g. 8 A-1030 Wien, Austria

Regulation of odorant receptor choice in Drosophila Melanogaster

One of the most intriguing problems in sensory neurobiology is the problem of odorant receptor (Or) choice. In the Drosophila olfactory system each of the 1300 odorant receptor neurons chooses to express one of the 63 Ors in the genome. The neurons expressing one particular odorant receptor are highly stereotype between animals in both antenna location and in numbers, thus suggesting that Or choice is a highly regulated deterministic process. We have performed an RNAi screen for genes involved in Or expression and identified several Transcription factors that give strong loss of Or expression phenotypes. Only one of these factors has been previously shown to regulate Ors, Acj6. At the meeting the possible combinatorial aspects of Or expression will be presented and discussed.

81

Carlos Ribeiro^{1 2} and Barry J. Dickson²

1. Behavior and Metabolism Laboratory, Champalimaud Neuroscience Programme, Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, P-2781-901 Oeiras, Portugal
2. Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, A-1030 Vienna, Austria

Carlos.Ribeiro@fchampalimaud.org

Sex peptide receptor and neuronal TOR/S6K signaling modulate value-based nutritional decisions in Drosophila

Animals evaluate the costs and benefits of alternative actions in order to choose the behavior which maximizes individual fitness and reproductive success. One of the most vital resources organisms need is food. Because food sources differ in their content of carbohydrate and protein, their value to the animal varies according to its current nutrient requirements. The chemical senses of olfaction and taste play a major role in the selection of the appropriate food sources needed to satisfy the specific nutritional requirements of the individual organism. Food choice thus provides a powerful model for exploring the neural mechanisms of value-based decision making and sensory processing. We have established a feeding preference assay which allows the study of nutritional value-based decision making in Drosophila melanogaster. Deprivation of nutrients induces specific changes in the feeding preferences of flies opening the possibility to analyze how a specific metabolic status of the fly affects food choice behavior. We show that in females mating status modulates changes in food preference. This modulation is mediated by the Sex peptide receptor (SPR) acting in a small number of internal ppk+ neurons. These neurons project to the suboesophageal ganglion a region of the central nervous system involved in taste information processing where they could act directly to modulate nutritional decision making. We furthermore show that by modifying neuronal TOR/S6K signaling we are able to specifically modulate feeding decisions. These results uncover this highly conserved nutrient sensing pathway as a likely direct neuronal sensor of the internal metabolic status of the fly. Together with a whole genome neuronal RNAi screen for genes controlling value-based nutritional decisions as well as Gal4 screens for involved neurons these results provide a framework for studying value-based decision making at the molecular, cellular and circuit level using the powerful neurogenetic tools available in Drosophila.

82

Volker Nehring¹, Fernando J. Guerrieri¹, Giovanni Galizia² & Patrizia d'Ettorre¹

1. Centre for Social Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark.
2. Lehrstuhl für Neurobiologie,Universität Konstanz, 78457 Konstanz, Germany

vnehring@bio.ku.dk

A new model for nestmate recognition: ants recognise foes, not friends

The stability of every society depends on the reliability of it's recognition system, as only discrimination against intruders can prevent the exploitation of the society's resources. In social insects, the basis for nestmate recognition is assumed to be the comparison of cuticular hydrocarbons (label) of an encountered individual with a neural representation of the recognition cues (template) of the discriminating individual. Insects whose labels match the template are accepted as nestmates, and those whose labels are dissimilar to the template are not accepted in the colony. This would mean that individuals had to achieve the label/template comparison carefully for each encountering individual, a complex process that would engross a number of neural resources.
Here, we propose a more parsimonious mechanism for nestmate recognition: the “undesirable cue present” model. Discriminators would not compare their template to every encountered label in its whole complexity, but rather “screen” encountering individuals for cuticular substances that are novel or present in higher abundances relative to the template. This implies that discriminators would overlook individuals whose labels are lacking substances relative to the discriminator template and would detect only intruders bearing substances that are not present in the template (or are lower in abundance). We tested the model by manipulating encountering nestmates of the carpenter ant Camponotus herculeanus whose labels have been modified to differ in the presence and absence of one hydrocarbon only.

83

Romina B. Barrozo, Christophe Gadenne & Sylvia Anton

INRA, UMR 1272 Physiologie de l'Insecte: Signalisation et Communication, Route de St Cyr, F-78026 Versailles Cedex, France. e-mail: rbarrozo@versailles.inra.fr

Post-mating sexual abstinence in an insect: a new case of olfactory plasticity

In animals, sex pheromones are generally considered as attractants and play a key role in the encountering of sexual partners, ultimately leading to copulation. Male reproductive success depends not only on the ability to locate and copulate with a female, but also on their ability to effectively transfer an ejaculate. However, males are limited with respect to the number of ejaculates they can deliver and the time required to restore depleted reserves. Mating is costly and males should avoid direct remating until they have replenished their reproductive tracts in order to remate without loss of efficacy. Although males of many species are known to enter a post-ejaculatory refractory period, the mechanisms that lead to this sexual abstinence are far from being understood. In the moth, Agrotis ipsilon, newly-mated males cease to be attracted to the female-produced sex pheromone, allowing them to “wait” until the next night. This behavioral plasticity is accompanied by a decrease in neuron sensitivity within the primary olfactory centre, the antennal lobe (AL). However, it was not clear if the absence of behavioral attraction after mating might on one hand result from insufficient sensory input due to the low sensitivity of central neurons (i.e. they don’t smell). On the other hand, even if high pheromone doses provide sufficient sensory input, the sex pheromone might be ignored by the male or might in turn suppress male attraction to females. To test these possible scenarios, we chose a strategy in which the sex pheromone was tested alone or in combination with a non-pheromonal type of attractant (flower odor) in virgin and newly-mated males. Our results provide evidence for a neuroethological mechanism inducing post-mating sexual abstinence in male moths, which helps us to understand how an individual may enhance its probability of surviving to the next reproductive opportunity and increase its amount of energy available to undergo a next reproductive event, by skipping unsuccessful reproduction.

84

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

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

remco.suer@wur.nl

Olfactory coding in trichoid sensilla of Anopheles gambiae s.s.

The malaria mosquito Anopheles gambiae is an anthropophilic mosquito highly adapted to transmit Plasmodium falciparum, the parasite causing the deadliest form of malaria. Resource-finding behaviour of mosquitoes, such as sugar feeding, host seeking, mating or egg laying, is mediated mainly by olfactory cues. Understanding olfactory coding in Anopheles gambiae will provide better means for the disruption of odour-mediated behaviour, a possible strategy to diminish malaria transmission.
Using single sensillum recording (SSR) the function of the olfactory receptor neurons innervating trichoid sensilla type B and D was studied. Based on an odour panel of more than 130 compounds, including known and potential infochemicals for female An. gambiae, 5 different functional types for trichoid sensilla type B and 4 functional types for trichoid sensilla type D were found. All functional types of trichoid sensilla D responded to more than 20 compounds. With the exception of one specific functional type, the response spectra of the other 4 functional types of B type trichoid sensilla were more broadly tuned than the D-sensilla, responding to more than 50 odours. Next to these characterization studies the responses of olfactory receptor neurons to binary blends were also studied to better understand the mechanisms of odour coding to odour mixtures in the peripheral olfactory system.

85

Mark Stopfer

National Institutes of Health, NICHD, Bethesda, MD, USA
stopferm@mail.nih.gov

Odor elicited oscillations in insects

Odor elicited oscillations have previously been characterized in the locust and the honeybee. Recently our lab has revealed that Manduca sexta and Drosophila also demonstrate very similar oscillatory responses that result from similar neural mechanisms. In all these species, we found that common odors at natural concentrations evoke neural oscillations. Upon odor stimulation, oscillations are generated by neural circuits in the antennal lobe, and are transmitted downstream to the mushroom bodies, where oscillating local field potentials can be recorded. Paired intracellular recordings from local and projection neurons within the antennal lobe revealed odor-elicited spikes and subthreshold membrane potential oscillations that were phase-locked to local field potentials oscillations recorded in the mushroom bodies. The oscillations were reversibly blocked by application of the GABAa receptor antagonist picrotoxin. In Drosophila, by conditionally and reversibly blocking the chemical transmission from genetically targeted populations of antennal lobe local neurons, we identified a specific class of widely-branching GABAergic neuron necessary for producing the oscillations. A computational model suggests these oscillations may be necessary for maintaining the sparseness of odor responses across wide ranges of odor concentration.

86

Giorgia Sollai¹, Paolo Solari¹, Francesco Loy², Carla Masala¹, Roberto Crnjar¹, Anna Liscia¹

1 Dept of Experimental Biology, University of Cagliari, Cittadella Universitaria di Monserrato, SS. 554 Km 4.500, I-09042 Monserrato (CA) ITALY.
2 Dept of Cytomorphology, University of Cagliari, Cittadella Universitaria di Monserrato, SS. 554 Km 4.500, I-09042 Monserrato (CA) ITALY.
liscia@unica.it

Olfactory responses to aggregation pheromones and kairomones in Culicoides imicola, vector of the blue tongue disease: electrophysiological and behavioural evidence.

The aim of this study was to examine the role of the olfactory system of the midge Culicoides imicola Kieffer, vector of the blue tongue virus (BTV) disease for sheep, as the major system mediating the search of potential hosts and of oviposition sites.
By means of electrophysiological techniques - electroantennogram (EAG), electrogram from the ovipositor (EOvG), single-sensillum recording (SSR) - and a behavioural approach, we found that both the olfactory antennal and ovipositor sensilla of C. imicola are involved in the search of the host and oviposition sites. In particular, our electrophysiological results show that both kairomones (L-lactic acid, 1-octen-3-ol, 3-ethylphenol, 4-propylphenol) and pheromones (heptadecane) stimulate the olfactory system of C. imicola, as previously suggested in C. impunctatus and C. nubeculosus (Mordue, 2003; Sollai et al., 2007).
Behavioural field tests strengthen the above mentioned results: in fact, the number of insects captured with traps baited with kairomones was higher than that of the control and, in the traps baited with heptadecane only females were captured, thus candidating this compound as an aggregation pheromone.
In conclusions, both the ovipositor and the antennal sensilla are involved in the search of hosts, oviposition sites and possibly act as an aggregation factor among conspecific females of C. imicola.

Mordue Luntz A.J. (2003). Arthropod semiochemicals: mosquitoes, midges and sealice. Biochem. Soc. Trans. 31:128-133.
Sollai G., Solari P., Masala C., Crnjar R., Liscia A. (2007). Effects of avermectins on olfactory responses of Culicoides imicola (Diptera: Ceratopogonidae). J. Med. Entomol. 44(4):656-659.

87

Yuqiao Gu, Philippe Lucas and Jean-Pierre Rospars

UMR1272, Physiologie de l’Insecte, INRA, 78026 Versailles Cedex, France

ygu@versailles.inra.fr

A model of the moth pheromone receptor neuron within its sensillar environment

Male moth olfactory receptor neurons (ORNs) sensitive to the sexual pheromone offer a favourable system to analyze the intricate biochemical and electrical events involved in olfactory transduction. Based on the wealth of experimental data available in our and other laboratories we are developing step by step a model of olfactory transduction processes in these ORNs.
Previously (Gu, Lucas and Rospars, PLoS Computational Biology 5(3): e1000321, 2009) we studied (i) the biochemical network involved in the perireception reactions (pheromone uptake, transport and deactivation), (ii) the initial membrane reactions (receptor opening a cationic channel either directly or indirectly via G-protein and phospolipase C producing second-messengers, IP3 and DAG), and (iii) the chemo-electrical network which includes depolarizing channels (notably receptor- and DAG-dependent cationic channels and calcium-dependent chloride channels), as well as various inactivating and regulating feedback mechanisms (e.g. calcium and voltage-dependent potassium channels). This model accounts quantitatively for the magnitude and time evolution of the experimentally measured receptor potential (RP).
In the present work we extend these investigations in three main directions. First, we go beyond the classical approach, which is restricted to a single patch of membrane, by considering the spatial organization of the system. The long outer dendrite, which contains the main transduction machinery, was modelled by a series of compartments. The location of channels in different ORN parts, the ligand-gated channels at the outer dendrite and the voltage-gated channels at the inner dendrite and soma, was taken into account. The global sensillum organization with ORNs, auxiliary cells, sensillar lymph and hemolymph, was also included. Second, we specified the most important mechanisms involved and so developed an equivalent but simpler model of RP generation. Third, we started to analyse patch-clamp data of ionic channels involved in action-potential (AP) generation. For both RP and AP submodels, new or better estimates of their unknown parameter values were obtained by fitting the models to experimental data.
These developments provide new insights on the moth olfactory transduction and, most importantly, suggest new experiments. They offer a useful reference in our continuing investigations of olfactory neural coding, its molecular mechanisms and information-processing significance.

This work was supported by ANR-BBSRC Pherosys and FP7 Neurochem.

88

Kübler LS, Olsson SB, Hansson BS

Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany.
lkuebler@ice.mpg.de

Mixture Interactions in the Moth Antennal Lobe

Natural odors cues are often multi component blends and animals typically perceive their olfactory environment as a complex mixture. Despite this fact, most studies concerning olfactory coding have considered single components. In insects, the initial representation of odors occurs in the first olfactory neuropil, the antennal lobe (AL), where three classes of neurons form synapses: olfactory sensory neurons (OSNs), projection neurons (PNs) and local interneurons (LNs). The resultant neural representation of an odor mixture in the AL may either retain simple single-odor information of blend components, or reveal mixture-specific interactions due to odor information processing in the AL. The goal of our study is to reveal mechanisms of host odor information processing in the AL of the hawk moth, Manduca sexta. Using a novel multicomponent stimulus system and intracellular recording, we analyze the fine representation of blends vs. single components in individual antennal lobe neurons (LNs, PNs) responding to combinations of up to seven behaviorally relevant host volatiles. We examined different temporal characteristics (e.g. spike frequency, latency) and observed that most neurons display different types of blend interactions. Moreover, we found that a single morphological type of neuron can exhibit diverse blend interactions including both inhibitory and excitatory response profiles.

89

Tina Boddum¹, Bill S. Hansson², Niels Skals³ and Ylva Hillbur¹

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

Tina.boddum@ltj.slu.se

Gall midge pheromone reception – a comparison of SSR from two species

Gall midges (Diptera: Cecidomyiidae) are tiny insects, only 1-2 mm. They have a short life span of 1-2 days. Within this limited time, male midges have to locate a female ready to mate and the females have to find a suitable oviposition site. In these tasks the gall midges are guided by olfaction (e.g. Birkett et al., 2004 and Hillbur et al., 1999).

The gall midge antenna has been reported to house different types of sensilla commonly found in other insects, e.g. s. trichodea, coeloconica and chaetica. However, gall midges also have a unique sensillum type called sensilla circumfila (e.g. Crook and Mordue, 1999). Sensilla circumfila consist of individual sensilla that bifurcate close to the antennal surface and then fuse with the neighboring sensilla, resulting in a looped structure that encircles each antennal segment. In some gall midge species s. circumfila exhibit a distinct sexual dimorphism, being highly enlarged in males, whereas the difference is less pronounced in other species. Sexual dimorphism is also observed in the number of s. trichodea. When the male s. circumfila is enlarged, s. trichodea occur in larger number in females than in males. In species where s. circumfila are similar in size and number in males and females, the number of s. trichodea is higher in males.

We tested the hypothesis that pheromone receptors are located in the sensillum type most prominent in the males. We studied two gall midge species, Contarinia nasturtii and Mayetiola destructor. C. nasturtii is a species with sexually dimorphic s. circumfila, whereas the numbers of s. trichodea differ between the sexes in M. destructor. Single sensillum recordings (SSR) were conducted on males from both species. Scanning (SEM) and transmission electron microscopy (TEM) were used for a detailed study of sensilla morphology and innervation. Our results confirmed that the two tested species utilize different sensilla for pheromone detection. Olfactory receptor neurons (ORNs) in C. nasturtii s. circumfila responded to the sex pheromone whereas ORNs in s. trichodea govern that function in M. destructor.

- Hillbur, Y., Anderson, P., Arn, H., Bengtsson, M. & Löfqvist, J. (1999). Naturwissenschafen 86. 292-294.
- Birkett, M., Bruce, T., Martin, J., Smart, L., Oakley, J. and Wadhams, L. (2004). Journal of Chemical Ecology. 30. 1319-1328.
- Crook, D. and Mordue, A. (1999). Entomologia Experimentalis et Applicata. 91. 37-50.

This study was supported by a Linnaeus Grant (Formas, Sweden).

90

Yufeng Sun^1,2, Huili Qiao², Paolo Pelosi², Tieniu Kang¹, Liang Sun¹, Yun Ling¹, Xinling Yang¹

1. Dept. of Chemistry, China Agricultural University, Beijing, China
2. Dept. of Agricultural Chemistry and Biotechnologies, University of Pisa, Italy

sunyufengcau@yahoo.com.cn, yangxl@cau.edu.cn

Analogues (E)-β-farnesene as novel aphids insecticides. Synthesis, binding to OBPs and biological activity

Aphids represent one of the major problems in agriculture, not only for the direct damage they produce to crops, but also because they can spread dangerous molds. Insecticides have been used to control their population, but alternative, environmental-friendly approaches are desirable. Besides, the resistance of aphids to insecticides poses important problems of difficult solution.
In a novel approach to control the populations of aphids in agriculture, we have combined in some new chemicals the repellent action of the aphid pheromone (E)-β-farnesene with molecules endowed with insecticidal properties. We thus synthesized 24 new compounds, where citronellol was esterified by various acids containing in their molecules one or two differently substituted aromatic or heteroaromatic rings. These compounds were tested for their insecticidal activity and for their binding affinity to three aphid OBPs. The ligand-binding experiments showed different affinities of the compounds for the three OBPs, suggesting that they are perceived as different, particularly as regards OBP3, the only one among the three OBPs tested to bind (E)-β-farnesene. The insecticidal activity of some of the new compounds proved as effective as products currently used in agriculture. We predict that the repellent effect added to these novel insecticides may strongly reduce the concentrations needed to control the number of aphids in the environment. Being (E)-β-farnesene widely used by many aphid species as an alarm pheromone, the derivatives here described could be effective on several kinds of aphids, that represent serious pests for different crops.

91

Immacolata Iovinella¹, Maria Antonietta Calvello¹, Maria Giovanna Carucci¹, Huili Qiao¹, Alberto Niccolini², Antonio Felicioli², Francesca Romana Dani³, Stefano Turillazzi³, Paolo Pelosi¹

1. Dept. of Agricultural Chemistry and Biotechnologies, University of Pisa, Italy
2. Dept. Physiological Sciences, University of Pisa, Pisa, Italy
3. CISM, University of Firenze, Firenze, Italy

rovinella@hotmail.it

Odorant-binding proteins and Chemosensory proteins of the honeybee: a proteomic approach

Chemical communication in the honeybee is among the most sophisticated systems, making use of several different kinds of pheromones to regulate interactions and exchange of information between castes and individuals. The recently sequenced genome of Apis mellifera contains about 200 genes encoding odorant and taste receptors, while only 21 for Odorant-binding proteins (OBPs) and 6 for Chemosensory proteins. Given the recently discovered essential role of OBPs in odour recognition, it is important to verify how many of these proteins are really expressed, as well as in which sensory organ and in which physiological condition, in order to understand the chemical language of these insects.
Therefore, based on genome information, we have adopted a proteomic approach to reveal the presence of predicted OBPs and CSPs in different parts of the body and in different castes and ages. MALDI-MS analysis performed on crude extracts showed that only few of the proteins predicted by the genome are actually expressed at relatively high levels. Significant differences in the expression patterns were found in different sensory organs, as well as in castes. Moreover, we found differences in the repertoire of expressed proteins also between workers of different ages, a phenomenon that can be related to different tasks performed by the honeybee during its life.
Extracts from antennae of workers and from larvae were also fractionated by 2D-electrophoresis and the protein spots were analysed by MALDI fingerprint after digestion, again revealing that only few OBPs and CSPs are expressed in sizeable amounts.
Selected OBPs and CSPs were also expressed in bacterial systems, purified and utilised in ligand-binding experiments, using several organic coumpunds. Specific spectra of affinities were observed with each protein. In particular, the best ligands for CSP1, and to a lesser extent for CSP3, were alcohols and acids of medium length, recognised components of the brood pheromone. In constrast, both OBP2 and OBP3 showed good specificity to 4-tert-butyl-benzophenone.

92

Huili Qiao¹, Elena Tuccori¹, Xiaoli He², Angelo Gazzano³, Linda Field², Jing-Jiang Zhou², Paolo Pelosi¹

1. Dept. of Agricultural Chemistry and Biotechnologies, University of Pisa, Italy
2. Department of Biological Chemistry, Rothamsted Research, Harpenden, UK
3. Dept. Physiological Sciences, University of Pisa, Pisa, Italy

qhlonline@hotmail.com

Discrimination of alarm pheromone (E)-β-farnesene by aphid odorant-binding proteins

Aphids are among the most serious pests in agriculture. Sex pheromones have been identified, but their use in population control is limited, due to the fact that sexual reproduction occurs once a year, while generally female produce youngs by parthenogenesis. An alarm pheromone, (E)-β-farnesene, is produced by most aphid species in the presence of danger and produces immediate esacape. However, this compound, difficult and expensive to synthesize, is rather volatile and highly susceptible to oxidation.
Odorant-binding proteins (OBPs) have been recently demonstrated to be required for odour perception in insects and directly involved in odour discrimination. In aphids they might represent new interesting targets for the control of their population in agriculture. Based on sequence information available in the EST database, we have cloned four genes encoding odorant-binding proteins (OBP) in Acyrthosiphon pisum and homologous genes in other aphid species. Unlike OBPs from other orders of insects, that are greatly divergent, in aphids these proteins have been found to be highly conserved, with differences between species limited to only few amino acid substitutions. On the contrary, similarities between OBP sequences of the same species are poor with 31% or less of identical amino acids. Three selected OBPs (OBP1, OBP3 and OBP8) have been expressed in bacteria and purified. Ligand-binding experiments have shown similar behaviour of the three proteins towards several organic compounds, but also some significant selectivities. In particular, (E)-β-farnesene, the alarm pheromone and structurally related compounds, such as farnesol and 3,7-dimethyloctyl acetate, exhibited good affinity to OBP3, but did not bind the other two proteins. We suggest that OBP3 could mediate response of aphids to the alarm pheromone.
These data may help designing new compounds acting as alarm pheromones, that could be easy and inexpensive to synthesize.

93

Marcus C. Stensmyr and Bill S. Hansson

Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, Hans Knöll Str. 8, 07745 Jena, Germany
mstensmyr@ice.mpg,de

Specialized noses for specialized lifestyles

The olfactory system directly interfaces with the environment, thus, changes in the environment, or a change in an animal’s habits, as e.g. specialization, would presumably also lead to changes in the olfactory system. Comparative studies on specialized animals, with known generalist ancestors, could hence be a way of revealing general processes shaping olfactory systems, as well as highlighting importance and function of specific chemosensory genes. In this presentation I will outline current work in our laboratory dealing with olfactory adaptations at the molecular, physiological, morphological and behavioral level in a set of highly specialized drosophilid flies.

94

Jesse Slone, Tetsuya Miyamoto, Joseph Daniels, and Hubert Amrein.

Dept. of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA, 27707.

Sweet Taste and Pheromone Perception in Drosophila

The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of sugars, bitter or toxic compounds and pheromones. However, with the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor, the functions of most GRs involved in feeding are unknown.  Gr5a is closely related to seven other Gr genes, six of which form a gene cluster on the left arm of the third chromosome (Gr64a-f). We recently generated a strain that lacks all six Gr64 genes (ΔGr64) and showed that these flies exhibit significantly diminished behavioral responses to all sugars tested, with the exception of fructose. Interestingly, response to trehalose is also abolished in ΔGr64 mutant flies, even though they contain a functional Gr5a gene. This observation indicates that two or more GRs are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes.  We are currently in the process of determining the composition of putative sugar receptor complexes and their ligand specificities using ΔGr64 mutant flies that are complemented with individual Gr64 genes, or combinations thereof. We and others have been unable to establish expression profiles for most Gr64 genes by means of the Gal4/UAS system, which presumably reflects complex arrangements of regulatory elements. Thus, we are using homologous recombination to generate Gal4 or LexA::VP16 knock-in alleles into all six Gr64 genes, as well as Gr5a. The Gr64a^Gal4 allele shows extensive expression in leg taste neurons.  However, Gr64a^Gal4 flies show a strong taste defect for sugars, and we are testing whether this knock in allele affects expression of other Gr64 genes in the locus.  The Gr5a^lexA::VP16 allele, on the other hand, shows extensive expression in the legs and labellum.  
We have also investigated the role of another group of Gr genes, most prominently Gr32a and Gr68a. Using homologous recombination to generate knock-outs for these genes, we have shown that they mediate courtship behaviors through and are directly involved in detecting specific pheromones. We will present an update on recent studies of these genes, as well as four related Gr genes suspected to be involved in social behaviors (Gr39a.a-d).
Finally, we are in the process to determine the topology of GR proteins.  Although GRs and the related olfactory receptor family were originally predicted to be seven-transmembrane domain (7TM) GPCR proteins, these chemoreceptor proteins were recently reported to have an inverted topology relative to GPCRs. Moreover, many protein prediction algorithms suggest that the majority of GRs have eight TMs, not seven.  We are using an in vivo protein complementation assay to examine this issue.
This work was supported by grant NIH-DC005606-02 to HA. JS was supported by an NSF graduate research fellowship

95

Thomas C. Baker

Center for Chemical Ecology, Department of Entomology, Penn State University, University Park, Pennsylvania, USA 16802
tcb10@psu.edu

Focusing of Pheromone Molecules by Trichoid Sensillar Cuticular Lipid Coatings

The lipid coatings on trichoid sensilla of male moths may be involved in both the selective adsorption of pheromone molecules from the air, as well as focusing these molecules onto sensillar pores. If the lipids on trichoid sensilla were better at adsorbing pheromone-type molecules than other surfaces of the antennae, pheromone molecules would be selectively focused onto the trichoid hairs. Secondly, different regions of the sensilla could be coated heterogeneously with different forms or orientations of lipids to expedite the movement of adsorbed pheromone molecules down the pores to the binding proteins and then to the receptor neurons in the sensillum lumen. We found that in Helicoverpa zea moths, there were in fact significant differences in the lipids on male antennae compared to those of females (1) and these could be attributed to the trichoid sensilla that exist in huge numbers on male antennae but not on female antennae. Thus at the macro-level we found evidence for differential coating of male trichoid sensillar surfaces that might selectively adsorb pheromone molecules.

We then used atomic force microscopy (AFM) on the sensilla to determine their individual nano-terrains, followed by chemical force microscopy (CFM) to contact the surfaces and probe for differences in chemical bonding forces that could indicate differences in lipid coatings on different local sensillar regions. We found that H. zea trichoid sensilla exhibited heterogeneity in their lipid coatings, with the multitudes of ridges on each sensillum possessing more hydrophobic coatings than the pores. These consistent differences between the chemical compositions coating the ridges versus the pores suggests that there is a lipid-based nano-focusing of the aldehyde pheromone molecules into the pores from non-pore-containing areas of the sensilla. A second species, Utethesia ornatrix, that uses hydrocarbon pheromone components, not aldehydes, exhibited no lipid heterogeneity on its trichoid sensillar surfaces.

1. J. Insect Physiol. 54: 1385-1391.

96

Marco Tasin, Claudio Ioratti and Gianfranco Anfora

FEM Research - San Michele all’Adige (TN) - Italy
marco.tasin@iasma.it
gianfranco.anfora@iasma.it

Volatile semiochemicals for insect control

The use of semiochemicals in pest control is nowadays a safe and low-impact mean used on over 600.000 ha worldwide. Limiting factors for a further implementation of semiochemicals are recognized both at a basic and at a field level. In the case of a major technique, the pheromone mating disruption, lack of knowledge on the mode of action hampers its efficacy and as a consequence its spread. As the mechanisms behind mating disruption are still not completely explained and different hypotheses have been put forward, an increase in the efficacy of this method may be reached by understanding the behaviour evoked or suppressed when males are exposed to synthetic pheromones. In addition, the emerging knowledge on other semiochemicals such as plant, food or microbial volatiles, may disclose the possibility of enhancing the efficacy of the current control strategies based on pheromones. We will show here experimental evidence of how the formulation of a synthetic pheromone impacts the mechanisms governing mating disruption and how additional volatiles beside insect pheromone influence the reproductive behaviour of a pest.

97

Bertram Gerber

Universität Würzburg, Department of Genetics and Neurobiology
bertram.gerber@biozentrum.uni-wuerzburg.de

Olfaction and olfactory learning in Drosophila

Our research strives for a comprehensive account of associative olfactory memory. The long-term goal is to account for the full circuitry involved in memory trace formation and the involved molecular mechanisms.

One present focus is on the contribution of two evolutionarily conserved presynaptic proteins, Synapsin and Sap47, for memory trace formation, in particular regarding their cellular site and molecular mode of action.

A second focus is the specificity of olfactory memory traces for odour-quality and -intensity, as well as in more general terms the relation between physiology and perception. I will present recent data regarding where along the olfactory processing streams physiological activity patterns are relevant for perception in Drosophila.

Gerber B, Stocker RF, Tanimura T & Thum A 2009 Smelling, tasting, learning: Drosophila as a study case. Results and Problems in Cell Differentiation 47, 1-47.
Gerber B & Stocker RF 2007 The Drosophila larva as a model for studying chemosensation and chemosensory learning: A review. Chemical Senses 32, 65-89

98

Krishna C. Persaud

School of Chemical Engineering and Analytical Science, The University of Manchester, M60 1QD, UK

Gas sensor arrays - moving towards biomimetic aspects of olfaction

krishna.persaud@manchester.ac.uk

Biological olfaction outperforms chemical instrumentation in specificity, response time, detection limit, coding capacity, time stability, robustness, size, power consumption, and portability. This outstanding performance is due, to a large extent, to the unique architecture of the olfactory pathway, which combines a high degree of redundancy, an efficient combinatorial coding along with unmatched chemical information processing mechanisms. These exceptional features of biological olfaction have not yet been successfully captured by artificial olfaction. We are attacking some of these aspects via a Collaborative Project: Biologically Inspired Computation for Chemical Sensing (NEUROCHEM). This uses information representation and processing of the olfactory pathway to overcome existent problems in chemical sensing. As a test-bench for the novel computing paradigms extracted, we are building biomimetic artefacts featuring very large number of receptors (sensors) exhibiting high levels of redundancy. This presentation focuses on approaches from various partners on:

• the encoding principles underlying the olfactory receptor neuron layer population,
• the role of redundancy and continuous population renewal towards signal-to-noise improvement and robust chemical sensing
• decorrelation implementation based on system dynamics
• odour segmentation in complex backgrounds,
• odour storage and retrieval from corrupted inputs - biologically inspired associative memories
• integration of a complete system implementing learning models of different modules of the olfactory pathway, both for vertebrates and insects.

Acknowledgements: Bio ICT Convergence Project – Grant Agreement no. 216916 Future and Emerging Technologies – FP7

99

Kei Ito and Takaaki Miyazaki

Institute of Molecular and Cellular Biosciences, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

itokei@iam.u-tokyo.ac.jp

Systematic mapping of the primary gustatory center in the Drosophila brain.

In both insects and mammals, neuronal circuits that handle gustatory information have been investigated less extensively compared to the olfactory system. Mammalian taste sensory cells do not innervate the brain by themselves and send signals indirectly via the taste neurons, making it difficult to analyze the functional projection map in the brain. The gustatory receptor neurons (GRNs) in the insect mouth, on the other hand, project their axons directly to the primary gustatory center (PGC) of the brain, which occupies distinct areas of the suboesophageal ganglion (SOG). Previous studies of the fruit flies have identified various GRNs and their axonal projections using promoters of the gustatory receptor genes and enhancer-trap strains, but the comprehensive and detailed structure of the PGC remains unknown. The insect taste sensillum is associated also with a taste-associated mechanosensory neuron to detect the encounter with foods. Their projection targets, however, remain unidentified.
To address these issues, we performed a large-scale screening of about 4,000 GAL4 enhancer-trap strains to obtain the lines that label specific subsets of the GRNs and mechanosensory neurons. Unlike the antennal lobe or the optic lobe, the PGC does not have easily identifiable landmark structures against which projection sites of each identified neuron types can be mapped precisely. To solve this problem we utilized a newly established collection of the LexA::VP16 (LexAV) enhancer-trap strains, which can induce gene expression independent from the GAL4 enhancer-trap strains. We identified a LexAV strain that visualizes various sensory neurons including including a large subset of the GRNs.
By double labeling specific GRNs and overall neurons with GAL4 and LexAV strains, we were able to distinguished 3 branches and 11 zones in the PGC, among which 1 branch and 5 zones were identified newly. Arborization areas of various known GRNs were also mapped in this framework. The putative taste-associated mechanosensory neurons terminate exclusively in 3 zones of them to which GRNs do not innervate. This supports the notion of the segregated primary centers that are specialized for chemosensory and mechanosensory signals associated with gustatory sensation.

100

Masaru K. Hojo¹, Ayako Wada-Katsumata², Mamiko Ozaki³, Toshiharu Akino¹, Susumu Yamaguchi⁴ and Ryohei Yamaoka¹

1. Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, 606-8585, Japan,
2. Division of Applied Life Science, Graduate of School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan,
3. Department of Biology, Graduate School of Science, Kobe University, Kobe, 657-8501, Japan and 4Wakamiko, Sutama-cho, Hokuto city, Yamanashi, 408-0112, Japan

m.k.hojo@gmail.com

Taste and olfactory strategies for parasitic butterfly-host ant chemical communication

The caterpillars of lycaenid butterfly Niphanda fusca develop as parasites inside the nests of their specific host ants Camponotus japonicus, where they grow by feeding on the worker trophallaxis. Because the ants often use chemicals to communicate information to their nestmates, the parasitic caterpillars will exploit host chemical communication systems. Here we show that N. fusca caterpillars use multiple chemical strategies to exploit the host ants.
The parasitic caterpillars attract their specific ant partners by secreting nutritious reward from specialized exocrine gland. The secretion of N. fusca contained trehalose and glycine as major component. We comparatively investigated gustatory preference for trehalose, glycine or a mixture of the two between host (C. japonicus) and non-host (Camponotus obscuripes) species of ants in behavioral and electrophysiological experiments. Glycine itself induced no taste sensation in either host or non-host ants. The mixture of trehalose plus glycine was chosen as much as pure trehalose by non-host ants. However, the host ants clearly preferred the mixture of trehalose plus glycine to trehalose alone. These behavioral data are supported by the electrophysiological responsiveness to sugars and/or glycine in the sugar-taste receptor cells of the ants. These results suggest that chemical composition of nutritious secretions is crucial for maintaining species-specificity of butterfly-ant associations.
After successful intrusion into the host ant nest, parasitic caterpillars are preferentially reared by the host workers rather than their own ant larvae. We hypothesized that N. fusca caterpillars chemically mimic some particular castes of the host ant, so that the caterpillars are accepted and cared for by the host workers. Behaviourally, it was observed that the host workers enthusiastically tended glass dummies coated with the cuticular chemicals of larvae or males and those of N. fusca caterpillars. Cuticular chemical analyses revealed that N. fusca caterpillars grown in a host ant nest acquired a colony-specific blend of cuticular hydrocarbons (CHCs). Furthermore, the CHC profiles of the N. fusca caterpillars were particularly close to those of the males rather than those of the host larvae and the others. We suggest that N. fusca caterpillars exploit worker care by matching their cuticular profile to that of the host males, since the males are fed by trophallaxis with workers in their natal nests for approximately ten months.
In summary, our results indicated the caterpillar’s chemical signals are precisely tuned to the host’s sensory systems, and multiple chemical strategies would be important to maintain complex butterfly-ant associations

101

Blanka Kalinová¹, Jirí Kindl¹, Irena Valterová¹, Jirí Popelár² and Milan Jílek²

1. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague
2. Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague

blanka@uochb.cas.cz

Chemical and acoustic communication in pre-mating behavior of A. sociella (Galleriinae, Pyralidae)

Bee moth, Aphomia sociella (L.), is an important parasite of bumblebees. The larvae destroy the host nest by consuming the brood combs and the offspring. Unlike the most Lepidoptera, reproductive behaviour of A. sociella and other members of the subfamily Galleriinae (family Pyralidae) is initiated by males and includes quite variable combinations of ultrasonic and pheromone signals.

We report on the analysis and identification of male sex pheromone and the role of chemical and acoustic communication in A. sociella pre-mating behavior.

Seven pheromone component candidates, namely 1-hexanol, 2-phenylethanol, [(R),(Z)]-nona-2,6-dien-4-olide, [(S),(Z)]-nona-6-en-4-olide, mellein and phytone were identified in male wing gland extracts and in emanations released by calling males (1). Behavioural experiments revealed that male sex pheromone mediates long range attraction, and the ultrasound is involved only in close range interaction (courtship) and male-male competition. Sound analysis showed two types of ultrasound signals, i.e. (i) randomly distributed nonperiodic pulses of ~ 95 dB amplitude and 10 - 40 kHz frequency, and (ii) ~100 μs periodic events with repeated frequency around 80 kHz and ~ 75 dB amplitude modulated into bursts. Bursts were grouped in trains and regular clusters further associated into chirps (~ 1 s) relatively randomly distributed with minimal interval of 1 s during courtship song. Ultrasound facilitates specific pre-copulatory behavioral response of female (wing fanning) and is essential for mating success (female choice).

Reference

1. B. Kalinová, J. Kindl, P. Jiroš, P. Zácek, S. Vašícková, M. Budešínský and I. Valterová: Composition and Electrophysiological Activity of Constituents Identified in Male Wing Gland Secretion of the Bumblebee Parasite Aphomia sociella (2009) Journal of Natural Products 72 (1) 8–13

102

Nicky Bos¹, Fernando J. Guerrieri² & Patrizia d’Ettorre¹

1. Centre for Social Evolution, University of Copenhagen, Denmark
2. Centre de Recherches sur la Cognition Animale, University of Toulouse, France

Making sense of complex odour blends

Animals have evolved perceptual strategies to extract significant information from a world full of stimuli. This is important for many aspects of an animal’s life, such as foraging, predator avoidance, mating and social behaviour. Social insects are well known for their ability to extract important information and learn from an enormous range of olfactory stimuli. We addressed the question of how ants perceive, process, and extract important information from a blend of odours in their environment. Ants were conditioned to individual odours, and tested for generalisation between the conditioned stimulus and a novel one. We used volatile molecules that are similar in structure but have different functional groups, such as 1-hexanol, 1-octanol, octanal and 2-octanone. We also tested if overshadowing occurred, where individual ants were conditioned to a blend of substances, and tested for reaction to the individual components of the blend. The results suggest that generalisation might follow an “inclusion criterion”, where any substance that elicits a sub-pattern of the conditioned odour will be generalised. Moreover, perception of odour blends appears to be elemental, where individual components of the blend are learned. The results give new insights into how ants perceive and process olfactory stimuli and might help understanding how multi-component chemical signals evolve.

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Last updated: May 05, 2009