ESITO VII

European Symposium for

Insect Taste and Olfaction

 

Talks
Posters
Schedule
ESITO Homepage


1

Thomas C. Baker, Sam Ochieng, Seong Gyu Lee

Department of Entomology
Iowa State University, Ames, Iowa 50011 U.S.A
[email protected]

Plant-odor and sex pheromone mixture interactions in moth antennal receptor neurons

We have been investigating the degree to which ORNs of heliothine moths respond to mixtures of pheromone components and behavioral antagonists. For Helicoverpa zea, when we presented mixtures of (Z)-11-hexadecenal, the major pheromone component, plus the behavioral antagonist, (Z)-11-hexadecen-1-ol-acetate, there was a doubling of action potential frequency by the ORN responding to the major pheromone component during the initial phasic portion of the response, as well as significant tonic enhancement in firing of this ORN, compared with its response to (Z)-11-hexadecenal alone. We also found similar phasic and tonic enhancement in response to blends of another antagonist, (Z)-11-hexadecen-1-ol, and also in response to the secondary pheromone component, (Z)-9-hexadecenal.

Because the enhancement of firing of this ORN appeared to occur somewhat unspecifically in response to both antagonistic and agonistic pheromone-related compounds, we decided to investigate further just how specific this mixture interaction phenomenon was by expanding the testing of mixtures to include various plant-related odors combined with the major pheromone component. Surprisingly, we found even greater mixture enhancement of response of this ORN when different plant-related volatiles were presented than when pheromone-related mixtures were tested. For both pheromone-component-related mixtures and pheromone component/plant volatile mixtures, the enhancement of firing frequency was not as great when the odorants were puffed simultaneously from separate odor cartridges as when the mixture was housed within and puffed from a single odor cartridge.

Supported by USDA/NRICGP grant #000-2930 to T.C.Baker


2

Bente G. Berg1,3, Dirk Müller2, Hanna Mustaparta3

1 Department of Psychology, Norwegian University of Science and Technology, 7491 Trondheim, Norway
2 Institute für Bilogie-Neurobiologie, Freie Universitet Berlin, Königin Luise Str.28-30, D-14195 Berlin, Germany
3 Department of Zoology, Norwegian University of Science and Technology, 7491 Trondheim, Norway
[email protected]

Novel types of antennal lobe neurons characterized in the female moth Heliothis virescens

The olfactory pathway from the periphery to higher integration centres is mainly ipsilateral in insects as well as in vertebrates. Thus, in most insect species the olfactory sensory neurons target the ipsilateral antennal lobe. Here they converge and make synapses with two morphologically, distinct classes of central neurons: local interneurons which are confined to the antennal lobe and projection neurons which form mainly ipsilateral pathways to higher integration centres of the brain as the calyces of the mushroom bodies and the lateral protocerebrum.

Based on studies of the female moth antennal lobe, combining electrophysiology with staining techniques and confocal microscopy, we present some projection neurons with unique morphological characterizations. One bilateral type directly connected the two antennal lobes by an axon projecting in the antennal commissure. The bilateral neurons targeted olfactory glomeruli in partly homotopic areas. The responses to plant odour stimulation of the ipsilateral antenna were recorded as excitation as well as inhibition. Interestingly, a similar pathway between the macroglomerular complexes of the well-studied male moth, has never been described. Whether the bilateral antennal lobe neurones hold a function specifically in the neuronal network processing plant odour information is not clarified.

 


3

Charles Derby, Paul Harrison, Holly Cate and Pascal Steullet

Department of Biology Georgia State
University Atlanta, Georgia USA
[email protected]

Continuous turnover of receptor neurons in the olfactory organ of crustaceans: mechanisms, regulation, and implications for functional organization

Many crustaceans, including the Caribbean spiny lobster Panulirus arus, grow throughout their lives, can live for many years, and are often in environmental conditions that can lead to stress or damage of their olfactory organ. Evolutionary adaptations to these conditions include continuous proliferation of olfactory receptor neurons (ORNs) to compensate for the increase in body size, and turnover of cells to replace damaged or dying cells with new ones, and complete regeneration of the olfactory organ following injury or removal (2,4). In vivo pulse labeling using bromodeoxyuridine (BrdU) shows that growth of the olfactory organ (the antennular lateral flagellum) of spiny lobsters occurs by proliferation of aesthetasc sensilla and their ORNs at one specific site, called the proximal proliferation zone (PPZ), which is immediately proximal to the existing ORNs. New cells are derived from precursors located in the antennular epithelium immediately proximal to the PPZ. Proliferation in the PPZ is continuous, and the zone spreads proximately as it generates new cells. Turnover occurs at the distal end of the antennule, where aesthetascs and their ORNs are lost at molting. Thus, the crustacean olfactory organ has a developmental axis, with proliferation at the proximal end, cell death at the distal end, and a region of mature, odor-responsive ORNs in between. These processes of proliferation, maturation, and death are highly dynamic, and can be modulated by central and peripheral factors. Central factors linked to the molt cycle regulate the size and shape of the PPZ, with greatest proliferation occurring in pre-molt when ecdysteroid levels peak. Local factors associated with peripheral injury to the antennule also modulate the size and shape of the PPZ. In fact, following ablation of all or part of the antennule, the extent of proliferation in the PPZ closely matches the amount of tissue damaged. In addition to the high rate of proliferation of ORNs in the PPZ, a much lower rate of proliferation occurs in the mature region of the antennule. This may serve to replace dying cells in the mature region. Death of ORNs in the mature region can up-modulate the proliferation in this region, presumably due to signals released from the dying ORNs. Such local death has no effect on proliferation in the PPZ. Thus, two types of peripheral signals appear to up-regulate proliferation in the antennule: one that functions only locally, and up-modulates the rate of local repair following ORN death; and a second that up-modulates proliferation in the PPZ following ablation of distant tissue. In conclusion, post-embryonic development and turnover of ORNs of lobsters is continuous but can be regulated by both central and local signaling mechanisms. Interestingly, these processes of growth, rejuvenation, and repair occur without a loss of the animal¹s ability to detect and discriminate odorants, learn about the odorant environment, and orient to odorant sources (1,3,5). Current cellular and molecular studies utilizing both in vivo and in vitro methods are aimed at identifying trophic factors and other gene products that regulate normal development of ORNs and regeneration following injury.

Supported by NIH DC00312 and NSF IBN0077474.

References:

1. Derby C.D. 2000. Learning from spiny lobsters about chemosensory coding of mixtures. Physiol. Behav. 69: 203-209.

2. Harrison P.J.H, Cate HS, Swanson ES, and Derby C.D. 2001. Post-embryonic proliferation in the spiny lobster antennular epithelium: rate of genesis of olfactory receptor neurons is dependent on molt-stage. J. Neurobiol. 47: 51-66.

3. Horner A.J., Ngo V., Steullet P, Keller T., Weissburg M., and Derby C.D. 2000. The role of different types of antennular sensilla in orientation by Caribbean spiny lobsters to natural odor stimuli under controlled flow conditions. Chem. Senses 25: 670-671.

4. Steullet, P., Cate H.S., and Derby C.D.. 2000. A spatiotemporal wave of turnover and functional maturation of olfactory receptor neurons in the spiny lobster Panulirus argus. J. Neurosci. 20: 3282-3294.

5. Steullet P., Cate H.S., Michel W.C., and Derby C.D. 2000. Functional units of a compound nose: aesthetasc sensilla house similar populations of olfactory receptor neurons on the crustacean antennule. J. Comp. Neurol. 418: 270-280


4

Klemens Störtkuhl, Raffael Kettler and Bernhard Hovemann

Ruhr Universität Bochum, Molekulare Zellbiochemie
Gebäude NC 5/173, 44780, Bochum Germany
[email protected]

First functional analysis of an insect olfactory receptor Or43a

59 candidate olfactory receptor (Or) genes have recently been identified in Drosophila melanogaster. In order to analyze functional properties of an Or of interest we reasoned that an ectopic expression of Ors in olfactory receptor neurons might show an increased amplitude in the electroantennogram (EAG) to specific volatile substances, identifying them as specific ligands. For our investigations we selected an Or that is expressed at the distal edge of the third antennal segment in about 15 olfactory receptor neurons. To identify ligands we used the Gal4/UAS system to misexpress this olfactory receptor in additional cells of the third antennal segment. Or-mRNA expression in the antenna of transformed and wild type flies was visualised by in situ hybridization with a digoxigenin - labeled probe. Cyclohexanol, cyclohexanone, benzaldehyde and benzyl alcohol evoke increased EAGs while responses to ethyl acetate, propionaldehyde, butanol and acetone were not significantly different from wild type. Our in vivo analysis reveals for the first time functional properties of one member of the recently isolated olfactory receptor family in Drosophila and will provide further insight into our understanding of olfactory coding (Störtkuhl & Kettler, 2001 PNAS inpress)

 


5

Karl-Ernst Kaissling

Max-Planck-Institut fuer Verhaltensphysiologie
Seewiesen, 82319 Starnberg, Germany
[email protected]

Possible functions of pheromone binding proteins in Bombyx mori and Antheraea polyphemus

Pheromone binding proteins occur in mM concentrations in the extracellular sensillum lymph surrounding the sensitive processes of olfactory receptor cells in insects. As proposed in the literature (reviewed in1), they may solubilize and carry the odorants to the receptor cells, protect the odorants from enzymatic degradation on their way to the receptor molecule, mediate the interaction between odorant and receptor molecule and, finally, deactivate the odorant, i.e. serve as a scavenger. Some of these functions seem to exclude each other: thus a carrier and mediator cannot, at the same time, serve as a scavenger. Therefore we use quantitative modeling to understand functional linkages of peripheral olfactory processes. The model parameters are based on morphometrical, radiometrical, biochemical, and electrophysiological data (reviewed in1) and, partially, on assumptions.

In our example of pheromone binding proteins (PBPs) in moths we discuss two model networks of chemical reactions. In both of the models the PBP first acts as a carrier which solubilizes the amphiphilic pheromone and transports it through the sensillum lymph over distances up to 1 µm to the receptor cell membrane. In the first model for the silkmoth Bombyx mori the pheromone is released when the pheromone-PBP complex approaches the receptor cell membrane. This is suggested by a decrease of binding capacity of the PBP in the proximity of the cell membrane according to binding studies at reduced pH and experiments with artificial membranes2. In this model the free pheromone interacts with the receptor molecules of the receptor cell whereas in the second model for the saturniid moth Antheraea polyphemus the pheromone-PBP complex interacts with the receptor molecules. In both models the pheromone accumulates on the olfactory hairs and needs to be prevented from permanently interacting with receptor molecules. Two ways of pheromone deactivation seem to occur: the enzymatic pheromone degradation and - while the pheromone is bound - a change of the PBP from the carrier to the scavenger function. The latter way of rapid pheromone deactivation seems necessary since the degradation of the pheromone adsorbed on the antenna is much too slow to account for the rapid decline of the receptor potential observed after stimulus offset. Two possible mechanisms of rapid pheromone deactivation are proposed1: In Bombyx the adsorbed pheromone might first bind to outer hydrophobic patches of the PBP (carrier mode) and later be 'swallowed' into the internal binding cavity of the PBP (scavenger mode). This idea was stimulated by the recently analyzed crystal structure of the PBP of Bombyx mori2. In A. polyphemus a switching of the PBP from the carrier to the scavenger form is suggested by a structural change (redox shift) of the pheromone-PBP complex observed in isolated hair preparations3. There is evidence that the shift requires the presence of an unknown agent (modeled by an enzyme) in the hairs3. Both models are able to simulate the rapid kinetics of the receptor potential. However, they differ in their reactions to changes of model parameters, for instance of the concentration of the pheromone degrading enzyme. The predicted lifetimes of the pheromone on the antenna also differ.

The modeling reveals that in both models the binding affinity of pheromone and PBP (60 nM) is several hundred times higher than that of the pheromone and the receptor molecule (38 µM). The rate constants of the pheromone-PBP interaction are about 1000-fold larger for the carrier (k2 = 0.17/(s x µM), k-2 = 0.01/s) than those for the scavenger form. Both models have the same rate constants of the pheromone-receptor interaction as derived from an analysis of the elementary receptor potentials elicited by single pheromone molecules (A. Minor, unpubl.1). Finally, the modeling allows calculation of the number of receptor molecules per receptor cell. The result indicates that the plasma membrane of the receptor cell dendrite is densely covered by receptor molecules, with a minimum value of 6000 units per µm2 (the density of rhodopsin molecules in the disc membrane of visual cells found to be 25000 ­ 40000 units per µm2).

References:

1. Kaissling, K. E. (2001) Chemical Senses, 26:125-150

2. Sandler, B.H., Nikonova, L., Leal, W.S. and Clardy, J. (2000) Chemistry & Biology, 7, 143-151.

3. Ziegelberger, G. (1995) Eur. J. Biochem., 232, 706-711.


6

Mattias C. Larsson1, Marcus C. Stensmyr1, Shannon B. Bice2, Walter S. Leal3, Bill S. Hansson1

1 Dept. of Ecology, Chemical Ecology
Lund University, SE-223 62 Lund, Sweden
[email protected]
2 Department of Neurobiology and Behavior
Cornell University, Ithaca NY 14853, USA
3 Department of Entomology
University of California at Davis Davis, CA 95616 USA

Neural interfaces to the odour world of scarab beetles

Several species of phytophagous scarabs are economically important pests, which has promoted the identification of attractants (both pheromones and plant odours) for use in monitoring or mass trapping. Plant odours used in field trapping of phytophagous scarabs have mainly consisted of floral compounds chosen more or less at random or due to their attractiveness to other scarab species. Similar compounds often attract unrelated scarabs all over the world. There is, however, still a high potential for finding new semiochemicals with relevance for different aspects of scarab biology.

Olfactory search strategies in phytophagous scarab beetles appear to be based on a limited set of key odorants emitted from desired resources. Each key odorant is likely selected for its high informational value, indicating the specific resource with high certainty. Certain single compounds, like some sex pheromones or typical floral or fruit compounds, are reliable enough indicators to merit attraction by themselves. In other cases several compounds in a blend may be needed to form a reliable, and thus attractive, signal.

Our results from single cell and GC-single cell studies with fruit extracts and synthetic odorants show that scarabs use specialised olfactory receptor neurons to detect the selected key compounds exploited in olfactory search behaviour. Each type of receptor neuron forms a specific input channel to the CNS, mediating information about a single compound or a few structurally related compounds. This is the case for pheromone neurons as well as for plant odour neurons. Key compounds for phytophagous scarabs typically include flowery terpenes like linalool and geraniol, fruit esters, short-chain acids like isovaleric acid, and phenylpropanoids like anethol or eugenol. Using scarab receptor neurons as odour detectors we have identified several more or less novel scarab attractants, like methyl salicylate, acetoin, and linalool oxide.


7

T.A. Christensen, H. Lei, V.M. Pawlowski and J.G. Hildebrand

Arizona Research Labs - Division of Neurobiology
Univ. of Arizona, Tucson, AZ 85721 USA
[email protected]

Integrating space and time in odor-coding circuits: evidence from neural-ensemble recordings

The computations underlying odor discrimination at the earliest stages of processing in the brain have been the subject of active inquiry for many decades (reviewed in 1). A number of activity labeling studies using both invertebrates and vertebrates show that different odors evoke distinct spatial patterns of activity across arrays of olfactory glomeruli in the antennal lobe or olfactory bulb. These studies suggest that a specific subset of functional "units" or "modules" together encode the chemical identity of each odor. But underlying this spatial map is an intricate network of intra- and inter-glomerular connections involving a physiological diverse population of excitatory and inhibitory neurons. Methods that permit higher temporal resolution of neural signals show that odors also evoke complex temporal patterns of firing in these processing circuits, and a growing number of studies suggest that multiple bits of information about the stimulus are encoded in the firing correlations between neurons in olfactory networks (1). How then are these spatial and temporal patterns integrated in the brain? To address this issue, we are using precisely spaced 3- or 4-pronged silicon multielectrode arrays in the moth antennal lobe to record population responses to odors with high spatial and temporal resolution (2). Simultaneous recordings from up to 25 neurons revealed a number of characteristics that were consistent across ensembles in different preparations. First, activity patterns were dependent on odor quality: pheromones evoked patterns localized to the macroglomerular complex, whereas host odors evoked more distributed patterns across the remaining glomeruli. Furthermore, molecularly different odors sometimes evoked very similar spatial patterns of activity, but the underlying temporal patterns were nevertheless quite distinct. Secondly, different neurons innervating the same glomerulus always showed similar tuning features, but they often displayed different firing dynamics. The third and most striking observation was that different odors (or even the same odor at different dosages) evoked correlated firing in distinct subsets of neurons across the recorded ensemble. These patterns of synchrony were furthermore not oscillatory, but instead, modulated by the dynamics of the odor pulses (3). Ensemble recordings in the antennal lobe have therefore uncovered precise temporal relationships between odor-encoding neurons, and these dynamics cannot necessarily be predicted from the spatial patterns of activity evoked by odors: thus, we see temporal codes for odors nested within spatial ones. Synchronized firing likely serves to strengthen the distributed representation of a given odor (or context) across an array of glomeruli at the earliest stage of processing in the brain.

References:

1. Christensen TA, White J (2000) Representation of olfactory information in the brain. In: TE Finger, WL Silver, D Restrepo (eds), Neurobiology of Taste and Smell, Vol II, pp. 201-232, Wiley, New York.

2. Christensen TA, Pawlowski VM, Lei H, Hildebrand JG (2000) Multi-unit recordings reveal context-dependent modulation of synchrony in odor-specific neural ensembles. Nature Neuroscience 3:927-931

3. Vickers NJ, Christensen TA, Baker TC, Hildebrand JG (2001) Odour-plume dynamics influence the brain¹s olfactory code. Nature 410:466-470

 


8

Robert A. Raguso, Tamaire Ojeda-Avila and Sheetal Desai

Department of Biological Sciences, University of South Carolina
Columbia SC 29208 USA
[email protected]

The Importance of Sensory Cues, Larval Diet and Adult Experience in Nectar Feeding by Manduca sexta

Manduca sexta hawkmoths in wild and laboratory settings utilize visual or olfactory floral cues to orient to flowers from a distance, but require the combination thereof in order to extend their probosces and feed. However, the sensory components of feeding behavior also can be modified by an insect¹s physiological state or associative learning. For example, through Pavlovian conditioning, K. Daly and B. Smith have shown that trained M. sexta will fly upwind and extend its proboscis to the conditioned odor stimulus in the absence of a visual cue. We examined two potential sources of behavioral modification in nectar feeding; larval diet and floral learning curves.

Since only 25-30% of lab-reared M. sexta show normal feeding responses, we proposed that adult fat-body mass inhibits adult nectar feeding; moths raised on a leaner diet should feed more as adults. Only animals reared on artificial diets with reduced sucrose or on tobacco leaves carried significantly lower fat stores than those reared on control, low water and low protein diets. These leaner animals, and those whose diet had been spiked with beta-carotene for enhanced visual accuity, did show greater interest in floral arrays, but variation between individual moths swamped any variation associated with larval diet. This experiment will be repeated using wind tunnel and GC-EAD assays to better examine olfactory responses.

The final stage of flower feeding occurs once a moth¹s proboscis is already extended, such that visual, tactile, olfactory and gustatory cues all play potential roles in its acceptance or rejection of a specific flower. We constructed artificial flowers that decoupled these cues, and measured the changes in handling time (from first contact with flower to probing the nectar tube) associated with each cue. We observed differences in the shapes of learning curves associated with these cues, especially when petal texture or taste (presence of nectar trace) were manipulated, but individual variation among moths remained high. Experiments such as these will lead to an eventual integration of the wealth of sensory and physiological mechanisms revealed through studies of M. sexta, within a complex behavioral context.

 


9

Veronica Rodrigues, Sheetal Bhalerao and Dhanisha Jhaveri.

Department of Biological Sciences, Tata Institute of Fundamental Research
Homi Bhabha Rd., Mumbai 400005 India
[email protected]

How do sensory neurons affect brain patterning in the olfactory system of Drosophila?

The study of how neural circuits develop is paramount to an understanding of how complex and precise behavioral patterns are generated in animals. Olfactory neurons from the periphery terminate in the glomeruli of the olfactory lobe. Afferent fibres are organized in three fascicles as they leave the third segment of the antenna. We have shown that the fasciculation pattern is regulated by peripheral glia; absence or alteration of these glia lead to alterations in fasciculation patterns.

Upon entry of the sensory afferents into the lobe, several cellular changes occur; viz. mitosis of the lobe glia and re-organization of the interneuronal populations. We have shown that the while the ability to project to the lobe is an autonomous property of all olfactory neurons, their segregation into glomeruli is not. Sensory axons from one of the three types of sensilla- that specified by the atonal gene- provide instructive cues for patterning the glomeruli. Complete loss of atonal function leads to an aglomerular lobe even though about 60% of the total sensory neuronal population are still present. We speculate that the neurons of the Atonal lineage provide instructive cues to the rest of the olfactory neurons to enable their arborization in distinct glomeruli. The signalling mechanism that allows communication between neurons of different type is being analyzed by loss-of-function and gain ­of-function genetics.

 


10

Sachse Silke, Galizia C. Giovanni & Menzel Randolf

Institut fuer Neurobiologie
FU Berlin, Koenigin-Luise-Str. 28/30, 14195 Berlin
[email protected]

Two inhibitory networks shape both temporal and spatial odor representation in olfactory output neurons: a calcium imaging study

The most important olfactory brain center - the antennal lobe (AL) in insects or the olfactory bulb in vertebrates - is a notable example of a neural network. While physiological properties of the input - the olfactory receptor neurons ­ have become clearer, the operation of the network itself remains cryptic. Therefore we measured spatio-temporal odor-response patterns in the output of the olfactory glomeruli using optical imaging in the honeybee Apis mellifera, and mapped these responses to identified glomeruli, the structural and functional units of the AL. We found that each odor evoked a complex spatio-temporal activity pattern of excited and inhibited glomeruli. These properties were odor- and glomerulus-specific and were conserved across individuals. The spatial pattern of excited glomeruli appeared more confined as compared to the previously published signals, which derived mainly from the receptor neurons, showing that inhibitory connections enhance the contrast between glomeruli in the AL. To investigate the underlying mechanisms, we applied GABA and the GABA-receptor antagonist picrotoxin (PTX). The results show the presence of two independent inhibitory networks: one is GABAergic and modulates overall AL activity, the other is PTX-insensitive and glomerulus-specific. Inhibitory connections of the latter network selectively inhibit glomeruli with overlapping response profiles, in a way akin to Œlateral¹ inhibition in other sensory systems. Selectively inhibited glomeruli need not be spatial neighbors. The net result is a globally-modulated, contrast-enhanced and predictable representation of odors in the olfactory output neurons.

 


11

Kristin Scott, Roscoe Brady Jr., and Richard Axe1

Howard Hughes Medical Institute
Columbia University, College of Physicians and Surgeons
701 W. 168th Street New York, NY 10032, USA
[email protected]

Characterization of a gene family encoding candidate gustatory and olfactory receptors in Drosophila

A novel family of candidate gustatory receptors (GRs) was recently identified identified by searching the Drosophila genome with an algorithm designed to detect genes encoding seven transmembrane domain proteins (Clyne et al., 2000). We have characterized the expression patterns of members of this family and find that GR genes are likely to encode both olfactory and gustatory receptors in adult flies and larvae. In situ hybridization and transgene experiments reveal that some receptors are expressed in topographically restricted sets of neurons in the proboscis, whereas other members are expressed in spatially fixed olfactory neurons in the antenna. Members of this gene family are also expressed in chemosensory bristles on the legs and in larval chemosensory organs. We have visualized the axonal projections of chemosensory neurons in the adult and larval brain and observe that neurons expressing different GRs project to discrete loci in the antennal lobe and subesophageal ganglion. These data provide insight into the diversity of chemosensory recognition and an initial view of the representation of gustatory information in the fly brain. We are interested in understanding the logic of taste discrimination in Drosophila and the ability to follow pathways of functionally distinct taste receptor neurons may ultimately allow us to unravel the neural circuitry involved in gustatory behaviors.

References:

1. Clyne, P.J., Warr, C.G. and Carlson, J.R. (2000). Candidate Taste Receptors in Drosophila. Science 287, 1830-1834.

 


12

Brian H. Smith, Ph.D.

Department of Entomology
1735 Neil Ave. Ohio State University Columbus, OH 43210-1220
[email protected]

Distributed plasticity in early olfactory processing

In recent years olfactory research has progressed at several levels of analysis. Behavioral analyses continue to reveal how animals (both invertebrate and vertebrate) modify responses to both pheromonal and nonpheromonal odorants based on experience, which implicates plasticity as an important principle in olfactory processing. Physiological investigations have now revealed in reasonable detail how odors are represented in the insect brain by spatial and temporal patterns of activity. There is also a growing body of evidence in mammals and in insects that neural analogs to behavioral plasticity are manifested in early (sensory and first-order synaptic processing) neural processing of odors. Yet the causal relationships between neural manifestations of olfactory processing ­ that is, spatial and temporal patterns of neural activity as well as plasticity ­ and established behavioral mechanisms remain unresolved. I will report on a variety of means that we have employed to measure and manipulate neural activity patterns invoked by exposure of moths and honey bees to nonpheromonal odorants. These types of manipulations sometimes reveal causal relationships across levels of analysis. In addition, I will propose that plasticity in the insect antennal lobe and vertebrate olfactory bulb is needed in order to fine tune those neuropils to recognize important odors and sort them out from an odor background. This "distributed" plasticity frees other higher-order processing centers to focus on associations of identified, important odors with contextual information processed by other sensory modalities.

 


13

Erich Städler

Eidg. Forschungsanstalt 334, Postfach 185
CH-8820 Wadenswil-Switzerland
[email protected]

Plant chemical cues important for oviposition of herbivorous insects.

Plant compounds are in general the most important sensory cues mediating oviposition. Depending on the insect mechanical and visual characters can play a significant role too, but plant compounds seem to be the major element of "search-" and / or "recognition images" of herbivores. The leaf surface and its waxes play an important role for host-plant selection by ovipositing females of different species. This seems understandable since ovipositing females rarely have access to the plant interior during first encounter with a potential host plant. The interaction of attracting or stimulating compounds with the waxes is not well understood and it is still open if chemical or mechanical properties of the waxes explain the observed synergistic effects.The so far identified attractants and / or stimulants enclose volatiles and non-volatiles with varying polarity. But, contrary to a common believe, volatiles acting as long range attractions are the exception and not the rule. Compounds with host-plant specific distribution as well as more generalised plant allelochemicals have been found to influence oviposition. Primary metabolites can induce the selection of plants or plant part within a plant species. Mixtures of compounds are the rule and may also contain compounds that alone or in higher concentration inhibit oviposition. Highly important stimulants or elements of mixtures can occur at extremely low concentrations. As expected in such cases the insect receptor neurons have been found to be extremely sensitive. Insects attacking the same host plant might perceive completely different spectrum of compounds or mixtures. This indicates that the association with specific host plants evolved differently. Some oligophagous insects are stimulated by different combination of plant compounds indicating an ability to recognise specific host-plant "search or recognition images". Plant compounds influencing oviposition of generalist (polyphagous) herbivores are still not very well known. The hypothesis that the generalists have receptor neurons with non-specific sensitivity spectrum seems wrong. But there are indications that plant compounds acting as repellents or deterrents play a more important role in polyphagous than oligo- or monophagous insects. Further "generalists" could be a collection of "specialists" that could explain some of the observed compound specific receptor neurons in these insects. The host-plant selection of ovipositing females can be influenced by prior experience. Most likely associative learning during alighting or selection of an oviposition site changes the preference for specific plants and plant volatiles seem particularly important in this respect.

 


14

M. C. Stensmyr1, I. Urru2, I. Collu2, A.M. Angioy2, B. S. Hansson1

1 Department of Ecology
Lund University, S-22362 Lund, Sweden
[email protected], [email protected]

2 Department of Experimental Biology
University of Cagliari, S.S. 554 Km 4500 Monserrato, Italy
[email protected]

Foul fragrances fool flies

Many plants have evolved elaborate means to deceive insects into acting as unrewarded pollinators1. Here we report the chemical basis of a plant deception, where the odour and the structure of a carcass is mimicked in order to attract carrion feeding blowflies.

The deceiving plant is the Dead horse arum, Helicodoceros muscivorus, native to the western Mediterranean region. The Arum plant produces a strong, to humans repugnant smell, strongly reminiscent of a rotten carcass. The unpleasant smell is accompanied by a meat coloured, hairy appearance.

We used gas chromatography with simultaneous flame ionization and electroantennographic detection3 to identify the compounds responsible for the chemical mimicry. Head space collections of volatiles from live, rooted Dead horse arum plants were performed in the field on the two islands Serpentara and Cavoli, off the coast of Sardinia. Head space collections of odours from rotten meat were performed in order to compare antennal detection of the flower odours with that of a natural food source. Antennae of the blowfly, Protophormia terranovae, a Dead horse arum pollinator species were used as electrophysiological odour detectors. Stimulation with Arum and meat odour collections produced identical reponse patterns. Our results show that to the fly the two different odours are inseparable, i. e. by relying on odours alone the flies cannot separate the flower from a carcass.

The chemicals eliciting antennal responses were identified using gas chromatography-mass spectrometric analysis, as three, structurally similar oligosulphides; dimethyl mono-, di- and trisulphide. These oligosulphides are typically found in the odour of decaying meat4, and are thus of crucial importance to the flies for food and oviposition site location. Dimethyl di- and trisulphide have previously been shown to be potent attractants to blowflies, including P. terranovae.5,6

References:

1. Schiestl, F. P. et al. Nature 399, 421-422 (1999).

2. Fridlender, A. Webbia 55, 7-35 (2000).

3. Arn, H., Städler, E. & Rauscher, S. Z. Naturforsch. 30, 722-725 (1975).

4. Gill, C. O. in Meat Microbiology (ed. Brown, M. H.) 225-264 (Applied Science Publishers, London & New York, 1982).

5. Mackley, J. W. & Brown, H. E. J. Econ. Entomol. 77, 1264-1268 (1984).

6. Nilssen, A. C., Tømmerås, B. Å., Schimd, R. & Evensen, S. B. Entomol. Exp. Appl. 79, 211-218 (1996).

7. Dardani M. & Collu I. Rend. Sem. Fac. Sci. Univ. Cagliari 2, 69-99 (2000).

 


15

Mark Stopfer and Gilles Laurent

California Institute of Technology
Pasadena, CA, USA
[email protected]

Spatial and Temporal Coding of Odors in Turbulent Air

In the locust and other insects, controlled odor pulses evoke fast oscillatory synchronization of ensembles of projection neurons (PNs). Superimposed upon these fast (~20Hz) oscillations are slower, odorant-specific spiking patterns that consist of sequences of excitation, inhibition and quiescence. These features have been shown to contain information about the identities of odorants. Further, the precision of encoding is enhanced when odor pulses are repeated.

To investigate whether these forms of temporal coding occur when odorants are carried in a more natural fashion, we presented odorants within a small wind tunnel (100 x 30 x 20cm; wind speed: 20-50cm/sec). Natural odor sources such as fresh flowers or vials of pure or diluted odorants ("green" chemicals such as 1-hexanol, 1-heptanol, and 1-hexen-3-ol or common blends such as cherry and mint) were placed at the upwind end of the tunnel, and a locust with a fixed antenna was placed at the downwind end. To monitor the arrival of windborne odor filaments near the locust's intact antenna, we recorded electroantennograms (EAGs) from a second, isolated antenna wired to a DC amplifier. Simultaneously, we made intracellular recordings from pairs of PNs and local neurons (LNs) and extracellular recordings of the local field potential (LFP) from the mushroom body, a PN projection site. (The LFP provides an indication of synchronized PN firing.)

Preliminary results from about 150 (3min) trials in 20 animals indicate: Odor filaments caused distinct, transient deflections in the EAG. In all trials, EAG deflections coincided with distinct bouts of ~20Hz oscillation in the LFP that were phase-locked to subthreshold oscillations in the LNs and PNs. When PNs responded to the odorant, spikes were generally phase-locked to the LFP. Odorant-specific slow temporal patterns were evident in the firing of PNs. Oscillations and phase-locking increased progressively with filament encounters. Thus, complex and disorderly stimuli can evoke odorant-encoding ensemble activity.

 


16

R.A. Steinbrecht*, S.R. Shanbhag, J. Carlson, C. Pikielny, D.P. Smith

*Max-Planck-Institut fuer Verhaltensphysiologie
82319 SEEWIESEN Germany
[email protected]

Smelling proteins - what can we learn about olfactory transduction from the fruitfly ?

The fruitfly, Drosophila melanogaster, has become one of the major model animals to study olfactory transduction. The fully decyphered genome of this species and the modern methods of engeneering relevant proteins directly from the genes are more than outweighing the disadvantages caused by the extreme smallness of the antennae. Another great advantage is the availability of mutants with certain defects in olfaction-guided behaviour. In Drosophila, meanwhile ~60 odorant-binding proteins are known and this is also the first insect species, in which olfactory receptor proteins have been characterized, interestingly their number is also ~60. On the antenna and maxillary palps we have described 17 morphological subtypes of sensilla with some 40 olfactory receptor neurons. Using antibodies against 5 different OBPs (OS-E, OS-F, LUSH. PBPRP2, PBPRP5) we have started to map the expression mosaic of these proteins and have found that: 1. OBP expression does correlate with morphological sensillum types and subtypes. 2. Several OBPs may be co-localized in the same sensillum. 3. OBP localization is not restricted to olfactory sensilla. The expression of PBPRP2 in antennal epidermis sheds some light on the possible evolution of OBPs. The compartmentalization of the insect antenna into different sensilla opens the possibility to study the elements of this "compound nose" by correlating immunolocalization data of OBPs with the expression mosaic of olfactory receptor proteins and, eventually, with electrophysiological data on the functional specificty of the different olfactory rceptor neurons.

 


17

Richard Vogt

Department of Biological Sciences
University of South Carolina Columbia, SC 29208, USA
[email protected]

OBPs: A divergent multigene family unique to holometabolous and hemipteroid insects and a genetic determinant of sensillum phenotype.

Odorant Binding Proteins (OBPs) represent a moderate sized multigene family; many but not all OBP homologues are olfactory. Olfactory OBPs are both highly divergent and differentially expressed among olfactory sensilla which have distinct odor sensitive phenotypes. Combinatorial expression of OBPs, olfactory receptor proteins (ORs), odor degrading enzymes (ODEs) and other gene products such as SNMPs contribute to the unique phenotypes of olfactory sensilla. The OBP gene family is not universal throughout insects; OBPs are only known from the holometabolous and hemipteroid lineages of neopterous insects. These two lineages are thought to share a common ancestor distinct from other neopterous insects including the orthopteroids; members of the OBP gene family are not known from any orthopteroid insect. Nevertheless, holometabolous and hemipteroid insects include 94% of known insect species belonging to 15 of the 29 extant insect Orders, emerging about 300 million years ago, and the OBP gene family may have contributed to the olfactory prowess of these species; through the divergence of these lineages, OBP genes have had ample opportunity to duplicate, diversify, and specialize. Manduca sexta has at least 7 and likely >20 OBP homologues. Drosophila melanogaster has at least 23 OBP homologues. Evolutionary relatedness of these genes can be seen in their genomic structures and chromosomal locations and associations, while functional relationships may be seen in the similarities and differences of their amino acid sequences. The regulation of OBP expression is tied to the developmental determination of sensilla phenotype, a determination which must occur early in antennal development, but in distinctly different manners in lepidoptera (e.g. M. sexta) and diptera (e.g. D. melanogaster). In adult male M. sexta, olfactory sensilla are distributed in two distinct zones; one zone is highly ordered and homogeneous containing the pheromone sensitive long trichoid sensilla, while another zone is highly disordered and heterogeneous containing a mixed population of sensilla types. These zones are apparent within hours of pupation, evident in the patterns of cellular arrangements and transcriptional gene expression. However, mitotic events which give rise to the cells comprising individual sensilla do not occur until 24-72 hrs after pupation, suggesting that zonal determination may occur before pupation within the imaginal disk, while phenotypic decisions within the zones may occur after pupation, during and immediately following mitotic activity. These developmental decisions are ultimately lead to the expression of specific combinations of OBPs, ORs, ODEs, SNMPs, cuticular form and other phenotypic determinants which define the functional characteristic of individual sensillar sensory organs.

 


18

Leslie B. Vosshall

Laboratory of Neurogenetics and Behavior
The Rockefeller University New York, NY 10021 USA
[email protected]

Olfactory Sensory Maps in Drosophila

How do insects recognize and discriminate hundreds of distinct odorous ligands in their environment and respond to these stimuli with appropriate behaviors? We have used a molecular approach to identify the receptor genes that underlie the process of odorant recognition in the fruit fly, Drosophila melanogaster. We and others have identified a large gene family encoding at least 60 different seven transmembrane domain G protein-coupled receptors. Each DOR gene is selectively expressed in a small non-overlapping subset of adult olfactory neurons in either the antenna or maxillary palp. The spatial distribution of neurons expressing a given receptor is conserved between individuals and generates a topographic map on the surface of the antenna. Neurons are likely to express only a single DOR gene along with a DOR gene that is expressed in most olfactory neurons, rendering them functionally distinct.

To understand the relationship between DOR gene expression in the periphery and glomeruli in the antennal lobe, we have mapped the patterns of projections of neurons expressing a given odorant receptor. Using genetic tracing techniques, we have marked subpopulations of olfactory neurons expressing a given DOR gene with the synaptic marker n-synaptobrevin-GFP. This N-terminal fusion of neuronal synaptobrevin to green fluorescent protein selectively associates with synaptic vesicles and efficiently labels axonal termini. We find that all neurons expressing a given receptor extend axons that converge upon one or two distinct antennal lobe glomeruli. The position and shape of the glomerulus corresponding to a particular population of afferent neurons is invariant between individuals. Olfactory axons innervate the ipsilateral glomerulus, then branch and form synapses with the bilaterally symmetric glomerulus in the contralateral antennal lobe.

Future experiments will address how this spatial map forms in development and whether spatial mapping is employed in Drosophila to encode odor quality.

 


19

Troy Zars, Martin Schwärzel, MAtthia Fischer and Martin Heisenberg

Lehrstuhl für Genetik und Neurobiologie
Biozentrum, Am Hubland D97074 Würzburg Germany
[email protected]

A sufficient circuitry approach for the localization of short-term memory in Drosophila.

The localization of memory traces is a major goal of neuroscientists. Toward that goal several techniques have been used, including surgical and chemical ablation as well a localized gene specific Œknock-outs¹. These approaches have been successful in determining necessary brain structures for a given memory task. However, in a highly integrated brain the determination of a necessary structure does not necessarily indicate the localization of a memory trace. We have asked if we could localize normal synaptic plasticity (largely thought to be a mechanism of memory formation) to a subset of the Drosophila brain and maintain normal memory formation. If flies could be found that both performed normally in a learning and memory task and had a sub-set of the brain with normal synaptic plasticity, then we conclude that the memory trace is at the site of that plasticity.

We have used different transgenes to localize an olfactory memory trace in Drosophila. The first is the rutabaga type 1 adenylyl cyclase. This cyclase type has been shown to be important for both synaptic plasticity and memory formation in several species and paradigms. Flies mutant in the rutabaga gene show defects in all memory forming tasks thus far tested and in synaptic plasticity. If a rutabaga transgene is expressed in the mushroom bodies of an otherwise mutant animal, olfactory short-term memory is restored to normal levels. Thus indicating a subset of the brain can be sufficient for memory formation. However, if memory is tested at later time points, memory scores for mushroom body rescued flies are intermediate between normal and mutant flies. Several possible explanations for this result are being addressed. In addition, a transgene that controls synaptic transmission in both a spatial and temporal fashion (a temperature sensitive shibire transgene) has been expressed in the mushroom bodies and its effects on memory acquisition, storage, and retrieval are under investigation.

Supported by grants from the DFG (He986/10-3) and the Fonds der Chemischen Industrie to M.H.


20

Walter S. Leal

Department of Entomology
University of California at Davis, Davis, CA 95616 USA
[email protected]

Molecular Mechanisms of Pheromonal Signals Perception in Insects

The remarkable fidelity of the insect olfactory system is unrivalled in nature, particularly its temporal precision during odor-oriented flights. While navigating en route to a pheromone source, an insect encounters intermittent chemical signals with the stimulus present in broken bursts and, consequently, the animal has to reset its detector in a millisecond timescale. In addition, the pheromones and other chemical signals (semiochemicals) are normally buried in complex mixture of odorants from a myriad of sources. This has led to the development of a remarkably selective and sensitive olfactory system, which approach the theoretical limit for a detector. There is growing evidence that the temporal precision of olfactory system in insects is determined by the early olfactory events (peripheral interactions) rather than by intracellular signaling processes (signal transduction), but the evidence with regard to signal inactivation is also dichotomous. One school favors the hypothesis that rapid inactivation of chemical signal is an enzymatic process regulated by pheromone-degrading enzymes (PDEs), whereas the other school favors the hypothesis that the chemical signals are first inactivated upon oxidation of pheromone-binding proteins (PBPs), the so-called redox shift. On the other hand, our recent structural studies suggest that odorant-binding proteins solubilize and transport pheromones to their receptors while protect them from pheromone (odorant)-degrading enzymes. The crystal structure of the pheromone-binding protein from Bombyx mori has six helices, which are held in place by three disulfide bridges. The ligand (bombykol) binds in a completely enclosed hydrophobic cavity formed by four antiparallel helices. The transport journey seems to end when the complex PBP-ligand arrives at "the destination", i. e., a negatively charged membranes. Our data suggest that the binding pocket opens upon interaction with these membranes. NMR studies confirmed the existence of two conformations, one at the sensillar lymph pH (B conformation), and the other at low pH (A conformation), probably a localised pH at the dendritic surface. That "B", but not to "A," binds bombykol suggests that this fast conformational change leads to the release of the ligand. Also, stopped-flow kinetics studies showed that the B-A reaction takes place in a millisecond timescale in agreement with the kinetics of odour-oriented navigation in insects.

 


21

W.L. Mechaber1, N.A. Scascighini1,2, J.G. Hildebrand1

1 ARL Division of Neurobiology, University of Arizona, Tucson, AZ, USA
2 Institute of Plant Sciences, Applied Entomology, Zürich, Switzerland

Female Moth Behavior Elicited by Collected Hostplant Volatiles

Crepuscular and nocturnal insects rely, in great measure, upon appropriate olfactory stimulation for host location and selection. As female moths fly to oviposition sites, they detect and respond to the appropriate volatiles released into the atmosphere by metabolically active plants. We have focused our research on examining (1) the composition of vegetative plant volatiles collected from intact, growing hostplants and (2) the effect of these volatiles, as stimulants, on the behavior of female moths. Our experiments were conducted with Manduca sexta (Sphingidae) as the model insect. We compared responses of female moths to headspace volatiles from proven hostplants belonging to two families: tomato (Lycopersicon esculentum; Solanaceae) and devil's claw (Proboscidea parviflora; Martyniaceae). Initial experiments tested headspace-volatile samples that were collected and presented as a solvent-based sample to mated, day-3 female moths in wind-tunnel bioassays. The behaviors observed in these bioassays were compared to behaviors observed when moths were presented with intact, potted plants. In response to volatile samples from both hostplant species, female moths displayed abdomen curling posture, with limited upwind oriented flight; responses to potted plants encompassed both behaviors. We have identified some of the compounds in common in the headspace mixtures from these two plant species. Our findings indicate that we can both collect behaviorally active volatiles and elicit appropriate female-specific behaviors using vegetative volatile samples alone.

 


22

Jürgen Krieger, Neil Oldham *, Stefanie Bette, Claudia Mohl and Heinz Breer

Universität Hohenheim, Institut für Physiologie, D-70593 Stuttgart, Germany
*Max Planck Institut für Chemische Ökologie, D-07745 Jena, Germany
[email protected]

Spectroscopic analysis of ligand binding to moths Pheromone binding proteins

To reach their specific receptors on the surface of the olfactory neurons, moths pheromone molecules enter the antennal sensilla through pores in the cuticle and have to traverse an aqueous barrier, the sensillum lymph. This transfer is supposed to be mediated by pheromone binding proteins (PBPs) of which several types from various moths species have been cloned and sequenced. In Bombyx mori and Heliothis virescens we have found only one PBP-subtype, whereas in Antheraea polyphemus and the sibling species Antheraea pernyi three subtypes each have been discovered. To analyse the binding features of PBPs and to approach the question, if PBPs may undergo structural changes upon ligand binding we have expressed PBPs in E.coli and purified the recombinant proteins from a periplasmic fraction. Binding of species specific pheromones and related compounds to recombinant PBPs was analyzed by native ESI-mass spectroscopy for the B.mori PBP and by fluorescence competition experiments using 1-amino-anthracene (1-AMA) and 2-(p-toluidinyl)-naphtalene-6-sulfonic acid (TNS) for A. polyphemus PBP1. In addition, the intrinsic fluorescence of ApolPBP1 and of mutated proteins, where single Tryptophans have been replaced by Alanin, was employed to monitor pheromone binding. Using the different spectroscopic approaches binding of pheromones and of related compounds with partly similar binding affinities was observed for both the B.mori and the A.polyphemus PBP. To register any structural changes of PBPs that may be induced by binding of pheromone ligands, assays using CD spectroscopy and UV-difference spectroscopy were performed. These experiments indicated that PBPs undergo structural changes upon interaction with pheromones.

This work was supported by the Deutsche Forschungsgemeinschaft.

 


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