Morphology and structure of insect antennae and sensilla
Insects - in contrast to vertebrates - have "everted noses", the antennae, which are studded with tens of thousands of tiny hairs and pegs. Each of these sensilla comprises the sensory processes of but a few olfactory receptor neurons. This partitioning and the accessibility of single receptor cells are two of the many advantages that insects offer for studies of the sense of smell. We study the following species:
· The silkmoths, Bombyx mori, Antheraea pernyi and A. polyphemus, the antennae of which represent multimodal but predominantly olfactory sense organs with emphasis on pheromone detection in the male sex. (Steinbrecht, Keil)
· The fruitfly, Drosophila melanogaster, which is the model animal of genetic and molecular approaches. (Steinbrecht, with S. Shanbhag)
· The malaria vector, Anopheles gambiae; which - in contrast to other mosquito species, such as Aedes aegypti - so far has not been adequately investigated (see host-odor binding proteins in the malaria mosquito Anopheles gambiae). (Hartlieb)
· The honey bee, Apis mellifera, which is one of the economically most important insects, although surprisingly little is known about their sensory equipment. (Keil in collaboration with Jürgen Tautz (Würzburg) and Gert Stange (Canberra)
Development and metamorphosis of insect antennae
The silkmoth antenna can be followed back into the feeding stage of the animal, the caterpillar. The tiny larval antenna bears half a dozen chemosensory pegs and two mechanosensitive bristles. A few additional chemo- and mechanosensilla are located on the maxillae, together with two receptors for CO2. During the fifth larval instar, the epidermis of the larval antenna retracts from the cuticle and forms the antennal imaginal disk. The disk grows considerably in the prepupal stage, finally giving rise to the large, annulated adult antennal anlage. During the pupal stage, the anlagen of about 70,000 adult olfactory sensilla are generated which send their axons towards the central nervous system. The leaf-shaped antennal anlage is transformed into the feathered adult antenna by lateral incisions, during which process the innervation pattern is completely rebuilt. A comparison between larval heads and antennae of Lepidoptera/Trichoptera, Hymenoptera, Mecoptera and Coleoptera shows remarkable structural similarities which strongly suggest a common ground plan. (Keil, C. Steiner)
Structure and composition of insect sensillar components
· Pore tubules: Odour molecules have access to the inside of olfactory hairs via the so-called pore tubules. These are tubular extensions of the outer lipid layer of the epicuticle which traverse the otherwise hydrophilic procuticle, which consists mainly of chitin and protein. As the tubules have a strong affinity to cationic markers, they most probably do not consist entirely of lipids, but also contain oligosaccharides. (Keil, Steinbrecht, Oldenburg)
· Olfactory dendrites: The olfactory dendrites are able to perform active movements and survive for a considerable time in culture medium. They have a cytoskeleton made up of microtubules mainly, but contain actin, IF-proteins, fodrin, kinesin, and myosin as well, which might explain their motility. Pore-like structures have been demonstrated in the ciliary membrane. (Keil, G. Kumar)
Immunocytochemical localization of proteins involved in signal transduction
Immunocytochemistry provides a link among biochemistry, molecular biology, morphology and function. An increasing number of proteins are presently being characterized in tissue homogenates or reconstructed in cloning experiments. Their precise localization within the sensory organ allows for hypotheses on their function. This is particularly true for insect olfactory sensilla, where electrophysiological recording can establish the stimulus specificity of different sensilla. We study the immunolocalization of:
· odorant-binding proteins and other transduction proteins in moths. (Steinbrecht, Maida, Ziegelberger)
· putative olfactory receptor molecules, odorant-binding proteins and other olfactory transduction proteins in Drosophila. (Steinbrecht, S. Shanbhag, in collaboration with J. R. Carlson, Yale Univ. New Haven CT, C. Pikielny, Piskataway, NJ. and D.S. Smith, Dallas)
· Host-odor binding proteins in the malaria mosquito Anopheles gambiae (Hartlieb)
· Carboanhydrase in CO2-receptors of the honey bee (Keil, in collaboration with G. Stange, Canberra, Australia)
Morphology of Drosophila sensory mutants
· Olfactory organs after "genetic surgery" (ablation of olfaction-specific genes) (Steinbrecht, S. Shanbhagh, in collaboration with C. Pikielny, Piscataway, NJ, USA)
· Mutants defective in mechanoreception (Keil, in collaboration with C. Zuker, La Jolla, USA)
Reception of plant volatiles
Insects use several types of olfactory sensilla to smell plant odours, which lead them to food sources or oviposition sites. In females of the moth species Bombyx mori the long olfactory hairs (sensilla trichodea) contain two receptor cells responding best to benzoic acid and linalool, respectively. The time course of the responses of these two receptor cell types, their specificity and possible inhibitors are under investigation. In many moth species the shorter olfactory hairs (sensilla basiconica) contain generalist receptor cells, responding to different odour classes, primarily various terpenes and green leaf volatiles. The sensilla coeloconica of insects are double walled multiporous sensilla, often located in pits. In moths they contain up to five receptor cells, most of which respond best to saturated aliphatic acids of different chain length. Former group members, involved in this project: Van Naters, T. Heinbockel, M. G. de Brito Sanchez, K. Haberkorn, H. Ding
The multiple role of odorant-binding proteins
Before airborne odorant molecules can stimulate the olfactory receptor cells of animals that live on land, they have to pass through an aqueous solution that contains high concentrations of soluble odorant-binding proteins (OBPs). In insect sensilla these OBPs play multiple roles in signal transduction. Sensillum lymph perfusion experiments in the moth Antheraea polyphemus implied a solubilizer and carrier function of the pheromone-binding protein (PBP) and supported the hypothesis that a complex of PBP and pheromone acts on the receptor cell membrane rather than the pheromone alone. Upon pheromone stimulation of isolated olfactory hairs, a redox-shift of the PBP from a reduced to an oxidized form, by forming a disulfide bridge, was observed. This shift only occured in the presence of receptor cell dendrites, suggesting that receptor activation might go along with PBP oxidation, the latter leading to a rapid pheromone deactivation (see Stimulus deactivation and degradation, or Perireceptor and receptor events).
In addition to the transporter and deactivator function described above, a participation of OBPs in odour discrimination was suggested. A few odorant binding studies support this idea, as do the increasing number of soluble OBPs that have been discovered within a given insect species and the distinct distribution pattern of different OBPs among different sensilla. (Ziegelberger, Maida, Steinbrecht, Kaissling, Pröbstl)
Stimulus deactivation and degradation
The sensory system of a male moth must, within fractions of a second, follow the fluctuations of pheromone concentrations within an odour plume. Consequently, pheromone molecules, once adsorbed on the sensilla of the antenna, should exert their stimulatory action only for a short time and the pheromone must be rapidly deactivated. Two possible mechanisms of pheromone deactivation are being investigated.
Firstly, we study pheromone degradation by specific enzymes like the sensillar esterase and aldehyde oxidase. Our data suggest that enzymatic pheromone degradation is not involved in the rapid pheromone deactivation, since:
· The metabolic half life of pheromone in intact A. polyphemus antennae is in the range of minutes;
· in individual males with similar electrophysiological responses, the esterase can be present in highly variable amounts, i.e. in some moths with at least 100-fold less activity;
· inhibitors of the sensillar esterase did not prolong the decline of the receptor potential. Secondly, the receptor-mediated redox shift of the pheromone-binding protein has provided experimental evidence of a non-enzymatic pheromone deactivation (see: The multiple role of odorant-binding proteins, or Perireceptor and receptor events).
At present we are investigating whether these findings are limited to the specialized case of pheromone deactivation or can be extended to general odours like linalool and benzoic acid. (Ziegelberger, Maida, Pröbstl, Pophof, Oldenburg, Kaissling)
A search for pheromone receptors
The highly specific and sensitive sex-pheromone response of the male silkmoth Antheraea polyphemus is believed to depend on the activation of specific receptor proteins present in the dendritic membrane of pheromone-sensitive receptor cells.
The strategy we have adopted in our search for receptor proteins was based on earlier investigations in which we removed the pheromone binding protein (PBP) from the sensillum lymph space. These experiments led to the idea that it is the complex of PBP-pheromone rather than the pheromone alone, that activates the postulated membrane-bound pheromone receptor. According to this working hypothesis, we tried to make use of a possible affinity between PBP and proteins isolated from the dendritic membranes of receptor cells. A protein with an apparent molecular weight of 70 kDa was eluted from a PBP-affinity column. This protein was present only in male sensory hairs, but not in control tissues like female antennae or brain, and showed binding of the tritiated main component of the female sex-pheromone blend. Thus, it may represent the putative membrane-bound pheromone receptor. Our experiments support the idea that binding of PBP, pheromone and receptor proteins is necessary to start Intracellular signalling. (Ziegelberger, Maida)
Host-odor binding proteins in the malaria mosquito Anopheles gambiae
Blood-sucking insects are the dominant vectors for infectious diseases in humans. One of the most dangerous species is the malaria vector A. gambiae. Worldwide 400 million people are infected by malaria and every year around 2 million, mainly children, die due to this disease.
CO2 and lactic acid are well known attractants for several host-seeking haematophagous insects. However, the human-specific mixture of volatiles attractive for A. gambiae has not been identified yet. The aim of our study is to identify proteins in olfactory sensilla of A. gambiae mosquitoes which participate in odour discrimination and host finding. The hope is to genetically modify such proteins and interfere with host finding by these mosquitoes. The project is supported by the WHO.
· Biochemical approach. Isolation and characterisation of putative odorant-binding proteins (OBPs) from antennae, head and head-appendages. All OBP candidates are N-terminal sequenced and the sequences used for the production of antisera, which are used for immunolocalization studies. Methods used: HPLC-gelfiltration, preparative isoelectrofocusing, native and SDS-PAGE, immunoblotting, binding studies.
· Morphological and immunocytochemical approach. The various olfactory sensilla types present in both sexes and their localization on the antennae are investigated. OBP-antisera are used to study the distribution of types of OBPs within sensilla responding to human odors.
Methods used: cryofixation, immunolabelling, transmission and scanning electron microscopy. (Ziegelberger, Proebstl, Hartlieb, Maida)
Intracellular signalling is a complex of biochemical events, including enzymatic reactions, that transduce chemical stimuli into electrical signals eventually sent to the brain. Transduction enzymes are present in very minute amounts; to overcome this drawback, we take advantage of the possibility to prepare homogenates from isolated olfactory sensilla containing the pheromone-sensitive receptor cell dendrites and the sensillum lymph only. Thus, this preparation is highly enriched in material participating in transduction processes. Molecules involved in the second-messenger transduction cascade, such as G-proteins, phospholipase C, nitric oxide synthase, protein kinase C, as well as proteins involved in calcium metabolism (calmodulin, calcineurin) have been identified by using specific antibodies. Their activity, the production of intracellular signal compounds controlled by pheromone stimuli, was studied in living antennae. Our knowledge of the antennal morphology allowed us to determine the intracellular concentrations of 'second messengers'. Finally, transduction proteins are immunolocalized at the level of the electron microscope within the olfactory sensilla, to associate their localization with a specific function. (Ziegelberger, Maida, Steinbrecht and Kaissling)
Perireceptor and receptor events
These events comprise the adsorptive uptake of stimulus molecules from the air space by the surface of the insect antenna, their diffusion towards the olfactory receptor cell, and a network of extracellular chemical reactions. The latter include the binding of stimulus molecules to odorant-binding proteins, the interaction of these complexes with receptor molecules of the receptor cell, a redox shift of the odorant-binding protein leading to odorant deactivation and, finally, the enzymatic degradation of the odorant.
We developed a quantitative mathematical model of the network reactions based on published data of this laboratory as well as data from the literature. From these studies we conclude that the receptor potential, the first electrical response of the receptor cell, reflects the kinetics of the network rather than those of intracellular transduction processes. The modeling leads to hypotheses about the extracellular processes which can be tested experimentally. The analysis was done using a computer model of the chemical network developed by J. Thorson, Oxford. (Kaissling, Minor, Thorson, Pophof)
Elementary receptor potentials
Elementary receptor potentials (ERPs) can be elicited by single pheromone molecules. They are produced by an increase of membrane conductance in the range of 30 pS. The fluctuations of these responses might reflect changes of the activation of olfactory receptor molecules. We study the kinetics of the ERPs and their modifications by various factors, such as ligand structure, inhibitors, electrolyte composition, and temperature. (Kaissling, Minor, Thorson, Pophof, Van Naters)
Humoral control of receptor cell function
Octopamine is a biogenic amine which functions as a neurotransmitter, neuromodulator and neurohormone in insects. Large amounts of octopamine are secreted into the antennal hearts of insects, pumping haemolymph into the antennae. Octopamine receptors were cloned from moth antennal tissue. In the wind tunnel octopamine improved orientation towards pheromone sources and blend discrimination in several moth species. Therefore, octopamine was chosen in a study of the possible humoral control of the sensitivity of antennal pheromone receptor cells. Octopamine and its antagonist epinastine had no effect on the transepithelial potential, the spontaneous nerve impulse discharge, or the amplitudes of receptor potentials elicited by pheromone stimulation. The peak nerve impulse frequency in response to pheromone was significantly decreased by epinastine and increased by octopamine. The enhanced responses caused by octopamine may contribute to the behavioural effects observed in the wind tunnel. Possible mechanisms of the modulation are either a direct action of octopamine on the receptor neurons, or indirect effects on the metabolism. (Pophof)
Carbon-dioxide receptors of larval and adult insectsare studied, their structure and immunocytochemistry. Many larval and adult insects have receptors for CO2. In the moth larva, we find a single chip-shaped sensillum on each maxillary palp, whereas in the adult hundreds of club-shaped sensilla are assembled in a deep apical cavity in the labial palp. Larvae orient towards a source of CO2. In adult moths, CO2 receptors are able to detect extremely small fluctuations in CO2 concentration, although no behavioural response has yet been found. In honeybees, electrophysiology has shown that the CO2 receptors are the pit organs on the antennae; their response can be blocked by acetazolamine, an inhibitor for the enzyme carbonic anhydrase. The aim is to localize this enzyme immunocytochemically, as well as characterize the structure of CO2 sensilla. (Keil, Ziesmann, In collaboration with G. Stange, Canberra)
Associative learning of odors in moths
In Lepidoptera olfactory signals are important for several different behaviours, such as mate-finding or searching of host plants for oviposition and feeding. It is unclear whether all behavioural responses are innate or whether they also include some behavioural plasticity is little understood. By observing proboscis extension in conditioning experiments I have shown that both sexes of Heliothis virescens, H. armigera and Spodoptera littoralis moths are able to associate a flower odour with a food reward after a few learning trials, indicating that moths have a learning ability comparable to that of honeybees.
Moths do not learn all odors to the same extent. The ability to learn a given odour depends on its behavioural significance. Experiments with male and female S. littoralis showed that both sexes learn individual pheromone components nearly as well as the flower odor geraniol, whereas the full female pheromone blend, which elicits an innate behaviour in males, is not learned by the males and is learned only occasionally by the females. The ability of moths to discriminate between odours is currently being investigated in a joint project with Mikke Carlsson (Lund University). (Hartlieb)
Behavioural responses of female silk-moths to plant volatiles
Though the behavioural response of male silk-moths to the female sex pheromones is a well-established and easily demonstrated phenomenon, how odours affect female behaviour is obscure. This is surprising in view of the clear electrophysiological responses in females to a wide range of plant odours including benzoic acid and terpenes (see Project Reception of plant volatiles). We plan to develop a behavioural paradigm for the female silk-moth. Initially we shall test the odours of mulberry leaves on which the females lay their eggs. If a reliable paradigm can be found, we shall try to determine which odour components in mulberry attract females and affect oviposition. (Kaissling, Oldenburg, Van Naters)