Rudolf Alexander Steinbrecht

Max Planck Institute for Ornithology · Department of Behavioural Neurobiology

The third antennal segment (funiculus) and the maxillary palp are the main and accessory olfactory sense organs of Drosophila melanogaster. Cryofixed antennae and palps revealed a superior preservation of the sensory dendrites and other cellular details as compared to conventional chemical fixation. Extensive cross-section series through funiculus...

Odorant-binding proteins (OBPs) are small soluble proteins present in the aqueous medium surrounding olfactory receptor neurones. In this study we examine the expression patterns of three Drosophila OBPs (LUSH=OBP76a, OS-E=OBP83b and OS-F=OBP83a), using post-embedding immunocytochemistry. All three OBPs are co-expressed in sensilla trichodea wherea...

Antennae of the silk moth, Bombyx mori, were frozen by immersion into propane at -180 degrees C, and further processed by (a) freeze substitution (FS) or (b) freeze etching (FE). Although no cryoprotectant was used, freezing damage was observed in deeper tissue regions only. Data from FS specimens closely resemble those from FE replicas. Therefore,...

Of all the invertebrate phyla, arthropods have invaded the terrestrial habitat most successfully; with the exception of Crustacea they are predominantly land dwellers. Without doubt, one of the main reasons for this achievement is the evolution of arthropod cuticle which provides mechanical support and protection against water loss at the same time...

Fine structure immunocytochemistry enables the in situ localization of odorant-binding proteins with the high resolution of the electron microscope. Protocols for successfully labeling these proteins in insect chemosensory sensilla are given and some pitfalls pinpointed as well as ways to avoid them. The major achievements accomplished by these met...

For almost 100 years, insects have been favorable "model systems" in biology. Just to mention a few examples: fruit flies in genetics and developmental biology; bugs and caterpillars in hormone research; houseflies, blowflies, and locusts in neurobiology; silk moths in pheromone research; honeybees and crickets in neuroethology. For more than 50 ye...

We have identified, cloned and expressed a new chemosensory protein (CSP) in the desert locust Schistocerca gregaria belonging to a third sub-class of these polypeptides. Polyclonal antibodies stained a band of 14 kDa, as expected, in the extracts of antennae and palps of the adults, but not in the 4th and 5th instars. In the related species Locust...

Four recombinant odorant-binding proteins of Bombyx mori, pheromone-binding protein (PBP), general odorant-binding protein 1 (GOBP1), general odorant-binding protein 2 (GOBP2) and antennal binding protein X (ABPX), were expressed in E. coli and used to raise polyclonal antisera. Immunoblots of antennal homogenates showed that these antisera were sp...

We investigate how the molecular and cellular maps of the Drosophila olfactory system are integrated. A correspondence is established between individual odor receptors, neurons, and odors. We describe the expression of the Or22a and Or22b receptor genes, show localization to dendritic membranes, and find sexual dimorphism. Or22a maps to the ab3A ne...

The olfactory organs on the head of Drosophila, antennae and maxillary palps, contain several hundred olfactory hairs, each with one or more olfactory receptor neurons. Olfactory hairs belong to one of three main morphological types, trichoid, basiconic, and coeloconic sensilla, and show characteristic spatial distribution patterns on the surface o...

Deciphering the genome of the fruitfly, Drosophila melanogaster, has revealed 39 genes coding for putative odorant-binding proteins (OBPs), more than are known at present for any other insect species. Using specific antibodies, the expression mosaic of five such OBPs (OS-E, OS-F, LUSH, PBPRP2, PBPRP5) on the antenna and maxillary palp has been mapp...

Odorant-binding proteins were studied in the noctuid moths Agrotis segetum, Autographa gamma, Helicoverpa armigera, Heliothis virescens and Spodoptera littoralis using antisera raised against the pheromone-binding protein (PBP) and general odorant-binding protein 2 (GOBP2) of Antheraea polyphemus (Saturniidae). Proteins immunoreacting with these an...

In Drosophila, as in most insects, gustation is mediated by sensory hairs located on the external and internal parts of the proboscis and on the legs and wings. We describe in detail the organization and ultrastructure of the gustatory sensilla on the labellum and legs and the distribution of PBPRP2, a putative odorant-binding protein, in the gusta...

SNMP-1 (sensory neuron membrane protein 1) is an olfactory-specific membrane-bound protein which is homologous with the CD36 receptor family. Previous light level immunocytochemical studies suggested that SNMP-1 was localized in the dendrites and distal cell body of sex-pheromone-specific olfactory receptor neurons (ORN); these studies further sugg...

Soluble, low molecular weight proteins were immunodetected in proboscis extracts of Mamestra brassicae males by Western blot, using antibodies raised against the general odorant-binding protein of the moth Antheraea polyphemus. The same antibodies weakly labelled the sensillum lymph and subcuticular space of sensilla styloconica on ultrathin sectio...

Antennae and maxillary palps of Drosophila melanogaster were studied with the electron microscope on serial sections of cryofixed specimens. The number of epidermal cells roughly equals the number of sensilla, except for regions where the latter are scarce or absent. Each epidermal cell forms about two non-innervated spinules, a prominent subcuticu...

The aqueous medium bathing the dendrites of olfactory neurons contains high concentrations of odorant-binding proteins (OBPs) whose role is still unclear. OBPs may facilitate interactions between odorants and their membrane-bound receptors, perhaps by increasing the water solubility of hydrophobic molecules. Alternatively, OBPs may be involved in t...

Identification of chitin in sensory hairs of olfactory sensilla of silkmoths was performed using two independent methods. Firstly, ultrathin sections were labelled with gold-conjugated wheat germ agglutinin and showed positive labelling in the cuticule of sensilla as well as in the antennal cuticle. Secondly, isolated sensory hairs and body scales...

Odorant-binding proteins (OBPs) are a major constituent of the aqueous perireceptor compartment in vertebrates and in insects. Although different in primary structure, they are supposed to serve similar functions in both animal groups: (i) OBPs may act as solubilizers and carriers of the lipophilic odorants in the aqueous mucus or sensillum lymph;...

Odorant-binding proteins (OBPs) are small, soluble proteins present in the aqueous medium surrounding olfactory receptor neurons. Their function in olfaction is unknown: they have been proposed to facilitate the transit of hydrophobic molecules to olfactory receptors, to deactivate the odorant stimulus, and/or to play a role in chemosensory coding....

The fine structure of coeloconic sensilla of Bombyx mori was studied in cryofixed specimens. These sensilla belong to the category of double-walled wall-pore sensilla. The pegs are approximately 10 microm long, located in pits on the dorsal side of the antennal branches, and longitudinally grooved in their distal half (grooved surface approximately...

Fine structure immunocytochemistry permitted the localization of proteins, which are presumably relevant to olfactory signal transduction in the olfactory sensilla of the silkmoths Bombyx mori (Lepidoptera: Bombycidae), Antheraea pernyi and Antheraea polyphemus (Lepidoptera: Saturniidae). Odorant-binding proteins of different classes, pheromone-bin...

An historical overview is given of the most important discoveries and hypotheses regarding stimulus transport in insect olfaction. The great structural similarity between the pore tubules of olfactory single-walled wall-pore sensilla and the epicuticular filaments of non-olfactory cuticle may reflect not only a similar composition but also a simila...

Odorant-binding proteins (OBPs) are small soluble proteins present in the aqueous medium surrounding olfactory receptor neurons. Their function in olfaction is still unknown: they have been proposed to facilitate the transit of hydrophobic molecules to olfactory receptors, to deactivate the odorant stimulus, and/or to play a role in chemosensory co...

Pheromone-sensitive sensilla trichodea of nine moth species belonging to six families and three superfamilies of Lepidoptera were immunolabelled with an antiserum against the pheromone-binding protein of Antheraea polyphemus. Strong immunolabelling of the sensillum lymph was observed in all long sensilla trichodea of A. polyphemus, A. pernyi (Satur...

Olfactory sensilla show a large diversification of sensillum types even in the same species. Thus, double-walled and single-walled sensilla with highly different wall pores are usually found on the same antenna, and these may appear in the form of long slender hairs, pore plates or pit pegs. The selective constraints leading to this diversification...

Research on insect olfaction was and still is dominated by studies on pheromones. Also, the first odorant-binding protein characterised in an insect was a pheromone-binding protein, the PBP of A. polyphemus (Vogt & Riddiford, 1981). The name of this soluble protein of low molecular weight and acidic isoelectric point was chosen because it was shown...

The distribution of odorant-binding proteins among olfactory sensilla of three moth species was studied by immuno-electron microscopy. Two polyclonal antisera were used in a post-embedding labelling protocol on sections of cryo-substituted antennae. The first was directed against the pheromone-binding protein (PBP) of Antheraea polyphemus, the seco...

The European cornborer antenna is filiform in both sexes, but exhibits a substantially larger diameter in the males. On the antenna of both sexes, the following sensillum types were characterized: sensilla trichodea, s. basiconica, s. auricillica, s. coeloconica, s. chaetica and s. styloconica. Long dorsal bristles were of a chaetic type. An interm...

Outer dendritic segments of olfactory receptor neurons tuned to sex pheromone components were measured morphometrically on the antenna of male European corn borers. Ostrinia nubilalis, to determine if a correlation exists between the diameter of the outer dendritic segment and the spike amplitude. The olfactory sensilla investigated each contained...

The tuft organs of the human body louse, Pediculus humanus corporis, display a complex cuticular apparatus without wall pores, consisting of the cuticular cone, cuticular pocket, and the sensillar peg with 5-6 tuft processes. The cuticular wall of the sensillar peg exhibits several layers of differing electron density tightly surrounding the dendri...

Odorant-binding proteins are small water-soluble proteins that have been detected in the perireceptor compartment of olfactory receptor cells of vertebrates and insects [1,2]. Although their definite physiological role in olfaction is still unclear, similar functions have been proposed in stimulus transport or inactivation [1–6].

Methods of plunge freezing and freeze-substitution (FS) for insect antennae and similar body appendages are described. In these more or less cylindrical specimens, usually a layer below the cuticular surface of 10-15 microns thickness is well preserved without freezing damage, further inwards ice-crystal ghosts of increasing size are encountered, b...

A pheromone-binding protein (PBP) has been purified from the antennae of male Bombyx mori. It is a single polypeptide chain with a molecular weight of 16 kDa and an isoelectric point of 4.9. The PBP is present in the male and, to a minor extent, also in the female antennae, but not in other parts of the body. Strong crossreactivity has been observe...

Odorant-binding proteins are supposed to play an important role in stimulus transport and/or inactivation in olfactory sense organs. In an attempt to precisely localize pheromone-binding protein in the antenna of moths, post-embedding immunocytochemistry was performed using an antiserum against purified pheromone-binding protein of Antheraea polyph...

The general morphology and methodological peculiarities of insect sensilla are briefly reviewed. The stimulus conducting pore-tubule systems of pheromone-sensitive sensilla of the silkmoths Bombyx mori and Antheraea polyphemus are described. Lipophilic tracers readily enter the hair lumen, while hydrophilic tracers do so only after prolonged extrac...

The thermo-/hygrosensitive sensilla styloconica of the silk moth Bombyx mori were studied using cryofixation and freez-substitution. These sensilla are characterized by a short poreless cuticular peg, which is double-walled in its distal part. The central lumen is innervated by the unbranched outer dendritic segments of the two presumed hygrorecept...

Thermo- and hygroreceptors are located in the sensilla styloconica at the tip of the antennal branches of the silkmoth Bombyx mort They usually contain three sensory cells, a moist-, a dry- and a cold-sensitive cell. The dendrite of the thermoreceptor ends below the cuticle and shows a peculiar lamellation and surface substructure. The dendrites of...

Contact chemoreceptors on the antennae of Bombyx mori are located in sen-silla chaetica. They are innervated by four contact chemoreceptive sensory cells and a fifth mechanoreceptive cell. Structures important for stimulus conduction are the apical pore complex and the tubular matrix between these pores and the dendritic endings. There are, however...

The thermo/hygroreceptive sensilla styloconica of the silkmoths Bombyx mori, Antheraea pernyi, and A. polyphemus were reconstructed from serial sections of cryofixed and chemically fixed specimens. The volume and surface area of the different sensillar cells were calculated from the area and circumference of consecutive section profiles. In additio...

The thermo-/hygroreceptive sensilla styloconica of the silkmoth Bombyx mori are located on the tips of the antennal branches. A small poreless cuticular peg is innervated by three sensory cells. The outer dendritic segments of two type-1 receptor cells, the presumed hygroreceptors, almost completely fill the peg lumen and are in close contact with...

The arista, a characteristic appendage of dipteran antennae, consists of 2 short segments at the base and a long distal shaft. A small sensory ganglion, from which arises the aristal nerve, is located proximally in the shaft. The fine structure of the aristal sensory organ was studied in detail in the fruitfly (Drosophila) and for comparison in the...

In insect sensory organs, the receptor endings are surrounded by an extracellular subcuticular matrix, the sensillum lymph. This matrix provides the correct aqueous and ionic milieu around the sensory processes, plays a role in the generation of the receptor current, and is also important for stimulus transport and/or stimulus inactivation in chemo...

The antenna of the male silkmoth has been a model system for olfactory physiology since the time when its response to the female sex pheromone could be recorded electrophysiologically (Schneider, 1957, 1984). The response triggers reproductive behaviour and is mediated by ten thousands of identical miniature sensory organs, the sensilla trichodea,...

An anomalous sensillum chaeticum is described in the silkmoth, Bombyx mori (Lepidoptera : Bombycidae), in which each of the 5 inner sensory dendrites gives rise to 2 ciliary and 2 outer dendritic segments. This is interpreted as an atavistic feature, because a double set of cilia is common in sensilla of apterygote insects and of non-insect arthrop...

This chapter discusses the functional morphology of pheromone-sensitive sensilla. It focuses on the variability of antennal and sensillar form. In terms of functional morphology, antennal and sensillar shape are important for stimulus uptake. The chapter discusses the internal structure of sensory hairs and also explores how pheromone molecules fin...

The distribution of diffusion barriers in silkmoth olfactory sensilla has been investigated with ionic lanthanum. The tracer was applied from the apical side of the sensory epithelium by first pinching off the hair tips and then dipping the antennal branches into the La(NO3)3 solution. The tracer neither passed the apical septate junctions between...

The interaction of the electron beam with the specimen not only provides information on its ultrastructure, but also on its elemental composition. In principle, the irradiating electron beam (probe) is focussed on the area of interest in the specimen, while the secondary radiation (e.g. X-rays) generated by the interaction with the specimen is proc...

Freeze-substitution (FS) and freeze-drying (FD) are dehydration techniques by which the water is gently removed from a frozen specimen. Both techniques can serve as a link between cryofixation and conventional thin sectioning at room temperature (Fig. 1). They are, therefore, hybrid techniques combining the advantages of the low temperature and the...

To preserve tissue by freezing is an ancient concept going back pre­ sumably to the practice of ice-age hunters. At first glance, it seems as simple as it is attractive: the dynamics of life are frozen in, nothing is added and nothing withdrawn except thermal energy. Thus, the result should be more life-like than after poisoning, tan­ ning and dryi...

In pupae of Euploea core and Amauris ochlea most of the cuticle displays a strong metallic golden or silvery lustre. The reflection colours appear between 24 and 48 h after pupation, first as blue and greenish hues which later turn into gold or silver, and vanish again about 24 h before emergence of the butterfly. If excised cuticle dries, the meta...

The sensory hairs of the silkmoth, Bombyx mori, are suitable test objects to check for recrystallization and secondary freezing damage in a biological object, because cryofixation by immersion into propane (90 K) routinely yields well-preserved specimens without noticeable freezing damage. After rewarming the frozen specimens for 10 min to 230 K (b...

Pupae of the butterflies Danaus chrysippus and Helioconius charitonius display characteristic patterns of golden spots, while the pupae of the genera Euploea and Amauris exhibit metallic lustre over most of their surface; E. core and midamus more golden, A. ochlea and niavius more silvery. The absolute reflectance exceeds 80% at wavelengths longer...

A technique is described which allows one to embed small frozen specimens for easy sectioning in a cryoultramicrotome. n-Heptane is recommended as a cryoembedding medium due to its suitable melting point (182.4K), high vapour pressure, chemical inertia, and good sectioning properties when solid. Freeze dried cryosections of cultured muscle cells, s...

The cellular organization of freeze-substituted antennal sensilla trichodea, which contain the sex pheromone receptors, was studied in male silkmoths of two species (Bombyx mori, Bombycidae; Antheraea pernyi, Saturniidae). The cellular architecture of these sensilla is complex, but very similar in both species. A three-dimensional reconstruction of...

Sensilla trichodea of the silk moths, Antheraea pernyi and Bombyx mori, were reconstructed from serial sections after freeze substitution. The volume and surface area of the different sensillar cells were calculated from the area and circumference of consecutive section profiles. A. pernyi and B. mori differ largely in the size of the sensory hair...

Insects are the most successful land dwellers among the invertebrates. They owe this achievement largely to their cuticle, which provides mechanical support and protects against water loss at the same time. This exoskeleton forms a barrier between the environment and the interior milieu, so that sense organs need unique adaptations to remain access...

Because accurate neuronal dimensions are essential for mathematical modeling of neuronal properties, the effects of a number of salines and fixative procedures on neuronal size were compared, including the non-chemical, freeze substitution method. Using an identified neuron we compared diameters and found some of the fixative-saline combinations ca...

Insect antennae can be easily frozen by immersion into propane at 90 K, freeze substituted at 194 K and, after warming up, embedded and sectioned at room temperature. Freezing damage (f.d.) may occur during cooling the specimen down (primary f.d.) or due to re-crystallization, during warming up, if substitution was not complete (secondary f.d.). Ex...

Sensilla trichodea, with porous walls and two receptor cells, are the most abundant sensilla on the antennae of male Spodoptera exempta. Two synthetic pheromone components, Z-9-tetradecen-l-yl acetate (Z-9-TDA) and Z-9,E-12-tetradecadien-l-yl acetate (Z-9,E-12-TDDA), produced identical electroantennogram (EAG) responses with male antennae, and stro...

This chapter discusses pheromone communication in moths with an emphasis on female sex pheromones. Male courtship pheromones are widely distributed in most lepidopteran groups, including butterflies, and differ largely from the female pheromones by their chemical composition, biosynthesis, and dissemination mechanisms. Although the era of pheromone...

Sensilla on the antenna of the bed bug, Cimex lectularius, were studied with the scanning and transmission electron microscope. Those which display a tubular body in the dendrite ending are presumed to have a mechanoreceptor function (bristles of type A, flat plate of type B). Bristles of type A1 contain additional dendrites which terminate at the...

When deprived of the terminal antennal segments, male and female bedbugs failed to respond to their alarm pheromone and to their assembling scent. Trans-oct-2-en-1-al or trans-hex-2-en-1-al, being the major constituents of the former, induce in adults and larvae of Cimex lectularius a typical alarm behaviour resulting in dispersal of assembled bedb...

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The sensilla (s). trichodea and s. basiconica on the antennae of the silk moth,Bombyx mori, were studied under the transmission electron microscope. Chemical fixation, freeze substitution and freeze etching methods were used. The following results have been obtained: Five sensillum types were distinguished, the olfactory function of which is known...