Odorant-binding proteins: expression and function.

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; (ii) OBPs may act in addition as peripheral filters in odor discrimination by selectively binding certain classes of odorants; (iii) OBPs may present the stimulus molecule in a particular way to the receptor proteins to facilitate signal transduction; (iv) OBPs may clean the perireceptor space from unwanted and toxic compounds; (v) OBPs may rapidly deactivate odorants after stimulation of the receptors. Experimental evidence in favor of this multiple role of OBPs is reviewed.

Olfactory coding in a compound nose. Coexpression of odorant-binding proteins in Drosophila.

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. We have examined the genomic organization and expression patterns of two olfactory-specific genes (OS-E and OS-F) of Drosophila melanogaster, the products of which are members of a protein family in Drosophila sharing sequence similarity with moth OBPs. We found that the OS-E and OS-F transcription units are located < 1 kb apart. They are oriented in the same direction and display a similar intron-exon organization. Expression of both OS-E and OS-F proteins is spatially restricted to the ventrolateral region of the Drosophila antenna. Within this region, both OS-E and OS-F proteins are expressed within two different types of sensory hairs: in most, if not all, sensilla trichodea and in approximately 40% of the interspersed small sensilla basiconica. We consistently observe that OS-E and OS-F are coexpressed, indicating that an individual sensillum can contain more than one odorant-binding protein. This finding has potential implications for the roles of odorant-binding proteins in olfactory coding.

Experimental morphology of insect olfaction: tracer studies, X-ray microanalysis, autoradiography, and immunocytochemistry with silkmoth antennae.

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 extraction with lipid solvents and/or pronase. X-ray microanalysis demonstrates a high potassium content of the sensillum lymph; calcium was only found in the haemolymph above detection limit. Auxiliary cells rapidly take up radioactive leucine administered via the haemolymph. Antibodies against pheromone-binding protein of Antheraea polyphemus label the sensillum lymph of sensilla trichodea, but not of sensilla basiconica in A. polyphemus as well as in B. mori. The cytoplasm of auxiliary cells of the sensilla trichodea is also labelled. The results are discussed in context with present hypotheses on the role of sensillum lymph in stimulus transport and inactivation.

Freeze-substitution for morphological and immunocytochemical studies in insects.

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, but in the very centre of antennal branches (diameter approximately 80 microns) of the silkmoth, Bombyx mori, freezing damage is usually reduced again. The frost-hardy species, Poecilocampa populi and Boreus hiemalis, exhibit regions free from freezing damage up to 40 microns below the cuticular surface. Secondary freezing damage in silkmoth sensory hairs is observed only after deliberately warming the specimens to -43 degrees C for >> 10 min before FS. Secondary artefacts due to the substitution process are investigated by comparison with freeze-etching and by comparing different FS media and protocols. Methanol is not recommended as a substitution medium for insect specimens. Structures particularly liable to substitution damage are the stimulus-conducting pore tubules of olfactory sensilla and the receptor cell membrane. Extraction of soluble components is more likely with pure organic solvents without added chemical fixing agents and with prolonged substitution at elevated temperatures. Such extraction may also be a possible artefact with soluble antigens in immunocytochemical studies. A review is given of the major achievements attained with these techniques is insect functional morphology and immunocytochemistry.

Expression mosaic of odorant-binding proteins in Drosophila olfactory organs.

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 mapped in the electron microscope. It was found that (1) OBP expression does correlate with morphological sensillum types and subtypes, (2) several OBPs may be co-localized in the same sensillum, and (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.

Fine structure and development of the silver and golden cuticle in butterfly pupae.

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 than 550 nm, but drops more or less steeply at shorter wavelengths (shown by microspectrophotometry for E. core and A. ochlea; in all species this effect is caused by constructive interference of the incident light at Multiple Endocuticular Thin Alternating Layers (METAL cuticle). Dense, cuticular D layers alternate with clear, watery C layers and form over 200 double layers. The thickness of the D layers is fairly constant throughout the stack, whereas the C layers systematically increase and decrease in thickness, thus causing the broad bandwidth of the reflector. Connecting filaments, traversing the C layers in zig zag course, probably secure the mechanical stability of the arrangement. After drying, the C layers have vanished and the lustre is lost; the cuticle is now perfectly transparent, except for D. chrysippus, where it is partly transparent and partly yellow. The metallic reflectance develops between 20 and 30 hr after pupal ecdysis, starting with blue colours which change via green to gold or silver. About half a day before emergence of the imago, the reflection fades again via the opposite colour sequence. Coincident with these colour changes, the METAL cuticle is being deposited and decomposed, respectively. The deposition zone immediately above the apical epidermal microvilli consists of about three helicoidal lamellae as in normal, non-reflecting cuticle. The METAL cuticle is formed abruptly at the outer border of the deposition zone, possibly during condensation of the cuticular microfibres. The periodicity it is suggested is controlled either directly by the epidermal cells or indirectly via appropriate self-assembling processes.

The tuft organs of the human body louse, Pediculus humanus corporis--cryofixation study of a thermo-/hygrosensitive sensillum.

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 dendrites. The sensillar peg is innervated by the unbranched dendrites of two receptor cells which do not reach into the tuft processes. A third receptor cell forms a lamellar outer dendritic segment which wraps around the two other dendrites and terminates before reaching the base of the sensory peg. This type of receptor cell is named stratoterminal while for those which end within the cuticular apparatus the term conoterminal is proposed. Five auxiliary cells are associated with each tuft organ. The pore organs display a porous cuticular plate innervated by the branching dendrites of four receptor cells. A fifth receptor cell terminates more proximal and resembles the stratoterminal cell of the tuft organ. From the fine structure it is evident that the tuft organs represent thermo/hygrosensitive sensilla whereas the pore organs may have an olfactory function.

Cryofixation without cryoprotectants. Freeze substitution and freeze etching of an insect olfactory receptor.

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, FS usually does not induce noticeable secondary artefacts by the preparation steps subsequent to freezing. When compared with chemically fixed antennae, the superior quality of cryofixation in this tissue is evident, particularly where cell surfaces and processes border the receptor lymph cavity; membranes are smooth following a steady course; dendrites and axons are round in cross-section with evenly distributed microtubules. The value of cryofixation is discussed with special reference to structures of presumed functional significance (e.g. stimulus conducting pore tubules, intramembrane particles of the receptor membrane, the ciliary segment of the dendrites, intercellular dilations, membrane junctions).

Fine structure of the antennal receptors of the bed bug, Cimex lectularius L.

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 tip of the bristle and may be gustatory receptors. Sensilla with pores in the hair wall are supposed to have an offactory, humidity and/or temperature receptor function (pegs and hairs of types C, D, E). Hairs of type E contain receptors for the alarm pheromones of the bed bug. Special attention has been paid to the pore structures and epicuticular layers of these sensilla. Possible differences in stimulus conduction are discussed between (i) sensilla with a simple wall and pores with pore tubules (types D and E) and (ii) the ribbed pegs (type C), which have a complex wall structure and spoke channels. The immersed cones of type F have a peculiar innervation, which has not been described previously. Two dendrites are held closely together by a third flat dendrite which wraps around them in the region of the outer segment. Coupling structures were found between the central dendrites, and between these and the third enveloping dendrite. Possible functions of this unique innervation are discussed. The dendrites innervating type D are grouped in three to eight bundles by multiple sheaths. The term thecogen cell is introduced to denote the innermost of the three sheath cells of a sensillum (the outer being the tormogen and the trichogen cell) which builds the dendrite sheath during ontogeny. Comparative morphometry revealed type-specific differences in the length and diameter of the dendrites. Some axons were found to lack any glial or perineurial sheath. Microorganisms were observed in the antennal tissue of several animals.

Diffusion barriers in silkmoth sensory epithelia: application of lanthanum tracer to olfactory sensilla of Antheraea polyphemus and Bombyx mori.

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(NO(3))(3) solution. The tracer neither passed the apical septate junctions between the dendrite and the thecogen cell nor those between thecogen, trichogen, and tormogen cells, nor the tight contact between the apical membrane of the tormogen cell and the cuticle. After perfusing the hemolymph space with La(NO(3))(3) solution, the tracer was found in the clefts between the thecogen, trichogen, tormogen, and epidermis cells, but not in those between the receptor cells and the thecogen cell, or between the axon and the glial envelope. Lanthanum neither entered the receptor-lymph space nor the subcuticular space. Therefore, (i) receptor-lymph space, subcuticular space, and hemolymph space are isolated from each other, and (ii) the cleft between thecogen and sensory cell is separated from the hemolymph as well as from the receptor-lymph spaces. Furthermore, the results indicate that pleated septate junctions form the diffusion barriers in silkmoth olfactory sensilla.

Functional morphology of a double-walled multiporous olfactory sensillum: the sensillum coeloconicum of Bombyx mori (Insecta, Lepidoptera).

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 30 microm(2)). The central lumen contains the outer dendritic segments of usually five receptor cells, and is surrounded by up to 15 partially fused cuticular fingers. The peripheral lumina of these cuticular fingers are filled with material resembling wax-canal filaments. Radial spoke channels (approximately 600 per peg), each 10-20 nm wide, connect the central lumen with the longitudinal groove channels. Groove and spoke channels are assumed to mediate the transport of odorant molecules from the outer epicuticular surface layers to the sensory dendrites. Thus the double-walled wall-pore sensilla represent a bauplan essentially different from single-walled wall-pore sensilla; the reason, however, why the two types are found together throughout the insect orders remains enigmatic. Other peculiar features of the coeloconic sensilla of the silkmoth are invaginations of the outer dendritic segments and direct contacts between the receptor cell somata. The latter may be the structural correlate to electrophysiological observations indicative of peripheral interaction between the receptor neurons. All three auxiliary cells have elaborately folded apical plasma membranes studded with portasomes and associated with an abundance of mitochondria; basally they often contact tracheal branches. As compared to the auxiliary cells of the single-walled olfactory sensilla of the same species, all the mentioned features are much more prominent and hint to a higher ion pumping activity at the border to the sensillum-lymph cavities.

Morphological characteristics of antennal sensilla in the European cornborer Ostrinia nubilalis (Lepidoptera: Pyralidae).

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 intermediate trichoid/basiconic type was found in low numbers on the ventral part of the antenna. In the male, three different morphological types of s. trichodea were observed, having one, two or three sensory cells, correlated with different dimensions of the hair. The s. trichodea with three sensory cells are most common in the basal part of the antenna, while sensilla with two cells are mainly found distally. Trichodea with one sensory cell are more evenly distributed over the length of the antenna. All cells present in the different s. trichodea respond to sex pheromone components or to a behavioural antagonist in electrophysiological sensillum recordings. S. basiconica and s. auricillica had 2-3 sensory cells, and a probable olfactory function. Sensilla coeloconica, also with a putative olfactory function, contained 3-5 sensory cells. S. chaetica of the taste/tactile type possessed 4 + 1 sensory cells. S. styloconica comprised three sensory cells with possible functions as thermo- and hygroreccptors.

Correlation between dendrite diameter and action potential amplitude in sex pheromone specific receptor neurons in male Ostrinia nubilalis (Lepidoptera: Pyralidae).

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 three receptor cells. Two cells were each specific for one of the two pheromone components, (Z)-11-tetradecenyl acetate (Z11-14:OAc) and (E)-11-tetradecenyl acetate (E11-14:OAc). Two strains of cornborers (Z and E) differ as to which of the two pheromone components is the main one. In both strains a large difference could be observed between the spike amplitudes elicited in the receptor cells by the two pheromone components, the main component always eliciting the large spike. In F1-hybrids (EZ) of these two strains, producing both pheromone components in similar quantities, the spike amplitudes were equal in the two pheromone-specific receptor cells. The third cell responded specifically to a behavioural antagonist. (Z)-9-tetradecenyl acetate (Z9-14:OAc) in both the parental and hybrid strains, and always showed the smallest spike amplitude. In a morphometric study, the outer dendritic segments were shown to differ more in diameter between the largest and second largest cell in the two parental strains than in the hybrid strain, while the smallest diameter cell did not differ between the different strains. These results imply that receptor cells with larger diameter produce spikes with greater amplitude. The data also show that all three types of receptor neurons display outer dendritic segments with strong variation in the diameter along the length of the segment, and with a pronounced taper towards the tip.

The expression pattern of four odorant-binding proteins in male and female silk moths, Bombyx mori.

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 specific. In Western blot analysis and immunocytochemical labelling experiments, the sera against recombinant PBP and GOBP2 of B. mori gave identical results as sera against native PBP and GOBP2 of Antheraea polyphemus, respectively, thus confirming earlier results obtained with the latter. Labelling consecutive cross sections of various sensillum types with all four antisera revealed different labelling patterns in male and female sensilla (s.) trichodea and s. basiconica. Long s. trichodea in males and females represented uniform labelling types, whereas for short s. trichodea, s. intermedia, and s. basiconica a great variety of labelling patterns was observed, some being more common than others. Long s. trichodea, which in males are uniformly tuned to the pheromone components bombykol and bombykal, all strongly expressed PBP; labelling with antisera against the other three odorant-binding proteins hardly was above background, only in some hairs GOBP1 was expressed somewhat more strongly. Long s. trichodea of females, which respond specifically to linalool and benzoic acid, showed a different labelling pattern. Here, we observed strong labelling with antibodies against GOBP2 and medium labelling with anti-GOBP1, sometimes with anti-ABPX. S. basiconica in both sexes most commonly co-expressed GOBP1 and GOBP2, but other patterns were occasionally found, with some of them showing PBP expression, also in females. The great variety of labelling types in short s. trichodea, s. intermedia, and s. basiconica suggests a similar variety of functional subtypes as observed in plant odour-sensitive sensilla of other moth species.

Expression and immunolocalisation of odorant-binding and chemosensory proteins in locusts.

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 Locusta migratoria, instead, the same antibodies cross-reacted only with a band of apparent molecular mass of 35 kDa in the extract of 1st-5th instars, but not in the adults. The recombinant protein binds the fluorescent probe N-phenyl-1-naphthylamine, but none of the compounds so far reported as pheromones for S. gregaria. The expression of the odorant-binding protein (OBP) and of CSPs of sub-classes I and II was also monitored in antennae, tarsi, palpi, wings and other organs of solitary and gregarious locusts in their nymphal and adult stages. OBP was found to be antenna specific, where it is expressed at least from the 3rd instar in both solitary and gregarious locusts. CSPs, instead, appear to be more ubiquitous, with different expression patterns, according to the sub-class. Immunocytochemistry experiments revealed that OBP is present in the sensillum lymph of sensilla trichodea and basiconica, while CSP-I and CSP-III were found in the outer sensillum lymph of sensilla chaetica and in the sub-cuticular space between epidermis and cuticle of the antenna. Sensilla chaetica on other parts of the body showed the same expression of CSP-I as those on the antenna.

Ions and mucoid substances in sensory organs--microanalytical data from insect sensilla.

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 chemoreceptors. Histochemical evidence indicates the presence of proteoglycans (probably hyaluronate and/or chondroitin sulfate) in the sensillum lymph. X-ray micro-analytical studies demonstrate a very low sodium content, whereas potassium is present in high concentrations, comparable to those in intracellular compartments. The total positive charges of cationic elements are not balanced by the negative charges of chlorine, but by polyanions of the mucoid substances present in sensillum lymph. These fixed negative charges probably play a key-role in the establishment of the non-neural transepithelial voltage of insect sensilla, which in turn is a major source of the receptor current. Moreover, the mucoid substances of the sensillum lymph are probably involved in water conservation, since sensilla are prone to water loss, because the overlying cuticle must be permeable to the chemical stimuli. Functional analogies are pointed out between insect sensillum lymph and vertebrate olfactory mucus and, in particular, the endolymph of the vertebrate inner ear.

Structure and function of insect olfactory sensilla.

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 are evident only in a few cases, e.g. the demand for extreme sensitivity in moth pheromone communication supported the evolution of long sensilla trichodea with high efficiency of capturing odour molecules. The structural diversity continues with the odorant-binding proteins (OBPs) in the sensillum lymph surrounding the sensory dendrites. These proteins may be subdivided into pheromone-binding proteins and two classes of general odorant-binding proteins according to their primary sequence. Different sensilla of the same morphological type may contain different OBPs of the same or of different subclasses. However, OBPs of different subclasses are not co-localized in the same individual sensory hair. The presence of a given OBP is related more to the functional specificity of the receptor cells than to the morphological type of the sensillum, suggesting a role of OBPs in stimulus recognition.

Are odorant-binding proteins involved in odorant discrimination?

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 (Saturniidae), Bombyx mori (Bombycidae) and Manduca sexta (Sphingidae). Very weak labelling was found with all sensilla trichodea of Dendrolimus kikuchii (Lasiocampidae) and Lymantria dispar (Lymantriidae). In three noctuid species, some long sensilla trichodea were labelled strongly, some only weakly and some were not labelled at all. The fraction of long sensilla trichodea that were strongly labelled was large in Helicoverpa armigera, but small in Spodoptera littoralis and Autographa gamma. The observed cross-reactivity was not correlated with taxonomic relatedness of the species but rather with chemical relatedness of the pheromones used by these species, as a high labelling density was consistently observed in sensilla tuned to pheromones with an alcyl chain of 16 carbon atoms. The highly divergent specificity of pheromone-receptor cells in Noctuidae appears to be mirrored by a similar diversity of the pheromone-binding proteins in the sensilla trichodea. These data support the notion that pheromone-binding proteins participate in odorant discrimination.