Olfactory Hairs Research Articles

Members of a family of drosophila putative odorant-binding proteins are expressed in different subsets of olfactory hairs

A polymerase chain reaction-based method was used to generate a Drosophila melanogaster antennal cDNA library from which head cDNAs were subtracted. We identified five cDNAs that code for antennal proteins containing six cysteines in a conserved pattern shared with known moth antennal proteins, including pheromone-binding proteins. Another cDNA codes for a protein related to vertebrate brain proteins that bind hydrophobic ligands. In all, we describe seven antennal proteins which contain potential signal peptides, suggesting that, like pheromone-binding proteins, they may be secreted in the lumen of olfactory hairs. The expression patterns of these putative odorant-binding proteins define at least four different subsets of olfactory hairs and suggest that the Drosophila olfactory apparatus is funnctionally segregated.

Morphological studies on the rodent main and accessory olfactory systems: The regio olfactoria and vomeronasal organ

The present study on the main olfactory system (MOS) and the accessory olfactory system (AOS) documents the functional morphology of the rodent olfactory region and that of the vomeronasal organ (VNO) using light and electron microscopical techniques. Special attention is given to the cytoarchitecture of the sensory epithelia, i. e. the olfactory epithelium (OE) of the regio olfactoria and the neuroepithelium of the VNO (VNO-NE). Both sensory epithelia consist of a pseudostratified columnar epithelium composed of three types of cells, i. e. receptor cells, supporting cells and progenitor cells. Even at the light microscopical level, however, distinctive morphological features can be distinguished which illustrate important differences between the two sensory epithelia. For example, the height of the respective epithelia differs considerably, the VNO-NE is approximately 170 microns tall and the OE is only about 90 microns. The receptors of the VNO-NE lack olfactory knobs which are typically found in the sensory cells of the OE. The perikarya of the receptor cells of the VNO-NE are very large when compared to those of the sensory cells of the OE. In contrast to the OE, blood vessels are found within the neuroepithelial layer of the VNO. The progenitor cells of the OE are located in a clearly distinguishable cell layer which is lacking in the rodent VNO-NE. The differences between the two epithelial layers become more obvious at the electron microscopical level. The olfactory knobs of the sensory cell dendrites of the OE reach the nasal cavity with numerous cilia. These olfactory hairs, on average 11 per knob, consist of a short proximal segment and a long and thin distal segment. This distal segment runs parallel to the epithelial surface and is embedded in the neuroepithelial mucosal layer. The dendrites of the receptor cells of the VNO-NE reach the lumen of the VNO with numerous branched microvilli which are also embedded in the mucous layer. Horizontal ultrathin sections through the apical portion of the OE reveal that each supporting cell completely envelopes several dendrites. This glia-like relationship is not found in the corresponding layer of the VNO-NE. The sensory cell perikarya of the OE contain only a few endoplasmatic reticulum (ER) profiles while the receptor cells of the VNO are characterized by an extensive smooth endoplasmatic reticulum (SER). In contrast to the fila olfactoria, numerous axons within the vomeronasal nerve show ellipsoidal varicosities without synaptic vesicles which may indicate the existence of at least two vomeronasal nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Sex pheromone reception in the moth, Mamestra thalassina. Characterization and distribution of two types of olfactory hairs

Two types of olfactory hairs were found on the antennae of the moth Mamestra thalassina (Lepidoptera: Noctuidae) by scanning electron microscopy and electrophysiology. Long olfactory hairs are arranged in 4–5 rows over the border of the sensory field of the flagellar segments. These sensilla were sensitive to (Z)-11-hexadecenyl acetate (Z11-16:Ac) and (Z)-11-hexadecenol (Z11-16:OH). Cross adaptation experiments showed that these two compounds are detected by two separate olfactory neurones. A ventro-medial sensillar field is composed of short hairs that respond only to (Z)-11-hexadecenal (Z11-16:Ald). No olfactory neurone specifically tuned to (Z)-7-dodecenyl acetate (Z7-12:Ac), a compound that elicited electroantennogram responses, was found. Z11-16:Ac, Z11-16:OH and Z11-16:Ald were found in hexane extracts from the female pheromone gland. The olfactory neurones specifically involved in the reception of the pheromone components in M. thalassina are housed in different sensillar types. Sensillar types are constant, morphologically identifiable and localized in precise areas at the surface of the antenna. The consequences of this arrangement of receptor cells are discussed.

Pheromone stimulation in perfused sensory hairs of the moth Antheraea polyphemus

Olfactory hairs of moths antennae were perfused with electrolyte during electrophysiological recording. The perfusate was drawn from the recording capillary electrode into the apically opened hair by applying suction on the haemolymph side. Due to suction, the receptor cell dendrite partially withdraws from the hair lumen. The withdrawal of receptor cell dendrites was examined by electron microscopy and after staining with lucifer yellow. In the withdrawn position the dendrite cannot be excited by airborne pheromone stimuli applied to the middle of the hair but it responds to pheromone dissolved in the perfusate. The normal position of the dendrite and its responses to external stimulation can be restored by applying pressure to the haemolymph side. These results show that pheromone molecules may stimulate olfactory cells directly without mediation by cuticular structures. Furthermore, the method of hair perfusion allows application of water-soluble agents to living dendrites of insect olfactory receptor cells.

On the function of the pheromone binding protein in the olfactory hairs of Antheraea polyphemus

Before a pheromone molecule of Antheraea polyphemus can stimulate a dendrite of a sensillum trichodeum, it has to pass through an aqueous solution, the “sensillum lymph”. Furthermore, after such a stimulation it has to be rapidly inactivated to prevent continuous stimulation of the dendrite. In the sensillum lymph a pheromone binding protein in mM concentrations is present. The literature describes two hypotheses concerning the function of the binding protein: one hypothesis claims that the latter serves to carry the pheromone through the sensillum lymph, while the other assumes a migration of the pheromone to the dendrite through the pore tubuli and an inactivator function of the binding protein. By means of a perfusion technique, the dendrites of sensilla trichodea were bathed in a solution of pheromone [(E,Z)-6,11-hexadecadienyl acetate] in Ringer with and without binding protein or bovine serum albumin for comparison. The responses of the cells were measured electrophysiologically. To elicit nerve impulses, a pheromone concentration of 180 nM in Ringer was necessary. Addition of binding protein or bovine serum albumin decreased the threshold concentration by a factor of 100. It was calculated that with such a pheromone concentration (1.8 nM) plus binding protein less than 1000 pheromone molecules within the hair produced a strong cell response, although in equilibrium 99.96% of these molecules are bound to the binding protein. It was therefore concluded that pheromone binding protein can act as a solubilizer or carrier.

Enzymatic Conversion of Sex Pheromones in Olfactory Hairs of the Male Silkworm Moth Antheraea polyphemus

Abstract The sex pheromone compound [6,7-3H]-6,ll-hexadecadienyl acetate is enzymatically converted to its corresponding fatty alcohol, aldehyde and acid in living and homogenized olfactory hairs and in other parts of the antennae of the male silk-moth Antheraea polyphemus. The pheromone is metabolized in vitro mainly in the inner hydrophilic compartments, but scarcely in the outer hydrophobic cuticle of the olfactory hairs.

Nodes on the large pheromone-sensitive dendrites of olfactory hairs of the male silkmoth, Antheraea polyphemus (Cramer) (Lepidoptera : Saturniidae)

Variation in dendritic diameter giving a distinct nodal appearance is a feature of arthropod olfactory dendrites. The morphology of such nodes on the large pheromone-sensitive dendrites of the long olfactory hairs of the male antenna of the silkmoth, Antheraea polyphemus (Lepidoptera : Saturniidae) has been investigated at the light microscopical level using Lucifer yellow and osmium-gallate staining methods. A simple method for obtaining living dendrites for observation under phase-contrast illumination was developed. Large conspicuous nodes, usually oval in shape, about 1 μm in diameter and 3 μm in length compared to the axis-fibre diameter of about 0.5 μm, were demonstrated, which are not artifacts of chemical fixation. Movement of these structures along the dendritic length has been observed. This study has established a suitable cellular preparation as a prelude to determining whether morphologically observed changes in the dendrite accompany and play a role in receptor cell physiology in general or in the transduction process in particular.

The Electrolyte Distribution in Insect Olfactory Sensilla as Revealed by X‐ray Microanalysis

X-ray microanalysis allows the quantitative analysis of elemental concentrations in situ with the high lateral resolution of the electron microscope.'Thus, it is now possible to measure the electrolyte distribution directly in the various cellular and extracellular compartments of insect sensilla. Eventually, this information will provide a link between our understanding of the complex ultrastructure of these sensory organs and their functional interpretation as derived mainly from electrophysiological experiments. Cryofixation and cryoultramicrotomy are essential prerequisites for X-ray microanalysis of electrolyte elements, because any solvent treatment might cause redistribution or even extraction of soluble elements. With antennae of the silkmoth, Bombyx mori, this has been achieved by quick immersion into liquid propane (at 190°C) and cryoembedding into heptane (at 80C).' Ultrathin cryosections were cut in a Reichert FC4 cryoultramicrotome at 1 10C, freeze dried and studied in a Siemens ST lOOF scanning transmission electron microscope. X-ray spectra were processed in an energy-dispersive spectrometer with multichannel analyzer (Link Systems). Dry weight concentrations of the analyzed elements were calculated according to the continuum method of Hall' after calibration with cryosections of salt solutions of known concentration (for a more detailed account of the procedure see Zierold4). The ultrastructure of frozen, dried cryosections allows the unequivocal identification of all important sensillar compartments, such as dendrites and receptor lymph in cross sections of olfactory hairs or, in the antenna1 branches, the receptor-cell somata, dendrites, axons, the different auxiliary cells, as well as the extracellular compartments of the receptor-lymph and hemolymph spaces. The plasma membranes around the cellular compartments and the organelles within them are clearly discernible (FIG. 1). X-ray microanalysis (TABLE 1 ) revealed a high concentration of potassium throughout the cellular compartments, but also in the extracellular receptor lymph, confirming earlier measurements on isolated receptor-lymph droplets? It is assumed

Sensillum-lymph proteins from antennal olfactory hairs of the moth Antheraea polyphemus (Saturniidae)

The soluble proteins in the antennae of the male and female saturniid moth Antheraea polyphenus were analyzed by isoelectric focusing in miniature ultrathin-layer polyacrylamide gels and by microgradient PAGE (μPAGE). Different preparations of sensillum lymph from male antennal olfactory hairs (isolated sensillum-lymph droplets, rinsing solution from isolated hairs) and homogenates of male and female antennae were compared. The pheromone-binding protein, PBP, and the pheromone-degrading sensillar esterase, SE, were detected only in male antennal preparations. Isolated sensillum lymph exhibited two proteins in Brilliant Blue staining: (1) the PBP appeared as a double band with a relative molecular mass of M r < 14,400 (α-lactalbumin); (2) an oligopeptide was detected as a faint band at a very small molecular mass ( M r ⪡ 14,000 ). The PBP in isolated sensillum lymph was identical to the PBP in the rinsing solution of isolated hairs and in male branch homogenates. Purified PBP from branch homogenates showed a third slightly smaller band. In reducing SDS-μPAGE, the denatured PBP from all preparations showed only one band at a M r of 14,400. The isoelectric point (pI) of the PBP was 4.7. The PBP appeared to be a non-glycosylated protein. Glycoproteins were present in the hemolymph in different patterns in both sexes. The concentration of PBP in sensillum-lymph droplets was ≈ 10 mM, corresponding to a total amount of ≈9 μg/55 μl sensillum lymph in one antenna. A third protein in the sensillum lymph, the sensillar esterase, was only visible by enzyme staining and had a M r of 55,000 and a pI of 3.0. The integumental esterases found in male and female antennae had a M r of 65,000, pI 5.85, and M r 90,000, pI 5.0. From body scales of both sexes, esterases in the range of M r 100,000 were solubilized by 0.01% Triton buffer and could be demonstrated in μPAGE.

Chemo-electrical transduction in insect olfactory receptors.

Insect behavior can be controlled by a single odor or by a blend of a few components in an exact ratio of concentrations. These odor compounds or odor mixtures are called pheromones if used for intraspecific communication. Each of the pheromone components is perceived by a particular type of receptor cell that is specifically tuned to its key compound and may respond to single odor molecules by firing single nerve impulses. The entire response range of a receptor cell may cover many decades of stimulus intensities. Each olfactory receptor cell is a bipolar neuron. Its dendrite innervates a specialized cuticular structure, usually a hollow cuticular hair that captures the stimulus molecules. The axon runs directly to the deutocerebrum-the first synaptic relay station of the central nervous system. Chemo-electrical transduction in these organs comprises the processes leading from the adsorption of odor molecules on the cuticular surface via stimulus transport to the generation of receptor potentials and nerve impulses and, eventually, to the inactivation of the stimulus molecules. The interconnections of these processes and their influences on the ultimate impulse response of the receptor cells are largely unknown. This review covers electrophysiological investigations of cellular response charac­ teristics as well as biophysical and biochemical work, much of which is based on radiolabeled pheromone compounds. These studies employed a few species of insects, mainly of moths with olfactory organs that are relatively large and accessible for experimental work. The olfactory hairs of

Interactions of pheromone with moth antennae: Adsorption, desorption and transport

Isolated antennae of the male moth Antheraea polyphemus adsorbed at least 32% of 3H-labelled pheromone molecules (E-6,Z-11 hexadecadienyl acetate) from an airstream passing the antenna. About 80% of the adsorbed molecules were caught by the long olfactory hairs (sensilla trichodea). The distal half of the hairs caught about twice as many molecules as the proximal half. About 40% of the molecules desorbed if the antennae were exposed to a clean airstream for 30 min. The adsorbed molecules were transported from the hairs towards the antennal branch. Transport due to diffusion would have a diffusion coefficient of 3 × 10 −7 cm 2/s. Forty per cent of the total radioactivity per hair could be detected in receptor lymph extruded from the olfactory hairs, after an incubation time of 2 min. Dried antennae showed an increased desorption and an increased velocity of the transport along the hairs. One interpretation is that the molecules enter the receptor lymph of the intact antennae and diffuse more slowly than those on the cuticular surface. Fractional elution of fresh antennae revealed a diminishing elutability of pheromone from antennae in pentane (DEP-effect) and almost constant elutability in more polar solvents (chloroform-methanol toluene). The DEP-effect could be reversibly abolished by dehydration of the antennae. It could be shown that the DEP-effect occurs mainly on the antennal branch rather than on the hairs. Residual (uneluted) radioactivity builds up mainly on the branch.

Dendritic membrane from insect olfactory hairs: isolation method and electron microscopic observations.

Sensory hairs from antennae of male saturniid moths (Antheraea polyphemus) were separated while deep-frozen by shaking antennal branches with glass beads. The hairs were collected through their differential adhesion to the surface of a petri dish. The yield, determined by the length of the isolated hair fragments, was about 38% of the estimated total hair length per antenna. The dendritic membrane was separated from the hair fragments by centrifugation through Sephadex and further purified by ultracentrifugation in sucrose buffers. Transmission electron microscopy was used to monitor the steps of the hair and membrane isolation and to investigate the membrane pellet. Some membrane vesicles bound cationized ferritin, thus indicating a negatively charged cell surface coat. Negatively stained membrane vesicles exhibited a pattern of repetitive substructures irregularly distributed over the vesicle surface. The units had a diameter of about 3 nm and a maximal density of 30,000/micron2.

Reconstruction and morphometry of silkmoth olfactory hairs: A comparative study of sensilla trichodea on the antennae of male Antheraea polyphemus and Antheraea pernyi (Insecta, Lepidoptera)

Olfactory trichoid hairs on the antennae of male Antheraea silkmoths were reconstructed with respect to the following parameters: number, shape, course, and dimensions of outer dendritic segments as well as the numbers of their microtubules; inner and outer dimensions of the cuticular hair shafts; and number and distribution of pores and pore tubules in the hair walls. The smallest distances between dendritic membranes and inner hair surfaces were determined with respect to the possibility of pore tubule contacts. It was shown that most hairs contain one thick and one, or frequently two, thin dendrites. The number of microtubules in the dendrites is correlated with dendrite diameter, which decreases towards the hair tip. The dendrites form numerous swellings and constrictions: this “beading” occurs especially along the thin dendrites. The dendrites do not run straight, but rather follow a sinuous course in the hairs. The density of wall pores is lowest in the basal region of the hairs. Only in relatively few places do the dendritic membranes get near enough the hair walls to come into the probable range of the pore tubules. In the sensilla trichodea of A. polyphemus, the hairs as well as the dendrites have markedly smaller diameters than in A. pernyi.

Freeze-fracture characteristics of insect gustatory and olfactory sensilla. II. Cuticular features.

The freeze-fracture technique has been used to obtain detailed information about cuticular constituents and outgrowths of the external skeleton of labella and antennae in the bluebottle fly Calliphora vicina and the antennae of the small moth (Yponomeuta spp.). The lamellated exoskeleton has a fibrous endocuticle and an exocuticle lacking fibers. Ductuli connecting the inside of the animal with the outside run perpendicularly through the endocutile and at angles of up to 45° in the exocuticle. Skeletal outgrowths lack fibers and display fracture features similar to those of the exocuticle. Among those having neuronal endings, gustatory, olfactory, and tactile hairs can be recognized. Noninnervated outgrowths can be subdivided into scales and pseudotrichia. Criteria such as shape, length/width-ratio of hairs, texture, presence and place of pores, shape of pores, and form of the socket or base are presented for further classification. Cuticular features of single-walled olfactory hairs of Calliphora are compared with those of several other species. Based on the shape of the pores, five types of hairs can be distinguished using literature data. It is concluded that the freeze-fracture technique is a valuable tool with which to describe the microarchitecture of the insect exoskeleton and supplements scanning electron microscopy, which is useful for describing the overall skeletal features.

Very tight contact of tormogen cell membrane and sensillum cuticle: Ultrastructural basis for high electrical resistance between receptorlymph and subcuticular spaces in silkmoth olfactory hairs

A very tight contact is present between the apical membrane of the tormogen cell and the cuticle of the hair base in olfactory sensilla trichodea of Antheraea polyphemus. The contact zone is characterized by numerous hemidesmosome-like structures of the cell membrane, which closely attach the latter to the cuticle. If apically opened hairs are incubated in a LaCl 3 solution, the tracer ions do not penetrate the contact zone. It is concluded that the tight contacts are the morphological correlates of the electrical isolation of the receptor-lymph space (cf. de Kramer et al., in press).

Surface coats of pore tubules and olfactory sensory dendrites of a silkmoth revealed by cationic markers

Negatively charged surface coats have been demonstrated on the pore tubules and dendritic membranes of olfactory hairs of male Antheraea polyphemus silkmoths by application of the cationic markers lanthanum (La 3+), ruthenium red (RR), and cationized ferritin (CF). Lanthanum and RR diffused readily into the apically opened hairs, whereas CF penetrated only for a relatively short distance. Deposits of the markers are distributed as follows: the inner surfaces of the hair walls are stained by RR and to a small degree by CF: the surfaces of the pore tubules and the dendritic membranes are stained by all three markers. The pore tubules have the strongest affinity for CF. The number of pore tubule-membrane contacts seems to be increased by the cationic dyes. The dendrites are often penetrated by RR, which forms deposits on the inner membrane leaflets, the cytoplasmic microtubules, and microfilaments, and by La 3+, but never by CF. The observations provide support for the assumption that, first, the pore tubule-membrane contacts are formed via surface coats of both structures, possibly influenced by cations and, second, that the dendrites remain intact after pinching off the hair tips.

Contacts of pore tubules and sensory dendrites in antennal chemosensilla of a silkmoth: Demonstration of a possible pathway for olfactory molecules

Antennal olfactory hairs of Antheraea polyphemus were investigated by means of transmission electron microscopy. Adequate preservation of dendrites and extracellular pore tubules is obtained by mechanical opening of the hair lumen and subsequent chemical fixation. The dendritic membrane has a cell coat. The dendrites contain microfilamentous structures in addition to their cytoplasmatic microtubules. The extracellular pore tubules traverse the hair cuticle and reach into the hair lumen for maximally 350 nm. Their diameter varies between 20 and 40 nm, depending on the preparation method. They consist of an electron-dense wall and an electron-lucent core. The wall has a helical substructure and is covered with a fuzzy coat. Contacts of pore tubules and dendritic membranes occur wherever dendrites are near the inner surface of the hair cuticle. Some of the pore tubules terminate approximately at right angles on the dendritic membrane, others lie against the membrane. The contact seems to be made via the surface coats of the tubules and the membrane. The structure of pore tubules which had been negatively stained with uranyl acetate is similar to the conventionally thin-sectioned material. The observations provide support for earlier assumptions that pore tubules are the pathways by which odor molecules reach the dendritic membrane.

Digitiform Sensilla on the Maxillar Palp of Coleoptera

The fine structure of the digitiform sensilla on the distal segment of the maxillar palps of Tenebrio and Dermestes is described. Each sensillum is associated with a single sensory cell and three enveloping cells, which enclose two receptor lymph cavities. The inner receptor lymph cavity of both species shows a different structural feature. Branches of the outer dendritic segments, which contain numerous microtubules, run to the tip of the hairshaft. A dendritic sheath extends to the apex of the peg. The hairshaft possesses a second canal, which is free of dendrites. The poreless hairshaft is inserted in a cuticular canal; the longer distal part of the shaft is positioned in a narrow superficial groove. The digitiform sensilla do not show the typical structures of mechanosensitive sensilla. The absence of pores in the setal wall does not point to a function as olfactory or gustatory hairs. The presumed function of the sensilla is discussed in relation to thermo-, hygro- and CO2-receptors.

Sex Attractant System in Polia pisi L. (Lepidoptera: Noctuidae)

Electrophysiological analysis of olfactory hair sensilla in male P. pisi has revealed four different types of presumed pheromone receptor cells, maximally responsive to (Z)-11-tetradecenyl acetate (Z11-14:Ac), (Z)-9-tetradecenyl acetate (Z9-14:Ac), (Z)-11-hexadecenyl acetate (Z11-14:Ac) and (Z)-7-dodecenyl acetate (Z7-12: Ac), respectively. These four compounds were tested, singly and in various combinations, for efficacy in attracting P. pisi males in the field. High trap catches were obtained with mixtures of Z11-14: Ac/Z9-14: Ac in the ratio 100/100, whereas the 100/30 and 30/100 mixtures of the two compounds were only slightly attractive. No male P. pisi were captured by single chemicals or binary combinations of Z11-14: Ac/Z11-16: Ac, Z11-14:Ac/Z7-12:Ac, Z9-14:Ac/Z11-16:Ac, Z9-14:Ac/Z7-12:Ac, or Z11-16:Ac/Z7-12:Ac. Various compounds, including Z11-16: Ac and Z7-12:Ac, were tried as third chemicals in addi­tion to 100 μg Z11-14: Ac + 100 μg Z9-14: Ac but none increased trap catches over the basic lure.

A Sex Attractant for the Green Budworm Moth, Hedya nubiferana

Abstract The two major components of the female pheromone blend of Hedya nubiferana have been identified as (E, E)-8,10-dodecadienyl acetate and (Z)-8-dodecenyl acetate. Each compound acts on a specialist receptor type in olfactory hair sensilla of the male antenna. In field screening tests, combinations of the two compounds and dodecanyl acetate were highly attractive to H. nubiferana males. The (Z, E)-8,10 isomer attracted males of Hedya ochroleucana.