antennal sensilla trichodea, which contain the sex phero- mone receptors, was studied in male silkmoths of two spe- cies (Bombyx mori, Bombycidae; Antheraea ...
Cell and Tissue Research
Cell Tissue Res (1984) 235:25-34
9 Springer-Verlag 1984
Pheromone receptors in Bombyx mori and Antheraea pernyi I. Reconstruction of the cellular organization of the sensilla trichodea R.A. Steinbrecht 1 and W. Gnatzy 2
1 Max-Planck-Institut ffir Verhaltensphysiologie, Seewiesen, Bundesrepublik Deutschland; 2 Zoologisches Institut, Universit/it Frankfurt/Main, Bundesrepublik Deutschland
Summary. 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 a sensillum trichodeum of B. mori is presented. Two receptor cells (in A. pernyi 1-3) and three auxiliary cells are present. Of the latter, only the thecogen cell forms a true sheath around the receptor cells. A unique thecogen-receptor cell junction extends over the entire area of contact. Septate junctions occur between all sensillar cells apically, and in the region of the axonal origin basally. Gap junctions are also found between all cells except the receptor cells. The trichogen and tormogen cells show many structural indications of secretory activity and are thought to secrete the receptor lymph. Their apical membrane bordering the receptor-lymph space is enlarged by microvilli and microlamellae, but only those of the trichogen cell show regularly arranged membrane particles (portasomes), indicating secretory specialization among the auxiliary cells. Epidermal cells are found as slender pillars between sensilla, but extend apically along the non-sensillar cuticle and basally along the basal lamina. Key words: Pheromone receptor - Silk moth - Sensillum
trichodeum - Cellular organization - Freeze substitution
The pheromone receptors of the silkmoths, Bombyx mori and Antheraea species are well known models of olfactory sense organs as a result of extensive morphological and physiological studies (for review, see Steinbrecht and Schneider 1980). However, there is still a gap in our knowledge of the spatial organization of the cellular elements of the sensilla trichodea serving this function. Quantitative data for Bombyx mori are restricted to the number and distribution of the sensilla trichodea (s. trichodea) on the antenna (Steinbrecht 1970), the dimensions of the outer receptor processes, and the size, number and distribution of the suggested stimulus-conducting pores in the cuticular hair (Steinbrecht 1973). For the genus Antheraea, despite its increasing importance in sensory physiology (cf. Kaiss-
Send offprint requests to: Dr. R.A. Steinbrecht, Max-Planck-Institut ffir Verhaltensphysiologie, D-8131 Seewiesen, Federal Republic of Germany
ling and Thorson 1980), morphological data on antennal sensilla are still scanty. Boeckh et al. (1960) described at the light-microscopical level the antenna and sensilla of A. polyphemus. A few fine-structural observations on the sensory hairs of the sensilla trichodea of A. pernyi are found in Schneider et al. (1964) and Schneider and Steinbrecht (1968). In a recent paper, Keil (1982) described the stimulusconducting pore tubules of A. polyphemus and their connections with the outer receptor dendrites. The present study is concerned with the spatial and cellular organization of the long s. trichodea on the antennae of male Bombyx mori (Bombycidae) and Antheraea pernyi (Saturniidae). Quantitative data concerning the volume and surface of the different cells that constitute these sensilla are provided in a following study (Gnatzy et al. 1984). The comparison of the two species will demonstrate to what extent sensilla of analogous function in two species of different families of Lepidoptera can be qualitatively and even quantitatively similar. The aim of this and the following study is to provide the necessary qualitative and quantitative fine-structural information that, when combined with adequate physiological data, may lead to a better understanding of the function of these receptor organs. Materials and methods
Bombyx mori L. and Antheraea pernyi (Gu~rin-Mrn~ville) were obtained as pupae from various suppliers. Four days after emergence of the male moths, antennae were excised and fixed by immersion in liquid propane ( - 1 8 0 ~ C). Freeze substitution was carried out for 5-7 days in acetone ( - 7 9 ~ C) containing 2% OsO 4. Subsequently, the specimens were warmed and embedded in Epon (Steinbrecht 1980, 1982). Oriented serial sections ( ~ 7 0 nm thick) were cut and collected on large area carbon-formvar double films. After uranyl-acetate and lead staining, micrographs were taken at regular intervals through the series of sections. The magnification of the Zeiss EM10 and Hitachi H 500 was calibrated by a cross-grating replica. Rather than producing montages of many micrographs taken at higher magnification, it was more convenient to enlarge a carefully focussed low magnification micrograph ( x 2500) photographically 5 to 15 times on copyline projection paper (Agfa P90). Individual sensilla were reconstructed from the section series by tracing the cell contours on transparent foils and composing a three-dimensional model by spacing these foils at the section intervals. Due to greater susceptibility
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to freezing damage, in A. pernyi far less specimens could be studied ( ~ 20 s. trichodea in 4 animals), than in B. mori ( ~ 75 s. trichodea in 18 animals). Three s. trichodea were completely reconstructed in each species. Results
1. Gross anatomy, specimen dimensions, and tissue preservation The bipectinate antennal flagella bear one pair of antennal branches per segment in B. mori, which are curved to form a basket-like structure, whereas in A. pernyi, two pairs of straight branches are found on each segment forming a feather-like antenna (Fig. 1 a, b). The pheromone-sensitive s. trichodea are arranged on the antennal stem and branches in a sieve-like array (see Fig. I c, for details, cf. Schneider and Kaissling 1957; Boeckh et al. 1960; Steinbrecht 1970).
Fig. I a--e. Bombyx mori (y and Antheraea pernyi ~; antennal gross anatomy, a, b Left antenna, frontal view of B. mori (a) and A. pernyi (b) with anterior (aB) and posterior (pB), upper (uB) and lower (IB) antertnal branches, respectively; S antennal stem. e A. pernyi, s. trichodea (ST) on antennal branch (B) (scanning electron micrograph), d, e Highly schematic drawings of antennal branches in cross section (d) B. mori, (e) A. pernyi; cuticle with sensilla stippled (dense stipples: mainly long s. trichodea; light stipples: mainly other sensilla); cuticle without sensilla drawn in black. E normal epidermis; H haemolymph sinus; N nerve; SE sensory epithelium; T trachea
Cross sections of the antennal branches reveal further differences between the two species (Fig. 1 d, e). While in B. mori there is a major and a minor sensory epithelium on opposing edges of the branch, in A. pernyi a single large complex of sensory epithelium occupies over three quarters of the cross-sectional circumference. Also the nerve has a different position in the two species. There is, however, much variation as to the relative size of sensory and nonsensory epithelia, and of the nerve and haemolymph sinus in different antennal regions. The diameter of the antennal branches is 50-100 ~tm in B. mori and 8(L125 lam in A. pernyi. In both species the sensory epithelium measures up to 20 lain in height; the cuticle, however, is much thicker in Antheraea (12 pm) than in Bombyx (3 p.m). In B. mori, many specimens did not show noticeable freezing damage in the outer 10 lam of the sensory epithelium, and even the inner parts had only moderate ice-crystal
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Fig. 2. Three-dimensional reconstruction of a s. trichodeum of B. mori from serial sections. Parts of the cuticle, auxiliary, and receptor cells cut away to show inner details of hair lumen, receptor-lymph spaces, and sheath around receptor cells. Note that the trichogen cell here is partly hidden behind the receptor cells (compare with Figs. 1, 2 in Gnatzy et al. 1984). The space between cuticle and basal lamina in vivo is filled with other sensillar and epidermal cells, which are omitted here for clarity (cf. Fig. 3b). A axons; BL basal lamina; C cuticle; CS ciliary segments of dendrites within inner receptor-lymph space; iDS, oDS inner and outer dendritic segments; HL, HS hair lumen and hair shaft; RCA, RCn the two receptor-cell somata; RL receptorlymph spaces formed by the tormogen (RL') and trichogen (RL") cells, connected with each other at RL*; TH thecogen cell; TO tormogen cell; TR trichogen cell
formation ( ~ 50 nm) without rupture o f cell membranes or other major distortions. In A. pernyi, due to the thicker isolating cuticle, even in the outermost tissue regions moderate freezing damage was observed. In deeper regions, cell membranes usually were forced into a zig-zag course by adjacent ice-crystal ghosts ( ~ 100 nm, Fig. 7b). However, even the limited number of well-preserved specimens in A. pernyi permits a generalization of most results for both species with the few exceptions stated.
2. Cellular organization and fine structure of the long sensilla trichodea a) The receptor cells. Qualitatively, the sensory hairs in both species are very similar: the lumen and the thick hair wall taper toward the hairtip; the outer segments o f the innervating dendrites remain unbranched. In Bombyx, the number of the bipolar receptor cells per sensillum is uniformly two, whereas in Antheraea one to three (most often two or three) receptor cells are found. One dendrite in each hair has a larger diameter, being usually twice as thick as the second (and third) one (for details, cf. Steinbrecht 1973; compare also Fig. 1 in Gnatzy et al. 1984). Below the hair base, the outer dendritic segments penetrate the antennal cuticle via the cuticular canal, which is more conspicuous in A. pernyi than in B. mori due to the thicker cuticle. In both species, about 7 Jam below the hair base the short ( ~ 1 tam) ciliary segments of the dendrites are encountered (Figs. 2, 3a, 5a). Proximal to these, the dendritic diameter increases rapidly (but not in a stepwise fashion as observed after chemical fixation; cf. Steinbrecht 1980), and the typical fine structure of inner dendritic seg-
ments is displayed (Figs. 2, 3b, 6a). Below the antennal cuticle the dendrites do not continue in the direction of the sensory hairs but are now running more or less in parallel to the main axis o f the antennal branch (Figs. 2, 3 b). The position of the ellipsoidal cell bodies of the two receptor cells of a sensillum is variable; they are observed sideby-side or in tandem position, resulting in a greater variability of the length o f the inner dendritic segments (10-17 ~tm). The axons leave the cell bodies opposite to the dendrites and pass the basal lamina at a distance of 5-12 txm from their origin to join the antennal nerve branch (Figs. 2, 4). The initial 10 ~tm of the axons are two to three times larger in caliber than later in the antennal nerve, and are separately wrapped by glial processes (see below).
b) Auxiliary cells. Three auxiliary cells are found without exception in the s. trichodea of A. pernyi and B. mori: the thecogen, trichogen, and tormogen cells. These cells form a concentric array o f sheaths, which is, however, restricted to the region around the ciliary segments (Figs. 2, 5 a). Only the innermost cell, the theeogen cell, forms a complete sheath around the inner segments and receptor cell bodies (Figs. 2, 3 b, 4 a, 6 a). This cell also forms a septum that separates the two receptor cells except for a short region immediately below the ciliary segment. In the ciliary region the thecogen cell borders the inner receptor-lymph cavity and apically terminates in a short dendrite sheath, which is not attached to the cuticle of the hair base (Fig. 3 a, 5a). The small, irregularly shaped nucleus in Bombyx is located proximal to the receptor cell bodies close to the axonal origin, while in Antheraea it is more often found neighboring the inner dendritic segments. Except for these
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Fig. 3a, b. B. mori; s. trichodeum in longitudinal section, a Hair base with outer dendritic segments (oDS) surrounded by the dendrite sheath (arrow); the ciliary segments (CS) are surrounded by an inner receptorlymph space (iRL) formed by the thecogen cell (TH), which is separate from the outer receptor-lymph space (oRL) bordered by the trichogen (TR) and tormogen cell (TO); septate junctions (arrowheads) apically seal the intercellular clefts between apical epidermal-cell processes (aE) and auxiliary cells (see also Fig. 4b). b Proximal parts of the sensillum with the receptor-cell somata (RCA, RCB) and inner dendritic segments (iDS) enveloped by the thecogen cell (TH). Note the outer receptor-lymph space (oRL) formed by the trichogen cell (TR) with extensive microlamellae (ML) and the basal labyrinth (asterisk) between the basal epidermal processes (bE). BL basal lamina; C cuticle; E epidermal cell with apical (aE) and basal processes (bE); H haemolymph, a • 12800; b x 6000 perikaryal regions, the envelope is generally very thin, sometimes less than 0.1 pm. Mitochondria are small; neither rough nor smooth endoplasmic reticulum is well developed (Figs. 5, 6). The trichogen cell surrounds the thecogen cell in the region of the inner dendritic segments only. Its shape is extremely variable, and largely depends on the space available between the densely packed receptor cell bodies of neighboring sensilla (Figs. 2, 3 b). The large ellipsoidal nucleus is usually found in the basal part of the cell, which lies close to the haemolymph sinus (Figs. 2, 3 b). In B. mori
the trichogen cell and all other sensillar cells are always separated from the basal lamina by thin epidermal cell processes (Figs. 3 b, 7 a). In A. pernyi the trichogen cell occasionally directly contacts the basal lamina (Fig. 7 b). Membrane invaginations are found along the basal membrane of the trichogen cell, forming a basal labyrinth with dilated extracellular spaces (Fig. 7). The apical cell membrane invaginates to form a deep pouch, the surface of which is extensively folded (Figs. 2, 3b, 6a). These microlamellae are of fairly constant thickness (80-100 nm), except where mitochondria are incorporated (mainly in the basal parts); their
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4a-e. Region of axonal origin. a The transition between the receptor-cell somata (RCA, RCB) and the axons (Aa, AB) is characterized by 9the replacement (arrows) of the thecogen cell sheath (TH) by a glial envelope (G). Arrowheads indicate the location of septate junctions between receptor, glia, and thecogen cell (B. mori). b Region similar to the encircled region in a at higher magnification. Septate (SJ) and gap (GJ) junctions are discernible; other symbols as above (B. mori), c Cross section showing the complex glial wrapping (G) and the caliber difference between the two axons (AA, AB) of a s. trichodeum (A. pernyi), a • 12000; b • 74100; c x 9400 Fig.
height is variable, some microlamellae may exceed 10 Ixm; branching and twisting is often observed, which accounts for extremely complex section profiles. Apically, the microlameUae sometimes transform into microvilli. In regions of dense packing, the centre-to-centre distance between the lamellae is 70-90 nm. In B. mori the entire pouch is filled with lamellae so that there is no free lumen wider than 2 lxm except close to the mouth of the pouch where it opens into the wider distal receptor-lymph cavity bordered by the tormogen cell. In A. pernyi the free receptor-lymph lumen is wider (up to 5 ~tm), and the micro-lamellae appear less densely spaced. In B. mori, the cytoplasmic side of the trichogen cell microlamellae is studded with 8-nm particles (15 nm centre-to-centre distance) (Fig. 6b). In A. pernyi, freezing damage did not allow the unequivocal demonstration of such particles. In both species, the most prominent organelles of the trichogen cell are extensive stacks of cisternae of the rough endoplasmic reticulum; there are several small Golgi apparatus; coated vesicles are encountered throughout the cytoplasm, pinocytotic vesicles are observed at the basal, lateral and apical cell membranes (Figs. 6a, 7). The tormogen cell forms a collar around the trichogen and thecogen cells in the ciliary region; it confines the large distal portion of the receptor-lymph cavity, and outlines the cuticular canal below the hair base (Figs. 2, 3a, 5a). This cell has its largest extension close to the cuticle, and there the large ellipsoidal or polymorphic nucleus is usually observed. Only slender cell processes may approach, but do not reach the basal lamina (Fig. 2). Its cell membrane is in intimate contact with the cuticle of the cuticular canal, but elsewhere is always separated from the antennal cuticle by thin epidermal processes. The apical membrane forms elaborate microlamellae and microvilli where it borders the outer receptor-lymph
space. As compared with the trichogen cell, the microlamellae of the tormogen cell are similar in thickness, but are less densely packed and of reduced height; mitochondria are less frequent and there are more microvilli. There are central filaments in the microvilli and a central dense layer in the microlamellae, but we could not demonstrate a regular array of particles on the cytoplasmic face of tormogencell microlamellae (Fig. 5 b). The cytoplasmic organization of the tormogen cell is very similar to that of the trichogen cell: rough endoplasmic reticulum is abundant, coated pits and vesicles are found at and near the apical and lateral membranes (Figs. 5 a, c).
c) Glia cells. Glial cell processes ensheath the axons from the point where they originate at the receptor-cell bodies (Fig. 4). One glia cell may serve the axons of several sensilla, even of different type (e.g.s. trichodea and s. basiconica). The nucleus is usually located within the antennal nerve branch. Therefore, these cells do not belong to the sensillar cells proper. As a rule, each axon is separately ensheathed - often by multiple glial wraps - for the initial 10 tam, i.e. as long as the axon has an increased caliber. More proximally, bundles of 5- > 40 slender, naked axons are observed within one glial sheath (Steinbrecht 1969). d) Epidermal cells. Epidermal cells have the form of slender pillars (often less than 1 ~tm in diameter), which span the sensory epithelium from the cuticle to the basal lamina (Fig. 3b). Apically and basally, large flat extensions are formed that line the non-sensillar cuticle and basal lamina, respectively. The polymorphic nuclei may be found in the apical or basal portion, or within the pillars. The apical membrane exhibits short irregular membrane folds; dense material fills the subcuticular space (Fig. 3 a). As in the trichogen and tormogen cells the rough endoplasmic reticu-
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Fig. 5a-e. B. mori. a Cross section of s. trichodeum at the level where the two ciliary segments (CS) within the inner receptor-lymph space (iRL) are concentrically surrounded by the thecogen (TH), trichogen (TR), and tormogen (TO) cells. At this level the outer receptor-lymph cavity (oRL) is bordered mainly by microlamellae and microvilli of the tormogen cell. C cuticle; E epidermis; ER rough endoplasmic reticulum. b Microlamellae (ML) and microvilli (MV) of tormogen cell at higher magnification (compare with encircled region in a). Note concentrically arranged filaments (arrow) in microvilli and central dense layer (double arrow) in microlamellae, e Pino-(exo-?)-cytotic vesicles (P) opening into the outer receptor-lymph space (oRL) and coated vesicles (CV) in the tormogen cell (compare with rectangle in a). Arrows point to repeating projections on the outer vesicle surface. a • 12 600; b • 80 000; c • 100 000
lum is prominent. Pinocytotic vesicles are found mainly along the basal extensions. The number of epidermal-cell nuclei was counted in two section series of the sensory epithelium of B. rnori. We observed 19 and 13 epidermal-cell nuclei in sensillum fields comprising 14 and 12 sensilla, respectively. Thus, the number of epidermal cells in the sensory epithelium roughly equals the number o f sensilla.
e) Junctions. Septate junctions display a very dense matrix in freeze-substituted material. Consequently, the septa are resolved only at high magnification and when sectioned exactly at right angles. The dense matrix, however, indicates the location o f these junctions also in low-magnification electron micrographs (Figs. 3a, 4a, b, 5a, 6a). A continuous belt of septate junctions connects all cell types of the sensory epithelium including the epidermal cells (Fig. 3a).
This belt is situated close to the apical end of these cells and defines the border between the basolateral and apical cell membrane. Septate junctions are also observed at the axonal origin between axons, glial cell processes, and the thecogen cell (Fig. 4). The basolateral parts of the enveloping and epidermal cells are devoid of junctions except for a few local desmosomes in the basal region (Fig. 7a). The thecogen cell, however, forms a special junction over the entire surface of the inner dendritic segments and the receptor-cell bodies (Figs. 6a, 7b; see also Steinbrecht 1980). Gap junctions have the appearance of fused pentalaminar membranes in freeze-substituted material, provided pretreatment with cryoprotectants is omitted prior to cryofixation (Raviola et al. 1980). Such pentalaminar membranes are found in close association with the septate junctions of the apical belt. They also form the major part of the mesaxon structures where the leaflets of the enveloping cells meet (autojunctions). These mesaxons are typical for the
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Fig. 6a, b. B. mori. a Cross section of s. trichodeum at the level of the inner dendritic segments (iDS) enveloped by the thecogen (TH) and trichogen cell (TR). At this level the outer receptor-lymph cavity (oRL) is entirely bordered by microlamellae (ML) of the trichogen cell. Arrows indicate pinocytotic and coated vesicles. GJ gap junction; SJ septate junction; TO remainder of tormogen cell. Inset: Thecogen-receptor cell junction (arrowhead) with narrow cleft; compare with normal intercellular cleft (asterisk). b Microlametlae of trichogen cell at higher magnification (see rectangle in a); the cytoplasmatic side is studded with dense particles (portasomes) at fairly regular intervals (arrowheads); mitochondria (M) are frequently found within the lamellae, a x 22000; inset • 90000; b x 94500 thecogen, tormogen and glia cells, while the trichogen cell is perforated by the thecogen cell (Figs. 4, 5 a, 6 a). Discussion
The origin of all sensillar cells from mother cells ("Stammzellen '~ differentiating within the epidermis early in development is generally accepted since the work of Henke (1953) and Wigglesworth (1953) (for review, see Gnatzy and Romer 1984). In the antennal sensory epithelium of B. mori, also the number and position of epidermal cells is possibly determined by early differential cell divisions as suggested by the 1 : 1 ratio of observed sensilla and epidermal cells (see also Steinbrecht 1970, on sensillar distribution patterns in Bombyx). A maximal packing density of sensilla can be reached if all epithelial cells differentiate into sensilla with no epidermal cells in between, as was observed with campaniform sensilla on the halteres (Pflugstaedt 1912) or with chemosensilla in antennal pits (Gnatzy and Weber 1978, and unpublished observations) of Calli-
phora. In these cases, the cuticular apparatus of neighbouring sensilla is directly contiguous, whereas in Bombyx and Antheraea a small ring of unspecialized cuticle underlain by epidermal cell processes is found around the base of each sensory hair. However, the receptor cells and the large auxiliary cells together comprise most of the volume of the sensory epithelia On B. mori > 80%), so that at deeper levels the epidermal cells are reduced to slender pillars extending toward the basement membrane. Thus, sensillar cells of neighboring sensory hairs are contiguous over most of their surface without intervening epidermal cells. It is intriguing that the slender epidermal pillars form extensive basal processes along the basal lamina. These closely resemble the epidermal feet as described by Locke and Huie (1981); hence a morphogenetic role of these extensions during the development of the complex cellular architecture of the sensory epithelia is possible. As far as the uptake of substances from the haemolymph by the auxiliary cells is concerned, we do not suppose that the intervening epidermal-ceU processes impose an effective barrier, since
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Fig. 7a, b. Basal region of sensilla. a In B. mori, the trichogen cell (TR) is always separated from the basal lamina (BL) by thin epidermal processes (bE), which form a basal labyrinth with dilated extracellular spaces (asterisks); D desmosome; ER rough endoplasmic reticulum, b In A. pernyi, the trichogen cell (TR) frequently contacts the basal lamina (BL) directly. Arrows indicate the basal labyrinth. Freezing damage is evident, especially in haemolymph (H), due to deeper plane of section (~ 25 pm below antennal surface); bE basal epidermal process; RC receptor cell; ML microlamellae of trichogen cell; TH thecogen cell. a • 32000; b • 16000
the intercellular clefts between these processes are open except for a few local desmosomes. Moreover, the numerous coated pits and vesicles in the epidermal processes indicate that there is also transcellular transport through these cells. Despite their obvious differentiation, sensilla are still an integral part of the epithelium, all types of cells being apically connected by septate junctions, which seal the intercellular cleft. Extensive gap junctions, on the other hand, are found between all cells except between the receptor cells and the thecogen cell. This may result in electric coupling; hence sensory epithelia can possibly be regarded as functional syncytia, like the ordinary epidermis (Caveney 1976). The function of the special large-area junction between the receptor cells and the thecogen cell still has to be revealed by appropriate electrophysiological experiments (see also Steinbrecht 1980). In accordance with the general rule (see Schmidt and Gnatzy 1971; Gnatzy 1978) we find three auxiliary cells, the thecogen, trichogen and tormogen cell, in olfactory sen-
silla of the two lepidopteran species. As revealed by the reconstruction, these three auxiliary cells form concentric sheaths only in a very limited region around the ciliary segments of the sensory processes. The only cell that forms a complete sheath around the receptor cells is the thecogen cell; it not only wraps the two receptor cells, but also separates them from each other by intervening processes. As compared with the other two auxiliary cells, the thecogen cell in the adult shows little structural signs of metabolic activity. Its function in the imago might be better understood, once we know more about the special thecogen-receptor cell junction. In some insect mechanoreceptors, the thecogen cell does not directly wrap the receptor cell somata, because glial cells intervene from the axonal pole (e.g., cercal filiform hairs of Periplaneta: Gnatzy 1976; see also Keil and Steinbrecht 1983). It is intriguing that these sensilla do not show the special thecogen-receptor cell junction or the septate junctions at the axonal origin, so that lanthanum ions can
33 penetrate from the haemolymph into the intercellular cleft around the receptor cells (Keil and Thurm 1979). The trichogen and tormogen cells both remain metabolically very active in the s. trichodea of adult B. mori and A. pernyi as indicated by their fine structure. Morphological signs o f a secretory function o f these cells are the well developed rough endoplasmic reticulum and Golgi apparatus, the pinocytotic vesicles opening frequently on the basolateral as well as on the apical cell membranes, coated vesicles inside the cytoplasm, and the extensive surface enlargement of the apical cell membrane by microvilli and especially by microlamellae. M a n y mitochondria were found to be closely associated with the microlamellae. The striking resemblance to transporting epithelia (Berridge and Oschman 1972) is further supported by the observation of membrane particles (" portasomes ", Harvey 1980) on the cytoplasmic side of the apical microlamellae. Harvey and co-workers (1981) showed that the activity of a K+-activated ATPase from Manduca sexta midgut cells is increased in plasmamembrane fractions bearing portasomes. Recently a membrane-bound, K§ ATPase has been detected in the proboscis of the fly Protophormia terraenovae (Wieczorek 1982). The presence Of this ATPase activity, which is clearly not of mitochondrial origin, is correlated with the occurrence of labellar taste hairs. Smith (1969) was the first t o demonstrate these membrane particles in an insect receptor, the mechanosensitive campaniform sensilla on the halteres of Calliphora (for Musca, see Thurm 1970). Later, they were observed in filiform hairs of Gryllus bimaculatus (Gnatzy 1975) and macrochaetae of Calliphora vicina (Keil 1978). Their existence in insect chemoreceptors was reported by Hansen and Heumann (1971) for the tarsal, bristles in Phormia regina and shown for basiconic sensilla on the funiculus of Calliphora (Gnatzy and Weber 1978). Portasomes, however, appear to be labile structures and their absence in electron micrographs o f conventionally fixed specimens does not allow one to exclude their presence in vivo (Altner and Prillinger 1980). With freeze-substituted s. trichodea o f B. mori, only the microlamellae o f the trichogen, but not those o f the tormogen cell are evenly studded with portasomes. The same holds true for the s. basiconica (R.A. Steinbrecht unpublished observations). In the s. coeloconica of B. mori, on the other hand, all three cells, the tormogen, trichogen, and even the thecogen cell show apical membrane folds with a dense array of portasomes, which are even more prominent than those in the s. trichodea (R.A. Steinbrecht unpublished observations). It is improbable that these differences have been brought about artificially, since the absence o f portasomes is not correlated with freezing damage. It should be mentioned, however, that the present methods do not allow a distinction between a low density of irregularly arranged portasomes and their complete absence. A special aspect of the secretory activity of the auxiliary cells has been pointed out by T h u r m and co-workers (for review, see Thurm and Kfippers 1980; Kiippers and Thurm 1982). The portasome-bearing apical microlamellae are supposed to be the site of an electrogenic potassium pump, the presumed source of a metabolism-dependent transepithelial voltage (TEV). It is postulated that this non-neural voltage amplifies the receptor current. The influence o f the TEV on the function of the receptor cell, however, is variable a m o n g different types o f sensilla, even within the range
of mechanoreceptors (Erler and Thurm 1978). For the olfactory s. trichodea of male Bombyx a metabolism-dependent TEV of 33 mV has been measured (Thurm and Wessel 1979), but its influence on receptor function (e.g., sensitivity) still remains conjectural. Studies on this topic are promising, especially with the detailed morphometric data now at hand (Gnatzy et al. 1984); refined electrophysiological experiments, if possible with intracellular recording, will certainly provide a better correlation of receptor function with the complex "Bauplan" of sensilla.
Acknowledgements. We thank Miss B. M/iller (Seewiesen), Miss U. Grfinert, Dipl.-Biol. (Frankfurt), Mrs. H. Henkel (Frankfurt) for technical assistance, and Mrs. M. Kreuder (Frankfurt) for drawing the reconstruction. We are grateful to Drs. J. Edwards, K.-E. Kaissling, T. Keil, and D. Schneider for their critical comments on the manuscript. Partly supported by the Deutsche Forschungsgemeinschaft (W.G., Gn 9/2). References
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