The ultrastructure of campaniform sensilla on the eye of the cricket, Gryllus campestris

Cell Tiss. Res. 195, 349-357 (1978) Cell and Tissue Research 9 by Springer-Verlag 1978 The Ultrastructure of Campaniform Sensilla on the Eye of the Cricket, Gryllus campestris* Marie-Luise Mtiller, H.-W. Honegger, Elvira Nickel and Christel Westphal** Fachbereich Biologie, Universit~itKonstanz, BundesrepublikDeutschland Summary. The structure of the campaniform sensilla of the cricket eye was investigated by light and electron microscopy. Each sensillum is innervated by a single bipolar neuron. Its axon extends through the retina into a side-branch of the n e r v u s t e g u m e n t a r i u s . The dendrite extends through a cuticular channel to the surface of the cornea. The distal part of the dendrite, the sensory process, contains a tubular body and is attached to a cuticular cap which is obliquely inserted into the exocuticle between the corneal lenslets. Some particular structural features as well as the function of the campaniform sensillum of the cricket eye are discussed. Key words: Campaniform sensilla - Mechanoreceptors - Ultrastructure Compound eye - Cricket. In a recent investigation Honegger et al. (in press) described two types of receptors found in the cricket eye which respond to mechanical stimuli. Scanning electron microscopic observations revealed, in addition to interommatidial bristles, ovalshaped structures very similar to campaniform sensilla. Campaniform sensilla were first described by Hicks (1856) in dipteran halteres. Pringle (1938) showed that campaniform sensilla are mechanoreceptors which respond to pressure and tension on the insect cuticle. The general morphology of campaniform sensilla has been described by Lees (1942) and their fine structure has been studied in various insects and in different areas of the body (Thurm, 1964, 1965; Uga and Kuwabara, 1967; Stuart and Satir, 1968; Schmidt, 1969, 1973; Smith, 1969; Chevalier, 1969; Moran et al., 1971; Corbiere-Tichan+, 1971; Schmidt and Gnatzy, 1971; Mclver and Siemicki, 1978). Send offprint requests to: H.-W. Honegger,FachbereichBiologie,Universit~itKonstanz,Postfach7733, 7750 Konstanz, Federal Republic of Germany * Supported by the Deutsche Forschungsgemeinschaft,grant Ho 463/10 ** The authors are indebted to Prof. H. Altner, Universityof Regensburg, and Mrs. EvelynThury, Contron GmbH, Mfinchenfor use of the scanning electron microscopefacilities 0302-766X/78/0195/0349/$01.80 350 M.-L. Mfiller et al. The present s t u d y was carried o u t to d e t e r m i n e w h e t h e r the structures o b s e r v e d in the scanning electron m i c r o s c o p e on the cricket eye ( H o n e g g e r et al., in press) show the characteristic u l t r a s t r u c t u r e o f functional c a m p a n i f o r m sensilla. Materials and Methods Crickets (Gryllus campestris) were obtained from a laboratory colony. They were anesthetized with chloroform prior to dissection of the eyes. For scanning electron microscopy the eyes were fixed in 2.5 ~glutaraldehyde in 0.1 M phosphate buffer, rinsed in 0.2 M phosphate buffer and dehydrated in ethanol. The eyes were dried by the critical point method, coated with a thin layer of gold and examined with the scanning electron microscopes Cambridge Stereoscan $4-10 and Jeol JSN-35. For light and transmission electron microscopy the eyes were divided into halves and fixed in Karnovsky's formaldehyde-glutaraldehyde solution (Karnovsky, 1965), pH 7.4, for 2 h at 4~C. They were then rinsed several times in cacodylate buffer, pH 7.4, 0.65 M, and postfixed in 2 % OsO4 in veronal acetate buffer, pH 7.0, at 4~C for 2 h. After several rinses in cold veronal acetate buffer the specimens were dehydrated in a graded series of ethanol. One part of the tissue blocks was double stained with 0.5% uranyl acetate and saturated lead acetate in 100% ethanol-acetone (1:1) for 2h at room temperature (Gnatzy, personal communication). All samples were embedded in Araldite. Semithin and thin sections were cut with a Reichert Om U2 ultramicrotome using glass knives. The semithin sections were examined with a phase contrast microscope in order to localize the receptors and to study their general organization. The thin sections were stained with lead citrate (Venable and Coggeshall, 1965) and examined in a Zeiss EM 10 electron microscope. Results In the s c a n n i n g electron m i c r o s c o p e the external structure o f c a m p a n i f o r m sensilla a p p e a r s as an o v a l - s h a p e d d e p r e s s i o n in the c o r n e a s u r r o u n d e d b y a wall o f raised cuticle (Fig. 1). A m o u l t i n g pit is l o c a t e d eccentrically in the depression. C a m p a n i f o r m sensilla were f o u n d exclusively in the clefts between the c o r n e a l facets a n d seem to be r a n d o m l y d i s t r i b u t e d over the surface o f the eye. In a scanning electron m i c r o s c o p i c m o n t a g e which spans one entire c o m p o u n d eye, 69 c a m p a n i f o r m sensilla were found. A t the light m i c r o s c o p i c level the m o r p h o l o g i c a l o r g a n i z a t i o n o f the c a m p a n i f o r m sensilla can be seen in l o n g i t u d i n a l sections (Fig. 2). T h e distal p a r t o f the sensillum consists o f a c a p o b l i q u e l y inserted in the exocuticle o f the cornea. The sensillum is bilaterally s y m m e t r i c a l (Fig. 2, inset). T h e cuticular channel runs 40 to 60 g m t h r o u g h the cornea. T h e l u m e n o f this extracellular channel is a b o u t 4 to 5 g m in d i a m e t e r a n d widens b e l o w the c o r n e a to the r e c e p t o r l y m p h cavity. This cavity a n d the channel are lined by accessory cells which also envelop the d e n d r i t e a n d the s o m a o f the sensory cell. T h e cell bodies o f the n e u r o n s are l o c a t e d b e n e a t h the r e c e p t o r l y m p h cavity between the crystalline cones. The fine structure o f the sensillum will be described b e g i n n i n g at the distal end. The cuticular c a p is a m o d i f i e d e x o c u t i c u l a r structure. It consists o f two layers: an electron dense h o m o g e n e o u s o u t e r layer a n d a fibrous l a m e l l a r inner layer (Figs. 3 a, 4a). In transverse sections the fibrous lamellae a p p e a r as concentric rings (Fig. 3 b). T h e b i p o l a r n e u r o n sends its d e n d r i t e to the inner layer o f the cap. The d e n d r i t e is d i v i d e d into a n inner a n d an o u t e r segment linked by a c o n n e c t i n g cilium Campaniform Sensilla on Cricket Eye 351 Fig. 1. Scanning electron micrograph of a campaniform sensillum (CS) and an interommatidial bristle (1B) located between the hexagonalcorneal facets, x 2,700. Inset: External structure ofa campaniform sensillum. It appears as an oval-shapeddepression surrounded by a raised cuticle (RC). The eccentrically located moulting pit (MP) is visible, x 3,100 (Fig. 5 b). The distal part of the outer segment is club-shaped and contains a tubular body. This body consists of microtubules arranged parallel to each other and embedded in an electron dense amorphous material. In cross sections the microtubules do not show a regular pattern or dense packing (Fig. 3c). Below the tubular body the microtubules have a less specific arrangement. The cytoplasm of the outer segment lacks other cell organelles. The outer segment of the dendrite is surrounded by an extracellular electron dense material, the cuticular sheath, which forms the moulting channel through the cap and connects the tip of the outer segment to the inner layer of the cap (Fig. 3 a). In the distal part of the dendrite, the cuticular sheath appears to be smooth. Proximal to the tubular body, the sheath possesses irregular infoldings which protrude into the outer segment (Fig. 4a). Predominantly at these infoldings of the cuticular sheath, the dendritic membrane is 352 M.-L. Miiller et al. Fig. 2. Light micrograph of a longitudinal sectionthrough a campaniform sensillum. The cap is inserted in the exocuticle (EX). The cuticular channel (CH) widens below the endocuticle (EN) to the receptor lymph cavity (LC). Further proximal the neuron (N) is visible. • 900. Inset: Transverse section about 3-4 ima below the surface of the eye. The cap (C) clearly shows bilateral symmetry, x 1,500 bordered by granules of approximately 20 nm in diameter. They consist of an electron dense amorphous material and are regularly spaced along the membrane (Fig. 4b). In some cases microtubules appear in close association with the granules. An extracellular space separates the dendritic membrane from the cuticular sheath. However, at some points, where the dendritic membrane is studded with the granules, it appears to be attached to the cuticular sheath. This is supported by observations on shrunken dendrites, where the dendritic membrane was suspended between the infoldings of the cuticular sheath (Fig. 4c). The inner segment of the dendrite is linked to the outer segment by the connecting cilium. The cytoplasm of the inner segment is more electron dense than that of the outer segment. It contains mitochondria, ribosomes, rough endoplasmic reticulum, a few vesicles and striated ciliary rootlets (Fig. 5 b). The cross striation of the rootlets has a periodicity of approximately 67 nm. The receptor lymph cavity is divided into an internal and an external chamber (Fig. 5 a) which contain extracellular fluid. The internal lymph cavity surrounds the connecting cilium and is bordered by the cytoplasmic membrane of an inner envelope cell. This inner envelope cell forms a cylinder around the internal lymph Campaniforrn Sensilla on Cricket Eye 353 Fig. 3. a Electron micrograph of a longitudinal section through the distal structures of a campaniform sensillum. Inner (iL) and outer layer (oL) of the cap are visible. The cuticular sheath (CSh) surrounds the outer segment of the dendrite (oSD) and forms the moulting channel (MC). The dendrite contains microtubules (M) and the tubular body (7) in its tip. x 17,000 b Oblique section through the cap. The lamellae (L) of the inner layer appear as concentric rings, x 12,600. c Cross section of the tubular body. Microtubules (M) are embedded in an amorphous material. Note that they do not show a regular pattern, x 75,600 Fig. 4. a Transverse section through the inner (iL) and outer layer (oL) of the cap and the outer segment of the dendrite (oSD) below the tubular body. Infoldings (/) of the cuticular sheath indent the dendrite. The accessory cells (AC) are connected by septate junctions (S J). x 16,600. b Dendritic membrane bordered by granules (G) 20 nm in diameter. The arrow indicates a microtubule closely associated with a granule, x 180,000. c Part of a shrunken dendrite. Its membrane seems to be attached to the infoldings (/) o f the cuticular sheath, x 94,500 Fig. 5. a Longitudinal section through the receptor lymph cavity. In its internal chamber (iCh) the outer dendrite segment (oDS) and the inner dendrite segment (iDS) are connected by a cilium (arrow). The external chamber (eCh) is lined by the accessory cells (AC) which send long slender microviUi (MV) into its lumen. • 8,000. b Higher magnification of the ciliary region. In the proximal direction striated ciliary rootlets (R) leave the connecting cilium (CC). • 28,000. e Septate junctions (S J) between the accessory cells. • 121,500 356 M.-L. Mfilleret al. cavity. The external lymph cavity is surrounded by outer envelope cells. In the literature these inner and outer envelope cells are frequently called accessory cells (Moran et al., 1971 ; Chevalier, 1969) and we have adopted the same terminology. The membranes of these cells are highly folded and possess long slender microvilli which protrude into the cavity (Fig. 5a). The cytoplasm of the accessory cells appears darker than that of their neighboring cells. The accessory cells are connected with each other and with the inner segment of the dendrite by septate junctions (Figs. 5c, 4a). Discussion The present light and electron microscopic observations show structures which are characteristic of functional campaniform sensilla: a bipolar neuron, a dendrite consisting of an inner and an outer segment and connected by a cilium, a tubular body located in the tip of the outer segment, a cuticular cap with a bilayered structure, to which the tip of the dendrite is attached, a cuticular sheath around the dendrite, and accessory cells surrounding the neuron and forming the receptor lymph cavity. Identical structures have been described for campaniform sensilla which have been shown to function as mechanoreceptors by electrophysiological recordings. Pressure or strain in the surrounding cuticle was the appropriate stimulus. Ultrastructural investigations and electrophysiological recordings were performed on campaniform sensilla of the cockroach leg by Moran et al. (1971 ) and Chapman and Duckrow (1975), on the campaniform sensilla of the halteres of dipterans by Smith (1969) and Thurm et al. (1975), on the campaniform sensilla of the cerci of crickets by Gnatzy and Schmidt (1971) and Dumpert and Gnatzy (1977). In addition to the general structural pattern the authors of the present paper observed granules 20 nm in diameter first described by Thurm (1964) and infoldings of the cuticular sheath which indent the dendrite membrane (Gnatzy and Schmidt, 1971). We conclude that the dendritic membrane is attached to these infoldings. Gnatzy and Schmidt (1971) suggested that the infoldings prevent the tubular body from moving in the proximal direction during stimulation. The caps of the campaniform sensilla described previously are inserted more or less parallel to the tangential plane of the cuticle. In the cricket eye these particular structures are always obliquely inserted between the facets. We assume that this oblique insertion exists because the caps may orient during growth parallel to a tangential plane of one corneal lens. An obliquely inserted cap could also provide the basis for directional sensitivity of the campaniform sensilla. Honegger et al. (in press) have shown, that large amplitude action potentials can be recorded when the cornea of the compound eye is indented. These action potentials are distinguishable from the much smaller amplitude spikes arising from interommatidial bristles, the only other mechanoreceptor in the cricket eye. Thus, ultrastructural and electrophysiological evidence is provided for the conclusion that functional campaniform sensilla exist on the cricket eye. When mechanical stimuli are applied to the cricket eye an eye cleaning behavior is evoked. The same stimuli excite interommatidial bristles as well as campaniform sensilla. Campaniform Sensilla on Cricket Eye 357 Therefore, it is concluded that both types of mechanoreceptors are involved in triggering eye cleaning behavior in crickets (Honegger et al., in press). References Chapman, K.M., Duckrow, R.B.: Compliance and sensitivity of a mechanoreceptor of the insect exoskeleton. J. Comp. Physiol. 100, 251-268 (1975) Chevalier, R.L.: The fine structure ofcampaniform sensilla on the halteres of Drosophila melanogaster. J. Morphol. 128, 443-464 (1969) Corbiere-Trichan~, G.: Ultrastructure de l'6quipement sensoriel de la mandibule chez la larve du Speophyes lucidulus Delar. (Col6opt6re cavernicole de la sous-famille des Bathysciinae). Z. Zellforsch. 112, 129-138 (1971) Dumpert, K., Gnatzy, W.: Cricket combined mechanoreceptors and kicking response. J. Comp. 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