The fine structure of haltere sensilla in the blowfly Calliphora erythrocephala (Meig.), with scanning electron microscopic observations on the haltere surface

TISSUE ~t CELL 1969 1 (3) 443-484 Published by Oliver Et Boyd Ltd, Edinburgh. Printed in Great Britain

DAVID S. SMITH*

THE FINE S T R U C T U R E OF HALTERE S E N S I L L A IN THE BLOWFLY, CALLIPHORA E R Y T H R O C E P H A L A (MEIG.), WITH S C A N N I N G ELECTRON M I C R O S C O P I C O B S E R V A T I O N S ON THE HALTERE SURFACE The dipteran haltere incorporates large numbers of regularly disposed mechanoreceptors providing the sensory input enabling the vibrating haltere to function as a gyroscopic organ of equilibrium. Campaniform sensilla of the basal and scapal regions have been investigated by light and transmission electron microscopy, and these observations are augmented by scanning electron studies of the cuticle overlying the groups of sensilla. Each sensillum possesses a specialized fan-shaped terminal containing a complex and ordered association of microtubules and filaments. The transmission of stress to this region via the cuticle, and its possible role in transduction is considered. The fine structure of apical and basal sections of the distal sensory process and associated sheath cells is described ; the functional significance of the distribution of mitochondria and other components is discussed. The organization of haltere chordotonal sensilla is described briefly, and compared with other mechanoreceptors with particular reference to microtubules and scolopale structures. ABSTRACT.

Introduction

AMONGSTinsect mechanoreceptors associated with the body surface, campaniform sensilla have been distinguished as possessing a simple or more complex cuticular dome 'into which the sense cell process is inserted like the clapper of a bell' (Snodgrass, 1935), while chordotonal or scolopophorous sensilla lack specially differentiated cuticular structures at their point of attachment to the integument. Pringle (1938) noted that campaniform sensilla occur generally in adult insects and often in thickened regions of the integument

* Papanicolaou Cancer Research Institute and School of Medicine, University of Miami, Miami, Florida 33136, Received 28 January 1969.

at the base of wings, near leg joints, at the base of stingbarbs, on mandibles, on dipteran halteres and so on. He summarized their distribution in stating that there is 'no known case of campaniform sensilla occurring in a part of the cuticle which would not be liable to strain'. Sense organs of this type are present in large numbers, together with chordotonal sensilla, in the halteres of flies, and form the subject of this report. Much of our previous knowledge of the structure of the halteres of Diptera stems from the work of Pftugstaedt (1912), who carried out a detailed examination of the cuticle-associated sense organs contained within these highly modified and reduced hind wings, and from the extensive studies of Brauns (1939) on the distribution of sense organs throughout the Order. In introducing 443

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a functional analysis o f the haltere, Pringle (19481) suggests that 'no insect organ of c o m parable complexity has given rise to so m u c h a r g u m e n t as to its true function' and follows earlier papers (Fraenkel and Pringle, 1938: Fraenkel, 1939) in s u p p o r t i n g and extending the hypothesis that the halteres perform a proprioceptive function, countering an alternative supposition (Buddenbrock, 1917, 1919) that activity o f these organs 'stimulates' muscular m o v e m e n t s . * Evidence supporting the former hypothesis included the disequilibrating effects o f haltere removal, and c o m p a r a b l e behavior of ' b i t h o r a x ' Drosophila in which increase in haltere size is acc o m p a n i e d by loss o f their n o r m a l function. E a c h haltere is du~nbbell-shaped, including a basal region articulating with the metathorax and containing m a n y sensory neurones, a n a r r o w hollow cuticular stalk and a distal dilated bulb, and the modified wing is vibrated rapidly at the frequency o f the forewings, in the vertical plane. Fraenkel

a n d Pringle suggested that the gyroscopic properties o f the vibrating haltere confer on it its special sensory features, a n d this possibility was e x t e n d e d and experimentally examined by Pringle (1948) in an elegant synthesis o f theoretical and physiological studies. I n the present study, sections of the haltere have been examined by light microscopy and transmission electron microscopy and, while the observations are primarily c o n c e r n e d with the c a m p a n i f o r m sensilla, some features o f adjoining c h o r d o t o n a l units are described for comparative purposes. Particular attention is given to structural features that may be concerned in transduction. In the case of c a m p a n i f o r m sensilla, where the distal sensory process is p o s i t i o n e d beneath variously sculptured regions o f the cuticle designed to transmit mechanical disturbance, information from sectioned material is a u g m e n t e d by observations on the cuticular surface m a d e with the scanning electron microscope.

* In the first edition of Micrographia (1665), Hooke figured the haltere of a gnat and noted that (p. 194) ~ . . . these little pendulums . . . the little creature vibrates to and fro very quick when it moves its wings, and I have sometilnes observ'd it to move them also, whil'st the wing lay still, but always their motion seem'd to further the motion of the wing ready to follow: of what use they are, as to the moving of the wing, or otherwise, I have not now time to exanline'. Following the subsequent observations of Derham (1711, Physico-Theology: cited by Fraenkel, 1939), a later edition of Micrographia (I 745) described the halteres as (p. 60) 'baiIances or poises', the function of which 'is undoubtedly to keep the body steady and upright in flying; for if one of them be cut off, the insect will fly as if one side was over-balanced, and ere long tumble to the ground; and if both be taken away, its flight is aukward and unsteady, manifesting the want of some necessary and essential part'. Barbut (Tile Genera lnsectorum of Linnaeus, 1781, p. vii) recorded that a spider he was observing in St James's Park ~ directed his course to the place from whence the sound proceeded, darted with impetuosity upon the unhappy fly, and seized him at the back of the thorax, where the halteres or poisers are placed and, having thrust his fangs into that tender part, he paused during the space of a second: the fly being strong and struggling vehemently, he as quickly retreated, and left the poor animal to exhaust its strength in its endeavours to escape. This manoeuvre of the spider prevented a retreat by flight, even if the fly had had vigour sufficient to break the web, by his having bitten off one of the halteres, a frequent custom in the spider . . .'!

Materials and Methods Halteres o f adult blowflies (Calliphora erythrocephala, Meig.) were employed in this study. Conventional electron microscopic studies utilized a culture maintained by D r M. J. Berridge in the D e p a r t m e n t of Biology, University o f Virginia, and haltere preparations for scanning microscopy were m a d e from the C a m b r i d g e D e p a r t m e n t o f Zoology culture. F o r fine structural observations, blowfly thoraces were isolated in a pool of fixative (chilled 2.5~ glutaraldehyde with 0.15 M sucrose in a 0-05 M cacodylate buffer at p H 7.4), and entire halteres and associated basal sclerites were dissected f r o m the thorax. The haltere stalk was cut, and this cuticular tube a n d the distal bulb o f the haltere were discarded. Fixation of the haltere base was continued for 4 h o u r s (at 4~ then the material was washed overnight in chilled cacodylate buffer containing 0-3 M sucrose, 'stained' in 1~o OsO~ in a p H 7"4 veronal-acetate buffer and d e h y d r a t e d in an ethanol series. Material was e m b e d d e d in Araldite (Duxford,

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S T R U C T U R E OF H A L T E R E S E N S I L L A Cambridge) via propylene oxide, and polymerized blocks were cut with glass knives on a Huxley microtome. Sections were stained for 10-20 minutes in saturated uranyl acetate in 50% ethanol, then for 1-3 minutes in lead citrate (Reynolds, 1963) and examined in a Philips EM 200. The progress of sectioning through each block was monitored with 1/~ sections stained with 1~-;; toluidine blue and 1~ borax, and the light micrographs included in this work have been prepared by this method. Material for scanning microscopy was freeze-dried and subsequently gold-coated by evaporation. Specimens were examined by Mr M. L. Peters in a scanning electron microscope in the Department of Engineering, University of Cambridge. Images were recorded on 35 mm film. Results

The results described in this paper relate not only to light microscopic studies on 1/z sections of cuticle-associated sensilla, but also to electron micrographs of thin sections and to electron scanning micrographs of the cuticle overlying the various sense cells packed into the haltere base. Previous accounts of the form and organization of halteres have employed direct observations of the cuticular surface of the organ including its articulations with the body, together with histoIogicaI studies on sections, which have served to illustrate the distribution of contained sensory cells and their distal cuticle-linked processes. In the present study, the surface contours of the haltere have been revealed by electron microscopy, and these observations are extended by correlated light and transmission electron microscopy. General reference may be made to the excellent morphological description of this intricate organ given by Pflugstaedt (1912) and Pringle (1948), which greatly facititated the present study. CAMPANIFORM SENSILLA

The campaniform sensilla are situated in precisely determined groups on the dorsal TISSUE 8- CELL 1969 1 (3)

and ventral surfaces of the proximal or basal regions of the hMtere. These groups are readily identified by the repeating special cuticular formations associated with them. But, in addition, the haltere base contains two further groups of mechanoreceptors-chordotonal sensilla, which do not reveal their presence by modification of the overlying cuticular surface_ The dorsal sm;[bce* (a) The basal plate is a domed thickened region of the basal region of the haltere bearing several rows of campaniform sensilla, disposed in an approximately longitudinal manner. (b) The dorsal scapal plate. Just distal to the basal plate lies a series of transversely oriented campaniform sensilla situated within a semi-cylindrical cuticular structure leading to the scape or stalk. (c) The dorsal Hick's papilla comprises a double row of sensilla adjoining the anterior side of the basal plate and placed at a slight angle to the longitudinal rows of the basal plate sensilla. The ventral stab~ace (d) The ventral scapal plate lies beneath that of the dorsal surface and is sirnilar in structure and orientation. (e) The ventral Hick's papilla, as its dorsal counterpart, includes a double row of sensilla situated proximal to the ventral scapal plate. The general form of the haltere base and the positioning of the campaniform sensilla is illustrated in the section shown in Fig. 1. The plane of this longitudinal section passes through several sensilla of the basal plate, but does not include the precise dorso-ventral axis and includes only the ventral component of the scapal plates. As in other anthropod mechanoreceptors, each sensillum comprises a single sensory neurone, the distal dendrite

* Brauns (1939) and Pringle (1948) described a circular 'undifferentiated papilla' adjoining the dorsal scapal plate. This sensillum has not been noted in the present study and its function is not known,

SMITH, D.S.

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or process of which is inserted beneath a region of specialized cuticle--a feature resolved in the light microscope. Figs. 2 and 3 represent transverse sections of the dorsal region of the haltere base: the former at the level of the dorsal scapal plate and the latter through the basal plate. It is clear in such preparations that while the cuticle overlying each basal plate sensillum is domed, that of the scapal plates is formed into a more complex series of regular corrugations. Furthermore, Fig. 3 includes a profile of the twin rows of the dorsal Hick's papilla, and in-

dicates that the cuticle of these sensory units resembles that of the scapaI plate, rather t ban that of the basal sensilla. The details of these cuticular regions wi]l be considered more fully later. The haltere campaniform units are regularly placed within each group--a feature illustrated (Figs. 4 and 5) in the tangentially sectioned basal plate. In Fig. 5, portions of four rows of sensilla are included, sectioned through the cuticular domes containing the tips of the distal sensory processes. The center-to-center distance between sensilla

Figs. 1 - 5 represent photomicrographs of 1 micron glutaraldehyde-osmJum fixed plastic embedded sections, stained with toluidine blue and mounted in /mmersiol? oil. Fig. 1. In this longitudinal section of the haltere, the basal region adjoining the thorax lies on the left, and the start of the slender stalk (s) on the right. The plane of section passes through the cuticle overlying the basal plate (bp) and ventral scapal plate (vsp) sensilla, and proximal (axon) processes of sensory neurones constituting part of the haltere nerve are included at ax. Dorsal and ventra~ surfaces of the haltere are indicated (D, V). x 680. Fig. 2. Transverse section passing through the haltere at the level of the dorsal scapa] plate (dsp)--parallel with the black arrows in the scanning electron micrograph shown in Fig. 6. In this light micrograph, the distal process of the sense ceils are included ( * ) inserted beneath troughs in the halteye cuticle. Details of the cuticular surface in this region of the haltere are seen at higher magnification in Fig. 7, and by transmission electron microscopy of a thin section in Fig. 13, :- 1920. Fig. 3. Transverse section passing through the haltere at the level of the basal plate sensilta (bp) (cf. Fig. 6), including the complex cuticular structures associated with the dorsal Hick's papilla (dhp), The distal sensory processes of the neurones are indicated by asterisks, and profiles of the nuclei of these cells are included at n. Axon processes of haltere nerve cells are seen in transverse section at ax. :< 1200.

Fig. 4. Tangential section of the haltere basal plate. At the edges of the field, the cuticle surrounding the tips of the distal processes is included (white asterisks), while beneath this the plane of section includes further profiles of the distal processes (arrows) separated by extracellular spaces (black asterisks), forming a lattice readily identified in the survey electron micrograph shown in Fig. 35. x 1900. Fig. 5, Section in a plane similar to the last, but grazing the haltere surface and not extending into the subcuticular region. The tips of distal sensory processes (arrows) occur at regular intervals in the center of the plate though placed closer together as the rows converge towards the base of the plate on the right of the field. Each process, at this level, lies in an elfipsoidal cavity bordered by cuticle (*). >:2900. TISSUE 8- CELL t989 1 (3)

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Fig. 6. Low power scanning electron micrograph of the basal region of a haltere of the blowfly Ca//iphora erythrocephala. The specimen is viewed from the dorsal aspect with respect to its insertion in the metathorax of the fly (cf. Fig. 1). Rows of cuticular domes (white arrows) overlying the sensilla of the basal plate (bp) are seen in the lower (basal) part of the field adjoined by the differentiated cuticle of the dorsal Hick's papillae (dhp). These cuticular structures are shown at higher magnification, respectively, in Figs. 1 0 - 1 2 and 9 and in thin section in Figs. 15 and 14. Rows of cuticular hoops associated with the sensilla of the dorsal scapal plate are included at dsp; these are seen at higher magnification in Fig. 7 and in a transmission micrograph in Fig. 13. :*: 750.

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450 along each row is c a . 7F in the center of the plate, and less as the rows converge towards the haltere base. In Fig. 4 the plane of section is similar, but less superficial, and passed through ranks of distal processes beneath the cuticular covering (cf. Fig. 35). The dorsal aspect of the haltere base is shown in a scanning electron micrograph in Fig. 6. From this aspect, the longitudinal rows of cuticular domes of the basal plate, with the dorsal Hick's papilla are resolved, together with the approximately transverse corrugations of the dorsal scapal plate sensilla. This view of the haltere corresponds to that obtainable in a dissecting microscope, but the scanning electron optics provide not only enhanced resolution, but also remarkable depth of focus. Further aspects of the haltere surface organization are included in Figs. 7 through 12. Dorsal scapal plate cuticular structure is illustrated in Figs. 7 and 8. The former includes portions of four rows of sensilla, viewed approximately from above. The spatial relationship between the alternating cuticular hoops along each corrugated row, and especially the positioning of the distal sensory processes with respect to these, is revealed by thin sections perpendicular and

longitudinal to the rows offioops (cf. Fig. 13). Fig. 8 represents a tangential scanning view of a corrugated cuticular row in the dorsal scapal plate, and clearly illustrates the 'hooped' disposition of the cuticle of the dorsal scapal plate sensilla (cf. Fig. 13), white the surface of the dorsal Hick's papilla (Fig. 9) differs in detail from that of the scapal plate, but is shown in section (Fig. 14) to possess a cuticular structure closely resembling that of the scapal plates. The basal plate region and dorsal Hick's papilla (cf. Fig. 6) is illustrated at higher magnification in Fig. 10. This aspect, in the scanning electron microscope, is normal to the surface, and reveals the elliptical domes overlying the basal plate sensilla and the more complex cuticular structures of the Hick's papillae. As in other micrographs, many fine cuticular hairs are resolved in the sensory areas and outside it; these elaborations of the cuticle are not equipped with sensory terminals and their function is unknown. In Fig. 11, the sharp doming of the cuticle over each sensillum is clearly illustrated, and this feature provides striking evidence of the value of the scanning system in revealing surface features of biological structures.

Fig. 7. Scanning electron micrograph of a portion of the haltere dorsaI scapal plate shown in Fig. 6, The scanning microscope reveals minute]y detailed image with great depth of focus, and the relationship between these details and the underlying structures is best appreciated with reference to a transmission electron rnicrograph of a thin section perpendicular to that shown here ; such as that shown in Fig. 13, The black asterisks in this figure correspond to those in Fig. 8 in marking the mid-points of the cuticular hoops that lie between the sensory cell attachments. The sensory attachments lie beneath the white asterisks here (beneath the arrows in Fig, 13). The arrows in this figure merely denote the edges of the specialized cuticular hoops providing the sensory cell insertions. :< 5000. Fig. 8. A scanning electron micrograph viewing the dorsal scapal plate cuticle in a near-tangential aspect (cf. the perpendicular view of Fig. 7). Arrows and black and white asterisks correspond to those of the last figure, and this aspect iiiustrates clearly the sharply hooped cuticular contours associated with scapal plate sensilla. These contrast with the simpler domed cuticle overlying the basal plate, shown in Figs. 1 0 - 1 2 and 15, ; 6500. F i g . 9. Surface scanning view of the cuticle overlying the row of dorsal Hick's papillae s h o w n in Fig. 6. This corresponds to a double row of squeezedtogether sensilla of the scapal plate type in which adjoining pairs of sensilla (a in Fig. 14) are inserted beneath adjoining cuticular hoops, indicated by white asterisks in this micrograph. ,: 4500. TISSUE & CELL 1969 1 (3)

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Fig. 10. Scanning electron micrograph of a field similar to that shown in Fig. 6 rotated and examined at higher magnification. The complex cuticle associated with the dorsal Hick's papillae is seen at the lower part of the field (dhp) (cf. Figs. 9, 14), while the remainder of the field included rows of regularly spaced elliptical cuticular domes overlying sense cells of the basal plate (cf. Figs. 11,12 and 15). The entire basal haltere surface, within and without the sensory area, is thickly supplied with non-innervated cuticular hairs, illustrated in this and other scanning micrographs, x 1700. TISSUE 8" CELL 1969 1 (3)

S T R U C T U R E OF H A L T E R E SENSILLA The fine structural observations on thin sections included in this paper may be correlated not only with light micrographs but also with scanning electron micrograpbs. Figure 13 illustrates the insertion of four distal sensory processes beneath special cuticular bars, slung beneath the general cuticle of the haltere. Comparison between this micrograph and Figs. 7 and 8 emphasizes the positioning of the sensory terminals with respect to the corrugations of the scapal plate. A comparable section through the dorsal Hick's papilla (Fig. 14) illustrates the similarity between this region and the dorsal scapal plate. Conversely, a section through a basal plate sensillum (Fig. 15) (cf. Fig. 3) reveals a simple domed protuberance of the cuticle, beneath which a distal sensory process is inserted. The organization of scapal plate and Hick's papilla differs from papilla of the basal plate primarily in the disposition of cuticular structures associated with the sensory tip. This difference is illustrated in Figs. 16 and 17. In the basal plate the sensory tip is inserted on a compact cuticular dome beneath which lies a contiguous dome of spongy cuticle surrounding the tip. Changes in the curvature of the dome would be transmitted mechanically to the sensory tip as stretch or compression effects. In campaniform sensilla of the scapal plate (Fig. 16) and Hick's papilla (Fig. 14) the sensory tip is inserted beneath a discrete band of amorphous cuticle approximately rectangular in transverse section that is slung by a narrow osmiophilic hinge, ca. 500-600 /~ in width, from the adjoining cuticle. Profiles such as those shown in Figs. 14 and 16 suggest that the bar to which the sensory tip is attached may respond to deforming strains in the general cuticle, transmitted via the hinge. In Figs. 13-17, the tip of the sensory cells is revealed as a narrow cellular profile, as little as 0.151~ in width, enclosed in an osmiophilic sheath that is produced into lateral flanges beneath the level of the cuticle. Light microscopic studies showed that the distal process of these campaniform sensilla is actually fan-shaped (Pflugstaedt, 1912; TISSUE 8- CELL 1969 1 (3)

453 Pringle, 1938, 1948) and the electron micrographs mentioned above represent transverse profiles of the slender fa,a. Pringle (1948) points out that the orientation of the fan-shaped lamellae is precisely determined and constant within each group of sensilla. In the scapal plates, the fan is almost parallel with the long axis of the haltere--that is, transverse to the rows of cuticular hoops, lying beneath the regions indicated with white asterisks in the scanning micrographs (Figs. 7 and 81. Scanning micrographs of basal plate sensilla do not directly reveal the orientation of the underlying fanshaped lamellae, but Pringle (11948) mentions light microscopic evidence that in this region each fan-shaped tip is oriented at an angle of about 30~ with respect to the longitudinally arranged rows of cuticular domes (cf. Fig. 6) - - t h a t is, along the long axis of each elliptical dome. Thin sections of the fan-shaped lamellae in the dorsal scapal plate, in the surface plane of the fan, are illustrated in Figs. 23(a) and 23(c), and a serial 1/z section is shown in a light micrograph in Fig. 23(b). These micrographs indicate that the fan includes an angle of about 110, and for purposes of three-dimensional reconstruction it should be mentioned that the field shown in Fig. 23(a) is approximately 1000 /~ in thickness, and cannot deviate by more than a few degrees from the cuticular hoop marked with a white asterisk in Figs. 7 and 8, or, that is, from a plane perpendicular to that shown in Figs. 13 and 16. The field shown in Fig. 23(a) is particularly instructive, and should be compared with the section perpendicular to this, shown in Fig. 16. Figure 23(c) passes very precisely through the surface plane of the fan, and also includes the cuticular hoop or band--seen in transverse section (Fig. 16) as a rectangular profile--and hinged to the general cuticle of the haltere. Movements of this specialized cuticular hoop are presumably transmitted to the underlying fan-shaped sensillum tip since these extracellular and cellular components seem to be structurally linked. Thin sections in the plane of the fan which are

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S M I T H , D.S. Fig. 11. A field similar to that shown in Fig. 10, but providing a more tangential aspect of the rows of cuticular domes and the curving hairs that arise from the valleys between them. • 2000. Fig. 12. A field similar to the last, at high magnification. The recurred cuticular hairs are precisely shaped and oriented with respect to the domes overlying the basal plate sensilla, but thin sections show that they are not innervated and their significance is unknown. • 3200. Fig. 13. A transmission electron micrograph in the region of the dorsal scapal plate, corresponding to the tight micrograph shown in Fig. 2, and to be interpreted by comparison with the scanning micrograph shown in Fig. 7. Regularly placed apical regions (a) of distal sensory processes ensheathed in extracellular caps and flanges (f) (cf. Fig. 16) are inserted beneath special cuticular structures (arrows), slung between and slightly beneath the intervening cuticle (*). The arrangement of these cuticular surfaces, as alternating hoops, may only be fully appreciated in surface aspect (Figs. 7 and 8). ~ 12,000. Fig. 14. A section similar to the last, but passing through the dorsal Hick's papilla. As mentioned already (Fig. 9), this region of the haltere corresponds to a squeezed-together double row of scapal plate sensilla. ~ 14,000. Fig. 15. A section through a sensillum of the basal plate, in which the distal sensory process is inserted beneath a domed protuberance of the haltere surface (cf. Figs. 10-12). This process, as in other haltere sensilla, is divided into an apical region (a) ending just beneath the cuticle, a basal region (b) extending into the perikaryon (cf. Fig. 38) ; the t w o being demarcated by a constricted region (arrow) including a fibrillar body (Fig. 26) beneath which lies a basal body and associated rootlet (Fig. 27). • 8000.

Fig. 16. A higher power electron micrograph of a thin section including the tip of a dorsal scapal plate sensillum and the neighboring cuticular structures (cf. Fig. 13). The general haltere cuticle is seen at 1, and the amorphous band of cuticle overlying the sensory tip (beneath white asterisks in Figs. 7, 8) is represented by 2. The latter is enclosed laterally by an osmiophilic layer (3), linked to the main cuticle surface by a similar narrow layer (4) which perhaps functions as a hinge, permitting relative movement of 1 and 2. The apical tip of the distal sensory process is encased in an opaque extracellular presumably cuticular sheath (c), which is separated from the overlying cuticle by a narrow layer of cuticle (5), more or less confluent with a spongy inner cuticular cap (6). >~38,000. Fig. 17. A section corresponding to the last, but including the tips of a basal plate sensillum. Here, the general cuticle (1) extends as a simple dome over the sensory element. An extremely narrow layer of finely filamentous cuticle (5) lies between the general cuticle and the extracellular casing of the sensory tip (c) which is produced into flanges (f) immediately beneath. The sensory tip is encircled by spongy cuticle (6) similar to that described in the last figure. The hinged cuticular bar illustrated in the last figure is conspicuously absent. ;," 31,000. Fig. 18. A longitudinal-oblique section through the fan-shaped tip of a basal plate distal sensory process in the haltere. Clearly defined groups of tubular and filamentous structures are seen on the right (black arrow), while distally, on the left, medial dense-walled lacunae (*) are flanked by closely packed bundles of tubules (white arrow) (cf. Figs. 23c, 24a-b, 25). • 92,000. Fig, 19. A field similar to the last, illustrating more clearly the medial and surface organization of the sensory tip. :~:92,000. TISSUE 8- CELL 1969 1 (3)

T H E A M P H I B I A N P A P I L L A OF T R I T U R U S the whole bundle. In all cases where the basal foot or the two central fibres of the kinocilium could be seen, this axis was approximately parallel to the direction of the basal foot and at right angles to a line joining the two central fibres. On this basis, it appears that the axes of all the hair bundles are roughly parallel to the long axis of the amphibian papilla (that running from the mesial end to the opening into the sacculus); the variation in orientation was not seen to exceed about 20 ~ on either side of the papillary axis. Moreover, the hair bundles in the saccular half of the macula have their kinocilium on the side nearest to the sacculus, whereas all those in the other half have their kinocilium on the opposite side. About halfway along the macula, where the junction between the two populations occurs, there is a zone where adjacent bundles may have their kinocilia on opposite sides. No orientations intermediate between the two were seen. Flock and Wers/ill (1962) describe two slightly different arrangements of the stereocilia of the receptor cells in the lateral-line neuromasts. In the amphibian papilla, the existence of two distinct arrangements is not apparent, although there is some variation between different cells in the number of stereocilia in the bundle and :in the number of rows in which they are arranged. The receptor cells are innervated by two different types of nerve terminal, corresponding to the granulated and non-granulated endings that have been described in many other labyrinthine organs (see, for example,

Smith and Sj6strand, 1961 a and b; Engstr6m and Ades, 1965). There is no evidence in these organs of the third type of ending described by Lowenstein et al. (1964) in the sensory epithelia of the labyrinth of Raia. The non-granulated endings contain large numbers of mitochondria and usually also a few vesicles (about 300 to 1000 ~ in diameter), with electron transparent contents; the supposed synaptic regions are associated with densely staining masses (about 0 2 to 0-6 t9 in diarneter) surrounded by small vesicles (about 300 to 500 /~ in diameter). The granulated endings are found less commonly than the other type. They are associated with membrane pairs in the base of the receptor cell (Fig. 10), and contain relatively few mitochondria but large numbers of small vesicles about 500 to 800 ~ in diameter. A few densely cored vesicles (of total diameter about 700 to 800 A, Fig. 10) are also found in the granulated endings; these vesicles resemble structures which in other nervous tissues are thought to contain amines (see, for example, Wood, 1966; Aghajanian and Bloom, 1967). Densely cored vesicles have occasionally also been seen in the nongranulated endings. (iv) The accessory cells" Each accessory cell bears one short 'cilium' and a large number of microvilli on the apical surface (Figs. 4, 5 and 14). The microvilli are much shorter (under 1 /~) and of smaller diameter than the stereocilia of the receptor cells. Moreover, they are arranged

Fig. 4. Oblique section passing through part of the macula and lumen of the amphibian papilla. In the upper region of the micrograph the section passes through several hair bundles. Each bundle is composed of many stereocilia and one kinocilium ( k ) ; all the kinocilia lie in similar positions relative to their associated stereocilia. The section also passes through many irregularly shaped vesicles which lie between the cupuia and the surface of the macula. The accessory cells (ac) bear microvilli (m) and 'cilia" (c), Dense material lines the cellular junctions near the lumenal border of the epithelium, b, basa~ b o d y ; cp, cuticular plate ; rc, receptor cell. ~-,:6200. Fig. 5. Oblique section through a hair bundle of a receptor cell. The foot (f) on the basal body points away from the stereoci[ia (s). ac, accessory cell; m, microvillus of accessory cell. ;,: 17,000. TISSUE & CELL 1969 1 (3)

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410 less regularly and are distributed over the entire apical surface of the cell. They contain a few longitudinally orientated fibres, but these are not as densely packed as the fibres in the stereocilia of the receptor cells. The cilium extends to about the same height as the microvilli but is of larger diameter. It differs from the kinocilium of the receptor cell in that it contains only nine fibrils. Near the base of the cilium these fibrils are arranged in a single ring (Fig. 12), but in the distal regions one of the fibrils lies in a central position with the other eight arranged as a ring around it. A similar arrangement of the fibrils in 'cilia' of rat cerebral cortex has been noted by Dahl (1963) and, in this case, study of serial sections showed that the central fibril is continuous with one of the outer nine in the basal portion of the cilium. In the amphibian papilla, serial sections through one cilium have not been made, but comparison of sections of many different cilia cut at various levels suggests a similar transformation in this case. At the extreme tip of the cilium the regular arrangement of the fibrils is Iost (Fig. 11). Cilia with only nine fibrils are often, though not always, associated with two basal bodies (see Barnes, 1961; Satir, 1965). In the present case, the cilium arises from one basal body with a basal foot, and a second basal body lies a little deeper in the cell, at right angles to the first (Fig. 14). There is considerable variation between different cells in the orientation of the basal feet; they may vary over an angle of about 180 ~ in a small area of the macula. In one cell, a basal body with two feet was observed.

At the lumenaI border of the epithelium the junctions between adjacent cells are lined by an accumulation of dense, granular material (Figs. 5, 6, and 15), similar to that found at a corresponding position in some other labyrinthine epithelia (Lowensteiu et al., 1964). Elsewhere i n the macula, the accessory cells interdigitate with each other and occasional desmosomes are seen between them. The cytoplasm of the accessory ceils is notable for its extensive array of expanded granular cisternae with electron transparent contents. There are dense inclusions in the apical regions of tbe cells.

Discussion (i) The mode of action of the amphibian

papilla From studies on various organs of the acoustico-lateralis system (see, for example, Lowenstein and Wersfill, 1959; Flock and WersN], 1962; Flock, Kimura, Lundquist and Wers/i./l, 1962; Wets/ill and Flock, 1965) it seems that the directional sensitivity of many acoustico-lateralis receptor cells is related to certain morphological features of their hair bundles. Displacements of the hair bundle towards the kinocilium (or the basal body in the mammalian cochlea) result in depolarization of the receptor cell membrane and increase in discharge frequency in the sensory nerve. Displacements in the opposite direction produce hyperpolarization and decrease in discharge frequency, while those at 90 ~ produce little or no effect (Flock, 1965).

Fig. 6. Longitudinal section through the apical portion of a receptor cell. The stereocilia are graded in height and arise from a cuticular plate (cp). d, dense materia~ lining intercellular junctions; m, microvillus. ;< 25,000. Fig. 7. Enlargement of part of Fig. 6 showing the base of a stereocilium, cp, cuticular plate ; f, fibres ; r, rodlet, x 94,000.

Fig. 8. Section showing dense rodlets (r) at the lateral border of the cuticular plate (co) of a receptor cell. • 62,000. TISSUE ~ CELL 1969 1 (3)

SMITH, D.S.

458 sufficiently precise to include the entire structure in a 1000 ~ section are difficult to obtain, and while the accompanying illustrations refer to scapal plate sensilla, they are probably equally applicable to sensilla of the basal plate. The cytoplasmic contents of the fan-shaped tips of the sensory cells are very complex and are illustrated here in several fields. The main features of the distal processes, proximal to their splaying out into the terminal fan, are shown in Figs. 20-22. At the level shown in Fig. 20, the distal process is still roughly cylindrical, prior to its terminal fanning out - - b u t the sensory process is contained with an amorphous osmiophilic sheath. The section shown in Fig. 21 is slightly proximal to the last--and here the sensory process is no longer enclosed within a cuticular covering, but is associated only with a nonmervous sheath cell. The principal fine structural features of these micrographs involve the organization of the sensory processes. The low power fields shown in Figs. 20 and 21 indicate the preponderance of microtubules in the cytoplasmic organization of the distal tip, but at higher magnification (Fig. 22) it becomes evident that precisely oriented microtubules are only one component of the minutely structured organization of the distal process of haltere sense cells. Fig. 22 il-

lustrates a transverse section of a scapal plate sensillum just beneath its expansion into the fan-shaped lamella. At this level, the plasma membrane is flanked by an osmiophilic layer of extracellular material which envelopes the fan (cf. Fig. 13). The sensory process contains microtubules, and also fine filaments. It is tempting to think of the regular organization of muscle cells at this point, but the arrangement of microtubules and filaments falls short of the regular disposition of striated muscle. As the fan-shaped extension of the distal sensory process is approached, microtubules and filaments become associated in special configurations: while no regularly precise arrangement has been detected, transverse sections indicate that the microtubules become grouped together, with intervening dense material, to form three-, four- and fivemembered associations in the midst of which are inserted thin (ca. 80 A) filaments. The elaborate insertion of these microtubules and filamentous structures into the fan-shaped lamella is documented in the accompanying electron micrographs. Within the fan, the arrangement of microtubules and filaments is very precise, and it seems likely that the organization of the filament tip may be concerned with the reception of stimulation transmitted to the sensory cell distal process from the overlying cuticle.

Figs. 20 & 21. Micrographs of transversely sectioned apical portions (a) of haltere distal sensory processes. The profile in Fig. 20 is ensheathed in an extracellular opaque material (cf. Fig. 17) surrounded by narrow tendrils (s) of the sheath cell accompanying the nerve process, while in the slightly more proximal Fig. 21 the cellular sheath alone is present. The edge of the surrounding cuticular rim is included (*) in each of these figures (cf. Fig. 5). Fig. 20, >,"40,000 ; Fig. 21, x 38,000. Fig. 22. Part of the distal sensory process shown in Fig. 20, at high magnification. Note the layer of opaque material (c) and the underlying plasma membrane (pm). The process contains parallel series of t w o components, tubular structures (1) and filaments (2). Many of the tubules are surrounded by a zone of dense material which may be more or less confluent from one tubule to the next, defining lacunae within which the filaments are situated. In this field, most of the tubules are grouped in threes and fours (arrows), while the left insert illustrates a lacuna defined by five tubules (arrow). Other tubules (3) lack the dense sheath. It appears that the number of microtubular structures is not entirely constant as the sensory tip is approached : the right-hand insert includes a group of four tubules, one of which is not represented in the adjoining section represented in the plate. • 100,000; right insert, :, 100,000; left insert, x 1 50,000. TISSUE 8 CELL 1969 1 (3)

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S T R U C T U R E OF H A L T E R E SENS1LLA There is evidently considerable variation in the number and disposition of microtubules of the sensory tip of different insect mechanoreccptors. In the campaniform units described by Stuart and Satir (1968) the distal sensory process contains a configuration of doublets over much of the length, giving way in the terminal dilation only to 'more microtubules than can be accounted for by simple separation of the 9 + 0 doublets', while this arrangement of doublets alone occurs in auditory sensilla (Gray, 1960). This region of the hair plate organ is more elaborate, containing 50-100 microtubules (Thurm, 1965). In haltere campaniform sensilla, the number of microtubules present in the apical portion of the distal process is much greater. The transverse section shown in Fig. 21 includes 450-500 microtubule profiles, and there is evidence that the complement of these structures may be somewhat reduced before the terminal fan is reached; the profile close to the origin of the fan (Fig. 20) contains about 350 microtubule profiles and examination of serial sections (Fig. 22) reveals the abrupt termination of some individual microtubules.

Fig. 23 represents a section of a scapal plate sensillum, passing almost exactly medially through the distal sensory tip perpendicular to the field shown in Fig. 16. In the narrow cylindrical region beneath the terminal fan, discrete microtubules are resolved, and these extend into the termination. At relatively low magnification in the electron microscope, the radial striations of the terminal fan described in light micrographs (Pflugstaedt, 1912; Pringle, 1948) are identifiable. Further details of this region of the sensillum are illustrated in Figs. 23(c), 24(a) and 24(b). Fig. 23(c) includes longitudinal profiles of tubules leading into the fan, and, within the fan itself, two distinct fine structural aspects: (i) a reticulum of opaque walls surrounding electron-lucent lacunae and (ii) superimposed on this, over half the field, radially disposed bundles of microtubular profiles. Fig. 24(b) indicates that these bundles are formed by clumping of microtubules entering the sensory tip, while sections of the tip perpendicular to this (Fig. 25) show that the microtubular bundles are restricted to the superficial regions of the fanshaped process, while the central portion of

Fig. 23(a). A section of a dorsal scapa] plate sensillum, passing medially through the distal sensory process, with labelling of cuticular structures corresponding to that in Fig. 16. In the plane of section shown in Figs. 13 and 16,the distal sensory processes are seen as narrow structures, but when sectioned normal to this plane the process is revealed as a fan-shaped terminal extension of the sensory cell. The overlying cuticle (the "hinged" rectangular profile 2 in Fig. 1 6) is here revealed as a hoop--also seen (white asterisks) in the scanning micrographs shown in Figs. 7 and 8. In the lower part of the field, where the sensory process is still more or less cylindrical, longitudinal profiles of tubules and filaments are evident (arrow), while towards the apex of the fan these give way to closely packed dark and light areas (black asterisk) superimposed upon which is a repeating radial pattern (white asterisks). Further details of similar sections are illustrated in Figs. 23(c), 24(a), (b), and 25. z 31,000. Fig. 23(b). Light micrograph of a section serial to that shown in the last figure. The fan-shaped sensory tip (*) and associated cuticular hoop (2) are clearly resolved. This hoop lies slightly beneath the level of the general cuticle, which is seen in grazing section (1) in this 1 micron section, but which is absent from the 1000 A section in Fig. 23(a). :-, 3500. Fig. 23(c). Illustrating, at higher magnification than in Fig. 23(a) and in a serial section, the organization of the distal sensory fan. The plasma membrane lying between the nerve process and the overlying cuticle is indicated (pm) ; otherwise labelling is as in Fig. 23(a). x 70,000. TISSUE 8- CELL 1969 1 (3)

SMITII, D.S.

462

the fan is occupied by dense walled lacunae. According to this interpretation, the field shown in Fig. 23(c) is very slightly diagonal with respect to the lateral surfaces of the fan: on the right, the section passes through the lateral microtubule bundles, while on the left only the 'honeycomb' of opaque and light areas are included--as in the central portion of Fig. 25. The origins of this 'light and dark' pattern is shown in Fig. 24(a), which passes exactly along the medial line of a scapal plate sensillum. | n the lower part of this field, just proximal to the fan-shaped sensory terminal, both microtubules and parallel filaments are resolved (cf. transverse section, Fig. 22). Dense material associated with the microtubules at this level takes the form of transverse bars, arranged in a more or less regular fashion. Within the fan, the hollow cores of the tubules (Fig. 22) become occluded, and the resulting rods become linked laterally to

form lacunae, into which pass the 80 ~ filaments that accompanied the microtubules along their course proximal to the distal fan. Near-transverse sections of the fan-shaped tip of a basal plate sensillum are illustrated in Figs. 18 and 19. These may be compared with Fig. 25, since they similarly illustrate peripheral grouping of microtubules, and the central core provided by confluence of material associated with them (cf. Fig. 24(a)). The remaining description of the distal sensory process concerns structural details situated beneath the distal fan-shaped process. Fig. 26 includes a junctional region between the distal and proximal (basal) sections of a distal sensory process of a scapal plate sensillum. Within the apical region, the most striking cytoplasmic structures are the microtubules, extending to the tip of the sensillum: these microtubules pass through a 'fibrillar body' situated in the constricted

Fig. 24(a), Just proximal to the fan-shaped terminal of the distal process, discrete tubules ( I ) and filaments (2) are evident--and are illustrated in transverse section at this level in Fig. 22. The dense material associated with the tubules (Fig. 22) appears initially to be represented by more or less regular transverse bars, indicated here by long arrows. In the apical micron or so, the arrangement of tubules and filaments becomes more complex. In the central region of the fan, illustrated in the plane of this section, the hollow cores of the tubules become occluded and they become more or less completely linked by dense material defining lacunae (*) along which pass the filaments (short arrows), obliquely sectioned in this micrograph. :. 100,000. Fig. 24(b). The spongework representing microtubule terminals shown above is characteristic of the extremely narrow central zone of the distal fan; just beneath each of the t w o flat surfaces of the fan, the tubules are instead drawn together into tight radially arranged bundles. In this micrograph, separate tubules are seen (thick arrow), while certain tubules (thin arrows) may be traced into the bundles (*). This radial organization is also evident in Figs. 23 (a) and 23(c). • Fig. 25. A micrograph of a basal plate distal sensory process passing transversely through the fan-shaped terminal, and corresponding to the sensilla profiles shown in Figs. 13-17. This field illustrates the distinction between the superficial tubule bundles (white asterisks)--resulting from close-packing of previously separate units (arrows)--and the central region (black asterisk) in which the individuality of the tubules is lost in a spongework of dense walled lacunae into which pass slender filaments. This field indicates that the width of the distal sensory tip is c a . 0.3#, of which each tubule bundles occupies c a . 500 A and the central spongework c a . 0.15/~. The field shown in Fig. 24(a) chances to pass very exactly through the medial axis of the fan; the lateral tubule bundles would have been included had the plane of section deviated by a few degrees from that shown--and would have then afforded an aspect such as that in Fig. 23(a) or 23(c). >', 94,000. TISSUE 8" CELL 1969 1 (3)

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S T R U C T U R E OF H A L T E R E SENSILLA region situated between the apical and basal section of the distal sensory process (cf. Figs. 33, 34). In longitudinal profiles of this region of the sensilIum, the components of this fibrillar body are preferentially sectioned transversely and are c a . 150 ~ in diameter. A transverse section at the level of the fibrillar body is shown in Fig. 33, and this again emphasizes the relationhsip between the intercellular gap between the distal process and the concentric sheath cell cylinder and the extracellular space within which the entire structure is situated. The large number of microtubules present in the distal tip is much reduced as the level of the constriction between apical and basal portions of the distal process, and, as illustrated in Fig. 33 and at higher magnification in Fig. 34, only about nine doublet microtubules traverse the fibrillar body. Longitudinal profiles of these microtubules are seen in Fig. 27, passing through the constriction and apparently contributing to the basal body. Fig. 27 includes a field slightly proximal to that shown in Fig. 26, and shows a longitudinal profile of the basal body of the distal process, together with the associated rootlet (cf. Figs. 36, 47)--characteristic of ciliary and flagellar structures elsewhere in the Animal Kingdom.

Fig. 28 represents a transversely sectioned sensillum tip at the level of the basal body. This field includes the non-nervous sheath of the sensillum, and illustrates the extensive intercellular spaces between the sensory tip and the surrounding sheath (glial) cell, and the wider extracellular space within which lie the regularly repeating sensory columns of the haltere (cf. Fig. 35). Two distinct types of sheath cell occur along the distal process. The outer of these is characterized by slender irregular microvilli extending distally almost to the tip of the process (Fig. 21) and proximally to the sensory cell body (Figs. 38, 39). In the basal region of the distal process an inner sheath cell is interposed. The fine structure of these is considered later. Beneath the level of the sensory cell basal body, the distal neuronal process is intimately connected with the enveloping inner sheath cell. Figure 29 illustrates the Organization of the basal section of this process and includes several profiles of the fragmented striated rootlet extending from the basal body, and also cylindrical sheath cell processes inserted into concentric invaginations of the nerve process. These junctions are fortified by desmosomal links: in Fig. 30,

Fig. 26. A field including the junctional region between the apical (a) and basal (b) regions of the distal sensory process of a scapal plate sensillum of the blowfly haltere. Microtubules extend to, and in small numbers (Figs. 33 and 34) pass through, the constriction between these regions, occupied by a compact "fibrillar body' (fb). Beneath this body extends a periodically banded rootlet (r), associated (Fig, 27) with a basal body. The sensory process is flanked by a cellular sheath (s), strengthened (cf. Figs. 28, 29, 30) by microtubulereinforced desmosomes (d). An intercellular gap (i) is interposed between the nerve process and the sheath, while extensive extracellular spaces (e) (cf. Fig. 35) lie between the sheath cells associated with the sensory columns. ;., 50,000. Fig. 27. A field slightly beneath the last, including the base of the fibrillar body, and passing through the underlying basal body (bb). Beneath the basal body extends a conspicuous rootlet (corresponding to that associated with other ciliary derivatives), seen in transverse section in Fig. 36 and at higher magnification in the plane of section shown here in Fig. 47. • 60,000. Fig. 28. A transverse section through a distal sensory process at the level of the basal body (bb)--identified in the last micrograph. Note the cytoplasm of the basal region of the process (b), the encompassing inner sheath cell (s') (cf. Fig. 26), while extensive extracellular spaces (e), in life presumably filled with haemolymph, surround the sheath cells of each sensory column, x 50,000. TISSUE ~ CELL 1969 1 (3)

SMITH, D.S.

466 microtubules within a sheath cell process flank a desmosomal junction with a distal sensory process. The regular spacing of groups of haltere campaniform sensilla is reflected in the regularity of the cuticular structures overlying the insertions of the sensory units, and this regularity is maintained as the distal processes pass inwards towards their cell bodies. This has been illustrated in a light micrograph of a tangential section beneath the surface of the basal plate (Fig. 4), and a comparable survey electron micrograph is shown in Fig. 35. It is immediately evident that the basal section of distal processes, beneath the constriction occupied by the fibrillar body, is more complex than the apical section, and even at this low magni-

fication the abundance of mitochondria is striking. The sheath around the nerve processes continues and here the outer cell surface is dissected into slender curving microvilli, and the surfaces of these contiguous glial components defines approximately cylindrical extracellular channels, filled with haemolymph in life, and containing filamentous structures of unknown nature, oriented parallel with the sensory columns. The cytoplasmic organization of the basal portion of the distal process is illustrated at higher magnification in Fig. 36. Small mitochondria with parallel cristae are profusely distributed within the distal process and each process contains a rootlet, extending from the basal body over much of the distance to the cell body. Clusters of small particles the size of

Fig. 29. A transverse section of the dorsal scapal plate sensory process below the level of the basal body. A profile of the ciliary rootlet is seen at r. The cytoplasm of the sensory process contains microtubules (mt) and small mitochondria (m). Intercellular spaces between the nerve process and the surrounding sheath cell are present (i), and these are linked by desmosomes (d), while linkage between these two cells is further strengthened by the invagination of slender inner sheath cell processes (arrows) into the sensory process--further illustrated in the next figure. • 60,000. Fig. 30. illustrating a desmosome linking an inner sheath cell extension (s') with the basal region of a distal cell process (b) (cf. Fig. 29). Each cell membrane bears a cytoplasmic layer of dense material (*), and this is flanked, on the side of the nerve cell process, by a row of microtubules (rot). A narrow layer of dense material (arrow) occupies the central region of the 200 A intercellular gap. >: 125,000. Fig. 31. A field illustrating linkage between the basal region of a distal cell process (b) and the surface of the enveloping inner sheath cell (s'). The gap between these cells is maintained at ca. 120 A, but this gap is periodically bridged by 'septa'--spaced at intervals of 120-180 ~,. A mitochondrion within the neurone process is included (m). • 170,000. Fig. 32. Desmosomes of the macula adhaerens type, including paired dense cytoplasmic plaques (*) occurring in addition to septate desmosomes along the mesaxon folds of the outer sheath cell (s) (cf. Fig. 36). /, 120,000. Fig. 33. A transverse section at the level of the fibrillar body of the sensillum. This is seen in transverse section (fb), situated in an extensive intercellular space (i) (cf. Fig. 26). At this level, the encircling sheath ceil layer (s) is thin, and outside this lies the general extracellular space (e) of the haltere. >: 40,000. Fig. 34. A portion of the last figure, at higher magnification. Two doublet microtubules (arrows) are included here: a large number of microtubules are present distal to the fibrillar body of the haltere sensilla, but only about nine pairs continue throughthefibrillar b o d y t o t h e underlying basal body. :,: 123,000. TISSUE ~t CELL 1969 1 (3)

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S T R U C T U R E OF H A L T E R E

469

SENSILLA

ribosomes also occur within the process, while longitudinally oriented microtubules are conspicuous both here and also in the enveloping inner sheath cell. As is shown in Fig. 31, the nerve and glial cell membranes are linked at intervals by restricted septate desmosomes and also by local desmosomes of the macula adhaerens type (Fig. 36). Similar cell-to-cell links have been described by Stuart and Satir (1968) in the campaniform sensilla of the termite sternal gland. Further structural details of the outer sheath cells are included in Fig. 37, which illustrates the slender microvilli stemming from the outer surface; these structures are ca. 350 in width, except where they are dilated to accornmodate numerous elongated mitochondria, commonly 0.25t~t in diameter. Higher magnification fields (Fig. 37 insert) reveal an interesting detail of these microvilli--small particles, ca. 70 ~ in diameter, attached to the cytoplasmic side of the unit plasma membrane. These particles resemble those described in Calliphora rectal papillae apical cell processes (Gupta and Berridge, 1966), the microvilli of lepidopteran midgut goblet cells (Anderson and Harvey, 1966; Smith et al., 1969) and the microvilli of Malpighian tubules (Berridge and Oschman, 1969), and it is interesting to note that each of the above cell surfaces is the site of transmembrane ion transport. The organization of the vertebrate and invertebrate perikaryon--the cytoplasm of the neurone cell body--is typically more complex than that of its axona[ or dendritic prolongations. As the distal processes of a haltere campaniform sensillum neurone passes into the perikaryon, a sharp transition is evident (Fig. 38), marked by the suddenly increased cytoplasmic density of the perikaryon--primarily attributable to the presence of large numbers of free ribosome clusters and small numbers of cisternae of

the .RNP-associated endoplasmic reticulum. Within the basal region of the distal process, ribosomes are sparsely distributed (Figs. 36, 38), and the most obvious cytoplasmic inclusions are microtubules and mitochondria, while multivesicular bodies (Fig. 39) are occasionally observed. The characteristic outer sheath microvilli seen in transverse sections of the sensory columns in Figs. 35-37 are seen in a longitudinal profile of the distal process in Fig. 39, while Fig. 38 includes a sheath cell nucleus adjacent to the perikaryon. The proximal (axon) processes of haltere campaniform sensilla are also contained within sheath cells. Fig. 40 includes part of the transversely sectioned nerve leading from the scapal plates; as in other insect peripheral nerves, the axons are invested with glial processes stemming from scattered glial cell bodies, disposed either in single sheets or in multiple layers involving loosely arranged mesaxon folds. Again as in other peripheral nerves, microtubules (neurotubules) and mitochondria are the most conspicuous axoplasmic components, and the ribosomes, present in the distal sensory process, are absent_ CHORDOTONALSENSILLA .In addition to the ranks of campaniform sensilla mentioned above, the region of the haltere proximal to the origin of the stalk contains two further series of mechanoreceptors, distinct from the basal and scapal plate sensilla by virtue of their disposition and structure. These are the chordotonal sensory receptors, differing from the campaniform units most obviously in lacking a differentiated cuticular region overlying the thin distal dendritic process. These sensilla are inserted beneath unmodified cuticle, and their distal processes are slung between this insertion and a second point of attachment provided by a cellular connective linking the

Fig. 35. A low pow er micrograph of the distal processes associated with several basal plate sensilla. Regularly disposed distal sensory processes are indicated by arrows : these are ensheathed by sheath (glial) cells (s). The extracellular spaces between sensory processes and gial elements are indicated (e), and these contain filaments (*) of u n k n o w n nature. >: 11,000.

TLSSUE 8 CELL 1969 1 [3)

SMITH, D.S.

470 base of the dendrite with the cuticle. While in the haltere both points of attachment are established beneath the cuticle, these proprioceptive units elsewhere in the body may be attached to apodemes, tracheae and internal organs, and it is supposed that their activation is triggered by stretch occurring between their attachment points. Chordotonal sensilla are grouped into two 'organs' in the haltere of Calliphora, and their orientation has been described by Pflugstaedt (1912) and Pringle (1948). The electron micrographs included here represent oblique transverse sections of units of the large chordotonal organ. These run at an angle of about 45 ~ to the long axis of the haltere, from the point of insertion on the ventral surface of the haltere, posterior and proximal to the ventral scapal plate--and, as Pringle (1948) points

out, parallel with the fan-shaped lamellae of the basal plate, but inserted on the opposite side of the haltere. The fine structure of the sensory cells of the abdominal auditory organs of Locusta described by Gray (1960) corresponds in some respects to the haltere chordotonal units, and in others to the campaniform sensilla described above. A series of chordotonal distal process are illustrated in Fig. 41. As in campaniform and other insect mechanoreceptors, the distal chordotonal dendrite is divided into apical and basal portions, characterized respectively by a ciliary derivative and a striated ciliary rootlet. As in the auditory sensilla (Fig. 41), the apex of the dendrite is inserted into a plug or 'cap' of opaque material: Gray described this as an extracellular structure, encircling the

Fig. 36. A micrograph illustrating the details of nerve-sheath cell association in a Calliphora haltere basal plate sensory unit. The transverse section shown here includes a profile of the basal portion of the distal sensory process (b), and the cytoplasm exhibits mitochondria (m), ribosomes (*), many microtubules (rot) and the ciliary rootlet (r). The inner sheath cell (s') is narrow and contains numerous microtubu~es (mt'). The outer sheath cell (s) includes a folded mesaxon, originating at the point indicated with an arrow, the opposed membranes of which are linked by desmosomes of the macula adhaerens (d) and septate (sd) type. The outer surface of this cell bears slender microvilli (mv) projecting into the extracellular space (e) between the sensory processes. :< 60,000.

Fig. 37. A field similar to the last, at higher magnification. This field includes a nucleus (n) and adjacent cytoplasm of an outer sheath cell. The outer surface of the cell is produced into microvilli (mv), dilated to include mitochondria (m), and these are separated by extensive extracellular spaces (e). The insert illustrates microvilli bearing cytoplasmic particles (arrow-heads), perhaps involved in the exchange of ions between the extracellular milieu and the cytoplasm of the sheath cell. :-', 50,900 ; insert, x 200,000.

Fig, 38, Micrograph illustrating the base of a haltere sensory process. A portion of the neurone nucleus is included at bottom left (n), and in the surrounding cytoplasm the most obvious componentsare unattached ribosomes (rb). Above the level of the perikaryon (above the arrows) extends the basal part of the distal process, encompassed by the cellular sheath (s). Ribosomes also extend, in smaller numbers, into the process (*). Part of a sheath cell nucleus is seen at n' on the right of the field. ;~ 30,000. Fig. 39. An electromicrograph similar to the last, further illustrating the distal process approaching the cell body. This contains microtubules (rot), occasional multivesicular bodies (rob) and clusters of ribosomes (*). Note the microvilli (my) of the outer sheath cell. • 40,000. TISSUE 8 CELL 1969 1 (3)

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474 terminal region of the dendrite, but in the haltere it appears to be an intracellular component. Observations on these sensory ceils are incomplete, and it is not known whether the sensory tip is functionally attached to a 'cap cell' or whether it penetrates this cell to a direct insertion on the overlying cuticle. Whereas the auditory sensilla of L o c u s t a involve a relatively typical ciliary structure, incorporating nine tubular components, the corresponding region of the haltere chordotonal sensillum resembles the neighboring campaniforln units in possessing large numbers of microtubules. In each instance, the apical portion of the distal dendrite is ensheathed by a scolopale cell--corresponding to the 'sheath cell' of the campaniform unit (Fig. 21). Gray described the arrangement of 5-7 scolopale rods of fibrous material, encircling the ciliary process, together with the partial branching of these structures as the junction with the cap is approached. In the profiles shown in Fig. 41, about 12 scolopale rods surround each sensory process, while further fine structural details of these are shown in Fig. 42: each rod comprises an irregular cylinder of opaque finely fibrillar material, fortified by numerous microtubules, generally arranged at the periphery of the rod and parallel with its long axis. i n auditory sensilla, the scolopale rods provide a robust framework proximal to the base of the ciliary structure, but in the haltere chordotonal sensilla this is not the case. The basal region of the latter (Fig. 41) are enclosed by a narrow cellular sheath, probably distinct from the distal scolopale cell, the cytoplasm of which contains a remarkably close packed complement of microtubules (Fig. 43), but lacks the opaque material characterizing the scolopale rods. At this magnification, many dendrite microtubules are observed, aligned alongside the nerve cell membrane (cf. Fig. 45).

As in the case of campaniform sensilla, the cytoplasm of chordotonal units show a marked apical-to-basal differentiation: in the basal region, even at low magnification, the presence of a single ciliary rootlet and numerous mitochondria is evident (cf. Fig. 36). Conversely, Gray described extensive splitting of the ciliary root into 30M0 rootlets in the basal part of the auditory dendrite. T h e conspicuous ciliary rootlet profiles are further illustrated in Fig. 44; these structures reach a diameter of ca. 0.75/2, and this detail of the organization of chordotonal cells is illustrated in more detail in Fig. 45. Unlike the compact ciliary rootlets of the campaniform sensilla (Fig. 36), these structures in the chordotonal units are irregularly reticular in transverse section (Fig. 45): they not only incorporate occasional microtubules within their internal cavities, but are often surrounded by regularly arranged ribbons of microtubules, resembling those adjacent to the cell membrane (Fig. 43). Despite the difference in complexity between baltere campaniform and chordotonal ciliary rootlets, their fine structure is comparable. In each case (Figs. 46, 47) these display a macroperiod repeat of ca. 600 ~. Discussion Pringle (1948) was unable to enlarge upon the observations of Pflugstaedt (1912) concerning the histological organization of the sensilla collected within the haltere base and scape. These observations have for the most part also been confirmed during the present study, and it is generally possible to relate unambiguously the components recognized by Pflugstaedt to their fine structural equivalents. It is interesting, at this point, to consider a few detailed aspects of correspondence between Pflugstaedt's account and the present. He divided the distal process of campaniform sensilla neurones into two portions;

Fig, 40. A transverse section of a nerve within the Cat/iphora haltere. This field includes part of the nerve from the scapal plates, and incorporates about 70 axons (ax). Axons of basal sensilla are added to this nerve before it leaves the haltere. Note the glial processes (g) surrounding the axons and the glial nucleus (n). x 12,000. TISSUE 8 CELL 1969 1 (3)

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S T R U C T U R E OF H A L T E R E SENSILLA distal and proximal to the structure (Knopf), identifiable in electron micrographs as the fibrillar body situated above the basal body of the ciliary derivative. The entire intracellular apparatus of the process distal to this structure is termed the mushroom body (pilzfdrmiges KOrperchens): Pflugstaedt accurately observed the ~rectional features of the distal process and its relationship with the overlying cuticle--the isodiametric stift,fJrmiges KSrperchens of Pflugstaedt is clearly represented by the closely associated microtubules and filaments of the distal segment, and he further identified the spatulate or fan-shaped sensory tip of these campaniform units together with its narrow aspect when sectioned normal to the surface of the fan. The disposition of the terminal fan beneath the basal and scapal plates and Hick's papillae was illustrated by Pflugstaedt, and the horseshoe disposition of the cuticle (huJ#iseHfb'rmiges Chitinstiick) seen in sections passing precisely parallel with the faces of the underlying fan has been illustrated here in electron micrographs. The radial striation of the fan figured by Pflugstaedt was a remarkably accurate observation and is represented in fine structural terms by the radial grouping of microtubules along the

lateral faces of the expanded distal terminal, illustrated here. The most significant emendation from Pflugstaedt's account concerns the relationship between the distal process and the non-nervous sheath that accompanies it. Both studies agree on the presence of an inner narrow sheath cell (Nervenhiille) surrounding the dista/process, while Pl]ugstaedt supposed that this cylinder was in turn contained within a second cell (Zwischenzelle) occupying the entire space between successive nerve processes, and reported to be a modified hypodermal unit slung from the cuticle. Pflugstaedt noted that this cell appeared to possess a striated border and that the cytoplasm has an empty appearance (plasmaarm) except in the vicinity of the nucleus, placed near the base of the cell. The electron microscope indicates that the limits and organization of this cell are quite different; it forms a slender outer sheath around the nerve process, stopping just short of the distal tip and at no point reaches the general cuticle. At least in the material examined here, the sensory columns are widely separated by extracellular cavities (the 'plasmaarm' regions) lined by the outer sheath cells, and it is possible that the 'thread' (Fiidchen) described as traversing the cell

Fig. 41. Transverse section of a group of chordotonal sensilla within the

Calliphora haltere. The upper part of the field passes through the apical portion (a) of the distal processes, The apex of the process of each nerve Cell is embedded in a plug of dense material--the "scolopale cap" (*). The apical portion of each sensillum is enclosed by a scolopale cell, characterized by numerous elongated stiffening elements, the scolopale rods (st), further illustrated in Fig. 42, The plane of section at lower right includes profiles of the basal region (b) of apical processes, and further details of this level of the sensory cells are shown in Figs. 4 3 - 4 6 . • 9000. Fig. 42. Transversely sectioned scolopale rods, associated with a chordotonal sensillum (cf. Fig. 41 ) at higher magnification. Each rod comprises an irregular, sometimes hollow cylinder of dense finely fibril Jar material (sf) closely associated with microtubules (arrows) oriented parallel with the long axis of the sensillum (cf. Fig. 30), • 1 35,000. Fig. 43. A portion of a basal region of a distal process similar to those showr, in Fig. 41, at higher magnification. The sensory process (b) is closely enveloped by a sheath cell (s) which lacks scolopale rods, but for part of its length possesses closely packed arrays of microtubules (mr). The adjoining distal process contains similar microtubules, aligned along the cell membrane (arrows) and elsewhere (Fig. 45). x 125,000o TISSUE 8 CELL 1969 1 (3)

SMITH, D.S.

478 from the cuticle to the base is represented by the extracellular filaments observed in the electron microscope. No hypodermal cells remain beneath the cuticle in the region of the basal and scapal plate sensilla in the mature adult material examined here, and it is possible that these filaments remain after the disorganization of the epidermal members responsible for laying down the specialized cuticle of the campaniform units. The chief contribution made by the electron microscope to current concepts in sensory physiology has concerned the clarification of points of similarity and divergence between the distal processes and associated structures of neurones adapted to respond to various stimuli. This information has in turn contributed to our knowledge of the structural features concerned with the primary receptor processes. In mechanoreceptors, excitation appears to be triggered by mechanical distortion of part of the distal process of the bipolar sensory cell, and in many arthropod receptors of this type, including campaniform sensilla, controlled deformation is conveyed to the neurone via specialized cuticular structures associated with the tip of the distal process. Functional interpretation of available fine structural information on sensory receptors must ultimately rely on electrophysiological

detection and analysis of the effect of appropriate mechanical stress applied to the sensillure, and in this respect the elegant studies of Thurm (1964, 1965) on cervical hair plate sensilla and head campaniform sensilla of the honey bee are especially ,~aluable. In each instance, Thurm describes the insertion of the approximately bilaterally symmetrical distal process of the sense cell onto a specialized region of cuticle--the 'joint membrane' and 'cap membrane' respectively in these two sensillum types--the last micron or two of the distal tip passing through a 'cap' of fibrous spongy material. In each case, the region surrounded by the cap contains a 'tubular body' comprising 50-100 parallel microtubules with an inner diameter of 90 ~ and a wall 30 A in thickness, possibly contributing to the dual rings of nine doublet microtubules observed in these sensilla at the level of the 'ciliary structure' (resembling a basal body) dividing the apical and basal portions of the distal sensory process. Thurm has suggested that the joint and cap membranes consist of the rubber-like protein resilin (Weis-Fogh, 1960; Andersen and Weis-Fogh, 1964) and constitute the region transmitting the cuticular deformations initiating excitation. With particular reference to hair plate sensilla, characterized by a fine moveable cuticular hair linked to the general cuticle via the joint

Fig. 44. A group of chordotonal sensilla sectioned through the basal region of the distal process--a level readily identified by the presence of rootlets (r) extending from the basal body. Profiles of sheath cells are seen at s, and of a sheath cell nucleus at n. • 18,000. Fig. 45. Unlike the narrow compact rootlets of campaniform sensilla of the haltere (Fig. 36), this structure (r) in the chordotonal sensillum is reticular in transverse section, incorporating numerous irregular cavities (*) which may include microtubules (short arrow). Microtubules also occur singly, in small groups and in conspicuous ribbons (long arrows) in the surrounding cytoplasm. • 110,000. Fig. 46. Longitudinal section of the rootlet of a haltere chordotonal sensillum. Arrowheads indicate the striation macroperiod lines, ca. 600 ,&, apart, separated by opaque and t~ansparent subperiods. "< 120,000. Fig. 47. A longitudinal section of a rootlet of a dorsal scapal plate campaniform sensillum. The macroperiod (arrowheads) is similar to that shown in the last figure. White arrows indicate the longitudinally oriented constituent filaments of the rootlet. These rootlets are irregularly cylindrical and the section grazes the central cavity (*). x 120,000. TISSUE Et CELL 1969 1 (3)

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480 membrane, Thurm has concluded, by monitoring the electrophysiological effects of directionally controlled hair displacement, that compression of the spongy cap beneath the joint membrane effects stimulation. The reduction by compression in the diameter of the nerve terminal produced by maximal bending of the hair was judged to amount to ca. 0"1/~, or a 15,% deviation from the resting position, while the great sensitivity of this receptor system is indicated by the finding that the threshhold for 100~.~ response may be as little as a 30 ~ (0-5~i;) change in the diameter of the sensory tip. As Thurm suggests, the structural features involved in stinmlus reaction should most reasonably be sought at the level of the neurone process affected by the perceived mechanical distortion that is, at the level of the tubular body. Both the cnticular specializations and the tubular body described by Thurm are readily identified in haltere campaniform sensilla, indeed in more striking form. In the simpler situation, occurring in basal plate sensilla, the relationship between sensory tip and cuticle in large measure resembles that of the campaniform units described by Thurm (1964), except that in place of a conical tip inserted via a concentric socket into the cap membrane, the distal extremity is here represented by a precisely oriented fan-shaped process ending just beneath a domed region of cuticle (corresponding to the 'cap membrane') within a layer of spongy cuticle corresponding to the 'cap' described in Thurm's electron micrographs. In scapal plate and Hick's papillae, the morphology of the neurone-cuticle link is more complex: the fan-shaped terminal is inserted beneath a bowed strip of cuticle attached to the adjoining cuticular ridges by electron-opaque hinges. In each instance, the dense extracellular sheath surrounding the distal tip is separated from the overlying cuticle by a narrow gap into which extends the spongy material of the neighboring cap. The cuticle overlying the basal sensilla is not noticeably demarcated from that lying between adjacent sensory endings, and has a finely fibrillar structure. Although Thurm suggests that the

SMITH, D.S. corresponding region in Apis scnsilla is composed of resilin, the optically amorphous characteristics of this protein (Andersen and Weis-Fogh, 1964) are striking and argue against this identification in basal plate haltere units. On the other hand, the homogeneous hinges of scapal plate and Hick's papillae mentioned above are sharply demarcated from adjoining cuticular structures and may well represent an elastic resilin coupling for transmission of mechanical stimuli, contrasting with the simpler basal plate cuticle which perhaps relies on distortion of the sensory tip transmitted directly via the cuticular dome. The tubular body described by Thurm in the hair plate sensillum comprises microtubules associated with an electron-opaque material, extending to the apex of the distal process as a roughly isodiametric structure. The corresponding region ofhaltere campaniform sensilla is more extensive, more regular and more complex, and in view of the possible importance of this region in transduction, these morphological features should be considered in some detail. Firstly, the terminal segment of the process dilates and flattens abruptly in the last two micra or so of its course into a narrow fan-shaped structure. Secondly, previous descriptions of the sensory tip have made no mention of the presence of a further component of the tubular body--fine (80 A) filaments, fitted more or less regularly into the array of microtubules and the dense material with which these become increasingly associated as their termination is approached. As has been mentioned earlier in this report, this dense material is arranged in transverse bars in the 'fiandle' of the fan but in the expanded terminal fuses to form a spongework into the interstices of which pass filaments extending from the proximal regions of the terminal segment. This picture is already complex enough, but as has been illustrated in Figs. 24(a) and 24(b) and 25, radially oriented clurnps of microtubules persist to form the outer 'ribs' of the terminal fan--one set alongside each face--providing the radially striated appearance of this part of the sensilTISSUE 8- CELL 1969 1 (3)

S T R U C T U R E OF H A L T E R E S E N S I L L A lure detected by Pflugstaedt in the light microscope. The extraordinary complexity of this region of the haltere campaniform sensilla re-emphasizes the problem of the structural basis of the transduction process. If the tubular body indeed plays a crucial part in this process, what are the features of this part of the sensory cell, so conspicuously devoid of mitochondria and other intracellular components, that permit it to respond to inecbanical distortion? We are familiar with the dual array of myofilaments in a muscle cell and with the evidence that rapid cyclic changes in the ionic environment (in this case, the concentration of Ca } ~) determines the biochemical and mechanical activity of the contractile system. In the sensory tip of campaniform sensilla we find an array of microtubules and filaments that approaches the regularity of the myofibril, and it is not inconceivable that, in reverse order to the sequence of events in muscle, deformation of the array may induce ionic changes involved in the transduction process. Pringle (1938b) considered the mechanical properties of cuticular elements of campaniform sensilla in terms of simple and instructive models. He pointed out that shearing stresses in the general cuticle can be resolved into compression and extension components, and showed how these directional deformations may be transferred to the nerve endings. He proposed that the sensilla are excited by stresses parallel with the fan-shaped sensory tip, and, drawing attention to the precise orientation of the ranks of haltere receptors, argued that since over a small area of cuticle the lines of compression and extension run approximately parallel, 'sensilla with parallel orientation will respond to the same type of shear force'. He further suggested (1938b, 1948), on the basis of studies on stress and orientation in tarsal and palp sensilla of the cockroach, together with a comparison between the directional features of campaniform and hair sensilla, that the orientation of the latter is always such that the compression component of shear is the adequate stimulus. According to the proposed model, this component inTISSUE 8- CELL 1969 1 (3)

481 creases the doming of the cap and, hence, supposedly stretches the underlying terminal. On the other hand, the demonstrated ineffectiveness of stretching as adequate stimulus in hair plate sensilla led Thurm to suggest that in both these and campaniform units, compression of the terminal is the crucial event in initiating the sensory response. As has been mentioned above, the campaniform sensilla described by Thurm are simpler than those of the haltere in possessing a cylindrical rather than a fan-shaped terminal, and while the insertion of the basal plate sensilla is comparable with that observed by Thurm, the cuticular structures associated with the terminal in Hick's and scapal plate units are considerably more complex. It may well be, however, that Thurm's hypothesis is of general application, and that the apparent discrepancy between this and Pringle's proposal is resolvable. In each of the haltere campaniform types described here, increased doming of the cap may not only elevate the sensory tip but simultaneously induce compression of the terminal micron or so, inserted into tile spongy cap (Figs. 16, 17, etc.) While this study is primarily concerned with the campaniform sensilla of the baltere, a less detailed study has been included on the chordotonal sensilla contained within the haltere base. In certain fine structural respects, these resemble the auditory chordotonal receptors of Locusta described by Gray (1960) but share other features with the neighboring campaniform units. As in many other sensory cells, the distal process of the chordotonal sensillum is divided into basal and apical regions, characterized respectively by microtubules and a ciliary rootlet. The apical region in the haltere units is approximately cylindrical and contains numerous microtubules, unlike the auditory sensillum which includes a cilium-like structure comprising nine doublets, or the sternal gland sensilla described by Stuart and Satir (1968) which incorporates a 9 + 0 grouping extending some distance from the basal body. As in other chordotonal organs, the apical tip ends some distance beneath the cuticle and is

482 inserted into a 'scolopale cap' beneath which the distal process is surrounded by a concentric sheathing sco[opale cell containing about 12 intracellular structures: in the haltore chordotonal receptor, many of the cytoplasmic microtubules are distributed in rows arranged preferentially around the ciliary rootlet and adjoining the plasma membrane. The rootlets of these cells are considerably wider than those of campaniform units of the haltere, and the former present an open reticulum in transverse section rather than the compact homologues of campaniform sensilla, but in each case the macroperiod of the rootlet is similar and divided into comparable electron-opaque and lucent subperiods. The extensive splitting of the rootlets described by Gray in auditory organs has not been noted in any of the sensilla of the Calliphora haltere. No clear picture of the functional significance of these differences in detail between various chordotonal sensilla has emerged, and it seems likely (Thurm, 1965) that these cells are triggered by compression of the sensory tip, as in the case of hair plate cells, in this instance transmitted via the conical scolopale cap distorted as a consequence of longitudinal stretching of the scolopale sheath. Although neither the course of the transduction process nor the site of generation of receptor potential is known in mechanoreceptors equipped with a ciliary derivative, Thurm (1965) suggests that these may occur in distinct regions, as in the vertebrate rod where the site of stimulus reception in the distal segment is separated from that of receptor potential generation, lying proximal to the ciliary structure. As in the analagous proximal (ellipsoid) region of the rod, the basal region of haltere chordotonal and campaniform sensilla is richly supplied with mitochondria. This feature, indeed, to varying degrees, seems to be a general property of sensory receptors in which the distal process is divided into two portions. Thurm points out that an oxygen-dependent receptor potential response seems to be a general feature of receptor excitability, and the observed distribution of mitochondria in

S M I T H , D.S. haltere sense cells may implicate the basal region of the distal process in this oxygenconsuming event. The non-nervous cells ensheathing the distal process of haltere sensory units deserve further mention. As in auditory sensilla (Gray, 1960), the apical part of a haltere chordotonal sensillum is ensheathed by a cell possessing several scolopale rods reinforced with microtubules, presumably providing a rigid framework for the enclosed ciliary derivative and, according to Thurm's proposal, inducing compression of the adjoining scolopMe cap and its enclosed sensory terminal when stretched longitudinally. Around the proximal region of the distal process, microtubules are present in close-packed arrays within a compact sheath (probably the 'Fasernzelle' of Pflugstaedt). The fibrous sheath cell occupying a similar position in an auditory sensillum encompasses three or more dendrites (Gray, 1960), while each haltere chordotonal unit possesses its own sheath, and moreover, the associated collagen fibrils described by Gray (1959) are absent. Campaniform sensilla lack well defined scolopale rods, and stimulation of the sensory tip is achieved directly by surface cuticular deformation, although at the level of the basal body and the origin of the ciliary rootlet at the junction of apical and basal regions the inner sheath cell is firmly linked to the sensory process by 'tongue and groove' interdigitations and by microtubulefortified desmosomes that resemble diminutive scolopale structures. However, the outer sheath cells of haltere campaniform sensilla possess other features of interest, notably the profuse coat of slender microvilli borne on their outer surface at the level of the basal and most of the apical regions of the distal process. The small particles situated on the cytoplasmic face of these microvilli resemble those described in several insect cells, and ,while the nature of these structures is not known, their distribution strongly supports the suggestion that they may play a part in transmembrane ion movements. The presence of numerous large mitochondria often dilating the microvilli of these sheath cells is likeTISSUE 8" CELL 1969 1 (3)

STRUCTURE

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483

SENSILLA

wise consistent with the possibility of active ion movements between the outer sheath and the extensive extracellular space between the sensory columns. It should be noted that proximally from the level of the fibrillar body the sensory process is separated from this outer sheath by an inner sheathing collar rich in microtubules but sparsely equipped with mitocbondria. While it is possible that ion regulation conducted by the outer sheath cell affects the immediate environment of the sensory process, it should also be noted that the cellular sheath does not extend to the terminal fan, which is surrounded only by an o p a q u e extracellular envelope, and it therefore seems unlikely that the sheath plays a part in any transduction process occurring

at the level o f the tubular body. It seems more probable that changes in the ionic environment of the basal portion may be related to receptor potential generation.

Acknowledgements This work was supported by Grants GB-5635 and GB-12117x from the National Science Foundation. The author gratefully acknowledges the help of Dr M. A. Message in preparing halteres for scanning microscopy, Mr L. Peters for his skillful operation of the scanning electron microscope, and Professor C. W. Oatley for generously providing research microscopic facilities in the Engineering Laboratory, Cambridge University.

References ANDERSEN, S. O. and WE~s-FOGH, T. 1964. Resilin. A rubber-like protein in arthropod cuticle. In Advances in Insect Physiology (J. W. L. Beament, J. E. Treherne and V. B. Wigglesworth, editors), Vol. 2, pp. 1-65. Academic Press, London and New York. ANDERSON, E. and HARVEY, E. R. 1966. Active transport by Cecropia midgut. 11. Fine structure of the midgut epithelium. J. Cell Biol., 31, 107-134. BERR1DGF,M. J. and OSCHMAN,J. L. 1969. A structural basis for fluid secretion by Malpighian tubules. Tissue & Cell, 1, 24~272. BRAUNS, A. 1939. Morphologische and physiologische Untersuchungen zum Halterenproblem unter besonderer Berticksichtigung brachypterer Arten. Zool. Jb. (Zool.), 59, 245-389. BUDDENBROCK, W. 1919. Die vermutliche L6sung der Halterenfrage. Pfliigers Arch. ges. Physiol., 175, 125-164. FRAENKEL, G. 1939. The function of the halteres of flies (Diptera). Proe. zool. Soc. Lond. A, 109, 69-78 FRAENKEL, G. and PRtN6LE, J. W. S. 1938. Halteres of flies as gyroscopic organs of equilibrium. Nature, Lond., 141, 909-920. GRAY, E. G. 1959. Electron microscopy of collagen-like connective tissue fibrils of an insect. Proc. R. Soc. B, 150, 233-239. GRAY, E. G. 1960. The fine structure of the insect ear. Pkil. Trans. R. Soc. Ser. B, 243, 75-94. GUPTA,B. L. and BERRIDGE,M. J. 1966. A coat of repeating subunits on the cytoplasmic surface of the plasma membrane in the rectal papillae of the blowfly, Catliphora erythrocephala (Meig.), studied in sittt by electron microscopy. J. Cell Biol., 29, 376-382. PFLUGSTAEDT,H. 1912. Die Halteren der Dipteren. Z. wiss. Zool., 100, 1-59. PRINGLE, J. W. S. 1938. Proprioception in insects, I1. The action of the campaniform sensilla on the legs. J. exp. Biol., 15, 114-131. PRINGLE, J. W. S. 1948. The gyroscopic mechanism of the halteres of Diptera. Phil, Trans. R. Soc. Set'. B, 233, 347-384. REYNOLDS,E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17, 208-212. SMITH, D. S., COM1"HER,K., JANNERS,M., LIPTON, C. and WITTLE, L. W. 1969. Cellular organization and ferritin uptake in the midgut epithelium of a moth, Ephestia kiihniella. J. Morph., 127, 41-72. SNODGRASS,R. E. 1935. Principles oflnsect Morphology, p. 521. McGraw-Hill, New York. STtJART, A. M. and SATIn, P. 1968. Morphological and functional aspects of an insect epidermal gland. J. Cell Biol., 36, 527-549. G TISSUE 8" CELL 1969 1 (3)

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THURM, U. 1964. Mechanoreceptors in the cuticle of the honey bee: fine structure and stimulus mechanism. Science, 145, 1063-1065. THURM, U. 1965. An insect mechanoreceptor. Part I: Fine structure and adequate stimulus. CoM Spring Harb. Syrup. quant. Biol., 30, 75-82. WEls-FoGH, T. 1960. A rubber-like protein in insect cuticle. J. exp. Biol., 37, 889-907.

TISSUE 8- CELL 1969 1 (3)