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Ultrastructural observations on the body wall of the leech, Batracobdella picta
TISSUE & CELL 1977 9 (1) 35-42 Published by Longman Group Ltd. Printed in Great Britain
SHERWIN
S. DESSER and IRIS WELLER
ULTRASTRUCTURAL OBSERVATIONS BODY WALL OF THE LEECH, BATRACOBDELLA PICTA
ON THE
ABSTRACT. A series of closely spaced annulations surround the surface of the body of Batracobdella picta. The epidermis is covered by a thin cuticle which is composed of several layers of orthogonally arranged, fibrous bundles. Numerous fine projections carpet the surface of the cuticle and appear to be derived from microvillar processes which extend through the cuticle from subjacent epithelial cells. Septate junctions occur between adjacent epithelial cells, and hemidesmosomes with associated tonofilaments appear to anchor the epithelium to the overlying cuticle and to the basal connective tissue. The epithelial cells contain abundant organelles including granular endoplasmic reticulum, mitochondria and Golgi complexes. The cytology of the body wall of B. pictu is compared with that of other annelids.
Introduction
tron microscopy for 2 days at room temperature, in a solution containing glutaraldehyde and osmium tetroxide buffered with sodium cacodylate (according to the method of Franke et al., 1969). They were then rinsed in 0.1 M sodium cacodylate buffer for 2 hr and dehydrated in a graded ethanol, ethanolfreon series, and subsequently placed in liquid freon TF before critical point drying through liquid carbon dioxide with a Denton critical point drying apparatus (Model DCP1). The leeches were then mounted on stubs and coated with approximately 100 8, of carbon and gold palladium and viewed in a Cambridge Stereoscan electron microscope. Three other young leeches were cut into three pieces and fixed for electron microscopy for 1 hr at 4°C in the above described solution. After post-osmication the tissues were dehydrated in a graded alcohol series, followed by propylene oxide, and embedded in a mixture of Epon-Araldite. The sections were stained in a saturated solution of uranyl acetate in methanol and in lead citrate. For ultrastructural localization of PAS-positive material, other sections were collected on gold grids and stained by the periodic acidthiosemicarbazide-silver proteinate (PATSC-Ag protein) method of Thiery (1967) using 30 min oxidation in 1% (w/v) periodic
considerable attention has been directed towards the ultrastructure and composition of the epidermis of earthworms and certain other annelids, little has been published on these features in leeches. To our knowledge the sole report on the ultrastructure of the body wall of leeches was that of Rutschke (1970) in which he briefly described the cuticles of seven species. In the present study the ultrastructure of the cuticle and epithelium of Butracobdellupicta (Verrill) is described and compared with that of other annelids. ALTHOUGH
Materials and Methods A specimen of Batracobdella picta with several young attached to its ventral surface was collected from Lake Sasajewan, Algonquin Park, Canada. Three of the young leeches which measured approximately 6 by 2 mm in a relaxed state, were removed from the brood leech and fixed for scanning elec--Department of Microbiology and Parasitology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1Al Received 18 October 1976. Revised 12 November 1976. 35
DESSER
36
acid, 1 hr in 1 y0 (w/v) semicarbazide, and 30 min in a 1 y0 (w/v) solution of strong silver proteinate (Protargol from Roboz Surgical Instrument Co., Washington, D.C.). A control sample was processed as above omitting the periodic acid oxidation. The sections were examined in a Zeiss EM 9A electron microscope. The photomicrograph (Fig. 4) was of a 1 p epoxy section stained in 0.5 ‘JOToluidine Blue buffered with 1% sodium borate. Results The following description is based mainly on observations made from the dorsal surface of B. picta, where the body wall appears as a series of closely spaced parallel ridges (segments), which surround the body and are separated from each other by deep grooves (Fig. 1). Each of the ridges possesses numerous infoldings, many of which completely traverse the ridge, giving it a striated appearance. Often a fine sulcus runs parallel to, and bisects each ridge (Fig. 2). Tiny projections can be discerned on the surface of the ridges when viewed at higher magnifications (Fig. 3). The outer surface of the epidermis consists of a cuticle approximately 1.5 p in thickness (Fig. 4). Beneath, and in contact with the cuticle is a single layer of large columnar epithelial cells, which at the lateral margins and in the groove between the epidermal ridges,
AND
WELLER
are considerably smaller than those beneath the crest of the ridges. Beneath the epithelial cells lie the circular and longitudinal body wall musculature. The external surface of the cuticle is carpeted by innumerable closely spaced, slender projections which measure approximately 320 rnp in length by 50 rnp in width at their base (Figs. 5, 6, 8). Each epicuticular projection is invested by a trilaminar membrane and possesses an electron-dense base and lighter distal region (Fig. 8). A fine filamentous material is associated with the projections which appear to be embedded in an homogeneous, granular band approximately 105 rnp wide. Beneath this granular band and comprising the bulk of the cuticle are a series of fibrous bundles arranged in layers, each of which is composed of parallel fibres and lying at approximately right angles to the layer above and below. In specimens stained with uranyl acetate and lead citrate the fibrous bundles are hardly discernible (Figs. 5, 8). In sections stained by the PA-TSC-Ag protein method, however, the cuticular bundles are densely stained and their fibrous nature is clearly revealed (Fig. 6). The cuticle contains approximately seven orthogonally arranged fibrous bundles, the outer and innermost of considerably smaller dimensions than the central ones. When stained by the above method, the granular band upon which the epicuticular projections sit, also stains intensely and is readily dif-
Abbreviations used in figures Bg, 81, C, Cf, G,
granular band of cuticle basal lamina cuticle collagen fibril Golgi complex
Dh. hemidesmosome mitochondria M, Mp, microvillar process Pe, epicuticular projections PC, pigment cell
Fig. I. Scanning electron micrograph illustrating the dorso-lateral surface of an immature Batracobdella picta, with its characteristic ridges or annulations. Numerous transverse infoldings in the ridges give them a striated appearance. x 63. Fig. 2. A fine sulcus frequently epidermal ridges. x 310.
runs parallel
Fig. 3. Highly magnified view of the surface spaced epicuticular projections. x 9400.
to and bisects
the highly
of the cuticle, illustrating
infolded
the closely
38
DESSER
ferentiated from a lesser granular matrix below, which is interspersed amongst the fibrous bundles (Fig. 6). Numerous dome-shaped, electron-dense structures project from the distal cytoplasm of the epithelial cells into the basal zone of the cuticle (Fig. 5). These structures resemble hemidesmosomes and presumably function to attach the epithelial cells to the cuticle. Tonofilaments appear to originate from electron-dense plaques lining the inner leaflet
AND
WELLER
of the plasmalemma of the epithelial cells, and extend backwards into their cytoplasm (Fig. 7). In intersegmental areas where the epidermal cells are narrow, the tonoftlament bundles often extend across the cell and insert into basal hemidesmosomes which anchor the epithelial cells to their connective tissue substrate (Fig. 9). In the region of the hemidesmosomes, electron-dense plaques approximately 50 rnp thick, lie beneath the plasmalemma of the
Fig. 4. Photomicrograph of a longitudinal section through the dorsal body wall of a contracted specimen, illustrating the outer cuticular layer and subjacent, darkly stained columnar, epithelial cells. Transected circular muscle cells (arrows) lie beneath the epithelium within each of the ridges. Longitudinal muscle cells (asterisk) run along the base of the ridges. x 640. Fig. 5. Electron micrograph of a longitudinal section through the crest of a ridge illustrating the general architecture of the epidermis. The epicuticular projections (Pe) are embedded in an homogeneous, granular band (Bg) which forms the outer surface of the cuticle (C). The cuticle is composed of several orthogonally arranged bundles of lightly stained material. Several dense, dome-shaped hemidesmosomes (Dh) are situated along the epithelial cell-cuticle interface. A microvillar process (Mp) extends from an epithelial cell into the cuticle. The epithelial cells possess a prominent nucleus, granular endoplasmic reticulum and free ribosomes. Between the nucleus and the apical margin of the cell are numerous mitochondria (M) and Golgi complexes (G). A homogeneous, granular basal lamina (Bl) lies beneath the epithelial cells and is continuous with a thin layer of connective tissue containing numerous collagenous fibrils. Two basal hemidesmosomes (arrows) can be seen in the lower right of the figure, attaching the epithelial cells to their basal connective tissue substrate. Portions of two pigment cells (PC) lie beneath the basal lamina in the connective tissue. x 29,000. Fig. 6. Portion of the cuticle and subjacent epithelial cell in a specimen stained by the PA-TX-Ag proteinate method. The structure of the orthogonally arranged fibrous bundles in the cuticle is clearly apparent. The bundles are embedded in a less dense, granular matrix. Numerous beta-glycogen particles are randomly scattered in the cytoplasm of the epithelial cell. Note the filamentous material (asterisk) associated with the epicuticular projections. x 71,200. Fig. 7. Three adjacent hemidesmosomes at the epithelial cell-cuticle interface. Numerous tonofilaments extend from a narrow, dense plaque on the inner leaflet of the plasmalemma, into the cytoplasm of the epithelial cell. Fibrous material (arrows) extends from the outer leaflet of the plasma membrane for a short distance into the cuticle. x 133,000. Fig. 8. A microvillar process (Mp) extending from the apical cytoplasm of an epithelial cell through the cuticle and terminating in a ‘bowling pin’-like projection. The epicuticular projections are invested by membrane and their bases are embedded in the granular band (Bg) of the cuticle. Note the dense material (arrows) associated with the microvillar process and apparently with adjacent epicuticular projections. x 57,000. Fig. 9. Portion of a narrow epithehal cell from the region of the groove between the body wall ridges. Bundles of tonofilaments extend from the apical to the basal hemidesmosomes (arrows). Electron-dense plaques lie on either side of the basal plasmalemma. Fibrous material (asterisk) extends from the modified basal lamina into the connective tissue. Banded collagen fibrils (Cf) occur in the latter region. Circular muscle can be seen beneath the connective tissue. x 62,000.
BODY
WALL
41
OF LEECH
epithelial cells (Fig. 9). Fibrous material radiates from these plaques into the collagenous connective tissue. In some cases tonofilaments from the surface hemidesmosomes terminate in the junctions at the lateral margins of the cells. Irregularly spaced microvillar processes project from the apical region of the epithelial cells into the cuticle (Fig. 5). These microvilli often appear to branch beneath the outer granular layer of the cuticle and give rise to several epicuticular projections; some of the microvilli terminate in peculiar ‘bowling-pin’like structures (Fig. 8). Interposed between the basal lamina of the epithelial cells and the body wall musculature is a narrow band of connective tissue. The latter contains numerous orthogonally arranged fibrils approximately 100 8, in diameter, which exhibit a faint cross-banding (Fig. 9). Scattered cells containing prominent electron-dense inclusions lie in the connective tissue (Fig. 5). These are presumably pigment cells. The epithelial cells of the epidermis are united by well-developed junctional complexes. At the surface of the epithelial cells and extending backwards for approximately 1 CL,the junctions are clearly of a septate nature (Fig. 5). Deeper in the cells the septa are absent and the plasma membranes of adjacent cells assume a highly convoluted appearance. The epithelial cells characteristically possess large, irregularly oval nuclei with prominent nucleoli (Fig. 5). The inner membrane of the nuclear envelope is often lined by a thin band of chromatin. The cytoplasm contains many free ribosomes and a welldeveloped granular endoplasmic reticulum. Elaborate Golgi complexes with their attendant vesicles generally occur in the area between the nucleus and the apical pole of the cell (Fig. 5). Multivesicular bodies were frequently observed between the Golgi complexes and the cell-cuticle interface. Numerous mitochondria are also concentrated primarily in the apical regions of the epithelial cells. Sections stained by the PA-TSC-Ag protein method, clearly revealed the presence of beta-glycogen particles, randomly scattered in the cytoplasm of the epithelial cells (Fig. 6). Occasionally cells containing two or more
cilia which extended through and beyond the cuticle were observed. Discussion
The ultrastructure of the cuticle and epithelium of B. picta is similar to that of the leech species described by Rutschke (1970). The latter author differed however in the interpretation of his material, most notably in his description of ‘air spaces’ and ‘canaliculi’ in the cuticle. These discrepancies may be due in part to differences in the fixation, and particularly to the staining of our material by Thiery’s method (1969), which clearly revealed the structure of the fibrous bands in the cuticle. The filamentous material associated with the epicuticular projections of B. p&a and other annelids resembles the mucopolysaccharide glycocalyx generally seen on the microvilli of intestinal epithelium. Richards (1974) suggested that the epicuticular projections in annelids may serve to ‘trap and mechanically stabilize the acid mucopolysaccharide coat which lies as a fine fibrous blanket around the worms’. The epicuticular projections appear to be a constant feature among the annelids. The origin of these projections has been a source of dispute as they are invested by membrane and sit upon a granular band on the outer surface of the cuticle, separated by more than 1 rnp from the underlying epithelium. Our observations support the view that the epicutitular projections originate from the microvillar processes of the epithelial cells which appear to branch in the distal cuticle, a situation similar to that described by Richards (1974) for several lumbricid species. In B. pictu, however, there are far fewer microvillar processes than in the lumbricids and they are insufficient to account for all the epicuticular projections. It has been suggested that after the formation of the epicuticular projections in annelid cuticles, the epithelial microvillar processes may either withdraw or degenerate (Hess and Menzel, 1967; Krall, 1968; Potswald, 1971). Ciliated sensory cells similar to those observed in this study were described from the epidermis of Enchytraeus by Richards (1974), and are probably a common feature among annelids. The well developed secretory apparatus in
42
DESSER
the epithelial cells of B. pictu suggest that they are responsible for the production and maintenance of the cuticle. Morphological similarity between the fibrous bundles in the cuticle of B. pictu and related worms (Coggeshall, 1966; Burke, 1974, Richards, 1974), coupled with the PAS positive reaction of the orthogonally arranged bundles suggest that the latter may be collagenous as has been demonstrated in other annelids (Gross, 1963; Fujimoto and Adams, 1964). In their recent radioautographic study, Burke and Ross (1975) revealed that the columnar epithelial cells of two species of oligochaetes were responsible for the synthesis of the overlying, collagenous cuticle. The epithelial cells were also implicated in the synthesis of a second type of collagen for the underlying basal lamina. Burke and Ross indicated, however, that the muscle cells might also be involved in the synthesis of collagenous material for the connective tissue layer. The sequence of deposition of the orthogonally arranged collagenous bands in anne-
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lid cuticle is presently not understood. Knowledge of this process must await a thorough developmental study. The intricate system of apical and basal hemidesmosomes and intervening tonofilaments probably serves a cytoskeletal function, as the epidermis must undergo considerable stress when the leeches undergo contraction and extension. A similar arrangement has been described in Lumbricus terrestris by Coggeshall (1966), Enchytraeus sp. by Richards (1974), and in the parasitic nematode Syphacia obveluta (see Dick and Wright, 1973). Thus the results of this study clearly reveal that the body wall of B. picta is in most respects similar to that of other species of annelids. Acknowledgements We are grateful to the Ministry of Natural Resources for the use of their facilities at the Wildlife Research Station, Algonquin Park, Ontario. This work was supported by NRC grant 6965.
References
BURKE, J. M. 1974. An ultrastructural analysis of the cuticle, epidermis and esophageal epithelium of Eisenia .foefida (Oligochaeta). J. Morph., 142, 301-320. BURKE, J. M. and Ross, R. 1975. A radioautographic study of collagen synthesis by earthworm epidermis. Tissue & Cell, 7, 631-650. COGGESHALL,R. E. 1966. Fine structural analysis of the epidermis of the earthworm, Lumbricus terrestris L. J. Cell Biol., 28, 95-108. DICK, T. A. and WRIGHT, K. A. 1973. The ultrastructure of the cuticle of the nematode Syphacio obvelatn (Rudolphi, 1802). I. The body cuticle of the larvae, males and females, and observations on its development. Can. J. Zool., 51, 187-196. FRANKE, W. W., KRIEN, S. and BROWN, R. M., Jr. 1969. Simultaneous glutaraldehyde-osmium tetroxide fixation with post-osmication-an improved fixation procedure for electron microscopy of plant and animal cells. Histochemie, 19, 162-164. FUJIMOTO,J. and ADAMS, E. 1964. Intraspecies composition differences in collagen from the cuticle and body of Ascaris and Lumbricus. Biochem. biophys. Res. Commun., II, 437-442. GROSS, J. 1963. Comparative biochemistry of collagen. In Comparative Biochemistry (eds. M. Florkin and H. Mason), Vol. 5, pp. 307-346. Academic Press, New York. HESS, R. and MENZEL, D. 1967. The fine structure of the epicuticular particles of Enchytraeus,fragmentosus. J. Ultrastruct. Res., 19, 487-497. KRALL, J. 1968. The cuticle and epidermal cells of Dero obtusa (Family Naididae). J. Ultrostruct. Res.. 25, 84-93. POTSWALD, H. 1971. A fine structural analysis of the epidermis and cuticle of the oligochaete Aeolosoma bengelense Stephenson. J. Morph., 135, 185-212. RICHARDS, S. K. 1974. The ultrastructure of the cuticle of some British lumbricids (Annelida). J. Zoo., Land.. 172, 303-3 16. RUYSCHKE, E. 1970. Zur Substruktur der Cuticula der Egel (Hirudinea). Z. Morph. iikol. Tirre, 67, 97-105. THIERY, J. P. 1967. Mise en evidence des polysaccharides sur coupes fin en microscopic &ctronique. J. Microscopic., 6, 987-1018.
SHERWIN
S. DESSER and IRIS WELLER
ULTRASTRUCTURAL OBSERVATIONS BODY WALL OF THE LEECH, BATRACOBDELLA PICTA
ON THE
ABSTRACT. A series of closely spaced annulations surround the surface of the body of Batracobdella picta. The epidermis is covered by a thin cuticle which is composed of several layers of orthogonally arranged, fibrous bundles. Numerous fine projections carpet the surface of the cuticle and appear to be derived from microvillar processes which extend through the cuticle from subjacent epithelial cells. Septate junctions occur between adjacent epithelial cells, and hemidesmosomes with associated tonofilaments appear to anchor the epithelium to the overlying cuticle and to the basal connective tissue. The epithelial cells contain abundant organelles including granular endoplasmic reticulum, mitochondria and Golgi complexes. The cytology of the body wall of B. pictu is compared with that of other annelids.
Introduction
tron microscopy for 2 days at room temperature, in a solution containing glutaraldehyde and osmium tetroxide buffered with sodium cacodylate (according to the method of Franke et al., 1969). They were then rinsed in 0.1 M sodium cacodylate buffer for 2 hr and dehydrated in a graded ethanol, ethanolfreon series, and subsequently placed in liquid freon TF before critical point drying through liquid carbon dioxide with a Denton critical point drying apparatus (Model DCP1). The leeches were then mounted on stubs and coated with approximately 100 8, of carbon and gold palladium and viewed in a Cambridge Stereoscan electron microscope. Three other young leeches were cut into three pieces and fixed for electron microscopy for 1 hr at 4°C in the above described solution. After post-osmication the tissues were dehydrated in a graded alcohol series, followed by propylene oxide, and embedded in a mixture of Epon-Araldite. The sections were stained in a saturated solution of uranyl acetate in methanol and in lead citrate. For ultrastructural localization of PAS-positive material, other sections were collected on gold grids and stained by the periodic acidthiosemicarbazide-silver proteinate (PATSC-Ag protein) method of Thiery (1967) using 30 min oxidation in 1% (w/v) periodic
considerable attention has been directed towards the ultrastructure and composition of the epidermis of earthworms and certain other annelids, little has been published on these features in leeches. To our knowledge the sole report on the ultrastructure of the body wall of leeches was that of Rutschke (1970) in which he briefly described the cuticles of seven species. In the present study the ultrastructure of the cuticle and epithelium of Butracobdellupicta (Verrill) is described and compared with that of other annelids. ALTHOUGH
Materials and Methods A specimen of Batracobdella picta with several young attached to its ventral surface was collected from Lake Sasajewan, Algonquin Park, Canada. Three of the young leeches which measured approximately 6 by 2 mm in a relaxed state, were removed from the brood leech and fixed for scanning elec--Department of Microbiology and Parasitology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1Al Received 18 October 1976. Revised 12 November 1976. 35
DESSER
36
acid, 1 hr in 1 y0 (w/v) semicarbazide, and 30 min in a 1 y0 (w/v) solution of strong silver proteinate (Protargol from Roboz Surgical Instrument Co., Washington, D.C.). A control sample was processed as above omitting the periodic acid oxidation. The sections were examined in a Zeiss EM 9A electron microscope. The photomicrograph (Fig. 4) was of a 1 p epoxy section stained in 0.5 ‘JOToluidine Blue buffered with 1% sodium borate. Results The following description is based mainly on observations made from the dorsal surface of B. picta, where the body wall appears as a series of closely spaced parallel ridges (segments), which surround the body and are separated from each other by deep grooves (Fig. 1). Each of the ridges possesses numerous infoldings, many of which completely traverse the ridge, giving it a striated appearance. Often a fine sulcus runs parallel to, and bisects each ridge (Fig. 2). Tiny projections can be discerned on the surface of the ridges when viewed at higher magnifications (Fig. 3). The outer surface of the epidermis consists of a cuticle approximately 1.5 p in thickness (Fig. 4). Beneath, and in contact with the cuticle is a single layer of large columnar epithelial cells, which at the lateral margins and in the groove between the epidermal ridges,
AND
WELLER
are considerably smaller than those beneath the crest of the ridges. Beneath the epithelial cells lie the circular and longitudinal body wall musculature. The external surface of the cuticle is carpeted by innumerable closely spaced, slender projections which measure approximately 320 rnp in length by 50 rnp in width at their base (Figs. 5, 6, 8). Each epicuticular projection is invested by a trilaminar membrane and possesses an electron-dense base and lighter distal region (Fig. 8). A fine filamentous material is associated with the projections which appear to be embedded in an homogeneous, granular band approximately 105 rnp wide. Beneath this granular band and comprising the bulk of the cuticle are a series of fibrous bundles arranged in layers, each of which is composed of parallel fibres and lying at approximately right angles to the layer above and below. In specimens stained with uranyl acetate and lead citrate the fibrous bundles are hardly discernible (Figs. 5, 8). In sections stained by the PA-TSC-Ag protein method, however, the cuticular bundles are densely stained and their fibrous nature is clearly revealed (Fig. 6). The cuticle contains approximately seven orthogonally arranged fibrous bundles, the outer and innermost of considerably smaller dimensions than the central ones. When stained by the above method, the granular band upon which the epicuticular projections sit, also stains intensely and is readily dif-
Abbreviations used in figures Bg, 81, C, Cf, G,
granular band of cuticle basal lamina cuticle collagen fibril Golgi complex
Dh. hemidesmosome mitochondria M, Mp, microvillar process Pe, epicuticular projections PC, pigment cell
Fig. I. Scanning electron micrograph illustrating the dorso-lateral surface of an immature Batracobdella picta, with its characteristic ridges or annulations. Numerous transverse infoldings in the ridges give them a striated appearance. x 63. Fig. 2. A fine sulcus frequently epidermal ridges. x 310.
runs parallel
Fig. 3. Highly magnified view of the surface spaced epicuticular projections. x 9400.
to and bisects
the highly
of the cuticle, illustrating
infolded
the closely
38
DESSER
ferentiated from a lesser granular matrix below, which is interspersed amongst the fibrous bundles (Fig. 6). Numerous dome-shaped, electron-dense structures project from the distal cytoplasm of the epithelial cells into the basal zone of the cuticle (Fig. 5). These structures resemble hemidesmosomes and presumably function to attach the epithelial cells to the cuticle. Tonofilaments appear to originate from electron-dense plaques lining the inner leaflet
AND
WELLER
of the plasmalemma of the epithelial cells, and extend backwards into their cytoplasm (Fig. 7). In intersegmental areas where the epidermal cells are narrow, the tonoftlament bundles often extend across the cell and insert into basal hemidesmosomes which anchor the epithelial cells to their connective tissue substrate (Fig. 9). In the region of the hemidesmosomes, electron-dense plaques approximately 50 rnp thick, lie beneath the plasmalemma of the
Fig. 4. Photomicrograph of a longitudinal section through the dorsal body wall of a contracted specimen, illustrating the outer cuticular layer and subjacent, darkly stained columnar, epithelial cells. Transected circular muscle cells (arrows) lie beneath the epithelium within each of the ridges. Longitudinal muscle cells (asterisk) run along the base of the ridges. x 640. Fig. 5. Electron micrograph of a longitudinal section through the crest of a ridge illustrating the general architecture of the epidermis. The epicuticular projections (Pe) are embedded in an homogeneous, granular band (Bg) which forms the outer surface of the cuticle (C). The cuticle is composed of several orthogonally arranged bundles of lightly stained material. Several dense, dome-shaped hemidesmosomes (Dh) are situated along the epithelial cell-cuticle interface. A microvillar process (Mp) extends from an epithelial cell into the cuticle. The epithelial cells possess a prominent nucleus, granular endoplasmic reticulum and free ribosomes. Between the nucleus and the apical margin of the cell are numerous mitochondria (M) and Golgi complexes (G). A homogeneous, granular basal lamina (Bl) lies beneath the epithelial cells and is continuous with a thin layer of connective tissue containing numerous collagenous fibrils. Two basal hemidesmosomes (arrows) can be seen in the lower right of the figure, attaching the epithelial cells to their basal connective tissue substrate. Portions of two pigment cells (PC) lie beneath the basal lamina in the connective tissue. x 29,000. Fig. 6. Portion of the cuticle and subjacent epithelial cell in a specimen stained by the PA-TX-Ag proteinate method. The structure of the orthogonally arranged fibrous bundles in the cuticle is clearly apparent. The bundles are embedded in a less dense, granular matrix. Numerous beta-glycogen particles are randomly scattered in the cytoplasm of the epithelial cell. Note the filamentous material (asterisk) associated with the epicuticular projections. x 71,200. Fig. 7. Three adjacent hemidesmosomes at the epithelial cell-cuticle interface. Numerous tonofilaments extend from a narrow, dense plaque on the inner leaflet of the plasmalemma, into the cytoplasm of the epithelial cell. Fibrous material (arrows) extends from the outer leaflet of the plasma membrane for a short distance into the cuticle. x 133,000. Fig. 8. A microvillar process (Mp) extending from the apical cytoplasm of an epithelial cell through the cuticle and terminating in a ‘bowling pin’-like projection. The epicuticular projections are invested by membrane and their bases are embedded in the granular band (Bg) of the cuticle. Note the dense material (arrows) associated with the microvillar process and apparently with adjacent epicuticular projections. x 57,000. Fig. 9. Portion of a narrow epithehal cell from the region of the groove between the body wall ridges. Bundles of tonofilaments extend from the apical to the basal hemidesmosomes (arrows). Electron-dense plaques lie on either side of the basal plasmalemma. Fibrous material (asterisk) extends from the modified basal lamina into the connective tissue. Banded collagen fibrils (Cf) occur in the latter region. Circular muscle can be seen beneath the connective tissue. x 62,000.
BODY
WALL
41
OF LEECH
epithelial cells (Fig. 9). Fibrous material radiates from these plaques into the collagenous connective tissue. In some cases tonofilaments from the surface hemidesmosomes terminate in the junctions at the lateral margins of the cells. Irregularly spaced microvillar processes project from the apical region of the epithelial cells into the cuticle (Fig. 5). These microvilli often appear to branch beneath the outer granular layer of the cuticle and give rise to several epicuticular projections; some of the microvilli terminate in peculiar ‘bowling-pin’like structures (Fig. 8). Interposed between the basal lamina of the epithelial cells and the body wall musculature is a narrow band of connective tissue. The latter contains numerous orthogonally arranged fibrils approximately 100 8, in diameter, which exhibit a faint cross-banding (Fig. 9). Scattered cells containing prominent electron-dense inclusions lie in the connective tissue (Fig. 5). These are presumably pigment cells. The epithelial cells of the epidermis are united by well-developed junctional complexes. At the surface of the epithelial cells and extending backwards for approximately 1 CL,the junctions are clearly of a septate nature (Fig. 5). Deeper in the cells the septa are absent and the plasma membranes of adjacent cells assume a highly convoluted appearance. The epithelial cells characteristically possess large, irregularly oval nuclei with prominent nucleoli (Fig. 5). The inner membrane of the nuclear envelope is often lined by a thin band of chromatin. The cytoplasm contains many free ribosomes and a welldeveloped granular endoplasmic reticulum. Elaborate Golgi complexes with their attendant vesicles generally occur in the area between the nucleus and the apical pole of the cell (Fig. 5). Multivesicular bodies were frequently observed between the Golgi complexes and the cell-cuticle interface. Numerous mitochondria are also concentrated primarily in the apical regions of the epithelial cells. Sections stained by the PA-TSC-Ag protein method, clearly revealed the presence of beta-glycogen particles, randomly scattered in the cytoplasm of the epithelial cells (Fig. 6). Occasionally cells containing two or more
cilia which extended through and beyond the cuticle were observed. Discussion
The ultrastructure of the cuticle and epithelium of B. picta is similar to that of the leech species described by Rutschke (1970). The latter author differed however in the interpretation of his material, most notably in his description of ‘air spaces’ and ‘canaliculi’ in the cuticle. These discrepancies may be due in part to differences in the fixation, and particularly to the staining of our material by Thiery’s method (1969), which clearly revealed the structure of the fibrous bands in the cuticle. The filamentous material associated with the epicuticular projections of B. p&a and other annelids resembles the mucopolysaccharide glycocalyx generally seen on the microvilli of intestinal epithelium. Richards (1974) suggested that the epicuticular projections in annelids may serve to ‘trap and mechanically stabilize the acid mucopolysaccharide coat which lies as a fine fibrous blanket around the worms’. The epicuticular projections appear to be a constant feature among the annelids. The origin of these projections has been a source of dispute as they are invested by membrane and sit upon a granular band on the outer surface of the cuticle, separated by more than 1 rnp from the underlying epithelium. Our observations support the view that the epicutitular projections originate from the microvillar processes of the epithelial cells which appear to branch in the distal cuticle, a situation similar to that described by Richards (1974) for several lumbricid species. In B. pictu, however, there are far fewer microvillar processes than in the lumbricids and they are insufficient to account for all the epicuticular projections. It has been suggested that after the formation of the epicuticular projections in annelid cuticles, the epithelial microvillar processes may either withdraw or degenerate (Hess and Menzel, 1967; Krall, 1968; Potswald, 1971). Ciliated sensory cells similar to those observed in this study were described from the epidermis of Enchytraeus by Richards (1974), and are probably a common feature among annelids. The well developed secretory apparatus in
42
DESSER
the epithelial cells of B. pictu suggest that they are responsible for the production and maintenance of the cuticle. Morphological similarity between the fibrous bundles in the cuticle of B. pictu and related worms (Coggeshall, 1966; Burke, 1974, Richards, 1974), coupled with the PAS positive reaction of the orthogonally arranged bundles suggest that the latter may be collagenous as has been demonstrated in other annelids (Gross, 1963; Fujimoto and Adams, 1964). In their recent radioautographic study, Burke and Ross (1975) revealed that the columnar epithelial cells of two species of oligochaetes were responsible for the synthesis of the overlying, collagenous cuticle. The epithelial cells were also implicated in the synthesis of a second type of collagen for the underlying basal lamina. Burke and Ross indicated, however, that the muscle cells might also be involved in the synthesis of collagenous material for the connective tissue layer. The sequence of deposition of the orthogonally arranged collagenous bands in anne-
AND
WELLER
lid cuticle is presently not understood. Knowledge of this process must await a thorough developmental study. The intricate system of apical and basal hemidesmosomes and intervening tonofilaments probably serves a cytoskeletal function, as the epidermis must undergo considerable stress when the leeches undergo contraction and extension. A similar arrangement has been described in Lumbricus terrestris by Coggeshall (1966), Enchytraeus sp. by Richards (1974), and in the parasitic nematode Syphacia obveluta (see Dick and Wright, 1973). Thus the results of this study clearly reveal that the body wall of B. picta is in most respects similar to that of other species of annelids. Acknowledgements We are grateful to the Ministry of Natural Resources for the use of their facilities at the Wildlife Research Station, Algonquin Park, Ontario. This work was supported by NRC grant 6965.
References
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