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Schistosoma mansoni: Description of the head gland of cercariae and schistosomules at the ultrastructural level
EXPERIMENTAL
PARASITOLOGY
39,
444-459
( 19%)
Schistosoma mansoni: Description of the Head of Cercariae and Schistosomules at the Ultrastructural Level H.
CHARLES Naval
Medical
Research (Accepted
Institute,
DORSEY
Bethesda,
for publication
Gland
Maryland 26 July
20014,
U. S. A.
1975)
DORSEY, CHARLES H. 1976. Schistosoma munsoni: Description of the head gland of cercariae and schistosomules at the ultrastructural level. Experimental Parasitology 39, 444-459. The head gland of the cercaria of Schistosoma mansoni appears to be a relatively large unicellular entity consisting of a fundus tapering into a system of multiple ducts that opens into the integument at the anterior end of the oral sucker. The fundus is located in the posteriodorsal area of the oral sucker and contains most of the secretory granules. The ducts are usually narrow and devoid of secretory granules especially near their integumental junctions in the cercaria. In schistosomules of S. munsoni the fundus is reduced and the ducts are distended as secretory granules move en masse into the integument during the penetration of cercariae into their host where they may provide material for repair of the integument of the oral sucker damaged during penetration. The head gland has a strong affinity for 1~1x01 fast blue and acid hematin stains which suggests the presence of phospholipids. INDEX DESCRIPTORS: Schistosoma mansoni; Cercaria; Schistosomule; Secretory granules; Secretory gland; Head gland; Penetration gland; Subtegumental gland cell; Secretory gland cell; Ultrastructure; Microscopy, electron.
For many years, the head gland has been an elusive organ first observed in the oral sucker of Schistosoma japonicum by Miyagawa ( Cort 1919), who described it as a massive organ located in the dorsal area of the oral sucker. Subsequently, Ebrahimzadeh ( 1970)) Ebrahimzadeh and Kraft ( 1971)) and Morris (1971) studied the head gland of cercariae of S. manson%. Ebrahimzadeh ( 1970) and Ebrahimzadeh and Kraft (1971) viewed the head gland as an irregularly shaped multi-nucleated gland complex with short ducts. Morris (1971) identified the head gland as “Type C penetration gland cells,” stating that their secretory granules are released through ducts into the tegument at the anterior end of the oral sucker. 444 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
No one (to the knowledge of the author) has described the ultrastructure of the head gland in schistosomulesof S. munsoni. This paper is concerned with the head gland as it appears in both cercariae and schistosomules of S. mansoni utilizing the light and electron microscope. MATERIALS
AND
METHODS
Unemerged and Light microscopy. emerged cercariae from laboratory reared Puerto Rican Biomphalaria glubrata were fixed 72 hr in Carnoy’s solution (Lillie 1965), or 24 hr in Karnovsky’s ( 1965) glutaraldehyde-paraformaldehyde solution. The cercariae were paraffi embedded and cut at 10 pm on a rotary microtome. Some of the tissues were stained with Luxol fast
STEIJCIUEE
OF HEAD GLAND IN SCHISTOSOME LARVAE
blue and counterstained with periodic acid-Schiff ( Shanklin and Nassar 1959). Others were stained with acid hematin ( Baker 1946). Electron microscopy. Unemerged and emerged cercariae were fixed 24 hr in Karnovsky’s solution as above and postfixed in 1% osmic acid made in 0.1 M sodium cacodylate buffered to pH 7.3 with potassium dichromate. Others were fixed 1 hr in the osmic acid solution only. Some emerged cercariae were placed in a protein medium (egg white of a chicken) for 2 hr before 2%hr fixation in Karnovsky’s solution (1965) and postosmification. Schistosomules were obtained after exposing mouse ears to cercariae, when the mice were sacrificed at intervals of 15 min, 30 min, and 1 hr. The schistosomules were either teased from ears floated in Hanks balanced salt solution and placed into the fixative, or the ears were cut into 2-mm squares and placed directly into the fixatives allotted to the cercariae above. The tissue was placed in 1% uranyl acetate (Terzakis 1968) for 1 hr, dehydrated, and embedded in Luft’s (1961) epoxy resin. Approximately l-pm thick sections were cut on a Porter Blum MT-2 microtome and stained with alkaline toluidine blue for orientation of tissue after which sections with an inference of pale gold to silver were cut, placed on one-hole grids, stained with lead citrate (Reynolds 1963) and viewed in a Siemen Elmiskop IA microscope with an accelerating voltage of 80 kV. RESULTS
Light Microscopy (1) Alkaline toluidine blue stain. The fundus of the head gland is easily recognized in the cone of the musculature of the oral sucker with this dye because the gland usually exhibits a different coloration because of the polychromatic staining properties of toluidine blue (Fig. 1). Nuclei are
445
FIG. 1. Light micrograph of the oral end of a cercaria stained with toluidine blue. Notice a part of the fundus of the head gland (g) surrounded by four nuclei (arrow heads). Muscular cone of the oral sucker (m). Ducts of the acetabular glands (d). One of the two large cells located in the oral sucker (asterisks). X1125. FIG. 2. Light micrographs of cercariae stained with Baker’s acid hematin. The head glands (arrows) stain an intense black. X950.
always seen surrounding the fundus of the head gland, but not a part of it, as indicated by electron microscopy. (2) Luxol fast blue-periodic ac&Schifl stain. In the aldehyde-fixed specimens, the head glands stained a distinct blue with Luxol fast blue contrasting sharply with the ducts of the postacetabular glands which stained magenta with the periodic acid-Schiff solution. The head glands of cercariae fixed in Carnoy’s solution did not stain with Luxol fast blue, but the ducts of the postacetabular glands remained Schiff positive. This staining procedure gives a clear picture of the head glands location which is dorsal to the ducts of the acetabular glands. (3) Acid hem&in. The head glands of aldehyde-fixed cercariae stained black with
16
CHARLES
H.
DORSEY
FIG. 3. Electron micrograph of a part of an unemerged cercaria. Notice the head gland which contains a nucleus (N) and secretory granules of different stages of development (asterisks) in its cytoplasm. Presumed granular ribosomal material (R). Mitochondria (M). Cytoplasmic extension of head gland ( arrow). X 18,800,
STRUCXJRE
OF
HEAD
GLAND
IN
SCHISTOSOME
LARVAE
447
FIG. 4. Electron micrograph of a cross section through the distal end of the oral sucker of a free swimming cercaria. Notice the dorsal position of the head gland (large arrow) with respect to the ducts of the acetabular glands (asterisks) and the digestive tract (D). Secretory granules contain particulate material of various electron densities typical of tissue fixed in osmic acid only (small arrows), Nerve processes ( N) . Cells of the muscular calyx ( M ). x 17,500.
Baker’s acid hematin (Fig. 2). The black gland contrasted strongly with the light brown color of the rest of the cercaria. The
head gland itself did not stain after fixation in Carnoy’s, but nuclei located in its immediate vicinity stained black.
448
CHARLES
H.
DORSEY
FIG. 5. Electron micrograph of part of a cercaria on cross section showing a portion of the head gland (G) draped around a large cell (C). Notice the tapering of the fundus into smaller processes ( arrows ). Secretory granules ( asterisks ) . Muscle (M ). X 21,400.
STRUCTURE OF HEAD GLAND IN SCHIST~S~ME
Electron Microscopy Cercariae. In unemerged cercariae the immature head gland is distinguished from other cells of the cercarial oral sucker by the secretory granules in its cytoplasm. The gland appears unicellular and has a nucleus with a prominent nucleolus (Fig. 3). The cytoplasm is laden with developing secretory granules intermingled with coarse granular material presumed to be ribosomes.The secretory granules seemto form from clear vacuoles which collect particulate material. A few mitochondria are also present, but no endoplasmic reticulum or Golgi were recognized at this stage of maturity (Fig. 3). In a matured head gland the nucleus usually appears well preserved. Its cytoplasm is filled with secretory granules that contain particulate material of various electron densities when fixed with osmic acid only (Figs. 4, 5, 7), but appear homogeneous and denser when prefixed in the aldehyde solution (Figs. 8, 11). Nuclei are often seen around the periphery of the fundus (Fig. I), but always appeared outside when observed at the ultrastructural level (Fig. 6). The fundus is located dorsally in approximately the posterior half of the oral sucker. A cross section through the muscular calyx of the oral sucker shows the fundus of the head gland above the two bundles of ducts of the acetabular glands and the esophageal tube of the digestive system (Fig. 4). Distally, the fundus drapes around one of two large cells located in the center of the oral sucker (Fig. 5). In this area, it separates into coarse elongated trunks that taper or divide into smaller processes (ducts) as they ramify radially toward the integument, but no connections to the integument were observed. Ducts extending along the longitudinal plane were seen to open into the integument in the vicinity of the ducts of the
LARVAE
449
acetabular glands at the anterior end of the oral sucker as previously stated by Morris ( 1971). They are usually small and difficult to trace in free swimming cercariae, but within 2 hr after placing them into the protein environment (egg white) the ducts dilated, revealing quite clearly the complexity of their distribution (Fig. 8). In Fig. 7 a typical relationship between the duct and integument is shown in an emerged cercaria. Both are quite narrow in a cercaria, but become distended when the organism transforms into a schistosomule (Fig. 12). The periphery of the duct becomes thicker a few micrometers before connecting to the integument, a feature that is similar to a duct of the head gland in a schistosomule; this is mainly because of microtubules which line the periphery (Fig. 12). The head gland is in intimate juxtaposition to two large cells which lie roughly end to end (Fig. 9). Together, these cells extend the entire length of the oral sucker occupying most of its central area. The cells usually contain well-defined oval nuclei in light cytoplasms filled with homogeneous granular material and a few mitochondria (Fig. 9). These cells apparently atrophy after host penetration, since they were never observed in schistosomules. The limiting membrane of the head gland appears extremely delicate and difficult to follow. In many instances secretory granules appear free in the viscera as previously indicated by Ebrahimzadeh and Kraft (1971). Schistosomules. The fundus of the head gland in schistosomules is reduced as it loses most of its secretory granules during cercarial penetration and appears to deteriorate completely within 24 hr. After the loss of secretory granules, mitochondria, ribosomal materials, and many small spherical inclusions are recognized in the
450
CHARLES
a
DORSEY
FIG. 6. Electron micrograph showing an area in the posterior region of the fundus of the head gland of a cercaria ( H) cut on a longitudinal plane. Notice the dense nucleus (N ) which is part of another cell laying against the fundus of the head gland. Limiting membrane of the head gland (arrows). X28,000.
~TRU~RE
0~ HEAD GLAND IN S~HIST~SOME
fundus (Fig. 10). Many of these inclusions are similar to the much larger secretory granules and may represent a dying effort by the head gland to replenish its lost secretory granules, since these inclusions are never observed in emerged cercaria. In addition, relatively large, membranebound bodies varying in electron-opaqueness and granularity are commonly seen in the head gland of schistosomules as well as cercariae ( Fig. 11). The ducts are enlarged as secretory granules move en massethrough them into the integument of the oral sucker during penetration (Fig. 12). Some of the secretory granules change into bodies with concentric membranous layers having electrondense cores (Figs. 12, 13), which are never seen in the head glands of cercariae; others remain homogeneously granular of different electron densities (Figs. 10, 12). Also, a notable enlargement or swelling of the integument at the anterior end of the oral sucker occurs after penetration while the rest of the integument of the oral sucker remains unchanged. This integumental change was observed whether the schistosomule was in skin (Fig. 14) or teased free of it (Figs. 12,13). When cercariae were immediateIy teased from the skin of their host (15 min postpenetration) and observed at the ultrastructural level, extensive disruption of the integument at the anterior tip of the oral sucker was seen (Fig. 13). However, within 30 min after penetration, areas of the oral sucker appeared to heal as patches of multilamellated membranes formed on the ex-
LARVAE
451
terior of its integument (Fig. 15). Secretory granules were found in the immediate vicinity of the healing (Fig. 15). DISCUSSION
This study shows that the head gland is a unicellular organ with multiple processes (ducts) that connect to the integument in the anterior region of the oral sucker. Ebrahimzadeh (1970) and Ebrahimzadeh and Kraft (1971) also recognized the multiple duct system of the gland, but did not observe the duct-integument connection. Ebrahimzadeh (1970) and Ebrahimzadeh and Kraft ( 1971) considered the gland to be a multinucleated syncytium rather than a unicellular entity, since nuclei situated around its periphery appeared to be a part of it. Nuclei were also observed around the periphery of the fundus of the head gland in this study (Fig. l), but were shown to be individual cells separated from the gland by a thin plasma membrane (Fig. 6). Morris ( 1971), was the first to observe the head gland in emerged cercariae of S. mans& at the subcellular level, identifying it as “Type C penetration gland cells.” He stated that when the granules from the head gland entered the integument they appeared to change into coarse tadpole-like filamentous material and released to the surface through vacuoles that often opened to the exterior. During this author’s many observations of free swimming cercariae, the head gland-integument connections were quite obvious, but in all casesonly a few scattered granules were observed in
FIG. 7. Electron micrograph of a section through the anterior end of the oral sucker showing a duct (D) of the head gland in communication with the integument (arrow head). Notice the narrowness of the duct ( D ) w h en compared with the expanded head gland duct of a schistosomule in Fig. 11. Notice the thickening of the duct walls approximately 2 pm before it opens into the integument (arrow), The integument is quite thin (arrow head) and lacks secretory granules as compared with the thickened integument which is crowded with secretory granules in Fig. 12. Secretory granules of head gland (G). Preacetabular gland (PR). Postacetabular gland PO). X28,000.
452
CHARLES
FIG. 8. Electron micrograph of the anterior in a protein medium. Notice the extremely narrowness of the duct in Fig. 7. Secretory glands that have released much of their x21,500.
H.
DORSEY
end of the oral sucker of a cercaria exposed 2 hr dilated ducts (arrow heads) as compared to the granules of head gland (g). Ducts of acetabular secretory granules (asterisk). Sensory cell ( S ).
the integument. However, after penetrating the host and becoming schistosomules, masses of secretory granules were seen in the integument, but no evidence of their
release to the exterior as tadpole-like filamentous material was observed. An observation of interest is the change of some secretory granules from dense,
STRUCXURE
OF
HEAD
GLAND
IN
SCHISTOSOME
LARVAE
453
FIG. 9. Electron micrograph of a cercaria showing part of one of the large cells in juxtaposition with the head gland. Nucleus (N) of the large cell. Mitochondria of the large cell (M). Notice the light, finely granular cytoplasm (C) of the large cell which appears translucent after staining with alkaline toluidine blue. Head gland ( asterisks ). Interdigitation of head gland-large cell interface (arrows). Ducts of the acetabular glands (arrow heads). X26,000.
homogeneous bodies into multilamellated inclusions with a dense central core. This change occurs within approximately 30 min after host penetration and probably helps to characterize the transformation of cercaria to schistosomule. The morphological change of the secretory granules was expressed in schistosomules fixed in either the aldehyde solution and postfixed in osmium, or osmium alone, suggesting that the change is a normal one rather than a fixation artifact. Perhaps the secretory granules of the head gland provide material for repair and
reorganization of the integument in the anterior end of the oral sucker injured during penetration, just as the lamellated bodies of the subtegumental cells (cytons) of schistosomules are believed to repair damaged integument as suggested by Rifkin (1971) and Hockley and McLaren (1973). The head gland is similar to the subtegumental cytons observed in schistosomules of S. mun.sti by Rifkin ( 1971) and Hockley and McLaren (1973) in that they both connect to the integument and contain secretory granules which are re-
454
CHARLES
10. FIG. schistosomule. nucleus (N) granules (S FIG. 11. Notice the membranes
x18,500.
H.
DORSEY
Electron micrograph showing an area of the fundus of a head gland of a Notice the thin plasma membrane (arrows) which is difficult to follow. The belongs to another cell. Mitochondrion (M). Spherical inclusions (V). Secretory ) . Ribosomes ( R) . X45,000. Electron micrograph of part of the fundus of a head gland of a schistosomule. large membrane-bound bodies (arrows). Nuclei (N) enclosed by multiple ( M) are not part of the head gland. Secretory granules ( S ) of the head gland.
STRUCTURE
OF
HEAD
GLAND
IN
SCHISTOSOME
LARVAE
FIG. 12. Electron micrograph showing a duct of the head gland in communication with the integument (arrow head) of the oral sucker of a 30-min postpenetrated schistosomule. Notice the thickened periphery of the duct near its integumental junction (large arrow); notice also, the thin plasma membrane (small arrow) of the ducts more distal from the integument. The morphologically different types of secretory granules (A, B, C). Microtubules (T). x42,000.
455
4t56
CHARLES
H.
DORSEY
FIG. 13. Electron micrograph of the anterior end of the oral sucker of a 15min postpenetrated schistosomule. Notice the disrupted and thickened integument (asterisks) caused by host penetration as compared with the thin, well-defined anterior end of the oral sucker of an emerged cercaria in Fig. 7. Disrupted ciliated sensory bulb (B). Ducts of acetabular glands (A). Ducts of the head gland (arrows). Concentric, multilamellated secretory granules of the head gland (arrow heads). X32.,500.
STRUCIZTRE
OF
HEAD
GLAND
IN
SCHISTOSOME
LARVAE
457
FIG. 14. Electron micrograph of a schistosomule (15-mm postpenetration) in the epidermis of mouse skin showing a part of the anterior end of the oral sucker. Notice the thickness of the integument at the top of the oral sucker (large arrow) in contrast to the integument of a noncrescent area (small arrow). Secretory granules of head gland (asterisks). Epidermis of ear (E). X12,500.
leased into the integument during and after host penetration. The head gland is also similar to the subtegumental cytons of matured worms reported by Smith et al. (1969), in that they both connect to the integument. Cercariae of Pamchis acanthus and larvae of other trematodes that encyst (Rees 1967, Cable and Schutte 1973) also have unicellular glands that release secretory granules into their integuments through ducts, but none is claimed to have a multiple duct system like the head gland of cercaria of S. munsoni observed in this study. Ebrahimzadeh (1970) showed that the head gland was strongly positive for the
azan stain of Heidenhain and Bielschowsky’s ammoniacal silver method, but weakly positive for the vital dyes: neutral red and nile blue sulfate. Since nile blue sulfate has an affinity for phospholipids (Menschik 1953), it was reasoned that other dyes which are considered positive for phospholipids (Luxol fast blue: Pearse 1955, acid hematin: Baker 1946) might also stain the secretory granules of the head gland. This led to the use of these dyes which did indeed stain the head gland quite strongly, suggesting the presence of phospholipids. However, more studies must be done using various phospholipases in order to verify this suggestion.
458
CHARLES
H.
DORSEY
15. Electron micrograph showing a damaged portion of integument at the anterior FIG. end of the oral sucker of a schistosomule which depicts the formation of multilamellated membranes (arrow) on its exterior. Notice the secretory granules (asterisks) in the immediate vicinity of the healing. Duct of the head gland (D). ~73,500. ACKNOWLEDGMENTS Supported by the Bureau of Medicine and Surgery Work Unit No. MR041.09.01.0130B6GJ. The opinions or assertions contained herein are the private ones of the author and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The animals used in this study were handled in accordance with the provisions of Public Law 89-54 as amended by Public Law 91-579, the “Animal Welfare Act of 1970” and the principles outlined in the “Guide for the Care and Use of Laboratory Animals,” U. S. Department of Health, Education and Welfare Publication No. (NIH) 73-23. REFERENCES J. R. 1946. The histochemical recognition of lysine. Quarterly Journal of Microscopical Science 87,441-470. CABLE, R. M., AND SCHUTTE, M. H. 1973. Comparative fine structure and origin of the metacercarial cyst in two philophthalmid trematodes, Parorchis acanthus (Nicoll 1906) and Philophthalmus megalurus (Cort 1914). Journal of Parasitology 59, 1031-1040. BAKER,
W. W. 1919. The cercaria of the Japanese japonicum, Katusrada. blood-fluke, Schistosoma University of California Publication of Zoology l&485-507. EBRAHIMZADEH, A. 1970. Beitrage zur Entwicklung, Histologie and Histochemie des Deiisenmansoni system der Cercarien von Schistosoma Sambon ( 1907). Zeitschrift fiir Parasitenkunde 34,319-342. EBRAHWZADEH, A., AND KRAFT, M. 1971. Ultrastrukturelle Untersuchungen Zur Anatomie der Cercarien von Schbtosoma mansoni. III Das fiir Parasitenkunde 36, Driisensystem. Zeitschrift 291-303. HOCKLEY, D. M., AND MCLAREN, D. J. 1973. Schistosoma mansoni: Changes in the outer membrane of the tegument during development from cercaria to adult worm. International Journal for Parasitology 3, 13-25. KARNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. Journal of Cell BZology 27, 137a. LILLIE, R. D. 1965. “Histopathologic Technic and Practical Histochemistry,” 3rd ed., p. 42. McGraw-Hill, New York. CORT,
STRUXURE
LUFT, J. H. 1961. Improvement embedding methods. Journal Biochemical
Cytology
OF
HEAD
GLAND
in epoxy resin Biophysics and
9, 409414.
MENSCHIK, Z. 1953. Nile blue histochemical method for phospholipids. Stain TechnoEogy 28, 13-18. MORRIS, G. P. 1971. The fine structure of the tegument and associated structures of the cercaria of Schistosoma mansoni. Zeitschrift fiir Parasitenkunde 36, 15-31. PEARSE, A. G. E. 1955. Copper phtalocyanins as phospholipid stains. Journal of Pathological Bacterblogy
70,
554-557.
REES, G. 1967. The histochemistry of the cystogenous gland cells and cyst wall of Pasorchis acanthus Nicoll, and some details of the morphology and fine structure of the cercaria. Parasitology 57, 87-110.
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SCHISTOSOME
LARVAE
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REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journul of Cell Biology 17, 208-212. RIFKIN, E. 1971. Interaction between Schistosoma munsoni schistosomules and penetrated mouse skin at the ultrastructural level. In “Aspects of the Biology of Symbiosis” (T. C. Cheng, Ed.), pp. 25-43. University Park Press, Baltimore. SHANKLIN, W. M., AND NASSAR, T. K. 1959. Luxol fast blue combined with the periodic acidSchiff procedure for cytological staining of kidney. Stain Technology 35, 257-260. SMITH, J. H. REYNOLDS, E. S., AND VON LICHTENBERG, F. 1969. The integument of Schistosoma marwoni. American Society of Tropical Medicine and Hygiene 18, 28-49. TERZAKIS, J. S. 1968. Uranyl acetate, a stain and a fixative. Journal of Ultrastructure Research 22, 168-184.
PARASITOLOGY
39,
444-459
( 19%)
Schistosoma mansoni: Description of the Head of Cercariae and Schistosomules at the Ultrastructural Level H.
CHARLES Naval
Medical
Research (Accepted
Institute,
DORSEY
Bethesda,
for publication
Gland
Maryland 26 July
20014,
U. S. A.
1975)
DORSEY, CHARLES H. 1976. Schistosoma munsoni: Description of the head gland of cercariae and schistosomules at the ultrastructural level. Experimental Parasitology 39, 444-459. The head gland of the cercaria of Schistosoma mansoni appears to be a relatively large unicellular entity consisting of a fundus tapering into a system of multiple ducts that opens into the integument at the anterior end of the oral sucker. The fundus is located in the posteriodorsal area of the oral sucker and contains most of the secretory granules. The ducts are usually narrow and devoid of secretory granules especially near their integumental junctions in the cercaria. In schistosomules of S. munsoni the fundus is reduced and the ducts are distended as secretory granules move en masse into the integument during the penetration of cercariae into their host where they may provide material for repair of the integument of the oral sucker damaged during penetration. The head gland has a strong affinity for 1~1x01 fast blue and acid hematin stains which suggests the presence of phospholipids. INDEX DESCRIPTORS: Schistosoma mansoni; Cercaria; Schistosomule; Secretory granules; Secretory gland; Head gland; Penetration gland; Subtegumental gland cell; Secretory gland cell; Ultrastructure; Microscopy, electron.
For many years, the head gland has been an elusive organ first observed in the oral sucker of Schistosoma japonicum by Miyagawa ( Cort 1919), who described it as a massive organ located in the dorsal area of the oral sucker. Subsequently, Ebrahimzadeh ( 1970)) Ebrahimzadeh and Kraft ( 1971)) and Morris (1971) studied the head gland of cercariae of S. manson%. Ebrahimzadeh ( 1970) and Ebrahimzadeh and Kraft (1971) viewed the head gland as an irregularly shaped multi-nucleated gland complex with short ducts. Morris (1971) identified the head gland as “Type C penetration gland cells,” stating that their secretory granules are released through ducts into the tegument at the anterior end of the oral sucker. 444 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
No one (to the knowledge of the author) has described the ultrastructure of the head gland in schistosomulesof S. munsoni. This paper is concerned with the head gland as it appears in both cercariae and schistosomules of S. mansoni utilizing the light and electron microscope. MATERIALS
AND
METHODS
Unemerged and Light microscopy. emerged cercariae from laboratory reared Puerto Rican Biomphalaria glubrata were fixed 72 hr in Carnoy’s solution (Lillie 1965), or 24 hr in Karnovsky’s ( 1965) glutaraldehyde-paraformaldehyde solution. The cercariae were paraffi embedded and cut at 10 pm on a rotary microtome. Some of the tissues were stained with Luxol fast
STEIJCIUEE
OF HEAD GLAND IN SCHISTOSOME LARVAE
blue and counterstained with periodic acid-Schiff ( Shanklin and Nassar 1959). Others were stained with acid hematin ( Baker 1946). Electron microscopy. Unemerged and emerged cercariae were fixed 24 hr in Karnovsky’s solution as above and postfixed in 1% osmic acid made in 0.1 M sodium cacodylate buffered to pH 7.3 with potassium dichromate. Others were fixed 1 hr in the osmic acid solution only. Some emerged cercariae were placed in a protein medium (egg white of a chicken) for 2 hr before 2%hr fixation in Karnovsky’s solution (1965) and postosmification. Schistosomules were obtained after exposing mouse ears to cercariae, when the mice were sacrificed at intervals of 15 min, 30 min, and 1 hr. The schistosomules were either teased from ears floated in Hanks balanced salt solution and placed into the fixative, or the ears were cut into 2-mm squares and placed directly into the fixatives allotted to the cercariae above. The tissue was placed in 1% uranyl acetate (Terzakis 1968) for 1 hr, dehydrated, and embedded in Luft’s (1961) epoxy resin. Approximately l-pm thick sections were cut on a Porter Blum MT-2 microtome and stained with alkaline toluidine blue for orientation of tissue after which sections with an inference of pale gold to silver were cut, placed on one-hole grids, stained with lead citrate (Reynolds 1963) and viewed in a Siemen Elmiskop IA microscope with an accelerating voltage of 80 kV. RESULTS
Light Microscopy (1) Alkaline toluidine blue stain. The fundus of the head gland is easily recognized in the cone of the musculature of the oral sucker with this dye because the gland usually exhibits a different coloration because of the polychromatic staining properties of toluidine blue (Fig. 1). Nuclei are
445
FIG. 1. Light micrograph of the oral end of a cercaria stained with toluidine blue. Notice a part of the fundus of the head gland (g) surrounded by four nuclei (arrow heads). Muscular cone of the oral sucker (m). Ducts of the acetabular glands (d). One of the two large cells located in the oral sucker (asterisks). X1125. FIG. 2. Light micrographs of cercariae stained with Baker’s acid hematin. The head glands (arrows) stain an intense black. X950.
always seen surrounding the fundus of the head gland, but not a part of it, as indicated by electron microscopy. (2) Luxol fast blue-periodic ac&Schifl stain. In the aldehyde-fixed specimens, the head glands stained a distinct blue with Luxol fast blue contrasting sharply with the ducts of the postacetabular glands which stained magenta with the periodic acid-Schiff solution. The head glands of cercariae fixed in Carnoy’s solution did not stain with Luxol fast blue, but the ducts of the postacetabular glands remained Schiff positive. This staining procedure gives a clear picture of the head glands location which is dorsal to the ducts of the acetabular glands. (3) Acid hem&in. The head glands of aldehyde-fixed cercariae stained black with
16
CHARLES
H.
DORSEY
FIG. 3. Electron micrograph of a part of an unemerged cercaria. Notice the head gland which contains a nucleus (N) and secretory granules of different stages of development (asterisks) in its cytoplasm. Presumed granular ribosomal material (R). Mitochondria (M). Cytoplasmic extension of head gland ( arrow). X 18,800,
STRUCXJRE
OF
HEAD
GLAND
IN
SCHISTOSOME
LARVAE
447
FIG. 4. Electron micrograph of a cross section through the distal end of the oral sucker of a free swimming cercaria. Notice the dorsal position of the head gland (large arrow) with respect to the ducts of the acetabular glands (asterisks) and the digestive tract (D). Secretory granules contain particulate material of various electron densities typical of tissue fixed in osmic acid only (small arrows), Nerve processes ( N) . Cells of the muscular calyx ( M ). x 17,500.
Baker’s acid hematin (Fig. 2). The black gland contrasted strongly with the light brown color of the rest of the cercaria. The
head gland itself did not stain after fixation in Carnoy’s, but nuclei located in its immediate vicinity stained black.
448
CHARLES
H.
DORSEY
FIG. 5. Electron micrograph of part of a cercaria on cross section showing a portion of the head gland (G) draped around a large cell (C). Notice the tapering of the fundus into smaller processes ( arrows ). Secretory granules ( asterisks ) . Muscle (M ). X 21,400.
STRUCTURE OF HEAD GLAND IN SCHIST~S~ME
Electron Microscopy Cercariae. In unemerged cercariae the immature head gland is distinguished from other cells of the cercarial oral sucker by the secretory granules in its cytoplasm. The gland appears unicellular and has a nucleus with a prominent nucleolus (Fig. 3). The cytoplasm is laden with developing secretory granules intermingled with coarse granular material presumed to be ribosomes.The secretory granules seemto form from clear vacuoles which collect particulate material. A few mitochondria are also present, but no endoplasmic reticulum or Golgi were recognized at this stage of maturity (Fig. 3). In a matured head gland the nucleus usually appears well preserved. Its cytoplasm is filled with secretory granules that contain particulate material of various electron densities when fixed with osmic acid only (Figs. 4, 5, 7), but appear homogeneous and denser when prefixed in the aldehyde solution (Figs. 8, 11). Nuclei are often seen around the periphery of the fundus (Fig. I), but always appeared outside when observed at the ultrastructural level (Fig. 6). The fundus is located dorsally in approximately the posterior half of the oral sucker. A cross section through the muscular calyx of the oral sucker shows the fundus of the head gland above the two bundles of ducts of the acetabular glands and the esophageal tube of the digestive system (Fig. 4). Distally, the fundus drapes around one of two large cells located in the center of the oral sucker (Fig. 5). In this area, it separates into coarse elongated trunks that taper or divide into smaller processes (ducts) as they ramify radially toward the integument, but no connections to the integument were observed. Ducts extending along the longitudinal plane were seen to open into the integument in the vicinity of the ducts of the
LARVAE
449
acetabular glands at the anterior end of the oral sucker as previously stated by Morris ( 1971). They are usually small and difficult to trace in free swimming cercariae, but within 2 hr after placing them into the protein environment (egg white) the ducts dilated, revealing quite clearly the complexity of their distribution (Fig. 8). In Fig. 7 a typical relationship between the duct and integument is shown in an emerged cercaria. Both are quite narrow in a cercaria, but become distended when the organism transforms into a schistosomule (Fig. 12). The periphery of the duct becomes thicker a few micrometers before connecting to the integument, a feature that is similar to a duct of the head gland in a schistosomule; this is mainly because of microtubules which line the periphery (Fig. 12). The head gland is in intimate juxtaposition to two large cells which lie roughly end to end (Fig. 9). Together, these cells extend the entire length of the oral sucker occupying most of its central area. The cells usually contain well-defined oval nuclei in light cytoplasms filled with homogeneous granular material and a few mitochondria (Fig. 9). These cells apparently atrophy after host penetration, since they were never observed in schistosomules. The limiting membrane of the head gland appears extremely delicate and difficult to follow. In many instances secretory granules appear free in the viscera as previously indicated by Ebrahimzadeh and Kraft (1971). Schistosomules. The fundus of the head gland in schistosomules is reduced as it loses most of its secretory granules during cercarial penetration and appears to deteriorate completely within 24 hr. After the loss of secretory granules, mitochondria, ribosomal materials, and many small spherical inclusions are recognized in the
450
CHARLES
a
DORSEY
FIG. 6. Electron micrograph showing an area in the posterior region of the fundus of the head gland of a cercaria ( H) cut on a longitudinal plane. Notice the dense nucleus (N ) which is part of another cell laying against the fundus of the head gland. Limiting membrane of the head gland (arrows). X28,000.
~TRU~RE
0~ HEAD GLAND IN S~HIST~SOME
fundus (Fig. 10). Many of these inclusions are similar to the much larger secretory granules and may represent a dying effort by the head gland to replenish its lost secretory granules, since these inclusions are never observed in emerged cercaria. In addition, relatively large, membranebound bodies varying in electron-opaqueness and granularity are commonly seen in the head gland of schistosomules as well as cercariae ( Fig. 11). The ducts are enlarged as secretory granules move en massethrough them into the integument of the oral sucker during penetration (Fig. 12). Some of the secretory granules change into bodies with concentric membranous layers having electrondense cores (Figs. 12, 13), which are never seen in the head glands of cercariae; others remain homogeneously granular of different electron densities (Figs. 10, 12). Also, a notable enlargement or swelling of the integument at the anterior end of the oral sucker occurs after penetration while the rest of the integument of the oral sucker remains unchanged. This integumental change was observed whether the schistosomule was in skin (Fig. 14) or teased free of it (Figs. 12,13). When cercariae were immediateIy teased from the skin of their host (15 min postpenetration) and observed at the ultrastructural level, extensive disruption of the integument at the anterior tip of the oral sucker was seen (Fig. 13). However, within 30 min after penetration, areas of the oral sucker appeared to heal as patches of multilamellated membranes formed on the ex-
LARVAE
451
terior of its integument (Fig. 15). Secretory granules were found in the immediate vicinity of the healing (Fig. 15). DISCUSSION
This study shows that the head gland is a unicellular organ with multiple processes (ducts) that connect to the integument in the anterior region of the oral sucker. Ebrahimzadeh (1970) and Ebrahimzadeh and Kraft (1971) also recognized the multiple duct system of the gland, but did not observe the duct-integument connection. Ebrahimzadeh (1970) and Ebrahimzadeh and Kraft ( 1971) considered the gland to be a multinucleated syncytium rather than a unicellular entity, since nuclei situated around its periphery appeared to be a part of it. Nuclei were also observed around the periphery of the fundus of the head gland in this study (Fig. l), but were shown to be individual cells separated from the gland by a thin plasma membrane (Fig. 6). Morris ( 1971), was the first to observe the head gland in emerged cercariae of S. mans& at the subcellular level, identifying it as “Type C penetration gland cells.” He stated that when the granules from the head gland entered the integument they appeared to change into coarse tadpole-like filamentous material and released to the surface through vacuoles that often opened to the exterior. During this author’s many observations of free swimming cercariae, the head gland-integument connections were quite obvious, but in all casesonly a few scattered granules were observed in
FIG. 7. Electron micrograph of a section through the anterior end of the oral sucker showing a duct (D) of the head gland in communication with the integument (arrow head). Notice the narrowness of the duct ( D ) w h en compared with the expanded head gland duct of a schistosomule in Fig. 11. Notice the thickening of the duct walls approximately 2 pm before it opens into the integument (arrow), The integument is quite thin (arrow head) and lacks secretory granules as compared with the thickened integument which is crowded with secretory granules in Fig. 12. Secretory granules of head gland (G). Preacetabular gland (PR). Postacetabular gland PO). X28,000.
452
CHARLES
FIG. 8. Electron micrograph of the anterior in a protein medium. Notice the extremely narrowness of the duct in Fig. 7. Secretory glands that have released much of their x21,500.
H.
DORSEY
end of the oral sucker of a cercaria exposed 2 hr dilated ducts (arrow heads) as compared to the granules of head gland (g). Ducts of acetabular secretory granules (asterisk). Sensory cell ( S ).
the integument. However, after penetrating the host and becoming schistosomules, masses of secretory granules were seen in the integument, but no evidence of their
release to the exterior as tadpole-like filamentous material was observed. An observation of interest is the change of some secretory granules from dense,
STRUCXURE
OF
HEAD
GLAND
IN
SCHISTOSOME
LARVAE
453
FIG. 9. Electron micrograph of a cercaria showing part of one of the large cells in juxtaposition with the head gland. Nucleus (N) of the large cell. Mitochondria of the large cell (M). Notice the light, finely granular cytoplasm (C) of the large cell which appears translucent after staining with alkaline toluidine blue. Head gland ( asterisks ). Interdigitation of head gland-large cell interface (arrows). Ducts of the acetabular glands (arrow heads). X26,000.
homogeneous bodies into multilamellated inclusions with a dense central core. This change occurs within approximately 30 min after host penetration and probably helps to characterize the transformation of cercaria to schistosomule. The morphological change of the secretory granules was expressed in schistosomules fixed in either the aldehyde solution and postfixed in osmium, or osmium alone, suggesting that the change is a normal one rather than a fixation artifact. Perhaps the secretory granules of the head gland provide material for repair and
reorganization of the integument in the anterior end of the oral sucker injured during penetration, just as the lamellated bodies of the subtegumental cells (cytons) of schistosomules are believed to repair damaged integument as suggested by Rifkin (1971) and Hockley and McLaren (1973). The head gland is similar to the subtegumental cytons observed in schistosomules of S. mun.sti by Rifkin ( 1971) and Hockley and McLaren (1973) in that they both connect to the integument and contain secretory granules which are re-
454
CHARLES
10. FIG. schistosomule. nucleus (N) granules (S FIG. 11. Notice the membranes
x18,500.
H.
DORSEY
Electron micrograph showing an area of the fundus of a head gland of a Notice the thin plasma membrane (arrows) which is difficult to follow. The belongs to another cell. Mitochondrion (M). Spherical inclusions (V). Secretory ) . Ribosomes ( R) . X45,000. Electron micrograph of part of the fundus of a head gland of a schistosomule. large membrane-bound bodies (arrows). Nuclei (N) enclosed by multiple ( M) are not part of the head gland. Secretory granules ( S ) of the head gland.
STRUCTURE
OF
HEAD
GLAND
IN
SCHISTOSOME
LARVAE
FIG. 12. Electron micrograph showing a duct of the head gland in communication with the integument (arrow head) of the oral sucker of a 30-min postpenetrated schistosomule. Notice the thickened periphery of the duct near its integumental junction (large arrow); notice also, the thin plasma membrane (small arrow) of the ducts more distal from the integument. The morphologically different types of secretory granules (A, B, C). Microtubules (T). x42,000.
455
4t56
CHARLES
H.
DORSEY
FIG. 13. Electron micrograph of the anterior end of the oral sucker of a 15min postpenetrated schistosomule. Notice the disrupted and thickened integument (asterisks) caused by host penetration as compared with the thin, well-defined anterior end of the oral sucker of an emerged cercaria in Fig. 7. Disrupted ciliated sensory bulb (B). Ducts of acetabular glands (A). Ducts of the head gland (arrows). Concentric, multilamellated secretory granules of the head gland (arrow heads). X32.,500.
STRUCIZTRE
OF
HEAD
GLAND
IN
SCHISTOSOME
LARVAE
457
FIG. 14. Electron micrograph of a schistosomule (15-mm postpenetration) in the epidermis of mouse skin showing a part of the anterior end of the oral sucker. Notice the thickness of the integument at the top of the oral sucker (large arrow) in contrast to the integument of a noncrescent area (small arrow). Secretory granules of head gland (asterisks). Epidermis of ear (E). X12,500.
leased into the integument during and after host penetration. The head gland is also similar to the subtegumental cytons of matured worms reported by Smith et al. (1969), in that they both connect to the integument. Cercariae of Pamchis acanthus and larvae of other trematodes that encyst (Rees 1967, Cable and Schutte 1973) also have unicellular glands that release secretory granules into their integuments through ducts, but none is claimed to have a multiple duct system like the head gland of cercaria of S. munsoni observed in this study. Ebrahimzadeh (1970) showed that the head gland was strongly positive for the
azan stain of Heidenhain and Bielschowsky’s ammoniacal silver method, but weakly positive for the vital dyes: neutral red and nile blue sulfate. Since nile blue sulfate has an affinity for phospholipids (Menschik 1953), it was reasoned that other dyes which are considered positive for phospholipids (Luxol fast blue: Pearse 1955, acid hematin: Baker 1946) might also stain the secretory granules of the head gland. This led to the use of these dyes which did indeed stain the head gland quite strongly, suggesting the presence of phospholipids. However, more studies must be done using various phospholipases in order to verify this suggestion.
458
CHARLES
H.
DORSEY
15. Electron micrograph showing a damaged portion of integument at the anterior FIG. end of the oral sucker of a schistosomule which depicts the formation of multilamellated membranes (arrow) on its exterior. Notice the secretory granules (asterisks) in the immediate vicinity of the healing. Duct of the head gland (D). ~73,500. ACKNOWLEDGMENTS Supported by the Bureau of Medicine and Surgery Work Unit No. MR041.09.01.0130B6GJ. The opinions or assertions contained herein are the private ones of the author and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The animals used in this study were handled in accordance with the provisions of Public Law 89-54 as amended by Public Law 91-579, the “Animal Welfare Act of 1970” and the principles outlined in the “Guide for the Care and Use of Laboratory Animals,” U. S. Department of Health, Education and Welfare Publication No. (NIH) 73-23. REFERENCES J. R. 1946. The histochemical recognition of lysine. Quarterly Journal of Microscopical Science 87,441-470. CABLE, R. M., AND SCHUTTE, M. H. 1973. Comparative fine structure and origin of the metacercarial cyst in two philophthalmid trematodes, Parorchis acanthus (Nicoll 1906) and Philophthalmus megalurus (Cort 1914). Journal of Parasitology 59, 1031-1040. BAKER,
W. W. 1919. The cercaria of the Japanese japonicum, Katusrada. blood-fluke, Schistosoma University of California Publication of Zoology l&485-507. EBRAHIMZADEH, A. 1970. Beitrage zur Entwicklung, Histologie and Histochemie des Deiisenmansoni system der Cercarien von Schistosoma Sambon ( 1907). Zeitschrift fiir Parasitenkunde 34,319-342. EBRAHWZADEH, A., AND KRAFT, M. 1971. Ultrastrukturelle Untersuchungen Zur Anatomie der Cercarien von Schbtosoma mansoni. III Das fiir Parasitenkunde 36, Driisensystem. Zeitschrift 291-303. HOCKLEY, D. M., AND MCLAREN, D. J. 1973. Schistosoma mansoni: Changes in the outer membrane of the tegument during development from cercaria to adult worm. International Journal for Parasitology 3, 13-25. KARNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. Journal of Cell BZology 27, 137a. LILLIE, R. D. 1965. “Histopathologic Technic and Practical Histochemistry,” 3rd ed., p. 42. McGraw-Hill, New York. CORT,
STRUXURE
LUFT, J. H. 1961. Improvement embedding methods. Journal Biochemical
Cytology
OF
HEAD
GLAND
in epoxy resin Biophysics and
9, 409414.
MENSCHIK, Z. 1953. Nile blue histochemical method for phospholipids. Stain TechnoEogy 28, 13-18. MORRIS, G. P. 1971. The fine structure of the tegument and associated structures of the cercaria of Schistosoma mansoni. Zeitschrift fiir Parasitenkunde 36, 15-31. PEARSE, A. G. E. 1955. Copper phtalocyanins as phospholipid stains. Journal of Pathological Bacterblogy
70,
554-557.
REES, G. 1967. The histochemistry of the cystogenous gland cells and cyst wall of Pasorchis acanthus Nicoll, and some details of the morphology and fine structure of the cercaria. Parasitology 57, 87-110.
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SCHISTOSOME
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REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journul of Cell Biology 17, 208-212. RIFKIN, E. 1971. Interaction between Schistosoma munsoni schistosomules and penetrated mouse skin at the ultrastructural level. In “Aspects of the Biology of Symbiosis” (T. C. Cheng, Ed.), pp. 25-43. University Park Press, Baltimore. SHANKLIN, W. M., AND NASSAR, T. K. 1959. Luxol fast blue combined with the periodic acidSchiff procedure for cytological staining of kidney. Stain Technology 35, 257-260. SMITH, J. H. REYNOLDS, E. S., AND VON LICHTENBERG, F. 1969. The integument of Schistosoma marwoni. American Society of Tropical Medicine and Hygiene 18, 28-49. TERZAKIS, J. S. 1968. Uranyl acetate, a stain and a fixative. Journal of Ultrastructure Research 22, 168-184.