JOURNAL OF MORPHOLOGY 224233-240 (1995)

Cyton II: A Subtegumental Cell Type in the Cercaria of Schistosoma mansoni

CHARLES DORSEY AND CAROLYN COUSIN Biology Department, University of the District of Columbia, Washington, D.C. 20008

ABSTRACT In Schistosoma mansoni cercaria, an aggregate of subtegumen- tal cells is found in a small, dorsoanterior area of the body (middivision). These cells are nestled between two laterally positioned flame cells and the muscle that delimits the anterior end of the body, and the anterior end of the central ganglion. This highly amorphous cell type, designated as cyton 11, has a heterochromatic nucleus and a cytoplasm that is elaborated into coarse, tortu- ous processes. Its cytoplasm contains ribosomes, mitochondria, sparse amounts of endoplasmic reticulum, and two types of circular-to-oval concentric membra- nous bodies. One type has an electron-dense core and measures 200-250 nm on the short axis, and the other is completely membranous and measures 100-125 nm on the short axis. The cell body of cyton I1 communicates with the tegument that covers a small, dorsoposterior area of the anterior organ (oral sucker); however, we could not confirm a tegumental connection with the body division. When cercariae transform into schistosomules, the concentric mem- branous bodies of cyton I1 migrate into the anterior organ's tegument via cytoplasmic processes of the cell. The major function of previously described cells that have similar membranous bodies is to supply additional membranes to the outer tegument during development into an adult worm. A multi- laminated outer membrane is an adaptation to the survival of the schis- tosomule and adult worm in the bloodstream of the vertebrate host (Hockley amd McLaren "731). The presence of membranous bodies from cyton I1 in the tegument does not confirm that this cell type participates in the forma- tion of multilaminated membranes. Its precise function remains to be determined. Q 1995 Wiley-Liss, Inc.

Three species of schistosomes are known to be the causative agents of schistosomiasis; one of these, Schistosoma mansoni, is en- demic to the western hemisphere. Several stages are present in its life cycle, and for hosts it alternates between a freshwater snail, Biomphalaria glabrata, and humans. The infectious stage to man, the cercaria, has a tegument that plays a major role in creating and sustaining a suitable parasite/host rela- tionship (Wilson and Barnes, '77). The tegu- ment of the schistosome is a syncytium whose outer portion is trilaminated in the cercaria and heptalaminated in the adult worm. The outer part of the tegument is connected to subtegumental cells in the body, and the head gland in the anterior organ (anterior body division). It was suggested by Hockley and McLaren ('73) that membranous bodies

are responsible for providing additional mem- branes to the tegument during in vivo devel- opment from cercariae to adult worms.

In the cercaria, two types of subtegumen- tal cells (cytons) in the body division and also the head gland in the anterior organ produce membranous bodies that move into the tegu- ment during transformation into a schistoso- mule. Bruce et al. ('701, Rifkin ('71), Morris ('711, Hockley and McLaren ('73), and Brink et al. ('77) reported information on the loca- tion, ultrastructure, and possible function of one type of subtegumental cell (designated cyton I in this paper). Morris ('711, Ebra- himzadeh and Kraft ('711, Dorsey ('76), and

Address reprint requests to Carolyn Cousin, Biology Depart- ment, University of the District of Columbia, 4200 Connecticut Avenue, Building 44, Room 203, Washington, D.C. 20008.

D 1995 WILEY-LISS, INC.

234 C. DORSEY AND C. COUSIN

Torpier et al. ('77) reported the ultrastruc- ture and activity of the unicellular head gland. Cousin et al. ('81) mentioned briefly both types of subtegumental cells and the head gland. Cyton I and the head gland have mem- branous bodies that play a role in survival and maturation. The membranous bodies found in these two cell types are similar t o those in the second type of subtegumental cell (designated cyton 11) mentioned by Cousin et al. ('81).

In this paper, the location and ultrastruc- ture of cyton I1 are described. A brief compari- son of the membranous bodies of cyton I1 with those of cyton I and the head gland is also presented. Information on cyton 11, coupled with the data provided in related studies, should contribute to a better under- standing of the role of membranous tegumen- tal bodies in maintaining a suitable host- parasite interface.

MATERIALS AND METHODS

Free-swimming cercariae, harvested from Biomphalaria glabrata and maintained at the Biomedical Research Institute in Rock- ville, Maryland, were fixed for 1 hr in 1% osmium tetroxide buffered, with 0.1 M so- dium cacodylate/potassium dichromate, pH 7.2. Some cercariae were fixed in 2% glutaral- dehydelparaformaldehyde, buffered with 0.1 M sodium cacodylate/hydrochloric acid, pH 7.2. The aldehyde-fixed organisms were post- fixed in osmium tetroxide, as above.

Schistosomules (postpenetration larvae) were obtained from the ears of anesthetized white laboratory mice (NMRI strain) at inter- vals of 1, 2, and 24 hr following cercarial penetration. The mice were sacrificed by cer- vical dislocation, the ears excised, and the inner and outer layers of their ears pulled apart. The inner layers were placed in Hank's balanced salt solution and teased into small pieces with fine forceps, which freed the schis- tosomules. Schistosomules were collected with a Pasteur pipette and fixed according to the above procedures. Subsequently, the or- ganisms were rinsed in 4.5% aqueous sucrose (3 changes, 5 min each) and placed in 0.1% aqueous uranyl acetate for 30 min. They were dehydrated through graded alcohols and propylene oxide, embedded in Luft's epoxy resin, and polymerized for 18 hr at 80°C. Thin sections (75-90 nm) were semiserially cut from blocks containing 1-3 organisms with a Porter-Blum MT2B ultramicrotome,

viewed with a JEOL 100 CX transmission electron microscope at 80 kV.

RESULTS

The body of the cercaria is divided into three divisions: the anterior organ (oral sucker), body (middle area), and tail. A group of cyton I1 cells is clustered in the dorsoante- rior end of the body (Figs. 1,2) and bounded by a pair of laterally-positioned flame cells of the osmoregulatory system (Fig. l), by the musculature which delimits the anterior end of the body (Fig. 31, and by the anterior end of the central ganglion (Fig. 3).

Cyton I1 is irregularly-shaped with a het- erochromatic nucleus (Figs. 2, 3) and cyto- plasm that is elaborated into coarse, tortuous processes that are loosely arranged around the musculature that delimits the anterior end of the body (Fig. 3). Its cytoplasm con- tains ribosomes, mitochondria, sparse amounts of endoplasmic reticulum, and two types of concentric membranous bodies (Fig. 4). In the cercaria, these bodies are numer- ous throughout the cell body of cyton 11. The larger type is elliptical-to-circular, contains

Fig. 1. Schistosoma mansoni. Transmission electron micrograph of a cross section through the anterior end of the body segment of a cercaria, showing an irregularly- shaped cyton I1 cell (C) located between two flame cells (arrowheads). Muscle (MI in the posterior end of the anterior organ, and laterally positioned acetabular glands

placed on one-hole Formvar-coated grids, and ducts (D), are shown. Scale bar, 10 bm.

CYTON I1 OF SCHISTOSOMA MANSON1 235

Fig. 2. Schisfosoma mansoni. Transmission electron micrograph showing several irregularly- shaped cyton I1 cells in a cercaria. Each cyton I1 has a single heterochromatic nucleus (N) surrounded by cytoplasm (c) . Membranes of the numerous membranous bodies (asterisks) are not discerned at this magnification. Scale bar = 2 fim.

an electron-dense core, and measures 200- 250 nm on short axis. The smaller type is membranous throughout, measures 100- 125 nm on short axis, and rarely contains dense cores (Fig. 4). The shape, size, and morphology of the smaller granule closely resembles the membranous bodies of cyton I (Fig. 5). Cytoplasmic processes are difficult to detect in the cercaria because they have con- stricted lumens.

Two hr after the cercaria becomes a schis- tosomule, the membranous bodies are re- duced in the cell body of cyton I1 (Fig. 6), because they have migrated into the distal cytoplasmic processes (Figs. 7,8). In the schis- tosomule, the cytoplasmic processes of cyton I1 are dilated, and extend from the body segment into the anterior organ where con- nections are seen with the anterior organ’s tegument. Although they may be present, tegumental connections via cytoplasmic pro-

cesses were not seen in the body division. Only a few membranous bodies (Figs. 7-9) are seen in cytoplasmic processes and in the tegument of the schistosomule of Schisto- soma mansoni. The smaller body is predomi- nant in the body tegument, while the large dense-core type is more prevalent in the tegu- ment of the anterior organ and is rarely seen in the body tegument (Fig. 10).

In the body division of the schistosomule, cyton I and cyton I1 are found subjacent to the tegument that contains groups of mem- branous bodies. Both cell types contain the small type of membranous body; therefore, unless the large dense-core membranous bod- ies from cyton I1 were present also, we could not determine the origin of these tegumental bodies (Fig. 8). In the anterior organ, a simi- lar problem exists in distinguishing between tegumental bodies from cyton I1 and the head gland. Both have the same type of large,

236 C. DORSEY AND C. COUSIN

Fig. 3. Schistosoma mansoni. Transmission electron micrograph of a cercaria through the anterior organ (A) and body (B), showing the musculature bordering the anterior organ and body (arrows). Note the nuclei (n), varicosed amorphous cytoplasm (4, and small cytoplas- mic processes (arrowheads) of cyton 11. Flame cell (F), neurons (asterisk), part of the neuropile (N), and the head gland (G) are shown. Scale bar = 5 pm.

Fig. 4. Schistosoma mansoni. Transmission electron micrograph of a cercaria through cyton I1 showing mem-

dense-core membranous bodies, and they may be released into the same area of the tegu- ment. In this case, however, the origin of the bodies can be easily determined, because the head gland contains homogeneous dense bod- ies in addition to the two types of membra- nous bodies. They occur in the distal cytoplas- mic processes of the head gland at the

branous bodies (arrowheads). Both small and large mem- branous bodies are shown (arrows); however, the large membranous bodies have dense cores. Scale bar = 0.5 pm.

Fig. 5. Schistosoma mansoni. Transmission electron micrograph through the cytoplasm of a cyton I of a cercaria. Note the membranous bodies (arrowheads) that are similar in size and shape to the smaller membranous bodies of the cyton I1 in Figure 4. Scale bar = 1 pm.

tegumental junction (Figs. 10, 11). Because these homogeneous dense bodies are present only in the cercarial head gland and not in cyton 11, their presence or absence deter- mines the origin. In both body division and anterior organ, it is possible that an area of the tegument could contain membranous bod- ies from both subtegumental cell types.

CYTON 11 OF SCHISTOSOMA MMSONI 237

Fig. 6. Schistosoma mansoni. Transmission electron micrograph of a section through a schistosomule which shows two cyton I1 cells. Each has a heterochromatic nucleus (N), ribosomes (r), mitochondria (m), and a few dense-core membranous bodies (arrowheads). Scale bar = 1 bm.

DISCUSSION

The location and ultrastructure of the cy- ton I1 cells of Schistosoma mansoni cercaria have been described. Because of the similari- ties of the membranous bodies of cyton I1 with those of cyton I and the head gland, a brief comparison will be made.

Cyton I1 cells are located in the dorsoante- rior region of the body segment, and probably discharge membranous bodies into the body tegument associated with that region; how- ever, this could not be confirmed. On the other hand, we were able to confirm that membranous bodies from cyton I1 do empty into the dorsoposterior tegument of the ante- rior organ. At least two other cell types re- lease membranous bodies into the tegument. Both cell types have membranous bodies simi- lar in size and morphology to those of cyton 11. One of these, cyton I, located subjacent to the peripheral musculature of the body, sup- plies membranous bodies to a large area of the tegument of the body division (Rifkin,

'71; Morris, '71; Hockley and McLaren, '73). Another cell type, the head gland, located in the median region of the anterior organ, re- leases membranous bodies only into the tegu- ment of the oral end of the anterior organ (Morris, '71; Dorsey, '76; Torpier et al., '77). In all three cell types, migration of the mem- branous bodies occurs only during and after transformation. Membranous bodies never migrate into the tegument in a nontransform- ing cercaria.

The membranous bodies of cyton I and the head gland provide additional membranes for the outer tegument. The bodies change the tegument from trilaminated in the cer- caria to multilaminated in the developing schistosomule. Hockley and McLaren ('73) demonstrated that the unraveling and depos- iting of the membranous bodies from cyton I provided additional membranes for the tegu- ment of the schistosomule, while Torpier et al. ('77) clearly showed that the large mem- branous bodies from the head gland depos-

238 C. DORSEY AND C. COUSIN

Fig. 7. Schistosoma mansoni. Transmis- sion electron micrograph of a schistosomule, showing a cross section in the body (B) and anterior organ (A) that is separated by a layer of connective tissue (J). Cytoplasmic processes (arrowheads), distributed around the connec- tive tissue layer (J), contain a few membra- nous bodies. One cytoplasmic process (arrow-

ited additional membranes on the apical sur- face of in vitro schistosomules. Rifkin ('71) believed that the membranous bodies of cy- ton I contribute to repair of tegumental abra- sions caused when the cercaria penetrates the vertebrate host. Hockley and McLaren ('73) postulated that the additional mem-

head with asterisk) is located at the junction between the anterior organ (A) and body (B). Several muscle complexes are shown: subtegu- mental peripheral muscle (M) in the body, muscle (m) forming the anterior wall of the body, and muscle (M*) forming the walls of the anterior organ. Scale bar = 2 pm.

branes serve as protection against environ- mental hazards that occur during matura- tion of the schistosomule into an adult worm. Dorsey ('76) felt that membrane repair was a function of the membranous bodies, also. He suggested that the dense bodies in the head gland of the cercaria help repair the heavily

Fig. 8. Schistosoma rnansoni. Transmission electron micrograph of an area through the junction of the body and anterior organ of a schistosomule. Shown are two cytoplasmic processes of cyton I1 in the connective tissue (J) that separates the body from the anterior organ. One cytoplasmic process is devoid of membranous bodies (b). The other cytoplasmic process (alpha and arrow) has two membranous bodies (arrowhead). A group of membra- nous bodies is seen in the tegument (T). Also, note cyton 11 with membranous bodies (cy with arrowhead). Scale bar = 1 um.

Fig. 9. Schistosoma mansoni. Transmission electron mi- crograph of a schistosomule, showing a cross section in the connective tissue (J) between the body (B) and the anterior organ (A). In the body (B), the distal end of a cytoplasmic process (alpha) with two membranous bodies (arrowhead) is seen. Subtegumental muscles (MI, muscle delimiting the

anterior organ (m), and tegumental membranous bodies (T with arrowhead) are shown. Scale bar = 1 pm.

Fig. 10. Schistosoma mansoni. Transmission elec- tron micrograph in the posterior region of the anterior organ of a schistosomule, showing a cytoplasmic process of cyton I1 (cy with arrowhead). This cytoplasmic process is invested in muscle (M) that joins the tegument (T) at a site where membranous bodies (arrowheads) are released from the apical end of the process (arrow). Note several large membranous bodies with dense cores. Scale bar = 1 pm.

Fig. 11. Schistosoma mansoni. Transmission electron micrograph of the apical area of the anterior organ, showing a cytoplasmic process h m w ) which connects the head gland (G) to the tegument (T). Note a mixture of dense homoge- neous bodies (hb) and membranous bodies, some of which have dense cores (arrowheads) in the head gland and tegu- ment. Scale bar = 1 pm.

CYTON I1 OF SCHISTOSOMA MANSONI 239

Figures 8-1 1

damaged apical surface of the schis tosomule caused by host penetration, and lie stated that the dense-core bodies of the hi :ad gland (which develop into membranous bo lies) con- tain phospholipid. These dense-co -e bodies

have a strong affinity for phospholipid stains (acid hematin and lux01 fast blue), and it is probable that these phospholipids play a role in the structural changes observed in the dense-core membranous bodies.

240 C. DORSEY AND C. COUSIN

Although the membranous bodies of cyton I1 are similar to those of cyton I and the head gland, we feel that cyton I1 plays only a minor role, if any, in depositing additional mem- branes upon the surface of the schistoso- mule’s tegument. There is only a small clus- ter of cyton I1 cells in the cercaria, and these cells communicate with a limited area of the tegument. In order for the membranous bod- ies of cyton I1 to have any significant role in multilaminated membrane formation, exten- sive migration into other regions of the tegu- ment would be required. Furthermore, the few membranous bodies present in the cyto- plasmic processes of cyton I1 cells and in the associated tegument would only contribute minimally, even if this dispersement does occur. Our observations do not suggest that such an event transpires. Thus, it is felt that cyton I1 probably plays some other role in the survival and early development of the schisto- somule, or that this cell type may simply be a remnant of a tegumental-associated cell of an ancestral platyhelminth.

ACKNOWLEDGMENTS

This investigation was supported by Bio- medical Research Support Grant RR08005 from Research Resources, National Insti- tutes of Health, US. Public Health Service, and by Agency for International Develop- ment grant DAN-5053-G-SS-9033-00.

LITERATURE CITED Brink, L.H., D.J. McLaren, and S.R. Smithers (1977)

Schistosoma mansoni: a comparative study of artifi- cally transformed schistosomula and schistosomula re- covered after cercarial penetration of isolated skin. Parasitology 74:73-86.

Bruce, J.J., F. Pezzelo, J.E. McCarty, and Y. Yama (1970) Migration of Schistosoma mansoni through mouse tis- sue. Ultrastructure of host tissue and integument of migrating larva following cercarial penetration. Am. J . Trop. Med. Hyg. 19:959-981.

Cousin, C.E., M.A. Stirewalt, and C.H. Dorsey (1981) Schistosorna mansoni: ultrastructure of early transfor- mation of skin- and shear-pressure derived schistoso- mules. Exp. Parasitol. 51:241-365.

Dorsey, C.H. (1976) Schistosoma mansoni: description of the head gland of cercariae and schistosomules at the ultrastructural level. Exp. Parasitol. 39:444-459.

Ebrabimzadeh, A., and M. Kraft (1971) Ultrastruk- turelle Unterschungen sur Anatomie der Cercarien von Schistosoma mansoni 111. Das Drusen-system. Z. Parasitenkd. 32: 157-1 75.

Hockley, D.J., and D.J. McLaren (1973) Schistosoma mansoni: changes in outer membrane of the tegument during development from cercaria to adult worm, Int. J . Parasitol. 3113-25.

Morris, G.P. (1971) The fine structure of the tegument and associated structures of the cercaria of Schisto- soma mansoni. 2. Parasitenkd. 36:15-31.

Rifkin, E. (1971) Interaction between Schistosoma man- soni schistosomules and penetrated mouse skin at the ultrastructural level. In T.C. Cheng (4): Aspects of the Biology of Symbiosis. London: Buttenvorths, pp. 2545 .

Torpier, G., M. Capron, and A. Capron (1977) Structural changes of the tegumental membrane complex in rela- tion to developmental stages of Schistosoma mansoni (Platyhelminthes: Trematoda). J . Ultrastruct. Res. 61: 309-324.

Wilson, R.A., and P.E. Barnes (1977) The formation and turnover of the membranocalyx on the tegument of Schistosoma mansoni. Parasitology 74:61-71.