J . Profozml.,39(4). 1992. pp. 480-484 0 1992 by the Society of Protozwlogistr

Surface Domains in the Pathogenic Protozoan Tritrichomonasfoetus MARLENE BENCHIMOL,* BECHARA KACHAR** *** and WANDERLEY DE SOUZA*,l *Deparrarnento de Parasitologia e Biofisica Celular. Instituto de Biofisica Carlos Chagas Filho, Unrversidade Federal do Rio de Janeiro, IIha do Fundtio, 21941, Rio de Janeiro. Brasil, **Departamento de Histologia, ICB, Universidade de Stio Paulo, 01051. S6o Paulo. B r a d and ***Laboratory of Molecular Otology, NIDCD, National Institutes of Health, Bethesda. Maryland 20892

ABSTRACT. The quick-freezing and freeze-etching techniques were used to analyze surface domains of Tritrichomonasfoetus. The surface of the protozoan body was not smooth, presenting surface projections, except on the flagellar surface. Images of the actual surface of the anterior flagella revealed the presence of intramembranous particles that form rosettes, as observed on the protoplasmic fracture face. They may represent integral transmembrane proteins exposed at the cell surface. Surface specializationswere also observed at the flagella base and where the recurrent flagellum attaches to the cell body. Key words. Electron microscopy, flagellar membrane, freeze etching.


itrichomonas foetus, a pathogenic protozoan of the urogenital tract of cattle, exerts its pathogenic effect when interacting with the surface of epithelial cells [lo]. Because the surface properties of parasitic protozoa play an important role in the process of parasite-host interaction, it is of interest to understand the structural organization of the surface of parasites. In previous studies, we identified some membrane domains in T. foetus using conventional freeze fracture [ 1, 21. In addition, mapping of lipid domains was done using filipin [3] and polymixin B[4]. These studies revealed special arrays of intramembranous particles localized in the membrane of the anterior flagella and in portions of the flagellar membrane involved in attaching the recurrent flagellum to the protozoan body. In the present study, we used quick-freezing and deepetching techniques to reveal surface domains of T. .foetus. MATERIALS AND METHODS The strain of Tritrichomonasfoetus used in the present work was isolated by Dr. H. Guida (EMBRAPA, Rio de Janeiro, Brazil) from the urogenital tract of a bull in Rio de Janeiro. The strain has been maintained by weekly transfers in the trypticaseyeast extract-maltose medium [6]. For the experiments, cells were grown in 1 50 x 1 50 m m tubes containing 20 ml of medium for 24-28 h at 37" C in a COz atmosphere. Cells were harvested by centrifuging at 1,000 g for 5 min in a clinical centrifuge and washed twice in phosphate-buffered saline solution. Some cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. After fixation, cells were rinsed four times in distilled water. Some living cells were frozen rapidly without chemical fixation or cryopreservation. In this case, cells were concentrated in a clinical centrifuge, and a small drop of cells was placed on a specimen support disk that was designed for the Balzers freezeetch apparatus. The specimen disks were affixed to the plunger of a Med-Vac rapid freezing device assembled for impact freezing against a copper block, which was cooled by liquid helium to -270" C or by liquid nitrogen to - 196" C. Rapidly frozen cells were stored in liquid nitrogen before freeze fracturing in a Balzers apparatus. Before etching, only one pass with the mi-

' To whom correspondence should be addressed.

crotome knife was made so that the fracture plane would be within 10-20 pm of the surface of impact to the copper block. After fracturing at - 1 1 5" C, the temperature was raised to - 100" C for 5-10 min for etching. Platinum was evaporated onto the specimen at an angle of 1 5" as the specimen was rotated. Carbon was evaporated at an angle of 90". Replicas were cleaned in a series of bleach and distilled water, mounted on 200-300 mesh grids, and viewed in a JEOL 100 CX electron microscope. Stereo pairs were taken at an angle of +lo" using a rotating-tilting specimen holder. Figures illustrating this technique were printed so that regions containing evaporated platinum were light and regions lacking metal were dark. RESULTS The main components of the anterior region of T..foetus can be identified in deep-etch images such as Fig. 1 that shows where the anterior flagella emerges. An etched view of the protozoan body shows that it is not smooth but has thin surface projections distributed throughout the surface (Fig. 1-3). It projects up to 8 nm from the membrane plane (Fig. 2). However, these projections were not seen in etched views ofthe flagellar membrane (Fig. 1 , 3, 5-10). We showed previously that the P (protoplasmic) face of the membrane lining the anterior flagella presents intramembranous particles forming rosettes [ 1,2]. This observation was confirmed in the present study (Fig. 1,4-7). We clearly observed that most particles on both fracture faces of the flagellar membrane are part of the rosettes. In rapidly frozen and freeze-etched flagella, the rosettes appeared as a ring of 9-12 distinct intramembrane particles (7-10 nm large) on the P face. On the E (extracellular) face they leave a circular furrow indicating an uprooting of particles from the external membrane leaflet or hemibilayer. In the freeze-etched surface view, these rosettes forming particles appear similar in height but slightly more regular and more confluent. Images of the actual flagellar surface also showed the presence of the rosettes (Fig. 5-7). At the etched surface, however, the particles were more closely associated (Fig. 6, 7). Some strands formed by clearly identified intramembranous particles were seen at the base of the anterior flagella, resembling the ciliary necklace (Fig. I , 4, 5 ) . This membrane specialization

+ Fig. 1 4 . Freeze etching of T. foetus. 1. Anterior region of the protozoan showing the emergence of the flagella and part of the cytoplasm (C). Etched view of the protozoan body (*) reveals the presence of a meshwork of filamentous structures. The arrow indicates transition region between fractured (left) and etched (right) surface of the flagellum where the ciliary necklace is evident. Bar = 1 pm. 2. Part of the cytoplasm (C) and actual surface of the protozoan. Filamentous structures are evident (arrows). Bar = 0.2 pm. 3. Region of attachment of the recurrent flagellum (F)to the protozoan body (B). Etched view of both surfaces (*) shows a meshwork of filamentous structures on the surface of the cell body. The flagellar surface is smooth. Bar = 0.2 pm.4. Basal portion of two anterior flagella showing the presence of intramembranous particles forming rosettes (long arrows) and others forming the flagellar necklace (short thick arrows). Bar = 0.2 pm.



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also was seen in etched views of the flagellar membrane, where the particles were more closely spaced (Fig. 5). Membrane specialization was seen in the region where the recurrent flagellum attaches to the cell body, as previously described in conventional freeze-fracture images [ 1, 2, 121. We could see clearly that specialization is also apparent on the actual flagellar surface (Fig. 8). In favorable views, the 0.3 nm periodicity of the array of particles at this region was evident (Fig. 9). Filamentous projections connected the smooth surface of the recurrent flagellum to the rugose surface of the protozoan body (Fig. 10). DISCUSSION Our present observations extend to a pathogenic protozoan, studies carried out mainly with mammalian cells that show the advantages of the quick-freeze and freeze-etching rotary replication techniques. This approach contributed to our present understanding of structural organization of the cytoskeleton, cilia, flagella, and other cellular components [8, 91. In the case of T. foetus, a pathogenic protozoa which has been the subject of several morphological studies using cytochemistry and freezefracture [ 1-5, 1 1, 121, our observations confirm previous studies and provide new information on organization of the cell surface and on some internal structures. We will center the discussion on the most relevant points. Previous cytochemical studies have revealed the presence of a carbohydrate-containing coat on the surface of T. foetus [S]. However, this coat is not seen in usual thin sections. In contrast, using the quick-freezing and freeze-etching rotary replication techniques a filamentous coat was seen that covered the entire surface of the protozoan, except for the membrane lining the three anterior flagella and the recurrent flagellum. This observation indicates the existence of differences between the membrane lining the cell body and that lining the flagella. One characteristic feature of the membrane which lines the three anterior flagella, but not the recurrent flagellum, is the presence of intramembranous particles forming rosettes [ I , 2, 121. Images obtained with a conventional freeze-fracture technique and in the present study show that the rosettes are formed by 9-1 2 clearly identified intramembranous particles. The rosettes on etched regions exposing the actual flagellar surface have particles that are so close to each other that they can no longer be individualized. Because the particles forming the rosettes can be seen on the P-fracture face of the flagellar membrane, as well as on the flagellar surface, they may represent integral transmembrane proteins. The intramembrane images of fast-frozen specimens are similar to those observed in conventional freeze-fracture replicas. In addition to the intramembrane view of the fractured surface, freeze etching exposes the true surface of the flagellar membrane where the extracellular domain of the proteins that forms rosettes can be visualized directly. Since the surface view and the


intramembrane view of the rosettes are practically identical, it is possible that the surface structure of the rosette forming particles corresponds to a surface domain of a transmembrane protein. During cleavage of the membrane, most of the surface domain of the transmembrane protein is being uprooted from the exoplasmic surface. The larger and more irregular appearance of the intramembrane view may be due to plastic deformation during cleavage. The nature of these rosette-forming proteins is not known, but their large size is compatible with, for example, sodium-channel proteins which are also formed of transmembrane proteins with a large surface domain. In almost all cilia and flagella analyzed by the freeze-fracture technique, a specialization has been observed at their base, forming the ciliary necklace [7].In T.foetus this specialization appears as rows of clearly distinguished intramembranous particles at the base of the flagellum. One important finding from the present study is that the intramembranous particles, which form the ciliary necklace, project toward the outer portion of the membrane so that they are seen on the flagellar surface exposed by etching. In T.foetus, as well as in other members of the Trichomonadidae family, one flagellum originates from the anterior region together with the other flagella, but bends and projects toward the posterior region, making contact with the protozoan body [ 1 11. Using the freeze-fracture technique, we showed previously the presence of a special array of membrane particles, which form parallel rows localized only at the region of the flagellum facing the cell body. Our present observations confirm these studies and show that these particles are exposed to the flagellar surface since,they can be identified in etched views of the membrane of the recurrent flagellum. In addition, we observed the presence of filamentous structures connecting the surface of the flagellum to the protozoan surface at this region. Further studies are necessary to determine the nature of this structure. ACKNOWLEDGMENTS The authors thank Miss Noemia Rodrigues Goncalves and Mr. Antonio Oliveira for technical assistance. This work was supported by Financiadora de Estudos e Projetos (FINEP), Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq), FundacBo de Amparo a Pesquisa do Rio de Janeiro (FAPERJ), and Brazil-USA Blue Ribbon Programme. LITERATURE CITED 1. Benchimol, M., Elias, C. A. & De S o u , W. 1981. Specializa-

tions of the flagellar membrane of Tritrichomonasfoetus. J. Parasitol.. 67:174-1 78.

2. Benchimol, M., Elias, C. A. & De Souza, W. 1982. Trifrichomonas foetus: fine structure of freeze-fractured membranes. J. ProtoZOO^., 19~348-353.

3. Benchimol, M. & De Souza, W. 1984. Tritrichomonas foetus: localization of filipin-sterol complexes in cell membranes. Exp. Parasitol., 58:356-364.


Fig. 5-10. Freeze etching of T.foetus. 5. Anterior region of the protozoan showing fractured and etched (*) views of the membrane lining the anterior flagella. The arrow indicates the point of transition. The particles forming the flagellar necklace are spaced more closely on the actual surface than in the membrane bilayer. Bar = 0.5 pm. 6 , 7. Fractured and etched (*) views of the membrane lining the anterior flagella. Several rosettes formed by 9-12 intramembranous particles are seen. In regions of transition (arrows), there is evidence that in the etched regions the particles are more closely associated to each other. Fig. 6,7, Bars = 0.2 pm. 8. Region of attachment of the recurrent flagellum (F)to the protozoan body (B) reveals both fractured and etched (*) views. The inner portions of the flagellum and cell body (C, cytoplasm) are also shown. Closely packed particles (arrowheads), seen on the actual flagellar surface exposed by etching, are observed only in the portion of the flagellum facing the protozoan body. Bar = 0.25 pm. 9. Region of attachment of the recurrent flagellum (F)to the protozoan body (B). The periodicity of the particle array on the protoplasmic fracture face of the flagellar membrane is evident (arrowheads). Bar = 0.25 pm. 10. Etched view reveals the presence of filamentous structures (arrows) connecting the smooth surface of the recurrent flagellum to the rugose surface of the protozoan body exposed by etching (*). Bar = 0.2 pm.



4. Benchimol, M. & De Souza, W. 1988. Tritrichornonasfoetus: freeze-fracture cytochemistry using polymyxin B. Exp. Parasitol.. 66: 35-43. 5. Benchimol, M.. Pereira, M. E. A,, Elias, C. A. & De Souza, W. 198 1. Cell surface carbohydrates in Trirrichomonas /oefus. J. ProtoZOO^., 28 1337-342. 6. Diamond, L. S. 1957. The establishment of various trichomonads of animals and man in axenic cultures. J. Parasirol., 43:488490. 7. Gilula, N. B. & Satir, P. 1972. The ciliary necklace. A ciliary membrane specialization. J. Cell Biol., 53:494509. 8 . Goodenough, U. W. & Heuser, J. E. 1982. Substructure of the outer dynein arm. J. Cell Biol., 99798-8 15. 9. Heuser, J. E. & Kirschner, M. W. 1980. Filament organization

revealed in platinum replicas of freeze-dried cytoskeletons. J. Cell Biol., 8 6 ~ 12-234. 2 10. Honigberg, B. M. 1978. Tritnchomonads of veterinary importance. In: Kreier, J. P. (ed.), Parasitic Protozoa, vol. 2. Academic Press, New York, pp. 164-273. 11. Honigberg, B. M., Mattern, C. F. & Daniel, W. A. I97 I. Fine structure of the mastigont systems in Tritrichornonasfoetus. J. ProtoZOO^., 18:183-198. 12. Honigberg, B. M., Volkmann, D., Entzeroth, R. & Scholtyseck, E. 1984. A freeze-fracture electron microscopic study of Trzchornonas vaginalis Don& and Trirrichornonus/oetus(Riedmuller). J. Protozool., 31:116-131. Received 12-10-90, 3-18-91; accepted 3-22-91

J. Prolozoo/., 39(4), 1992. pp. 484-494 0 1992 by the Society of Prolozoologirls

Life Cycle of Goussia pannonica (Molnar, 1989) (Apicomplexa, Eimeriorina), an Extracytoplasmic Coccidium from the White Bream Blicca bjoerkna JULIUS LUKES Instiruie of Parasitology. Czech Academy of Sciences, BraniSovskri 31, 37005 CeskP Budfijovice, Czechoslovakia

ABSTRACT. Life cycle stages of Goussia pannonica from naturally-infected white bream Blicca bjoerkna were studied by light and electron microscopy. Fourteen of the sixteen fish examined were infected, with developmental stages found in all parts of the intestine. Merogonial. gamogonial, and sporogonial stages were localized intracellularly and extracytoplasmically in the microvillous region of enterocytes. They were separated from the gut lumen by closely apposed enterocyte and parasitophorous vacuole membranes. There were two types of extracytoplasmic attachment: I ) monopodial, with a single zone ofattachment, and 2) spider-like, with several isolated zones of attachment to the host cell. First-generation merozoites were formed by ectomerogony. Second- or third-generation merozoites were formed by endodyogeny and endopolygeny. Thirty to 50 biflagellated microgametes developed at the periphery of a microgamont. Macrogamonts contained lipid inclusions, amylopectin and dense granules; however, granules comparable to wall-forming bodies type I and 11 were absent. At the beginning of sporogony, the sporont cytoplasm detached from two layers which subsequently became constituents of the oocyst wall. After the rupture of enterocyte and parasitophorous vacuole membranes, the sporont was released into the water where exogenous sporulation was completed within 48 h. The thin sporocyst wall contained a small longitudinal suture. Sporocyst and oocyst walls were of similar structure. Key words. Cr.vptosporidiuni. enterocyte, fusion, invasion, microvillus, ultrastructure.


S fish coccidia have been given more attention in the last decade, it has become obvious that they differ in many respects from coccidia parasitizing warm-blooded vertebrates [ 101. Cryptosporidium appears to be the only coccidium inhabiting the microvillous region of the intestine of higher vertebrates [43]. However, in both freshwater and marine fish, the same region is parasitized by a wide spectrum of morphologically different species [22]. In addition t o at least two Cwptosporidiutn species [ 13, 191, one of which considerably extends the range of morphological types within this genus [19], members of three other genera (Eimeria, Epieimeria, and Goussia) have been found in the microvillous zone o f fish intestine [22]. Their developmental stages are localized among the microvilli in the apical part of the enterocyte [ 17, 20, 22, 271. Although most members of the genus Goussia live intracytoplasmically, G. ariprnseris [25], G. girellae [ 171, G. janae [22], G. langdoni [28]. a n d G. zarnowskii [ 151 were recently found in the microvillous region of the intestine. Moreover, G. spraguei was described from the microvillous border of kidney tubule epithelium [29]. In his review o n coccidiosis in Hungarian fishes, Molnar [26] published a short description of a new species, G. pannonica, from the intestine o f white bream. Blicca bjoerkna. based o n light microscopic observations of developmental stages and the morphology of oocysts. This paper presents new data on the

ultrastructure of merogonial, gamogonial and sporogonial stages o f this interesting parasite. MATERIALS A N D METHODS Sixteen white bream, B. bjoerkna, from Cernovick? Potok Brook, Sobeslav, South Bohemia were sampled in February, March and April 1990 a n d April a n d May 199 1. Mucosa of the anterior, central and posterior parts of the intestine and the gut contents were examined fresh. Infected tissue was fixed in 10% neutral buffered formalin and paraffin sections were stained with haematoxylin-eosin. For transmission electron microscopy (TEM), small pieces of intestine were fixed in 2% osmic acid in 0.2 M cacodylate buffer a t 4" C for I h, dehydrated a n d embedded in Epon (Serva, Heidelberg)-Araldite (Polysciences, Wanington, PA). Sections were cut with glass knives o n an 8800 Ultratome I11 (LKB, Bromma). Semithin sections were stained with toluidine blue, thin sections were stained either with uranyl acetate a n d lead citrate [42] or with modified Sato's lead stain solution [ 1 11 a n d examined with a Philips 420 electron microscope. To study exogenous sporulation, feces and fecal casts were incubated in tap water at 20" C for several days. Feces containing sporonts and oocysts were fixed in 2% osmic acid in 0.2 M cacodylate buffer, frozen in this buffer by liquid N, for 30 s [3], and then thawed and processed for T E M as described above. Thin sec-

Surface domains in the pathogenic protozoan Tritrichomonas foetus.

The quick-freezing and freeze-etching techniques were used to analyze surface domains of Tritrichomonas foetus. The surface of the protozoan body was ...
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