Ultra Structure of Balantidium

ULTRA STRUCTURE OF BALANTIDIUM

V. ZAMAN Department of Parasitology, Faculty of Medicine, University of Singapore.

Parasitic ciliate Balantidium has a very microscope. For light microscopy, the wide distribution in nature and has been ciliates were transferred to starch-free medium reported from 50 or more species of animals for 7-8 days and then examined under phase­ (Moulton, et aI, 1961). The parasite is also contrast. Starch containing parasites were of medical importance as it is the only ciliate quite unsuitable for this purpose as starch which infects man. This is the first publica­ grains obscured the internal structure of the tion on the ultra structure of the parasite. parasite. The parasites were photographed using a Leitz "Orthomat" microscope, with In this study, a species of Balantidium, an electronic flash. isolated from turtle, has been used as this species grows luxuriantly in vitro. The exact RESULTS taxonomic position of the species is, however, uncertain. Balantidium has been described The surface of the parasite is covered with previously in tortoise by Chagas (1911) and thousands of cilia arranged in linear manner Wenyon (1926). and the parasite has a striped appearance under phase-contrast. (Fig. 3). The lines MATERIAL AND METHODS of cilia start from the oral cavity and extend backwards over the body of the parasite to The parasite was first isolated in hsm + S the posterior end. (Jones, 1946) and maintained in this medium. This is a simple monophasic medium con­ In the electron microscope the surface of taining horse serum and yeast autolysate. the parasite shows alternating ridges and The parasite was kept at room temperature furrows both in the longitudinal and cross (25-30° C) and subcultured at intervals of 7 sections (Fig. 1, 2). The ridges are an exten­ days. The cultures contained a mixed bac­ sion of the ectoplasm of the parasite and the terial flora and also Entamoeba terrapinae. cilia arise from the furrows (Fig. 2-4). The The Entamoeba grew luxuriantly along with cell membrane or the pellicle is double-layered the Balantidium and neither parasite adversely and covers the ridges and the furrows. The affected each other's growth. For electron outer layer of the cell membrane is reflected microscopy, sediment from 3-4 day-old to the protruding cilium at each ciliary exit cultures was pooled from a number of tubes (Fig. 5). The cilia on cross-section display and washed by centrifugation in phosphate the universal 9+2 pattern of tubular fibrils buffered saline (pH 7.2). The washed para­ (Fig. 4). The basal region of the cilium or sites were fixed in buffered glutaraldehyde kinetosome is tubular and opens proximally (pH 7.2) at 5° C for 12 hours and subsequently (Fig. 5) and maintains an almost constant post-fixed in buffered osmium tetroxide, diameter throughout its length. In the centre dehydrated in graded alcohols and embedded of the distal part of the kinetosome is an in Araldite. Sections were cut on a Porter­ electron dense oval body or the central Blum microtome with .glass knives and corpuscle. From this central corpuscle 2 mounted on formvar coated grids. The central fibrils of the cilium extend up to the examination was made in a Hitachi HS-8 base of the kinetosome. In some sections,

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Fig, 1 - b = bacteria Fig, 2 - er = ectoplasmic ridges er = ectoplasmic ridges f = fibrils MN = macronucleus c = cilia mn = micronucleus s = starch m = mitochondria 226 Vol. I No,2 June 1970 ULTRA STRUCTURE OF Balantidium .

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Fig. 3 - Phase-contrast. Showing the striped Fig~ 5 - cm = cell membrane appearance of Balantidium. cc = central corpuscle cf =. central fibril pf = peripheral fibril Fig. 4 - er = ectoplasmic ridges k '" kinetosome cf = central fibril m = mitochondria pf = peripheral fibril t = tubules b= bacteria Vol. I No.2 June 1970 227 SOUTHEAST ASIAN J. TROP. MED. PUB. HLTH.

Fig. 6 - m = mitochondria Fig. 7 - MN = macronucleus b = bacteria mn = micronucleus mn = nuclear membrane MN = macronucleue nl = nucleoli Fig. 8 - MN = macronucleus mn = micronucleus mn = micronucleus s = starch s = starch

228 Vol. I No.2 June 1970 ULTRA' STRUCTURE OF Balantidium fibrils are also seen arising from the base of is large and displays a very dense rope-like the kinetosome and running posteriorly (Fig. network of chromatin, unlike many other 2). In the cross-section tubules are seen in ciliates where the chromatin material is the peripheral region of the parasite (Fig. 5). arranged as oval or round bodies. A number In the cytoplasm the starch grains are clearly of nucleoli are seen in between this network visible as large crystalline bodies. The bac­ of chromatin, a feature which had not been teria are seen as smaller dense electron bodies recognized in previous light microscopic having a smooth outline (Fig. 1, 6). The studies. The structure of the micronucleus mitochondria appear as structures with very is also unusual as it does not contain a great low electron density situated mainly in the deal of electron dense material usually seen ectoplasm or the periphery of the parasite in other ciliates (Roth, 1957; Seshachar, (Fig. 1, 6, 5). The mitochondrial contents 1964; Tsujita, et ai, 1957). In some sections 2 are homogenous and do not display any cris­ micro nucleoli have been observed, a finding tae or microvilli. The most prominent which has not been recorded in previous light structure in the cytoplasm of the parasite is microscopic studies. The structure of the the macronucleus (Fig. 1, 6). This is sur­ mitochondria in Balantidium is also of parti­ rounded by a nuclear membrane which does cular interest as it shows no internal structures not have pores. The nucleoplasm contains such as microvilli or cristae. As is well­ a very distinct network of electron dense known mitochondria are of great importance chromatin. In between this network are a in the oxidative phosphorylation in aerobic number of electron dense oval or slightly cells. Balantidium is essentially an anaerobic irregular bodies which are identified as cell living in the caecum and the large intestine. nucleoli. The micronucleus is found close Even in cultures, it grows at the bottom of the to the macronucleus in a depression on its tubes in relatively anaerobic conditions. All surface (Fig. 1,6,7,8). The internal structure the free-living protozoa have very distinct of the micronucleus is of low electron density mitochondria but in Balantidium the structure and it lacks the chromatin network seen in the has probably lost its significance during the macronucleus. The micronucleus tends to course of evolution from the free-living to appear triangular in cross sections (Fig. 1,7,8). parasitic life. Mitochondria with poorly In a few sections 2 micronuclei are seen lying developed internal structure have also been close to each other (Fig. 8). described previously in other parasitic ciliates (Rudzinska, et aI, 1966). Entamoeba which DISCUSSION has similar cultural requirements and lives in the same habitat as Balantidium, mitochon­ The study shows a number of features dria are completely absent (Deutsch and which are common to other free-living and Zaman, 1959; Miller, et aI, 1961). In this parasitic ciliates. These include the ridges respect Balantidium seems TO occupy an and furrows on the surface, the structure of intermediate position between the free-living the cilium consisting of 9+2 pattern of the protozoa and Entamoeba. peripheral and central fibrils, the double­ layered cell membrane and the tubular SUMMARY structure of the kinetosome (Randall, 1956; Grimstone, 1961; Pitelka, 1963). The ultrastructure of Balantidium has been studied. It was found that the surface consists There are, however, some unusual features of alternating ridges and furrows. The cilia not previously recognized. The macronucleus arise from the furrows and display the univer­

Vol. I No.2 June 1970 229 SOUTHEAST AsIAN J. TRap. MED. PUB. HLTH. sal 9 + 2 pattern of internal fibrils on cross GRIMSTONE, A.V., (1961). BioI. Rev., 36:97. section. The cytoplasm consists of food JONES, W.R., (1946). Ann. Trop. Med.Parasit., vacuoles containing starch grains and bacteria. 40:130. The mitochondria which are located mostly at the periphery are indistinct and do not MILLER, J.H., SWARTZWELDER, J.C. and have any organized internal structures. The DEAS, J.E., (1961). J. Parasit., 47:577. macronucleus shows electron d(!nse rope-like MOULTON, J.E., HEUSCHELE, W.P. and SHERI­ network of chromatin with interspaced DAN, B.W., (1961). Cornell Vet., 51 :350. nucleoli. The micronucleus is situated in a depression on the macronucleus. Occasional­ PlTELKA, D.R., (1963). Electron-microscopic ly two micronuclei were observed. structure of Protozoa. Pergamon Press. London. ACKNOWLEDGEMENTS RANDALL, J.T., (1956). Nature, 178:9.

During the study, technical assistance was ROTH, L.E., (1957). Biophys. Biochem. Cytol., provided by Mr. Loh Ah Keong. The ultra­ 3:985. microtome was obtained through the China RUDZINSKA, M.A., JACKSON, J.G. and TUFF­ Medical Board of New York. RAU, M., (1966). J. Protozool., 13:440. SESHACHAR, B.R., (1964). J. Protozool., REFERENCES 11 :402. CHAGAS, C., (1911). Mem. inst. O. Cruz., TSUJITA, M., WATABE, K. and TSUDA, S., 3: 136. (1957). Cytologia., 22:322. DEUTSCH, K. and ZAMAN, V., (1959). Exptl. WENYON, C.M., (1926). Protozoology. Bail­ Cell Res., 17:3lO. liere, Tindall. and Cox. London.

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