Megalodiscus temperatus: Scanning electron microscopy of the tegumental surfaces

EXPERIMENTAL PARASITOLOGY 36, 123-130 (1974)

Megalodiscus

Scanning Electron Microscopy of the Tegumental Surfaces

temperatus:

PAUL M. NOLLEN Depadment of Biological Sciences, Western I&rois Macomb, Illinois 61455

University,

AND MATHEW

J. NADAKAWKAREN

Department of Biological Sciences, I&&s State Uniuersity, Normal, Illinois 61761 (Submitted for publication July 16, 1973) NOLLEN, PAUL M., AND NADAKAVTIUREN, MATHEW J. 1974. Megalodiscus ternperatus: .Scanning Electron Microscopy of the tegumental surfaces. Experimental Parasitology 36, 123-130. The tegumental surface of Megalodiscus temperatus forms cobblestonelike areas with rows of indentations encircling the worm. This pattern merges in several areas into folds and ridges, some of which represent the musculature of the posterior sucker and genital pore. Papillae surrounding the base of the oral sucker appear as two types: one with a bulb-like base and a short apical knob; the second typified by a hair-like structure (cilium?) of variable length projecting from a pit. From their location on the oral sucker and the resemblance to previously described structures, they are presumed to be sensory receptors. A circle of papillae on the closed posterior sucker was also observed. Rod-shaped bacteria were seen adhering to some of the worms observed but they were not found to be in any special association with the tegument or in any specific areas of the worm’s surface. INDEX DESCFUPTOJB: Megalodiscus tempera&s; Scanning Electron Microscopy; Sensory receptors; Tegument; Fscherichia coli.

INTRODUCTION

The structure and physiology of the tegument of Megalodiscus temperatus has been studied extensively by Bogitsh ( 1968) and Shannon and Bogitsh (1971) with the transmission electron microscope ( TEM ) . However, in those studies no surface details were described. The presence of bacteria adhering to the tegument of M. temperatus has been observed (B. J. Bogitsh, Vanderbilt University, Nashville, Tennessee; and G. P. Morris, Queens University, Kingston, Ontario, Canada, personal com-

munications) but never reported in the literature. Nollen et al. (1974) isolated Escherichia coli from the tegument of these frog intestinal flukes. They also observed clusters of silver grams just outside the tegument on autorediograms of M. temperatus incubated with tritiated tyrosine, thymidine, and adenosine, which may be emanating from radioisotopes incorporated by the bacteria adhering to the tegument. Treatment with antibiotics did not appreciably remove the peripheral labeling, leading them to believe these silver grains may be due to a drying artifact.

123 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Recent ultrastructural studies of the surfaces of adult monogenetic trematodes and of adults, miracidia, and cercariae of digenetic trematodes have revealed a variety of sensory receptors (Lyon 1972). It is possible that similar structures may be present in adult M. tempera&s. The present study was undertaken to examine the general structure of the tegumental surfaces of adult M. temperatus, to search for tegumentary sensory structures, and to investigate the location and density of bacteria on the tegument. A scanning electron microscope (SEM) was utilized for these observations because of its greater depth of field, resolution, and magnification than that possible with a light microscope. MATERIALS AND METHODS Megalodiww temperatus, freshly isolated from infected frogs (Runu pipiens) were fixed in phosphate buffered 5% glutaraldehyde (pH 7.3) for 30 min. The worms were rinsed in phosphate buffer and post-fixed in phosphate buffered 1% osmium tetroxide (pH 7.3) for 30 min. They were dehydrated through a graded series of ethyl alcohol and mounted on SEM stubs using conductive silver paint. They were then coated with gold and examined in a Cambridge Stereoscan SEM Model II-A. &SULTS

The typical piump nature of Megalodb cus temperatus is shown in Fig. 1. The large posterior sucker is partially closed and shows striations, probably due to the musculature of this organ, The oral sucker seen en face is much smaller and does not show the striations noted on the posterior sucker. The tegument is composed of cobblestonelike areas with definite indentations

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at regular intervals encircling the worm (Fig. 2). When the worm is viewed from the anterior end, these indentations appear as concentric circles surrounding the oral sucker (Fig. 1) . This pattern occurred on all worms viewed and was more extreme in some areas of the tegument, which can be seen in Fig. 3, where indentations are much deeper and the cobblestone appearance not so apparent. The genital pore can be seen in Fig. 3 as a protuberance from the surface of the worm with striations leading into the pore opening. At higher magnifications the oral sucker (Figs. 44) shows a bead-like tegumental surface with numerous papillae surrounding the base of the sucker like a collar. These appear to be of two types, here designated as A and B for sake of convenience (Figs. 5 and 6). Type A has a basal bulb with a short apical knob while Type B is located in a pit and has a long hair-like (cilium?) structure projecting from the base of the pit. The hair-like structure varies in length in the different papillae, indicating that it may be somewhat extensible. In Fig. 6, A and B may show two stages of the Type A receptor while C indicates a Type B receptor with an extremely long cilium. Enlarged views of the posterior sucker are shown in Figs. 7 and 8. In the closed position (Fig. 7) structures that appear to be papillae are seen encircling the sucker. These are too regular in distribution to be caused by random folding of the tegument. However, they do not show knobs or hairlike projections similar to the papillae described from the oral sucker. The texture of the inner lining of the posterior sucker (Fig. 8) is much different from the tegument covering the sucker and the surfaces of the worm. Rod-shaped bacteria in large numbers were found on some worms (Fig. 9) but

FIG. 1. A low magnification view of the worm showing the large posterior sucker and the smaller oral sucker. x100. FIG. 2. The tegument of the adult showing the typical pattern of indentations. x550.

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not on all worms observed in the scanning scope. Bacteria were found on various areas of the worm’s surface but were not restricted to specific tissues or organs. Since the bacteria were not washed off during processing for SEM, it is assumed they adhere by some means to the outer surface of M. temper&m. No special association of the bacteria to the tegument, such as localization in pits or indentations, was observed. DISCUSSION

Observations with SEM show the tegumental surface of Megalodiscus temperatus to be folded into a cobblestone-like pattern of indentations. Similar depressions although not as deep as observed for M. temperatus, have been reported in SEM micrographs of ,the surface of adult Schistosoma mansoni males and females (Silk et al. 1970; Race et al. 1971) and Paragonimus sadoensk cercariae (Ishii and Miyazaki 1968). These surface patterns may represent the internal musculature of the worms located just below the tegument. Two types ‘of papillae (A and B) were observed on the oral sucker of M. temperatus. These papillae (Figs. 5 and 6), from their basic structure and location on the oral sucker, appear to be sensory receptors. Type A papillae are similar to receptors described in SEM studies from the adhesive organ of Apatemon gracilis minor (Erasmus 1969) and the oral sucker of the cercariae of Paragonimus sadoensk (Ishii and Miyazaki 196S) and Schistosomatium douthitti (Johnson and Moriearty 1969). All of these sensory structures have a basal bulb topped by a short apical knob. Type

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B papillae resemble those reported from SEM and TEM studies in both adults and cercariae of Schktosoma mansoni (Morris and Threadgold 1967; Robson and Erasmus 1970; Silk and Spence 1969; Silk et al. 1970; Race et al. 1971; Nuttman 1971; Miller et al. 1972), the cercariae of Fa.scioZu hepatica (Dixon and Mercer 1965) and Himasthlu secunda (Chapman and Wilson 1970), the miracidia of F. hepatica (Wilson 1970), and the tegument of adult Cyathe cotyle bushiensis (Erasmus 1967). These tegumentary structures have a cilium protruding from a pit or surrounded by tegumental tissue. Lyon (1972) has classiiied sensory receptors from monogenetic and digenetic trematodes into the following types based on TEM studies: “( 1) Single receptors consisting of a nerve bulb and a terminal non-motile 9 + 2 cilium with rootlets, (2) Compound (uniciliate) receptors consisting of a number of associated nerve terminals, each of which bears only a single terminal cilium, (3) Compound (mu&iciliate) receptors made up of one or a few nerve endings only each of which bears many modified cilia.” Type B papillae would fit into types 1 and 2 of Lyon if the hairlike structure protruding from them is interpreted as a cilium. The function of these cilium containing receptors is not known but both tangoreception and rheoreception have been postulated (Morris and Threadgold 1967; Lyon 1972). Since the oral sucker of M. temperatus is active in feeding, mechanoreceptors to detect currents and tissue surfaces would prove valuable to the worm.

FIG. 3. The tegumental surface composedof deeper indentations, some of which extend into the genital pore. X480. FIG. 4. The oral sucker with sensory receptors encircling the base of
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FIG. 9. Rod-shaped

ELECTRON

bacteria

adhering

MICROSCOPY

to the tegument

M. tempera&s is known to feed on tissues and blood cells of the intestinal lining of the frog’s rectum. Bogitsh (1972) identified unsheathed sensory receptors in the pharyngeal pouches of M. tempemtw and indicated they were chemoreceptors that may be used “to control contractions of the cecal area and/or to stimulate the secretion or activation of hydrolytic enzymes in the ceca .” It is possible that Type A papillae may be protruded from a tegumental pit. This is illustrated8 in Fig. 6 where the retracted form is represented at A and the protruded form at B. A TEM study of the oral sucker area would resolve this issue by showing the internal structure of the papillae. It would also reveal the internal structure of the papillae found on the posterior sucker

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to the posterior

sucker.

and give some indication of their function, Such a study is underway in our laboratories. The rod-shaped bacteria adhering to the tegument of M. temperatus are not found on all worms or on a specific portion of the worm. Nollen et al. (1974) isolated this bacteria and identified it as Escherich;a coli, which is a common inhabitant of the frog’s rectum. The ability of these bacteria to stick to the tegument through processing for SEM would indicate that they are more than chance associates of the tegument. Shannon and Bogitsh ( 1971) ,demonstrated that the tegument secretes a glycocalyx on the surface of adult M. tempera&x This mucopolysaccharide secretion may be the adhesive substance that allows the bacteria to stick to the tegument. Autoradiograms

FIG. 7. A view of the partially closed posterior sucker with a circle of papillae-like structures (P). x500. FIG. 8. The posterior sucker open showing a different type of tegumental folding. X1230.

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of M. tempera&s exposed to tritiated tyrosine, thymidine, and adenosine show clumps of silver grains on the surface of the tegument (Nollen et al. 1974). This labeling was not found on all autoradiograms and comd not be associated with specific areas of the worm’s surface. The observations of bacterial location by SEM and peripheral labeling on autoradiograms correlates to a high #degree. The labeling is thus probably due to radioisotopes incorporated by bacteria adhering to the tegument. With this in mind, it is important for future studies on uptake and incorporation of metabolites of hehninth parasites using radioisotopes to consider the possibility of erroneous results from assimilation of these compounds by surface-adhering bacteria. REFERENCES BOGITSH, B. J. 1968. Cytochemical and ultrastructural observations on the tegument of the trematode Megalodiscus temperatus. Transactions of the American Microscopical Society 87, 477-486. BOGITSH, B. J. 1972. Cytochemical and biochemical observations on the digestive traots of digenetic trematodes. IX. Megalodiscus temperatus. Experimental Parasitology 32, 24P 260. CHAPMAN, H. D., AND WILSON, R. A. 1970. The distribution and fine struoture of the integumentary papillae of the cercariae of Himasthla secunda (Nicoll). Pamsitobgy 61, 219227. DIXON, K. E., AND MERCER, E. H. 1965. The fine structure of the nervous system of the cercaria of the liver fluke, Fasciola hqrtica L. Journal of Parasitology 51, 967-976. ERASMUS, D. A. 1967. The host-parasite interface of Cyathocotyle bushier&s Khan, 1962 m(Trematoda; Strigeoidea) II. Electron microscope studies of the tegument. Journal of Parasitology 53, 703-714. ERASMUS, D. A. 1969. Studies on the host-parasite interface of strigeoid trematodes. V. Regional differentiation of the adhesive organ of Apatenwn gracilis minor Yamaguti, 1933. Pamsitology 59, 245-256. Isrm, Y., AND MIYAZAKI, I. 1968. Preliminary observations on the ultrastructure of the body surface of Paragonimus cercaria. IaTapanese Journal of Parasitology 17, 487493.

JOHNSON, D., AND MORJEARTY, P. 1969. Examination of schistosome cercariae and schistosomules by scanning electron microscopy. Eleck~ Microscopy Society of America 27, 50-51. LYON, K. M. 1972. Sense organs of monogeneans. In: “Behavioral Aspects of Parasite Transmission” (E. U. Canning and C. A. Wright, Eds. ), Zoological Journal of the Linnean Society 51, Suppl. 1, 181-199. MILLER, F. H., TULLOCH, G. S., AND KUNTZ, R. E. 1972. Scanning electron microscopy of the integumentary surface of Schistosoma mansoni. Journal of Parasitology 58, 693-698. MORRIS, G. P., AND THREADGOLD, L. T. 1967. A presumed sensory structure associated with the tegument of Schistosoma mansoni. Journal of Parasitology 53, 537-539. NOLLEN, P. M., PYNE, J. L., AND BAJT, J. E. 1974. Megalodiscus tempera&x Absorption and incorporation of tritiated tyrosine, thymidine, and adenosine. Experimental Parasitalogy 35, 132-140. N-MAN, C. J. 1971. The fine structure of ciliated nerve endings in the cercaria of Schistosoma mansoni. Journal of PaTasitology 57, 855-859. RACE, G. J., MARTIN, J. H., MOORE, D. V., AND LARSH, J. E., JR. 1971. Scanning and transmission electron microscopy of Schistosoma mansoni eggs, cercariae, and adults. American Journal of Tropical Medicine and Hygiene 20, 914-924. R OBSON, R. T., AND ERASMUS, D. A. 1970. The ultrastructure, based on stereoscan observations, of the oral sucker of the cercaria of Schistosoma munsoni with special reference to penetration. Zeitschrift fur Parasitenkunde 35, 76-86. SHANNON, W. A., JR., AND BOGITSH, B. J. 1971. Megalodiscus temperatus: Comparative radioautography of glucose-3H and galactose-3H incorporation. Experimental Parasitology 29, 309-319. SILK, M. H., AND SPENCE, I. M. 1969. Ultrastructural studies of the blood fluke-Schistosoma munsoni. III. The nerve tissue and sensory structures. South African Journal of Medical Science 34, 93-104. SILK, M. H., SPENCE, I. M., AND Buca, B. 1970. Observation of Schistosoma munsoni blood flukes in the scanning electron microscope. South African Journal of Medical Science 35, 23-29. WILSON, R. A. 1970. Fine structure of the nervous system and specialized nerve endings in the miradicium of Fasciola hepatica. Parasitology 60, 399410.