The infrastructure of the tegument of Moniliformis dubius (Acanthocephala)

Transcription

1 137 The infrastructure of the tegument of Moniliformis dubius (Acanthocephala) By W. L. NICHOLAS and E. H. MERCER (From the Department of Zoology and the John Curtin School of Medical Research, Australian National University, Canberra, Australia) With s plates Summary The ultrastructure of the body wall of Moniliformis dubius has been studied in the light and electron microscope. It consists of an apparently syncytial tegument, overlaid by a tenuous cuticle in the form of a finely fibrous extracellular fringe and is backed by a basement membrane and fibrous connective tissue. The tegument contains a framework of fibres, which, distally, is connected to a dense fibrous meshwork separated from the cuticle by two membranes. Within the syncytial tegument are found the usual cytoplasmic organelles: mitochondria (often degenerate in structure), Golgi clusters, small amounts of other smooth membranes, and numerous dense particles (glycogen and perhaps ribosomes). Many mitochondria contain dense particles. Evidence of vacuole formation at the surface of the tegument suggests that pinocytosis plays a part in assimilation. Introduction THE ultrastructure of the acanthocephalan body wall has not been described in detail. Rothman (1961) published a brief note on the appearance of the body wall of Macranthorhynchus hirudinacens under the electron microscope and Nicholas and Hynes (1963) included an electron micrograph of a section through the body wall of Moniliformis dubius in a review of the group. Some observations have also been made on the acanthor with the electron microscope by Burnham (1957). Baer (1961) has described the acanthocephalan body wall as it appears under the light microscope. According to him it consists of a cuticle of two layers, overlying a complex hypodermis, also divisible into two layers. The outermost layer of the cuticle is thin and apparently structureless. Beneath this, the second layer, also thin, has fine striations perpendicular to the surface. The outer part of the hypodermis contains a complex system of fine, interwoven fibres, which may appear felt-like, or as layers of fibres parallel to the surface, alternating with felt-like layers. The inner part of the hypodermis contains fibres orientated perpendicularly to the surface, its elements inserted into the felt layer, distally, and into a basal membrane, proximally. A system of fluid-filled canals lies within the deeper parts of the hypodermis. The whole of the hypodermis, like many of the other body tissues, appears syncytial. The body wall contains few large nuclei in relatively fixed positions. In some acanthocephala, including M. dubius, these become very [Quart. J. micr. Sci., Vol. 106, pt. 2, pp , 1965.]

2 138 Nicholas and Mercer Tegument of Moniliformis large and ramified; in others the nuclei of the body wall fragment during larval development. Von Brand (1939) included some histochemical observations on the distribution, of lipid and glycogei. in a study of the chemical composition of Macranthorhynchus hirudinaceus, and Bullock (1949) made further observations on both in a number of other species. Crompton (1963) made a comprehensive histochemical study of Polymorphic minutus and, in addition, identified a thin layer of mucopolysaccharide outside the tegument, which he termed the epicuticle. The distinctions drawn in the past between cuticle and epicuticle on the one hand, with the implication that these are successive layers of a secreted non-living structure, and the hypodermis, on the other, implying a living cellular structure, are misleading. As we shall show, both the cuticle and the hypodermis recognized by Baer (1961) are cytoplasmic. We shall use the term tegument for both. Rothman (1959) and Threadgold (1963) in their work on platyhelminthes also adopted the term tegument, for analogous reasons. It would then seem logical to rename the epicuticle of Crompton (1963) as cuticle, although again, as we shall show, it hardly constitutes a tough outer covering as is sometimes implied by the term. M. dubius lives, as an adult, in the intestine of the rat, and in the larval stages, in the haemocoel of cockroaches and other insects. Its life cycle has been described by Burlingame and Chandler (1941) and Moore (1946). Material and methods Moniliformis dubius was maintained in the laboratory with rats as the definitive host and cockroaches, Periplaneta americana, as the intermediate host, following the technique described by Burlingame and Chandler (1941). We selected for our study immature worms, taken from rats which had been infected for two to three weeks, and sexually mature worms of both sexes from infections of more than six weeks standing. The worms were transferred from the intestine of the rat to Ringer's solution, rapidly rinsed, and cut into small pieces. Small segments of the body wall were then rapidly transferred to fixative. Most of the specimens were fixed in 1% osmium tetroxide (Palade, 1952), dehydrated in a series of ethanol solutions of increasing strength and embedded in Araldite. Sections, cut with diamond or glass knives, were stained with either 3-5% aqueous uranyl acetate or lead citrate (Karnovsky, 1961) before examination with a Siemens Elmiskop I electron microscope. Some specimens were fixed in 1% potassium permanganate (Luft, 1956) followed by staining with uranyl acetate, and others, fixed in osmium tetroxide, were treated with 1 % phosphotungstic acid in absolute alcohol before embedding. Glutaraldehyde was used to fix tissues prior to treatment with enzymes (Sabatini, Bensch and Barrnett, 1963). A 3% solution in o-i M sodium phosphate buffer of ph 7-3, containing a trace of CaCl 2, was employed. So that the structures seen under the electron microscope could be corre-

3 Nicholas and Mercer Tegument of Moniliformis 139 lated with gross morphology, thicker sections (about 2-5 /x) were cut from the same blocks and examined in the phase-contrast microscope. These in turn were compared with conventional sections prepared from paraffin-embedded tissue, stained in haematoxylin and eosin. To identify glycogen deposits and ribosomes small pieces of the body wall from immature female worms were subjected to enzymic hydrolysis and then compared with water-treated controls by light and electron microscopy. For glycogen, tissues fixed in glutaraldehyde, washed in water, and digested in human saliva (7 h at room temperature) were transferred to the osmium fixative and prepared for electron microscopy. For light microscopy, paraffin sections, prepared from Carnoy-fixed tissue, were treated with saliva and stained by the PAS reaction (Pearse, i960). Ribonuclease (Sigma) digestion in water (nine hours) at 37 0 C was used for ribosomes (Pearse, i960). Tissue fixed in glutaraldehyde before digestion and transferred to the osmium fixative afterwards was prepared for electron microscopy. For the light microscope, frozen sections of unembedded tissue were examined in acridine orange by fluorescent microscopy (Bertalanffy, Masin, and Masin, 1958). Diminution in orange fluorescence following digestion of the sections was taken as an indication of RNA. For lipids, frozen sections were cut from tissue fixed in formaldehyde-calcium (Baker, 1944), embedded in gelatine, and stained with Sudan black. Results The tegument of Moniliformis dubius, as seen in paraffin sections stained with haematoxylin and eosin, under the light microscope corresponds closely with the tegument of other Acanthocephala described by Baer (1961) and Crompton (1963). Its structure as revealed by the light microscope is shown diagrammatically (fig. 1). Because of some confusion in nomenclature, which has arisen from the limitations of the light microscope, we have preferred to identify the successive layers of the cuticle and tegument by the Roman numerals I to VI. The principle features observed under the electron microscope are shown in two low power electron micrographs (figs. 2 and 3). The outermost layer I, which was named the epicuticle by Crompton (1963) in his study of Polymorphus minutus and which he concluded on histochemical grounds was formed from mucopolysaccharide, is clearly visible under the electron microscope (figs. 2 and 4). We have confirmed that in M. dubius layer I is PAS-positive and that the reaction is not diminished by salivary digestion. It consists of a meshwork of fine fibres (less than 100 A in diameter), apparently randomly orientated. It shows no sharp outer boundary and varies a good deal in thickness in different specimens, but is generally about 1 fx thick in adult worms and somewhat thinner in immature specimens. Layer I is separated from the deeper layers of the tegument by a typical plasma membrane ca 80 A thick and resolvable into 2 lines at high resolution (fig. 8). Beneath this and adjacent to it, there is a second more electron-dense

4 140 Nicholas and Mercer Tegument of Moniliformis membrane, the sub-plasma membrane. Both membranes are shown under high magnification in fig. 8. The layers between the outer plasma membrane and the basement membrane, i.e. layers II to V are intracellular and contain the usual cell organelles as well as a system of coarser fibres. A further doublecontoured plasma membrane separates layer V from the connective tissue (VI). However, though the tegumentary layers II to V are cytoplasmic, the tegument cannot be considered cellular, in the strict sense, since it is not o\-. %\r>;i!/ih^:i--7^^r/vl [egument Sub-tegumentary muscle - pseudocoel FIG. I. Highly diagrammatic representation of the body wall of Moniliformis dubius to show the relative positions and approximate thickness of the successive layers. Its relative dimensions are those of an adult female worm, cm, circular muscles; Ic, canals of lacunar system; I, lipid inclusion; Im, longitudinal muscles; «, nucleus; ni, nuclear inclusion; tf, tegumentary fibres; v, vacuole. divided into discrete cells, but is a syncytium formed from a small number of large cells. The tegument contains relatively few large nuclei which can be found quite readily under the light microscope in paraffin sections stained with haema- FIGS. 2 and 3. Low-power views of a transverse section of the tegument of an immature worm (osmium tetroxide fixation and uranyl acetate stain) to show the appearance of each of the layers I to VI. FIG. 2 shows the outer layers and Fig. 3 the deeper layers.

5 FIGS. 2 and 3 W. L. NICHOLAS and E. H. MERCER

6 FIGS. 4 and 5 W. L. NICHOLAS and E. H. MERCER

7 Nicholas and Mercer Tegument of Moniliformis 141 toxylin and eosin, but are difficult to find under the electron microscope in the adult worm because they are widely separated in the tegument and the area surveyed electron-microscopically is so small. They are easier to find in the smaller immature worms. The nuclear membrane, the nucleolus and other nuclear contents resemble those described in higher animals. Immediately beneath the sub-plasma membrane, in layer II there is a dense meshwork with long, tapering interstices running perpendicularly to the surface (figs. 2, 4, and 8). The structure can only be recognized as a meshwork in partially (fig. 4) or wholly tangential sections. In strictly transverse or longitudinal sections (i.e. sections at right angles to the surface) the structure appears to consist of branching septa perpendicular to the body surface, interspersed with less dense pores which taper as they approach the body surface. It is these septa which have been interpreted as fine striations by previous workers using the light microscope. The septa in a large adult worm are about 3-3 \i deep. The pores between the septa taper from about o-6 ju,, proximally, to about 0-06 \L at the outer surface. The interstices of the mesh contain membrane-bounded vesicles of various sizes, many of them large enough to occlude the pores (fig. 8). Frequently, both the plasma membrane and the sub-plasma membrane appear to dip down into the pore (fig. 8). The sub-plasma membrane cannot be followed very far into the pores, but the plasma membrane often extends well down into the pore (layer II), and it seems likely that the larger vesicles between the septa and those in the deeper regions of the tegument are formed from an invagination and pinching-off of the plasma membrane, their limiting membrane being derived from the plasma membrane (fig. 8). Rarely, two concentric double-contoured membranes were seen to enclose a vesicle, and possibly the outer of these two membranes is formed from the sub-plasma membrane, though the latter has not been shown to be double-contoured at the surface of the tegument (fig. 8). The fibres which form a prominent part of the tegument under the light microscope can be resolved in the electron microscope into bundles of finer fibres, which we shall term tegumentary fibres. The fibre bundles of the tegument follow an open spiral path and constitute a mechanical framework, distally continuous with the base of the meshwork (fig. 4) and proximally joined to the basement membrane (fig. 7). The individual fibres composing the bundles are strongly stained by osmium tetroxide, potassium permanganate, uranyl acetate, and phosphotungstic acid, but poorly by lead citrate. They look tubular since only the periphery of the fibre shows affinity for FIG. 4. This shows the peripheral region of the tegument (layers I to III) of an immature worm in transverse section (osmium tetroxide fixation and uranyl acetate stain). The septa have been cut somewhat tangentially. c, cuticle; mi, mitochondrion; rf, radial tegumentary fibres; s, septa; v, vesicles. FIG. 5. This shows layer IV in a transverse section from an immature worm (osmium tetroxide fixation and uranyl acetate stain). er, endoplasmic reticulum; g, Golgi cluster; ff, feltwork of tegumentary fibres; /, lipid inclusion; mi, mitochondrion; rf, radial tegumentary fibres.

8 142 Nicholas and Mercer Tegument of Moniliformis heavy metals and a 'core' does not (fig. 9). No banding has been observed in these fibres. No sharp distinction can be made between the meshwork and the fibre bundles, which resemble one another in their affinity for heavy metal stains, and, although at the periphery of the tegument the meshwork appears to be formed from closely packed and extremely fine fibrous material (fig. 8), at its base they aggregate into larger fibres continuous with those of the tegumentary bundles. The fibre bundles containing relatively few fibres are predominantly radial in their orientation in layers III (fig. 2) and V (fig. 3). In layer IV they are more numerous, contain more fibres, and form a feltwork (figs. 2 and 9). Layer III, lying between the septa and the felt layer IV, from which it is not sharply delimited, is filled with vesicles to the virtual exclusion of all other structures, apart from some radially orientated fibres (figs. 2 and 4). Many of these vesicles resemble those found in the pores and are probably derived from them. Similar vesicles are present in the deeper layers of the tegument though they are less numerous than at the base of the septa. Mitochondria of somewhat atypical appearance are present throughout the tegumentary layers III to V (figs. 4, 5, 6, and n). In specimens taken from immature worms they were identified by the presence of two concentric double-contoured membranes, the inner membrane forming few poorly developed cristae. In specimens taken from adult worms mitochondria were difficult to identify with certainty, partly, we believe, because of difficulty in ensuring rapid fixation of adult tissues, and partly because cristae were very poorly developed. Vesicles containing myelinic forms were not infrequent and it seems possible that mitochondria show an increasing tendency to degenerate in the adult. Lipid droplets are present throughout layers III, IV, and V ranging in size from droplets which are clearly seen under the light microscope (in frozen sections stained with Sudan black) to very small ultra-microscopic droplets (figs, 5 and 6). Their numbers varied greatly in different specimens and may be very considerable (as observed in frozen sections under the light microscope and in Araldite sections under the electron microscope). Other membranous organelles (elements of the reticulum) are sparsely distributed throughout layers III, IV, and V. Small stacks of parallel paired membranes (Golgi clusters) are common in immature specimens and less so in adults (figs. 5 and 6). Small dense particles, 150 A to 400 A in diameter, are common throughout the cytoplasm. The larger particles are aggregates of smaller particles. From Fie. 6. Layer V of an immature worm in transverse section (osmium tetroxide fixation and uranyl acetate stain), er, endoplasmic reticulum; g, Golgi cluster; /, lipid inclusion; mi, mitochondrion; m, myelinic forms. An arrow points to intramitochondrial ribosome-like particles. Fie. 7. This shows part of layer V, the basement membrane, and the connective tissue forming layer VI, in transverse section from an immature worm (osmium tetroxide fixation and uranyl acetate stain), bm, basement membrane; cf, connective tissue fibres; rl, radial tegumentary fibres; v, vesicles. The arrows point to the plasma membrane.

9 i I FIGS. 6 and 7 ^*-</.'7.z-,w i x::;*j.-*i-y i s>w W. L. NICHOLAS and E. H. MERCER

10 W. L. NICHOLAS and E. H. MERCER

11 Nicholas and Mercer Tegument of Moniliformis 143 their affinity for lead and their size the larger particles were thought to be glycogen. The diminution in their density when stained with lead following salivary digestion supports this identification. Some of the larger particles are present in vesicles and their density is not reduced following digestion. The presence of glycogen in tegumentary layers III to V was confirmed by a decrease in the PAS reaction following digestion. The smaller particles are perhaps ribosomes. The presence of RNA in the tegument was established by fluorescence microscopy. An orange fluorescence characteristic of RNA in acridine orange was reduced following ribonuclease treatment. The appearance of the tissue in the electron microscope was not substantially altered by ribonuclease digestion. Small groups of dense particles are often found within structures which we have identified as mitochondria (fig. 6). At the base of the tegument there is a double-contoured cell membrane, backed by a basement membrane (figs. 7 and 10.) Beyond this lies an extracellular space containing fine fibrils, groups of which show differing orientations (figs. 3 and 7). These fine fibrils often show evidence of periodicity but it was not possible to identify them certainly with known fibrous structures. The coarser, radially-orientated tegumentary fibres appear attached to the plasma membrane, the point of insertion being marked by an electron-dense thickening of the surface (fig. 7). Narrow projections of the plasma membrane, together with the fibre attachments, extend distally from the basement membrane. In some places, vesicles, resembling those found through the body of the tegument, line the distal surface of the plasma membrane (figs. 7 and 10). Sometimes the plasma membrane is drawn out to form an incompletely enclosed vesicle (fig. 10). It seems logical to interpret the static picture seen here as part of a dynamic process by which vesicles are either being pinched off from the plasma membrane, or, alternatively, are coalescing with it. Beneath the extracellular space lie circular and longitudinal muscle cells. In their basic structure, the body walls of male, female, and immature worms resemble one another. A very considerable increase in size accompanies maturation, the female worms used in our study having about ten times the diameter of the immature worms. Males are slightly smaller than the females. It is difficult to be precise, because the body is elastic and moniliform, but all the layers (I to VI) increased in thickness, although we have made no attempt to determine whether their relative proportions remained constant. Other differences between the immature worm and the adult are that FIG. 8. A high-power view of the outer surface of the tegument of an immature worm in transverse section (osmium tetroxide fixation and uranyl acetate stain). It shows structures, indicated by arrows, which have been tentatively identified as pinocytotic vacuoles in the process of formation. Also indicated is a vesicle («i) enclosed by two concentric plasma membranes, c, cuticular fibres; pm, plasma membrane; sm, sub-plasma membrane; s, septa in slightly tangential section; v, vesicle. FIG. 9. A high-power view of the feltwork of fibres seen in layer IV of an adult worm in transverse section (osmium tetroxide fixation and uranyl acetate stain).

12 144 Nicholas and Mercer Tegument of Moniliformis membranous organelles (elements of the reticulum, Golgi clusters, mitochondria) were less prominent in the latter. The tegumentary fibres on the other hand became more numerous. Discussion The tegument of M. dubius shows many interesting features, some of which cannot be satisfactorily interpreted by the purely descriptive approach adopted in this paper. In this sense, our account must be considered as exploratory and our interpretations as tentative. It confirms previous descriptions of the tegument (Baer, 1961) at the level resolvable by the light microscope. The cytoplasmic fibres, which form so prominent a feature of the tegument, are unlike any previously described. From their architecture they clearly serve a mechanical purpose. The dense meshwork found at the periphery of the tegument, layer II, is perhaps formed from the same material as the tegumentary fibres. The much finer fibres beneath the basement membrane are different in appearance and superficially resemble fine collagen fibres. The cuticle layer I (epicuticle of Crompton, 1963) is composed of still finer filaments probably mucopolysaccharide in nature. The invagination of the external surface membranes to form long tongues within the meshwork of fibres (layer II) of the tegument and the numerous vesicles seen immediately below this layer are strongly suggestive of pinocytosis. The formation of pinocytotic vacuoles over the whole surface of the body could provide a means of assimilating nutrients in an animal not provided with an alimentary canal, but, in the absence of experimental evidence for this hypothesis, it must be considered tentative. A similar hypothesis has been advanced by Rothman (i960; 1963) for assimilation in cestodes which like acanthocephala lack an alimentary canal, and by Threadgold (1963) to explain vacuolar structures seen in the tegument of another endoparasite, Fasciola hepatica, though this animal possesses a functional alimentary canal. Bjorkman and Thorsell (1964) studied the uptake of ferritin by F. hepatica with the electron microscope, but, although they found ferritin had been taken up by the tegument, they considered that the process was not pinocytosis, and preferred the term 'transmembranosis'. Crompton (1963) suggested that the epicuticular material in the acanthocephalan P. minutus, which we have identified with layer I in M. dubius, was secreted over the surface from the pores. If this view is correct, the structures FlG. 10. This shows a high-power view of the basement membrane, which separates the layers V and VI. Balloon-like extensions of the plasma membrane into layer V are shown. Transverse section of immature worm (osmium tetroxide fixation and uranyl acetate stain). Roman numerals identify the tegumentary layers and an arrow indicates the plasma membrane, bm, basement membrane; ct, connective tissue fibres; v, vesicle, apparently either in the process of formation or of coalescing with the plasma membrane. FIG. 11. This shows vesicles, with one double-contoured membrane, and mitochondria with two concentric membranes. Transverse section of immature worm, layer III (osmium tetroxide fixation and uranyl acetate stain), mi, mitochondrion; v, vesicle.

13 VI f.u FIGS, IO and W. L. NICHOLAS and E. H. MERCER n

14 Nicholas and Mercer Tegument of Moniliformis 145 which we have suggested are pinocytotic vacuoles in the process of formation might be secretory vacuoles in the act of discharging. These seem, however, rather too numerous to serve this purpose alone. Whatever the nature of vacuolar structures the epicuticle must affect the passage of nutrients to the tegument. Mitochondria show few poorly developed cristae, although these are better developed in the immature worms. There are signs (myelinic figures and loss of inner membrane) that mitochondria may degenerate in the adult. M. dubius is a facultative anaerobe (Laurie, 1957,1959) and mitochondria may lose some definitive structural features in anaerobically respiring cells. Linnane, Vitols, and Nowland (1962) found that under anaerobic conditions, the yeast Torulopsis utilis lacked mitochondria. Instead a multi-membrane system was present, which when the cell was returned to aerobic conditions, was involved in the formation of more typical mitochondria. Similarly Polakis, Bartley, and Meek (1964) found that the yeast Saccharomyces cerevisiae, when grown anaerobically, lacked mitochondria, though they developed under aerobic conditions in the presence of suitable substrates. Bjorkman and Thorsell (1962, 1964) found three types of mitochondria in the liver fluke, F. hepatica. In the tegument they were small and had very few cristae which they associated with anaerobiosis and a low cytochrome content. Cell boundaries were not apparent in the tegument under the light microscope, and, although it is not very practicable to search large cells under the electron microscope for cell membranes, none was encountered, apart from the proximal and distal bounding membranes of the tegument. The absence of recognizable cell membranes in the tegument would not be surprising in view of the ontogenetic development of the tegument. The tegument arises in development by the considerable enlargement of a few cells, without concomitant cell division. The nuclei of M. dubius become enlarged as the animal develops in the definitive host but do not divide. Study of the development of the tegument from the embryonic stage seems necessary to establish its true character (cellular or syncytial). References BAER, J. G., In Traiti de Zoologie, edited by P. P. Grass6, vol. s, pp Paris (Masson et Cie). BAKER, J. R., Quart. J. micr. Sci., 85, 1. BERTALANFFY, L. VON, MASIN, M., and MASIN, F., Cancer, n, 873. BJORKMAN, N., and THORSELL, W., Exp. Cell Res., 27, 342. BJORKMAN, N., and THORSELL, W., Exp. Cell Res., 33, 319. BRAND, T. VON, J. Parasit., 25, 329. BULLOCK, W. L., J. Morph., 84, 201. BURLINGAME, P. L., and CHANDLER, A. C, Amer. J. Hyg., 33D, 1. BURNHAM, K. D., Ph.D. Thesis. Iowa State University. CROMPTON, D. W. T., Parasitology, 53, 663. KARNOVSKY, M. J., J. biophys. biochem. Cytol., 11, 729. LAURIE, J. S., Exp. Parasit., 6, 245. LAURIE, J. S., Exp. Parasit., 8, 188. LINNANE, A. W., VITOLS, E., and NOWLAND, P. G., J. Cell Biol., 13, 345.

15 146 Nicholas and Mercer Tegument of Moniliformis Lwr, J. H., J. biophys. biochem. Cytol., 2, 799. MOORE, D. B., J. Parasit., 32, 257. NICHOLAS, W. L., and HYNES, H. B. N., In The Lower Metazoa, edited by E. C. Dougherty. Berkeley (University of California Press). PALADE, G. E., J. exp. Med., 95, 285. PEARSE, A. G. E., i960. Histochemistry. London (Churchill). POLARIS, E. S., BARTLEY, W., and MEEK, G. A., Biochem. J., 90, 369. ROTHMAN, A. H., J. Parasit., 45 (4, suppl.), 28. ROTHMAN, A. H., i960. J. Parasit., 46 (5, suppl.), 10. ROTHMAN, A. H., J. Parasit., 47 (4, suppl.), 25. ROTHMAN, A. H., Trans. Amer. micr. Soc, 82, 22. SABATINI, D. D., BENSCH, K., and BARRNETT, R. J., J. Cell Biol., 17, 19. THREADGOLD, L. T., Exp. Cell Res., 30, 238.