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, over- laid 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 micro- scope 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, inter- woven 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 hypo- dermis. 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. 137-146, 1965.] 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 dis- tribution, of lipid and glycogei. in a study of the chemical composition of Macranthorhynchus hirudinaceus, and Bullock (1949) made further observa- tions on both in a number of other species. Crompton (1963) made a com- prehensive 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 de- finitive 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 CaCl2, was employed. So that the structures seen under the electron microscope could be corre- 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 fixa- tive 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 370 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 histo- chemical 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 speci- mens. 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 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 double- contoured 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 dimen- sions 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.