Electron microscope and histochemical studies on the eggs of Hymenolepis diminuta.

The ultrastructure and histochemistry of the mature eggs of Hymenolepis diminuta were studied. The oncosphere is surrounded by several "protective layers"; the outer coat, the outer envelope, the inner envelope, and the embryophore. The outer coat appears to be cellular in origin and consists of an inner zone of electron dense material with an irregular surface of budlike projections and an outer zone of granular material. Histochemically, a mucopolysaccharide or mucoprotein composition is indicated. The outer envelope represents a clear space in most eggs, but lipid accumulations were noted in some. The inner envelope and embryophore are a continuous morphological unit with a structural protein composition. Exterior to the inner envelope is a degenerating cytoplasmic zone. This is separated from the embryophore by a zone of finely granular material. The embryophore is composed of electron-dense homogeneous material. An outer syncytial layer with numerous cytoplasmic extrusions surrounds the oncosphere. It is separated by a basal lamina from the underlying body wall musculature. The oncospheral hooks consist of 3 layers. The middle fibrous layer elicits a positive reaction for keratin. Structural and liquefaction changes occur in the cytoplasm and the granules of the penetration gland as the oncosphere reaches the infective state. The eggs of hymenolepidid cestodes have been studied by a number of investigators (Moriyama, 1961a, b; Ogren, 1955, 1961a, b, 1962, 1967, 1968; Ogren and Magill, 1962; Rybicka, 1966a, b, 1967a, b, c, d; Swiderski, 1967). However, many of the anatomical details of these eggs are beyond the resolution of the light microscope. Collin (1968, 1969) studied the hatched oncospheres of Hymenolepis citelli with the electron microscope, but there have been no studies on the fine structure of the mature eggs of these cestodes. Electron microscopy has revealed many new and interesting features in the eggs of the dilepidid cestode, Dipylidium caninum (Pence, 1967), and of the taeniid cestodes (Inatomi, 1962; Morseth, 1965; Nieland, 1968). ConReceived for publication 13 June 1969. * Supported in part by PHS Research Grant AI02347 from NIH. t NIH Predoctoral fellow, 5F01 GM-38,070. Part of a dissertation to be submitted to the Graduate School of the Louisiana State University Medical Center for the degree of Doctor of Philosophy. sequently, this investigation was undertaken in order to elucidate the fine morphology of the mature eggs of the hymenolepidid cestode, Hymenolepis diminuta. Histochemical tests were employed to demonstrate the composition of some of the structures observed. MATERIALS AND METHODS Adults of Hymenolepis diminuta were obtained from experimentally infected laboratory rats. Gravid proglottids were cut into small pieces and immediately fixed at 4 C in 5.28% glutaraldehyde bufferred to pH 7.4 (Pease, 1964). After 1 to 6 days fixation, the tissue was washed briefly in ice-cold Millonig's buffer, and postfixed in cold 2.0% osmium tetroxide. The tissue was washed again in cold buffer, rapidly dehydrated in graded concentrations of ethyl alcohol, and passed to a 1:1 mixture of propylene oxide and either Araldite 502 or Epon 812 for 12 to 24 hr. Embedment was in Araldite 502 and Epon 812 according to the methods of Luft. Sections were cut with a diamond knife on a Porter-Blum MT-1 microtome and mounted on formvar membrane carbon-coated 200-mesh copper grids. These were stained 30 min in lead hydroxide (Watson, 1958). Preparations were examined with an RCA EMU 2B or a Hitachi HU 11A electron microscope at 50 kv. FIGURES 1-3. Electron micrographs of the eggs of Hymenolepis diminuta. 1. Section through outer coat (OC), outer envelope (OE), inner envelope (IE), embryophore (E), and part of oncosphere (ON) of the mature egg. Note wide clear space representing the outer envelope. 2,900 X. 2. Section through outer layers of egg demonstrating material in outer envelope and polar thickening of the embryophore (P). Note material of apparent cellular origin at exterior of the outer coat (CM). 4,370 X. 3. Outer coat of mature egg demonstrating outer granular layer (OL) and inner layer (IL) with its budlike projections. 16,240 X.