Cytological Studies on the Absorptive Surfaces of Cestodes. VI. Cytochemical Evaluation of Electrostatic Charge

Abstract

Adsorption of acid anionic and cationic colloidal iron, "H-polyamino acids, and "Ca to the tegument plasmalemma of Hymenolepis diminuta was examined by electron microscopy, radioassay, and autoradiography. Observations are consistent with the presence of a relatively high electronegative charge density at the outer surface of the membrane, which appears capable of repelling anionic materials of high molecular weight. Possible microenvironmental implications include the exclusion of potentially deleterious organic materials of like charge and the concentration of physiologically important inorganic counterions proximal to the parasite's surface. A layer of complex carbohydrates at the surface of a variety of microorganisms, metazoan and higher plant cells has been demonstrated in numerous histochemical, ultrastructural, and biochemical investigations (see reviews by Bennett, 1963; Rambourg and Leblond, 1967; Ito, 1969; Martinez-Palomo, 1970; Winzler, 1970). Not infrequently, these coats are polyelectrolytes (Katchalsky, 1964) with significant influence on the electrokinetic charge of the cell surface (Haydon and Seaman, 1967; Ward and Ambrose, 1969). Tapeworms are similarly invested with a carbohydrate-rich surface coat (Lumsden et al., 1970a; Rothman and Elder, 1970) which binds ionic colloidal tracers both in vitro and in vivo (Lumsden et al., 1970b). We have suggested that ionogenic functions present in the tegument membrane glycocalyx might serve as electrostatic binding sites of significance in the physiology of the host-parasite relationship. However, it is known that carbohydrates can also chelate metal ions, apparently by covalent interactions involving their hydroxyl and carbonyl oxygen atoms (Charley et al., 1963; Davis, 1965; Deller, 1966). In order to evaluate the relative charge density of the tapeworm surface membrane the binding of oppositely charged metallic and nonmetallic tracers was compared. Received for publication 9 August 1971. *This work was supported by research grants from the NIH (AI 08673) and the NSF (GB 17992), and Career Development Award K04AI 23449 from the U. S. Public Health Service, NIH, NIAID. As colloidal materials do not cross the tegument membrane of adult hymenolepid cestodes (Lumsden et al., 1970b), they make suitable tracers for studies of surface adsorption by these parasites. Micellar suspensions of cationic and anionic metal oxides, and tritiated polybasic and polyacidic peptides were therefore selected as markers which could be directly imaged by electron microscopy or quantitatively detected by radioassay. In most instances, glutaraldehyde-fixed worms were used instead of live material, since this form of fixation was found to stabilize surface membrane structure against the otherwise cytolytic effects of the polyamino acid tracers (Mamelak et al., 1969) and the charge blocking chemical procedures carried out in conjunction with these incubations. MATERIALS AND METHODS Adult Hymenolepis diminuta were obtained from laboratory-infected rats according to Read et al. (1963). After rinsing in tris-maleate-buffered Krebs Ringers saline (KRT) (Read et al., 1963), intact worms were placed in vials containing icecold, 1.0 M cacodylate-buffered 3% glutaraldehyde, followed 30 min later by thorough washing with cacodylate buffer, then KRT. Cationic ferric oxide was prepared by adding 1 volume 0.5 M ferric chloride to 12 volumes boiling distilled water, followed by dialysis against 10 volumes of distilled water (Mowry, 1958). A stock suspension containing approximately 1 g of iron per liter was prepared by appropriate dilution of the dialyzed suspension with distilled water, the initial iron concentration therein being determined by titration with sodium diphenylamine sulfonate or permanganate after stannous chloride reduction (Griffin, 1954; Willard and Fuhrman, 1947). Anionic ferric oxide was prepared by mixing 1