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Neutral lipids were detected histochemically in mother and daughter sporocysts of Schistosoma mansoni cultured in vitro. These lipids progressively increased with prolonged culture. There was little phospholipid and no fatty acid, esterases, or lipases found in sporocysts by the methods employed. Mother and daughter sporocysts incorporated labeled acetate from the culture medium but no further information was obtained on the complex lipid-synthesizing capabilities of these organisms.

Athymic nude mice (Nu/Nu), heterozygous littermates (Nu/+), and thymus reconstituted homozygous animals (Nu/Nu TxR) were exposed to Schistosoma mansoni cercariae. A marked impairment in the development of peripheral blood eosinophilia and hepatic granuloma was observed in the Nu/Nu animal. In addition, host morbidity judged by organ weight, portal pressures and reticuloendothelial activity were less in the Nu/Nu animal. Thymus-reconstituted animals demonstrated eosinophilia, increased granuloma size, and morbidity equivalent to that shown by the Nu/+ heterozygous littermates. These studies indicate that the eosinophilia, granulomatous hypersensitivity, and ultimate host morbidity, associated with murine schistosomiasis, is dependent on thymic dependent lymphocyte function.

The distribution of acetylcholinesterase in mother and daughter sporocysts of Schistosoma mansoni was studied histochemically. In young mother sporocysts derived from miracidia cultured in vitro the miracidial neural mass and flame cells were shown to persist. The nerve trunks and commissures, as well as papillae, are apparently lost in the transformation process. In young daughter sporocysts freshly dissected from mother sporocysts there was little enzyme activity except for a sparse distribution in the tegument. After cultivation, intense enzyme activity was associated with developing cercarial embryos. A similar distribution of activity was observed in older daughter sporocysts obtained from the digestive gland of snails. No evidence of flame cells, neural mass, or commissures was detected in daughter sporocysts by the methods employed.

Whole egg masses or separated capsules containing trochophore-stage embryos of Biomphalaria glabrata were axenized in dilute hypochlorite with or without antibiotics. Embryos were extracted in snail saline and teased into fragments which were dissociated (generally with trypsin) and seeded into media. Of numerous formulations used, 4 were equally good: 3 are based upon diluted medium 199 or NCTC-135, with additions, and the other was formulated from analyses of hemolymph and published values; 3 are used under 5% CO2 and the other in air. Osmolalities vary from 116 to 220 mOsm/Kg H20. Fetal bovine serum is added to all and appears necessary to sustain cell growth. Cultures were not enhanced by use of collagen coatings, plasma clots, or adult snail extracts. Snail hemolymph is toxic when fresh and ineffective after heat inactivation. Five cell types were seen: fibroblast-like, principally in the first 2 weeks; epithelial-like, sometimes making sheets or monolayers; attenuated contractile muscle cells which sometimes became broad and binucleate; macrophage-like; and hemocytes. Numerous mitotic figures have been observed in aceto-orcein squash preparations, especially after incubation with colcemid. Cultures were usually healthy until terminated up to 6 weeks old.

Early cercarial embryos of Schistosoma mansoni developed to swimming cercariae within seven days in vitro, via the same sequence of stages as reported in vivo. The presence of living Biomphalaria glabrata cells (Bge line) was indispensable for proper development. Cultured cercariae were water-sensitive, gave no cercarienhullen reaction, and appeared to lack the surface glycocalyx. Further development was not detected in mice or hamsters.

Miracidia of Schistosoma mansoni, hatched aseptically, readily shed their ciliated epidermal cells in a culture medium. Electron microscopy shows that within 200 min in culture a new sporocyst tegument has formed, presumably from material mobilized from the subepidermal region. Granules resembling the alpha and beta particles of glycogen are involved in this process, passing up into the new tegument where they appear to disintegrate. The surface at this time bears simple microvilli, which become abundant and branched by 20 hr incubation. Mother sporocysts cultured for 6, 8, or 10 days were injected into Biomphalaria glabrata snails in which some at each age continued development leading to production of cercariae. Within the first 24 to 48 hr after miracidial penetration, focal tissue responses in the snail, Biomphalaria glabrata, act to destroy certain individual sporocysts of Schistosoma mansoni while others proceed to normal development (Newton, 1954; Pan, 1963). Elicitation or absence of this host foreign body response is probably associated with surface features of very young mother sporocysts (Wright, 1971). Information about the nature and changes in the parasite surface during the critical period of transformation from miracidium to sporocyst is therefore basic to comprehension of specificity between host and parasite. It is difficult to follow these events in vivo because the precise moment of miracidial penetration usually cannot be determined, and the tiny organisms are hard to find within the snail tissue. Induction of the same changes in vitro, with viability of sporocysts demonstrated by subsequent normal development upon implantation into snails, offers a means whereby these phenomena may be readily observed. We present here a simple culture method together with a description of surface changes between miracidium and sporocyst of S. mansoni in vitro. MATERIALS AND METHODS Golden hamsters, Mesocricetus auratus, were infected with 150 to 200 cercariae of S. mansoni. After about 8 weeks an animal was killed by rapid cervical dislocation, rinsed with 82% alcohol, its liver removed aseptically and dropped into a Received for publication 8 July 1974. * Supported by Grant AI-10271 from the NIAID, NIH, Bethesda, Maryland. sterile, ice-cold stainless steel semi-micro blender container (Eberbach Corporation) along with 100 ml of sterile ice-cold distilled water containing 200 units of Penicillin G per ml. All subsequent procedures were done aseptically using sterile glassware and reagents. The liver was minced for about 10 sec at high speed and the container placed in the refrigerator for 20 to 30 min. The top 80 ml of fluid was then carefully decanted, and 100 ml of fresh water with antibiotics added. The container was again refrigerated for 20 to 30 min. Meanwhile 2 X 105 units of Penicillin G were added to each of 2 1-liter volumetric flasks which had earlier been filled to the base of the neck with filtered aquarium water, capped (aluminum 35-mm film cans were used), and autoclaved. After the second sedimentation of the comminuted liver, 10 ml of sediment was transferred by pipette to each volumetric flask. The body of each flask was covered with aluminum foil, sterile water added up to the liter mark, and the upper neck illuminated with a small desk lamp. When miracidia were abundant in the top few cm of the water column, 5-ml aliquots, usually containing several hundred organisms, were withdrawn at intervals and transferred to 15-ml screwcapped conical centrifuge tubes, which were then placed into shaved ice in an insulated bucket. After miracidia had concentrated at the bottom of the tubes, as much water as possible was removed with a drawn-out Pasteur pipette, while observing through a binocular dissecting microscope. Two to 3 ml of sporocyst culture medium (DiConza and Basch, 1974) was added and the tubes incubated at room temperature (25 ?+ 1 C). Medium was made up with horse or fetal calf serum (Grand Island Biological Co.) or human serum from our laboratory staff. All sera were inactivated (56 C, 30 min) before use. Some cultures were transferred to 10by 75-mm disposable glass culture tubes which were tightly sealed with 00 silicone stoppers. Medium usually was not changed during the culture period. Obser

Tetracotyles of Cotylurus lutzi were incubated to adults in a basic medium of 40% NCTC135 and 40% chicken serum, supplemented with 20% extract of chicken heart, liver, muscle, intestine wall, or mucosa. Ten per cent or more of eggs produced in media containing intestine extracts appeared normal, but miracidial development occurred only when upper small intestine mucosal extract was used. Five Biomphalaria glabrata snails became infected by such miracidia, shedding cercariae which encysted as tetracotyles and developed into normal adults in zebra finches. Dialysates of the effective mucosal extract failed to stimulate production of normal eggs when added to the basic medium. The strigeid trematode Cotylurus lutzi Basch, 1969, can be grown in vitro from tetracotyle to adult by the method of Voge and Jeong (1971). Their medium contains equal parts of NCTC-135 and chicken serum (CS), plus 100 units of penicillin and 100 tug of streptomycin per ml. Worms develop well in this but produce only fragile, shell-less eggs incapable of embryonation. We have modified this medium so that normal eggs, yielding infective miracidia, are produced by worms grown entirely in vitro. MATERIALS AND METHODS Young adult snails, Biomphalaria glabrata (Say), were exposed to large numbers of cercariae of C. lutzi. After 1 to 2 months the snails were washed and their shells gently crushed in 0.34% NaCl in distilled water. The ovotestes, containing encysted tetracotyles, were removed and teased apart in sterile 0.34% saline containing 10 ,ug gentamicin, 50'0 units of penicillin, and 500a ,g streptomycin per ml. Freed organisms were rinsed 4 times in this solution. Cultures were set up in plain-lipped 25by 150-mm glass test tubes containing 5 ml of medium. About 15 well-encysted tetracotyles were placed into each tube, which was closed by a silicone rubber stopper and then sealed by a tightly wound strip of parafilm. No special gas phase was used. Basic culture medium contained 40% NCTC-135 and 40% CS (Grand Island Biological Co.), plus 100 units of penicillin, 100 jug streptomycin, and 10 jug gentamicin per ml. This was supplemented Received for publication 29 August 1972. * Supported by Grant AI-10271 from the NIAID, NIH, Bethesda, Maryland. t At present student, School of Medicine, UCLA, Los Angeles, California. with various extracts of chicken origin. From 4 freshly killed 2to 3-kg adult white leghorn chickens, separate extracts of liver, breast muscle, heart, and intestinal wall muscle were prepared. About 15 g of tissue was finely minced, then ground with a mortar and pestle, and finally squeezed through silk bolting cloth. The resulting liquid was made up to 20 ml with normal saline and centrifuged for 60 min at 3,000 rpm at 5 C. The supernatant fluid was filtered twice through paper, then through 0.65and 0.45-Am Millipore filters for sterilization. For extracts of chicken intestinal mucosa, the small intestine was cut at the gizzard and at the beginning of the large intestine (as determined by texture changes and the appearance of fecal masses). This portion, approximately 70 cm, was divided into 2 equal lengths, referred to as "upper" and "lower" small intestine. Each half was cut open longitudinally and washed in several changes of cold (5 C) normal saline. A glass microscope slide was used to scrape off the mucosal cell layer, which was then collected and homogenized in a glass tissue grinder with teflon plunger. Mucosal homogenates from the upper and lower small intestine were then treated as described above. Dialysis of the upper small intestine mucosal extract was done through cellulose tubing (pore size 2.4 nm) overnight at 5 C against 1,000 ml of normal saline or of distilled water. In the former case, 6 ml of extract was used; in the latter, 7 ml. Following dialysis against distilled water, NaCl was added to restore the saline concentration of the dialysate, which in both cases was sterilized using 0.65and 0.45-utm Millipore filters. After filter sterilization, all extracts were stored at -10 C until used. Egg yolk medium was prepared as described by Berntzen and Macy (1969). Extracts were added to the basic medium as shown in Table I. Media were changed on alternate days. In any one tube, organisms were maintained in the same formulation throughout; when extract prepared from one chicken was used up, the homologous material from the next chicken