Joseph J. DiConza

The miracidium-sporocyst transition in Schistosoma mansoni: surface changes in vitro with ultrastructural correlation.

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-

In vitro development of Schistosoma mansoni cercariae.

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 cercarienhüllen reaction, and appeared to lack the surface glycocalyx. Further development was not detected in mice or hamsters.

Histochemical demonstration of acetylcholinesterase in sporocysts of Schistosoma mansoni (Trematoda)

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.

Accumulation of lipids in Schistosoma mansoni sporocysts cultured in vitro

Abstract 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.

Cotylurus lutzi (Strigeidae): pre-and postadult stages cultured in vitro.

P. F. Basch, J. J. DiConza, and B. E. Johnson (J. Parasitol. 59, 319-322, 1973) reported cultivation of Cotylurus lutzi from tetracotyle to adult, with production of eggs yielding miracidia infective to snails. We have now extended in vitro cultivation to all stages from postcercaria to mother sporocyst. Biomphalaria glabrata, 12-15 mm diam, were exposed to a few dozen cercariae of C. futzi. On the following day the snails were cleaned and soaked for 3 hr in 500 units/ml penicillin G and 500 mCig/ml streptomycin sulfate in sterile distilled water, changed once. Shells were crushed and ovotestes, separated from the rest of the snails, were teased apart in sporocyst culture medium (J. J. DiConza and P. F. Basch, J. Parasitol. 60, 757-763, 1974). Postcercariae so obtained were rinsed in several changes of medium to remove host cells, and 16 organisms were transferred to a sterile 10 x 75 mm culture tube with 1.5 ml of medium. The tube was tightly stoppered and maintained at 25” & 1°C. Figures l-6 show these larvae at intervals up to 32 days, when half of them appeared to be well-formed tetracotyles. The cyst wall was still deformable by