Portions of digestive gland and ovotestis were removed from aquarium-reared Australorbis glabratus about 40 days after infection with Schistosoma mansoni. The tissues were minced and explanted in flasks under sterile conditions. The cultures were maintained at 26 to 28 C either on a complex medium, or, with generally superior results, on a balanced salt solution containing glucose and trehalose (BSS). Medium was changed daily and cultures were evaluated by counting the emerged cercariae and by histologic study of the tissues. Apparently normal cercariae emerged daily in relatively large numbers during the 1st week, and in decreasing numbers during the succeeding 1 to 2 weeks. Omission of the sugars from BSS adversely affected the yields of cercariae; addition of amino acid mixtures to BSS did not increase the amount or duration of cercarial emergence. At least in part, cercarial emergence was found to be influenced by light. A small proportion of culture-derived cercariae was infective for mice by skin penetration or intraperitoneal injection. Tissues harboring "immature" infections were also explanted, i.e., from snails 19 days after exposure to S. mansoni. It was demonstrated that cercarial embryos were capable of undergoing maturation in vitro when cercariae appeared in these cultures after a lag of 8 to 13 days. Intramolluscan larval trematodes have received little study in vitro. The few published accounts indicate that immature rediae of Cyclocoelum microstomum from miracidia survived at most for 14 days in complex media (Ingersoll, 1956), and that rediae of Fascioloides magna from snails survived up to 10 days in solutions of individual substrates (Friedl, 1961). The axenic rediae did not grow or produce succeeding larval stages in vitro. Techniques are now described for the study in vitro of larval Schistosoma mansoni in tissues from parasitized Australorbis glabratus. It has been demonstrated, apparently for the first time, that trematode cercariae can develop and emerge in vitro and that under certain circumstances some of them are infective. MATERIALS AND METHODS Maintenance and infection of snails Snails were from a Puerto Rican strain (PR-2) of A. glabratus colonized in our laboratory since 1957 (Chernin and Schork, 1959) and maintained on watercress and romaine lettuce. Groups of 100 snails, 4 to 8 mm in diameter, were mass-exposed in finger bowls to miracidia of a Puerto Rican strain of S. mansoni (Chernin and Dunavan, 1962). Snails were then maintained on lettuce at 25 C, in darkness, in covered, aerated, stainless-steel pans. The snails were isolated in vials and examined for cercarial emergence 38 to 41 days postexposure, except as noted. Snails yielding cercariae at this time were used in experiments within 24 hr; these usually measured 12 to 16 mm in diameter. Composition of solutions Two solutions, previously developed in snail tissue culture studies (Chemrnin, 1963), were compared as media. The first is a complex nutrient medium (TC-NM) consisting mainly of a balanced salt solution and sugars (BSS), plus small amounts of vertebrate serum, amniotic fluid, embryo extract, lactalbumen hydrolysate, and yeast extract. The second solution is the BSS which contains several salts and 100 mg each of glucose and trehalose per 100 ml. The TC-NM was adjusted before use to pH 7.4 to 7.5 with 0.1 N NaOH; the BSS required no adjustment for a similar pH. All media were supplemented with 200 units penicillin G and 200 pg streptomycin sulfate per ml. Securing and processing snail tissues Glassware and instruments were sterile, all procedures were done in a hood, and a stereomicroscope was used in dissection. Snails were blotted dry, wiped with 70% alcohol, and kept temporarily Received for publication 27 April 1964. * These studies were supported in part by research grant AI-00513 and by a Research Career Award from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service.

Maintenance in vitro of Larval Schistosoma mansoni in Tissues from the Snail, Australorbis glabratus.

IN THIS PAPER I propose to bring together significant facts already known to have some bearing on the mechanisms which govern or influence host compatibility or incompatibility during parasit ism, to add information based on my own recent research, and to present some speculations. From the broad viewpoint, host compatibility may be defined as the expression of the physical and physiological (including chemical) states of the host and parasite which enable the parasite to invade and carry out its life processes, includ ing perpetuation of the species. Incompatibi lity refers to those factors which completely or part ially prevent the establishment and normal development of the parasi te. Both of these are highl y complex phenomena influenced to one degree or another by a series of separate but commonly int errelated factors. As workin g models from which we can seek evidence to support or reject various working hypotheses, we have chosen to examine the relationship between molluscs and digenetic trematodes, since this category of association, unl ike a num ber of others, is an example of parasitism whether the definition of \"parasitism\" is couched in nutritional, pathological, ecological, or immunological terms. Molluscs, except in a very few unusual instances, serve as interm ediate hosts for the Digenea, but this fact does not render them less appropriate as exper imental tools. In fact, inasmuch as it is generally accepted that molluscs were the original hosts

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The compatibility and incompatibility concept as related to trematodes and molluscs.

The egg of Fasciola hepatica has a smooth surface with a slightly elevated circle marking the fracture of the operculum. The operculum and the aperture have crenated edges. The epithelial cells of the miracidium are covered with long cilia. When miracidia are vibrated in an ultrasonic cleaner the cilia of the epithelial cells of the four posteroir tiers are broken off only leaving longitudinal rows of cilium stubs, whereas the cilia of the first tier are still retained. The apical papilla is provided with a dorso-ventral furrow, multiciliated pits and isolated sensory cilia. The narrow intercellular ridge is smooth, whereas the epithelial cells have small cytoplasmic knobs between the cilia. The penetration into the snail (Lymnaea truncatula) and the transforamtion into sporocyst may be separated into three phases. (1) Less than 1 min after attachment to the snail the ciliated cells of the anterior tier are shed and swim away. (2) The cilia of the remaining cells beat violently and after about 5 min most cilia are broken off near the cell surface. The miracidium remains for about 15 min embedded as far as the intracellular ridge receptors (lateral papillae and sheathed ciliated nerve endings). During this period extensive contraction and relaxation of the body are performed. (3) The final penetration of the snail epithelium takes about 15 min. Simultaneously with the penetration into the snail tissue the "bald" cells (epithelial cells with cilium stubs only) of the four posterior tiers loosen, florm globules and fall off. The surface below the cells is smooth and in cytoplasmic continuity with the intercellular ridge and the apical papilla, and this syncytium forms the later tegument of the sporocyst. After a few days the tegument of the sporocyst is provided with microvillus-like projections and the apical papilla and sensory structures are lost.

Stereoscan studies of eggs, free-swimming and penetrating miracidia and early sporocysts of Fasciola hepatica.

Metamorphosis of miracidia into cercariae of Schistosoma mansoni in vitro.

A Bge cell co-culture system, previously shown to support the in vitro production of daughter sporocysts from mother sporocysts of Schistosoma mansoni and S japonicum, has proven capable of supporting the in vitro development of intramolluscan stages of the deer liver fluke, Fascioloides magna Miracidia commenced transforming within 4 h of incubation with Bge cells, and had completely shed their epidermal plates within 18-24 h Redial stages were visible inside in vitro-transformed mother sporocysts after 12-16 days of co-culture with Bge cells, and emerged as fully-developed larvae starting at 14-20 days post-cultivation Rediae survived over 60 days of in vitro culture, and reached a maximum size of 150-170 microns Although particulate material was visible in their cecae, rediae were not observed to actively feed on Bge cells Bge cells did not attach to or encapsulate larval stages at any time throughout the incubation period Unlike Schistosoma spp, in which a high percentage of miracidia spontaneously shed their ciliated epidermal plates and transformed into mother sporocysts in Chernin's balanced saline solution (CBSS), transformation of F magna was dependent on Bge cell products Less than 5% of F magna miracidia transformed spontaneously in either CBSS or Bge medium with 10% fetal bovine serum (complete or C-Bge) However, incubation of miracidia in either Bge cell-conditioned C-Bge medium or a greater than 30 kDa fraction concentrated from conditioned CBSS increased transformation rates to 67 and 83%, respectively This secretory Bge cell factor(s) appeared to be protein in nature since its activity was completely abrogated by heat or proteinase K treatments Overall, these results demonstrate that Bge cells are required for stimulating in vitro miracidial transformation and supporting early larval development of a fasciolid trematode under culture conditions This is the first report of in vitro development of rediae from miracidia for a digenetic trematode

Biomphalaria glabrata embryonic (Bge) cell line supports in vitro miracidial transformation and early larval development of the deer liver fluke, Fascioloides magna.

A. C. Todd

Papers

In vitro metamorphosis of the miracidium of Fascioloides magna (Bassi, 1875) Ward, 1917.