Schistosoma mansoni: emergence of progeny-daughter sporocysts in monoxenic culture

Daughter sporocysts of Schistosoma mansoni released from mother sporocysts from Biomphalaria glabrata were cultured monoxenically for 21 days at 27.4 C in a medium consisting of a balanced salt-sugar solution supplemented with lactalbumin hydrolysate, inactivated fetal calf serum, serum Fraction A, and egg ultrafiltrate. The sporocysts were retained in transparent porous membranes so that their germinal development could be observed without interference from the underlying cells of Aedcs albopictus tissue cultures; the membrane permitted interchange of medium between sporocysts and cell cultures. The greatest increase in length, fivefold, occurred by 18 days. Progeny-daughters began to emerge at 9 days.The effects of pH, osmolality, and antibiotics were evaluated in axenizing solutions. Optimal conditions were a pH range of 6.5–7.6 and 113–153 mOsm. Antibiotics were not toxic.     

Role of pleated septate junctions in the epithelium of miracidia of Schistosoma mansoni during transformation to sporocysts in vitro

The surfaces of miracidia of Schistosoma mansoni were examined ultrastructurally during in vitro transformation to sporocysts. Before transformation, the surface was composed of ciliated epithelial plates (EP) that were set into a reticulum of narrow syncytial ridges (SR). The EP were attached to SR by extensive pleated septate junctions that had 18-24 strands of intramembrane particles (IMP) on the protoplasmic faces and complementary pits on the ectoplasmic faces. These junctions also appeared to separate the EP plasma membrane into apical and basolateral domains with a larger number of IMPs on the latter. Transformation was induced by placing the miracidia in salt containing medium which also halted ciliary beating. In 2-5 hr, the SR expanded until they formed a syncytium covering the parasite surface, while the EP retracted and rounded up. During this time, the EP and SR were held in contact with one another by the septate junctions which became progressively convoluted. Subsequently, the EP detached from the parasite. When transforming miracidia were returned to fresh water, the cilia resumed beating and the EP detached from the parasite surface and exposed the underlying basement membrane. Those EP that remained attached were connected only by septate junctions to the expanded SR. These studies demonstrate that the formation of the syncytium occurs gradually with contact maintained between EP and SR via the septate junctions. Further, the septate junctions are similar to occluding junctions in mammalian epithelia since they segregate the plasma membrane of the EP and they have an adhesive function.     

Schistosoma mansoni: axenic culture of daughter sporocysts

Daughter sporocysts of Schistosoma mansoni were cultured axenically for up to 13 days in media conditioned with Aedes albopictus tissue cultures. Sporocysts increased in length, processes appeared on the tegument, and small embryos developed. Two media, differing in ionic balance and source of amino nitrogen, were compared. No development occurred in either medium when freshly prepared.     

Morpho-anatomic and functional sectorization of Schistosoma mansoni daughter sporocysts

During the larval development of S. mansoni in the snail host, morpho-anatomic changes occur in the daughter sporocyst by a sectorization of this larval stage. Three sectors can be distinguished: an anterior zone with a well-differentiated birth pore; dilated zones containing the developing cercariae; constricted zones without cercarial embryo. The photonic and electronic microscopical study shows variations in the tegumental structure of these sectors. This evolution of the daughter sporocysts is discussed in relation with the dynamics of larval stages and the replication process of sporocysts.     

Analysis of tegumental surface proteins of Schistosoma mansoni primary sporocysts

Axenically transformed primary sporocysts of Schistosoma mansoni (NMRI strain) were labeled with 125I in an effort to identify sporocyst proteins exposed at the tegumental surface. Using the 125I activating reagent, 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril, in conjunction with SDS-PAGE and autoradiography, up to 12 bands were radiolabeled out of 60 components visualized by silver staining. Labeled proteins ranged in apparent Mr from greater than 200 to less than 12 kDa. Pronase treatment of living sporocysts after radioiodination removed all labeled material, suggesting that only surface proteins were being iodinated. Western blot analysis employing 5 monoclonal antibodies (MAB's) to sporocyst surface antigens revealed a wide range of reactivities which produced banding patterns closely reflecting autoradiograms of identical samples. The concomitant removal by Pronase of immunoreactive and radiolabeled surface proteins with identical Mr in the range of 90-130 kDa suggests that epitopes recognized by these antibodies are associated with these higher molecular weight surface proteins. However, although Pronase removes all labeled surface proteins, substantial nonradiolabeled, immunoreactive material with Mr less than 90 kDa still remains on enzyme-treated parasites. This indicates that MAB-reactive epitopes, in addition to their occurrence with surface proteins, are also associated with either unlabeled, protease-resistant surface components or internal antigens. The immunohistochemical localization of antibody-reactive material in gland-like structures within sporocysts supports an internal source for nonradiolabeled, immunoreactive components. Finally, the periodate sensitivity of the epitopes recognized by all tested MAB's suggests that carbohydrate moieties may represent a common and extremely immunogenic constituent of the sporocyst surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity of praziquantel on in vitro transformed Schistosoma mansoni sporocysts

Praziquantel (PZQ) is effective against all the evolutive phases of Schistosoma mansoni. Infected Biomphalaria glabrata snails have their cercarial shedding interrupted when exposed to PZQ. Using primary in vitro transformed sporocysts, labeled with the probe Hoechst 33258 (indicator of membrane integrity), and lectin of Glycine max (specific for carbohydrate of N-acetylgalactosamine membrane), we evaluated the presence of lysosomes at this evolutive phase of S. mansoni, as well as the influence of PZQ on these acidic organelles and on the tegument of the sporocyst. Although the sporocyst remained alive, it was observed that there was a marked contraction of its musculature, and there occurred a change in the parasite's structure. Also, the acidic vesicles found in the sporocysts showed a larger delimited area after contact of the parasites with PZQ. Damages to the tegument was also observed, as show a well-marked labeling either with Hoechst 33258 or with lectin of Glycine max after contact of sporocysts with the drug. These results could partially explain the interruption/reduction mechanism of cercarial shedding in snails exposed to PZQ.     

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.     

Characterization of excretory-secretory proteins synthesized in vitro by Schistosoma mansoni primary sporocysts

Excretory-secretory (E-S) products released by larval schistosomes have been implicated in the interference of host snail defense systems. Because of the potentially important role that E-S products play in the parasite-host relationship, total and newly synthesized E-S proteins from in vitro-cultured Schistosoma mansoni primary sporocysts were characterized using incorporation of [35S]methionine followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Total E-S protein decreased more than 5-fold from day 1 to day 3 of culture and remained constant until day 8 when protein concentrations began to increase. Release of newly synthesized protein, however, increased from day 1 through day 8. Both silver staining and fluorography of SDS-PAGE-separated E-S products revealed a wide variety of polypeptides ranging in Mr from 13 to greater than 200 kDa. The dynamics of the release of individual polypeptides, both total and newly synthesized, varied over time. Although certain polypeptides decreased in concentration, others remained constant or increased with time in culture. Culture conditions were found to be important for sporocyst viability and growth, and for release of newly synthesized proteins. Sporocysts cultured in medium containing fetal bovine serum (complete) grew significantly larger and had a significantly greater viability than did sporocysts cultured in medium lacking serum (incomplete). Also, sporocysts cultured in complete medium synthesized and released significantly more protein than did sporocysts cultured in incomplete medium. These sporocysts continued to produce a 54-kDa polypeptide, whereas sporocysts in incomplete medium stopped producing this protein by day 3 of culture. The present study has shown that S. mansoni primary sporocysts, cultured in vitro, synthesize and secrete a wide variety of glycoproteins and that the type and quantity of glycoproteins released are dependent on culture conditions.     

Axenic culture of Schistosoma mansoni sporocysts in low O2 environments.

Recent successes in culturing intramolluscan larval stages of Schistosoma mansoni have relied on synxenic culture with a cell line (Bge) developed from embryos of a molluscan host Biomphalaria glabrata. To further facilitate progress toward control of schistosomiasis, a system for axenic in vitro culture of the parasite has now been developed. When culture media were preconditioned by Bge cells, sporocysts lived longer in vitro and produced more offspring. Because Bge-derived components could be protecting sporocysts from oxidative stress, axenic sporocysts were cultured at lowered O2 levels. In an hypoxic environment, S. mansoni sporocysts grew well and produced daughter sporocysts continuously under axenic conditions and in a medium completely lacking host molecules. Sporocyst production occurs independently of host influence.     

Capacity of irradiated echinostome sporocysts to protect Schistosoma mansoni in resistant Biomphalaria glabrata

Three closely related species of Echinostoma flukes each has a distinctive pattern of protection of Schistosoma mansoni in schistosome-resistant Biomphalaria glabrata host snails. Protection of developing S. mansoni by irradiated E. paraensei sporocysts in the schistosome-resistant snail host was strong; protection induced by irradiated E. lindoense and E. liei sporocysts was weak or not measurable. The capacity of irradiated E. paraensei sporocysts to interfere with the host's innate anti-schistosome response also differed between strains of B. glabrata. Protection of S. mansoni strain Lc-1 was greater in B. glabrata strain 10-R2 than it was in strain M-RLc snails. Irradiated E. paraensei sporocysts also induced a different response to the two schistosome strains in a single host strain. Irradiated E. paraensei sporocysts induced in B. glabrata 10-R2 snails a stronger protection of S. mansoni strain PR-1 than of strain Lc-1. Exposure of each snail to the irradiated E. paraensei miracidia usually protected the following challenge schistosome infection better when 30 rather than 10 irradiated echinostome miracidia were used.     

Schistosoma mansoni: characterization of clones maintained by the microsurgical transplantation of sporocysts

Five male and 5 female clones of Schistosoma mansoni were established and maintained for 3 yr by the serial microsurgical transplantation of sporocysts from infected to uninfected Biomphalaria glabrata snails. The clones were initially derived from 10 randomly selected snails with monomiracidial infections. Clones were characterized by several criteria, including their infectivities for mice and snails, their cercarial outputs, and their ability to produce immunity in mice. The mean infectivities of individual clones in mice ranged from 26 to 44%, and were highly consistent within each clone. The infectivities of cloned sporocysts in snails ranged from 44 to 100% and were also highly consistent within clones. Mean cercarial outputs from individual clones ranged from 450 to 4,300 per snail. In mice, clones differed significantly from each other in their ability to immunize and in their susceptibility to immunity. Each clone was unique and did not appear to differ with time or subpassaging through snails, suggesting that the differences had a genetic basis.     

Lectin binding to cells of Schistosoma mansoni sporocysts and surrounding Biomphalaria glabrata tissue

The binding of different lectins to the surface of mother and daughter sporocysts of Schistosoma mansoni (Trematoda) and to cells of its intermediate host Biomphalaria glabrata (Gastropoda) was investigated. The test system consisted of several biotin-labeled lectins, an avidin-biotin-peroxidase complex and 3,3'-diaminobenzidine. The fixatives used were Formalin, Bouin's and Zenker's solutions; unfixed material was also studied. Most lectins reacted equally with host tissue and parasite tissue. However, receptors for Ulex europaeus I (most probably fucose) were only demonstrated on daughter sporocysts. Thus, a method was found to specifically mark Schistosoma mansoni daughter sporocysts in the digestive gland tissue of its intermediate host. Mother sporocysts and surrounding host tissue differed in their distribution of galactosyl groups. Both lack fucose and N-acetyl-galactosamine. The differences in lectin binding of galactosyl determinants were also observed during the in vitro development of mother to daughter sporocysts.