Schistosoma mansoni: Long-term maintenance of clones by microsurgical transplantation of sporocysts

Schistosoma mansoni sporocysts originally derived from monomiracidially infected Biomphalaria glabrata snails were serially transplanted into the cephalopedal sinus of anesthetized snails by the microsurgical implantation of fragments of parasitized hepato-pancreas and ovotestis. Three to six passages each of five male and five female clones were maintained for as long as 2.0 years. Of the recipient snails which survived surgery, 87% released cercariae, usually beginning 5–7 weeks after surgery. The percentage of snails which released cercariae increased with successive passages. The mean survival time of surgically infected snails after cercarial emergence began was 9.2 ± 0.5 weeks, nearly the same as that of miracidially infected snails. Longevities of snails infected with male or female clones were similar. Recipient snail size and age did not influence cloning success. Beginning 5 weeks from the onset of cercarial emergence large numbers of cercariae (a mean of 3900/snail from male clones and 1300/snail from females) were obtained during each shedding period. These results clearly demonstrate that the microsurgical transplantation of sporocysts is a practical means of maintaining and expanding populations of genetically homogeneous schistosomes (clones).     

Schistosoma mansoni: Cloning by microsurgical transplantation of sporocysts

Development of a method of infecting of the molluscan host by microsurgical transplantation of the parasite's sporocysts enables the researcher to maintain the host cycle of Schistosoma mansoni exclusively by asexual means and without the participation of a vertebrate host. After transplantation, larval morphogenesis becomes altered to form an additional generation of sporocysts. These invade the digestive gland of the recipient mollusc progressively, producing normally infective cercariae. The maintenance of the life cycle of S. mansoni in the laboratory for 1 year, solely in the mollusc, has been obtained through six successive transplantations. Thus, a true cloning of S. mansoni has been achieved, the original transplant material being derived from a monomiracidial infection. From the practical viewpoint, this transplantation technique is of definite utility in the maintenance of the cycle, the vertebrate stage having been eliminated. From the theoretical viewpoint, unexpected analogies become apparent with the two types of larval demography found in Digenea (Digenea with sporocyst and Digenea with rediae).     

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.     

Rescue of sporocysts of Schistosoma mansoni in nonsusceptible Biomphalaria by head-foot transplantation into susceptible snails

To measure the longevity of sporocysts of Schistosoma mansoni in nonsusceptible snails (13-16-R1 and Salvador strains of Biomphalaria glabrata, and Biomphalaria obstructa), the head-foot (HF) of miracidia-exposed snails was transplanted into the hemocoel of a susceptible NIH albino recipient at 1-36 days postexposure (DPE). Recipient snails which were not exposed to miracidia then were monitored for infection transferred by the implant, and infection prevalences in recipients of HF transplants from nonsusceptible donors were compared to those in snails implanted with an HF from NIH albino donors. Transplants from NIH albino snails between 1 to 15 DPE infected 98% of recipients. Similarly, at 1 DPE, 69-85% of transplants from nonsusceptible snails contained viable sporocysts, as shown by resulting patent infections in the recipients. Recipient infection prevalence, and presumably numbers of transplants containing viable sporocysts, declined as a function of DPE, and by 5-9 DPE this decrease was significant for all 3 types of nonsusceptible donors. However, viable sporocysts still occurred in B. obstructa and 13-16-R1 B. glabrata as late as 19 and 20 DPE, respectively, and in Salvador B. glabrata as late as 33 DPE. Thus, sporocysts persist in nonsusceptible snails considerably longer than suggested by results of previous histological studies.     

Cloning of Schistosoma mansoni sporocysts in vitro and detection of genetic heterogeneity among individuals within clones

The establishment of in vitro cultivation techniques to maintain larval and adult stages of the trematode Schistosoma mansoni has facilitated research on diverse aspects of the biology of this parasite. Because of the difficulty in obtaining defined intramolluscan stages of this parasite, one aim of this study was to develop an in vitro technique for the generation of defined clonal daughter sporocyst (DSp) generations that originate from a single mother sporocyst. Sporocysts died when cultured singly; however, when single sporocysts were cultured in inserts within wells with about 1,000 others, the single individuals produced daughters asexually. In recent years, evidence has been accumulating for variability among, and within, schistosome populations. Such variability has been seen in both larval and adult stages. Even within clonal cercariae, genomic and biochemical heterogeneity has been observed, indicating the existence of a yet unknown mechanism that generates variability during larval development. Therefore, another aim of this study was to examine clonal DSps generated in vitro for diversity regarding the presence or absence of a specific repetitive DNA element (W1). Such sporocysts were found by molecular analysis to be heterogeneous with respect to the occurrence of W1. This phenomenon had previously been observed in clonal schistosome populations and described as genomic instability. In this study, we provide the first molecular evidence that variability can be generated within sporocyst generations, supporting the hypothesis of mitotic recombination events during the asexual life stage of schistosomes.     

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.     

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.     

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.     

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.     

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)     

The invasion of Biomphalaria pfeifferi by Schistosoma mansoni miracidia and the development of daughter sporocysts

Laboratory experiments have been carried out to determine the susceptibility of Gezira Biomphalaria pfeifferi snails to S. mansoni miracidia and the relationship between miracidia and daughter sporocyst production at the 10–17 day development stage. The relationship between snail numbers, miracidia numbers and water volume has also been studied. Two non susceptible snails, Bulinus truncatus and Cleopatra bulimoides, both of which occur naturally in Gezira canals, were tested to see if they act as decoys for S. mansoni miracidia.The results showed that the B. pfeifferi are 100% susceptible to S. mansoni invasion, at least to the daughter sporocyst development stage. The more miracidia that penetrated the more daughter sporocysts were produced, however individual variation and overlap were great. When one miracidium was released to find one snail it succeeded in low water volumes (5 m, 50 ml), but failed in 5 litres. When 100 miracidia were released mortality of snails was high suggesting superinfection particularly when only one or five snails were available. Among survivors daughter sporocyst counts were very high. Cleopatra and Bulinus snails do have a decoy effect when present in large numbers. In their presence the number of infected snails was marginally reduced and the number of daughter sporocysts greatly reduced. However, if superinfection is reduced by decoy effect, it is conceivable that Biomphalaria may be protected by decoy snails in circumstances where miracidia counts are high.