Involvement of nitric oxide in killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata

In strains of the snail Biomphalaria glabrata (Gastropoda) that are resistant to the parasite Schistosoma mansoni (Trematoda), hemocytes in the hemolymph are responsible for elimination of S. mansoni sporocysts. The defensive role of reactive nitrogen species was investigated in in vitro interactions between hemocytes derived from the resistant 13-16-R1 strain of B. glabrata and the parasite. The nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) and the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide reduced cell-mediated killing of S. mansoni sporocysts. To determine if peroxynitrite (ONOO-) is involved in killing, assays were run in the presence of the ONOO- scavengers uric acid and deferoxamine. These did not influence the rate of parasite killing, indicating that NO is directly responsible for mediating cytotoxicity, but ONOO- is not. The combination of the NOS inhibitor L-NAME and catalase, an enzyme that detoxifies hydrogen peroxide (H2O2), reduced average sporocyst mortality to a greater extent than L-NAME alone. Killing of the sporocysts was, however, not totally inhibited. It is suggested that NO and H2O2 are both involved in hemocyte-mediated toxicity of 13-16-R1 B. glabrata against S. mansoni sporocysts.     

Killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata: role of reactive oxygen species

The fate of Schistosoma mansoni (Trematoda) sporocysts in its molluscan host Biomphalaria glabrata (Gastropoda) is determined by circulating phagocytes (hemocytes). When the parasite invades a resistant snail, it is attacked and destroyed by hemocytes, whereas in a susceptible host it remains unaffected. We used 3 inbred strains of B. glabrata: 13-16-R1 and 10-R2, which are resistant to the PR-1 strain of S. mansoni, and M-line Oregon (MO), which is susceptible to PR-1. In an in vitro killing assay using plasma-free hemocytes from these strains, the rate of parasite killing corresponded closely to the rate by which S. mansoni sporocysts are killed in vivo. Hemocytes from resistant snails killed more than 80% of S. mansoni sporocysts within 48 hr, whereas sporocyst mortality in the presence of hemocytes from susceptible snails was <10%. Using this in vitro assay, we assessed the involvement of reactive oxygen species (ROS) produced by resistant hemocytes, during killing of S. mansoni sporocysts. Inhibition of NADPH oxidase significantly reduced sporocyst killing by 13-16-R1 hemocytes, indicating that ROS play an important role in normal killing. Reduction of hydrogen peroxide (H2O2) by including catalase in the killing assay increased parasite viability. Reduction of superoxide (O2-), however, by addition of superoxide dismutase or scavenging of hydroxyl radicals (*OH) and hypochlorous acid (HOCl) by addition of hypotaurine did not alter the rate of sporocyst killing by resistant hemocytes. We conclude that H2O2 is the ROS mainly responsible for killing.     

Effects of exogenous interleukin-1β on primary sporocysts of Schistosoma mansoni (Trematoda) incubated with plasma and hemocytes from schistosome-susceptible and resistant Biomphalaria glabrata (Gastropoda)

Abstract. The cytokine interleukin-1β (IL-1β) mediates interactions of immune and inflammatory cells in mammals. Previous reports also have linked plasma (cell-free hemolymph) levels of IL-1β in the snail Biomphalaria glabrata to resistance against Schistosoma mansoni . In the present study, fluorescent probes were used to study larval schistosome and snail hemocyte viability during in vitro encounters. Hemolymph (plasma and hemocytes) from schistosome-susceptible (M-line) and resistant (13–16-R1) B. glabrata was added to sporocysts of S. mansoni and the viability of hemocytes and parasites was assessed. Next, IL-1β was added to sporocyst-hemolymph samples, the viability of sporocysts and hemocytes determined and then compared to control assays. The number of live sporocysts present after incubation for 1 h with hemolymph from M-line snails was significantly greater than the number seen when hemolymph from 13–16-R1 snails was tested. Nearly all sporocysts survived the 1 h incubation with M-line hemolymph, and most of the hemocytes attached to sporocysts were dead. In contrast, nearly all sporocysts were dead when hemolymph from 13–16-R1 snails was tested, and most attached hemocytes were alive. Addition of IL-1β to M-line hemolymph resulted in a dramatic increase in sporocyst death. Addition of IL-1β to 13–16-R1 hemolymph produced a small but significant increase in the rate of sporocyst death. These results indicate that the concentration of IL-1β present in hemolymph from B. glabrata is directly related to the ability of this snail to kill S. mansoni sporocysts in vitro.     

Serotonin-induced muscular activity in Schistosoma mansoni larval stages: importance of 5-HT transport and role in daughter sporocyst production

Mother sporocysts of Schistosoma mansoni transport exogenously supplied serotonin (5-hydroxytrypamine; 5-HT), and respond to it with increases in motility. In the present study, we investigated the importance of 5-HT transporter activity in the manifestation of these 5-HT-induced motility changes, and further examined the role of 5-HT in the development of daughter sporocysts in vitro. Serotonin-induced motility of in vitro-derived sporocysts is not inhibited by antidepressant compounds, e.g., fluoxetine, that block 5-HT transport, suggesting that the receptors responsible for motility responses to 5-HT are surface exposed. Using a sporocyst in vitro culture system, we show that depletion of larval stores of 5-HT reduces production of daughter sporocysts, the second intramolluscan larval stage. Moreover, we demonstrate a strong correlation between endogenous 5-HT levels and basal mother sporocyst muscle activity. Overall, these data suggest that larval stages of S. mansoni can detect exogenous 5-HT via surface-exposed receptors, and they are consistent with the hypothesis that endogenous stores of 5-HT are important for the proper regulation of muscular contractions in mother sporocysts, and for the successful emergence of daughter sporocysts.     

Echinostoma paraensei: hemocytes of Biomphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni

Earlier in vivo work by Lie et al. (1977) indicated that the innate resistance of the 10R2 strain of Biomphalaria glabrata to PR1 Schistosoma mansoni could be interfered with if the snails were infected previously with another trematode, Echinostoma paraensei. We have studied this interference phenomenon using in vitro methods in an attempt to understand its mechanistic basis. Hemolymph, derived from 10R2 snails infected with E. paraensei for 14-28 days, killed 25% of S. mansoni sporocysts in vitro, significantly less (P less than 0.001) than the 90% killing rate observed with hemolymph from uninfected, control 10R2 snails. Hemolymph from the infected 10R2 snails and from schistosome susceptible M line snails did not differ significantly (P greater than 0.1) in their relative inability to kill S. mansoni sporocysts in vitro. The defect in sporocyst killing exhibited by echinostome infected 10R2 snails was traced to the cellular, rather than the humoral, component of the hemolymph. Preparations containing uninfected 10R2 snail hemolymph and echinostome daughter rediae exhibited significantly less (P less than 0.001) killing of S. mansoni sporocysts than did controls containing only 10R2 hemolymph and S. mansoni sporocysts. Our results suggest that echinostome larvae release factors that interfere with the ability of B. glabrata hemocytes to kill S. mansoni sporocysts.     

Lectin-binding properties of the surfaces of in vitro-transformed Schistosoma mansoni and Echinostoma paraensei sporocysts

As carbohydrates on the surfaces of sporocysts of digenetic trematodes may be targets of attack by the molluscan internal defense system, the lectin-binding patterns of living, in vitro-transformed sporocysts of Schistosoma mansoni and Echinostoma paraensei were characterized. Schistosoma mansoni sporocysts specifically bound 8 and E. paraensei 6 of 11 lectins examined. Sporocysts of the 2 species responded differently to 7 of the 11 lectins. Lectins inhibitable by mannose, galactose, and N-acetylgalactosamine were bound by both species. Lectins inhibited by fucose and N-acetylglucosamine bound uniquely to S. mansoni, and an N-acetylneuraminic acid (NeuNAc)-inhibitable lectin bound only to E. paraensei. Preincubation of sporocysts of either species in the plasma of the host snail Biomphalaria glabrata for as long as 24 hr only marginally altered the subsequent binding of lectins. Pretreatment of S. mansoni sporocysts with pronase E and trypsin substantially altered subsequent lectin binding, but similar treatment of E. paraensei sporocysts had little effect. A neuraminidase enzyme derived from Clostridium perfringens diminished binding of the NeuNAc-inhibitable lectin to E. paraensei sporocysts. This study indicates that lectin-binding monosaccharides are expressed abundantly on sporocyst surfaces, they vary considerably between 2 species parasitizing the same host, and they are not obscured readily or altered by exposure to host plasma.     

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.     

Lectin and human blood group determinants of Schistosoma mansoni: alteration following in vitro transformation of miracidium to mother sporocyst.

A mixed agglutination assay method was employed to detect the presence of surface determinants for various lectins and human blood group antibodies on Schistosoma mansoni miracidia and cultured mother sporocysts. Miracidia were found to possess surface receptors for the lectins Con A (concanavalin A), anti-Heel (eel serum agglutinin), and anti-ADb (Dolichos seed extract), as well as human anti-A antibodies. Following in vitro transformation of the miracidium to mother sporocyst, anti-Heel and human anti-A receptors were no longer detectable on the sporocyst surface, while determinants for Con A and anti-ADb remained essentially unaltered. It is concluded that transition of the miracidium to the sporocyst results in the alteration of surface molecular structures on schistosome larve. Furthermore, since determinants for Con A, anti-Heel, anti ADb, and human anti-A have been found associated with macromolecules in the hemolymph of the snail Biomphalaria glabrata (Stnislawski et al., 1976), there is now evidence that miracidia and mother sporocysts of S. mansoni and their snail host share molecules with common lectin and human blood group determinants.     

日本血吸虫胞蚴期超微结构的初步观察

本文用扫描与透射电镜观察了我国大陆品系37日和45日龄日本血吸虫母胞蚴及其体内未成熟子胞蚴体被的结构。同时观察了取自螺肝组织的62日龄成熟子胞蚴。初次揭示日本血吸虫胞蚴期体被的超微结构,基本上与曼氏血吸虫胞蚴期相似。日本血吸虫母胞蚴和成熟子胞蚴除体被无体棘外,其他很相似。比较未成熟的子胞蚴与未成熟的尾蚴,揭示外质膜由两层结构构成;随后,外层结构溶化消失,而同时出现微绒毛。构成这样母子二代胞蚴及其体内胚胎既相同又有差别,认为与幼虫寄生部位及生殖生理状态有关。     

Schistosoma mansoni: origin and expression of a tegumental surface antigen on the miracidium and primary sporocyst

A monoclonal antibody, recognizing a carbohydrate epitope associated with several tegumental surface components on Schistosoma mansoni primary sporocysts, was used to follow tegumental formation during transformation of the miracidium to sporocyst and its subsequent development in vitro and in vivo. Indirect fluorescent antibody and direct immunogold labeling methods confirm a structural connection between the intercellular ridges and a submuscular, multinucleate syncytium in the miracidium. Immunoreactive vesicles within this latter system directly contribute to elaboration of the tegumental surface membrane, through the process of membrane fusion. Lateral expansion of intercellular ridges by vesicular fusion ultimately result in fully transformed sporocysts exhibiting vesicular membrane epitopes as prominent tegumental surface components. Light microscopical and ultrastructural observations, together with Western immunoblot analyses, suggest a gradual depletion of intracellular and surface immunoreactive material of vesicular origin in primary sporocysts grown in culture for up to 12 days. In contrast, similar immunoreactive vesicles appear to be continuously synthesized throughout in vivo primary sporocyst development. Monoclonal antibody reactive epitopes appear to be uniquely expressed in the miracidium/primary sporocyst since similar molecules are absent from daughter sporocysts, cercariae, adults, and snail tissues.     

Schistosoma mansoni: relationship between cercarial production levels and snail host susceptibility

Two populations of Biomphalaria glabrata snails differing slightly in their susceptibility to Schistosoma mansoni infection showed dramatic differences in cercarial output per snail. Exposed to five or more miracidia, snails from a group with a 90-100% susceptibility rate (Group A) produced nearly twice the number of cercariae as those from a group with a 70-80% susceptibility rate (Group B). Exposure of individual snails to known numbers of miracidia resulted in higher numbers of primary (mother) sporocysts in Group A snails than in Group B snails. However, monomiracidial exposure of snails from both groups resulted in equivalent numbers of cercariae produced per positive snail, indicating that, once established, all primary sporocysts possess a similar reproductive potential. Morphometric analysis of serially sectioned 9-day-old primary sporocysts supported this conclusion; the size of the primary sporocysts and the size and numbers of secondary (daughter) sporocysts within each primary sporocyst were comparable in snails from both groups. The data indicate cercarial production in this system is regulated prior to, and/or during, early development of the primary sporocyst.     

Sequence of reconversion of the sporocysts of Schistosoma mansoni producing cercariae, to the production of new generations of sporocysts (author's transl)

During the life cycle of Schistosoma mansoni the production of sporocysts of a higher order than secondary is a normal mode of larval multiplication which intervenes in asexual reproduction of the parasite. The sequence of reconversion of sporocysts producing cercariae to those producing sporocysts III, IV, etc... can be divided into three principal steps: (1) cessation of cercariae production; (2) degeneration of cercariae contained in the sporocyst, and (3) production of the new generation of sporocysts. Degeneration of intrasporocystic larval material seems to be an indispensable step for the new orientation of production. The signifance of this method os multiplication in the ecology of transmission is discussed.     

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.     

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.     

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.     

Mechanisms underlying digenean-snail specificity: role of miracidial attachment and host plasma factors

Digenetic trematodes usually show a high degree of specificity for their molluscan intermediate hosts. A panel of 4 digenean species (Echinostoma paraensei, E. trivolvis, Schistosoma mansoni, and Schistosomatium douthitti) and 5 snail species (Biomphalaria glabrata, Helisoma trivolvis, Lymnaea stagnalis, Stagnicola elodes, and Helix aspersa representing 3 gastropod families) was used to assess the relative contributions of miracidial behavior, host plasma osmolality, and host plasma factors in dictating specificity. Additional experiments were undertaken with a fifth digenean, Echinoparyphium sp. Expected patterns of compatibility were first confirmed; each parasite species produced patent infections in its known snail host, but not in the other snail species. One exception was S. douthitti, which unexpectedly did not infect L. stagnalis. As judged by direct observation and by noting their disappearance after exposure to snails, miracidia were generally less likely to attach to or penetrate incompatible than compatible hosts. However, over half of the miracidia of each parasite species attached to or attempted penetration of both compatible and incompatible hosts, suggesting that under the experimental conditions used, miracidial host location and attachment behaviors were not of overriding importance in dictating observed patterns of specificity. For each digenean species, the percentage of larvae that became immobile, rounded, showed tegumental damage, or died over a 6-hr interval in plasma of the various snails was assessed. In no case was plasma from a compatible host harmful to sporocysts or rediae. In contrast, in 8 of 16 (50%) incompatible combinations, snail plasma had a significant negative effect on sporocyst condition. In 4 of 12 (33%) incompatible combinations, plasma had a significant negative effect on rediae. In 9 of 10 combinations tested, lymnaeid plasma was toxic for the parasites of planorbid snails and in 2 of 4 combinations, planorbid plasma was toxic for the parasites of lymnaeid snails. Toxicity was not attributable to differences in plasma osmolality between snail species. The ability of plasma from incompatible snails to affect viability of both sporocysts and rediae was surprisingly strong, suggesting that humoral factors play a greater role in dictating patterns of digenean-snail specificity than previously appreciated.     

Surface membrane proteins of Biomphalaria glabrata embryonic cells bind fucosyl determinants on the tegumental surface of Schistosoma mansoni primary sporocysts

Previous observations that in vitro adherence of Biomphalaria glabrata embryonic (Bge) cells to sporocyst larval stages of Schistosoma mansoni was strongly inhibited by fucoidan, a sulfated polymer of L-fucose, suggested a role for lectinlike Bge cell receptors in sporocyst binding interactions. In the present investigation, monoclonal antibodies with specificities to 3 major glycan determinants found on schistosomes, LacdiNAc, fucosylated LacdiNAc (LDNF), and the Lewis X antigen, were used in adhesion blocking studies to further analyze the molecular interactions at the host-parasite interface. Results showed that only the anti-LDNF antibody significantly reduced snail Bge cell adhesion to the surface of sporocysts, suggesting that fucosyl determinants may be important in larval-host cell interactions. Affinity chromatographic separation of fucosyl-reactive Bge cell proteins from fucoidan-bound Sepharose 4B revealed the presence of polypeptides ranging from 6 to 200 kDa after elution with fucoidan-containing buffer. Pre-elution of the Bge protein-bound affinity column with dextran (Dex) and dextran sulfate (DexS) before introduction of the fucoidan buffer served as controls for protein binding based on nonspecific sugar or negative charge interactions. A subset of polypeptides (approximately 35-150 kDa) released by fucoidan elution was identified as Bge surface membrane proteins, representing putative fucosyl-binding proteins. Far-western blot analysis also demonstrated binding reactivity between Bge cell and sporocyst tegumental proteins. The finding that several of these parasite-binding Bge cell proteins were also fucoidan-reactive suggests the possible involvement of these molecules in mediating cellular interactions with sporocyst tegumental carbohydrates. It is concluded that Bge cells have surface protein(s) that may be playing a role in facilitating host cell adhesion to the surface of schistosome primary sporocysts through larval fucosylated glycoconjugates.     

Schistosoma mansoni: lectin-dependent cytotoxicity of hemocytes from susceptible host snails, Biomphalaria glabrata

Normally benign hemocytes from a strain (M-line) of the snail, Biomphalaria glabrata, susceptible to Schistosoma mansoni, became cytotoxic toward the sporocyst stage if the parasite was first treated with the lectin, concanavalin A. Concanavalin A binding was inhibitable with alpha-methyl mannoside and killing was dose-dependent. Maximal levels of concanavalin A-induced cytotoxicity were comparable with levels observed when hemocytes from a resistant snail strain (13-16-R1) encountered untreated sporocysts. Induction of the cytotoxic response did not occur if hemocytes alone were pretreated with the lectin. A unique method incorporating ultraviolet microscopy and the vital fluorescent dye, eosin Y, was used for discriminating between live and dead sporocysts. This model may prove useful in understanding mechanisms used by invertebrate effector cells in recognition and killing of invading organisms.