Interaction between primary and secondary sporocysts of Schistosoma mansoni and the internal defence system of Biomphalaria resistant and susceptible to the parasite

The outcome of the interaction between Biomphalaria and Schistosoma mansoni depends on the response of the host internal defence system (IDS) and the escape mechanisms of the parasite. The aim of this study was to evaluate the responsiveness of the IDS (haemocytes and soluble haemolymph factors) of resistant and susceptible Biomphalaria tenagophila lineages and Biomphalaria glabrata lineages in the presence of in vitro-transformed primary sporocysts and secondary sporocysts obtained from infected B. glabrata. To do this, we assayed the cellular adhesion index (CAI), analysed viability/mortality, used fluorescent markers to evaluate the tegumental damage and transplanted secondary sporocysts. B. tenagophila Taim was more effective against primary and secondary sporocystes than the susceptible lineage and B. glabrata. Compared with secondary sporocysts exposed to B. tenagophila, primary sporocysts showed a higher CAI, a greater percentage of dead sporocysts and were labelled by lectin from Glycine max and Alexa-Fluor 488 fluorescent probes at a higher rate than the secondary sporocysts. However, the two B. tenagophila lineages showed no cercarial shedding after inoculation with secondary sporocysts. Our hypothesis that secondary sporocysts can escape the B. tenagophila IDS cannot be confirmed by the transplantation experiments. These data suggest that there are additional mechanisms involved in the lower susceptibilty of B. tenagophila to S. mansoni infection.     

Differential lectin labelling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection

Lectins/carbohydrate binding can be involved in the Schistosoma mansoni recognition and activation of the Biomphalaria hemocytes. Therefore, expression of lectin ligands on Biomphalaria hemocytes would be associated with snail resistance against S. mansoni infection. To test this hypothesis, circulating hemocytes were isolated from B. glabrata BH (snail strain highy susceptible to S. mansoni), B. tenagophila Cabo Frio (moderate susceptibility), and B. tenagophila Taim (completely resistant strains), labelled with FITC conjugated lectins (ConA, PNA, SBA, and WGA) and analyzed under fluorescence microscopy. The results demonstrated that although lectin-labelled hemocytes were detected in hemolymph of all snail species tested, circulating hemocytes from both strains of B. tenagophila showed a larger number of lectin-labelled cells than B. glabrata. Moreover, most of circulating hemocytes of B. tenagophila were intensively labelled by lectins PNA-FITC and WGA-FITC, while in B. glabrata small hemocytes were labeled mainly by ConA. Upon S. mansoni infection, lectin-labelled hemocytes almost disappeared from the hemolymph of Taim and accumulated in B. glabrata BH. The role of lectins/carbohydrate binding in resistance of B. tengophila infection to S. mansoni is still not fully understood, but the data suggest that there may be a correlation to its presence with susceptibility or resistance to the parasite.     

Biomphalaria tenagophila: dynamics of populations of resistant and susceptible strains to Schistosoma mansoni, with or without pressure of the parasite

Resistant (Taim, RS) and susceptible albino (Joinville, SC) Biomphalaria tenagophila populations were kept together, at different proportions, throughout a 18-month-period. Some of the snail groups were submitted to Schistosoma mansoni infection. The targets of this study were (a) to analyze the populational dynamics among resistant and susceptible individuals to S. mansoni; (b) to study the resistance phenotype in descendants of cross-breeding; (c) to observe whether the parasite could exert any kind of selection in those snail populations. Throughout the experiment it could be observed that the susceptible B. tenagophila strain (Joinville) underwent a selective pressure of the parasite that was negative, since the individuals showed a high mortality rate. Although B. tenagophila (Taim) population presented a higher mortality rate without pressure of the parasite, this event was compensated by a reproductive capacity. B. tenagophila Taim was more fecund than B. tenagophila Joinville and was able to transmit the resistance character to their descendants. F1 generation obtained by cross-breeding between resistant and susceptible lineages was completely resistant to S. mansoni infection, irrespective of the Taim proportion. Moreover, less than 5% of F2 progeny were susceptible to S. mansoni infection.     

Effect of gamma radiation on the activity of hemocytes and on the course of Schistosoma mansoni infection in resistant Biomphalaria tenagophila snails

High doses of gamma radiation (10 Krad) in Biomphalaria tenagophila snails (Taim strain), which have been found to be resistant to Schistosoma mansoni, were not sufficient to impair their resistance to the parasite. The number of hemocytes, as well as their phagocytic activity, were not affected by irradiation, thus showing resemblance with mammal macrophages, which are resistant to gamma irradiation also.     

Specific tyrosine phosphorylation induced in Schistosoma mansoni miracidia by haemolymph from schistosome susceptible, but not resistant, Biomphalaria glabrata

Molecular interplay during snail-schistosome interactions is poorly understood and there is much to discover concerning the effect of snail host molecules on molecular processes in schistosomes. Using the Biomphalaria glabrata - Schistosoma mansoni host-parasite system, the effects of exposure to haemolymph, derived from schistosome-resistant and susceptible snail strains, on protein tyrosine phosphorylation in miracidia have been investigated. Western blotting revealed several tyrosine phosphorylated proteins in this larval stage. Exposure of miracidia to haemolymph from susceptible snails for 60 min resulted in a striking, 5-fold, increase in the tyrosine phosphorylation of a 56 kDa (p56) S. mansoni protein. In contrast, haemolymph from resistant snails had little effect on protein tyrosine phosphorylation levels in miracidia. Confocal microscopy revealed that tyrosine phosphorylation was predominantly associated with proteins present in the tegument. Finally, treatment of miracidia with the tyrosine kinase inhibitor genistein significantly impaired their development into primary sporocysts. The results open avenues for research that focus on the potential importance of phospho-p56 to the outcome of schistosome infection in snails, and the significance of protein tyrosine kinase-mediated signalling events to the transformation of S. mansoni larvae.     

Localization of carbohydrate determinants common to Biomphalaria glabrata as well as to sporocysts and miracidia of Schistosoma mansoni

The presence of antigenic carbohydrate epitopes shared by Biomphalaria glabrata as well as by the sporocysts and miracidia representing snail-pathogenic larval stages of Schistosoma mansoni was assayed by immunohistochemical staining of paraformaldehyde-fixed tissues. To this end, both polyclonal rabbit antiserum raised against soluble egg antigens (SEA) of S. mansoni and monoclonal antibodies recognizing the carbohydrate epitopes LDN [GalNAc(beta1-4)GlcNAc(beta1-)], F-LDN [Fuc(alpha1-3)GalNAc(beta1-4)GlcNAc(beta1-)], LDN-F [GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-)], LDN-DF [GalNAc(beta1-4)[Fuc(alpha1-2)Fuc(alpha1-3)]GlcNAc(beta1-)] and Lewis X [Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-)] were used. Intriguingly, anti-SEA serum as well as anti-F-LDN antibodies displayed significant binding in the foot region, anterior tissue and the hepatopancreas of uninfected snails, whereas the Lewis X epitope was only weakly detectable in the latter tissue. In contrast, increased binding of antibodies recognizing LDN, LDN-F and LDN-DF was observed in infected snail tissue, in particular in regions involved in sporocystogenesis, in addition to an enhanced binding of anti-SEA serum and antibodies reacting with F-LDN. A pronounced expression of most of these carbohydrate antigens was also observed at the surface of miracidia. Hence, the detection of shared carbohydrate determinants in uninfected snail tissue, sporocysts and miracidia may support the hypothesis of carbohydrate-based molecular mimicry as a survival strategy of S. mansoni.     

Molecular and functional characterization of a tandem-repeat galectin from the freshwater snail Biomphalaria glabrata, intermediate host of the human blood fluke Schistosoma mansoni

In the present study, a tandem-repeat type galectin was characterized from an embryonic cell line (Bge) and circulating hemocytes of the snail Biomphalaria glabrata, intermediate host of the human blood fluke Schistosoma mansoni. The predicted B. glabrata galectin (BgGal) protein of 32 kDa possessed 2 carbohydrate recognition domains, each displaying 6 of 8 conserved amino acids involved in galactoside-binding activity. A recombinant BgGal (rBgGal) demonstrated hemagglutinating activity against rabbit erythrocytes, which was specifically inhibited by galactose-containing sugars (lacNAc/lac>galNAc/gal). Although native galectin was immunolocalized in the cytoplasm of Bge cells and the plasma membrane of a subset of snail hemocytes (60%), it was not detected in cell-free plasma by Western blot analysis. The findings that rBgGal selectively recognizes the schistosome-related sugar, lacNAc, and strongly binds to hemocytes and the tegument of S. mansoni sporocysts in a sugar-inhibitable fashion suggest that hemocyte-bound galectin may be serving as a pattern recognition receptor for this, or other pathogens possessing appropriate sugar ligands. Based on molecular and functional features, BgGal represents an authentic galectin, the first to be fully characterized in the medically-important molluscan Class Gastropoda.     

Dominant character of the molecular marker of a Biomphalaria tenagophila strain (Mollusca: Planorbidae) resistant to Schistosoma mansoni

Biomphalaria tenagophila population from Taim (state of Rio Grande do Sul, Brazil) is totally resistant to Schistosoma mansoni, and presents a molecular marker of 350 bp by polymerase chain reaction and restriction fragment length polymorphism of the entire rDNA internal transcriber spacer. The scope of this work was to determine the heritage pattern of this marker. A series of cross-breedings between B. tenagophila from Taim (resistant) and B. tenagophila from Joinville, state of Santa Catarina (susceptible) was carried out, and their descendants F1 and F2 were submitted to this technique. It was possible to demonstrate that the specific fragment from Taim is endowed with dominant character, since the obtained segregation was typically mendelian.     

Compatibility of Biomphalaria tenagophila with Schistosoma mansoni: a study of homologous plasma transference

This study aims to investigate the importance of the serum factors present in the plasma of resistant Biomphalaria tenagophila snails, when transferred to susceptible conspecific. Susceptible B. tenagophila (CF) received plasma from resistant B. tenagophila (Taim), and both were later infected with Schistosoma mansoni. We noticed that the plasma transfer showed an increase on the resistance of susceptible snails of about 86% when compared to the non-immunized group (p < 0.001).     

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.     

A potential vector of Schistosoma mansoni in Uruguay

Susceptibility experiments were carried out with a Biomphalaria straminea-like planorbid snail (Biomphalaria aff. straminea, species inquirenda) from Espinillar, near Salto (Uruguay), in the area of the Salto Grande reservoir, exposed individually to 5 miracidia of Schistosoma mansoni (SJ2 and BH2 strains). Of 130 snails exposed to the SJ2 strain, originally infective to Biomphalaria tenagophila, 30 became infected (23%). The prepatent (precercarial) period ranged from 35 to 65 days. The cercarial output was irregular, following no definite pattern, varying from 138 to 76,075 per snail (daily average 4.3 to 447.5) and ending up with death. Three specimens that died, without having shed cercariae, on days 69 (2) and 80 after exposure to miracidia, had developing secondary sporocysts in their tissues, justifying the prospect of a longer precercarial period in these cases. In a control group of 120 B. tenagophila, exposed to the SJ2 strain, 40 became infected, showing an infection rate (33.3%) not significantly different from that of the Espinillar snail (chi 2 = 3.26). No cercariae were produced by any of the Espinillar snails exposed to miracidia of the BH2 strain, originally infective to Biomphalaria glabrata. Four specimens showed each a primary sporocyst in one tentacle, which disappeared between 15 and 25 days post-exposure, and two others died with immature, very slender sporocysts in their tissues on days 36 and 54. In a control group of 100 B. glabrata exposed to BH2 miracidia, 94 shed cercariae (94%) and 6 remained negative. Calculation of Frandsen's (1979a, b) TCP/100 index shows that "Espinillar Biomphalaria-SJ2 S. mansoni" is a vector-parasite "compatible" combination.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

Freshwater snails and Schistosomiasis mansoni in the state of Rio de Janeiro,Brazil: III--Baixadas Mesoregion

In this paper, the third of a series dealing with the survey of freshwater gastropods of the state of Rio de Janeiro, the results of collections carried out in the Mesoregion Baixadas from 2000 to 2002 are presented. Twenty-two species, belonging to seven families, were found. As to the snail intermediate hosts of Schistosoma mansoni, the most frequent species was Biomphalaria tenagophila besides some new findings of Biomphalaria straminea. No specimens were found harboring larval forms of S. mansoni although different kinds of cercariae had been observed.     

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.     

Schistosoma mansoni: continuous variation in susceptibility of the vector snail of schistosomiasis, Biomphalaria tenagophila I. Self-fertilization-lineage.

Mascara, D., Kawano, T., Magnanelli, A. C., Silva, R. P. S., Sant' Anna, O. A., and Morgante, J. S. 1999. Schistosoma mansoni: continuous variation in susceptibility of the vector snail of schistosomiasis, Biomphalaria tenagophila I. Self-Fertilization-Lineage. Experimental Parasitology 93, 133-141. Artificial selection of Biomphalaria tenagophila snails for susceptibility to infection by Schistosoma mansoni (Brazilian SJ strain) was carried out from natural populations. After five self-fertilization generations, two lineages were isolated and were designated as SUSC (highly susceptible 93-100%) and RES (nonsusceptible 5-0%). Length of the prepatent period, cercarial production, and mortality of the hosts in postexposure were determined in all generations (F(1)-F(8)) and were analyzed as quantitative traits related to host susceptibility. Distribution patterns of frequencies were observed within snail families (samples derived from one F(0) snail), these traits showing a significant influence by selection applied to susceptibility. The multiple quantitative classes were described in terms of continuous variation. During the selection of SUSC lineage, classes with higher values of prepatent length and lower cercarial production were eliminated, and the heritability calculated for these two traits was 0.811 and 0.709, respectively. Experimental results were correlated with an increase in the level of susceptibility in the generations selected and are discussed in relation to inheritance patterns as well as the quantitative variation of susceptibility.     

Genetic markers from Biomphalaria tenagophila (Gastropoda: Pulmonata: Planorbidae) obtained by the double stringency polymerase chain reaction technique

Biomphalaria tenagophila, one of the intermediate hosts of the trematoda Schistosoma mansoni, is a simultaneous hermafrodite snail species. In order to analyse the genetic structure of these populations, we performed a double-stringency PCR technique to obtain genetic markers with microsatellites and arbitrary primers in a single reaction.     

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.     

Freshwater snails and Schistosomiasis mansoni in the state of Rio de Janeiro, Brazil: IV - Sul Fluminense Mesoregion

In this paper, the forth of a series dealing with the survey of freshwater gastropods of the state of Rio de Janeiro, the results of collections carried out in the Sul Fluminense Mesoregion from 2000 to 2002 are presented and revealed the occurrence of 18 species: Antillorbis nordestensis; Biomphalaria glabrata; Biomphalaria peregrina; Biomphalaria straminea; Biomphalaria tenagophila; Drepanotrema anatinum; Drepanotrema cimex; Drepanotrema lucidum; Ferrissia sp.; Gundlachia ticaga; Gundlachia sp.; Heleobia sp.; Lymnaea columella; Melanoides tuberculatus; Physa acuta; Physa marmorata; Pomacea sordida and Pomacea sp. As to the snail hosts of Schistosoma mansoni the most frequent species was B. tenagophila, found in all municipalities surveyed, except Parati. Besides new records the present study extends the distribution of B. peregrina and B. straminea in the state. No specimens were found harbouring larval forms of S. mansoni although different kinds of cercariae had been observed. An account about the current schistosomiasis transmission sites in this Mesoregion is presented as well.