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.     

Schistosoma mansoni modulation of phagocytosis in Biomphalaria glabrata

Both short-term (3 hr) exposure of Biomphalaria glabrata snails (M-line and 13-16-R1) to Schistosoma mansoni (PR1) miracidia and in vitro incubation of parasite sporocysts with host hemolymph components altered host phagocytic ability. Hemocytes obtained from susceptible (M-line) snails that had been exposed to parasite miracidia for 3 hr showed reduced levels of phagocytosis of yeast cells in vitro compared to hemocytes from unexposed individuals. Incubation of whole hemolymph with sporocysts in vitro also reduced yeast phagocytosis in this susceptible strain. In contrast, resistant (13-16-R1) hemocytes showed increased levels of yeast phagocytosis after in vitro incubation with the parasite, and the opsonic properties of 13-16-R1 plasma were greater after exposure of snails to miracidia. These strain-specific effects of S. mansoni on host hemocyte phagocytosis and plasma opsonization were seen only when both plasma and hemocytes were present at the time of exposure to the parasite.     

Schistosoma mansoni in Susceptible and Resistant Snail Strains Biomphalaria tenagophila: In Vivo Tissue Response and In Vitro Hemocyte Interactions

Schistosomiasis is a parasitic disease that is highly prevalent, especially in developing countries. Biomphalaria tenagophila is an important invertebrate host of Schistosoma mansoni in Brazil, with some strains (e.g. Cabo Frio) being highly susceptible to the parasite, whereas others (e.g. Taim) are completely resistant to infection. Therefore, B. tenagophila is an important research model for studying immune defense mechanisms against S. mansoni. The internal defense system (IDS) of the snail comprises hemocytes and hemolymph factors acting together to recognize self from non-self molecular patterns to eliminate the threat of infection. We performed experiments to understand the cellular defenses related to the resistance and/or susceptibility of B. tenagophila to S. mansoni. During the early stages of infection, fibrous host cells of both snail strains were arranged as a thin layer surrounding the sporocysts. However, at later stages of infection, the cellular reactions in resistant snails were increasingly more intense, with thicker layers surrounding the parasites, in contrast to susceptible strains. All parasites were damaged or destroyed inside resistant snails after 10 h of infection. By contrast, parasites inside susceptible snails appeared to be morphologically healthy. We also performed experiments using isolated hemocytes from the two strains interacting with sporocysts. Hemocyte attachment started as early as 1 h after initial infection in both strains, but the killing of sporocysts was exclusive to hemocytes from the resistant strain and was time course dependent. The resistant strain was able to kill all sporocysts. In conclusion, our study revealed important aspects of the initial process of infection related to immune defense responses of strains of B. tenagophila that were resistant to S. mansoni compared with strains that were susceptible. Such information is relevant for the survival or death of the parasites and so is important in the development of control measures against this parasite.     

The influence of hydrocortisone on cellular defence mechanisms of Biomphalaria glabrata

Since the internal defense system of mollusks consists of cellular and humoral mechanisms, we examined the role of hydrocortisone in mollusks defense cells and the influence of this steroid on the development of Schistosoma mansoni in its intermediary host. Hydrocortisone had an immunosuppressive action in Biomphalaria glabrata, as reflected in the reduced number of defense cells and the altered cell physiology. Histopathological analysis showed that hydrocortisone facilitated the intramolluscan development of S. mansoni, by reducing the extent of the inflammatory response, seen as a greater number of viable sporocysts with no surrounding hemocytes.     

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.     

Primary culture of the region of the amebocyte-producing organ of the snail Biomphalaria glabrata, the intermediate host of Schistosoma mansoni

Biomphalaria glabrata snails are major hosts for the digenetic trematoda Schistosoma mansoni, the causative agent of human schistosomiasis. The success or failure of the infection will be dependent on the mobilization of the molluskan internal defense system, where a major role will be played by circulating hemocytes produced by the APO (amebocyte-producing organ) of the snail. In this report, the primary culture of the APO region of B. glabrata was obtained for the first time, as well as a control culture of the ovotestis. Three different cell populations migrated easily from the explants in culture, with no need of any dispersion agent. The cells grew in suspension at an incubation temperature of 15 degrees C and the cultures were maintained viable for up to two weeks. Two of these cell populations obtained resembled cell types known to be present in the hemolymph of Biomphalaria. The availability of APO cells in culture may contribute to a better understanding of the internal defense in mollusks, in general, as well as the specific response of B. glabrata to S. mansoni infection.     

Respiratory burst of Biomphalaria glabrata hemocytes: Schistosoma mansoni-resistant snails produce more extracellular H2O2 than susceptible snails

The production of reactive oxygen species by hemocytes from the gastropod Biomphalaria glabrata has been linked to their ability to kill the trematode parasite Schistosoma mansoni. For 2 laboratory strains of the snail, 1 resistant (13-16-R1) and 1 susceptible (MO) to the PR1 strain of S. mansoni, we compared hemocyte production of extracellular hydrogen peroxide when stimulated with the protein kinase C agonist phorbol myristate acetate (PMA). The time course of the PMA-induced response is similar in both strains with respect to onset, peak production, and termination of the respiratory burst. However, the magnitude of the response differs between strains, in that hemocytes from resistant snails generate significantly more hydrogen peroxide. These findings suggest that the capacity to produce hydrogen peroxide could be critical in determining susceptibility or resistance to S. mansoni.     

Schistosoma mansoni: passive transfer of resistance by serum in the vector snail, Biomphalaria glabrata

Passive transfer of natural resistance to Schistosoma mansoni (PR-1 strain) has been successfully accomplished in the snail intermediate host, Biomphalaria glabrata (PR albino, M-line strain). Injection of serum (cell-free hemolymph) from a naturally schistosome-resistant strain of B. glabrata (10-R2) into PR albino snails induced a complete protection from a primary infection with the parasite in 29 of 48 snails (60.4%). In comparison, inoculation of homologous PR albino serum or heterologous proteins (fetal calf serum) had no effect. Moreover, this protection could be induced 24 hr prior to, or 24 hr after, exposure to the parasite, although heating of 10-R2 serum to 70 C for 30 min destroyed its protective ability. When in vitro transformed sporocysts were preincubated in 10-R2 or PR albino serum and then were injected into susceptible snails, a high level of infection (88.5 and 83.3%, respectively) was produced in both groups. Thus, the 10-R2 serum factor does not appear to be mediating specific parasite recognition by host hemocytes. Alternatively, our results suggest that 10-R2 serum possesses a heat-labile factor which specifically activate B. glabrata hemocytes to encapsulate and destroy sporocysts whereas PR albino serum lacks this factor.     

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.     

Identification of a genetic marker associated with the resistance to Schistosoma mansoni infection using random amplified polymorphic DNA analysis

In schistosomiasis, the host/parasite interaction remains not completely understood. Many questions related to the susceptibility of snails to infection by respective trematode still remain unanswered. The control of schistosomiasis requires a good understanding of the host/parasite association. In this work, the susceptibility/resistance to Schistosoma mansoni infection within Biomphalaria alexandrina snails were studied starting one month post infection and continuing thereafter weekly up to 10 weeks after miracidia exposure. Genetic variations between susceptible and resistant strains to Schistosoma infection within B. alexandrina snails using random amplified polymorphic DNA analysis technique were also carried out. The results showed that 39.8% of the examined field snails were resistant, while 60.2% of these snails showed high infection rates.In the resistant genotype snails, OPA-02 primer produced a major low molecular weight marker 430 bp. Among the two snail strains there were interpopulational variations, while the individual specimens from the same snail strain, either susceptible or resistant, record semi-identical genetic bands. Also, the resistant character was ascendant in contrast to a decline in the susceptibility of snails from one generation to the next.     

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.     

Extra-cellular matrix changes in Schistosoma mansoni-infected Biomphalaria glabrata

Reactivity of snails against parasites exhibits a primitive focal reaction, with encapsulation, phagocytosis and destruction of parasite larvae by macrophage-like cells - the hemocytes. This reaction mimics granulomatous inflammation seen in higher animals. However, different from the latter, little is known about the participation of extra-cellular matrix in such snail defense reactions. Normal and Schistosoma mansoni-infected Biomphalaria glabrata of different strains were submitted to cytological, histological, ultrastructural and biochemical methods in order to investigate the behavior of extra-cellular tissues at the site of anti-parasite reactions. In spite of the presence of two cell-types in peripheral hemolymph, only one cell-type was present at the sites of tissue reactions. Although pre-existent collagen and elastic fibers and microfibrils sometimes appeared slightly compressed around focal reactions, no evidences of duplication, synthesis or deposition of connective-tissue extra-cellular components were observed within or around the zones of reactive cell accumulations. Thus, tissue reactions against S. mansoni in the snail B. glabrata appeared exclusively dependent on one specific population of hemocytes.     

The mitochondrial genome of Biomphalaria glabrata (Gastropoda: Basommatophora), intermediate host of Schistosoma mansoni

The complete mitochondrial (Mt) genome of the gastropod Biomphalaria glabrata, a major intermediate host for the human parasite Schistosoma mansoni, was sequenced. The circular genome, the first determined from a basommatophoran snail, is AT rich (74.6%) and the smallest Mt genome (13,670 nucleotides [nt]) characterized from mollusks to date. Sequences from 2 B. glabrata strains, M-line and 1742, differed by only 18 nt. Phylogenetic analysis of 16S and ND1 sequences confirmed the Brazilian ancestry of both B. glabrata strains. Gene predictions indicated 22 transfer RNA, 12S and 16S ribosomal RNA (rRNA), and 13 protein-encoding genes, as is typical for metazoans. Of the mollusk Mt genomes currently known, the gene order was most similar to that of stylommatophoran gastropods, concordant with the monophyly of pulmonate gastropods. Screening of GenBank (expressed sequence tags database [dbEST]) with the Mt sequence identified 108 entries from B. glabrata as Mt-derived sequences, including 12S and 16S rRNA sequences. Moreover, 11 sequences originating from the Mt genome of B. glabrata were identified among EST entries ascribed to intramolluskan stages of S. mansoni. The availability of this Mt sequence will facilitate further molecular investigations into the biology of Biomphalaria sp. and interactions between this intermediate host and intramolluskan stages of S. mansoni.     

Changes induced in Biomphalaria glabrata (Say, 1818) following trials for artificial stimulation of its internal defense system

Biomphalaria glabrata can react through different pathways to Schistosoma mansoni miracidium penetration, according to the degree of resistance/susceptibility presented by different snail strains, which is a genetically determined character, resistance being the dominant feature. However, it has been observed that previous susceptible snail strain may change its reactive behavior along the course of infection, exhibiting later a pattern of cercarial shedding and histopatopathological picture compatible with high resistance. Such observation suggests the possibility of B. glabrata to develop a sort of adaptative immunity face a schistosome infection. To explore on this aspect, the present investigation looked for the behavior of S. mansoni infection in B. glabrata previously subjected to different means of artificial stimulation of its internal defense system. Snails previously inoculated with irradiated miracídia (Group I); treated with S. mansoni antigens (Group II) or with a non-related parasite antigen (Group III) were challenged with 20 viable S. mansoni miracidia, and later looked for cercarial shedding and histopathologic changes at different times from exposition. Nodules of hemocyte accumulations were found at the site of antigen injection. These nodules resembled solid granulomas, and were larger and more frequent in snails injected with S. mansoni products as compared to those injected with Capillaria hepatica. However, the presence of such granulomas did not avoid the S. mansoni challenge infection from developing in a similar way as that seen in controls. The data are indicative that hemocytes are able to proliferate locally when stimulated, such capacity also remaining localized, not being shared by the population of hemocytes located elsewhere within the snail body.     

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.     

Lysosomal enzyme activities in susceptible and refractory strains of Biomphalaria glabrata during the course of infection with Schistosoma mansoni

The distribution and abundance of the lysosomal enzyme markers, acid phosphatase (AP), peroxidase (PO), and nonspecific esterase (NE), within circulating blood cells (hemocytes) were examined in a schistosome-susceptible (PR albino M-line) and a resistant (10-R2) strain of Biomphalaria glabrata during the course of infection with Schistosoma mansoni. The dynamics of serum (cell-free hemolymph) AP activities and total hemocyte numbers in infected snails also were investigated. Hemocyte subpopulations, as determined by these enzyme markers, responded differently to parasite infection between snail strains. Generally, the hemocyte subpopulations within PR albino snails remained largely unchanged, whereas the same subpopulations in 10-R2 snails fluctuated considerably. The distribution of AP in the hemocytes of 10-R2 snails decreased by 1 hr postexposure (PE) to the parasite and remained low through 12 hr before increasing to control values at 24 hr and 2 wk PE. In comparison, PO activity increased by 1 hr PE and peaked at 12 hr before dropping to 0 hr values by 2 wk PE. The NE activity exhibited still another pattern with the percentage of NE-positive cells decreasing from 0 to 12 hr PE followed by a recovery to 0-hr values by 24 hr. The abundance of these hemocyte enzymes followed a similar pattern to that of their distribution, although some differences were observed. Serum AP values varied little in PR albino snails except for a significant increase at 2 wk PE, indicating a possible response to tissue damage resulting from migrating daughter sporocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free radical scavengers in susceptible/resistant Biomphalaria alexandrina snails before and after infection

The activities of catalase (Cat), superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione transferase (GST), glucose-6-phosphate dehydrogenase (G6PD) and glyceraldehyde3-phosphate dehydrogenase (G3PD) were studied in tissue and hemolymph of susceptible (S) (EgBS(2)) and resistant (R) (EgBR(2)) Biomphalaria alexandrina snails. The results showed that CAT and GST were higher in the hemolymph of snails susceptible to Schistosoma mansoni than in that of snails resistant to infestation, while SOD and G3PD were lower in the susceptible snails. The role of these enzymes as free radical scavengers was traced 1 and 24 h after infection of the two snail lines with S. mansoni. Moreover, the activities of SOD and G3PD were also measured 2 and 4 weeks post infection. The results revealed that the overall enzymatic activities were higher in susceptible than in resistant snail tissues. After 1 h of infection, all enzymes were increased in R and S snails except GST and G6PD which decreased in S snails. After 24 h of infection, GST increased in S snails and G3PD decreased in both S and R snails while other enzymes reached normal levels.     

SELECTION AND STRAIN SPECIFICITY OF COMPATIBILITY BETWEEN SNAIL INTERMEDIATE HOSTS AND THEIR PARASITIC SCHISTOSOMES

Many theoretical models of host-parasite coevolution assume that variation in host resistance to parasite infection is, at least partially, genetically determined and specific to the strain of infecting parasite. However, very few experimental studies have been conducted to test this assumption in animal-parasite systems. Biomphalaria glabrata snails serve as the intermediate hosts of Schistosoma mansoni. Although some snails are resistant to infection, there is no evidence of fixation of resistance in field populations. Two possible explanations for this are high fitness costs associated with resistance and a dynamic coevolution between parasite and host, perhaps involving matching alleles or gene-for-gene interactions. Two strains of B. glabrata were artificially selected for either resistance or susceptibility to each of two strains of S. mansoni parasite for three generations. Third-generation snails were then were exposed to either the parasite strain to which they had been selected or to a different parasite strain. In both host strains, resistance and susceptibility (compatibility) were found to be heritable. Moreover, compatibility to one parasite strain was not associated with compatibility to another strain, implying no genetic trade-off. Our results are discussed in terms of potential mechanisms of resistance in this host-parasite system and their implications to general coevolutionary theory.