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Planorbid freshwater snails are important intermediate hosts for parasitic diseases caused by parasitic worms, most notably schistosomiasis. There are numerous reports of snails, specifically Biomphalaria glabrata, having compromised defences against schistosomes after being exposed to thermal stress. Environmental modifications to the defenses of schistosome transmitting snails could have negative ramifications for human disease risk in the context of climate change. Here the effects of heat shock on the production of hydrogen peroxide, a primary anti-microbial effector in many molluscs, were examined. The present findings show that heat shock increases NADPH oxidase 2 mRNA levels and hydrogen peroxide produced by snail hemocytes, and that both of these phenotypes could be reversed by an HSP-90 inhibitor. These findings indicate that snail defense systems are altered by heat shock at a molecular level in B. glabrata, and that snail immunity to many pathogens may be altered by the rapid variations in temperature that are associated with global climate change., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Freshwater snails are obligate intermediate hosts for numerous parasitic trematodes, most notably schistosomes. Schistosomiasis is a devastating human and veterinary illness, which is primarily controlled by limiting the transmission of these parasites from their intermediate snail hosts. Understanding how this transmission occurs, as well as the basic immunobiology of these snails may be important for controlling this disease in the future. Allelic variation in the Guadeloupe resistance complex (GRC) of Biomphalaria glabrata partially determines their susceptibility to parasitic infection, and can influence the microbiome diversity and microbial defenses in the hemolymph of these snails. In the present study, we examine the most abundant proteins present in the hemolymph of snails that are resistant or susceptible to schistosomes, as determined by their GRC genotype. Using proteomic analysis, we found that snails with different GRC genotypes have differentially abundant hemolymph proteins that are not explained by differences in transcription. There are 13 revealed hemolymph proteins that differ significantly between resistant and susceptible genotypes, nearly 40% of which are involved in immune responses. These findings build on the mounting evidence that genes in the GRC region have multiple physiological roles, and likely contribute more extensively to the general immune response than previously believed. These data also raise the intriguing possibility that the GRC region controls resistance to schistosomes, not directly, but indirectly via its effects on the snail's proteome and potentially its microbiome., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The immune system of snails is highly sensitive to pollutants, which can suppress its immune response. We investigated the effects of exposure to the glyphosate-based herbicide Roundup® Original on the snail Biomphalaria glabrata infected by the platyhelminth Echinostoma paraensei by evaluating changes in the snail's internal defense system. Four cohorts were studied: control group, infected snails, snails treated with Roundup®, and snails infected and treated with Roundup®. The hemocyte viability was assessed, morphological differentiation of cells was observed and flow cytometry was performed to determine the morphology, viability and the lectin expression profiles. The frequencies of dead hemocytes were lower in the infected group and higher in both pesticide treated groups. Three cell types were identified: blast-like cells, hyalinocytes and granulocytes. The highest number of all types of hemocytes, as well as the highest number of dead cells, were observed in the infected, pesticide-treated group. The association between infection and herbicide exposure greatly increased the frequency of dead hemocytes, suggesting that this condition impairs the internal defense system of B. glabrata making the snails more vulnerable to parasitic infections., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Freshwater snails are the intermediate hosts for numerous parasitic worms that are detrimental to human and agricultural health. Understanding the immune responses of these snails could be vital for finding ways to block transmission of those parasites. Allelic variation in a recently discovered genomic region in the snail, Biomphalaria glabrata, influences their susceptibility to schistosomes. Here we tested whether genes in that region, termed the Guadeloupe Resistance Complex (GRC), are involved in recognition of common pathogen-associated molecules that have been shown to be stimulants of the hydrogen peroxide defense pathway. We show that hemocytes extracted from individuals with one of the three GRC genotypes released less hydrogen peroxide than the other two genotypes, after stimulation with galactose. This difference was not observed after stimulation with several other microbial-associated carbohydrates, despite those ligands sharing the same putative pathway for hydrogen peroxide release. Therefore, we conclude that allelic variation in the GRC region may influence the recognition of galactose, rather than the conserved downstream steps in the hydrogen peroxide pathway. These results thus are consistent with the hypothesis that proteins produced by this region are involved in pathogen recognition., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Climate change may affect the internal defense system (IDS) of freshwater snails, and as a result their capacity to transmit disease. We examined effects of short-term exposure to supra- and sub-optimal temperatures or starvation on 3 parameters of the IDS of the schistosome-resistant Salvador strain of Biomphalaria glabrata - hemocyte concentrations, cell division in the amebocyte-producing organ (APO), and resistance to infection with Schistosoma mansoni. Adult snails were exposed to 1 of 3 temperatures, 20°C, 27°C (controls), or 33°C, for 1 or 2weeks, with food. A fourth group was maintained at 27°C, but without food. Compared to the controls, starved snails had significantly higher hemocyte counts at both 1 and 2weeks, although mitotic activity in the APO was significantly lower at both time periods. Exposure to 20°C or 33°C for 1 or 2weeks did not affect hemocyte numbers. However, APO mitotic activity in snails exposed to 20°C was significantly higher at both 1 and 2weeks, whereas mitotic activity in snails exposed to 33°C was significantly lower at 1week but normal at 2weeks. None of the treatments altered the resistance phenotype of Salvador snails. In a follow-up experiment, exposure to 33°C for 4-5h, a treatment previously reported to both induce expression of heat shock proteins (Hsps) and abrogate resistance to infection, caused immediate upregulation of Hsp 70 and Hsp 90 expression, but did not alter resistance, and Hsp expression levels returned to baseline after 2weeks at 33°C. Results of this study indicate that abnormal environmental conditions can have both stimulatory and inhibitory effects on the IDS in adult B. glabrata, and that some degree of acclimation to abnormal temperatures may occur., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This review summarizes the research progress made over the past decade in the field of gastropod immunity resulting from investigations of the interaction between the snail Biomphalaria glabrata and its trematode parasites. A combination of integrated approaches, including cellular, genetic and comparative molecular and proteomic approaches have revealed novel molecular components involved in mediating Biomphalaria immune responses that provide insights into the nature of host-parasite compatibility and the mechanisms involved in parasite recognition and killing. The current overview emphasizes that the interaction between B. glabrata and its trematode parasites involves a complex molecular crosstalk between numerous antigens, immune receptors, effectors and anti-effector systems that are highly diverse structurally and extremely variable in expression between and within host and parasite populations. Ultimately, integration of these molecular signals will determine the outcome of a specific interaction between a B. glabrata individual and its interacting trematodes. Understanding these complex molecular interactions and identifying key factors that may be targeted to impairment of schistosome development in the snail host is crucial to generating new alternative schistosomiasis control strategies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fibrinogen-related proteins or FREPs constitute a large family of molecules, defined by the presence of a fibrinogen-related domain (FReD). These molecules are found in all animals and are diverse in both form and function. Here, we review the current understanding of gastropod FREPs, which are characterized by the presence of a fibrinogen domain connected to one or two immunoglobulin superfamily domains by way of a short interceding region. We present a historical perspective on the discovery of FREPs in gastropods followed by a summary of advances made in the nearly two decades of research focused on the characterization of FREPs in Biomphalaria glabrata (BgFREPs). Topics covered include BgFREP genomic architecture, predicted structure and known functions, structural comparisons between BgFREPs, and evidence of somatic diversification. Also examined are the expression patterns of BgFREPs during snail development and immunological challenges. Recent functional characterization of the role BgFREPs play in the defence response against digenean trematodes is also presented, as well as new data investigating the nucleotide-level genomic conservation of FREPs among Pulmonate gastropods. Finally, we identify areas in need of further research. These include confirming and identifying the specific binding targets of BgFREPs and elucidating how they later engage snail haemocytes to elicit an immunological response, precise mechanisms and importance of BgFREP diversification, characterizing the tissue expression patterns of BgFREPs, as well as addressing whether gastropod FREPs retain immunological importance in alternative snail-trematode associations or more broadly in snail-pathogen interactions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Snails' susceptibilities to infection with Schistosoma mansoni were determined through observation of infection rates, total cercarial production and tissue responses of the first generation (F1) of Biomphalaria alexandrina snails, originally collected from different Egyptian governorates (Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta) and responses were compared between groups. The emergence of cercariae for a 3-month period and the calculation of survival and infection rates, in control (Schistosome Biological Supply Center; SBSC) and infected snails were evaluated. SBSC and Giza snails showed greater susceptibilities to infection and lower mortality rates. In addition, at 6 and 72 h post-exposure to miracidia all the snail groups showed no difference in the anatomical locations of sporocysts. The larvae were found in the head-foot, the mantle collar and the tentacles of the snails. Sporocysts showed normal development with low tissue reactions in SBSC and Giza snail groups infected with S. mansoni miracidia (SBSC). However, in Fayoum, Kafr El-Sheikh, Ismailia and Damietta snail groups, variable tissue responses were observed in which numerous hemocytes made direct contact with S. mansoni larvae forming capsules. The results suggested that, different responses of B. alexandrina snail's hemocytes towards S. mansoni are related to the degree of susceptibility of these snails. So this is important in planning the strategy of schistosomiasis control., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Comparative analyses of the N-glycosylation pattern of hemolymph glycoproteins from Biomphalaria glabrata strains Puerto Rico (BgPR) and Salvador (BgBS-90), differing in their susceptibility towards Schistosoma mansoni infection, were performed by Western blotting, enzyme-linked immunosorbent assays, two-dimensional high-performance liquid chromatography and mass spectrometry. Obtained data demonstrated an enhanced expression of serologically cross-reacting, fucosylated carbohydrate epitopes by the highly susceptible BgPR-strain in comparison to the resistant BgBS-90-strain. In particular, glycoproteins of BgPR snails exhibited larger amounts of glycans with (β1-2)-linked xylose or terminal Fuc(α1-3)GalNAc(β1-4)[±Fuc(α1-3)]GlcNAc(β1-)-units which are known to mediate cross-reactivity with schistosomal glycoconjugates. This finding could be corroborated by immunohistochemical studies showing again an enhanced expression of such carbohydrate epitopes in BgPR tissue. Hence, our results provide evidence for a correlation of B. glabrata susceptibility towards S. mansoni infection and the expression of carbohydrate determinants shared by the parasite and its intermediate host.

Schistosomes develop successfully in susceptible snails but are encapsulated and killed in resistant ones. Mechanism(s) shaping these outcomes involves the parasites ability to evade the snail's defenses. RNA analysis from resistant (BS-90), non-susceptible (LAC2) and susceptible (NMRI) juvenile Biomphalaria glabrata to Schistosoma mansoni revealed that stress-related genes, heat shock protein 70 (Hsp 70) and reverse transcriptase (RT), were dramatically co-induced early in susceptible snails, but not in resistant/non-susceptible ones. These transcripts were, however, down regulated upon exposure to irradiated parasites although penetration behavior of irradiated vs. normal parasites were the same, indicating that Hsp 70 and RT regulation was elicited by infection and not injury. Understanding molecular events involved in stress response transcriptional regulation of Hsp 70 in juvenile snails could pave a way towards the identification of genes involved in schistosome/snail interactions.

The snail Biomphalaria glabrata (Gastropoda, Mollusca) is an important intermediate host for the human parasite Schistosoma mansoni (Digenea, Trematoda). Anti-pathogen responses of B. glabrata were studied towards a better understanding of snail immunity and host-parasite compatibility. Open reading frame ESTs (ORESTES) were sampled from different transcriptomes of M line strain B. glabrata, 12h post-challenge with Escherichia coli (Gram-negative), Micrococcus luteus (Gram-positive) bacteria or compatible S. mansoni, and controls. The resulting 3123 ORESTES represented 2129 unique sequences (373 clusters, 1756 singletons). Of these, 175 (8.1%) were putative defense factors, including lectins, antimicrobial peptides and components of various immune-effector systems. Comparison of biological processes (GO-terms) within different transcriptomes indicated that B. glabrata increased oxygen transport and metal binding in reaction to all challenges. Comprehensive comparisons of transcriptomes revealed that responses of B. glabrata against bacteria were similar to each other and differed from the ineffective response to S. mansoni. Furthermore, the response to S. mansoni infection was less comprehensive than that to bacteria. Many novel (unknown) sequences were recovered in association with particular challenges. B. glabrata possesses multi-faceted, potent immune defenses. This agrees with the notion that S. mansoni is capable of immune-evasion and prevents effective host defense responses in order to survive in B. glabrata. Future analysis of the numerous unknown sequences recovered from challenged snails may reveal novel immune factors and provide increased understanding of immunity of B. glabrata in relation to parasite-host compatibility.

The three populations of hemocytes and the fixed phagocytes of Biomphalaria glabrata show different affinities for the following injected foreign materials: ferritin, horseradish peroxidase, zymosan particles, latex beads, and live bacteria. Ferritin and horseradish peroxidase, which are internalized by micropinocytosis, induce a moderate activation of hemocytes. Particles trigger a strong activation that consists of mobilization of the cytoskeleton, with formation of pseudopodia and filopodia and modifications of the plasma membrane that enable hemocytes to adhere together, as in encapsulations. Phagocytosis of zymosan and live bacteria induces alterations in mitochondria that may reflect changes in the respiratory activity. Phagocytosis of particles follows different processes: fixed phagocytes ingest latex beads of two different sizes according to the "zipper model", which implies sequential binding between phagocyte receptors and opsonized ligands; zymosan particles are ingested by medium-size hemocytes by a variant of the zipper model in which a limited close contact between cell and particle resembles focal contacts in cell spreading; bacteria or large latex beads are ingested by large hemocytes without apparent close contact following a "trigger model" which resembles macropinocytosis., (Copyright 1999 Academic Press.)

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., (Copyright 1999 Academic Press.)

Injection of foreign particles (zymosan, latex beads, living bacteria) and substances (ferritine, horseradish peroxidase) in Biomphalaria glabrata induces the rapid appearance of tubular double-helical filaments in the hemolymph. This rapid induction may be due to the polymerization of precursor proteins of the hemolymph. Tubular helical filaments are also observed in normal snails, especially in the hemal spaces of the proximal kidney; they seem to belong to the extracellular matrix and form bundles of a few parallel units associated with fine fibrils. Hemocytes adhere to these bundles by means of the filopodia tip or by small areas of the cell surface; consequently, they may become temporarily sedentary in the hemal spaces. The deposition of helical filaments seems increased in ageing snails and in snails parasitized by Echinostoma caproni., (Copyright 1999 Academic Press.)

Previous studies have shown that Schistosoma mansoni excretory-secretory polypeptides (ESP) inhibit various internal defense functions of hemocytes from Biomphalaria glabrata and that plasma also may exert a modulatory effect on hemocyte activity. To better understand how plasma may influence hemocyte-schistosome interactions in inbred strains of snails, biotinylated ESP (b-ESP) were used as probes to identify ESP-reactive plasma components and partially characterize the nature of their binding interactions. In a plasma binding assay, b-ESP bound in a dose-dependent fashion to immobilized snail plasma, although no quantitative differences in ESP reactivity to plasma of schistosome-susceptible (M-line) and -resistant (13-16-R1) snail strains were detected. Moreover, co-incubation of b-ESP with homologous plasma or pretreatment of plasma with nonbiotinylated ESP in the plate assay significantly reduced b-ESP binding to immobilized plasma, indicating a specific interaction between ESP and plasma. Pretreatment of b-ESP with mannose, porcine stomach mucin, fetuin, and asialofetuin resulted in a significant inhibition of b-ESP binding to plasma, whereas pretreatment of immobilized plasma with a cocktail of monosaccharides (including mannose), porcine stomach mucin, and fetuin had no inhibitory effect. These data suggest the presence of a carbohydrate-binding protein(s) in sporocyst ESP that is targeted to plasma glycoconjugates. Based on the carbohydrate content of the major inhibitory glycoproteins, galactosyl and n-acetyl-galactosaminyl sugars may represent putative determinants for the lectin-like ESP molecule(s). However, ESP binding to plasma from M-line and 13-16-R1 B. glabrata strains exhibited similar sugar and glycoprotein inhibition patterns. SDS-PAGE and electroblot analyses further demonstrated that ESP bound to a subset of separated plasma polypeptides, most prominently to a doublet of 52 and 54 kDa, and a complex of proteins with molecular masses greater than 150 kDa. Other polypeptides exhibiting weaker binding interactions included components of 34, 60, 64, 86, and 125 kDa. Although both M-line and 13-16-R1 snail strains contained a similar complement of ESP-binding plasma proteins, ESP binding to the 34- and 86-kDa proteins occurred at higher frequencies in susceptible M-line plasma. It is concluded that ESP, released during in vitro transformation of S. mansoni miracidia, contain carbohydrate-reactive proteins capable of selectively binding to components of snail plasma. Quantitative differences between snail strains in the occurrence of ESP-binding plasma molecules was documented.

Decreases in the number of Schistosoma mansoni cercariae released from susceptible M-line Biomphalaria glabrata were detected following injection with the recombinant human cytokine, interleukin-1. No differences in either the time post-exposure at which shedding began or the percentage of snails shedding cercariae were detected between interleukin-1 injected, heat-inactivated interleukin-1 injected, or sham injected controls. However, sham injected and heat-inactivated interleukin-1 injected snails maintained significantly higher (approximately three-fold) levels of cercarial production compared to interleukin-1 injected snails over 8 weeks of cercarial shedding. Injection of interleukin-1 into schistosome-susceptible (M-line) and resistant (13-16-R1) strains of B. glabrata increased hemocyte phagocytosis of target particles and phagocytosis stimulated O2- production in both snail strains at 24 hr postexposure to the parasite. Resistant 13-16-R1 snails maintained, on average, 2.4 times the number of O2- producing phagocytic cells than did M-line susceptible snails, indicating that the incomplete abrogation of cercarial shedding in M-line snails may be due to an inadequate number of activated circulating effector cells in these snails. These data strongly support the contention that the evolutionarily conserved cytokine, interleukin-1, or a molecule in snail plasma with interleukin-1-like immunospecificity, biological activity, and function plays a significant role in the maintenance of susceptibility or resistance to S. mansoni infection in B. glabrata. Finally, these data also supply evidence for the evolutionary conservation of the function and role of interleukin-1, O2-, and antioxidant defense mechanisms in this host-parasite relationship.

Biomphalaria glabrata snails which were not susceptible as juveniles to infection by Schistosoma mansoni were selectively bred (by self-fertilization) from the highly susceptible NMRI laboratory snail stock. The susceptibility rate among juvenile snails derived from interbreeding NMRI parents was initially 85-95%, but after several generations of selection, less than 5% of exposed snails became infected by the parasite. Selection for low susceptibility also resulted in a large proportion of snails that displayed low fecundity and produced abnormal egg masses. Individual adult snails which were isolated from an interbreeding population of nonselected NMRI snails usually produced well-developed egg masses each containing 15-30 embryos. However, when juvenile snails from this same population were reared in isolation and not allowed to cross-fertilize, many displayed a pattern of low fecundity and abnormal egg production similar to that observed in the selected low susceptible line. Furthermore, it was found that many of the isolated snails which exhibited low egg production were also not susceptible to parasitic infection.

Polyclonal antibodies were raised in rabbits to two groups of diffusely staining M line Biomphalaria glabrata plasma polypeptides, of 150-210 and 70-120 kDa, designated as Group 1 molecules (G1M) and group 2 molecules (G2M), respectively. G1M and G2M are known to increase in abundance and to become more diverse following infection of B. glabrata with the digenetic trematode Echinostoma paraensei. These antibodies were used in conjunction with immunoblotting and slot blotting procedures to document binding of G1M/G2M from plasma of unexposed control snails (C plasma) or plasma from snails with 8-day infections of E. paraensei (I plasma) to various targets. Binding of G1M/G2M to both gram-positive and gram-negative bacteria, human A, B, and O and rabbit erythrocytes, and to sporocysts and rediae of E. paraensei was documented. Immunoblots revealed that erythrocytes are bound by a particular G2M band of 100-120 kDa present in I plasma that is in low concentration or lacking in C plasma. This explains previous results indicating that I plasma agglutinates all 4 erythrocytes examined whereas C plasma agglutinates only rabbit erythrocytes. More G1M/G2M binding to sporocysts occurred if I plasma rather than C plasma was used for incubations. Also, monosaccharide inhibition of G1M/G2M binding to sporocysts was observed in I plasma but not in C plasma. The results indicate that infection with E. paraensei induces production by B. glabrata of unique plasma polypeptides and that molecules present in I plasma can bind to the surfaces of non-self objects including E. paraensei larvae in a lectin-like fashion.

Monoclonal antibodies (mAb) directed against Schistosoma mansoni tropomyosin isoform, SMTM (Xu et al. Experimental Parasitology 69, 373-392, 1989), were used to test for cross-reactivity with Biomphalaria glabrata antigens. One mAb (1F10) recognized antigens of 39, 41, and 80 kDa in a snail head/foot antigen preparation but not a hepatopancreas antigen preparation. Another mAb (1C1) cross-reacted with a 39-kDa antigen in the head/foot extract but not in the hepatopancreas extract. Epitope mapping revealed the 1F10 epitope to be between amino acids 135 and 188 of both Bg39 (Dissous et al. Molecular and Biochemical Parasitology 43, 245-256, 1990) and BgTMII (Weston and Kemp, Experimental Parasitology 76, 358-370, 1993), while the 1C1 epitope was located between amino acids 189 and 213 of BgTMII. Various invertebrate species, including members from Trematoda, Pulmonata, Annelida, and Arthropoda, were tested for cross-reactivity with the monoclonal antibodies. While the 1F10 mAb displayed broad invertebrate cross-reactivity, the 1C1 mAb cross-reactivity was restricted to schistosomes, F. hepatica, and the pulmonate snails B. glabrata and Physa sp.

Rabbit antisera, raised against whole, homogenized hepatopancreas from uninfected Biomphalaria glabrata, were used to screen a cDNA library prepared from adult Schistosoma mansoni. Of 1.8 x 10(5) recombinants screened, 34 clones were specifically immunoreactive with the antisera. Twenty of the clones were subcloned for further analysis, and 13 of the 20 were demonstrated to hybridize to a cDNA probe encoding schistosome tropomyosin I (pSMTM) at high stringency (Xu et al. Experimental Parasitology 69, 373-392, 1989). One partial cDNA clone (pSM4), which failed to hybridize to pSMTM, was used to rescreen the adult worm cDNA library and a 1381-bp cDNA clone, SmTMII, was identified and characterized. SmTMII contained a single 284-amino acid open reading frame and was shown to be 65.8% homologous to SMTM. Computer-assisted analysis of the predicted protein structure depicted a hydrophilic molecule with the extensive alpha helical secondary structure characteristic of tropomyosins. SmTMII was expressed in Escherichia coli and the resulting 45-kDa fusion protein was recognized by both a polyclonal antiserum specific for SMTM and the antiserum of mice chronically infected with S. mansoni. Northern analysis showed the SmTMII mRNA in the adult stage to be about 1.6 kb in size. Analysis of the SmTMII gene by Southern blot revealed a complex hybridization pattern suggesting the presence of introns or multiple gene copies. The 650-bp EcoRI fragment of SmTMII was used to screen a cDNA library prepared from uninfected Biomphalaria glabrata and two clones, each encoding a novel snail tropomyosin, BgTMII, were isolated and characterized. The two clones, consisting of 2816 and 2314 bp, respectively, differed from each other only in the polyadenylation signal used and each contained a single and identical 284-amino acid open reading frame having 94.7% homology with the previously reported snail tropomyosin, BG39 (Dissous et al. Molecular and Biochemical Parasitology 43, 245-256, 1990). BgTMII was shown to have a higher amino acid homology with SMTM (68.3%) than with BG39 (65.1%) and a higher amino acid homology with SMTM than the two schistosome tropomyosins (SMTM and SmTMII) have with each other (65.8%). Implications of these observations in relation to the concept of host-parasite antigen mimicry are discussed.

Hemocytes derived from a strain (13-16-R1) of Biomphalaria glabrata resistant to Schistosoma mansoni were significantly more likely to bind untreated latex beads than hemocytes from the schistosome-susceptible M line strain. Beads preincubated in 13-16-R1 plasma were more readily bound by both 13-16-R1 and M line hemocytes than beads preincubated in M line plasma. Beads preincubated in plasma derived from snails of either strain infected with the trematode Echinostoma paraensei were more readily bound by hemocytes than beads preincubated in plasma from control snails of the corresponding strain. Plasma from snails exposed to S. mansoni did not have a similar effect. Throughout these experiments, beads receiving a particular treatment were consistently bound at higher rates by 13-16-R1 than M line hemocytes. SDS-PAGE of plasma components eluted from beads revealed differences between treatments, particularly in diffuse bands falling into two groups, of 75-130 and 150-220 kDa. The results indicate that both hemocytes and plasma components from the two host strains differ and identify plasma molecules deserving of additional study as possible modulators of hemocyte effector functions. Also, S. mansoni and E. paraensei provoked different responses in the same host snail.

In vitro phagocytosis of erythrocytes by hemocytes of B. glabrata, intermediate host of S. mansoni, is strongly influenced by calcium, several lectins, and plasma factors. Our results indicate that two different mechanisms of non-self-recognition in B. glabrata may occur: (1) In the presence of calcium, phagocytosis occurs in noninfected and in infected snails without involvement of any other substances, and hemocytes of schistosome resistant as well as those of susceptible snails are able to recognize and phagocytose the target cells. (2) In the absence of calcium, phagocytosis occurs if bridging molecules (heterologous lectins in our assays) were present for which effector and target cells possess binding sites or if target cells were plasma coated prior to the assays. In suspensions in homologous plasma, hemocytes of both snail strains, infected or noninfected, subsequently showed phagocytic activities of about 70-80%. Preincubation of target cells in homologous plasma resulted in similar high phagocytic activities of hemocytes even in the absence of plasma during the standard assay. In these assays, a significantly higher proportion of hemocytes of resistant snails phagocytosed plasma-opsonized erythrocytes, whereas hemocytes of susceptible snails internalized less erythrocytes per cell and needed 60 min to phagocytose at percentages equivalent to that of resistant hemocytes within 10 min. Preincubation of erythrocytes in resistant plasma significantly increased the subsequent phagocytic activity of susceptible hemocytes, whereas preincubation of erythrocytes in susceptible plasma decreased the phagocytosis level of resistant hemocytes.

Excretory-secretory (E-S) products obtained during in vitro Schistosoma mansoni miracidium-to-sporocyst transformation were found to contain a 108-kDa polypeptide capable of scavenging both exogenously produced and M-line Biomphalaria glabrata hemocyte-derived superoxide (O2-) anions. Separation of crude transformation E-S products using HPLC and ion exchange chromatography resulted in the separation of two isoforms of the 108-kDa molecule. Using an in vitro phagocytosis assay, both isoforms were found to be capable of reducing O2- production by phagocytically stimulated M-line B. glabrata hemocytes without cell loss and without a concomitant reduction in phagocytosis. Although parasite antioxidant molecules appear to play a role in the evasion of host oxidative defense systems in several parasite-vertebrate systems, no previous reports of a parasite antioxidant capability against the potential of oxidative killing by invertebrate defense systems has been reported. In conjunction with the previously confirmed production of O2- by B. glabrata hemocytes and reports of reactive oxygen metabolite production by hemocytes from several molluscan species, these results indicate that reactive forms of oxygen and parasite antioxidant systems may play an important role in the determination of compatibility in the trematode-mollusc relationship.

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.

The resistance or susceptibility of Biomphalaria glabrata strains to strains of Schistosoma mansoni, the human blood fluke, are evidenced by the responses of snail hemocytes to sporocysts of the schistosome, both in vivo and in vitro. It is now reported that living sporocysts of the PR1 strain of S. mansoni agglutinate in the plasma of all tested strains of B. glabrata, in contrast to fixed sporocysts which agglutinate only in plasma from resistant snail strains. The agglutinating activity in resistant plasmas is not divalent cation dependent, and was not inhibited by the 26 carbohydrates and four amino acids tested. In addition, the observation that gelatinous deposits develop on transforming miracidia-sporocysts in B. glabrata plasmas is also reported. Both the agglutination and gel-formation phenomena may facilitate recognition of, and attacks on, sporocysts, thereby contributing to susceptibility and resistance in this host-parasite system.

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.

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.

The reaction of amoebocytes in the hemolymph of the infected intermediate host, Biomphalaria glabrata, to Schistosoma mansoni antigens has been investigated using the indirect immunofluorescence antibody test. Monolayers of amoebocytes, prepared from hemolymph of infected and normal snails, were first fixed and then reacted with antisera obtained from mice infected for 7 to 9 weeks. Nonspecific and cross-reactions between the antisera and monolayers of amoebocytes were eliminated by absorbing the antisera with tissues from uninfected snails. The liberation of detectable schistosomal antigens in the hemolymph in soluble and particulate forms coincided with completion of the infection cycle 3 to 4 weeks after exposure to miracidia. The schistosomal antigens were demonstrable in the cytoplasm of amoebocytes and in the center of amoebocyte aggregates.