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Freshwater snails of the genus Biomphalaria serve as obligatory hosts for the digenetic trematode Schistosoma mansoni, the causative agent for the most widespread form of intestinal schistosomiasis. Within Biomphalaria, S. mansoni larvae multiply and transform into the cercariae form that can infect humans. Trematode development and proliferation is thought to be facilitated by modifications of host behavior and physiological processes, including a reduction of reproduction known as "parasitic castration." As neuropeptides participate in the control of reproduction across phylogeny, a neural transcriptomics approach was undertaken to identify peptides that could regulate Biomphalaria reproductive physiology. The present study identified a transcript in Biomphalaria alexandrina that encodes a peptide belonging to the gonadotropin-releasing hormone (GnRH) superfamily. The precursor and the predicted mature peptide, pQIHFTPDWGNN-NH 2 (designated Biom-GnRH), share features with peptides identified in other molluscan species, including panpulmonates, opisthobranchs, and cephalopods. An antibody generated against Biom-GnRH labeled neurons in the cerebral, pedal, and visceral ganglia of Biomphalaria glabrata. GnRH-like immunoreactive fiber systems projected to all central ganglia. In the periphery, immunoreactive material was detected in the ovotestis, oviduct, albumen gland, and nidamental gland. As these structures serve crucial roles in the production, transport, nourishment, and encapsulation of eggs, disruption of the GnRH system of Biomphalaria could contribute to reduced reproductive activity in infected snails., (© 2020 Wiley Periodicals LLC.)

Quantum dots have generated great interest because of their optical properties, both to life sciences and electronics applications. However, possible risks to the environment associated with these nanoparticles are still under investigation. The present study aimed to evaluate the toxicity of suspensions of cadmium telluride (CdTe) quantum dots to Biomphalaria glabrata mollusks, a very sensitive aquatic environmental bioindicator for physical and chemical agents. Toxicity was examined by using embryos and adult mollusks as well as hemocytes. The distribution of cadmium in the organs of adults was also assessed. Effects of the stabilizing agent of the quantum dots were also evaluated. Animals were exposed to suspensions of quantum dots for 24 h, at concentrations varying from 1.2 to 20 nM for embryos and from 50 to 400 nM for adult mollusks. Results showed that suspensions of quantum dots induced malformations and mortality in embryos and mortality in adults, depending on the concentration applied. In the cytotoxicity study, hemocyte apoptosis was observed in adults exposed to the highest concentration of quantum dots applied as well as to the stabilizing agent. Cell binucleation and micronucleus frequencies were not significative. Bioaccumulation evaluation revealed that quantum dots targeted the digestive gland (hepatopancreas). Taken together, outcomes suggested that specific nano-effects related directly not only to composition but also to the aggregation of quantum dots may be mediating the observed toxicity. Thus B. glabrata was determined to be a very sensitive species for interpreting possible nano-effects in aquatic environments. Environ Toxicol Chem 2019;38:2128-2136. © 2019 SETAC., (© 2019 SETAC.)

Background: Schistosomiasis is a harmful neglected tropical disease caused by infection with Schistosoma spp., such as Schistosoma mansoni. Schistosoma must transition within a molluscan host to survive. Chemical analyses of schistosome-molluscan interactions indicate that host identification involves chemosensation, including naïve host preference. Proteomic technique advances enable sophisticated comparative analyses between infected and naïve snail host proteins. This study aimed to compare resistant, susceptible and naïve Biomphalaria glabrata snail-conditioned water (SCW) to identify potential attractants and deterrents., Methods: Behavioural bioassays were performed on S. mansoni miracidia to compare the effects of susceptible, F1 resistant and naïve B. glabrata SCW. The F1 resistant and susceptible B. glabrata SCW excretory-secretory proteins (ESPs) were fractionated using SDS-PAGE, identified with LC-MS/MS and compared to naïve snail ESPs. Protein-protein interaction (PPI) analyses based on published studies (including experiments, co-expression, text-mining and gene fusion) identified S. mansoni and B. glabrata protein interaction. Data are available via ProteomeXchange with identifier PXD015129., Results: A total of 291, 410 and 597 ESPs were detected in the susceptible, F1 resistant and naïve SCW, respectively. Less overlap in ESPs was identified between susceptible and naïve snails than F1 resistant and naïve snails. F1 resistant B. glabrata ESPs were predominately associated with anti-pathogen activity and detoxification, such as leukocyte elastase and peroxiredoxin. Susceptible B. glabrata several proteins correlated with immunity and anti-inflammation, such as glutathione S-transferase and zinc metalloproteinase, and S. mansoni sporocyst presence. PPI analyses found that uncharacterised S. mansoni protein Smp_142140.1 potentially interacts with numerous B. glabrata proteins., Conclusions: This study identified ESPs released by F1 resistant, susceptible and naïve B. glabrata to explain S. mansoni miracidia interplay. Susceptible B. glabrata ESPs shed light on potential S. mansoni miracidia deterrents. Further targeted research on specific ESPs identified in this study could help inhibit B. glabrata and S. mansoni interactions and stop human schistosomiasis.

The human disease schistosomiasis (or bilharzia) is caused by the helminth blood fluke parasite Schistosoma mansoni, which requires an intermediate host, the freshwater gastropod snail Biomphalaria glabrata (the most common intermediate host). The free-swimming parasite miracidia utilise an excellent chemosensory sense to detect and locate an appropriate host. This study investigated the biomolecules released by the snail that stimulate changes in the behaviour of the aquatic S. mansoni miracidia. To achieve this, we have performed an integrated analysis of the snail-conditioned water, through chromatography and bioassay-guided behaviour observations, followed by mass spectrometry. A single fraction containing multiple putative peptides could stimulate extreme swimming behaviour modifications (e.g. velocity, angular variation) similar to those observed in response to crude snail mucus. One peptide (P12;-R-DITSGLDPEVADD-KR-) could replicate the stimulation of miracidia behaviour changes. P12 is derived from a larger precursor protein with a signal peptide and multiple dibasic cleavage sites, which is synthesised in various tissues of the snail, including the central nervous system and foot. P12 consists of an alpha helix secondary structure as indicated by circular dichroism spectroscopy. This information will be helpful for the development of approaches to manipulate this parasites life cycle, and opens up new avenues for exploring other parasitic diseases which have an aquatic phase using methods detailed in this investigation., Competing Interests: The authors have declared that no competing interests exist.

Objective: To establish a method for the quantitative determination of serotonin and dopamine in the nervous system of Biomphalaria glabrata by using ultra high performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC MS/MS) ., Methods: The B. glabrata nervous system was broken in the pure methanol solution after obtaining it by dissecting with microscope. Then, the supernatant containing the target substance after twice high speed centrifugation was got. The extraction was separated on an ACQUITY UPLC BEH Amide column with Waters TQ-XS series mass spectrometry detector, with ESI source and positive electrospray ionization mode when the machine testing., Results: The detection limit of serotonin was 0.03 ng/ml and the limit of quantification was 0.1 ng/ml. The detection limit of dopamine was 0.05 ng/ml and the limit of quantification was 0.15 ng/ml. The recoveries of serotonin ranged from 90.68% to 94.72% over the range of 1 to 40 ng/ml. The recoveries of dopamine ranged from 91.68% to 96.12% over the range of 1.0 ng/ml to 40 ng/ml., Conclusions: The established UPLC MS/MS method is simple, stable and reproducible. It can be used for the quantitative analysis of serotonin and dopamine in the nervous system of B. glabrata snails.

The freshwater snail Biomphalaria glabrata has drawn much research interest by virtue of it being one of the intermediate hosts of the parasitic flatworm Schistosoma mansoni, a causative agent of human schistosomiasis. Schistosomiasis is a chronic disease that affects over 260 million people globally, particularly in tropical and sub-tropical regions. One strategy that has been proposed as a way to prevent human infection by the parasite, involves the use of pheromone traps to lure the snail host away from areas of human activity. This requires an understanding of chemosensory communication in B. glabrata, especially of the chemoattractive factors. Although evidence indicates that specific chemical communication takes place, little is known about chemoattractants produced by the snail itself. Here, we report on the functional characterization of an endogenously produced temptin-like protein (BgTemptin) from B. glabrata and demonstrate that recombinant BgTemptin is attractive to this snail. Exposure of B. glabrata to BgTemptin results in 81% (lane maze) and 70% (T-maze) time spent near to the BgTemptin source. This effect, which is dependent on the concentration of the protein, provides another tool that can be further developed and used in efforts to control and eliminate schistosomiasis.

Background: Molluscs, including snails, are prone to parasite infection, which can lead to massive physiological and behavioural changes, yet many of the molecular components involved remain unresolved. Central to this point is the neural system that in snails consists of several ganglia that regulate the animals' physiology and behaviour patterns. The availability of a genomic resource for the freshwater snail Biomphalaria glabrata provides a mean towards the high throughput analysis of changes in the central nervous system (CNS) following infection with Schistosoma miracidia., Results: In this study, we performed a proteomic analysis of the B. glabrata CNS at pre-patent infection, providing a list of proteins that were further used within a protein-protein interaction (PPI) framework against S. mansoni proteins. A hub with most connections for both non-infected and infected Biomphalaria includes leucine aminopeptidase 2 (LAP2), which interacts with numerous miracidia proteins that together belong to the immunoglobulin family of cell adhesion related molecules. We additionally reveal the presence of at least 165 neuropeptides derived from the precursors of buccalin, enterin, FMRF, FVRI, pedal peptide 1, 2, 3 and 4, RYamide, RFamide, pleurin and others. Many of these were present at significantly reduced levels in the snail's CNS post-infection, such as the egg laying hormone, a neuropeptide required to initiate egg laying in gastropod molluscs., Conclusions: Our analysis demonstrates that LAP2 may be a key component that regulates parasite infection physiology, as well as establishing that parasite-induced reproductive castration may be facilitated by significant reductions in reproduction-associated neuropeptides. This work helps in our understanding of molluscan neuropeptides and further stimulates advances in parasite-host interactions.

Schistosomiasis remains a disease of major global public health concern since it is a chronic and debilitating illness. The widely distributed Schistosoma mansoni that causes intestinal schistosomiasis represents a great threat. Its world-wide distribution is permitted by the broad geographic range of the susceptible species of its intermediate host, Biomphalaria, which serves as an obligatory host for the larval stage, at which humans get infected. The objectives were to identify the proteins responsible for the snails’ compatibility outcome through differentiation between the total proteins among Biomphalaria alexandrina snails at different ages. The work was conducted on snails that differ in age and genetic backgrounds. Four subgroups (F1) from the progeny of self-reproduced susceptible and resistant snails (F0) were studied. Infection rates of these subgroups (young susceptible, adult susceptible, young resistant and adult resistant) were 90 %, 75 %, 40 % and 0 %, respectively. Using Sodium Dodecyl Sulphate Polyacrylamide Gel electrophoresis (SDS-PAGE), differences in protein expression were evaluated between adult and young snails of different subgroups. Dice similarity coefficient was calculated to determine the percentage of band sharing among the experimental subgroups. The results showed that the combination of similarities between age and compatibility status of the snails, lead to the highest similarity coefficient, followed by the combination of similarities of both genetic origin and age, even though they differ in the compatibility status. On the other hand, the differences in the genetic background, age and compatibility status, lead to the least similarity index. It was also found that the genetic background in young snails plays a major role in the determination of their compatibility, while the internal defense system has the upper hand in determining the level of adult compatibility. In conclusion, the findings of the present work highlight the great impact of the snail age in concomitance with the genetics and the internal defense in the determination of B. alexandrina/S.mansoni compatibility. Future works are recommended, as further characterization of the shared protein bands among the studied subgroups is needed to clarify their role in host-parasite relationship.

Angiostrongylus cantonensis is considered the main agent responsible for human eosinophilic meningoencephalitis. This parasite has low specificity for mollusk hosts and it can also use aquatic snails as auxiliary hosts. Studies based on the metabolic profile of Biomphalaria spp. infected by A. cantonensis have been conducted to observe parasite-host interactions. In the present study, the glucose content in the hemolymph and glycogen content in the digestive gland and cephalopedal mass of Biomphalaria tenagophila and Biomphalaria straminea experimentally infected by A. cantonensis were evaluated, along with the activity of LDH. The snails were dissected from 6 to 21days after infection to collect the hemolymph and separate the tissues. Decreases of 96% and 6.4% in the glucose content triggered a transition from aerobic to anaerobic metabolism in the two infected snail species, B. straminea and B. tenagophila, respectively. That finding was confirmed by high-performance liquid chromatography. These results indicate that when infected, these snails are able to change their metabolic profile, suggesting a strategy to maintain their homeostatic balance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The effects of 5, 20, and 40 miracidia dose exposures of Echinostoma caproni on the amino acid contents of Biomphalaria glabrata were studied using high performance thin-layer chromatography-densitometry. Amino acids were identified and quantified in whole bodies of exposed snails and in the uninfected matched controls at 2 and 4 weeks post-exposure. Using cellulose layers with the mobile phase 2-butanol-pyridine-glacial acetic acid-deionized water (39:34:10:26) and ninhydrin detection reagent [2% ninhydrin in acetone-n-butanol (1:1)], five amino acids were identified, i.e., leucine/isoleucine, valine, alanine, glycine, and ornithine, by hRF value comparison and color differentiation. Quantitatively, there was a marked elevation in the amounts of four of these five amino acids (isoleucine/leucine, valine, alanine, and ornithine) across dose levels at 4 weeks post-infection (P<0.05). Elevation of the amino acid content in the high dose snail group suggested that some changes occurred in the amino acid metabolism of the snails in that group as a function of miracidia dose.

O-glycosylation is a widely occurring posttranslational modification of proteins. The glycosylation status of a specific site may influence the location, activity and function of a protein. The initiating enzyme of mucin-type O-glycosylation is UDP-GalNAc:polypeptide GalNAc transferase (ppGalNAcT; EC 2.4.1.41). Using electron-transfer dissociation mass spectrometry, ppGalNAcT from the snail Biomphalaria glabrata was characterized regarding its ability to glycosylate threonine and serine residues in different peptide sequence environments. The preferences of the snail enzyme for flanking amino acids of the potential glycosylation site were very similar to vertebrate and insect members of the family. Acceptor sites with adjacent proline residues were highly preferred, while other residues caused less pronounced effects. No specific O-glycosylation consensus sequence was found. The results obtained from synthetic peptides were in good correlation with the observed glycosylation patterns of native peptides and with the order of attachment in a multi-glycosylated peptide. The snail enzyme clearly preferred threonine over serine in the in vitro assays. No significant differences of transfer speed or efficiency could be detected using a mutant of the enzyme lacking the lectin domain. This is the first characterisation of the substrate specificity of a member of the ppGalNAcT family from mollusc origin.

In spite of using aquatic snails as bioindicators for water pollution, little attention has been paid to the effect of parasitism upon the concentration of heavy metals (Al, Cd, Cu, Fe, Mn, Pb and Zn) in these organisms. The present study therefore aimed to compare the concentrations of heavy metals in trematode-infected Biomphalaria alexandrina collected from Kafer Alsheikh and Menofia provinces, Egypt, with uninfected snails from the same sites, in order to assess the effect of parasitism on the use of these snails as bioindicators. The concentrations of heavy metals in the soft parts and shells of snails were measured by flame atomic absorption spectrometry. The results showed that the heavy metal profile in snails infected with Echinostoma liei was very different from that in snails infected with Schistosoma mansoni. The total concentration of heavy metals in E. liei-infected snails collected from Kafer Alsheikh or Menofia province was greater than in uninfected snails. In contrast, the total concentration of heavy metals in S. mansoni-infected snails was reduced compared with uninfected snails. In conclusion, the status of snails with respect to parasitic infection must be taken into consideration when these snails are used as bioindicators.

The effects of a 5 versus 25 miracidia exposure of Echinostoma caproni on the lipid composition of Biomphalaria glabrata was studied using high performance thin layer chromatography (HPTLC)-densitometry. A 50 miracidia dose was not used because such a high level of exposure caused severe snail mortality by 3 weeks post-exposure (PE). Lipids were determined in the digestive-gland gonad complex (DGG) of the exposed snails and in the uninfected matched controls at 2 and 4 weeks PE. Extraction of lipids from DGGs was carried out by the Folch method with chloroform-methanol (2:1), and extracts were analyzed on Analtech HPTLC-HLF pre-adsorbent silica gel plates with measurement of separated bands using a CAMAG Scanner 3. For neutral lipids the mobile phase was petroleum ether-diethyl ether-glacial acetic acid (80:20:1) and the detection reagent was 5% ethanolic phosphoric acid, and for polar lipids chloroform-methanol-deionized water (65:25:4) mobile phase and 10% cupric sulfate in 8% phosphoric acid detection reagent were used. No significant differences in the concentrations of free sterols, free fatty acids, triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine were seen at 2 weeks PE in any of the groups. At 4 weeks PE, the free fatty acid concentration increased significantly in the snails exposed to 25 miracidia compared to that of the 5 miracidia/snail group or the controls. Elevation of the free fatty acid fraction in the high dose snail group suggested that some changes occurred in the lipid metabolism of the snails in that group as a function of miracidia dose.

High performance thin-layer chromatography was used to determine the concentration of β-carotene and lutein in the whole body and digestive gland-gonad complex (DGG) of uninfected Biomphalaria glabrata snails and those infected with Schistosoma mansoni for 6 and 8 weeks. Pigments were extracted from the snails using acetone and separated on EMD Millipore reversed phase C-18 plates with concentration zone using petroleum ether-acetonitrile-methanol (1:1:2) mobile phase. After development, two yellow pigment zones, lutein and β-carotene, were identified with respective Rf values of 0.55 and 0.13 and then quantified by densitometry. Statistical analysis of the weight percentages of each pigment showed a significant decrease (P < 0.05) in the concentration of β-carotene in the DGGs of infected B. glabrata at 6 and 8 weeks post-infection compared to the uninfected snails. No significant differences were seen in the concentrations of β-carotene in the whole body of the uninfected versus infected snail samples. Changes in the lutein concentration of the infected DGG and whole snail bodies were insignificant compared to the uninfected controls. In conclusion, larval S. mansoni infection caused a significant decrease in the β-carotene concentration of the DGG at 6 and 8 weeks post infection.

Background: Biomphalaria glabrata is the mollusc intermediate host for Schistosoma mansoni, a digenean flatworm parasite that causes human intestinal schistosomiasis. An estimated 200 million people in 74 countries suffer from schistosomiasis, in terms of morbidity this is the most severe tropical disease after malaria. Epigenetic information informs on the status of gene activity that is heritable, for which changes are reversible and that is not based on the DNA sequence. Epigenetic mechanisms generate variability that provides a source for potentially heritable phenotypic variation and therefore could be involved in the adaptation to environmental constraint. Phenotypic variations are particularly important in host-parasite interactions in which both selective pressure and rate of evolution are high. In this context, epigenetic changes are expected to be major drivers of phenotypic plasticity and co-adaptation between host and parasite. Consequently, with characterization of the genomes of invertebrates that are parasite vectors or intermediate hosts, it is also essential to understand how the epigenetic machinery functions to better decipher the interplay between host and parasite., Methods: The CpGo/e ratios were used as a proxy to investigate the occurrence of CpG methylation in B. glabrata coding regions. The presence of DNA methylation in B. glabrata was also confirmed by several experimental approaches: restriction enzymatic digestion with isoschizomers, bisulfite conversion based techniques and LC-MS/MS analysis., Results: In this work, we report that DNA methylation, which is one of the carriers of epigenetic information, occurs in B. glabrata; approximately 2% of cytosine nucleotides are methylated. We describe the methylation machinery of B. glabrata. Methylation occurs predominantly at CpG sites, present at high ratios in coding regions of genes associated with housekeeping functions. We also demonstrate by bisulfite treatment that methylation occurs in multiple copies of Nimbus, a transposable element., Conclusions: This study details DNA methylation for the first time, one of the carriers of epigenetic information in B. glabrata. The general characteristics of DNA methylation that we observed in the B. glabrata genome conform to what epigenetic studies have reported from other invertebrate species.

Carboxylic acids play an important role in both aerobic and anaerobic metabolic pathways of both the snail and the parasite. Monitoring the effects of infection by schistosome on Biomphalaria alexandrina carboxylic acids metabolic profiles represents a promising additional source of information about the state of metabolic system. We separated and quantified pyruvic, fumaric, malic, oxalic, and acetic acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to detect correlations between these acids in both hemolymph and digestive gland gonad complex (DGG's) samples in a total of 300 B. alexandrina snails (150 infected and 150 controls) at different stages of infection. The results showed that the majority of metabolite pairs did not show significant correlations. However, some high correlations were found between the studied acids within the control group but not in other groups. More striking was the existence of reversed correlations between the same acids at different stages of infection. Some possible explanations of the underlying mechanisms were discussed. Ultimately, however, further data are required for resolving the responsible regulatory events. These findings highlight the potential of metabolomics as a novel approach for fundamental investigations of host-pathogen interactions as well as disease surveillance and control.

Recent evidence supports the involvement of inducible, highly diverse lectin-like recognition molecules in snail hemocyte-mediated responses to larval Schistosoma mansoni. Because host lectins likely are involved in initial parasite recognition, we sought to identify specific carbohydrate structures (glycans) shared between larval S. mansoni and its host Biomphalaria glabrata to address possible mechanisms of immune avoidance through mimicry of elements associated with the host immunoreactivity. A panel of monoclonal antibodies (mABs) to specific S. mansoni glycans was used to identify the distribution and abundance of shared glycan epitopes (glycotopes) on plasma glycoproteins from B. glabrata strains that differ in their susceptibilities to infection by S. mansoni. In addition, a major aim of this study was to determine if larval transformation products (LTPs) could bind to plasma proteins, and thereby alter the glycotopes exposed on plasma proteins in a snail strain-specific fashion. Plasma fractions (< 100 kDa/> 100 kDa) from susceptible (NMRI) and resistant (BS-90) snail strains were subjected to SDS-PAGE and immunoblot analyses using mAB to LacdiNAc (LDN), fucosylated LDN variants, Lewis X and trimannosyl core glycans. Results confirmed a high degree of glycan sharing, with NMRI plasma exhibiting a greater distribution/abundance of LDN, F-LDN and F-LDN-F than BS-90 plasma (< 100 kDa fraction). Pretreatment of blotted proteins with LTPs significantly altered the reactivity of specific mABs to shared glycotopes on blots, mainly through the binding of LTPs to plasma proteins resulting in either glycotope blocking or increased glycotope attachment to plasma. Many LTP-mediated changes in shared glycans were snail-strain specific, especially those in the < 100 kDa fraction for NMRI plasma proteins, and for BS-90, mainly those in the > 100 kDa fraction. Our data suggest that differential binding of S. mansoni LTPs to plasma proteins of susceptible and resistant B. glabrata strains may significantly impact early anti-larval immune reactivity, and in turn, compatibility, in this parasite-host system.

The calcium content in the hemolymph and shell of Biomphalaria glabrata (Say, 1818) was determined after exposure to different parasite burdens (5 and 50 miracidia) of Echinostoma paraensei (Lie and Basch, 1967). The snails were dissected 1, 2, 3, and 4 weeks after infection to collect the hemolymph and shell. An increase in calcemia was observed in snails infected with both miracidial doses. A significant decrease in the calcium ions in the shell was observed, coinciding with the calcemia peak in the hemolymph. This indicates greater mobilization of calcium between the shell and hemolymph to regulate the calcium content in the body when the snail is exposed to stress conditions, as has also been observed in some other infected snail species. The results obtained indicate that in this model, the calcium metabolism depends on the miracidial dose used., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The effect of experimental exposure of Biomphalaria glabrata to different doses (5 and 50) of Echinostoma paraensei miracidia on the total levels of cholesterol and triglycerides circulating in the hemolymph and the neutral lipids in the digestive gland-gonad (DGG) complex were studied. The snails were dissected one, two, three and four weeks after infection to collect the hemolymph and DGG tissue, to measure the levels of cholesterol and triglycerides in the hemolymph and neutral lipids in the tissue. The results for the hemolymph showed a similar order of variation for both substrates tested in the first week after infection. The reduced levels of these lipids in the infected snails indicate intense use of these substrates both by the intermediate host and the parasite, suggesting its probable participation in the energy metabolism and structural construction of the developing larval stages. Alterations in the profile of neutral lipids in the DGG were also found. The results obtained indicate that in this model, the lipid metabolism depends on the miracidial dose used., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The extracellular hemoglobin multimer of the planorbid snail Biomphalaria glabrata, intermediate host of the human parasite Schistosoma mansoni, is presumed to be a 1.44 MDa complex of six 240 kDa polypeptide subunits, arranged as three disulfide-bridged dimers. The complete amino acid sequence of two subunit types (BgHb1 and BgHb2), and the partial sequence of a third type (BgHb3) are known. Each subunit encompasses 13 paralogus heme domains, and N-terminally a smaller plug domain responsible for subunit dimerization. We report here the recombinant expression of different functional fragments of BgHb2 in Escherichia coli, and of the complete functional subunits BgHb1 and BgHb2 in insect cells; BgHb1 was also expressed as disulfide-bridged dimer (480 kDa). Oxygen-binding measurements of the recombinant products show a P(50) of about 7 mmHg and the absence of a significant cooperativity or Bohr effect. The covalently linked dimer of BgHb1, but not the monomer, is capable to form aggregates closely resembling native BgHb molecules in the electron microscope., (Copyright © 2011 Wiley Periodicals, Inc.)

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.

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.

All fibrinogen (FBG)-bearing proteins documented to date in the freshwater snail Biomphalaria glabrata, the intermediate host of the human blood fluke Schistosoma mansoni, possess the same molecular structure; one or two immunoglobin superfamily (IgSF) domains at the N-terminus and a FBG domain at the C-terminus (named as FBG-related protein (FREP)). Here we report two novel genes that encode FBG-bearing proteins from B. glabrata. Different from all known FREPs, the first gene encodes a protein (657 amino acids (aa)) composed of a long N-terminal region with no sequence homology to any known protein, a middle epidermal growth factor (EGF) repeat region and a C-terminal FBG domain, designated FBG-related molecule (FReM). Differential expression at 2 days post-exposure (dpe) to the trematode S. mansoni or Echinostoma paraensei was found in the S. mansoni susceptible M line and resistant BS-90 snail strains. The second gene is a new member of the FREP family, designated FREP14, which encodes a 399 aa putative secreted protein. FREP14 is different from known FREPs in that it is encoded by a single locus and is not upregulated in early or late stage S. mansoni exposure, but is upregulated in late stage E. paraensei infection. Furthermore, gene expression during the snail's ontogenesis and at a late stage of trematode infection (52 dpe) has been investigated in the two newly identified genes (FReM and FREP14) described in this paper and five representative members of known FREPs (FREPs 2, 3, 4, 12, and 13). A variety of expression patterns were observed, suggestive of functional diversity among the members of FBG-bearing proteins. Our findings further broaden our understanding of the diversity and function of the FBG-bearing protein encoded genes in B. glabrata.

Flame atomic absorption spectrometry was performed to determine the alteration of calcium concentration in the soft parts and shells of Biomphalaria alexandrina and Bulinus truncatus due to the infection with Schistosoma mansoni and S. haematobium, respectively. The results showed significant lowering in the calcium content of the shells of cercariae shedding B. alexandrina and B. truncatus relative to the calcium content in the shells of uninfected ones. In contrast, the calcium content in the soft parts of cercariae shedding snails was higher than in the soft parts of uninfected snails; the differences were statistically significant. Generally, calcium content was significantly higher in the shells than in the soft parts of the snails, regardless infected or uninfected. The results obtained and the hypothesis of hypercalcification in shells of infected snails were discussed.

High-performance liquid chromatography (HPLC) was used to determine the effects of a patent Schistosoma mansoni infection on certain carboxylic acids in the digestive gland gonad complex (DGG) and hemolymph of Biomphalaria glabrata. An analysis of DGG samples was done using extraction with 50% Locke's solution, cleanup of the extract by anion exchange solid phase extraction (SPE), and ion exclusion HPLC with ultraviolet detection. Hemolymph was applied directly to the SPE column. Acetic, fumaric, malic, and pyruvic acids were detected, confirmed, and quantified at concentrations ranging from 12 to 280 ppm in the DGG and less than 124 to 8,000 microg/dl in the hemolymph. Infection with S. mansoni caused a significant reduction (Student's t test, P < 0.05) in the concentrations of acetic, fumaric, malic, and pyruvic acids in the DGG but not the hemolymph of B. glabrata compared to uninfected cohort snails. The significant reduction of certain carboxylic acids in the DGG of B. glabrata patently infected with S. mansoni suggests that these acids are utilized by the sporocysts and cercariae in the snail tissue, or that the infection stimulates reduced production or increased utilization by the snail tissue.

The effects of aestivation or starvation on the neutral lipid and phospholipid content of Biomphalaria glabrata patently infected with Schistosoma mansoni were determined by high-performance thin-layer chromatography-densitometry. Infected-aestivated snails were maintained in a moist chamber at 24 +/- 1 C and a relative humidity of 98 +/- 1%. Infected-starved snails were maintained in artificial spring water (ASW) at 23 +/- 1 C without exogenous food. Infected snails (the controls) were maintained in ASW at 23 +/- 1 C and fed lettuce ad libitum. The 3 groups were maintained in the laboratory for 7 days, and then the lipids from the digestive gland-gonad complex (DGG) were extracted and analyzed by class. Infected-aestivated snails exhibited greater mortality rate and weight loss after 7 days than did the infected-starved snails. The steryl ester concentration in the infected-starved snails was significantly increased (P = 0.010) compared with the controls but not compared with infected-aestivated snails; the concentration of phosphatidylcholine in infected-aestivated snails was significantly decreased (P = 0.007) compared with the controls but not when compared with the infected-starved snails. Aestivation or starvation had a significant effect on the concentration of certain lipid classes in the DGG of B. glabrata infected with S. mansoni.

The human parasitic trematode Schistosoma mansoni has a complex life cycle that includes the freshwater snail Biomphalaria glabrata as intermediate host. Within each stage, the parasite synthesizes a wide array of glycoconjugates, exhibiting, in part, unique carbohydrate structures. In addition, the parasite expresses definitive host-like sugar epitopes, such as Lewis X determinants, supporting the concept of carbohydrate-mediated molecular mimicry as an invasion and survival strategy. In the present study, we investigated whether common carbohydrate determinants occur also at the level of the intermediate host. To this end, a structural characterization of hemolymph glycoprotein-N-glycans of B. glabrata was performed. N-glycans were released from tryptic glycopeptides and labeled with 2-aminopyridine. Sugar chains serologically cross-reacting with S. mansoni glycoconjugates were isolated by immunoaffinity chromatography using a polyclonal antiserum directed against schistosomal egg antigens and fractionated by Aleuria aurantia lectin affinity chromatography and high-performance liquid chromatography. Obtained glycans were analyzed by different mass spectrometric techniques as well as by monosaccharide constituent and linkage analysis. The results revealed a highly heterogeneous oligosaccharide pattern. Cross-reacting species represented about 5% of the total glycans and exhibited a terminal Fuc(alpha1-3)GalNAc unit, a (1-2)-linked xylosyl residue, or both types of structural motifs. In conclusion, our study demonstrates the presence of common carbohydrate epitopes also at the level of S. mansoni and its intermediate host.

Thin layer chromatography was used to analyze the glucose and maltose concentrations of the digestive gland-gonad complex (DGG) of uninfected-estivated Biomphalaria glabrata snails and estivated B. glabrata patently infected with Schistosoma mansoni. All snails were estivated in a most chamber at a relative humidity of 98+/-1% and a temperature of 23+/-1 degrees C for 14 days. Carbohydrates were extracted from the DGG with 70% aqueous ethanol, and extracts were analyzed on silica gel preadsorbent plates using ethyl acetate-glacial acetic acid-methanol-water (60:15:15:10) mobile phase, alpha-naphthol-sulfuric acid detection reagent, and quantification by densitometry. The concentrations of glucose and maltose were significantly reduced in both uninfected-estivated snails and infected-estivated snails.

This study examined the effects of larval trematode infection on the neutral lipid and phospholipid content of Biomphalaria glabrata patently infected with the daughter rediae of Echinostoma caproni. Uninfected snails were used as matched controls. As determined by qualitative high-performance silica gel thin-layer chromatography (HPTLC), the major neutral lipids present in the whole bodies and digestive gland-gonad complexes in both snail populations were free sterols, free fatty acids, and triacylglycerols, and the major polar lipids were phosphatidylcholine and phosphatidylethanolamine. Quantitative analysis by HPTLC with visible and UV scanning reflectance densitometry showed no significant differences in the concentrations of these lipids in whole bodies of infected snails vs the controls, but the concentration of triacylglycerols in the infected digestive gland-gonad complex was significantly less than that of the uninfected. No qualitative differences in neutral lipids and phospholipids in shell or plasma samples were found between infected vs uninfected snails.

Nuclear receptors form an important class of transcription regulators in metazoans. To learn more about the evolution of these proteins, we have initiated structural studies on nuclear receptor ligand-binding domains from various animals. Here we present the crystal structure of the ligand-binding domain (LBD) of the retinoid X receptor (RXR) from the mollusc Biomphalaria glabrata. The structure reveals a novel tetrameric association in which each monomer is complexed to the human RXR ligand 9-cis retinoic acid and to a human co-activator-derived peptide. The ligand and the co-activator peptide are bound in essentially the same manner as observed in previously reported human RXR LBD structures, suggesting that the mechanisms of RXR-mediated transcription regulation are very similar in mollusc and human. The structure shows further that binding of ligand and co-activator peptide does not necessarily lead to the typical holo-conformation in which helix 12 (H12) folds back and packs against the LBD. Within a canonical dimer, only one monomer is in this closed agonist conformation. The other monomer is in an open conformation with H12 protruding from the LBD core, occupying the H12 interaction groove of another open monomer in an adjacent dimer in a domain swapping fashion, thus resulting in a tetrameric association. Additional tetramer interfaces are formed between H11 of the closed LBD and H6 of the open LBD. This novel holo-tetramer configuration may have a biological role in activating genes whose promoters are poorly recognised by dimers but much more efficiently by the corresponding tetramers.

Synchrotron-radiation-based computer microtomography (SRmicroCT) was applied to three biomineralised objects First, embryonic snails of the freshwater snail Biomphalaria glabrata, second, rhopalia (complex sense organs) of the medusa Aurelia aurita, and third, human teeth. The high absorption contrast between the soft tissue and mineralised tissues, i.e. the shell in the first case (consisting of calcium carbonate) and the statoliths in the second case (consisting of calcium sulphate hemihydrate), makes this method ideal for the study of biomineralised tissues. The objects can be non-destructively studied on a micrometre scale, and quantitative parameters like the thickness of a forming a snail shell or statolith crystal sizes can be obtained on a length scale of 1-2 mum. Using SRmicroCT, the dentin-enamel border can be clearly identified in X-ray dense teeth.

Seven biotinylated lectins were tested with fluorescein labeled Avidin D for binding to haemocyte surfaces of non-inflected and infected Biomphalaria alexandrina snails with Schistosoma mansoi at 24 to 72 hours post infection (PI). Five lectins (Con A, DBA, SBA, RCA120 and WGA) yielded positive results. Two lectins (PNA and UEA-I) were negative. For the five positive lectins, the use of specific inhibition sugar indicated presence of glucose/mannose, galactose, N-acetyl-galactosamine (GalNAc) and N-acetyl-glucosamine (GlcNAc). The UEA-I lectin was negative and this indicates the absence of L-Fucose. There were no changes in the carbohydrates of haemocyte surfaces during the short periods (0 hr to 72 hrs) PI with S. mansoni. Electrophoresis of haemocyte homogenates of both non-infected and infected snails with S. mansoni yielded a complex pattern of polypeptides. The separated profiles demonstrate the occasional appearance or absence of certain bands in infected snails through four weeks PI. Post S. mansoni infection, dominant proteins appeared only through four weeks PI which were of average molecular masses 36, 9.3 and 5.3 kDa. At fourth week PI a remarkable decrease in the number of haemocyte protein fractions as well as the protein densities were reduced in compared to control snails. The results showed that qualitative and quantitative differences were detectable in the haemocyte proteins between non-infected and infected snails during four weeks of infection.

Two freshwater invertebrate organisms, Biomphalaria glabrata and Lumbriculus variegatus, were tested as potential experimental animal models to assess Pb exposure using acute laboratory bioassays. Since long, the enzyme delta-aminolevulinic acid dehydratase (ALA-D) has been recognised as a useful biomarker of Pb exposure and effect. Therefore, determinations of ALA-D activity were performed in the whole body soft tissues of pigmented and non-pigmented gastropods B. glabrata and in the oligochaete L. variegatus. The organisms were exposed to varying concentrations of Pb for 48 h. Levels of Pb in the invertebrates were also analysed. Highly significant negative correlations were found between the enzymatic activity and the levels of Pb exposure, and also between the enzymatic activity and the metal incorporated by the invertebrates. No significant differences were found in the enzyme activity nor in the resulting metal accumulation based on gastropod pigmentation. The values of Pb concentration that produce 50% of inhibition on the enzyme activity (EIC50) were 0.023 and 0.029 mg Pb/L for pigmented and non-pigmented B. glabrata, respectively. A much higher value was found for L. variegatus (0.703 mg Pb/L). The non-observed effect concentration (NOEC) on enzyme activity for the oligochaetes was 0.05 mg Pb/L, about twice the EIC50 calculated for the gastropods. These data showed that both pigmented and non-pigmented B. glabata were much more sensitive organisms than the oligochaetes. The differences in enzyme inhibition could be attributed, at least partly, to differences in the metal body burden found between the organisms, since levels of Pb accumulated by B. glabrata were approximately three times higher compared to those observed in L. variegatus. Therefore B. glabrata showed to be a more suitable and reliable bioindicator organism for assessing Pb contamination in aquatic ecosystems, especially at low levels of metal exposure.

The calcium carbonate concentrations in the shells of Helisoma trivolvis and Physa sp. naturally infected with larval trematodes and Biomphalaria glabrata experimentally infected with larval trematodes were analyzed quantitatively. The larval trematode-snail relationships studied were H. trivolvis infected with larval Echinostoma trivolvis and Physa sp. infected with various larval digeneans, and B. glabrata infected with Echinostoma caproni or Schistosoma mansoni. The calcium carbonate concentrations of the shells of infected snails and uninfected cohorts and of the water in which the snails were maintained were determined by ion exchange chromatography. No significant differences in the calcium carbonate concentrations of shells of infected versus uninfected snails were found. The shells of B. glabrata infected with E. caproni contained significantly less calcium carbonate than the shells of uninfected B. glabrata. The hypercalcification hypothesis, i.e., larval trematodes induce an increase in the calcium concentrations in the shells of their snail hosts, was not upheld in any of the snail-larval digenean systems studied herein.

Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used to study element ions in whole bodies of uninfected Biomphalaria glabrata snails and those experimentally infected with larval Schistosoma mansoni trematodes. Infected snails were analysed 8 weeks post-infection. Cohort snails that were left uninfected were analysed at the same time as the infected snails. Sixteen elements (aluminum, boron, barium, calcium, cadmium, copper, iron, potassium, magnesium, manganese, sodium, nickel, lead, selenium, tin and zinc) were found to be present in infected and uninfected whole bodies at concentrations above the detection limit of the ICP-AES analysis. Of these, calcium, cadmium, manganese and sodium were present in significantly higher amounts (Student's t-test, P<0.05) in whole infected versus whole uninfected snails. Variations in the present results compared with other studies reflect intrinsic differences in the larval trematode-snail systems used.

Thin-layer chromatography was used to analyze the free-pool amino acids of the digestive gland-gonad complex (DGG) of Biomphalaria glabrata infected with Echinostoma caproni and uninfected (control) snails. Qualitative analysis revealed the presence of histidine, lysine, serine, alanine, valine, and isoleucine or leucine in all samples. Quantitative analysis of lysine and valine gave mean weight percentages of 0.00699 +/- 0.0022 and 0.00174 +/- 0.00056, respectively, in the DGG of uninfected snails, and 0.00504 +/- 0.0014 and 0.00254 +/- 0.00033, respectively, in the DGG of infected snails. The differences in values between infected and uninfected snails were not statistically significant (Student's t-test, P > 0.05).

The hemoglobin from Biomphalaria glabrata is an extracellular respiratory protein of high molecular mass composed by subunits of 360 kDa, each one containing two 180 kDa chains linked by disulfide bridges. In this work, small angle x-ray scattering (SAXS) measurements were performed with the hemoglobin at pH 5.0 and 7.5. Radii of gyration of 98.6 +/- 0.5 and 101.8 +/- 0.2 A and maximum diameters of 300 +/- 10 and 305 +/- 10 A, respectively, were obtained from Guinier plot extrapolation and analytical curve fitting. The pair distance distribution functions p(r) corresponded to globular particles with a somewhat anisotropic shape for both preparations. Computer analysis of the low angle part of the scattering curve led to the determination of the low resolution envelope of the protein, revealing a P(222) symmetry. Shape reconstruction from ab initio calculations using the complete scattering curve furnished a compact prolate three-dimensional (3D) bead model for the protein. Hydrodynamic parameters were obtained from experiments and theoretical calculations using the 3D model. The results of the structural and biochemical studies reported herein indicate that the multisubunit structure of this hemoglobin is compatible with a tetrameric arrangement., (Copyright 2003 Wiley Periodicals, Inc. Biopolymers 69: 470-479, 2003)

In a previous study, when the snail Biomphalaria glabrata was infected with Schistosoma mansoni and maintained on a diet of hen's egg yolk, it produced fully developed cercariae in about one-half the time taken by snails fed Romaine lettuce. Increased lipids were also noted in the snails fed the yolk diet. The purpose of the present study was to further investigate nutritional effects of a high-lipid diet on larval schistosome development and to reexamine the time to cercarial patency in infected snails maintained on either the yolk or lettuce diet and to use high-performance thin-layer chromatography (HPTLC) to analyze the neutral lipids in the digestive gland-gonad complex (DGG) of snails maintained on both diets. Infected snails maintained at 26 C and fed either diet produced fully developed cercariae by 4 wk postinfection (PI). Likewise, infected snails maintained at 23 C and fed either diet produced fully developed cercariae by 6 wk PI. The contention that the yolk diet enhanced the time to cercarial patency was not confirmed. The HPTLC analysis of neutral lipids showed that the DGG of infected snails fed the yolk diet contained significantly greater amounts of free sterols and cholesteryl esters but not triacylglycerols than that of the infected snails fed the lettuce diet.

Freshwater snails belonging to the genus Biomphalaria are intermediate hosts of the trematode Schistosoma mansoni in the Neotropical region and Africa. In Brazil, one subspecies and ten species of Biomphalaria have been identified: B. glabrata, B. tenagophila, B. straminea, B. occidentalis, B. peregrina, B. kuhniana, B. schrammi, B. amazonica, B. oligoza, B. intermedia and B.t. guaibensis. However, only the first three species are found naturally infected with S. mansoni. The classical identification of these planorbids is based on comparison of morphological characteristics of the shell and male and female reproductive organs, which is greatly complicated by the extensive intra-specific variation. Several molecular techniques have been used in studies on the identification, genetic structure as well as phylogenetic relationships between these groups of organisms. Using the randomly amplified polymorphic DNAs (RAPD) analysis we demonstrated that B. glabrata exhibits a remarkable degree of intra-specific polymorphism. Thus, the genetics of the snail host may be more important to the epidemiology of schistosomiasis than those of the parasite itself. Using the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) in intra-populational and intra-specific studies we have demonstrated that snails belonging to the B. straminea complex (B. straminea, B. kuhniana and B. intermedia) clearly presented higher heterogeneity. Using the low stringency polymerase chain reaction (LS-PCR) technique we were able to separate B. glabrata from B. tenagophila and B. tenagophila from B. occidentalis. To separate all Brazilian Biomphalaria species we used the restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer region (ITS) of the DNA gene. The method also proved to be efficient for the specific identification of DNA extracted from snail eggs. Recently we have sequenced the ITS2 region for phylogenetic studies of all Biomphalaria snails from Brazil.

The mineralized shell (consisting of calcium carbonate) of the tropical freshwater snail Biomphalaria glabrata was investigated with high resolution synchrotron X-ray powder diffractometry and X-ray absorption spectroscopy (EXAFS). Parts from different locations of the snail shell were taken from animals of different age grown under various keeping conditions. Additionally, eggs with ages of 60, 72, 120, and 140 hours were examined. Traces of aragonite were found as first crystalline phase in 120 h old eggs, however, Ca K-edge EXAFS indicated the presence of aragonitic structures already in the X-ray amorphous sample of 72 h age. The main component of the shell of adult animals was aragonite in all cases, but in some cases minor amounts of vaterite (below 1.5%) are formed. The content of vaterite is generally low in the oldest part of the shell (the center) and increases towards the mineralizing zone (the shell margin). In juvenile snails, almost no vaterite was detectable in any part of the shell.

Using high performance liquid chromatography (HPLC) analysis it was possible to determine simultaneously the concentration of organic acids (pyruvate, lactate, succinate, fumarate, malate, acetate, propionate, acetoacetate, and ss-hydroxybutyrate) in the digestive gland and the extracellular concentration of these same acids in the hemolymph of estivating Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. After a 7 day period of estivation, there was a significant increase in the tissue levels of lactate, succinate, malate and acetate compared to non-estivating snails. After 14 days of estivation, the levels of lactate and acetate were also significantly elevated. The hemolymph concentrations of pyruvate and acetate increased significantly after 7 days and acetate concentrations continued to be significantly increased up to 14 days of estivation. The other organic acids studied, such as ketone body acetoacetate and ss-hydroxybutyrate or the volatile acid propionate, did not accumulate. Their tissue concentrations, however, increased on the 7th day of estivation and reached normal levels within two weeks of estivation for some of them. One should take into consideration how the reduction in metabolism can be handled under aerobic conditions, and what role anaerobic pathways may play in both energy formation and redox balance processes.

A cDNA encoding an integrin subunit was cloned and structurally characterized from an embryonic cell line derived from Biomphalaria glabrata, snail intermediate host of the human blood fluke Schistosoma mansoni. Cells of the B. glabrata embryonic (Bge) snail cell line were initially tested for their sensitivity to the integrin-specific tetrapeptide inhibitor Arg-Gly-Asp-Ser (RGDS). Washed Bge cells when exposed to 0.5 to 2.0mM of RGDS were significantly inhibited in their ability to spread on a glass substrate. Spreading inhibition was specific, since a control peptide Arg-Gly-Glu-Ser (RGES) did not have the same effect. RT-PCR was performed using previously reported degenerate oligonucleotide primers to the ligand binding domain (LBD) of known beta integrin subunits and Bge cDNA. A 137 bp fragment was amplified, TA-cloned, sequenced, and the na and deduced aa sequences were compared with other beta integrins. Databank analysis showed that the 137 bp product shared >/=55.6% aa similarity to other beta integrin LBDs. Southern and northern blot analyses using the 137 bp sequence as a probe revealed binding to Bge genomic DNA restriction fragments and to an approximately 8 kb poly-(A)+RNA transcript, respectively. An exact 5' primer synthesized to the 137 bp product and an oligo-d(T) primer then were used to amplify from Bge cDNA, a partial beta integrin sequence of 2285 bp that contained a 1971 bp ORF. The remaining upstream coding region was obtained using 5' RACE methods. The complete ORF, consisting of 2364 bp, encoded a 788 aa sequence with shared similarity to other known beta integrins (44.6-61.5%). Sequence and structural comparisons, which include a characteristic LBD, a series of three homologous cysteine-rich repeats, membrane proximal sequence (LLTFIHD), cytoplasmic NPXY motifs, and predicted domain lengths of the molluscan protein, clearly identifies it as an integrin homologue. This report represents the first cloning of a cDNA putatively encoding an integrin subunit from molluscan cells, and establishes the Bge cell line as a model for studying cellular adhesion in molluscs at the molecular level.

The freshwater snail Biomphalaria glabrata is an intermediate host of the trematode Schistosoma mansoni. However, some strains of B. glabrata are resistant to successful infection by S. mansoni larvae. The present work examines the profile of organic acids present in S. mansoni-resistant and -susceptible strains of B. glabrata, in order to determine whether the type of organic acid present is related to susceptibility. The organic acids were extracted from the hemolymph of two susceptible B. glabrata strains (PR, Puerto Rico and Ba, Jacobina-Bahia from Brazil), and from the resistant strains 13-16-R1 and 10R2, using solid phase extraction procedures followed by high performance liquid chromatography. The organic acids obtained were analyzed and identified by comparison with known standards. Pyruvate, lactate, succinate, malate, fumarate, acetate, propionate, beta-hydroxybutyrate and acetoacetate were detected in all hemolymph samples. Under standard conditions, the concentration of each of these substances varied among the strains tested and appeared to be specific for each strain. An interesting variation was the low concentration of pyruvate in the hemolymph of PR-snails. Only the concentration of fumarate was consistently different (p < or = 0.05) between resistant and susceptible strains.

Utilizing a Biomphalaria alexandrina-derived lectin (BaSII) of proven specificity to a Schistosoma mansoni-associated fucosyllactose [(Fuc alpha 1-2)Gal beta 1-4 Glc] determinant, a 37-kDa determinant-bearing glycoprotein (Sm 37) was identified selectively on adult male schistosomes. Sm 37 was purified to homogeneity from extracts of adult male worms metabolically radiolabeled with [35S] methionine by BaSII affinity chromatography followed by separation on an HPLC column. Treatments with endoglycosidases, alkaline borohydride, as well as serial lectin affinity chromatography and analysis on 2-dimensional gels indicated that Sm 37 is synthesized as a 33-kDa polypeptide backbone that expresses the fucosyllactose determinant on the outer chain of a single N-linked complex-type glycan unit of either the biantennary or, to a lesser extent, the tri- or tetra-antennary types. The distinct structures of the complex oligosaccharides accounted for the expression of 2 isomorphs of Sm 37. the glycoprotein lacks other conventional high mannose-type or O-linked oligosaccharides and, as deduced from the N-terminal amino acid sequence, the Sm 37 polypeptide may be distinct from other schistosome polypeptides of known sequence. Based on the structural relatedness of the Sm 37-associated fucosyllactose determinant to the antigenic blood group H trisaccharide, these observations may have implications for mechanisms of these host-parasite interactions.

A novel family of isolectins that selectively recognize a schistosome-associated fucosyllactose determinant was identified in the hemolymph of Biomphalaria alexandrina, a snail vector of Schistosoma mansoni. Three lectins of this family were purified by serial affinity chromatography on a column of L-fucose and elution with a gradient of 0.1-1 M L-fucose (designated BaSII and BaSIII), followed by a column of D-glucose and elution with 0.3 M D-glucose (designated BaSI). Assessment of the structural characteristics by one- and two-dimensional gels indicated that, inspite of similarities in native molecular weights, the three lectins were tetramers of noncovalently-associated subunits that were of different sizes and pIs in BaSI, and of equal size but distinct pIs in BaSII and BaSIII. Comparisons of two-dimensional gels of the glycosylated and deglycosylated forms were consistent with the presence of an invariant alpha subunit (13.2 kDa, pI 7.2) constituting the three deglycosylated lectins, which associates with other subunits unique to each lectin, namely a beta subunit (10.1 kDa, pI 5.8) in BaSI, an alpha 1 subunit (13.2 kDa, pI 6.8) in BaSII and BaSIII, and an alpha 2 subunit (13.2 kDa, pI 7.0) in BaSIII. Each of these subunits is subjected to differential post-translational N-linked glycosylations, which accounts for the additional heterogeneity expressed by the glycosylated lectins. Based on miracidial glycoprotein binding and inhibition assays, the three lectins exhibited optimum binding at similar pH and temperature, but were distinct in their binding affinities towards the fucose moiety constituting the fucosyllactose target. These observations indicate that an oligomorphic family of recognition molecules may have evolved to regulate the snails' response to schistosomes.

Snail plasma serves as both a sink for metabolites and a source of nutrients for parasites developing within their intermediate hosts. It also contains molecules involved in immunological events like non-self recognition, phagocytosis and encapsulation. In this study we present improved protocols for the separation and partial characterization of plasma proteins of schistosome-susceptible and resistant strains of Biomphalaria glabrata. Within each strain, the plasma of snails 12, 24, 48 and 72 h post-exposure to Schistosoma mansoni and of non-exposed snails was compared. Protein concentrations in hemolymph of all snail strains, non-exposed or parasite-exposed, were about 29 mg/mL and were not found to differ significantly. The dominant plasma molecule (80-85%) is extracellular hemoglobin (Hb) with a native mass of > 1 M Da, and subunits of 190 kDa. It is the only protein bearing heme as shown after separation by native-PAGE and LDS-PAGE. The relatively large amounts of Hb and its large size cause problems if native plasma components are to be separated in PAGE. To obtain satisfactory separation, we used short-term ultracentrifugation to deplete Hb from plasma without qualitative loss of other proteins. Using this methodology, we have examined proteins by native polyacrylamide gel electrophoresis, in the presence of SDS or LDS only or SDS and mercaptoethanol, and by isoelectric focusing. Proteins have been detected in gels by silver stains and staining for heme groups, and, after transfer to membranes, by means of lectins and neoglycoproteins. Molecular weights of plasma proteins range between 10 and > 450 kDa, and isoelectric points are from pH 4 to 9.4. All strains show similar protein patterns, although minor inter- and intrastrain differences occur. These differences are quantitative rather than qualitative, not consistent, and cannot be correlated with the snail's ability to effectively attack and kill S. mansoni sporocysts. In all snail strains, plasma proteins remained qualitatively stable during 3 days after exposure to S. mansoni. New proteins were not evident, and none was lost as a consequence of exposure to parasites. Our new Hb-depletion technique is an excellent approach to separate and examine Biomphalaria plasma proteins in their native state. The use of lectins to probe for the presence of carbohydrates showed that the majority of plasma proteins is glycosylated. Mannose, galactose, and N-acetylgalactosamine are their major carbohydrate components; fucose was not detected. Several lectins apparently in the molecular mass range of 330-500 and 56-135 kDa with major carbohydrate-specificities for N-acetyl-galactosamine, N-acetylglucosamine, mannose, glucose, galactose and fucose were detected in the plasma of both resistant and susceptible snails by using neoglycoproteins as probes.

The miracidia of Schistosoma mansoni and S. haematobium approach their host snails by increasing their rate of change of direction (RCD) in increasing gradients of snail-conditioned water (SCW), and they perform a turnback response in decreasing gradients. After contact with the host "repeated investigation" is the typical host-specific response. Both species show no significant directed chemotactical orientation towards their snail hosts. All three host-finding responses (increased RCD, turnback response, and "repeated investigation") seem to be stimulated in both species by a similar component of SCW, a macromolecular glycoconjugate with a molecular weight > 30,000. The saccharide chains seem to be O-glycosidically linked via serine and N-acetylgalactosamine. The glycoconjugate is sensitive to lysozyme which may suggest that muramic acid as a gastropod-specific component is involved in the recognition process. Small molecular components of SCW, as well as magnesium chloride offered as pure chemical, may cause a moderate increase in the RCD. Therefore a minor contribution of these components to the host-finding response of schistosome miracidia cannot be excluded. That schistosome miracidia respond to complex macromolecules as host cues may indicate an adaptation to avoid interference of the host-finding with ubiquitous small molecular mud components and it might enable the miracidia to achieve a high degree of host-specificity in their host-finding.

As an approach to better understand the molecular mechanisms underlying the differential cellular response to larval Schistosoma mansoni observed in Biomphalaria glabrata, we have analyzed hemocyte membrane-associated polypeptides from inbred susceptible and resistant strains of B. glabrata. A combination of techniques involving biotin-labeling of cell surface-exposed polypeptides and a highly sensitive chemiluminescent detection technique has allowed the identification of major polypeptides expressed on nonadherent and adherent hemocyte subpopulations from individual snails and an assessment of the variability in their expression in response to S. mansoni exposure and latex bead injection. Results showed that S. mansoni exposure and latex bead injection differentially modulate the expression of 4 of 10 major hemocyte surface polypeptides with estimated molecular weights of 155/130, 120/110, 85, and 66 kDa. Little correlation was observed between the expression of any given polypeptide and a specific response to S. mansoni exposure. Moreover, the complex pattern of hemocyte surface polypeptides observed in snails subjected to the two treatments suggests that multiple signals may be influencing either the expression of these four polypeptides at the hemocyte surface or the hemodynamic of specific peptide-bearing hemocyte subpopulations released into the circulation. The fact that a natural and an artificial insult in the two snail strains elicit a differential response in the occurrence of these four polypeptides suggests that they may be involved in defense-related activities.

High performance thin-layer chromatographic analysis was done on the hemolymph and digestive gland-gonad complex (DGG) of Biomphalaria glabrata snails experimentally infected with the intramolluscan stages of Echinostoma caproni. The major sugars detected in both the DGG and hemolymph of infected and uninfected snails were glucose and trehalose. Quantitative analysis by scanning densitometry showed a significant reduction in glucose in both the hemolymph and DGG of infected snails at 4, 6, and 8 wk postinfection. A similar analysis on trehalose showed that this sugar was significantly reduced at 6 wk postinfection in the hemolymph and DGG of infected snails and could not be detected from these sites in infected snails by 8 wk postinfection. Findings from this study were compared with information on sugars in B. glabrata infected with larval stages of Schistosoma mansoni.

1. Carbohydrates were extracted from total tissue extracts of Biomphalaria alexandrina snails and were analyzed to their monosaccharides using GLC. 2. The snails were chemically treated with thioxanthone derivatives (compounds I, II, III) and the change in the monosaccharide constituents of their carbohydrates was investigated. 3. The isolated monosaccharides from native and chemically pretreated snails were injected into mice and their protective effects were examined after infection of mice with cercariae of Schistosoma mansoni. 4. The results showed that the main monosaccharides in carbohydrates of snails were galactose, glucose, fucose and mannose and that chemical treatment caused a drop in the galactose content. 5. Moreover, monosaccharide fractions from snails treated with compound III were the most effective in inducing protection against Schistosoma infection in mice.