Identification and localization of a gonadotropin-releasing hormone-related neuropeptide in Biomphalaria, an intermediate host for schistosomiasis.

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-NH2 (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.

Comparative study of excretory-secretory proteins released by Schistosoma mansoni-resistant, susceptible and naïve Biomphalaria glabrata.

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.

Studies on Toxicity of Suspensions of CdTe Quantum Dots to Biomphalaria glabrata Mollusks.

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.

A Biomphalaria glabrata peptide that stimulates significant behaviour modifications in aquatic free-living Schistosoma mansoni miracidia.

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.

[Quantitative determination of biogenic amine from Biomphalaria glabrata nervous system by UPLC MS/MS].

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 protein pheromone temptin is an attractant of the gastropod Biomphalaria glabrata.

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.

Changes in the neuropeptide content of Biomphalaria ganglia nervous system following Schistosoma infection.

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.

Total protein composition of young and adult Biomphalaria alexandrina snails with different compatibilities to Schistosoma mansoni infection / Composición total de proteína en caracoles Biomphalaria alexandrina juveniles y adultos con distintas compatibilidades a la infección por Schistosoma mansoni

Abstract: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. Rev. Biol. Trop. 64 (4): 1747-1757. Epub 2016 December 01.

Resumen:La esquistosomiasis es una enfermedad crónica y debilitante que constituye una problemática de salud pública a nivel mundial. Debido a que Schistosoma mansoni está ampliamente distribuida y a que es el causante de la esquistosomiasis intestinal representa una gran amenaza. Biomphalaria es el hospedero intermedio y obligatorio para el estado larval, presenta una amplia distribución geográfica e infecta al ser humano. El objetivo fue identificar las proteínas responsables del efecto de compatibilidad en caracoles Biomphalaria alexandrina de distintos estadios a través de la diferenciación del total de proteínas en ellos. La investigación se llevó a cabo con caracoles de diferentes edades y antecedentes genéticos. Se estudiaron cuatro subgrupos (F1) de la progenie de caracoles susceptibles y resistentes reproducidos asexualmente (F0). Las tasas de infección de estos subgrupos (juvenil susceptible, adulto susceptible, juvenil resistente, adulto resistente) fueron 90 %, 75 %, 40 % y 0 %, respectivamente. Con la electroforesis en gel de poliacrilamida en presencia de dodecilsulfato sódico (SDS-PAGE) se evaluaron las diferencias en la expresión proteica entre los caracoles juveniles y adultos de los distintos subgrupos. Se calculó el coeficiente de similitud de Dice para determinar el porcentaje de bandas compartidas entre los subgrupos experimentales. Los resultados mostraron que la combinación de similitudes entre la edad y el estado de compatibilidad de los caracoles genera el mayor coeficiente de similitud seguido por el de la combinación de similitudes tanto de la edad como del origen genético aunque varían en el estado de compatibilidad. Por otra parte, las diferencias en los antecedentes genéticos, la edad y el estado de compatibilidad generan el índice de similitud más bajo. También se encontró que el antecedente genético en caracoles juveniles es importante en la determinación de su compatibilidad, mientras que el sistema de defensa interno es el que determina el nivel de compatibilidad en adultos. En conclusión, los resultados de este trabajo resaltan la importancia de la edad del caracol en conjunto con la genética y la defensa interna para determinar la compatibilidad de B. alexandrina/S.mansoni. Se recomienda realizar futuros trabajos así como una mayor caracterización de las bandas proteicas compartidas entre los subgrupos estudiados para esclarecer su papel en la relación hospedero-parásito.

The influence of Angiostrongylus cantonensis (Nematoda, Metastrongylidae) infection on the aerobic metabolism of Biomphalaria straminea and Biomphalaria tenagophila (Mollusca, Gastropoda).

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.

Effects of Echinostoma caproni miracidia dose on the amino acid contents of Biomphalaria glabrata as determined by high-performance thin-layer chromatography.

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.

Total protein composition of young and adult Biomphalaria alexandrina snails with different compatibilities to Schistosoma mansoni infection.

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.

UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferase from the snail Biomphalaria glabrata - substrate specificity and preference of glycosylation sites.

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.

Heavy metal concentrations in the freshwater snail Biomphalaria alexandrina uninfected or infected with cercariae of Schistosoma mansoni and/or Echinostoma liei in Egypt: the potential use of this snail as a bioindicator of pollution.

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.

Effects of Echinostoma caproni miracidia dose on the neutral and polar lipids of Biomphalaria glabrata as determined by high-performance thin-layer chromatography.

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.

Effects of Schistosoma mansoni infection on lutein and ß-carotene concentrations in Biomphalaria glabrata snails as determined by quantitative high performance reversed phase thin-layer chromatography.

High performance thin-layer chromatography was used to determine the concentration of ß-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 ß-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 ß-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 ß-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 ß-carotene concentration of the DGG at 6 and 8 weeks post infection.

5-methyl-cytosine and 5-hydroxy-methyl-cytosine in the genome of Biomphalaria glabrata, a snail intermediate host of Schistosoma mansoni.

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.

Potential correlation between carboxylic acid metabolites in Biomphalaria alexandrina snails after exposure to Schistosoma mansoni infection.

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.

Glycotope sharing between snail hemolymph and larval schistosomes: larval transformation products alter shared glycan patterns of plasma proteins.

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.

Potential Correlation between Carboxylic Acid Metabolites in Biomphalaria alexandrina Snails after Exposure to Schistosoma mansoni Infection

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.

Influence of Echinostoma paraensei (Lie and Basch, 1967) infection on the calcium content in Biomphalaria glabrata (Say, 1818).

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.