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Seven species of Solanum were screened for their molluscicidal properties against Biomphalaria glabrata, the intermediated host of Schistosoma mansoni, Solanum agrarium, S. jabrense, S. melissarum, S. megalonyx, S. paludosum, S. paraibanum and S. stipulaceum. Four extracts showed molluscicidal activity with LC(50) from 22 to 56 microg/ml.
Snail immune responses towards a trematode infection are known to rely on both plasmatic and cellular host factors. As an approach to further investigate the suspected involvement of plasmatic factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns of plasma collected from susceptible and resistant snails. This proteomic approach revealed that 13 plasmatic proteins exhibited significant differences in their apparent representativity. The genes corresponding to five of them were characterised by a combination of mass spectrometry and molecular cloning. They encode two isoforms of a glycolytic enzyme, two isoforms of a calcium binding protein and an inhibitor of cysteine protease. Furthermore, we investigated gene expression in parasite-exposed or -unexposed snails as well as in various tissues by quantitative PCR. This study showed that: (i) differential representation of plasma proteins between the snail strains was correlated with a differential level of transcripts; (ii) expression of these genes after parasite exposure was differentially regulated in the two strains; and (iii) these genes were expressed predominantly in the albumen gland.
Following infection with Schistosoma mansoni larvae, haemocytes of resistant Biomphalaria glabrata snails execute a rapid defence during which they migrate towards and encapsulate the parasites. Such immediate and precise responses are thought to depend on signal transduction cascades though the signalling components involved remain largely unknown. It is proposed that mitogen-activated protein kinases may play a role in B. glabrata immune signalling, in particular p38 mitogen-activated protein kinases, which are known to be associated with stress and inflammatory signalling. Using degenerate PCR followed by Rapid Amplification of cDNA Ends a full-length p38 mitogen-activated protein kinase-like cDNA was cloned from both the B. glabrata embryonic (Bge) cell line (Bge-p38) and haemocytes (Bgh-p38). In addition, B. glabrata p38 mitogen-activated protein kinase activation was examined at the protein level in Western blot analyses using an antibody that specifically recognises activated/diphosphorylated p38 mitogen-activated protein kinase. Results showed that Bge cell p38 mitogen-activated protein kinase was activated/phosphorylated following 30 min incubation with anisomycin, an established p38 mitogen-activated protein kinase activator. Furthermore, p38 mitogen-activated protein kinase was also activated after only 5 min exposure to either the beta-glucan polymer laminarin or S. mansoni larval excretory-secretory products. In a comparative study, activated haemocyte p38 mitogen-activated protein kinase could also be detected using the anti-phosphorylated p38 antibody following cell treatment with anisomycin. However, in contrast with Bge cells, haemocyte p38 was not activated by either excretory-secretory products or laminarin treatments, suggesting fundamental differences in the role of p38 mitogen-activated protein kinase in signal transduction pathways between haemocytes and Bge cells.
Because susceptibility or resistance of Biomphalaria glabrata to the trematode Echinostoma caproni correlates with differential hemocytic adhesive properties, we compared the expression of genes involved in adhesion processes between hemocytes from susceptible and resistant snails. Quantitative reverse transcriptase-PCR analysis revealed four genes whose transcripts were differentially represented between hemocytes from resistant and susceptible snails. These genes encode two dermatopontin-like, one matrilin-like and one cadherin-like proteins. Expression analyses performed following parasite exposure suggested that dermatopontins may be involved in the compatibility differences between these strains. We also investigated expression levels on whole snails of different genes potentially involved in extracellular matrix structure or coagulation. Our results support the hypothesis that susceptible snails possess a hemolymph coagulation-like system that is more potent than that of resistant snails. This system may prevent hemocyte migration towards the parasite larvae and therefore facilitate parasite settlement in susceptible snails.
The putative expression of distinct terminally fucosylated glycoconjugates among murine lymphocyte subpopulations was sought using a Biomphalaria alexandrina-derived lectin (BaSI), of proven specificity to a fucosyllactose determinant, and the fucose-binding lectin from Lotus tetragonolobus seeds. Direct labeling of isolated lymphocyte subsets in suspension as well as immuno-histochemical and two-dimensional Western blotting assays demonstrated the exclusive expression of the BaSI-reactive ligand among multiple isoforms of two major 95 and 92 kDa and a minor 82 kDa acidic glycoproteins, selectively localized to the splenic marginal zone B lymphocytes of adult mice. The expression of the L. tetragonolobus lectin-reactive ligand was, on the other hand, restricted primarily to a single homogeneous 50 kDa acidic glycoprotein associated with a subset of the mature (PNA-) medullary thymocytes of adult mice as well as a minority of the immature (PNA +) thymocytes within the deep cortical region in newly born mice. The significance of these findings is discussed in relation to mechanisms that govern lymphocyte development and homing.
Continuous exposure of Biomphalaria glabrata snails to 0.1% colchicine resulted in a significant increase, relative to non-exposed snails, in the number of arrested mitotic figures in the amebocyte-producing organ (APO) as soon as 4 h, with peak numbers after 12 h of exposure. The number of circulating hemocytes was significantly elevated at 24 h. However, by 72 h both the number of mitotic figures in the APO and the concentration of circulating hemocytes in the hemolymph had returned to control levels. Hemocytes appeared to possess normal morphology throughout this exposure, including the formation of long filopodia with supporting rodlike structures that have been reported to contain microtubules. Snail survival decreased as a function of exposure time. Significantly fewer snails, relative to controls, survived a 48-h exposure, and only 1 out of 30 snails recovered from a 72-h exposure to 0.1% colchicine. Colchicine-exposed snails displayed intoxicated behavior, even upon removal from the colchicine solution, although no histopathology was evident in the CNS of snails exposed for 72 h.
The investigation of the response of Biomphalaria glabrata snails to Echinostoma paraensei (digenea) at 2 days post-exposure by suppression subtractive hybridization yielded a partial sequence of the anti-oxidant enzyme manganese superoxide dismutase (MnSOD). Full-length MnSOD (669nt) from M line and BS-90 strains of B. glabrata differed by one synonymous nucleotide replacement. B. glabrata has 1-4 MnSOD loci (Southern hybridization). Both snail strains expressed MnSOD at equal baseline levels (quantitative PCR). Susceptible snails increased expression of MnSOD following infection with E. paraensei or Schistosoma mansoni, and expression was reduced in the incompatible combination (BS-90 B. glabrata and S. mansoni). Thus, MnSOD did not determine resistance or susceptibility for these parasites, but expression of MnSOD is consistent with its involvement in a stress response of B. glabrata.
Although Schistosoma mansoni occurs mainly in the tropics, where intense levels of solar radiation are present, the impact of ultraviolet (UV) light on schistosome transmission is not known. The purpose of this study was to investigate potential effects of UVB (290-320nm) on juvenile Biomphalaria glabrata, the snail intermediate host of S. mansoni. Albino and wild-type snails were exposed to doses of UVB from UV-fluorescent lamps, and the following were measured: survival, photoreactivation (light-mediated DNA repair), effects on feeding behavior, and morphological tissue abnormalities. Irradiation with UVB is lethal to B. glabrata in a dose-dependent manner. Exposure to white light subsequent to UVB irradiation enhances survival, probably by photoreactivation. The shell offers some, but not complete, protection. Experiments in which UVB transmittance through the shell was blocked with black nail polish suggest that injury to both exposed (headfoot) and shell-enclosed (mantle and visceral mass) tissues contributes to mortality in lethally irradiated snails. Wild-type (pigmented) snails are less susceptible to lethal effects of UVB than albino snails, and they may be more capable of photoreactivation. UVB exposure inhibits snail feeding behavior, and causes tentacle forks and growths on the headfoot. Thus, UVB may influence the life cycle of S. mansoni by both lethal and sub-lethal damage to the snail intermediate host. However, the ability of snails to photoreactivate may mitigate these effects.
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.
Genetic strains of the snail Biomphalaria glabrata vary in their resistance to the parasite Schistosoma mansoni. Phagocytic cells (hemocytes) circulating in the hemolymph of B. glabrata play an essential role in the snail's innate immune response. Hemocytes of resistant B. glabrata kill S. mansoni in vitro via a mechanism which involves a respiratory burst. Reactive oxygen species (ROS), which are products of the respiratory burst, can act as mediators of both oxidative damage and of immune-related intracellular signaling pathways. One specific ROS, hydrogen peroxide (H2O2), has been shown to be involved in hemocyte-mediated sporocyst killing. We tested the hypothesis that Cu/Zn superoxide dismutase (SOD), a cytosolic enzyme that catalyzes the conversion of superoxide anion to H2O2, is somehow different between resistant and susceptible snail strains. We report a hemocyte transcript with all the features of a typical cytosolic Cu/Zn SOD (GenBank accession numbers AY505496 and AY505497). The amount of Cu/Zn SOD mRNA in hemocytes from resistant snails was double that of hemocytes from susceptible snails, and this correlated directly with an increased Cu/Zn SOD enzymatic activity in resistant hemocytes. Additional experiments determined that in vitro interaction/encapsulation of sporocysts did not influence Cu/Zn SOD mRNA levels in hemocytes from either snail strain. Thus, resistance in this host-parasite system does not appear to depend on a transcriptional response of hemocyte Cu/Zn SOD, but may be due, at least in part, to a constitutively elevated enzymatic level of Cu/Zn SOD.
Variation in susceptibility of the snail Biomphalaria glabrata to infection by the parasite Schistosoma mansoni is, at least in part, genetically determined. Functional studies have demonstrated that hemocyte-mediated killing of the parasite involves hydrogen peroxide, the enzymatic product of superoxide dismutase (SOD). The present study identifies alleles of the gene coding for cytosolic copper/zinc SOD (SOD1). The resistance/susceptibility phenotypes and SOD1 genotypes were determined for 354 snails of the predominantly resistant 13-16-R1 strain of B. glabrata. Resistance to the parasite was found to be significantly associated with one allele of the SOD1 gene. Conversely, a separate SOD1 allele was significantly associated with susceptibility.
Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, a medically important schistosome. In order to identify transcripts involved in snail-schistosome interactions, subtractive cDNA libraries were prepared, using suppression subtractive hybridization (SSH) between a parasite-exposed schistosome-resistant and a susceptible strain of B. glabrata, and also between schistosome-exposed and unexposed snails from the resistant snail line. Separate libraries were made from both haemocytes and the haemopoietic organ. Subtraction was performed in both directions enriching for cDNAs differentially expressed between parasite-exposed resistant and susceptible samples and up or down-regulated in the resistant line after challenge. The resulting eight libraries were screened and eight genes, differentially expressed between the haemocytes of resistant and susceptible snail strains, were identified and confirmed with reverse transcriptase PCR, including two transcripts expected to be involved in the stress response mechanism for regulating the damaging oxidative burst pathways involved in cytotoxic killing of the parasite: the iron-storage and immunoregulatory molecule, ferritin, and HtrA2, a serine protease involved in the cellular stress response. Transcripts with elevated levels in the resistant strain, had the same expression patterns in the subtracted libraries and unsubtracted controls; higher levels in exposed resistant snails compared to susceptible ones and down-regulated in exposed compared with unexposed resistant snails. Differential expression of two of the transcripts with no known function from the susceptible strain, was independently confirmed in a repeat exposure experiment.
Dominant lethal effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were evaluated in the freshwater snail Biomphalaria glabrata. Wild-type snails were exposed during 10 days to 50, 75 and 100ppm of 2,4-D dimethylamine salt (2,4-D DMA) and paired with non-exposed albino snails 1, 11, 25 and 40 days after the exposure. The offspring of the non-exposed albino snails was scored for lethal malformations. One day after the exposure, a significant effect was observed at 75 and 100ppm without a dose-response relationship. After 11 days, the effect was observed only at the highest dose. After 25 days, significant increases in the dominant lethal effects occurred at 50 and 75ppm; effects were directly related to the doses. Background levels of lethal malformations were resumed after 40 days. Although the major and direct measure of dominant lethal mutations is the rate of lethal malformations in the heterozygous offspring of the albino snails, the sensitivity of the assay was substantially increased with the evaluation of all non-viable embryos, that are the sum of those with lethal malformations, identified or not as wild-type.
In 2001, ideas for a snail genome project were discussed at the American Society of Parasitologists meeting (New Mexico) and a snail genome consortium was subsequently established (the first consortium meeting was held in 2005). A proposal for sequencing the snail genome was submitted to the National Human Genome Research Institute, and Biomphalaria glabrata was prioritized as a non-mammalian sequencing target in 2004. The sequencing of the genome of this medically important snail is now underway.
Biomphalaria glabrata, a freshwater gastropod mollusc, was tested as biondicator organism to assess cadmium, lead and arsenic exposure using acute laboratory bioassays. Modifications of glycogen levels were measured in different anatomical regions of B. glabrata in order to test the usefulness of this parameter as a general biomarker of chemical stress. The snails were exposed 96 h to different concentrations of the following contaminants: 0.1 and 0.05 mg Cd/L; 0.5, 0.1 and 0.05 mg Pb/L; 0.5, 0.1 and 0.05 mg As/L. Significant decreases in the polysaccharide content were observed in gonadal region for all treated animals. Arsenic and lead at 0.1 and 0.5 mg/L level of exposure were also able to decrease the levels of glycogen in the pulmonary and digestive gland region. Glycogen content in the cephalopedal region of treated animals presented a significant decrease (p<0.05) when compared with control organisms only for arsenic at the highest level of exposure. To establish possible correlations between glycogen and contaminants accumulated by snails, analyses of the elements bioaccumulated in the different anatomical regions of B. glabrata were also performed. Cadmium and lead followed a similar pattern of bioaccumulation with highest values in the digestive gland region. Arsenic bioaccumulation, however, was highest in the gonadal region.
Under laboratory conditions, latex from Euphorbia splendens has shown promise as a plant molluscicide for control of Biomphalaria species, intermediate hosts for Schistosoma mansoni. The purpose of this study was to evaluate its efficiency under field conditions. Application of filtered latex at 12 ppm to one stream in an endemic rural area in Minas Gerais state, Brazil, in September 1995, did result in a reduction in snail density as compared to an untreated stream but the snail population recovered quickly. However, two applications with a two-week interval of unfiltered E. splendens latex at 5 ppm in November 1996 in the same stream resulted in complete disappearance of B. glabrata and snails did not reappear until the 14th month after the applications. In the control stream, without treatment, the snails were found during all months. Laboratory studies confirmed that unfiltered latex is a more potent molluscicide than filtered latex. Considering the advantages of the latex such as its low toxicity to other aquatic animals and its photobiodegradability, as well as the simple method of application, this natural product is promising as an effective molluscicide.
The genetic variability of Brazilian Biomphalaria glabrata populations was studied using SSR-PCR. This technique is a variant of the polymerase chain reaction (PCR), which consists of using a single primer directed towards microsatellite regions under high stringency reaction conditions. Twenty snails of each population from eight distant Brazilian localities were analyzed. Morphology and PCR-RFLP were used for previous specific identification of the snails. Bands generated after gel electrophoresis of the SSR-PCR products of each snail were used to study intra- and interpopulation genetic variability. Fifty-five prominent bands were considered in a pairwise band comparison for the determination of genetic variability. Genetic variability was greater between populations than within populations. Snail populations from the field and the laboratory presented almost no genetic differences. No relationship between genetic variability and geographic distance was found. SSR-PCR proved to be a good alternative molecular tool for the population study of B. glabrata.
The biochemical processes linked up to cyclic nucleotides related to the phenomenon of phagocytosis in Biomphalaria glabrata hemocytes were studied. In hemocytes the results suggest the presence of a phagocytic capacity similar to that observed in human cells related to regulation by cyclic adenylate monophosphate (cAMP), but not by cyclic guanylate monophosphate (cGMP). This similarity and differences in the metabolic process of phagocytary capacity between human phagocytary cells and hemocytes could represent an interesting model aimed at studying the phylogenetic evolution of enzymatic complexes.