The galactan-degrading enzymes in the snail Biomphalaria glabrata.

1. Embryonic snails incorporate from the perivitelline fluid in which they are embedded a polysaccharide, called galactan, which is composed entirely of D-, or D- and L-galactose. In this investigation the p-nitrophenyl-beta-D-galactoside degrading enzyme of Biomphalaria glabrata which was assumed to be involved in the degradation of the galactans was purified almost to homogeneity and its specificity was studied. 2. It has a mol. wt of 135,000 and is composed of two identical subunits. 3. It could be shown that p-nitrophenyl-beta-D-fucoside was hydrolysed eight times faster, but native galactan was neither decomposed nor was it inhibitory for the hydrolysis of p-nitrophenyl-glycosides. 4. Thus, it is most likely that this galactosidase is not involved in the galactan metabolism. 5. However, a membrane-bound enzyme complex was revealed which was able to metabolize the native galactan of Biomphalaria glabrata completely and which showed graded reactivity towards galactans of other species. 6. Since no intermediate degradation products were found it must be assumed that they were metabolized further in the mitochondria.     

Bacterial flora of the schistosome vector snail Biomphalaria glabrata.

The aerobic heterotrophic bacterial flora in over 200 individuals from 10 wild populations and 3 laboratory colonies of the schistosome vector snail Biomphalaria glabrata was examined. Internal bacterial densities were inversely proportional to snail size and were higher in stressed and laboratory-reared snails. The numerically predominant bacterial genera in individual snails included Pseudomonas, Acinetobacter, Aeromonas, Vibrio, and several members of the Enterobacteriaceae. Enterobacteriaceae seldom predominated in laboratory colonies. Our data suggest that Vibrio extorquens and a Pasteurella sp. tend to predominate in high-bacterial-density snails. These snails may be compromised and may harbor opportunistic snail pathogens.     

Bacterial flora of the schistosome vector snail Biomphalaria glabrata

The aerobic heterotrophic bacterial flora in over 200 individuals from 10 wild populations and 3 laboratory colonies of the schistosome vector snail Biomphalaria glabrata was examined. Internal bacterial densities were inversely proportional to snail size and were higher in stressed and laboratory-reared snails. The numerically predominant bacterial genera in individual snails included Pseudomonas, Acinetobacter, Aeromonas, Vibrio, and several members of the Enterobacteriaceae. Enterobacteriaceae seldom predominated in laboratory colonies. Our data suggest that Vibrio extorquens and a Pasteurella sp. tend to predominate in high-bacterial-density snails. These snails may be compromised and may harbor opportunistic snail pathogens.     

The snail (Biomphalaria glabrata) genome project

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.     

Monoamines and their metabolites in the freshwater snail Biomphalaria glabrata

Metabolism of the major monoamines and their functions were studied in the freshwater snail Biomphalaria glabrata. In both juvenile and adult snails, the plasma (cell-free hemolymph) appears to act as a reservoir for most of these monoamines and their metabolites including among others, L-dopa and dopamine as major constituents. Significant quantities of L-tryptophan, precursor of indoleamines, also was found in the plasma. L-dopa, serotonin, homovanillic acid and dopamine were prominently represented in the central nervous system of the snail, while serotonin and its metabolites, 5-hydroxyindole acetic acid and 5-hydroxytryptophol were found in the ovotestis. Catecholamines such as L-dopa, dopamine and homovanillic acid were identified in the albumen gland. Functional aspects of both dopamine and serotonin were studied using in vitro cultures of albumen glands, the site of perivitelline fluid and galactogen synthesis in B. glabrata. Dopamine was found to stimulate the release of secretory proteins when exogenously added to gland cultures and this process was inhibited by chlorpromazine, a dopamine receptor antagonist. Similarly, exogenous serotonin stimulated in vitro protein secretion by albumen glands. Thus, these results suggest that monoamines may play important roles in regulating reproductive activity of this snail and provides an excellent model for studying neurotransmitter function and metabolism in molluscs.     

Equations for describing growth of the schistosome host snail Biomphalaria glabrata

Biomphalaria glabrata and Stagnicola palustris were raised in aquaria under crowded and uncrowded conditions. Measurements of greatest shell diameter were taken at regular intervals. Von Bertalanffy and logistic growth curves were fitted by least squares and maximum likelihood methods. The resulting parameter estimates produced better fits for the logistic equation than for the von Bertalanffy equation.     

Genetic variation among laboratory strains of the planorbid snail Biomphalaria glabrata

Isozymes of laboratory strains of Biomphalaria glabrata have been studied by starch gel electrophoresis. Methods are outlined for adaptation of this technique to the genetic study of these snails. Twenty-eight presumptive gene loci have been identified. Twelve invariant enzymes were observed. Sixteen loci displayed some polymorphism within or among the strains. These polymorphisms were generally widespread among strains from Brazil, Puerto Rico, St. Lucia, and the Dominican Republic. A high degree of intrastrain polymorphism was noted even in some presumably inbred laboratory strains. Crosses between strains were used to demonstrate the genetic basis for the patterns observed at 9 of the 16 polymorphic loci.     

Glucose transport across the intestinal wall of the freshwater snail Biomphalaria glabrata

• 1.1. Isolated midguts of the freshwater snail Biomphalaria glabrata were mounted in an incubation chamber in saline containing 2 mM glucose and perfused with the same solution. External and internal media were continuously gassed with carbogen gas (95% O₂, 5% CO₂). In order to measure the flux rates of glucose [¹⁴C]glucose was applied in the perfusion medium or in the incubation medium. Net fluxes of glucose were calculated as the differences between unidirectional in- and effluxes.
• 2.2. A directed net flux from the mucosal to the serosal side of the intestine was demonstrated (mucosal to serosal = 50 ± 10 nmol cm⁻²hr⁻¹(N = 6) serosal to mucosal 7 ± 1 nmol cm⁻²hr⁻¹ (N = 6), net flux = 43 nmol cm⁻²hr⁻¹).r
• 3.3. The active transport of glucose was reduced by the presence of metabolic inhibitors, cyanide (1 mM) and dinitrophenol (1 mM) on the mucosal as well as on the serosal side. Ouabain (1 mM) inhibited the transport rate only when it was added on the serosal side. Amiloride (1 mM) had no effect on the transport rate whether added on the mucosal or on the serosal side.
• 4.4. Inhibition of glucose transport by oubain, a specific inhibitor of Na⁺/K⁺-ATPase, suggests that glucose transport is secondary active and coupled to Na⁺-transport.

     

Molecular identification of symbionts from the pulmonate snail Biomphalaria glabrata in Brazil

The icthyosporean, Capsaspora owczarzaki, a known predator of Schistosoma mansoni sporocysts in vitro, is more prevalent in laboratory-reared strains of the intermediate snail host, Biomphalaria glabrata resistant to S. mansoni, than from the susceptible M line strain. We examined whether B. glabrata resistant to the NIH-PR-1 strain of S. mansoni from 2 regions in Brazil were also host to C. owczarzaki. Symbiont presence was examined using hemolymph culturing and nested polymerase chain reaction of snail genomic DNA with primers designed to specifically amplify sequences from relatives of the Icthyosporea. All B. glabrata of the resistant Salvador strain from the laboratory of Dr. Lobato Paraense in Rio de Janeiro, Brazil (n = 46) tested negative for symbionts. Three of 18 semiresistant 10-R2 B. glabrata from the laboratory of Dr. Barbosa in Recife, Brazil tested positive for C. owczarzaki. Another icthyosporean, Anurofeca sp., was identified from 1, 10-R2 snail and from 2 of 12 field-collected B. glabrata from Praia do Forte Orange, Ilha de Itamaracá. Snails from 2 other sites, Hotel Colibri, Pontezinha and Praia do Sossego, Ilha de Itamaracá, were negative for Anurofeca. Two genera of ciliates were also identified. Paruroleptus sp. was found in 4, 10-R2 snails and Trichodina sp. was identified in 2 field-collected snails from Praia do Forte Orange and Praia do Sossego.     

Effects of Schistosoma mansoni infection on inorganic elements in the snail Biomphalaria glabrata

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.     

Intraspecific variations in the hemolymph of Biomphalaria glabrata, a snail host of Schistosoma mansoni.

An attempt was made to characterize the hemolymph of Biomphalaria glabrata with reference to "normal" intra-specific variation, i.e., both inter- and intra-strain differences. Total protein concentration, per cent hemoglobin, pH, and osmolarity were studied. Seven geographic strains of B, glabrata were examined. In addition, observations were made on the hemolymph of Biomphalaria straminea, several strains of Helisoma caribaeum, and on B. glabrata subjected to infection with Schistosoma mansoni or to periods of starvation. Intra-strain differences in total protein concentration and total hemoglobin concentration in B. glabrata appeared to be more closely related with snail size than with absolute age. Inter-strain variation in B. glabrata was also noted, but the differences were of the same magnitude as those from intra-strain samples. Significant differences in total protein concentration were observed, however, between the means of similar size B. glabrata, B. straminea and H. caribaeum. The osmolatity of the hemolymph from different size B. glabrata was similar as were the osmolalities of the hemolymph from similar size snails of different strains. However, all B. glabrata strains exhibited hemolymph osmolalities lower than observed in strains of H. caribaeum. Infection with S. mansoni reduced the protein concentration of B. glabrata hemolymph. Differences were noted as early as 1.5-24 hr post-infection, with significant alterations occurring at about 11 days post-infection. To a lesser extent, starvation also depleted the protein content of the hemolymph.     

Echinostoma lindoense: larval antigens from the snail intermediate host, Biomphalaria glabrata

Passive hemagglutination using chromic chloride proved to be a rapid and useful method for a study of minute quantities of antigen extracted from larval Echinostoma lindoense (Sandground and Bonne), a trematode that develops in the snail intermediate host, Biomphalaria glabrata (Say). Parasite rediae were initially fragmented by three different procedures. Their soluble proteins were separated into two bands by electrophoresis on cellulose acetate, and into three fractions by molecular sieve chromatography. Rabbit antiserum was prepared from six weekly intramuscular injections of soluble redial protein in complete Freund's adjuvant, followed after 1 month by a single inoculation of alum-precipitated antigen. Antiserum was absorbed free of anti-snail antibodies and the immune complexes were removed by ion-exchange chromatography over DEAE-cellulose, producing an immunochemically pure IgG. Study of the rabbit anti-trematode antibody by precipitation, complement fixation, hemagglutination (HA), and inhibition of HA revealed a specific and high titered anti-larval antibody. These methods offer an approach to the problem of measuring the snail host's protective response against trematode reinfection; they also can be used to study the antigenic maturation of successive larval stages in the intermediate host.     

Calcium binding constituents of the organic shell matrix from the freshwater snail Biomphalaria glabrata

The Ca2+ binding of an EDTA-free water-soluble (SM) and -insoluble (IM) organic matrix of the freshwater snail Biomphalaria glabrata was investigated, using a 45Ca2+ autoradiography after SDS-electrophoretical separation and a calcium binding assay. Electrophoresis of the SM showed a considerable amount of Alcian blue and Stains all positive material, regarded as glycosaminoglycans (GAGs) or proteoglycans (PGs). This part of the SM was slightly positive after 45Ca2+ autoradiography at pH 6.8. The Ca2+ binding increased, raising the pH to 7.4 and 8.0 and was especially strong when simulating the real conditions of the extrapallial space with a carbonate buffer of pH 7.4. The Ca2+ binding assay of the IM showed the same pH-dependency that was observed in the SM. The titration of the IM with Ca2+ at pH 8.0 lead to a dissociation constant of 7.5 x 10(-5) M. While Mg2+ displaced 45Ca2+ in the same way as nonradioactive Ca2+, an approximately 400-fold amount of Na+ was necessary to reduce the binding of 45Ca2+ to 50%. The Ca2+ binding of the organic matrix from the B. glabrata shell appears to be a process of low specificity, medium affinity and high pH-dependency. Apparently, acidic carbohydrate-rich PGs are the only calcium binding constituents of the organic shell matrix.     

Linkage relationships of seven enzyme and two pigmentation loci in the snail Biomphalaria glabrata

Crossing experiments with inbred stocks of the snail (Biomphalaria glabrata) demonstrated that variants at two loci determining pigmentation and seven enzyme-determining loci exhibited normal Mendelian segregation ratios in F2 progeny. Among 39 pairwise comparisons for joint segregation, there was evidence of genetic linkage between a locus controlling mantle pigmentation (S) and 6-phosphogluconate dehydrogenase (Pgd) and confirmation of a previously described linkage between esterase-2 (Est-2) and catalase (Cat). Recombination fractions were estimated to be 17 +/- 4 for S-Pgd and 33 +/- 5 for Est-2-Cat. The remaining five loci--Acon-1, Pgm-1, Lap-1, Lap-2, and Pgd--assorted independently. This brings to 17 the number of loci examined for segregation and assortment in this medically important species. As Biomphalaria has a chromosome number n = 18, markers should soon be available for most or all of the linkage groups.     

Further characterization of passively transferred resistance to Schistosoma mansoni in the snail intermediate host Biomphalaria glabrata.

A heat-labile plasma factor from genetically resistant 10-R2 Biomphalaria glabrata snails confers passively transferred resistance (PTR) to Schistosoma mansoni when injected into susceptible snails within 24-hr of exposure to miracidia. However, no additional details on PTR have emerged since the initial 1984 report, nor has the plasma resistance factor been characterized. In the present study, new information is provided on the occurrence of resistance factor in plasma of additional types of snails, effect of "priming" resistant plasma donors by prior exposure to miracidia, duration of PTR, molecular weight of resistance factor, and fate of sporocysts in snails with PTR. Susceptible NIH albino snails injected 24 hr prior to exposure to miracidia with individual samples of plasma from a different strain (Salvador B. glabrata) or a different species (B. obstructa) of nonsusceptible snail displayed infection prevalences of 49% or 59% of control levels, respectively, whereas injections of homologous plasma had no effect. PTR was not enhanced by prior exposure of resistant Salvador plasma donors to miracidia. Unexpectedly, PTR induced by injections of Salvador plasma persisted for at least 21 days. The molecular weight of the resistance factor(s) was between 10 and 30 kDa, based on results of centrifugal ultrafiltration. A significantly higher proportion of dead sporocysts occurred in histological sections of tentacles from snails injected with Salvador plasma than in tentacles of snails injected with NIH albino plasma at 7 days postexposure to miracidia. Most dead sporocysts in Salvador plasma-injected snails were undergoing gradual degeneration, rather than rapid, hemocyte-mediated destruction, as occurred in Salvador snails.     

The role of the osphradium in chemoreception by the snail Biomphalaria glabrata (Say)

The possible involvement of the osphradium of Biomphalaria glabrata (Say) in food finding has been investigated by using behavioural assays to determine whether snails with cauterized osphradial canals can locate a source of food extract in still water. A new technique for anaesthetizing the snails for the cautery experiments is described. The experimental results indicate that a functional osphradium is not essential for directional movement towards a source of food extract. The present results are in conflict with those of Michelson (1960). Reasons for the differences between the present results and those of Michelson and the possible functional roles of the osphradium are discussed.