Surface antigens of Biomphalaria glabrata (Gastropoda) hemocytes: evidence for linkage-independence of some hemolymph-like surface antigens and Con A receptor-bearing macromolecules

The structural relationship between hemolymph-like surface antigens and concanavalin A (Con A)-reactive macromolecules on circulating hemocytes of Biomphalaria glabrata was assessed using a double-ligand labeling method. It was determined that Con A-induced clearance of its own receptor complexes resulted in a significant reduction, but of complete elimination, of hemolymph-like antigens, i.e., antigens cross-reactive with an anti-B. glabrata hemolymph antiserum, suggesting the presence of at least two separate antigenic populations with anti-hemolymph reactivity; one group structurally linked and another group structurally independent of Con A-binding membrane components. The latter group of surface antigens appears to be (1) chemically related to the higher-molecular-weight hemolymph components, most probably hemoglobin, (2) Pronase resistant, and (3) partially composed of a subpopulation of cryptic (hidden) cross-reacting antigens uncovered at the cell surface as a consequence of Con A-receptor clearance. Results of this study demonstrate not only that individual hemocytes of B. glabrata may possess different populations of hemolymph-like antigens, but also that the interaction between some membrane components and appropriate ligands (e.g., carbohydrate-binding lectins) could result in a modulation in expression of other groups of surface antigens.     

Surface antigens of Biomphalaria glabrata (Gastropoda) hemocytes: functional heterogeneity in cell subpopulations recognized by a monoclonal antibody

A hemocyte surface membrane marker (BGH₁) has been identified using hemocyte-specific monoclonal antibodies (mABs) generated by somatic cell fusion methods. The BGH₁ epitope was expressed on a subpopulation of circulating, glass-adherent blood cells from two strains of the snail, Biomphalaria glabrata. Approximately 40% of the circulating hemocytes from the PR albino (M-line) B. glabrata strain were BGH₁^?, compared to a prevalence of 10% BGH₁⁺ cells in the 10-R2 snail strain. When hemocytes were firmly attached and spread on a glass surface, BGH₁⁺ cells were morphologically distinguishable from BGH₁^? cells by their ovoid shape and the presence of short, thin filopodial projections along the ectoplasmic border. In contrast, BGH₁^? hemocytes were more pleomorphic and possessed long, spike-like filopodia. Moreover, the BGH₁ epitope was trypsin-resistant and retained its antigenic reactivity with probe mABs following fixation with paraformaldehyde or paraformaldehyde/MeOH. Fixation with glutaraldehyde, however, significantly reduced mAB binding to the BGH₁ surface epitope. There was no apparent age-dependent expression of the BGH₁ determinant since circulating hemocyte populations in very young (1–2 mm) to adult (10–12 mm) snails were composed of both BGH₁⁺ and BGH₁^? subpopulations. Quantitative shifts in the prevalence of epitope-bearing hemocytes between the smallest snail size class (1–2 mm) and the larger snails (3–4 and 10–12 mm) are believed to be due to a differential production and/or release of BGH₁^? hemocytes within the blood circulation rather than a gradual age-related change in the expression of surface antigens on individual cells. Experiments designed to assess the in vitro phagocytic capability and lysosomal acid phosphatase (APase) activity of mAB-reactive hemocytes revealed that BGH₁⁺ cells, when compared to those lacking the surface marker, were significantly reduced in both their phagocytic and APase-producing activities. Since the PR albino strain of B. glabrata possesses a higher proportion of BGH₁^? hemocytes and a lower total concentration of circulating cells than do snails of the 10-R2 strain, PR albino snails are thus potentially reduced in their natural capacity to mount cellular reactions against foreign materials.     

Biomphalaria glabrata (Gastropoda): effect of urethane on the morphology and function of hemocytes, and on susceptibility to Schistosoma mansoni (Trematoda)

Urethane (ethyl carbamate) anesthesia of Biomphalaria glabrata resulted in several rapid changes to the snail's blood picture. At 2 hr postexposure (PE) to the drug, there was an elevation in the prevalence of acid phosphatase (APase)-positive hemocytes, a significant increase in the number of APase granules per cell, a three fold increase in circulating blood cell number, and a decrease in the percentage of hemocytes expressing a cell subpopulation-specific surface membrane epitope (BGH₁). However, urethane had little effect on erythrophagocytosis by host hemocytes. All of the observed changes returned to control levels by 12 hr PE to the drug. Blood cells cultured with various concentrations of the anesthetic in vitro did not exhibit any alterations in the parameters described above. Since circulating hemocytes represent the primary effector component involved in internal defense reactions, the effect of urethane-induced changes in the composition of circulating blood cells on susceptibility to a larval trematode, Schistosoma mansoni, was examined. Such changes had no apparent effect on host susceptibility since the rate of infection for urethane-exposed snails (88.2%) was the same as for nonurethane-treated B. glabrata (82.4%). However, the effects of urethane-induced changes in hemocyte number and composition on other invading organisms is not known. Therefore, it is suggested that such alterations should be considered when internal defense reactions are studied in snails exposed to this drug and other commonly used anesthetics.     

Neotropical schistosomiasis: African affinities of the host snail Biomphalaria glabrata (Gastropoda: Planorbidae)

Schistosoma mansoni , the blood fluke responsible for human intestinal schistosomiasis in the Neotropics, was imported repeatedly with African slaves during the period 1500–1800. This trematode, and its intermediate host snails of the genus Biomphalaria , are widely distributed across Africa and the disease is thought to have quickly become established in South America and the West Indies because of the presence of an endemic susceptible congener, B. glabrata. We compared B. glabrata with four other Neotropical and three African species of Biomphalaria using 20 allozyme loci and found that it is phenetically and phylogenetically more like the African species; both parasite and American host snail are apparently of historically or geologically recent African origin. Furthermore, genetic distances, cladistic analyses and fossil data suggest the African Biomphalaria species may themselves have evolved from Neotropical founders following an initial trans-Atlantic dispersal in the reverse direction 2.3–4.5 Mya. Interpretation of existing patterns remains problematic as few African snails have been characterized genetically and both B. glabrata and African B. pfeifferi appear to comprise several cryptic species.     

Effects of exogenous interleukin-1β on primary sporocysts of Schistosoma mansoni (Trematoda) incubated with plasma and hemocytes from schistosome-susceptible and resistant Biomphalaria glabrata (Gastropoda)

Abstract. The cytokine interleukin-1β (IL-1β) mediates interactions of immune and inflammatory cells in mammals. Previous reports also have linked plasma (cell-free hemolymph) levels of IL-1β in the snail Biomphalaria glabrata to resistance against Schistosoma mansoni . In the present study, fluorescent probes were used to study larval schistosome and snail hemocyte viability during in vitro encounters. Hemolymph (plasma and hemocytes) from schistosome-susceptible (M-line) and resistant (13–16-R1) B. glabrata was added to sporocysts of S. mansoni and the viability of hemocytes and parasites was assessed. Next, IL-1β was added to sporocyst-hemolymph samples, the viability of sporocysts and hemocytes determined and then compared to control assays. The number of live sporocysts present after incubation for 1 h with hemolymph from M-line snails was significantly greater than the number seen when hemolymph from 13–16-R1 snails was tested. Nearly all sporocysts survived the 1 h incubation with M-line hemolymph, and most of the hemocytes attached to sporocysts were dead. In contrast, nearly all sporocysts were dead when hemolymph from 13–16-R1 snails was tested, and most attached hemocytes were alive. Addition of IL-1β to M-line hemolymph resulted in a dramatic increase in sporocyst death. Addition of IL-1β to 13–16-R1 hemolymph produced a small but significant increase in the rate of sporocyst death. These results indicate that the concentration of IL-1β present in hemolymph from B. glabrata is directly related to the ability of this snail to kill S. mansoni sporocysts in vitro.     

Schistosoma mansoni and Biomphalaria glabrata share epitopes: antibodies to sporocysts bind host snail hemocytes

Using an independent protocol, we have confirmed that sporocysts of the human blood fluke, Schistosoma mansoni, synthesize antigens which stimulate rabbit antibody activity to epitopes on infermediate snail host hemocytes. This molecular mimicry may aid S. mansoni to escape the innate immune system of this host, Biomphalaria glabrata.     

Larval Schistosoma mansoni excretory-secretory glycoproteins (ESPs) bind to hemocytes of Biomphalaria glabrata (Gastropoda) via surface carbohydrate binding receptors

Flow cytometric analysis of circulating blood cells (hemocytes) of Biomphalaria glabrata, molluscan intermediate host of Schistosoma mansoni, revealed the presence of 2 overlapping hemocyte subpopulations, designated R1 and R2. R1 hemocytes are characterized by their smaller size, reduced granularity, and the presence of the BGH1 surface epitope, whereas R2 cells are larger, more granulated, and generally lack the BGH1 cell marker. Both hemocyte subpopulations bound fluorescent dye (Oregon Green)-conjugated excretory-secretory glycoproteins (fESPs), although the specific fESP binding signal (geometric mean value), after correction for cellular autofluorescence, was greater in the R1 hemocyte subpopulation compared to that of the R2 subset. Partial inhibition of fESP binding to hemocytes consistently was achieved using various glycoconjugates (mucin, asialo-mucin, asialo-fetuin, heparin) and polysaccharides (fucoidan, dextran sulfate 8000), suggesting the involvement of hemocyte carbohydrate-binding receptors (CBRs) in reactions with ESP-associated glycans. Although sulfation of carbohydrate ligands contributed significantly to ESP blocking activity of some inhibitory polysaccharides and heparin, other sulfated proteoglycans (chondroitins A and B, heparan sulfate) were noninhibitory, indicating that charge alone was not solely responsible for the observed inhibition of hemocyte binding by fESPs. A similar blocking effect by desialylated glycoproteins (asialo-mucin, asialo-fetuin) further supports the contention that ESP-hemocyte interactions are mediated primarily through CBRs. The glycoconjugate inhibitors of ESP binding were only partially effective over a range of concentrations and their glycan moieties (oligosaccharides or long-chain polymers) comprised a diversity of major sugar groups, suggesting that hemocyte CBRs and S. mansoni larval ESPs likely represent a multiple receptor-ligand system. Previously reported findings of differential effects of ESPs on a variety of in vitro hemocyte functions are consistent with such a hypothesis.     

Effects of intermittent starvation on mature Biomphalaria glabrata (Gastropoda: Planorbidae)

Mature Biomphalaria glabrata, submitted to four weeks of varied starvation times (0, 1, 3, 5, 6 & 7 d.week^–1, were thereafter refed during four weeks. The different intermittent starvation times had no significant effect on snails survival. As weekly starvation increased, the rate of change in body weight and fecundity decreased. In snails fed one or two d.week^–1, the rate of change in body weight was negative, while fecundity remained at a low level. Continuous hunger stopped oviposition.Starvation had no further effect on body growth after the first week of refeeding; however, its effect on fecundity remained significant over the two first weeks.     

Concanavalin A-mediated phagocytosis of yeast by Biomphalaria glabrata hemocytes in vitro: Effects of temperature and lectin concentration

Exposure of either Biomphalaria glabrata granulocytes or yeast to concanavalin A (Con A) prior to being incubated together in a “microhemadsorption assay” affected the number of yeast attached to or endocytosed by the granulocytes after 60 min. The yeast-granulocyte interaction was sensitive to temperature, Con A concentration, and the cell treated. Numbers of associated (attached + endocytosed) and endocytosed yeast either increased or decreased, as compared with nontreated controls, depending on the protocol. The effect of a given protocol was not necessarily similar for attachment and endocytosis. By separately enhancing these two stages of phagocytosis, Con A may be a useful tool for studying this process of molluscan granulocytes.     

Experimental exposure of Helisoma trivolvis and Biomphalaria glabrata (Gastropoda) to Ribeiroia ondatrae (Trematoda)

Experimental infections provide an important foundation for understanding host responses to parasites. While infections with Ribeiroia ondatrae cause mortality and malformations in a wide range of amphibian second intermediate host species, little is known about how the parasite affects its snail first intermediate hosts or even what species can support infection. In this study, we experimentally exposed Helisoma trivolvis, a commonly reported host of R. ondatrae, and Biomphalaria glabrata, a confamilial snail known to host Ribeiroia marini, to increasing concentrations of embryonated eggs of R. ondatrae obtained from surrogate definitive hosts. Over the course of 8 wk, we examined the effect of parasite exposure on infection status, time-to-cercariae release, host size, and mortality of both snail species. Helisoma trivolvis was a highly competent host for R. ondatrae infection, with over 93% infection in all exposed snails, regardless of egg exposure level. However, no infections were detected among exposed B. glabrata, despite previous accounts of this snail hosting a congener parasite. Among exposed H. trivolvis, high parasite exposure reduced growth, decreased time-to-cercariae release, and caused marginally significant increases in mortality. Interestingly, while B. glabrata snails did not become infected with R. ondatrae, individuals exposed to 650 R. ondatrae eggs grew less rapidly than unexposed snails, suggesting a sub-lethal energetic cost associated with parasite exposure. Our results highlight the importance of using experimental infections to understand the effects of parasite exposure on host- and non-host species, each of which can be affected by exposure.     

Echinostoma paraensei: hemocytes of Biomphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni

Earlier in vivo work by Lie et al. (1977) indicated that the innate resistance of the 10R2 strain of Biomphalaria glabrata to PR1 Schistosoma mansoni could be interfered with if the snails were infected previously with another trematode, Echinostoma paraensei. We have studied this interference phenomenon using in vitro methods in an attempt to understand its mechanistic basis. Hemolymph, derived from 10R2 snails infected with E. paraensei for 14-28 days, killed 25% of S. mansoni sporocysts in vitro, significantly less (P less than 0.001) than the 90% killing rate observed with hemolymph from uninfected, control 10R2 snails. Hemolymph from the infected 10R2 snails and from schistosome susceptible M line snails did not differ significantly (P greater than 0.1) in their relative inability to kill S. mansoni sporocysts in vitro. The defect in sporocyst killing exhibited by echinostome infected 10R2 snails was traced to the cellular, rather than the humoral, component of the hemolymph. Preparations containing uninfected 10R2 snail hemolymph and echinostome daughter rediae exhibited significantly less (P less than 0.001) killing of S. mansoni sporocysts than did controls containing only 10R2 hemolymph and S. mansoni sporocysts. Our results suggest that echinostome larvae release factors that interfere with the ability of B. glabrata hemocytes to kill S. mansoni sporocysts.     

Echinostoma paraensei and Schistosoma mansoni: adherence of unaltered or modified latex beads to hemocytes of the host snail Biomphalaria glabrata.

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

Nitrogenous compounds in the excreta of the germfree snail Biomphalaria glabrata (mollusca: Planorbidae)

• 1.1. Nitrogen excretion of germfree snails kept in sterile saline solution during 14 days consists mainly of ammonia and urea in approximately equal amounts.
• 2.2. Uric acid is excreted intermittently being accumulated in snail nephridia.

     

Characterization of the bacterial microbiota of Biomphalaria glabrata (Say, 1818) (Mollusca: Gastropoda) from Brazil

Roughly 200 000 000 people in 74 countries infected with schistosomes all share the fact that they came in contact freshwater harbouring infected snails. The aim of the study is to characterize the microbiota of wild and laboratory‐reared snails of Biomphalaria glabrata from Pernambuco, Brazil. The microbiota of these molluscs was identified biochemically by the VITEK 2 automated microbiological system. Antimicrobial susceptibility testing was carried out by the disc diffusion method with ß‐lactam antibiotics, aminoglycosides, quinolones, folate pathway inhibitors, fenicols and tetracyclines. The results showed that all bacteria identified were gram‐negative, including 11 bacterial genera: Aeromonas, Citrobacter, Enterobacter, Cupriavidus, Rhizobium, Stenotrophomonas, Pseudomonas, Klebsiella, Acinetobacter, Vibrio and Sphingomonas. Regarding the antimicrobial susceptibility, all the isolates exhibited resistance to amoxicillin and sensitivity to meropenem (beta‐lactam antimicrobials). The microbiota of the wild snails consisted predominantly of Enterobacter cloacae, while the laboratory‐reared snails predominantly showed Citrobacter freundii and Aeromonas sobria.

Significance and Impact of the Study

Biomphalaria glabrata is a Brazilian freshwater Planorbidae of great medical relevance as an intermediate host of Schistosoma mansoni. About a month after being infected by one or more miracidia larvae of a compatible schistosome, B. glabrata sheds thousands of cercariae into the water where they seek human skin and, if successful, penetrate to establish infection, eventually taking up residence and maturing in blood vessels of the small intestine. Results obtained from this study aim at targeting novel biological control strategies for schistosomiasis such as paratransgenesis. This is the first study on the microbiota of B. glabrata from Brazil.     

Effects of environmental calcium deprivation on the egg masses of Physa marmorata Guilding (Gastropoda: Physidae) and Biomphalaria glabrata Say (Gastropoda: Planorbidae)

Eggs of the basommatophoran snails Physa marmorata and Biomphalaria glabrata were cultured in low concentrations of calcium to determine effects on growth and development. In both species there was some development in media with 0.12 mg/l Ca²⁺ but embryos were unable to hatch. 61.04% of embryos of P. marmorata could develop to hatching in 0.22 mg/l Ca²⁺ but those of B. glabrata required a level of 0.42 mg/l Ca²⁺, to attain even a 31.07% hatch. Marked effects on growth rate, embryo size and on time taken to achieve hatching were noted in both species at very low calcium levels. The possibility of cation-controlling mechanisms in the egg membrane is discussed.     

Effects of starvation on growth and reproductive apparatus of two immature freshwater snails Biomphalaria pfeifferi and Biomphalaria glabrata (Gastropoda: Planorbidae)

Starvation of immature snails of Biomphalaria pfeifferi and B. glabrata results in an arrest of growth and animals remain immature. Spermatogenesis is limited to spermatogonia (B.g.) or spermatocytes 1 (B.p.). The number and the size of oocytes remain inferior to that of controls. Animals show reduced genital tracts.Once feeding is restored, growth is resumed but wet weight and shell diameter do not reach the same level as in controls. Fecundity and gametogenesis do not differ from that in controls. Genital tracts weight is proportional to body weight.