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Characterization of an insulin receptor-related receptor in Biomphalaria glabrata embryonic cells.

Tyrosine kinase receptors play a key role in the communication of cells with their environment. Growth hormone receptors, such as insulin receptors, are involved in the regulation of cell growth, differentiation and metabolism in multicellular organisms. Insulin-related peptides and members of the insulin receptor subfamily have been described in a wide variety of invertebrates, including freshwater molluscs. In this paper, we describe the metabolic effect of insulin on a mollusc cell line (Bge) derived from embryos of the snail Biomphalaria glabrata. Using a PCR strategy, we have cloned from Bge cells a cDNA encoding a protein (BgIR) homologous to, and exhibiting all of the typical features of insulin receptors. Northern blot analysis confirmed the expression of BgIR in B. glabrata snails and suggested its wide distribution in the snail body. Bge cells have been shown to provide the environmental conditions necessary for the in vitro development of the sporocysts of Schistosoma mansoni, a trematode parasite that uses B. glabrata as an intermediate host. The possible implication of BgIR in the activating and proliferating processes observed in Bge cells during their coculture with S. mansoni larvae is discussed.

Amino Acid Sequence↗

The respiratory properties of Biomphalaria glabrata exposed to Schistosoma mansoni infection, starvation, CO, and choices of different oxygen concentrations.

The oxygen consumption rate (VO(2)) of Biomphalaria glabrata populations, using polarometric and manometric methods, when plotted against dried body mass as logarithmic co-ordinates, respectively, fell on a regression line with a slope between 0.933 and 1.02. The slope of the regression line for non-infected Schistosoma mansoni populations was found to be 1.04 with no differences in the VO(2) between infected and non-infected snails. The VO(2) of CO-treated snails was the same as for the control snails. The VO(2) of starved snails declined after 3 days and was half the original value after 10 days starvation at 27 degrees C. The P(50) value for snail haemolymph containing haemoglobin suspended in a Tris-HCl buffer was 5.57(+/-0.73)mmHg at a pH of 7.51 and 25 degrees C. For Sephadex-75 cleaned haemolymph the P(50) value was 1.72(+/-0.07)mmHg at 25 degrees C and pH 7.51. Snails exposed to oxygen fs and to choices of different oxygen concentrations in water did not exclusively prefer high (130mmHg), low (15mmHg), or normal (80mmHg) oxygen tensions. The oxygen consumption rate of 782 cercariae at 27 degrees C was measured as 0.0092 microl O(2)/h per single cercaria. The results, when compared with the data in the literature [Z. Vergl. Physiol. 46 (1963) 467;; S. A. J. Zool. 14 (1979) 202], indicate that the mantle cavity gas bubble plays an insignificant or no role at all when pulmonate snails are kept in water with high partial pressures of oxygen and at low temperatures.

Animals↗

Schistosoma mansoni: lectin-dependent cytotoxicity of hemocytes from susceptible host snails, Biomphalaria glabrata.

Normally benign hemocytes from a strain (M-line) of the snail, Biomphalaria glabrata, susceptible to Schistosoma mansoni, became cytotoxic toward the sporocyst stage if the parasite was first treated with the lectin, concanavalin A. Concanavalin A binding was inhibitable with alpha-methyl mannoside and killing was dose-dependent. Maximal levels of concanavalin A-induced cytotoxicity were comparable with levels observed when hemocytes from a resistant snail strain (13-16-R1) encountered untreated sporocysts. Induction of the cytotoxic response did not occur if hemocytes alone were pretreated with the lectin. A unique method incorporating ultraviolet microscopy and the vital fluorescent dye, eosin Y, was used for discriminating between live and dead sporocysts. This model may prove useful in understanding mechanisms used by invertebrate effector cells in recognition and killing of invading organisms.

Animals↗

Male-female larval interactions in Schistosoma mansoni-infected Biomphalaria glabrata.

This paper investigates Schistosoma mansoni male-female larval interactions in simultaneous bi-miracidial Biomphalaria glabrata infections. Larval interactions were analysed at four levels of infection: (i), miracidial infectivity, estimated by the prevalence of mollusc infection; (ii), mollusc pathology, measured by the mollusc growth and survival; (iii), dynamics of the cercarial sex ratio; and (iv), cercarial infectivity, measured as the success of development into adulthood. Our results showed that larval interactions exist in S. mansoni-infected B. glabrata. These interactions do not occur in miracidial infectivity, but occur in mollusc pathology, cercarial sex ratio dynamics and cercarial infectivity. Regarding mollusc pathology, we showed that the bi-miracidially-infected molluscs were smaller than the mono-miracidially-infected ones. This could be the result of larval competition. Regarding the dynamics of the cercarial sex ratio, we showed larval female superiority as compared with male larvae inducing the shedding of female-biased cercarial sex ratios. These sex ratios were rhythmic and could be the reflection of an external expression of the intramolluscan development. Regarding cercarial infectivity, we showed that the simultaneous presence of both sexes in a mollusc increased the cercarial infectivity. This result could be due to male-female larval synergism.

Animals↗

The symbiont Capsaspora owczarzaki, nov. gen. nov. sp., isolated from three strains of the pulmonate snail Biomphalaria glabrata is related to members of the Mesomycetozoea.

While investigating the resistance of some strains of Biomphalaria glabrata to infection with Schistosoma mansoni, a unicellular eukaryotic symbiont was noted in the snail haemolymph. It was similar in appearance to Nuclearia sp. reported from B. glabrata. Sequences comprising the 18S, ITS1, 5.8S, ITS2 and the beginning of the 28S rDNA gene regions were obtained from symbionts isolated from three strains of B. glabrata, and compared with the same sequences obtained from a culture of Nuclearia sp. 18S rDNA sequences were identical for all four isolates. 18S rDNA sequences were used in a phylogenetic analysis to produce minimum evolution, maximum parsimony, maximum likelihood and Bayesian trees. All four analyses indicated that the B. glabrata symbiont is not closely related to Nuclearia but instead to the Mesomycetozoea, a recently recognised clade of symbiotic eukaryotes. Based on phylogenetic analysis, life history and morphological differences, the symbiont is described as a new genus and species, Capsaspora owczarzaki. Distinguishing characters are the presence of life cycle stage(s) that occur within snail haemolymph; ability to kill and ingest digenetic trematode larvae; ability to undergo asexual fission to produce daughter cells; absence of flagella, a mucous sheath and membranes containing chitin, elastin, or collagen; and presence of long unbranching pseudopodia and a penetration process. Using both polymerase chain reaction (PCR) and culturing techniques, the S. mansoni-resistant Salvador and 13-16-R1 strains were found to be significantly more likely to harbour the symbiont than the susceptible M line strain. Small but consistent sequence differences were noted among symbiont isolates from different snail strains, raising the possibility that the symbiont has diverged in different snail lineages. This suggests further that the symbiont is not restricted to albino lab-reared snails. A role, if any, of the symbiont in resistance awaits further study.

Animals↗

Single-species infections of Echinostoma caproni cercariae in pulmonate snails and concurrent infections of E. caproni and Echinostoma trivolvis cercariae in Biomphalaria glabrata.

Single-species infections of Echinostoma caproni cercariae in various pulmonate snails showed that a higher percentage of cercariae encysted successfully in Biomphalaria glabrata than in other snail species and that the percentage encystment was significantly greater in B. glabrata than in either Helisoma trivolvis or Lymnaea elodes. There were significant differences in cyst diameter, thickness of outer and inner cysts, and diameter of excretory concretions between E. caproni and Echinostoma trivolvis metacercariae. These morphological differences helped to distinguish each species of cyst in concurrent cercarial infections of E. caproni and E. trivolvis in B. glabrata. Moreover, in concurrent infections E. trivolvis localised only in the saccular kidney, whereas E. caproni occurred in both the saccular kidney and the pericardium.

Animals↗

Molecular studies of Biomphalaria glabrata, an intermediate host of Schistosoma mansoni.

The freshwater gastropod Biomphalaria glabrata is one of the most important invertebrate hosts of the helminth parasite Schistosoma mansoni. Investigators are using different strategies to determine the molecular basis of this snail-parasite relationship. Of particular interest are the identification of parasite resistance genes in the snail, and the application of molecular probes to better understand the epidemiology of schistosomiasis. This review will focus on recent advances that have been made on genome analysis of B. glabrata. Much of this work has centred on the use of random amplification of polymorphic DNA-PCR-based technology, with restriction fragment length polymorphism analysis and the generation of expressed sequence tags from the snail. A brief discussion of how parasite products may complicate this analysis is also given, along with an indication of the scope of the problems that lie ahead.

Amino Acid Sequence↗

Short-term immunoisolation of incompatible xenografts in a snail, Biomphalaria glabrata.

Hearts of the snails Physa virgata and Biomphalaria glabrata were implanted into the hemocoel of B. glabrata. Implants either were microencapsulated in 2.5% agarose or were unencapsulated. Unencapsulated xenografts from P. virgata underwent complete necrosis within 3 days post-implantation (DPI), whereas allografts were still alive at 7 DPI. In the case of microencapsulated implants, both allografts and xenografts were alive at 3 DPI, showing 4.8 and 14.9% pyknosis among myofiber nuclei, respectively. These results suggest that direct cytotoxicity by hemocytes, rather than toxic plasma factors or donor-recipient physiological incompatibility, is responsible for rapid xenograft death. However, both types of microencapsulated grafts were necrotic at 7 DPI. This necrosis may have resulted from oxygen deprivation brought about both by the agarose and by the heterotopic implantation site, inasmuch as microencapsulated allografts and xenografts cultured in saline for 7 days showed only 1.98 and 30% pyknosis, respectively.

Animals↗

Production of reactive oxygen species by hemocytes of Biomphalaria glabrata: carbohydrate-specific stimulation.

Recognition of specific carbohydrate structures, which occur commonly on the surfaces of invading pathogens, is thought to elicit internal defense mechanisms in invertebrates. To investigate the nature of carbohydrates that evoke a defensive response in hemocytes of the gastropod Biomphalaria glabrata, we tested eight different carbohydrates, conjugated to bovine serum albumin (BSA), for generation of reactive oxygen species (ROS). Six of the carbohydrate moieties tested are thought to be present on the S. mansoni sporocyst surface (mannose, galactose, fucose, N-acetyl-glucosamine, N-acetyl-galactosamine, and lactose); the other two carbohydrates tested were glucose and melibiose. ROS generation was measured using the fluorescent probe - 2',7'-dichlorofluorescein-diacetate (DCFH-DA). Hemocytes were derived from two different strains of B. glabrata: one of the strains used (MO) is susceptible to infection by the trematode Schistosoma mansoni (PR-1 strain), while the other snail strain (13-16-R1) is resistant to infection with PR-1. Three of the BSA-carbohydrate conjugates (BSA-galactose, BSA-mannose, and BSA-fucose), stimulated generation of reactive oxygen species in the molluscan hemocytes. The responses of the hemocytes were similar whether they were derived from susceptible or resistant snails. If the carbohydrate structures we found, to stimulate ROS generation are involved in parasite recognition, our results suggest that parasite killing may involve either qualitative differences in production of reactive oxygen species, or additional factors.

Animals↗

Hematopoietic tissue allografts in Biomphalaria glabrata (Mollusca: Pulmonata) induce humoral immunity to Schistosoma mansoni.

We examined a potential mechanism for adoptively transferred resistance against Schistosoma mansoni in schistosome-susceptible snails receiving allografts of the hematopoietic amebocyte-producing organ (APO) from a schistosome-resistant strain of Biomphalaria glabrata. Susceptible NIH albino snails first were implanted with the APO from resistant Salvador strain donors. At 14 days post-implantation, cell-free plasma was isolated from APO recipients and injected into a second group of NIH albino snails. Controls were injected with plasma from NIH albino snails that previously had received implants of Salvador mantle, an immunologically inert tissue. Finally, plasma recipients, along with untampered (non-injected) controls, were exposed to miracidia of S. mansoni and subsequently monitored for infection. A significantly lower prevalence of infection occurred in the group injected with plasma from APO recipients (53%) in comparison with that in untampered controls (100%) or in snails receiving plasma from mantle recipients (97%). These results suggest that adoptively transferred resistance in B. glabrata results from secretion of soluble resistance factor(s) by the implant, rather than, or in addition to, formation of a hemocyte chimera having cytotoxic donor hemocytes.

Adoptive Transfer↗

Heterotopic heart transplants in Biomphalaria glabrata (Mollusca: Pulmonata). Fate of congeneric xenografts.

Hearts were implanted heterotopically into the hemocoel of NIH albino Biomphalaria glabrata from three xenogeneic donor snails, including two New World (B. obstructa, B. tenagophila) and one Old World (B. alexandrina) species, as well as from wild type allogeneic donors. Recipients were examined histologically at 1, 3, 7, 10, 15, 30, 60, 120, and 180 days postimplantation (PI). Grafts from all four donor species were temporarily infiltrated by recipient hemocytes at early intervals PI and in most instances also became encapsulated. Furthermore, the grafts subsequently showed histopathological alterations and abnormal heartbeat relative to preimplantation controls. However, hemocytic reactions eventually subsided, the implants remained structurally intact, and implant myocardial cells both maintained high levels of intracellular glycogen and continued to contract rhythmically for 6 months. No major differences occurred in fates among different xenografts, or between xenografts and allografts. Our observations of prolonged xenograft survival differ from those of previous investigators, most of whom have reported rapid destruction of implanted xenogeneic tissues in molluscs.

Animals↗

Amebocytic accumulations in Biomphalaria glabrata: fine structure.

Internal pathologic conditions in four stocks of Biomphalaria glabrata involve amebocytes. Most snails exhibiting these conditions are also nonsusceptible to infection with Schistosoma mansoni. Fine structure studies reveal that periaortic and atrial amebocytic accumulations show two responses that involve heart tissue: a generalized response in which amebocytes have infiltrated the myocardium and focal areas of nodules in which heart tissue is encapsulated and destroyed. In pericardial accumulations amebocytes are subspherical at the periphery with their numerous filopodia intertwining; centrally they elongate and flatten to form a nodule. Hemocoelic accumulations are similar to pericardial ones but more compact. Hyalinocytes and granulocytes were both found in the four types of amebocytic accumulations; each contained phagocytized material. Acid phosphatase reaction product was detected in the Golgi bodies, primary phagosomes, multivesicular bodies and myelin bodies of granulocytes.

Acid Phosphatase↗

Cellular response to Echinostoma caproni infection in Biomphalaria glabrata strains selected for susceptibility/resistance.

The Biomphalaria glabrata/Schistosoma mansoni system represented the only model available so far, for investigating snail resistance to parasites. A new host parasite model has been recently provided by selection of B. glabrata strains that are genetically resistant and susceptible to Echinostoma caproni. As a first approach in investigating resistance mechanisms in this model, we compared the hemocytic response and its effect on E. caproni development in a susceptible and a resistant strains. Histological analysis revealed that resistance does not prevent penetration or migration through the snail tissues. However, all mother sporocysts (MS) settled in the ventricle and the aorta of resistant snails were encapsulated and killed by 4 days post-exposure. MS abnormally settled in the pericardial cavity were not encapsulated and could survive for 5 days. Regardless of their level of encapsulation, all live MS observed during the first days of infection in resistant snails showed abnormal development and degeneration, raising the question of the possible role of humoral factors in the resistance to E. caproni. Finally, we observed an increased number of adherent hemocytes in the resistant as compared with the susceptible snails after parasite exposure. This different effect on circulating hemocyte subpopulations may reflect the failure of E. caproni to interfere with the hemocytic response of resistant snails.

Animals↗

Comparative gene analysis of Biomphalaria glabrata hemocytes pre- and post-exposure to miracidia of Schistosoma mansoni.

The internal defense mechanism of the snail Biomphalaria glabrata during a schistosome infection is activated and mediated via the immune effector cells known as hemocytes. Since resistance and susceptibility to schistosome infection is known to be genetically determined, our interest was to use the EST approach as a gene discovery tool to examine transcription profiles in hemocytes of resistant snails pre- and post-exposure to Schistosoma mansoni. Comparative analysis of the transcripts suggested that parasite exposure caused an active metabolic response in the hemocytes. The most abundant transcripts were those showing 23-74% similarity to known reverse transcriptases (RT). Further characterization by RT-PCR indicated the RT transcripts were expressed in normal snails, parasite exposed snails, and the embryonic cell line Bge. To determine whether the occurrence of RT transcripts correlates to the presence of functional enzyme activity in the snails, RT assays were performed from both resistant and susceptible snails, pre- and post-exposure to miracidia, using protein extracts from the head-foot and posterior region tissues. Results indicated that in the resistant snail, RT activity was greater in the posterior region than in the head-foot. After exposure, however, RT activity increased dramatically in the head-foot, with peak activity at 24 h post-exposure. The detection of RT activity in B. glabrata was unexpected and the role of this enzyme in the hemocyte-mediated killing of parasites is not yet known. However, identification of this and other transcripts from these cells by the EST approach provides a useful resource towards elucidating the molecular basis of resistance/susceptibility in this snail-host parasite relationship.

Amino Acid Sequence↗

Protein kinase C regulation of cell spreading in the molluscan Biomphalaria glabrata embryonic (Bge) cell line.

Cellular adhesion and spreading are critical components involved in the processes of cell and tissue development, and immune responses in molluscs, but at present, little is known regarding the signaling pathways involved in these basic cellular functions. In the present study, the molluscan Biomphalaria glabrata embryonic (Bge) cell line was used as an in vitro model to study the signal transduction pathways regulating molluscan cell adhesion and spreading behavior. Western blot analysis using antibodies specific to mitogen-activated protein kinase (MAPK) revealed the presence of an MAPK-like immunoreactive protein in Bge cells, that was phosphorylated upon exposure to phorbol myristate acetate (PMA). Moreover, Bge cell treatment with inhibitors of protein kinase C (PKC), Ras and MAPK kinase (Mek) suppressed PMA-induced expression of activated MAPK, suggesting that PKC-, Ras- and Mek-like molecules may be acting upstream of MAPK. Similarly, in vitro Bge cell-spreading assays were performed in conjunction with the same panel of inhibitors to determine the potential involvement of PKC, Ras and Mek in cellular adhesion/spreading. Results revealed a similar pattern of inhibition of cell-spreading behavior strongly implying that the Bge cell spreading also may be regulated through a MAPK-associated signal transduction pathway(s) involving proteins similar to PKC, Ras and Mek.

Alkaloids↗

Isolation and characterization of phenoloxidase from egg masses of the gastropod mollusc, Biomphalaria glabrata.

Previous studies have shown that phenoloxidase activity is present in the albumen gland and egg masses of Biomphalaria glabrata, and its potential role in egg formation in this snail has been proposed. In the present study, a phenoloxidase enzyme has been isolated from the supernatant of egg mass homogenates using a combination of hydrophobic interaction chromatography and gel filtration high-performance liquid chromatography (GF-HPLC). The isolated phenoloxidase eluted as a single peak of activity upon GF-HPLC (representing a 132-fold purification) and subsequently was detected as a single band with an estimated molecular mass of 35 kDa by SDS-PAGE analysis. Phenylthiourea-inhibitable mono- and diphenoloxidase activities were demonstrated for the isolated enzyme suggesting that both enzyme activities are associated with a single, tyrosinase-type molecule.

Ammonium Sulfate↗

Structure of two FREP genes that combine IgSF and fibrinogen domains, with comments on diversity of the FREP gene family in the snail Biomphalaria glabrata.

Upon exposure to infection with digenetic trematodes such as Echinostoma paraensei, the freshwater snail Biomphalaria glabrata produces increased quantities of hemolymph lectins, some of which are unique polypeptides containing both immunoglobulin superfamily (IgSF) and fibrinogen domains. These unusual lectins have been termed fibrinogen-related proteins (FREPs), and recognize and precipitate digenean antigens. We here report 11 distinct FREP-encoding sequences from B. glabrata, and provide the complete genomic sequence for two of the most frequently recovered FREPs. The unique juxtaposition of IgSF and fibrinogen domains, previously known only from incomplete cDNAs, is confirmed. Sequences corresponding to known peptides derived from FREPs from hemolymph were found in one of these genes. Both genes contain four exons, the first encodes a putative signal peptide, the second and third a portion of an IgSF-type loop, and the fourth a fibrinogen domain. Cysteines, postulated to form an intrachain loop, are present in the IgSF domain and are separated from one another by 78 or 79 residues. The IgSF sequences most closely resemble V (variable)-type Ig domains, based on canonical and hydrophobic residues and predicted secondary structure. Some minor differences in genomic fragments isolated for each of the two sequences were noted and may represent allelic variants. The results may be of relevance in understanding the role of B. glabrata in transmission of Schistosoma mansoni, a digenean parasite that infects nearly 100 million people in the tropics.

Amino Acid Sequence↗