Biomphalaria glabrata amoebocytes: Effect of Schistosoma mansoni infection on in vitro phagocytosis

The rate of phagocytosis by amoebocytes obtained from hemolymph of the pulmonate Biomphalaria glabrata infected with the trematode Schistosoma mansoni for 24 hr and 2, 4, and 6 weeks has been determined using the monolayer assay system. Amoebocyte preparations from snails infected for 4 and 6 weeks showed a gradual decrease in the phagocytic rates compared to those from uninfected controls. Snails harboring the parasite for 4 and 6 weeks also showed a significant increase in the number of amoebocytes in the hemolymph. No significant changes were detected in the rate of phagocytosis or number of amoebocytes in snails infected for 2 weeks or less. Alterations in the morphology and behavior of amoebocytes from infected snails were also noted.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.     

Resistance to schistosome infection in Biomphalaria glabrata induced by gamma radiation

Two strains of Biomphalaria glabrata were studied with respect to the effects of ionizing radiation on their susceptibility to Schistosoma mansoni infection. Gamma radiation at levels of 3.5 and 5 krad did not induce susceptibility in the resistant S-3 strain, but was found to initiate resistance in the susceptible PR-1 strain. In an attempt to understand the induced resistance in irradiated snails, histopathologic examinations and analyses of snail hemolymph were performed. Results indicated that miracidia invading irradiated snails were quickly surrounded and encapsulated by amoebocytes. Similarly, alterations in the hemolymph of irradiated snails suggested that radiation induced aging. It is suggested that radiation-altered snails may be of value in studying the defense mechanisms of these organisms.     

Schistosoma mansoni: immunofluorescent detection of its antigen reacting with Biomphalaria glabrata amoebocytes

The reaction of amoebocytes in the hemolymph of the infected intermediate host, Biomphalaria glabrata, to Schistosoma mansoni antigens has been investigated using the indirect immunofluorescence antibody test. Monolayers of amoebocytes, prepared from hemolymph of infected and normal snails, were first fixed and then reacted with antisera obtained from mice infected for 7 to 9 weeks. Nonspecific and cross-reactions between the antisera and monolayers of amoebocytes were eliminated by absorbing the antisera with tissues from uninfected snails. The liberation of detectable schistosomal antigens in the hemolymph in soluble and particulate forms coincided with completion of the infection cycle 3 to 4 weeks after exposure to miracidia. The schistosomal antigens were demonstrable in the cytoplasm of amoebocytes and in the center of amoebocyte aggregates.     

Pathology and host responses induced by Schistosomatium douthitti in the freshwater snail Lymnaea catascopium.

Most Schitosomatium douthitti miracidia penetrated the esophageal wall of Lymnaea catascopium without provoking amoebocyte encapsulation responses or extensive pathological changes. Amoebocytes frequently attached to developing mother and daughter sporocysts, but did not encapsulate or destroy them. Pressure resulting from extensive growth of mother sporocysts ruptured the transverse membrane of some snails. After releasing daughter sporocysts, mother sporocysts in some snails were destroyed by amoebocytes. Many migrating cercariae became trapped in the tissues of L. catascopium, particularly in the posterior portion of the foot, and were encapsulated and destroyed. Large increases in numbers of amoebocytes in the anterior portion of the lung roof of infected snails were noted, even before cercarial production had been initiated. Atrophy of the digestive gland occurred in infected snails.     

Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

Differences between blood cells of juvenile and adult specimens of the pond snail Lymnaea stagnalis

Summary Blood cells (amoebocytes) of juvenile and adult specimens of the pond snail Lymnaea stagnalis were compared. Juvenile snails contain fewer circulating amoebocytes per l haemolymph. However, a higher percentage of these cells shows mitotic activity, as determined by incorporation of ³H-thymidine in vitro. Relatively more amoebocytes of juvenile snails have the characteristics of less differentiated cells: they are small and round with few inclusions, a high nucleus-to-cytoplasm ratio, and a high pyronin stainability. Enzyme cytochemical studies showed that acid phosphatase (AcP), non-specific esterase (NSE), and alkaline phosphatase (AlP) are present in all amoebocytes of juvenile and adult snails. AcP activity is relatively weak. NSE activity is dispersed throughout the cytoplasm and occasionally found in granules, whereas AlP is clearly localized in granules. Differences between the two age groups were found only for the enzyme peroxidase (PO). In juvenile snails a lower percentage of the cells is positive and the granules that contain the activity are less abundant than in amoebocytes of adults. It is suggested that, due to the above-mentioned characteristics of the amoebocytes, the activity of the internal defence system in juvenile L. stagnalis is on a lower level than that in adult snails. This might be an explanation for the fact that juvenile L. stagnalis are highly susceptible to infection by the schistosome Trichobilharzia ocellata, whereas adult snails are less susceptible.     

Recognition of foreignness by blood cells of the freshwater snail Lymnaea stagnalis, with special reference to the role and structure of the cell coat

The structure and function of the cell coat of the blood cells (amoebocytes) of the freshwater snail Lymnaea stagnalis were studied with ultrahistochemical tests, including concanavalin A (Con A) labeling, and with in vitro phagocytosis experiments. The cell coat is intensely stained by ruthenium red and tannic acid. The cells possess binding sites for Con A. Proteolytic enzymes destroy the receptors for Con A and totally inhibit the phagocytic activity of amoebocytes. Incubation experiments with proteases, carbohydrases, and inhibition sugars revealed that (1) the Con A binding sites are anchored in the plasma membrane by proteins, and (2) glucose, fructose, mannose, and to a lesser extent N-acetylglucosamine and N-acetylgalactosamine, inhibit the binding of Con A to amoebocytes, suggesting that these carbohydrates might form part of these binding sites.     

Total protein and carbohydrate content and protease and disaccharidase activities in the hemolymph of Lymnaea stagnalis naturally infected with digenean larvae

Lymnaea stagnalis is an intermediate host of many Digenea. The infestation affects host metabolism. The aim of the work was to investigate hemolymph biochemical indicators of L. stagnalis infected with four species of trematodes: Diplostomum pseudospathaceum, Paryphostomum radiatum, Plagiorchis elegans or Opisthioglyphe ranae. The protein profiles and proteinase activity in the hemolymph of sexually mature individuals of Lymnaea stagnalis maintained at 19°C were tested. As the carbohydrates are main substrates for energetic metabolism of the great pond snail their content and disaccharidase activity were also studied. Hemolymph samples were collected during weeks 3 and 4 of rearing. No significant differences in the total protein content between uninfected individuals and snails infected with the first three trematode species were detected. In the snails infected with O. ranae the quantity of total proteins was near twice higher than in those uninfected. A higher share of 70 kDa proteins in infected than in uninfected snails as well as reduction of the low molecular weight fractions of proteins for snails infected with D. pseudospathaceum and P. radiatum were detected. During week 3, carbohydrate content in the infected snails did not differ from that in the controls while during week 4 it was significantly lower in snails infected with P. elegans or O. ranae. The content of the major soluble carbohydrate in the hemolymph - saccharose — changed in a similar way. No activity of trypsin or pepsin in the hemolymph sample was detected while the activity of chymotrypsin was lower in infected snails vs. controls. On the other hand, saccharase and maltase activities were higher in infected than in uninfected snails. The biochemical hemolymph indicators in naturally infected host-snails show some differences depending on the parasite species but they are not sufficiently species-specific to offer the basis for establishing the model unique for a particular parasitosis. Our results from the field did not always coincide with those from the laboratory.     

Opsonic involvement in phagocytosis by mollusk hemocytes.

Macrophages from the gastrophod mollusk Otala lactea are capable of in vitro recognition and phagocytosis of foreign particles such as yeast, mammalian erythrocytes, and bacteria. The degree of intensity of the phagocytic response, in certain instances, is governed by the surface characteristics of the particle in question as well as by the presence of opsonic factors.Hemagglutinins have been implicated as opsonins in certain invertebrates, including mollusks. Otala lacks serum lectins; however, its hemolymph stimulates phagocytosis of formalized yeast but not erythrocytes and bacteria. Hemagglutinin-containing extracts of Otala albumin gland were shown to opsonize formalized red cells. The rate of ingestion of the bacteria used in this study by Otala hemocytes was variable and was not influenced by the presence of hemolymph in the medium.     

Inducement of miracidia-immobilizing substance in the hemolymph of Biomphalaria glabrata

Lie K. J., Jeong K. H. and Heyneman D. 1980. Inducement of miracidia-immobilizing substance in the hemolymph of Biomphalaria glabrata. Intemational Journal for Parasitology10: 183–188. More than 85% of echinostome-infected albino B. glabrata laboratory strain snails develop miracidia-immobilizing substance(s) (MIS) in the hemolymph, while less than 5% of control uninfected snails show this ability. Snails infected with Echinostoma lindoense show a strong miracidial immobilizing test (MIT) when homologous miracidia are exposed to the hemolymph and a moderate response when E. liei and Paryphostomum segregatum miracidia are used. Infection with E. paraensei results in a high level of hemolymph MIS with E. lindoense miracidia, a moderate one with P. segregatum miracidia, and a weak one when hemolymph is tested against E. liei as well as the homologous E. paraensei miracidia. Infection with E. liei induces a strong MIT with E. lindoense miracidia whereas only a moderate one was observed when using homologous or P. segregatum miracidia. Infection with P. segregatum gives a moderate MIT reaction to miracidia of the homologous species, as well as to E. lindoense and E. liei, and only a weak response to E. paraensei miracidia. Infection with S. mansoni fails to induce hemolymph that shows MIS to any of the parasites tested. Production of hemolymph MIS is temporary. It begins one day postexposure, reaches its maximum 10–14 days postexposure, and declines to the preinfection level several weeks later. Infection of snails with irradiated parasites also results in a temporary production of hemolymph MIS.Uninfected snails show a tissue-extract MIS, which is especially strong when digestive gland extracts are used. However, these snails give little or no evidence of a hemolymph MIS.     

Induction of locomotor behavior in the giant African snail,Achatina fulica

• 1.1. The locomotor-inducting factor of the giant African snail, Achatina fulica, was examined.
• 2.2. Snails showed nocturnal circadian behavior in relative humidity at least over 50%. Although the rhythmicity was independent of light and darkness, it was disturbed easily by hydration, and hydrated snails continued to locomote throughout the day. For induction of locomotor behavior, relative humidity over 50% was the fundamental factor and water is shown to be the limiting factor for the endogeneous circadian oscillator.
• 3.3. The integument of snails showed a higher water permeability. Through the integument, hemolymph osmolality changed easily according to hydration and dehydration from about 120 to 400 mOsm/kg H₂O. Circadian behavior was induced in snails in which hemolymph osmolality ranged from about 130 to 230 mOsm/kg H₂O.
• 4.4. By hydration, hemolymph osmolality in quiescent and estivated snails which have higher osmolality decreased gradually and then they began to locomote according to the degree of dilution, and vice versa. The induction of behavior in these snails was controlled by low hemolymph osmolality.
• 5.5. Together with the endogeneous rhythmicity, water environment was shown to be the key factor for the induction of locomotor behavior.
• 6.6. Based on these results, the mechanisms of the induction of locomotor behavior in terrestrial pulmonates are proposed.

     

Schistosoma mansoni: Cytotoxicity of hemocytes from susceptible snail hosts for sporocysts in plasma from resistant Biomphalaria glabrata

Sporocysts of Schistosoma mansoni (PR1 strain) survive and grow in Biomphalaria glabrata PR albino strain snails, whereas they are encapsulated and die in B. glabrata 10R2 strain snails. These processes also occur in an in vitro system in which the only living cells are those of sporocysts and snail hemolymph. Hemocytes of the susceptible snail are normally not effective in damaging sporocysts. However, when the encounter occurred in the presence of cell-free plasma from resistant snails, previously impotent hemocytes severely damaged sporocysts in 24 hr. The cytotoxic capacity of resistant strain hemocytes was not altered by plasma from susceptible snails. Furthermore, it was retained even when plasma was replaced by culture medium free of snail components. The nature of the plasma factor(s) which facilitated damage by otherwise impotent hemocytes is discussed, and evidence is evaluated for the hypothesis that snail resistance is dependent upon the specificity of cytophilic factors present both in the plasma and on the hemocyte plasma membranes.     

Release of lysozyme from hemolymph cells of Mercenaria mercenaria during phagocytosis

Activity of the lysosomal enzyme, lysozyme, has been quantitatively determined in the serum and cells of the hemolymph of Mercenaria mercenaria which had been exposed to known quantities of Bacillus megaterium and also in the serum and cells of hemolymph which had not been exposed to bacteria. The results indicate that the level of enzyme activity is greater in serum of hemolymph that had been exposed to B. megaterium and concurrently, there is an equivalent decrease in the level of activity in the cells. This evidence indicates that the amount of lysozyme released from cells into serum is enhanced during phagocytosis of the bacteria.It has also been demonstrated that the release of lysozyme from cells occurs during the process of phagocytosis and is not a delayed phenomenon.Enzyme release by secondary phagosomes is reflected morphologically by what is commonly referred to as degranulation. This process does not involve the rupture of the plasma membrane of the hemolymph cells since biochemical studies have revealed that there is no release of the cytoplasmic enzyme, lactate dehydrogenase.     

A comparative morphological and enzyme histochemical study on blood cells of the freshwater snails Lymnaea stagnalis,Biomphalaria glabrata,and Bulinus truncatus

The morphology and ultrastructure of the blood cells of the freshwater snails Lymnaea stagnalis, Biomphalaria glabrata, and Bulinus truncatus were studied. By performing in vitro experiments and enzyme histochemical studies, special attention was paid to the role of the blood cells in phagocytosis of foreign particles. No fundamental differences were found in the ultrastructure, lysosomal enzyme contents, and phagocytic capacities of the blood cells of these species. It is concluded that only one type of blood cell, the amoebocyte, exists in the freshwater snails. Amoebocytes constitute a morphologically and functionally heterogeneous population of cells, ranging from round (electron-dense) cells with the morphological characteristics of young cells to highly phagocytic spreading cells with a prominent lysosomal system. In addition to acid phosphatase, nonspecific esterase and peroxidase were found within the lysosomes. The presence of enzyme activity in the RER and the Golgi bodies indicates that amoebocytes are able to synthesize lysosomal enzymes continuously.     

Carboxylic Acids as Biomarkers of Biomphalaria alexandrina Snails Infected with Schistosoma mansoni

Biomphalaria alexandrina snails play an indispensable role in transmission of schistosomiasis. Infection rates in field populations of snails are routinely determined by cercarial shedding neglecting prepatent snail infections, because of lack of a suitable method for diagnosis. The present study aimed at separation and quantification of oxalic, malic, acetic, pyruvic, and fumaric acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to test the potentiality of these acids to be used as diagnostic and therapeutic biomarkers. The assay was done in both hemolymph and digestive gland-gonad complex (DGG) samples in a total of 300 B. alexandrina snails. All of the studied acids in both the hemolymph and tissue samples except for the fumaric acid in hemolymph appeared to be good diagnostic biomarkers as they provide not only a good discrimination between the infected snails from the control but also between the studied stages of infection from each other. The most sensitive discriminating acid was malic acid in hemolymph samples as it showed the highest F-ratio. Using the Z-score, malic acid was found to be a good potential therapeutic biomarker in the prepatency stage, oxalic acid and acetic acid in the stage of patency, and malic acid and acetic acid at 2 weeks after patency. Quantification of carboxylic acids, using HPLC strategy, was fast, easy, and accurate in prediction of infected and uninfected snails and possibly to detect the stage of infection. It seems also useful for detection of the most suitable acids to be used as drug targets.     

Schistosoma mansoni: Lysozyme activity in Biomphalaria glabrata during infection with two strains

Levels of lysozyme activity were determined in the hemolymph, digestive gland, and headfoot extracts of M-line stock of snails, Biomphalaria glabrata, during infection with the PR-1 and Lc-1 strains of the trematode, Schistosoma mansoni. At 3 hr postexposure there was a 10-fold increase in the levels of enzyme activity in the hemolymph of snails infected with the Lc-1 strain to which the snail is resistant. This increase was considerably higher when compared to the threefold increase in the PR-1-infected snails. The infection also induced a gradual depletion of lysozyme activity in the headfoot muscles of the two groups of infected snails. There were no changes in the levels of enzyme activity in the digestive gland extracts of the control and the two groups of infected snails. Similar changes in the levels of enzyme activity in the hemolymph and headfoot extracts of infected snails suggest a nonspecific response to a parasite infection and do not indicate that lysozyme is primarily responsible for the destruction of schistosome parasite in a resistant snail host.     

Aminopeptidase and lysozyme activity levels and serum protein concentrations in Biomphalaria glabrata (Mollusca) challenged with bacteria.

The total serum protein concentrations and levels of aminopeptidase and lysozyme activities in the sera of the gastropod Biomphalaria glabrata have been determined. The groups of snails from which hemolymph samples were taken for study included (1) untampered controls, (2) sham-injected snails, (3) heat-killed Bacillus megaterium-injected, and (4) live B. megaterium-injected ones. Our results indicate that there are significant elevations in the levels of aminopeptidase activity in 2 hr in the sera of snails that had been sham-, dead bacteria-, and liver bacteria-injected. The levels of lysozyme activity were not altered in sham-, dead bacteria-, and live bacteria-injected snails. This is contrary to an earlier finding (T. C. Cheng, M. J. Chorney, and T. P. Yoshino, 1977. J. Invertebr. Pathol., 29, 170–174), and the difference is believed to be due to the age of the snails employed. Comparisons of total serum proteins have revealed that the concentration in snails injected with live B. megaterium is significantly higher than in sham-injected ones. This may be due to increase of some yet undetermined serum protein fraction.     

Schistosoma mansoni modulation of phagocytosis in Biomphalaria glabrata

Both short-term (3 hr) exposure of Biomphalaria glabrata snails (M-line and 13-16-R1) to Schistosoma mansoni (PR1) miracidia and in vitro incubation of parasite sporocysts with host hemolymph components altered host phagocytic ability. Hemocytes obtained from susceptible (M-line) snails that had been exposed to parasite miracidia for 3 hr showed reduced levels of phagocytosis of yeast cells in vitro compared to hemocytes from unexposed individuals. Incubation of whole hemolymph with sporocysts in vitro also reduced yeast phagocytosis in this susceptible strain. In contrast, resistant (13-16-R1) hemocytes showed increased levels of yeast phagocytosis after in vitro incubation with the parasite, and the opsonic properties of 13-16-R1 plasma were greater after exposure of snails to miracidia. These strain-specific effects of S. mansoni on host hemocyte phagocytosis and plasma opsonization were seen only when both plasma and hemocytes were present at the time of exposure to the parasite.     

Comparative mortality studies on Biomphalaria glabrata (mollusca: Pulmonata) exposed to copper internally and externally

Various concentrations of copper in the form of CuSO₄ were injected into the hemocoel of Biomphalaria glabrata, and mortality of this snail was subsequently monitored. The concentrations of copper in the hemolymph of injected snails were calculated, and specimens were incubated in these concentrations. Greater mortality was observed when snails were incubated in concentrations of copper than when they were injected with a sufficient amount of copper to attain these same concentrations in the hemolymph. Injection of copper into the hemocoel of B. glabrata resulted in the formation of a noncellular hemolymph precipitate, most likely denatured proteins, at the injection site, which was most noticeable with higher concentrations of copper. It has been concluded that external concentrations of copper are more cidal to B. glabrata than are internal, i.e., injected, concentrations. These data support the hypothesis that the cidal action of copper on B. glabrata is due to an attack on the mollusc's surface epithelia.