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Lymnaea stagnalis is a common freshwater gastropod. Importantly, the snail serves as the intermediate host for more than one hundred species of digenetic trematodes, including the avian schistosome Trichobilharzia szidati, a causative agent of cercarial dermatitis in humans. Infection of L. stagnalis by T. szidati initiates a dynamic confrontation between the host and the parasite that culminates in immunocompatibility ensuring survival and development of larvae. Unfortunately, the molecular mechanisms determining this immunocompatibility remain poorly characterised. By employing a variety of immune elicitors, including chemical compounds, PAMPs and bacteria, research in the last two decades has elucidated some of the molecular processes that regulate the snail internal defence response such as haemocyte signalling pathways. These discoveries provide a framework for future studies of molecular interactions between T. szidati and L. stagnalis to help elucidate factors and mechanisms enabling transmission of schistosome parasites. Moreover, support from recently available next generation sequence data and CRISPR-enabled functional genomics should further enable L. stagnalis as an important model for comparative immunology and contribute to a more comprehensive understanding of immune functions in gastropod molluscs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This is a pioneering study in the ecotoxicological assessment of immunotoxic effects of the three selected drugs of concern to a freshwater gastropod species. Lymnaea stagnalis was exposed in the laboratory for 3 days to three drugs used for immune systems: diclofenac (nonsteroidal anti-inflammatory drug), cyclophosphamide (anti-cancer immunosuppressive drug) or cyclosporine A (anti-xenograft immunosuppressive drug). Exposure ranges included environmental realistic (1-10μgL -1 ) and therapeutic concentrations (100-1000μgL -1 ). At the end of exposure times, the immune parameters of individual snails were measured: hemocyte density and viability, hemocyte phagocytosis capacity and hemocyte-related oxidative activities (basal and NADPH-oxidase stimulated with zymosan particles). Diclofenac and cyclosporine A induced immune responses, although the effects were not strong. No immunosuppression was observed. Such subtle immunomodulations bring further interrogations regarding their long-term immunotoxicity and possible resulting tradeoffs with life-history traits. On the other hand, the prodrug cyclophosphamide did not induce significant immune responses. Since metabolism pathways differ greatly between vertebrates and invertebrates, this study also suggests that relevant vertebrate metabolites should be included in the immunotoxicity assessment of pharmaceuticals in non-target invertebrate species. Finally, the possible interactive effects of these pharmaceuticals sharing similar modes of action or effects features should also be explored., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Predicting the evolution of phenotypic traits requires an understanding of natural selection on them. Despite its indispensability in the fight against parasites, selection on host immune defense has remained understudied. Theory predicts immune traits to be under stabilizing selection due to associated trade-offs with other fitness-related traits. Empirical studies, however, report mainly positive directional selection. This discrepancy could be caused by low phenotypic variation in the examined individuals and/or variation in host resource level that confounds trade-offs in empirical studies. In a field experiment where we maintained Lymnaea stagnalis snails individually in cages in a lake, we investigated phenotypic selection on two immune defense traits, phenoloxidase (PO)-like activity and antibacterial activity, in hemolymph. We used a diverse laboratory population and manipulated snail resource level by limiting their food supply. For six weeks, we followed immune activity, growth, and two fitness components, survival and fecundity of snails. We found that PO-like activity and growth were under stabilizing selection, while antibacterial activity was under positive directional selection. Selection on immune traits was mainly driven by variation in survival. The form of selection on immune defense apparently depends on the particular trait, possibly due to its importance for countering the present parasite community., (© 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.)

Radix lagotis is an intermediate snail host of the nasal bird schistosome Trichobilharzia regenti. Changes in defence responses in infected snails that might be related to host-parasite compatibility are not known. This study therefore aimed to characterize R. lagotis haemocyte defence mechanisms and determine the extent to which they are modulated by T. regenti. Histological observations of R. lagotis infected with T. regenti revealed that early phases of infection were accompanied by haemocyte accumulation around the developing larvae 2-36 h post exposure (p.e.) to the parasite. At later time points, 44-92 h p.e., no haemocytes were observed around T. regenti. Additionally, microtubular aggregates likely corresponding to phagocytosed ciliary plates of T. regenti miracidia were observed within haemocytes by use of transmission electron microscopy. When the infection was in the patent phase, haemocyte phagocytic activity and hydrogen peroxide production were significantly reduced in infected R. lagotis when compared to uninfected counterparts, whereas haemocyte abundance increased in infected snails. At a molecular level, protein kinase C (PKC) and extracellular-signal regulated kinase (ERK) were found to play an important role in regulating these defence reactions in R. lagotis. Moreover, haemocytes from snails with patent infection displayed lower PKC and ERK activity in cell adhesion assays when compared to those from uninfected snails, which may therefore be related to the reduced defence activities of these cells. These data provide the first integrated insight into the immunobiology of R. lagotis and demonstrate modulation of haemocyte-mediated responses in patent T. regenti infected snails. Given that immunomodulation occurs during patency, interference of snail-host defence by T. regenti might be important for the sustained production and/or release of infective cercariae.

The effects of trematode Plagiorchis mutationis parasitism on the cellular immune responses of the snail host Lymnaea stagnalis were investigated. The number of spreading blood cells (hemocytes) from infected snails was significantly less (69.5%) than in uninfected individuals (79.2%). The phagocytic activity of blood cells in infected snails was also significantly less (17.2%) compared to uninfected snails (27.8%). The hemocytes from the infected snails did not form a complete capsule around Sephadex beads in vitro. The protective reactions of the snail hosts were independent of the parasite load (daily cercariae production). In vitro, dead cercariae of P. mutationis were encapsulated by hemocytes from uninfected snails. The hemocytes of the infected snails formed a complete capsule around only 20% of dead cercariae in vitro, with remaining cercariae either unencapsulated (50% of cercariae) or incompletely encapsulated (30% of cercariae). The total number of hemocytes in the infected snails was twofold less than in uninfected individuals. The results of this study showed that the cellular response of snail host L. stagnalis to P. mutationis trematode infection is similar to the previously studied snail-trematode model systems.

Understanding the outcomes of host-parasite interactions in nature is in high demand as parasites and pathogens are important for several ecological and evolutionary processes. Ecological immunology (ecoimmunology) has a key role in reaching this goal because immune defence is the main physiological barrier against infections. To date, ecoimmunological studies largely lean on measuring constitutive immune defences (components of defence that are always active). However, understanding the role of inducible components of immune function is important as the immune system is largely an inducible defence. Measuring such defences can be complicated as different parasites may activate different immune cascades, and expression of different immune traits may not be independent. We examined the suitability of different immune activation techniques for the freshwater snail Lymnaea stagnalis. By experimentally challenging snails with different immune elicitors [injection with snail saline (i.e. wounding), lyophilized Escherichia coli cells, lyophilized Micrococcus lysodeikticus cells, healthy snail gonad, and trematode-infected snail gonad; maintenance in microorganism-enriched water] and measuring phenoloxidase-like and antibacterial activity of their haemolymph, we found increased immune activity against some immune elicitors, but also decreased activity. Our findings suggest potentially complicated relationships among immune traits, and propose suitable techniques for ecological studies in this study system.

Pharmaceuticals are pollutants of potential concern in the aquatic environment where they are commonly introduced as complex mixtures via municipal effluents. Many reports underline the effects of pharmaceuticals on immune system of non target species. Four drug mixtures were tested, and regrouped pharmaceuticals by main therapeutic use: psychiatric (venlafaxine, carbamazepine, diazepam), antibiotic (ciprofloxacine, erythromycin, novobiocin, oxytetracycline, sulfamethoxazole, trimethoprim), hypolipemic (atorvastatin, gemfibrozil, benzafibrate) and antihypertensive (atenolol, furosemide, hydrochlorothiazide, lisinopril). Their effects were then compared with a treated municipal effluent known for its contamination, and its effects on the immune response of Lymnaea stagnalis. Adult L. stagnalis were exposed for 3 days to an environmentally relevant concentration of the four mixtures individually and as a global mixture. Effects on immunocompetence (hemocyte viability and count, ROS and thiol levels, phagocytosis) and gene expression were related to the immune response and oxidative stress: catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), Selenium-dependent glutathione peroxidase (SeGPx), two isoforms of the nitric oxide synthetase gene (NOS1 and NOS2), molluscan defensive molecule (MDM), Toll-like receptor 4 (TLR4), allograft inflammatory factor-1 (AIF) and heat-shock protein 70 (HSP70). Immunocompetence was differently affected by the therapeutic class mixtures compared to the global mixture, which increased hemocyte count, ROS levels and phagocytosis, and decreased intracellular thiol levels. TLR4 gene expression was the most strongly increased, especially by psychiatric mixture (19-fold), while AIF-1, GR and CAT genes were downregulated. A decision tree analysis revealed that the immunotoxic responses caused by the municipal effluent were comparable to those obtained with the global pharmaceutical mixture, and the latter shared similarity with the antibiotic mixture. This suggests that pharmaceutical mixtures in municipal effluents represent a risk for gastropods at the immunocompetence levels and the antibiotic group could represent a model therapeutic class for municipal effluent toxicity studies in L. stagnalis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Owing to global climate change, the extreme weather conditions are predicted to become more frequent, which is suggested to have an even greater impact on ecological interactions than the gradual increase in average temperatures. Here, we examined whether exposure to high ambient temperature affects immune function of the great pond snail (Lymnaea stagnalis). We quantified the levels of several immune traits from snails maintained in a non-stressful temperature (15°C) and in an extreme temperature (30°C) that occurs in small ponds during hot summers. We found that snails exposed to high temperature had weaker immune defence, which potentially predisposes them to infections. However, while phenoloxidase and antibacterial activity of snail haemolymph were reduced at high temperature, haemocyte concentration was not affected. This suggests that the effect of high temperature on snail susceptibility to infections may vary across different pathogens because different components of invertebrate immune defence have different roles in resistance.

Natural populations often show genetic variation in pathogen resistance, which is paradoxal because natural selection is expected to erode genetic variation in fitness-related traits. Several different factors have been suggested to maintain such variation, but their relative importance is still poorly understood. Here we examined if environmental heterogeneity and genetic trade-offs could contribute to the maintenance of genetic variation in immune function of a freshwater snail Lymnaea stagnalis. We assessed the immunocompetence of snails originating from different families and maintained in different feeding treatments (ad libitum feeding, no food) by measuring the density of circulating hemocytes, phenoloxidase activity, and antibacterial activity of snail hemolymph. Food limitation reduced snail immune function, and we found significant among-family variation in hemocyte concentration and PO activity, but not in antibacterial activity. Interestingly, food availability modified the family-level variation observed in PO activity so that the relative immunocompetence of different snail families changed over environmental conditions (G x E interaction). We found no evidence for genetic trade-offs between snail growth and immune defense nor among immune traits. Thus, our findings support the idea that environmental heterogeneity may promote maintenance of genetic variation in immune defense, but also suggest that different immune traits might not respond similarly to environmental variation.

Species-specificity and dynamics of 90Sr, 137Cs and some transuranic elements accumulation in bivalve and gastropod freshwater molluscs of the Chernobyl exclusion zone during 1997-2008 was analyzed. The results of radiation dose and chromosome aberration rate estimation and the analysis of hemolymph composition of freshwater snail (Lymnaea stagnalis L.) was produced. The absorbed dose rate was registered in the range of 0.3-85.0 microGy/h. In closed water bodies the heightened chromosome aberration rate (up to 27%) in embryo tissues, and also the change of haematological indexes for the adult individuals of snails was registered.

The disturbance of plasma membrane carbohydrates and of lipopolysaccharide (LPS) ligands in relation to cytoskeletal transformations of haemocytes has been investigated after chronic exposure of pond snails (Lymnaea stagnalis) to the peroxidizing toxicant fomesafen. Neither of the two lectins used (concanavalin A and wheat germ agglutinin) showed any binding modification after incubation of the snails in the presence of the toxicant. However, after exposure of the snails to fomesafen, a clear and persistent reduction in LPS labelling of haemocytes occurred. The actin cytoskeleton of the same cells also appeared to be sensitive to the toxicant. The reduction in LPS-binding sites was related to actin staining, leading to the hypothesis that LPS ligands and actin could be similarly modulated by the toxicant. Damaged cells showed non-adherent membrane portions with reduced filopodial extrusions, exhibiting a smooth surface free of microvilli. These changes could lower the spreading and adhesion of the cells and could therefore account for the loss in their phagocytic capabilities.

The effects of a xenobiotic on the circulating haemocytes of Lymnaea stagnalis were investigated after short-term (24 h, 96 h) and long-term (504 h) exposure of snails to environmental concentrations. Fomesafen, a pro-oxidant generator led to the activation of the haemocyte apoptotic program by promoting reactive oxygen species (ROS). Cells entering apoptosis underwent a series of events, both on the plasma membrane and in the mitochondria; these events were quantified by flow cytofluorometry. The data showed a loss of mitochondrial transmembrane potential (Deltapsim), which was dose-dependent and time-dependent and related to an increased release of superoxide anions. The phosphatidylserine that was exposed at the outer plasma membrane was not related to the disruption of either ROS or Deltapsim but was strongly correlated with the haemocyte concentration (total haemocyte count). This cascade of apoptotic processes occurred in a dose-independent manner and was not strengthened over time. The increase of circulating haemocytes depended upon the life span of the cells and might have reflected either facilitated cell turn-over or the accompanying presence of haemocytes phagocytosing apoptotic cells.

Responses of circulating hemocytes were studied in Lymnaea stagnalis exposed to 10, 30, 90, and 270 microg/L fomesafen for 24 and 504 h. Flow cytometry was used to quantify fomesafen-induced production of reactive oxygen species (ROS), phagocytic activity on Escherichia coli, and oxidative burst when hemocytes were challenged by E. coli or phorbol 12-myristate-13-acetate (PMA). Lysosomal membrane damage was assessed, using the neutral-red retention time (NRRT) assay. Exposure to fomesafen for 24 h resulted in increase in ROS levels and decreases in phagocytosis and the oxidative burst in PMA-stimulated hemocytes. After 504 h, intracellular levels of ROS returned to normal, but phagocytosis of E. coli was still inhibited and the associated oxidative burst significantly reduced. After both durations of exposure, decreases of NRRT indicated that lysosome membrane fragility increased with fomesafen concentration. Potential implications for the health and survival of the snails and consequences on populations are discussed.

To characterize molecular mechanisms regulating snail cellular immune responses, the contributions of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3-K) were examined in hemocytes of the trematode intermediate host snails Biomphalaria glabrata and Lymnaea stagnalis. Simultaneous measurement of phagocytosis/encapsulation and H2O2 production by hemocytes in the presence or absence of specific signal transduction inhibitors was used to assess the role of extracellular-signal regulated kinases 1 and 2 (ERK1/2), p38, JNK and PI3-K. Hemocyte spreading was significantly reduced in a dose-dependent manner by the ERK inhibitor, PD098059, and by wortmannin, a potent PI3-K inhibitor. The JNK inhibitor, SP600125, and the p38 kinase inhibitor, SB203580, had no effect on hemocyte spreading. Sheep red blood cell phagocytosis was significantly impaired by PD098059, SP600125, and SB203580. Hydrogen peroxide production during phagocytosis was severely inhibited by PD098059. Additionally, PD098059, but not the other inhibitors, significantly impaired the cellular encapsulation of trematode larvae and H2O2 production during encapsulation. These results suggest that MAPK and PI3-K signal transduction pathways play a pivotal role in the immune responses of snail hemocytes. PI3-K and ERK appear to strongly regulate cell motility. ERK, JNK and p38 contribute to phagocytosis-mediated signal transduction. ERK also play a major role in oxidative burst activation and the encapsulation of trematode larvae by snail hemocytes.

Integrins play a key role in cellular immune responses in a variety of organisms; however, knowledge of integrins and their effects on cell signalling and functional responses in molluscan defence reactions is poor. Using integrin-mediated cell adhesion kits, alphaVbeta3 and beta1 integrin-like subunits were identified on the surface of Lymnaea stagnalis haemocytes. Haemocyte binding via these integrins was found to be dependent on Ca2+/Mg2+. Western blotting with an anti-phospho (anti-active) focal adhesion kinase (FAK) antibody revealed a 120-125 kDa FAK-like protein in these cells; this protein was transiently phosphorylated upon haemocyte adhesion over 90 min, with maximal phosphorylation occurring after 30 min binding. Also, integrin engagement with the tetrapeptide Arg-Gly-Asp-Ser (RGDS) resulted in a rapid increase in phosphorylation of the FAK-like protein; however, RGDS did not affect the phosphorylation of extracellular signal-regulated kinase. Treatment of haemocytes with RGDS (2 mM) inhibited phagocytosis of E. coli bioparticles by 88%. Moreover, at this concentration, RGDS reduced cell spreading by 61%; stress fiber formation was also impaired. Taken together, these results demonstrate a role for integrins in L. stagnalis haemocyte adhesion and defence reactions and, for the first time, link integrin engagement to FAK activation in molluscs.

Background Information: Nitric oxide (NO) is an important molecule in innate immune responses. In molluscs NO is produced by mobile defence cells called haemocytes; however, the molecular mechanisms that regulate NO production in these cells is poorly understood. The present study focused on the role of cell signalling pathways in NO production by primary haemocytes from the snail Lymnaea stagnalis., Results: When haemocytes were challenged with PMA (10 microM) or the beta-1,3-glucan laminarin (10 mg/ml), an 8-fold and 4-fold increase in NO production were observed after 60 min respectively. Moreover, the NOS (NO synthase) inhibitors L-NAME (N(G)-nitro-L-arginine methyl ester) and L-NMMA (N(G)-monomethyl-L-arginine) were found to block laminarin- and PMA-induced NO synthesis. Treatment of haemocytes with PMA or laminarin also increased the phosphorylation (activation) status of PKC (protein kinase C). When haemocytes were preincubated with PKC inhibitors (calphostin C or GF109203X) or inhibitors of the ERK (extracellular-signal-regulated kinase) pathway (PD98059 or U0126) prior to challenge, significant reductions in PKC and ERK phosphorylation and NO production were observed following exposure to laminarin or PMA. The greatest effect on NO production was seen with GF109203X and U0126, with PMA-induced NO production inhibited by 94% and 87% and laminarin-induced NO production by 50% and 91% respectively., Conclusions: These data suggest that ERK and PKC comprise part of the signalling machinery that regulates NOS activation and subsequent production of NO in molluscan haemocytes. To our knowledge, this is the first report that shows a role for these signalling proteins in the generation of NO in invertebrate defence cells.

Snails are intermediate hosts to schistosome parasites, some of which are the main cause of human schistosomiasis (bilharzia), and have been used as models for parasite-host interactions for a long time. Here, we have characterized a novel internal defense peptide of the snail Lymnaea stagnalis, of which the relative abundance in brain tissue increases upon infection with the avian schistosome Trichobilharzia ocellata. This protein, named granularin, is secreted by granular cells, which are numerous in the connective tissue surrounding the brain. The protein is unique because it comprises only a single Von Willebrand factor type C domain that is normally found in large transmembrane and secreted extracellular matrix proteins. The granularin gene is twice up-regulated during parasitation. Purified granularin stimulates phagocytosis of foreign particles by blood hemocytes. Together, our data indicate that granularin represents a novel protein that acts as an opsonin in the molluscan internal defense response.

Immunotoxicological effects of environmentally relevant concentrations (10, 23, 50, 100 microg/l) of atrazine were studied in Lymnaea stagnalis. Individual hemolymph sampling was performed at 0, 24, 48, 72, 96, 168, 336, 504 and 672 h during exposure. Every atrazine concentration induced a significant increase in the mean number of circulating hemocytes, without any concentration-response relation. A peak (1.6-fold increase) of hemocyte density was observed after 96 h of exposure. After 504 h, the number of hemocytes remained higher only in the snails exposed to the two highest concentrations. Granulocytes contributed most to the increase in hemocyte density in herbicide-exposed snails. Both short- (24 and 96 h) and long-term (504 h) exposures resulted in significant inhibition of hemocyte phagocytic activity upon E. coli. Over the long-term, phagocytosis recovered for the two lowest concentrations. After 504 h of exposure, every herbicide level resulted in a significant reduction of reactive oxygen species production in E. coli-stimulated hemocytes, which was not observed for short-term exposures.

Our knowledge of cell signalling pathways in the molluscan immune system and their response to immunological challenge is currently poor. The present study focused on the Protein Kinase C (PKC) pathway in the immune cells (haemocytes) of Lymnaea stagnalis and its response following exposure to bacterial lipopolysaccharide (LPS). Western blotting of haemocyte proteins with either anti-PKC (pan) or anti-phospho-PKC (Ser 660) antibodies revealed the presence of two PKC-like immuno-reactive proteins of approximately 76 and 85 kDa. Challenge of haemocytes with LPS transiently increased the phosphorylation of the 85 kDa isoform, with a 2.2-fold increase in phosphorylation levels at 5 min and a return to basal levels after 20 min. This LPS-mediated response was blocked following treatment of haemocytes with GF109203X. PKC activities measured in anti-phospho-PKC immunocomplexes following haemocyte treatment with LPS and GF109203X correlated well with the observed PKC phosphorylation levels. These data show for the first time that the activity of the PKC pathway in molluscan immune cells is modulated by LPS, as it is in mammals, and suggest that cell signalling in the innate immune response may have been conserved through evolution.

In a previous study, monoclonal antibodies (mAbs) against different haemolymph molecules of the marine shrimp Penaeus monodon were produced and characterised. It was suggested that these mAbs could be used in studying haemocyte differentiation, behaviour and function in P. monodon. In the present study, the reaction of these mAbs on P. monodon was compared with other crustaceans and disparate taxa. The mAbs also reacted with haemolymph components of three freshwater crustaceans, a terrestrial isopod crustacean and with coelomic fluid of an annelid. No reactions were observed with haemolymph of an insect and a mollusc, nor with blood cells of two vertebrates. This comparative study shows reactivity of the mAbs with a wide range of crustaceans and related animals and suggests that well conserved molecules are recognised, which may indicate functional importance. Well-described mAbs can be used in studies of the crustacean defence system and may finally result in a better insight into this system.

The results of the studies on our model combination Trichobilharzia ocellata-Lymnaea stagnalis, presented in this review, lead to the conclusion that schistosomes use multiple strategies to reach their goals, i.e. to propagate and to continue their life cycle. They have to escape from being attacked by the internal defence system (IDS) of the snail host and to profoundly affect the host's energy flow, of which reproduction and growth are the main determinants, for their own benefit. These physiological changes they establish mainly by interfering with the two regulatory systems in the snail host, the IDS and the neuroendocrine system (NES). Moreover, these two regulatory systems clearly interact with each other. Parasitic E/S products affect the host's IDS both in a direct and an indirect way. The neuropeptides or neuropeptide-like substances that are secreted by parasite glands into the host directly suppress haemocyte activity in the snail. The indirect effects include effects of (1) peptides from connective tissue cells and (2) neuropeptides from NES and/or IDS. Parasitic E/S products also induce the effects on energy flow in the host. These E/S products act either directly on a target, as shown for the inhibiting effect of the parasite on the development of the male copulation organ, or on the NES regulating reproductive activity, e.g. on gene expression. Indirect effects of E/S products on the NES (hormone-receptor interaction, electrical activity) are mediated by a factor from connective tissue cells, presumably belonging to the IDS. The physiological changes in the snail host are obviously of vital importance for the parasites, since they make use of different strategies to bring them about.

Organisms are often confronted by both predators and pathogens. Defending against such widely divergent enemies requires more than one type of defence. Multiple defences, however, raise the possibility of trade-offs among defences. We tested for such trade-offs by manipulating the level of predator-avoidance behaviour and immune function in the freshwater snail Lymnaea stagnalis (Gastropoda: Pulmonata). Our results show that predator avoidance and immune function had clear costs in terms of reproduction and survival. Further, we show that increased levels of predator-avoidance behaviour reduced the snails' ability to defend against potential pathogens. Predator-avoidance behaviour may thus have the additional indirect cost of reduced immunocompetence and increased susceptibility to pathogens. Our results suggest that ecological factors (e.g. predator density) may considerably modify the expression and costs of immune defences.

Morphological characteristics and functions of hemocytes were used to compare the immunological effects of biological and chemical stress in the freshwater snail Lymnaea palustris. Animals were either infected by a trematode parasite (Metaleptocephalus sp.), or exposed to environmental contaminants, namely atrazine and hexachlorobenzene (HCB). Three populations of circulating hemocytes, morphologically and cytochemically distinct (round cells, hyalinocytes, granulocytes), were identified in both control and parasitized or pesticide-exposed snails. After 6 h of exposure, HCB and atrazine resulted in 8-fold increases in the mean total number of hemocytes, whereas only a 2.2-fold increase was observed 6 h after cercaria emission in parasitized snails. The impact of HCB was limited to the first 24 h of exposure, whereas long-lasting effects of atrazine were observed. Hyalinocytes and, to a lesser extent, round cells contributed most to the increases in hemocyte density in pesticide-exposed snails. Parasitism and atrazine treatment resulted in significant increases of lectin-stained hemocytes, whereas exposure to HCB did not affect the percentages of stained and unstained cells. Hemocyte phagocytic activity increased in HCB-exposed snails but with no concomitant change of the oxidative burst. Opposite results were obtained in atrazine-treated snail hemocytes, with unchanged phagocytosis and decreased phorbol 12-myristate 13-acetate-stimulated production of reactive oxygen intermediates. No increase in phagocytosis, or in the production of reactive oxygen intermediates, was observed in hemocytes from parasitized snails. Infection with the immunologically compatible trematode parasite Metaleptocephalus sp. and exposure to atrazine generated similar reactions from circulating hemocytes, whereas a different response pattern was observed in HCB-exposed snails.

We have identified a core alpha1-->3-fucosyltransferase activity in the albumin and prostate glands of the snail Lymnaea stagnalis. Incubation of albumin gland extracts with GDP-[(14)C]Fuc and asialo/agalacto-glycopeptides from human fibrinogen resulted in a labeled product in 50% yield. Analysis of the product by 400 MHz (1)H-NMR spectroscopy showed the presence of a Fuc residue alpha1-->3-linked to the Asn-linked GlcNAc. Therefore, the enzyme can be identified as a GDP-Fuc:GlcNAc (Asn-linked) alpha1-->3-fucosyltransferase. The enzyme acts efficiently on asialo/agalacto-glycopeptides from both human fibrinogen and core alpha1-->6-fucosylated human IgG, whereas bisected asialo/agalacto-glycopeptide could not serve as an acceptor. We propose that the enzyme functions in the synthesis of core alpha1-->3-fucosylated complex-type glycans in L. stagnalis. Core alpha1-->3-fucosylation of the asparagine-linked GlcNAc of plant- and insect-derived glycoproteins is often associated with the allergenicity of such glycoproteins. Since allergic reactions have been reported after consumption of snails, the demonstration of core alpha1-->3-fucosylation in L. stagnalis may be clinically relevant.

Factors which may determine trematode-snail interactions were assessed in the present study. Compatibility was examined using a bacterial clearance assay to detect the modulatory effects of both compatible and incompatible trematode infections on the activity of haemocytes from Lymnaea stagnalis, during the early stages of infection. Exposure to and injection with Trichobilharzia ocellata, a compatible trematode, or the incompatible Schistosoma mansoni, resulted in modulation of haemocyte activity. However, T. ocellata activated haemocytes 1.5 h post-infection (p.i.) and then suppressed activity 24-72 h p.i. whereas with S. mansoni no suppression, only activation of haemocytes was observed throughout the test period (1.5-72 h p.i.). In previous studies, modulation of the haemocyte clearance activity by T. ocellata was found to be mediated by 2 E-S fractions, an activating fraction and a suppressing one. Investigations to assess whether the lack of suppression of haemocyte activity, observed in the S. mansoni-L. stagnalis incompatible trematode-snail interaction studied, was due to either the absence or ineffectiveness of the suppressing E-S fraction, were performed on a second incompatible combination, T. ocellata-Planorbis corneus. Using this combination it was revealed that only the activating E-S fraction had modulatory effects on P. corneus haemocytes, indicating that the suppressing E-S fraction, which actively interferes with the clearance activity of haemocytes from L. stagnalis, appears to act in a host-specific manner. In conclusion, the suppressing E-S fraction determines, at least in part, compatibility in the trematode-snail association studied. This is also probably likely in other trematode-snail combinations.

Neural intracerebral allo- and xenografts in pulmonate gastropods demonstrated a variation in the tolerance of neural xenogeneic grafts that was dependent on the phylogenetic distance between the donor and the host. Like allografts, neural congeneric xenografts (Hp/Haa and H1/Haa) of cerebral ganglia (CG) were tolerated and restored growth in juvenile mesocerebrum-deprived (Haa) snails. However, CG neural xenografts between different genera of stylommatophorans (Achatina fulica/Haa) or between genera of different orders (Lymnaea stagnalis: Basommatophora/Haa: Stylommatophora) revealed an interspecific histoincompatibility. These results, compared with those described by other authors, suggest that gastropods possess mechanisms for the recognition of non-self that depend on the organ considered and the phylogenetic distance separating host and donor. Research should now attempt to identify the factors responsible for graft destruction.

To survive the attacks of the internal defence system (IDS) of their host, parasites have developed various strategies to manipulate the IDS. We present evidence that the avian schistosome parasite Trichobilharzia ocellata affects gene expression in the granular cells, a cell type of the IDS of the intermediate host, the mollusc Lymnaea stagnalis. From a differential screening, a clone was isolated encoding a protein named molluscan defence molecule (MDM), which encompasses five C2-like immunoglobulin (Ig) domains. The protein shares a domain organization and high amino acid sequence identity with hemolin, an Ig-family member of the insect IDS. Interestingly, both MDM and hemolin have highest sequence identity with neural cell adhesion molecules, but lack the typical fibronectin repeats and motifs for membrane anchors. We find that the expression of the MDM gene is gradually down-regulated during the course of parasitosis to approximately 21% compared to the non-parasitized level, 8 weeks post-infection. Based on our findings, we suggest that MDM is involved in the proper function of the Lymnaea IDS, and that down-regulation of MDM is part of the parasite-induced disabling on non-self recognition.

Macrophage-like defence cells (haemocytes) of the pond snail Lymnaea stagnalis mediate cytotoxicity through reactive oxygen intermediates (ROIs). This activity is NADPH-oxidase dependent, as in mammalian phagocytes during the respiratory burst. In this study, mother sporocysts of schistosomes, the compatible Trichobilharzia ocellata and the incompatible Schistosoma mansoni evoke in vitro ROI activities (detected by luminol dependent chemiluminescence, LDCL) from L. stagnalis haemocytes. S. mansoni is encapsulated by haemocytes and eliminated, whereas T. ocellata escapes encapsulation and survives. Both schistosomes were equally susceptible to in vitro oxidative damage from exposure to hydrogen peroxide and to ROIs generated by a xanthine/xanthine oxidase system. Protocatechuic acid, a specific antagonist of NADPH-oxidase, delayed the killing of T. ocellata and S. mansoni sporocysts by haemocytes of resistant snails (Biomphalaria glabrata and L. stagnalis, respectively). We conclude that ROIs take part in haemocyte-mediated cytotoxicity. However, neither a snail's capability to generate ROIs, nor a schistosome's susceptibility to ROIs, determine snail/schistosome incompatibility. Snail/schistosome compatibility is rather determined by the parasite's ability to modulate haemocyte behaviour such that effective encapsulation and the generation of lethal concentrations of ROIs are prevented.

The ability of haemocytes, from the haemolymph of the gastropod mollusc Lymnaea stagnalis, to recognize and eliminate the bacterium Aeromonas salmonicida was shown using an in vitro bacterial clearance assay. The assay employs a dye which is reduced by A. salmonicida in direct proportion to the number of viable bacteria resulting in a colour change which can be determined spectrophotometrically. Addition of cytochalasin B resulted in a marked decrease in bacterial clearance, implicating both intracellular and extracellular cytotoxicity of haemocytes. A comparison of haemocytes from uninfected snails and snails infected with the avian schistosome parasite Trichobilharzia ocellata showed that both juveniles and adults of L. stagnalis were susceptible to infection with T. ocellata. After exposure to the trematode for 1.5 h the haemocytes from these infected snails had an enhanced clearance capacity, whilst cells obtained from snails with 24-96 h infections showed decreased clearance of the bacteria, indicating suppression by the parasite. Haemocytes, as well as plasma, which was tested on haemocytes from uninfected snails, were used and hence a distinction was made between cell and humoral-associated effects. The results show that both cellular and humoral components of immunity were activated, then suppressed, following exposure to the parasite. Infection with T. ocellata seems to have a modulating effect on the bactericidal activity of the internal defence system of the snail host, L., stagnalis.

Levels of protein bound hexose, hexosamine and proteins in ethanol precipitate of the body fluid and protein bound hexose and proteins in the perchloric acid filtrate of the body fluid of the snail L. luteola which received a xenoplastic implant in its cephalopedal sinus, are determined. The significant increase in protein bound hexose in the ethanol precipitate of bodyfluid in recipient snail as compared to sham operated and control snails is interesting. The results are discussed in the light of what is known about glycoproteins and their role in invertebrate immunity.

In this in vitro study we investigated whether previously described in vivo plasma-associated effects, that occurred in the period shortly after penetration of Trichobilharzia ocellata into the snail host Lymnaea stagnalis (1.5-72 h post-exposure; p.e.) were direct and/or indirect effects of parasite-derived factor(s). It was investigated whether the effect is mediated by the central nervous system (CNS) of the host. Phagocytic activity of the haemocytes was taken as a parameter for the activity of internal defence of the host. A number of preliminary experiments were performed. When the supernatant of in vitro cultured parasites (33 h; corresponding with their developmental stage in vivo when plasma-associated activation was found) was applied directly to monolayers of haemocytes, it appeared to enhance their phagocytic activity. No direct effect, however, was found with a supernatant of parasites cultured for a longer period of time (72 h; when, in vivo, a plasma-associated suppression was found). In this case, indirect suppression was detected: the parasites appeared to have released a factor that induced the CNS of the host to release material suppressing the activity of the internal defence system of the host. To date the nature of this factor is unknown.

Effects of infection with the avian schistosome Trichobilharzia ocellata on the activity of the internal defence system of the intermediate snail host Lymnaea stagnalis were studied, utilizing an in vitro phagocytosis assay for determining haemocyte activity. A distinction was made between plasma- and cell-associated effects. The period immediately after penetration of the parasite into the snail host (1.5-72 h post-exposure (p.e.)) was extensively studied. In addition, several time-points coinciding with the later-successive-stages of parasite development (2, 4, 6, 8 and 10 weeks p.e.) were investigated. Plasma-associated enhancement of defence activity was found between 1.5 and 6 h p.e., followed by plasma-associated suppression between 12 and 72 h p.e. A cell-associated activation was found between 1.5 and 6 h p.e. and also at 8 and 10 weeks p.e. How these effects on the defence system may be related to phenomena observed in infected snails at these time-points is discussed.

The presence of immunoreactive ACTH molecules on phagocytic cells from the freshwater snails Planorbarius corneus and Lymnaea stagnalis was shown by cytofluorimetric analysis. The role of ACTH in phagocytosis and in the release of biogenic amines, two biological responses that may be taken as ancestral types of immune response and stress, respectively, has also been evaluated. ACTH markedly increased the phagocytosis of Staphylococcus aureus by P. corneus haemocytes and caused the release of biogenic amines from such cells into the serum. These data, as well as tracing the ancestral physiological role of ACTH, favour the hypothesis that immune and neuroendocrine systems share a common evolutionary origin.

We analyzed the effects of infection with Trichobilharzia ocellata on hemocytes of its snail host, Lymnaea stagnalis, and correlated them with successive stages of parasite development. Circulating hemocytes were studied at 0, 2, 4, 6, and 8 weeks post exposure (p.e.) with respect to cell number, distribution of subpopulations (as characterized by morphology, determinants recognized by either of two lectins and a monoclonal antibody) and to proliferative, phagocytic and endogenous peroxidase activity. Infection results in a net elevated level of activity of circulating hemocytes at 2 weeks p.e., when mother sporocysts are present in the head-foot-mantle region, as well as at 4 weeks p.e., when daughter sporocysts are migrating to and growing in the digestive gland region. A lower level of activity was observed at 6 weeks p.e., when cercariae are differentiating within daughter sporocysts. A net activation was again found at 8 weeks p.e., when cercariae are escaping. So, infection with T. ocellata results in a net general activation of the internal defense system of L. stagnalis, during several stages of development of the parasite.

Monoclonal antibodies were raised against the small molecular weight fraction (less than 30 kilodaltons) of an extract from 200 central nervous systems (CNS) of the freshwater snail Lymnaea stagnalis. In a first screening step the supernatants of the 297 emerging hybridomas were immunocytochemically tested on sections of the CNS of L. stagnalis. Sixty-six appeared to produce neuron-specific antibodies, five reacted with non-neuronal elements. In a second step the 66 neuron-specific antibodies were tested on sections of the CNS of the guppy. Three reacted positively. In the third step the three antibodies were tested on the CNS of the rat. One antibody (Mab4H5) appeared to give positive results. In the snail brain Mab4H5 stains two identified giant neurons, one in the visceral ganglion (VD1), and one in the right parietal ganglion (RPD2)--these neurons form part of the network controlling the respiratory system--and a small number of cells in the cerebral ganglia (in the anterior and ventral lobes). Ultrastructural observations using immunogold labelling in VD1 showed the antigen to be localized to the secretory vesicles. In the guppy Mab4H5 stains fibres in the tectum and cell bodies in the reticular formation. In rat CNS staining was observed in Purkinje neurons of the cerebellum, in cortical pyramidal neurons and in neurons and fibres in other brain areas. Subsequent Mab4H5 staining of the CNS of the lizard, the cockroach and parts of the human CNS showed that these tissues also contain Mab4H5-positive neurons. In the human cortex and cerebellum the staining pattern appeared to be similar to that of the rat. On the basis of the results it is hypothesized that the antibody reacts with phylogenetically ancient amino acid sequences.

Fasciola hepatica miracidia labelled in vivo with radioselenium have been used in studies on the capacity of the miracidia to attach to and penetrate the tegument of infected compared with non-infected host snails (Lymnaea truncatula). The results show that the penetration of the larvae into the snail tissue is not influenced by an already existing infection, regardless of its developmental stage.

Evidence obtained earlier with immunofluorescence and immunoelectron microscopy was interpreted to imply molecular mimicry of the host Biomphalaria glabrata by the parasite Schistosoma mansoni. Using Western Blotting, we find that "mimicry" is due to widespread shared epitopes. Furthermore, at least one individual plasma protein of B. glabrata shares epitopes with the majority of plasma proteins in the mollusc. The widespread antigenic determinants may be carbohydrates. Caution is warranted when antisera, raised in mammals against heterogeneous invertebrate extracts, are used to screen for related antigenic determinants.

In order to investigate whether the schistosome Trichobilharzia ocellata interferes with defence activities in its snail intermediate host Lymnaea stagnalis, aspects of the immune system of infected snails and of non-infected controls were compared. The elimination of injected live Staphylococcus saprophyticus bacteria starts at a lower rate in infected snails 1 and 5 weeks after exposure to the parasite, but then proceeds faster than in control snails. During the first 3 weeks of infection, when only mother sporocysts are present, the haemocytes of the infected snails have an increased capacity to phagocytose rabbit red blood cells in vitro. From 5 weeks onwards, when mother and daughter sporocysts are present but cercariae are not yet mature, the phagocytic activity decreases to below control level. The number of circulating haemocytes is also higher in infected snails than in controls at this time. Moreover, the cells are larger, have more inclusions and an increased surface area with many long, branched, spiked pseudopods. The development of the parasite is retarded in a subpopulation of snails in which the haemolymph plasma agglutinates erythrocytes with high titres, compared to a subpopulation with low haemagglutinating activity. The haemagglutinating activity in infected snails of the first decreases significantly from 6 weeks onwards.

By means of in vitro phagocytosis it was demonstrated that macrophage-like blood cells of an invertebrate (amoebocytes of the pond snail Lymnaea stagnalis) possess cytophilic receptors for foreignness. These receptors are lectin molecules which are reversibly bound to the surface of the cells. Phagocytosis is dependent, beyond recognition of foreignness, on temperature and pH, and on the feeding condition of the snails.

The immunological immaturity of juvenile specimens of some snail species, e.g. Lymnaea stagnalis, may contribute to their greater susceptibility to infection by schistosome parasites. In a comparison between juvenile and adult specimens of the pond snail L. stagnalis, we have shown that the blood cells (amoebocytes) of juvenile snails are less efficient at phagocytosing: fewer amoebocytes are competent and the average number of particles engulfed per cell is lower. This functional immaturity seems to correlate with morphological immaturity of the amoebocytes. Opsonic and haemagglutinating activities are low in juvenile snail plasma, but much higher in adult plasma. Finally, however, the initial rate at which injected bacteria are eliminated from the circulation seems only slightly slower in juvenile snails than it is in adults.

Monolayers of blood cells (amoebocytes) and sections of connective tissue and of amoebocyte pellets of the freshwater pulmonate gastropod Lymnaea stagnalis were stained immunocytochemically with antisera to snail agglutinin/opsonin. The presence of this substance was demonstrated light microscopically both in the cytoplasm and on the cell membrane of amoebocytes. This suggests that amoebocytes synthesize agglutinin/opsonin, and bear it at their surface as receptors for foreign materials.

In Lymnaea stagnalis injections with dead Escherichia coli or Staphylococcus saprophyticus bacteria resulted in an enhanced clearance of both live S. saprophyticus and E. coli injected 4 days later. A non-specific activation of the internal defence system was concluded from these findings. The activation was dose-dependent: pre-injections with high doses resulted in a higher increase in the clearance capacity of the snails than pre-injections with low doses of bacteria. The state of increased activity of the defence system, induced with injection of dead E. coli, lasted at least 64 days. The heightened responses of the defence system were probably due to an activation of the blood cells (amoebocytes) since: 1) amoebocyte numbers increased faster in bacteria pretreated snails than in control animals; 2) ultrastructural observations revealed that the amoebocytes of bacteria pre-treated animals had a more ruffled outline than those of control snails; 3) amoebocytes from sensitized snails showed a higher phagocytic activity in vitro; 4) mitotic activity of amoebocytes increased after snails had been injected with bacteria.

Mother and daughter sporocysts of Tricholbilharzia ocellata, developing in the snail host Lymnaea stagnalis, were searched for substances with antigenic similarities to the snail's haemolymph. Antisera to cell-free snail haemolymph and fractions thereof were used in three different immunocytochemical staining methods, applied on sections of parasitized snails. Snail tissue was consistently stained; cercariae were stained, indicating that the applied methods were successful. Most sections through mother and daughter sporocysts were completely unstained. It is concluded that neither mother nor daughter sporocysts are masked by the antigens studied or substances mimicking these. The relevance of the present observations is discussed.

In the central nervous system of the pond snail Lymnaea stagnalis a large number of elements (cells and fibers) can be identified with antisera (a-FM) to the molluscan cardioactive tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2). Of these elements some are also reactive to antivasotocin (a-VT) and/or anti-gastrin (a-Gas). These observations suggest that the a-FM positive elements belong to more than one type. Previous results had already indicated that the immunoreactivity of many a-FM positive cells is influenced by the type of fixation. Taking into account the effects of three fixatives on the reactivity of the cells, and their staining characteristics with the two other antisera used, 8 a-FM positive types could be distinguished. Homologous and heterologous adsorptions were carried out to test the specificity of a-FM, a-VT and a-Gas. After homologous adsorptions no staining was obtained. After heterologous adsorptions only part of the multiple staining cells were identified. This indicates that in a-FM, a-VT and a-Gas in addition to (more) selective IgG molecules, less specific IgG molecules occur that can bind to other peptides than those used to raise the antisera (cross-reaction). The (more) selective IgG molecules in a-FM bind to 6 of the a-FM positive types, suggesting that in L. stagnalis a family of FMRFamide-like substances occurs. This conclusion is sustained by results obtained with a-FM adsorbed with fragments of FMRFamide.(ABSTRACT TRUNCATED AT 250 WORDS)

The mouse monoclonal antibody LS1 recognizes a membrane epitope present on circulating hemocytes of the gastropod mollusc Lymnaea stagnalis. In both juvenile and adult pond snails, LS1+ (LS1 positive) hemocytes have the morphology of immature cells. The percentage of LS1+ hemocytes is higher in juveniles (ca. 39%) than it is in adults (ca. 14%). Functional characteristics of LS1+ hemocytes and lectin binding to these cells were studied. In both age groups, the proliferative activity, as measured by the incorporation of deoxybromouridine, is much higher for LS1+ hemocytes than it is for LS1- (LS1 negative) cells. LS1+ hemocytes are phagocytically less active and have a lower lysosomal enzyme (peroxidase) content as compared to hemocytes that lack the epitope. Histochemical staining of the total population of circulating hemocytes shows that the lectins DBA, BS-l-A4 and BS-l-B4, PNA, SBA and ECA do not react with the hemocytes. LTA, APA, WGA, Con A and LCA bind to all hemocytes. RCA and STA recognize surface carbohydrate moieties present on subpopulations of hemocytes only. The LS1+ hemocyte population virtually lacks the carbohydrate residues recognized by STA, whereas the LS1- population never shows binding of RCA. Our results support the findings that the LS1 epitope is a membrane marker of less differentiated hemocytes in both juvenile and adult L. stagnalis. Furthermore, they suggest a correlation between the presence of the LS1 epitope and the absence of STA binding, whereas absence of the LS1 marker may correlate with the presence of a sugar recognized by RCA.