Snail defence responses to parasite infection: The Lymnaea stagnalis-Trichobilharzia szidati model.

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.

Immune responses in the aquatic gastropod Lymnaea stagnalis under short-term exposure to pharmaceuticals of concern for immune systems: Diclofenac, cyclophosphamide and cyclosporine A.

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.

Natural selection on immune defense: A field experiment.

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.

Influence of Trichobilharzia regenti (Digenea: Schistosomatidae) on the defence activity of Radix lagotis (Lymnaeidae) Haemocytes.

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 influence of Plagiorchis mutationis larval infection on the cellular immune response of the snail host Lymnaea stagnalis.

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.

Activation of the immune defence of the freshwater snail Lymnaea stagnalis by different immune elicitors.

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.

Effects of short-term exposure to environmentally relevant concentrations of different pharmaceutical mixtures on the immune response of the pond snail Lymnaea stagnalis.

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.

Immune defence under extreme ambient temperature.

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.

Maintenance of genetic variation in immune defense of a freshwater snail: role of environmental heterogeneity.

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.

[Radioecological studies of freshwater mollusks in the Chernobyl accident exclusion zone].

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.

Effect of a toxicant on phagocytosis pathways in the freshwater snail Lymnaea stagnalis.

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.

Haemocyte apoptosis as a general cellular immune response of the snail, Lymnaea stagnalis, to a toxicant.

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.

Hemocyte-specific responses to the peroxidizing herbicide fomesafen in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata).

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.

Specific inhibitors of mitogen-activated protein kinase and PI3-K pathways impair immune responses by hemocytes of trematode intermediate host snails.

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.

Integrin engagement modulates the phosphorylation of focal adhesion kinase, phagocytosis, and cell spreading in molluscan defence cells.

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.

Regulation of nitric oxide production in snail (Lymnaea stagnalis) defence cells: a role for PKC and ERK signalling pathways.

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.

Granularin, a novel molluscan opsonin comprising a single vWF type C domain is up-regulated during parasitation.

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.

Effects of environmental concentrations of atrazine on hemocyte density and phagocytic activity in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata).

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.

Bacterial lipopolysaccharide modulates protein kinase C signalling in Lymnaea stagnalis haemocytes.

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.

Monoclonal antibodies against haemocyte molecules of Penaeus monodon shrimp react with haemolymph components of other crustaceans and disparate taxa.

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.