W.P.W. van der Knaap

The role of serum factors in phagocytosis of foreign particles by blood cells of the freshwater snail Lymnaea stagnalis

Abstract Monolayers of blood cells (amoebocytes) of the freshwater snail Lymnaea stagnalis were incubated in vitro in snail Ringer as well as in snail serum. Sheep erythrocytes and yeast cells were added and allowed to react with the amoebocytes. The results showed that one or more serum factors promote recognition and subsequent phagocytosis of foreign particles by amoebocytes. Moreover, amoebocytes may possess plasmamembrane receptors for foreign particles.

Invertebrate blood cells: Morphological and functional aspects of the haemocytes in the pond snail Lymnaea stagnalis

The internal defence system (immune system) of the pond snail Lymnaea stagnalis is reviewed. Humoral defence activities are agglutination, opsonisation and inhibition of bacterial growth. Cellular defence is exerted by antigen trapping endothelial cells, foreign protein engulfing pore cells, phagocytic reticulum cells and mobile haemocytes. The haemocytes contribute most to defence and are therefore treated in more detail. There is one type of haemocyte; morphological heterogeneity of the haemocyte population is due to varying states of differentiation of the cells. Haemocytopoiesis is through mitosis of haemocytes, both in the pool of tissue-dwelling cells and in circulation. Haemocytes resemble monocytes/macrophages, they are typical phagocytes equipped with recognition factors (lectins), lysosomal enzymes and a cytotoxicity mechanism using reative oxygen intermediates. A comparison is made of the internal defence systems of the three main metazoan taxa, insects, molluscs and vertebrates.

Immunocytochemical demonstration of a humoral defence factor in blood cells (amoebocytes) of the pond snail, Lymnaea stagnalis

SummaryMonolayers 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.

Schistosomicidal activities of Lymnaea stagnalis haemocytes: the role of oxygen radicals

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 snails capability to generate ROIs, nor a schistosomes susceptibility to ROIs, determine snail/schistosome incompatibility. Snail/schistosome compatibility is rather determined by the parasites ability to modulate haemocyte behaviour such that effective encapsulation and the generation of lethal concentrations of ROIs are prevented.

Elimination of bacteria from the circulation of the pond snail Lymnaea stagnalis

Abstract Doses of 5 × 10 7 living bacteria ( Staphylococcus saprophyticus and Escherichia coli ), injected into the freshwater pulmonate, Lymnaea stagnalis , are rapidly removed from the circulation. Two hrs after injection, 99% of the bacteria have been cleared. In the initial stage of the clearance process, numbers of circulating amoebocytes decreased rapidly. Light microscopy on whole snails and electron microscopy on blood cell pellets and connective tissue showed that in addition to circulating amoebocytes, connective tissue amoebocytes and fixed phagocytes play a role in endocytosis and subsequent digestion of bacteria. The clearance of bacteria seems to be due to phagocytic activity alone, as substances with bactericidal properties were not found in the haemolymph. The role of opsonic factors in in vivo phagocytosis is discussed.

NADPH-oxidase activity: the probable source of reactive oxygen intermediate generation in hemocytes of the gastropod Lymnaea stagnalis.

Macrophage‐like defense cells (hemocytes) of the pond snail Lymnaea stagnalis generate reactive oxygen intermediates (ROIs) upon contact with non‐self, following kinetics similar to those of ROI production by mammalian leukocytes during respiratory burst. In this study, several inhibitors of NADPH‐oxidase, the key enzyme of the respiratory burst in mammalian phagocytes, were tested for their effect on oxidative activities [as demonstrated by nitroblue tetrazolium (NBT) reduction and luminol‐dependent chemiluminescence (LDCL)] of phagocytosing snail hemocytes. In the presence of di‐ phenylenc iodonium, zymosan‐stimulated hemocytes of L. stagnalis failed to reduce NBT and showed a markedly reduced LDCL response. Also, compounds that prevent assembly of functional NADPH‐oxidase complexes in activated mammalian cells were effective; preincubation of hemocytes with 1,4‐naphthoquinone inhibited the LDCL response and NBT reduction upon phagocytic stimulation. Furthermore, coincubation but not preincubation with five different catechol‐like phenols inhibited oxidative activities of zymosan‐stimulated hemocytes. These results imply similarities in composition and regulation of the ROI‐generating mechanisms of both mammalian and snail defense cells. It is postulated that in L. stagnalishemocytes, (1) NADPH‐oxidase activity is responsible for ROI production, (2) an active NADPH‐oxidase enzyme complex has to be assembled from putative cytosolic and membrane‐associated components, and (3) continuous replacement of active NADPH‐oxidase enzyme complexes is necessary to sustain respiratory burst‐like oxidative activities during interactions with non‐self.

Blood cell types and blood cell formation in gastropod molluscs

Abstract The results of previous and recent investigations on the structure, function and formation of blood cells of the freshwater snail Lymnaea stagnalis are described and discussed. Two hypotheses on blood cell formation and blood cell types are presented.

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

Abstract 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.

Specificity and memory in increased defence reactions against bacteria in the pond snail Lymnaea stagnalis

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.

Effect of saccharides on plasma mediated haemagglutination and in vitro phagocytosis by haemocytes of the snailLymnaea stagnalis

To study the role of lectins in the defence system of the snailLymnaea stagnalis, saccharides were tested for their capacity to inhibit plasma-mediated haemagglutination of trypsinised and fixed mouse red blood cells (M-rbc) and fixed rabbit red blood cells (R-rbc).d-Arabinose,N-acetylneuraminic acid, fucoidan andd-melezitose were good agglutination inhibitors using both types of rbc.d-Fructose, however, stimulated haemagglutination. Based on these results, in vitro phagocytosis assays were performed withd-arabinose (an agglutination inhibitor) andd-fructose (an agglutination stimulator). In co-incubation experiments both sugars inhibited adherence and phagocytosis of M-rbc in a concentration-dependent manner. Adherence and phagocytosis of R-rbc was stimulated at the lowest concentrations of D-arabinose and most concentrations ofd-fructose. Preincubation experiments showed decreased adherence and phagocytosis after preincubation of M-rbc with either sugar. Adherence and phagocytosis of R-rbc was reduced after preincubation of haemocytes withd-fructose. Preincubation of R-rbc had little effect. Our results support former studies indicating the presence of lectins both in the snail plasma and on the haemocytes.