The roles of haemocytes and the lymphoid organ in the clearance of injected Vibrio bacteria in Penaeus monodon shrimp.

In order to study the reaction of Penaeus monodon haemocytes, live Vibrio anguillarum bacteria were injected and the shrimp were periodically sampled. Immuno-double staining analysis with specific antisera against the haemocyte granules and bacteria showed that large numbers of haemocytes encapsulated the bacteria at the site of injection. A rapid decrease of live circulating bacteria was detected in the haemolymph. Bacterial clearance in the haemolymph was induced by humoral factors, as observed by agglutinated bacteria, and followed by uptake in different places in the body. Bacteria mainly accumulated in the lymphoid organ (LO), where they, or their degradation products, could be detected for at least 7 days after injection. The LO consists of folded tubules with a central haemal lumen and a wall, layered with cells. The haemolymph, including the antigens, seemed to migrate from the central tubular lumen through the wall, where the bacteria are arrested and their degradation is started. Electron microscopy of the LO revealed the presence of many phagocytic cells that morphologically resemble small-granular haemocytes. It is proposed that haemocytes settle in the tubule walls before they phagocytose. Immunostaining suggests that many of the haemocytes degranulate in the LO, producing a layer of fibrous material in the outer tubule wall. These findings might contribute to the reduced haemocyte concentration in the haemolymph of diseased animals or following injection of foreign material. It is proposed that the LO is a filter for virtually all foreign material encountered in the haemolymph. Observations from the present study are similar to clearance mechanisms in the hepatic haemolymph vessel in most decapod crustaceans that do not possess a LO. The experimental shrimp appeared to contain many LO spheroids, where bacterial antigens were finally observed as well. It is proposed that the spheroids have a degradation function for both bacterial and viral material, and that their presence is primarily related to the history of the infectious burden of the shrimp.

Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon.

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture in the past decade. In contrast to extensive studies on the morphology and genome structure of the virus, little work has been done on the defence reaction of the host after WSSV infection. Therefore, we examined the haemocyte response to experimental WSSV infection in the black tiger shrimp Penaeus monodon. Haemolymph sampling and histology showed a significant decline in free, circulating haemocytes after WSSV infection. A combination of in situ hybridisation with a specific DNA probe for WSSV and immuno-histochemistry with a specific antibody against haemocyte granules in tissue sections indicated that haemocytes left the circulation and migrated to tissues where many virus-infected cells were present. However, no subsequent haemocyte response to the virus-infected cells was detected. The number of granular cells decreased in the haematopoietic tissue of infected shrimp. In addition, a fibrous-like immuno-reactive layer appears in the outer stromal matrix of tubule walls in the lymphoid organ of infected shrimp. The role of haemocytes in shrimp defence after viral infection is discussed.

The role of the haematopoietic tissue in haemocyte production and maturation in the black tiger shrimp (Penaeus monodon).

The haematopoietic tissue (HPT) of the black tiger shrimp (Penaeus monodon) is located in different areas in the cephalothorax, mainly at the dorsal side of the stomach and in the onset of the maxillipeds and, to a lesser extent, towards the antennal gland. In young and in experimentally stimulated animals, the HPT is expanded in relatively larger and more numerous lobules throughout the cephalothorax. Four cell types could be identified in the HPT by electron microscopy. The type 1 cells are the presumed precursor cells that give rise to a large- and a small-granular young haemocyte, denominated as the type 2 and type 3 cells, respectively. A gradient of maturation from the type 1 towards the type 2 or 3 cells could frequently be observed. The presumed precursor cells are located towards the exterior of the lobules and maturing young haemocytes towards the inner part, where they can be released into the haemal lacunae. The type 4 cells show typical features of interstitial cells. Different stimulation experiments were carried out and various techniques were used to study the HPT in relation to the (circulating) haemocytes. The majority of the cells in the HPT are able to proliferate and proliferation can be increased significantly after the injection of saline and, to a much higher extent, after LPS injection. The circulating haemocytes of crustaceans are generally divided into hyaline (H), semigranular (SG) or granular (G) cells, of which large- and small-granular variants of each of these were suggested in the present study. Even after stimulation in this study, the circulating haemocytes scarcely divide. The high variations that were found in the total haemocyte count in the stimulation experiments were not accompanied by significant differences in differential haemocyte count and, therefore, appeared to be a less useful indicator of stress or health in P. monodon. Light and electron microscopical observations support the regulation of the populations of the different haemocyte types in the circulation by (stored) haemocytes from the connective tissue. In conclusion, according to morphological and immuno-chemical criteria, it is proposed in the present study to divide the haemocytes into a large-and a small-granular developmental series. After extensive morphological observations, it is suggested that the hyaline cells are the young and immature haemocytes of both the large- and small-granular cell line that are produced in the HPT, and can be released into the haemolymph. Indications were found that the granular cells, of at least the large-granular cell line, mature and accumulate in the connective tissue and are easily released into the haemolymph. Combining the results of the present study with literature, this proposed model for haemocyte proliferation, maturation and reaction will be discussed.

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.

Characterisation of different morphological features of black tiger shrimp (Penaeus monodon) haemocytes using monoclonal antibodies.

Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.

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 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.

Identification and characterization of a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalactosaminyltransferase from cercariae of the schistosome Trichobilharzia ocellata. Catalysis of a key step in the synthesis of N,N'-diacetyllactosediamino (lacdiNAc)-type glycans.

Three different stages of the avian schistosome Trichobilharzia ocellata appeared to contain a novel N-acetylgalactosaminyltransferase activity. To investigate its function in the biosynthesis of schistosome glycoconjugates, the enzyme was partially purified from cercariae, a free-living stage of the parasite, by affinity chromatography on UDP-Sepharose. Acceptor specificity studies showed that the enzyme catalyses the transfer of N-acetylgalactosamine (GalNAc) from UDP-GalNAc to oligosaccharides, glycopeptides and glycoproteins carrying a terminally beta-linked N-acetylglucosamine (GlcNAc) residue, regardless of the underlying structure. Analysis of the products obtained with GlcNAc and a desialylated and degalactosylated diantennary glycopeptide by 400 MHz 1H-NMR spectroscopy revealed that a GalNAc beta 1-->4GlcNAc (N,N'-diacetyllactosediamine,lacdiNAc) unit was formed. The enzyme can therefore be described as a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalactosaminytransferase (beta 4-GalNAcT). Using specific acceptors, the enzyme could be distinguished from all other beta 4-GalNAcTs described to date, including the one from pituitary gland that is involved in the specific glycosylation of pituitary glycohormones. By contrast, the enzymatic properties of the schistosome beta 4-GalNAcT (except for the sugar-donor specificity) strongly resemble those of the beta 4-galactosyltransferase of higher animals, an enzyme which is known to control the synthesis of Gal1-->4GlcNAc (lacNAc)-type oligosaccharide chains. By analogy, the beta 4-GalNAcT is concluded to control the key step in the synthesis of lacdiNAc-type chains. LacdiNAc-type glycans are also common to the mollusc Lymnaea stagnalis, which is the intermediate host of T.ocellata. It is proposed that the schistosome beta 4-GalNAcT functions in the expression of specific carbohydrate structures that contribute to a molecular mimicry, enabling the schistosome to evade the defence system of the snail host.

Separation of Lymnaea stagnalis hemocytes by density gradient centrifugation.

A chelating anti-clumping (alpha-C) buffer allowed blood cells (hemocytes) of a gastropod, Lymnaea stagnalis to be separated by discontinuous Percoll density gradient centrifugation. The hemocytes of L. stagnalis were separated into five fractions, having a density lower than 10, 20, 30, 40, and 50% Percoll, respectively. Trypan blue exclusion assays showed viability of separated hemocytes to be between 81 and 89%. Cytospin preparations of these hemocytes were examined. Small cells were mainly observed at high densities; at lower densities medium and large hemocytes were also present. No absolute separation was achieved. Some density fractions were enriched for hemocytes with regard to the distributions of two endogenous lysosomal enzymes (alpha-naphthyl acetate esterase and acid phosphatase).

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 diphenylene 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. stagnalis hemocytes, (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.

Effects of Trichobilharzia ocellata on hemocytes of Lymnaea stagnalis.

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.

Generation of oxygen radicals in hemocytes of the snail Lymnaea stagnalis in relation to the rate of phagocytosis.

The kinetics of oxygen radical production by phagocytosing hemocytes of the pond snail Lymnaea stagnalis were investigated. After contact had been established between zymosan and hemocytes in monolayers at 0 degrees C, phagocytosis was initiated by a shift to room temperature. Until the internalization phase of phagocytosis was completed, oxidative activity was detected mainly extracellularly (superoxide dismutase inhibitable cytochrome C reduction and peroxidase-catalyzed phenol red oxidation were used for the detection of superoxide and hydrogen peroxide, respectively). Thereafter, extracellular oxygen radical production by phagocytosing hemocytes decreased. Luminol-dependent chemiluminescence activity grew and, after the internalization phase of phagocytosis, remained at a high level, suggesting continued oxygen radical activity intracellularly. These results indicate that contact between zymosan and the hemocyte's plasma membrane stimulates a membrane-bound system to generate and release oxygen radicals. After internalization, this system appears to continue oxygen radical production inside the phagosome. So far, oxygen radical production in snail hemocytes shows many similarities to the mechanism in mammalian leucocytes.

Analysis of surface carbohydrates of Trichobilharzia ocellata miracidia and sporocysts using lectin binding techniques.

Miracidia and in vitro-derived primary sporocysts of the avian schistosome Trichobilharzia ocellata were studied for the expression and the characteristics of glycoconjugate moieties comprising the surface coat. Using a panel of 9 peroxidase labelled lectins, several different lectin binding sites were demonstrated on the larvae. Fixed miracidia have binding sites for 7 of the lectins; wheat-germ agglutinin binds to both the ciliated plates and the tegumental ridges between them; the other 6 lectins bind to the plates only. Three of the miracidia-binding lectins, wheat-germ agglutinin, concanavalin A and peanut agglutinin, also bind to fixed sporocysts. Since the miracidial ridges are devoid of binding sites for concanavalin A and peanut agglutinin, whereas the sporocyst tegument binds these lectins, it appears that these sites become exposed during or shortly after transformation. In saturation experiments, low concentrations of peanut agglutinin and concanavalin A are bound more avidly by sporocysts than by miracidia, indicating a higher binding affinity of the former. The two larval forms do not differ in affinity for wheat-germ agglutinin but they have different binding capacities; when offered in high concentrations, more of this lectin is bound by sporocysts than by miracidia. Lectin binding was competitively inhibited by adding the appropriate free saccharides. Live larvae showed the same lectin binding pattern as did fixed specimens. Proteinase treatment reduced lectin binding to living and, to a lesser extent, to fixed larvae, suggesting that binding sites are constituents of proteoglycoconjugates. After SDS-PAGE of extracts from miracidia and sporocysts and subsequent Western blotting, some of the lectins failed to bind glycoproteins, others reacted with an array of bands. The patterns differed among the lectins and each lectin gave different patterns for miracidia and sporocysts. The results obtained with these two lectin-binding techniques support the conclusion that stage-specific proteoglycoconjugates occur at the surface of T. ocellata larvae.

Immune mechanisms in trematode-snail interactions.

Digenetic trematodes, including several species of medical and veterinary significance, nearly all depend on molluscs (usually gastropods) as hosts for asexual reproduction. The molluscan internal defence system, although lacking many of the familiar features of the vertebrate immune system, may pose a substantial obstacle to digenean development. In this article, Wil van der Knaap and Eric Loker discuss the manner in which this system is mobilized to kill larval trematodes and the real and putative mechanisms of digenean evasion of molluscan immune responses. These two possible outcomes of trematode-snail associations make them intriguing model systems for exploring processes of parasite infectivity and host resistance.

Monoclonal antibody recognized hemocyte subpopulations in juvenile and adult Lymnaea stagnalis: functional characteristics and lectin binding.

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.

The production of toxic oxygen metabolites by hemocytes of different snail species.

The phagocytic hemocytes of four snail species were investigated for their ability to generate reactive oxygen metabolites upon stimulation by foreign material. Hemocytes of the pond snail Lymnaea stagnalis and of the garden snail Helix aspersa showed a luminol-dependent chemiluminescence (CL) when they phagocytosed zymosan particles. This CL was inhibited by superoxide dismutase (SOD) and sodium azide, indicating the involvement of oxygen intermediates. Hemocytes of the planorbid snails Planorbarius corneus and Biomphalaria glabrata did not give a detectable CL response. This is probably due to the presence of hemoglobin in the hemolymph; after isolation of the cells and subsequent stimulation, however, still no CL could be measured. Hemocytes of all four snail species showed a SOD-sensitive nitroblue tetrazolium (NBT) reduction, indicating the generation of superoxide anions. Regarding the NBT reaction, no differences were observed between strains of B. glabrata that were susceptible or resistant to PR-1 Schistosoma mansoni; neither did exposure to the parasite have an effect on the ability of the hemocytes to reduce NBT. Also, hemocytes from Trichobilharzia ocellata-infected L. stagnalis did not react differently from hemocytes of uninfected snails. It is now clear that phagocytically stimulated hemocytes of several molluscan species can generate reactive forms of oxygen; the relevance of this fact for the phylogeny of killing systems operative in leukocytes is discussed.

Possible role of reactive forms of oxygen in in vitro killing of Schistosoma mansoni sporocysts by hemocytes of Lymnaea stagnalis.

The interactions between sporocysts of Schistosoma mansoni and hemocytes of the pond snail Lymnaea stagnalis were studied in an in vitro system. All sporocysts were encapsulated and killed in the hemolymph of juvenile or adult snails within 24 h; the killing patterns of both age groups were not very different. The separation of hemolymph into hemocytes and plasma and subsequent incubation of sporocysts showed that the cells, not the plasma, were responsible for parasite killing. The possible involvement of reactive oxygen intermediates and the enzyme peroxidase in the cytotoxic mechanism was studied. Chemiluminescence (CL) could not be detected during the hemocyte-parasite encounter. Histochemical staining with nitroblue tetrazolium (indicative for superoxide) and diaminobenzidine (indicative for hydrogen peroxide) showed that oxygen intermediates were produced by snail hemocytes that encapsulated sporocysts. Histochemistry also suggested the presence of peroxidase activity at the hemocyte-sporocyst interface. The cytotoxic mechanism underlying parasite killing by hemocytes and the role of reactive forms of oxygen and peroxidase are discussed.

Alterations in the internal defence system of the pond snail Lymnaea stagnalis induced by infection with the schistosome Trichobilharzia ocellata.

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.