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Size determination of hemocytes from the American oyster, Crassostrea virginica, and the description of a phagocytosis mechanism
... Thus, the opsonic properties of a serum agglutinin (which stimulated the phagocytosis of yeast cells in Ca 2 +-free buffer) were demonstrated, together with the role of a haemocyte-associated lectin in phagocytosis of unopsonised yeast cells in Ca 2 +-containing buffer (Renwrantz and Stahmer 1983). A role for serum factors in recognition of non-self substances and the phagocytosis of unopsonised target cells in Ca 2 +-containing media has also been reported for a number of other molluscs, including the pond snail, Lymnaea stagnalis (Sminia et al. 1979; van der Knaap et al. 1981), the oyster, Crassostrea virginica (Renwrantz et al. 1979 ), the Asian clam, Corbiculafluminea (Tuan and Yoshino 1987 ), and the freshwater snail, Biomphalaria glabrata (). This dual recognition system may, therefore, prove to be widespread in molluscs. ...
... In the vineyard snail, Helix pomatia, we previously demonstrated that a homologous blood-group A-specific HPA from the albumin gland stimulated the elimination of injected rbc from the snail's circulation (Harm and Renwrantz 1980), A rbc being cleared much faster than B or rabbit rbc (Renwrantz and Mohr 1978; Renwrantz et al. 1981 ). Phagocytosis of sinus-trapped rbc subsequently occurred and particleladen haemocytes were detected in the circulation 40-60 h later (Renwrantz 1979; Renwrantz et al. 1981). Since attachment (= recognition) to haemocytes is a prerequisite for internalisation, to further our understanding of these events in Helix, in vitro phagocytosis assays with haemocytes were performed . ...
... , unless otherwise stated, were obtained from Sigma Chemical Co., and were of the highest reagent grade possible. Blood-group substance A (peptone A) was isolated as described by Renwrantz (1979) from peptone S (Brunnengr/iber, Lfibeck, Fed. ...
Haemocytes of the gastropod mollusc, Helix pomatia, possess on their surface a membrane-integrated GalNac-specific lectin which binds to and stimulates phagocytosis of GalNac-bearing target cells (human A erythrocytes) only in the presence of extracellular calcium ions. Target cells without GalNac moieties on their surface (human B and bovine erythrocytes) are not recognised. Helix haemocytes also possess a Ca2+-independent mannose-6-phosphate-specific lectin on their surface which, in the absence of extracellular calcium ions, enables recognition and phagocytosis of A rbc opsonised with agglutinins isolated from either the snail's albumin gland or serum. These opsonins, however, bind to host haemocytes only after binding to GalNac moieties on the surface of test particles. Our results indicate that such a ligand-specific opsonin/target cell interaction apparently induces a conformational change in the opsonin, resulting in exposure of mannose 6-phosphate moieties that are recognised by the Ca2+-independent lectin on the surface of the haemocytes.
... Iterative updates on their progress were provided by Cheng (e.g., Cheng 1967, 1981. Combined, these studies have detailed the fates of a diverse array of foreign particles and microorganisms in oysters and characterized the process of phagocytosis (e.g., Stauber 1950, Stauber 1961, S. Y. Feng 1958, Tripp 1958, 1960, J. S. Feng 1966, Tripp & Kent 1967, Acton et al. 1969, Fries & Tripp 1970, Feng et al. 1971, 1977, Cheng & Cali 1974, 1977, Cheng & Rudo 1976, Renwrantz et al. 1979, Hinsch & Hunte 1990. Most living microorganisms are killed upon ingestion by phagocytic amebocytes, including viral particles (J. S. , Fries & Tripp 1970) bacteria, phytoplankton (e.g., Navicula ostrearia), and metazoans. ...
... Ingestion, or endocytosis, of microorganisms by eastern oyster amebocytes reportedly can occur in three ways; some microbes adhere to the amebocyte filopodia (Bang 1961), some are taken into funnel-shaped psuedopods (Renwrantz et al. 1979), and some are enveloped in a vesicle on the amebocyte surface (Cheng 1975). Regardless of the path of entry, the targeted microorganism is captured and drawn into a cytoplasmic invagination (Fig. 21) that encircles it with concentric lamellae to form a "phagosome" (Cheng 1975). ...
The distribution of eastern oysters Crassostrea virginica near terrestrial watersheds has led to a general impression that low or variable salinity is imperative for survival. However, freshwater runoff contains numerous mineral elements from geologic deposits that could play significant roles in oyster physiology. Two metals of terrestrial origin, copper and zinc, are accumulated to extremely high concentrations in eastern oysters, even in the absence of anthropogenic sources. As yet, there has been no defendable demonstration of a physiologic function for such high concentrations. Both copper and zinc, however, are accumulated almost exclusively in the amebocytes and calcareous shell of oysters, a unique distribution that implicates a role in the functions of amebocytes. Amebocytes are migratory, diapedetic cells generally recognized to provide nutriment and defense through phagocytosis, killing, and digestion of invading or ingested microorganisms. There is sufficient evidence in existing literature to suggest that copper and zinc directly contribute to these antimicrobial activities. This review presents historical and recent findings that demonstrate a strong affinity of oyster amebocytes for copper and zinc (even in low ambient concentrations), prolonged retention of the metals despite a potential route of elimination, and strong circumstantial evidence of antimicrobial activity by accumulated copper and zinc. It is proposed that oysters actively concentrate copper and zinc as antimicrobial agents to be used in intracellular and extracellular killing (direct toxicity) as well as extracellular clot formation (precipitation of hemolymph). This potential, combined with evidence of amebocyte involvement in deposition of oyster shell, provides an alternative framework for understanding amebocyte functions, defense activities, and coastal distributions of oyster populations. It also affords some resolution to the apparent contradiction of eastern oysters thriving at seemingly polluted locations.
... Changes in hemocyte subpopulations were found between January and May, with fewer granular hemocytes and more agranular cell types as temperature increased in the May samples. Such a shift could increase susceptibility to disease, since granulocytes are thought to be the main effectors in the oyster cellular defense response (Foley & Cheng 1975, Renwrantz et al. 1979. In contrast, Chu & LaPeyre (199313) reported higher percent granulocytes as well as increased total hemocyte counts in hemolymph samples drawn from oysters held in the laboratory at 25°C as compared to 10°C. ...
... Correlational analysis showed positive associations between % granulocytes and NBT reduction in both March and October, while correlations between % granulocytes and % mobility, and O/O granulocytes and particle binding, were positive in March and negative in October. These results indicate that the association of granularity with phagocytosis and locomotion may not exist year-round (Foley & Cheng 1975, Renwrantz et al. 1979. The intuitive association between NBT reduction activity and granularity is perhaps the most plausible because granules contain enzymes that produce cytotoxic molecules, including OF, that effect intracellular killing and digestion of phagocytosed material (Badwey & Karnovsky 1980, Auffrct 1988. ...
Oysters Crassostrea virginica from Chesapeake Bay, Virginia, and Apalachicola Bay, Florida, USA, were collected in March and October 1992 to investigate possible differences in defense-related hemocyte activities between individuals from geographically separate populations. In March, hemolymph drawn from Chesapeake Bay oysters contained an average of 1.08 x 10⁶ hemocytes ml⁻¹ hemolymph, significantly lower than the average 1.63 x 10⁶ hemocytes ml⁻¹ hemolymph obtained from Apalachicola Bay oysters. Hemocyte number did not differ significantly in the October comparison. At both times of year, Chesapeake Bay oyster hemolymph samples contained significantly greater proportions of granular hemocytes compared to Apalachicola Bay hemolymph samples. Hemocyte samples from Chesapeake Bay oysters demonstrated a higher percentage of mobile hemocytes and greater particle binding ability than Apalachicola Bay oyster hemocytes when tested in March, but the reverse was found in the October experiments. Chesapeake Bay oyster hemocytes produced significantly more superoxide anion as measured by nitroblue tetrazolium reduction than did Apalachicola Bay oyster hemocytes in both March and October. Oyster hemolymph levels of the protozoan parasite Perkinsus marinus did not differ significantly between the 2 sites at either time of year. These results demonstrate the importance of background studies to characterize site-specific differences in oyster hemocyte defense-related functions.
... N: nucleus; GR: granule; G: golgl complex; mi: mitochondria. (x15 200) virginica and Mercenana mercenaria by Foley & Cheng (1975) and Renwrantz et al. (1979) from C. virginica. In contrast, Ruddell (1971) indicated that in C. virginica the agranular amebocytes appeared to have greater phagocytic ability. ...
... A similar model was described by Cheng (1975). Two more models of the same event were described by Bang (1961) and Renwrantz et al. (1979). ...
Phagocytosis of foreign materials by haemocytes is an important aspect of the internal defence of bivalve molluscs. Two main haemocyte types can be distinguished in the haemolymph of the clam Ruditapes decussatus: granulocytes and hyalinocytes. The ability of clam haemocytes to phagocytose zymosan particles, Vibrio P1 cells and trophozoites of the protistan parasite Perkinsus atlanticus was demonstrated by means of in vitro assays. However, clam haemocytes did not phagocytose P. atlanticus zoospores in the assays. Granulocytes showed the highest phagocytic capacity in each assay. Phagocytic capacity of haemocytes was not significantly affected by clam age. An ultrastructural study of phagocytosis showed the following sequence of events: engulfment of particles by pseudopods, formation of a phagocytic vacuole, fusion of lysosomes with the phagocytic vacuole, and digestion of the particles giving rise to residual bodies that might be discharged.
... D'autres études ont également rapporté un autre mode d'ingestion. Par exemple, chez l'huitre creuse et la moule bleue, les hémocytes sont capables d'étendre un unique pseudopode en forme de cône qui engloutit le microenvahisseur (Renwrantz et al., 1979;Stefano, 1990). Il a été suggéré que le mode d'internalisation varie en fonction des récepteurs responsables de l'induction de la phagocytose mais aussi en fonction de la taille et la forme des particules visées (Champion and Mitragotri, 2006). ...
Mytilus edulis est un mollusque bivalve de grand intérêt économique et écotoxicologique. Cette espèce sentinelle est connue pour sa résistance aux contaminants chimiques et biologiques. Néanmoins, depuis quelques années la moule bleue est touchée par des mortalités dans les élevages des Pertuis Charentais ayant pour dénominateur commun la présence de bactéries virulentes de type Vibrio. Le premier axe de cette thèse décrit les interactions des isolats de V. Splendidus avec la moule bleue au niveau cellulaire et physiologique. Les infections expérimentales ont permis la sélection de deux isolats bactériens affiliés à V. splendidus/V. hemicentroti : une souche virulente codée 10/068 1T1 et une souche inoffensive codée 12/056 M24T1. Ces deux bactéries ont été marquées à la GFP et validées en tant que modèles authentiques d’exposition à travers leurs caractéristiques de croissance et de virulence. Par ailleurs, V. hemicentroti 10/068 1T1 est capable d’altérer différentes fonctions hémocytaires incluant la motilité, l’adhésion, l’internalisation, la production de ROS, la maturation du phagosome et la viabilité contrairement à la bactérie non virulente. Les produits extracellulaires bactériens semblent être toxiques et inhibent certaines réponses cellulaires (internalisation et production de ROS). Enfin, nous avons reproduis avec succès l’infection des animaux par le pathogène via un modèle expérimental de cohabitation. Le suivi de l’infection montre que V. hemicentroti 10/068 1T1 a pour cible principale les branchies. Le deuxième axe explore le fonctionnement du système MXR (MultiXenobiotic Resistance) chez la moule bleue. La séquence codante complète d’un nouveau transporteur ABCG2 a été établie et la protéine résultante a été identifiée. La caractérisation moléculaire montre la présence du transcrit dans les hémocytes ainsi que dans les branchies et son homologie avec les autres protéines appartenant à diverses espèces. L’utilisation des sondes fluorescentes bodipy prazosin et pheophorbide A, combinées avec des bloqueurs spécifiques, démontre l’activité d’efflux de ce transporteur et son hétérogénéité dans les tissus et cellules. Par ailleurs, il est également démontré que l’expression des trois transporteurs ABC (abcb, abcc, abcg2) identifiés chez la moule bleue est modulée par les contaminants chimiques. Les animaux exposés au BaP au laboratoire ou prélevés sur un terrain contaminé montrent une surexpression des transcrits abc dans les branchies et une sous expression dans les hémocytes. La saisonnalité, sur le terrain, a également un effet sur les niveaux des transcrits et interfère avec les réponses liées aux contaminants. Seul le transporteur abcb exprimé dans les branchies n’est pas affecté par des variations saisonnières et montre une surexpression dans le site contaminé tout au long de l’année. En conclusion, nos résultats démontrent la vulnérabilité de la moule bleue face un pathogène. L'impact immunotoxique des xénobiotiques et le rôle que peuvent jouer les transporteurs ABC dans le fonctionnement du système immunitaire des moules reste à explorer.
... Variations have been reported on the morphology of engulfment. For example, molluscan phagocytes take up vertebrate erythrocytes by means of funnel-like pseudopods (Renwrantz et al. 1979). Phagocytic blood cells, which are always capable of amebalike movement, are more easily manipulated when they adhere to a glass or plastic surface than when they are free, so assays for phagocytosis often exploit adherent cells. ...
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... On the contrary, the hyalinocytes showed a limited phagocytic ability and lower levels of hydrolytic enzymes. Similar results were reported in most bivalves (Foley & Cheng, 1975;Moore & Lowe, 1977;Bayne et al., 1979;Renwrantz et al., 1979;Tripp, 1992;Mourton et al., 1992;Hine & Wesney, 1994;Russell-Pinto et al., 1994). The role of bivalve hyalinocytes is unknown. ...
In vitroassays were performed to assess the phagocytic ability of mussel haemolymph cell types. Among the haemocyte types, the granulocytes showed an important phagocytic activity whereas the hyalinocytes did not, thus indicating that mussel haemocytes are functionally heterogeneous. Phagocytic activity of acidophilic granulocytes was higher than basophilic ones. Ultrastructural study showed thatVibrioP1 cells were internalised in phagosomes and then digested. As a result of the digestive process, concentric lamellae and other degraded materials were produced inside the phagosomes, and non degraded materials gave rise to residual bodies which could be discharged from the cells. Similar events were observed in the phagocytic process of zymosan particles. The study of influence of temperature on phagocytosis activity revealed that the percentage of phagocytic cells was lower at 10°C than at 20°C and 30°C. Phagocytosis activity was higher in the assays performed at 15ppt than those at 25ppt and 36ppt. No significant differences on percentage of phagocytic cells were found between young and adult mussels. Generation of superoxide anion by haemocytes using zymosan as a phagocytosis stimulant was demonstrated through the nitroblue tetrazolium reduction test.
... However, the proportion of granulocytes and hyalinocytes may be highly variable within the same species. For instance, in C. virginica, [31] observed a higher number of hyalinocytes whereas [32] and [17] noticed a higher number of granulocytes. In the Sydney rock oyster, in contrast to results from the present study, [29] found 15% of small agranulocytes, 46% of hyalinocytes and 38% of granulocytes, however the oysters used by [29] were not infected by M. sydneyi. ...
Sydney rock oysters (SRO) Saccostrea glomerata suffer mass mortalities during summer and autumn as a result of infection by a protozoan parasite Marteilia sydneyi (QX disease). Mass selected disease resistant (QXR) lines have been used with some success in affected estuaries in recent years, with resistance attributed to oxidative defense systems. However, the role of hemocytes in resistance to QX by SRO has not been fully explored. In the present study, fifty QXR and fifty wild caught (WC) oysters were collected from a lease at Pimpama River during a QX outbreak in January 2011. Hemocytes characteristics (type, morphology) and functions (mortality, phagocytosis and oxidative activity) from both oyster lines were analyzed by flow cytometry in the context of infection intensity and parasite viability (determined histologically). Amongst the QXR oysters, 20% were diseased containing viable parasite, 74% had killed M. sydneyi and 6% were uninfected. In contrast, 86% of WC oysters were diseased, 2% had killed M. sydneyi and 12% were healthy. Significant differences in hemocyte number and physiology between the two oyster lines were found (ANOVA). Phagocytosis rate and the mean oxidative activity per cell were similar between both oyster lines. Higher numbers of infiltrating and circulating hemocytes, higher percentage of circulating granulocytes, their higher size and complexity in QXR oysters, and the production of reactive oxygen species were associated with the ability to kill the parasite. High abundance of M. sydneyi in the digestive tubule epithelium of both oyster lines implied inability to kill the parasite at the beginning of the infection. However, QXR oysters had the ability to kill M. sydneyi at the stage of sporangiosorae in the epithelium of digestive tubules. The similar phagocytic ability of hemocytes from both oyster lines, the size of the parasite at this infection stage, and its localization suggested that encapsulation is likely to be the main process involved in the eradication of M. sydneyi by QXR oysters.
... On the contrary, the oysters O. edulis and C. gigas (Chagot, 1989) had more circulating hyalinocytes than granulocytes. In C. virginica, Feng et al. (1971) reported a higher number of hyalinocytes whereas Renwrantz et al. (1979) did the contrary. Discrepancy among results from different authors may be due to interspecific differences, but also to differences in classification of hemocyte types, pathological or physiological stage of the animals, methods employed in differential counting, and number of specimens studied. ...
The study of hemogram characteristics in a population of cultured mussels through a period of 18 months revealed that the concentration of circulating Mytilus galloprovincialis hemocytes varied, following a seasonal pattern, being positively correlated with water temperature. Most mussels had between 1 and 5 x 10(6) hemocytes/ml of hemolymph. Differential counts showed that M. galloprovincialis hemolymph contained more granulocytes than hyalinocytes. The hemolymph cell concentration was not associated with size or weight of mussels. Circulating hemocyte concentration did not decrease in mussels with reabsorbing gonad, despite the hemocytic accumulation inside and around gonad follicles. No significant difference in hemolymph cell concentration was found between two groups of mussels of different ages. Nevertheless, older mussels had higher mean values of circulating granulocytes although these differences were only significant in the winter sample. Infestation by the protistan Marteilia refringens caused a significant increase of hemocytes in the hemolymph. In addition, the occurrence of some parasites induced significant changes in the proportion of circulating hemocyte types. Results suggested that a hemocytic response was detected in hemolymph when the foreign organisms are pathogenic and evoke a heavy inflammatory reaction. Mussels with large or numerous granulocytomas had a higher number of circulating granulocytes and hyalinocytes than those without granulocytomas. Copyright 1998 Academic Press.
... The morphological heterogeneity of bivalve hemocytes has been well documented (Cheng, 1981;Fisher, 1986). Phagocytic activity variations between hemocyte types were also observed in several bivalve species (Renwrantz et al., 1979;Cheng, 1990;Tripp, 1992;Carballal et al., 1997b;Lopez et al., 1997a,b). Thus, there may be other differences between hemocyte subpopulations, such as enzymatic activities. ...
Enzymatic activities in the hemolymph of healthy and Bonamia-infected Ostrea edulis and Crassostrea gigas were studied with a commercial kit for the detection of 19 enzymes: 15 and 16 enzymes, respectively, were detected in the hemolymph of O. edulis and C. gigas and 10 of them showed relatively high activity levels. Most of them existed in both the cell-free fraction of the hemolymph and in the hemocytes. The cell-free hemolymph fraction of Bonamia ostreae-infected European flat oysters showed an elevated enzymatic activity level compared with that of healthy individuals. C. gigas hemocytes possessed higher enzymatic activity levels than O. edulis hemocytes. Differences in enzymatic activities existed in granulocytes and hyalinocytes in both oyster species. The enzyme release from oyster hemocytes seemed to be selective. The infection by B. ostreae induced enzymatic activity variations in European flat oysters. Higher enzyme levels within hemocytes may contribute partly to the natural resistance of C. gigas to the infection by B. ostreae.
... 1955: both cited in Cheng 1981 ). Nevertheless, the existence of 2 haemocyte types was proposed by Huffman & Tripp (1982) in Mya arenaria and by Auffret (1985) in Ruditapes philippinarum. In the present work, we determined a percentage of 16.5% for granulocytes and 79.2% for hyalinocytes; these data are not in agreement with those of other authors. Renwrantz et al. (1979) found about 70% granulocytes and 30% agranulocytes in Crassostrea virginica, confirming a previous study by Galtsoff (1964) ; these percentages are typical for bivalves (Huffman & Tripp 1982). In our species, hyalinocytes were more abundant than granulocytes. The cytoplasm of some haemocytes exhibited yellow-orange inclusions larger tha ...
A morphological and enzymatic characterization of Chamelea gallina haemocytes was carried out as a prerequisite for further studies on venus clam immunobiology. Two main types of circulating haemocytes were identified (1) hyalinocytes (79.2%), agranular cells with a central nucleus surrounded by a little cytoplasm, and (2) granulocytes (16.5%), smaller granular cells with smaller nuclei. Small cells with a strongly basophilic nucleus and a thin layer of peripheral cytoplasm, probably undifferentiated blast cells (4.3%), were also observed. Both granulocytes and hyalinocytes can assume a spreading or round morphology. The enzymatic activities of haemocytes were also investigated. Some of the granulocytes and hyalinocytes were positive for hydrolytic enzymes, suggesting a role for these cells in phagocytosis; no oxidative enzymes were detected in C. gallina haemocytes. Granulocytes and hyalinocytes can easily adhere to the substratum and exhibit a low phagocytosis activity towards foreign particles (6.3%), whereas the fraction of cells containing ingested material significantly increased after pre-incubation of test particles with cell-free haemolymph, which suggests the presence of opsonin(s) in the haemolymph.
... The flow cytometer is likely to be an important tool for investigating these critical issues. For instance, the small granulocytes found in C. virginica had been described from microscope observations [1,34,35], but they are relatively very scarce. The large number of cells that we were able to analyse made this small subgroup stand out as being characteristic of the oyster. ...
Haemocyte subpopulations from three bivalve species (the clams Ruditapes philippinarum and Mercenaria mercenaria and the oyster, Crassostrea virginica) were characterised using light-scatter flow cytometry and a standard set of methods. Two parameter (forward and side scatter) plots for the three species were very similar and resembled plots for mammalian white blood cells. Two haemocyte groups (granulocytes and agranulocytes) were found in both the haemolymph and the extrapallial fluid of the clams while those two groups and an additional third group were found in the haemolymph of the oyster. All subpopulations were sorted on to glass slides, identified, photographed, and measured microscopically. Sorting of the bivalve granulocyte and agranulocyte groups indicated varying degrees of heterogeneity within each population in terms of either size or granularity, or both. However, subsorting of selected regions within the major groupings produced highly pure haemocyte populations. The comparison showed both similarities and differences among species. For instance, a distinct subpopulation of small granulocytes was present only in oysters and a subpopulation of spindle-shaped haemocytes, only in M. mercenaria. The haemocyte subpopulations delineated by light-scatter flow cytometry underscore questions about cell lineages, but the instrument also offers a powerful technique for answering them.
... In previous reports, bivalve granulocytes have exhibited high phagocytic abilities against various foreign particles [39,50,51]. On the other hand, bivalve agranulocytes have been shown to have no or less phagocytic ability compared to granulocytes in Mytilus galloprovincialis [52], Tridacna crocea [53], C. virginica [54], Mercenaria mercenaria [55], and Mytilus edulis [56]. Takahashi and Mori [39] examined the phagocytic ability of C. gigas granulocytes and agranulocytes against three different species of bacteria, formalinized erythrocytes, heat-killed yeast cells and latex beads. ...
Integrins play a key role in immunoresponses such as attachment, spreading, and phagocytosis in invertebrate hemocytes. This study was designed to identify integrin expression patterns at the hemocyte subpopulation level, and correlate the expression levels with phagocytic ability. First, we cloned a beta integrin from Crassostreagigas hemocytes and used real-time RT-PCR to analyze the quantitative expression level of its encoding mRNA. The expression level in hyalinocytes was significantly higher than that in granulocytes and agranulocytes. Subsequently, we investigated the phagocytic ability of each subpopulation using anti-alpha(5)beta(1) integrin antibody, and found that phagocytosis of hyalinocytes was inhibited by neutralization with the antibody but enhanced against the antibody-conjugated microspheres. In contrast, phagocytic abilities of granulocytes and agranulocytes showed high and zero levels, respectively, regardless of the antibody. These results suggest that phagocytosis of hyalinocytes is regulated by an integrin-dependent mechanism and that of granulocytes is elicited by other functional receptors.
... With respect to whether gastropods possess one or more types of blood cells, no workers have presented a uniform classification (Ratcliffe and Rowley, 1979; Sminia, 1979; Adamowicz and Bolaczek, 2003). While Sminia and Barendsen (1980) have suggested that only one type of blood cell, the amoebocyte, exists in the freshwater snails, many authors have argued that granular and agranular hemocytes could be readily recognized in other molluscs as well (Cheng, 1975; Renwrantz et al., 1979; Fisher, 1986; Auffret, 1988). This study compared hemocytes of three gastropod molluscs , namely Trachea vittata, Pila globosa and Indoplanorbis exustus, and found morphological distinctions that could represent functional differences. ...
Light microscopic observations were made on the hemocytes of three gastropod species namely Trachea vittata, Indoplanorbis exustus and Pila globosa. It revealed two basic types of hemocytes. They are agranulocytes and granulocytes. Agranulocytes are hyalinocytes which are round, unspread hemocytes and have a large nucleo-cytoplasmic ratio. Granulocytes are spreading hemocytes, forming numerous pseudopodia. For the purpose of differential counting, we present a categorization of the granulocytes into three sub-categories based on cell dimensions, nucleo-cytoplasmic ratio, distribution of granules in the cytoplasm and position of the nucleus. The smaller granulocytes are younger cells, and are termed Granulocytes I (Progranulocytes). The larger ones are fully developed cells that have been differentiated into Granulocyte II (basophilic) and Granulocyte III (eosinophilic).
Clam Ruditapes philippinarum is one of the most important commercial aquaculture species in China. The haemocytes play vital roles in internal defense of the calm. In this investigation, classification and immune functions of R. philippinarum haemocytes were identified. The haemocyte density was (8.28 ± 1.42) × 106/mL and two major haemocyte types basophilic hyalinocytes and eosinophilic granulocytes were recognized based on the presence or absence of granules and staining affinities of their cytoplasm. Granulocytes were the most common cell type (73.08 ± 3.23%). The hyalinocytes and granulocytes could be divided into eight subtypes respectively according to N/C ratio as well as the nucleus shape and number by light microscope. Fourteen types of granules were identified and the multivesicular body and R-body were first found in bivalve, moreover, transmission electron microscope observation was consistent with the results from light microscope. Also eight different external forms of haemocytes could be identified by scanning electron microscope. Both granulocytes and hyalinocytes showed the abilities of phagocytosis and reactive oxygen species (ROS) production which were higher in granulocytes than that in hyalinocytes. The phagocytic rate of the total haemocytes and the granulocytes was about 45.06% and 40.74% respectively. The ROS production of the total haemocytes and granulocytes was 58.7% and 51.19% respectively. Although the hyalinocytes showed less ability in phagocytosis and ROS production, they played important roles in agglutination. This investigation provided a fundamental knowledge for future study of the immune function of haemocytes in clam R. philippinarum.
For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker's yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1-5, 6-10, 11-15, and ≥16), and the phagocytic frequencies were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or β-laminarin and then allowed to phagocytose the three types of the particles. The percentage of phagocytic cells of β-laminarin-treated granulocytes decreased significantly for zymosan and zymocel, but not for yeast. These results suggest that C. gigas might possess at least two types of hemocytes, and that one type of the hemocytes (granulocytes) is more active for phagocytosis. The granulocytes were found to have multiple subtypes with different phagocytic abilities and multiple phagocytic receptors. Some of the granulocyte subtypes revealed a much stronger phagocytic ability, depending on the presence of β-glucan receptors for phagocytosis.
Cellular inflammatory response to nonself materials is a universal phenomenon in the Animal Kingdom. To refresh our memories, inflammation, as generally defined, is the reaction of tissues to insult. The process is characterized by local heat, swelling, redness, and pain. These characteristics have been defined as a result of observations on vertebrates, particularly mammals. As our knowledge of inflammation as revealed by studying invertebrates, especially molluscs, has progressed, it is becoming increasingly evident that these characteristics need not all be present, especially conspicuous swelling and redness. Also, I would like to advance the idea that nonspecific inflammatory response does not occur in molluscs or any other group of animals. In other words, there is some degree of specificity at the molecular level in all instances of cellular inflammation. The evidences for this statement are presented later. At this point, I wish to review in a conceptual manner the various phenomena which collectively comprise cellular response to insult in molluscs. Specifically, as depicted in Fig. 1, I intend to point out that cellular infiltration and subsequent events in molluscs, as in other groups of animals, involves (1) chemotactic attraction of reaction cells to the insulting agent, (2) surface recognition of self from nonself on the part of reaction cells and attachment of the nonself insulting agent to such cells, (3) endocytosis or encapsulation, and (4) intracelluar events leading to degradation and elimination of the insulting agent. As will become apparent at a later point, the reason for reviewing these phases of cellular reaction to challenge is that in terms of modern cell biology, there appears to be specific recognition sites involved at each phase, hence my thesis that nonspecific inflammatory response per se does not occur.
By the time the oldest fossiliferous rocks were laid down, the molluscs were already represented by many different patterns (Runnegar and Pojeta, 1974). Few phyla show such wide diversity imposed on such a uniform body plan. The estimated 47,000 living molluscan species (Boss, 1971) evolved from a group related to the same stock as that from which the arthropods and annelids derive. The gastropods are the most abundant class, representing 80% of the total species, while the lamellibranchs represent 16% and the cephalopods only 1%.
There are a number of characteristics which should be considered in a definitive classification of hemocytes [40], but the initial criterion is most often morphology. Although no general agreement on the number of different cell types in oyster hemolymph has been reached, most investigators divide the hemocytes into at least two major classifications: the granular and the agranular [7,40]. There are apparent differences in the roles that granular and agranular cells play in any organism, and those roles are not necessarily the same for each species. The granular hemocytes are generally larger than the agranular cells and contain walled vesicles (granules) in the cytoplasm. Using light microscopy, investigators have also been able to distinguish acidophilic and basophilic granulocytes [7], stem cells [2], slightly granular cells [40], and differences based on nuclear size [28]. But even with ultrastructural investigation, these studies of oyster hemocytes have not led to a generally accepted scheme of nomenclature and classification. Rather, they have emphasized differences between oyster species and raised conflicting evidence within species. For example, there are major disagreements between invertigations examining the ultrastructure of Crassostrea virginica agranular hemocytes [27,34,48]. Also, the easily distinguishable C. virginia granulocytes was first beleived to be two cell types [30] due to its altered morphology upon degranulation [7].
Molluscan hemocytes serve a variety of functions. It is known that these cells are involved in wound repair (Pauley and Sparks, 1965; des Voignes and Sparks, 1968; Pauley and Heaton, 1969; Ruddell, 1971b), shell repair (Wagge, 1951, 1955), nutrient digestion and transport (Yonge, 1923, 1926; Takatsuki, 1934a; Yonge and Nicholas, 1940; Zacks and Welsh, 1953; Wagge, 1955; Zacks, 1955; Owen, 1966; Purchon, 1968; Cheng and Cali, 1974; Cheng and Rudo, 1976; Cheng, 1977), excretion (Durham, 1892; Canegallo, 1924; Orton, 1923), and internal defense, i.e., cellular immunity (Stauber, 1950, 1961; Tripp, 1958a; b, 1960; Feng, 1959, 1965; and critical reviews by Cheng, 1967; Cheng and Rifkin, 1970; Cheng, 1979).
Worldwide concern over threats to natural resources and public health has led to increased efforts to monitor and assess environmental conditions. This has stimulated the need for development and application of select biological and ecological measurements, or indicators, that are responsive to environmental stress. Measures of bivalve mollusc defence activities, such as haemocyte density, phagocytic activity, locomotion and production of cytotoxic molecules; and haemolymph constituents, such as agglutinins and lysozyme, have potential as indicators and appear to be responsive to xenobiotic chemical insults in the aquatic environment. However, basic research on the relevance of these measurements in inferring resistance to disease or enhanced survival is currently insufficient, reducing their value as potential biomarkers to address environmental objectives. In addition, variation in defence activities caused by seasonal temperature and reproductive cycling, salinity changes, nutritional status, diseases and parasites, and genetic stocks is high and may limit applicability of bivalve defence-related measurements as indicators. This review examines these sources of variability and their possible implications for interpreting changes in bivalve defence activity as an indicator of stress. Examples of contaminant-induced changes in bivalve defence functions are described.
Eastern oysters (Crassostrea virginica) were collected monthly from two sites approximately 15 km apart in Apalachicola Bay, FL. during a 1-y period. Hematologic and serologic measurements were made on hemolymph withdrawn from the adductor muscle. The two sites experienced nearly identical temperature patterns during the study period, but salinity and other physical factors fluctuated. Significant differences attributable to sampling date were found for circulating hemocyte density, phagocytic activity, and superoxide union (O2)-producing ability and for serum protein, lysozyme, and agglutinating activity, with data from both sites combined. This variability was most likely related to temperature or temperature-influenced reproductive cycling. Oyster hemocyte locomotion did not vary significantly with time over the study period, nor were significant differences found between sites. Significant differences between site means (combined for all dates) were found for O2, protein and lysozyme, and significant date * site interactions were found for phagocytosis, agglutination, and lysozyme, indicating that local conditions, such as salinity fluctuations, influenced these measurements. An accurate description of variability in oyster defensive functions will require more frequent sampling and a better understanding of local environmental influences.
Foreign elements, molecules, or organisms naturally or experimentally introduced into naive molluscs are, although with exceptions, phagocytosed (see reviews by Cheng, 1975, 1981). Of the two categories of hemocytes common to all bivalves, hyalinocytes and granulocytes (Cheng, 1981), the latter in Crassostrea virginica and Mercenaria mercenaria are found to be the most active from the standpoint of phagocytosis (Foley and Cheng, 1975). There are three ways, involving semi-permanent filopodia, classical endocytosis and afunnel-like pseudopodia, by which bacteria are engulfed by hemocytes. The uptake mechanism has been studied at the electron microscope level in Crassostrea gigas (Ruddell, 1971; Feng et al., 1977), C. virginica (Cheng and Cali, 1974; Cheng, 1975), and Mytilus coruscus (Feng et al., 1977). Furthermore, as a rule, such phagocytosed materials, if digestible, are degraded by intracellularly, and some are removed when cells containing such materials migrate to the exterior through epithelial borders (Cheng, 1977a). Since granulocytes are more phagocytic than hyalinocytes, and one of the major differences between these two types of cells is the occurrence of large numbers of cytoplasmic granules in granulocytes, further studies to resolve the nature of these granules revealed that those in C. virginica are electron-lucid vesicles each of which possesses a complex wall (Feng et al., 1971; Cheng and Cali, 1974; Cheng et al., 1974; Cheng, 1975). However, the granules of M. mercenaria granulocytes, which are membrane-bound vesicles containing a homogeneously electron-dense substance (Cheng, 1975; Cheng and Foley, 1975), have subsequently been demonstrated to be true lysosomes (Yoshino and Cheng, 1976). Furthermore, it has been established that when challenged with foreign materials, there is hypersynthesis of certain lysosomal hydrolases within the hemocytes and their subsequent release into serum (Cheng et al., 1975; Cheng and Butler, 1979; Cheng and Mohandas, 1985; Mohandas and Cheng, 1985). These lysosomal hydrolases, which are associated with intracellular degradation (Cheng and Cali, 1974; Cheng et al., 1974), have also been demonstrated to have antimicrobial properties (McDade and Tripp, 1967; Cheng, 1978).
The Pacific oysters, Crassostrea gigas, were stressed with different concentrations of benzo(a) pyrene and depurated to determine the hemocyte lysosomal membrane stability and hydrolytic enzymatic activity as a biomarker candidate to the chemical, using NRR (neutral red retention) and API ZYM System, respectively. The membrane damage measured as NRR decrease was significant with the increase of chemical concentration and exposure time (P-galactosidase, \beta-glucuronidase, and N-acetyl- \beta-glucosaminidase. Of them, only two enzymes, acid phosphatase and alkaline phosphatase, showed some potential available for the generation of enzymatic biomarker in the oyster. The results are suggestive of the potential availability of the cellular and enzymatic properties as a biomarker. However, considering that a robust biomarker should be insensitive to natural stress coming from normal physiological variation, but sensitive to pollutants, a concept of intrinsic stress the animal possesses should be taken into consideration. This reflects the necessity of further research on the intrinsic stress affecting the cellular and enzymatic properties of the chemical?stressed oysters prior to using the data as a biomarker.
Worldwide concern over threats to natural resources and public health has led to increased efforts to monitor and assess environmental conditions. This has stimulated the need for development and application of select biological and ecological measurements, or indicators, that are responsive to environmental stress. Measures of bivalve mollusc defence activities, such as haemocyte density, phagocytic activity, locomotion and production of cytotoxic molecules; and haemolymph constituents, such as agglutinins and lysozyme, have potential as indicators and appear to be responsive to xenobiotic chemical insults in the aquatic environment. However, basic research on the relevance of these measurements in inferring resistance to disease or enhanced survival is currently insufficient, reducing their value as potential biomarkers to address environmental objectives. In addition, variation in defence activities caused by seasonal temperature and reproductive cycling, salinity changes, nutritional status, diseases and parasites, and genetic stocks is high and may limit applicability of bivalve defence-related measurements as indicators. This review examines these sources of variability and their possible implications for interpreting changes in bivalve defence activity as an indicator of stress. Examples of contaminant-induced changes in bivalve defence functions are described.
The blood cells of Acteon tornatilis (L.) were examined by light microscope to provide descriptions of cell types, their size distributions, “in vitro” reactions,
and cytochemical properties. The haemolymph contains hyalinocytes (diameter 6.0 sd 0.8μ) which represent about 20% of the
total cell population, and stain acidophilic. Haemocytes with basophilic granules (4–19μm in diameter) represent about 80%
of the total. Some of these granulocytes (about 15%) contain large acidophilic globules. Comparable globule-containing cells
have been described from the digestive glands of other opisthobranch molluscs, and their biological role is discussed.
Today, mollusc farming represents the fourth part of the mundial aquaculture production. These results have been permitted by the improvement of farming techniques commercially important but also by advances in the biology of a species. Valuable reproductive studies have been carried out in s.a. hatcheries on triploids and tetraploids animals. Since the last decade, advances in the knowledge of metabolism and growth mechanisms has helped the improvement of the brood stock. Nevertheless, the mundial expansion of bivalve farming increases the outbreak of strong epizootics. Investigations allow a valuable knowledge on the scope of molluscan defence mechanisms against pathogen agents. The results are discussed in relation to a possible selection for bivalve farming.
Light and electron microscopical studies were carried out to characterize the hemocytes of the oyster, Crassostrea ariakensis. Three types of hemocytes were recognized: granulocytes, large hyalinocytes and small hyalinocytes. The large hyalinocytes and small hyalinocytes were agranular cells. The large hyalinocytes presented thin cytoplasm and high nucleus: cytoplasm ratios. The small hyalinocytes were the most homogeneous in shape and showed round or oval. They have the highest nucleus: cytoplasm ratios and contained very thin cytoplasm. The granulocytes showed abundant cytoplasm and low nucleus: cytoplasm ratios. At the ultrastructural level, the large hyalinocytes cytoplasm contained a variable number of mitochondria, Golgi complex and endoplasmic reticulum. They showed a total absence of cytoplasmic granules or a few small electron-lucid vesicles of different sizes. The granulocytes showed more polymorphic than the agranular cells, with numerous pseudopodia sprouting off their surface. The granulocytes showed similar organelles, but on the contrary they had abundant electron-dense particles or electron-lucent granules in the cell cytoplasm. The small hyalinocytes presented a total absence of cytoplasmic granules and few organelles and only one or two mitochondria were sometimes observed in the cytoplasm. Total hemocyte counts gave a mean (±SE) concentration of 2.06 ± 0.20 × 107 cells mL−1 of hemolymph. Differential hemocyte counts identified the granulocytes as the predominant cell type, followed by the large hyalinocytes and the small hyalinocytes. The percentages of the three cell types in the hemocyte population of C. ariakensis were 68.4%, 21.6% and 9.0%, respectively. The quantitative study of phagocytosis showed that the granulocytes were phagocytic cells and the agranular cells showed a limited phagocytic ability. After the separation in Percoll density gradient, the granulocytes were separated from agranulocytes and pure granulocytes were obtained. However, the separation of different agranular cell types (large hyalinocytes and small hyalinocytes) was not achieved by Percoll or Ficoll gradient centrifugation. Our study provides a morphofunctional basis for the cellular defense mechanisms in C. ariakensis.
Hemocytes of the soft shell clam, Mya arenaria, based on appearance after Romanowsky-type staining, can be shown to be either granulocytes or agranulocytes comprising 76.5 and 23.5% of the total cell population, respectively. Cytoplasmic granules are basophilic, eosinophilic, or refractile. Cytochemical studies indicate that these cells are markedly heterogeneous with respect to certain hydrolytic lysosomal enzymes and in cytoplasmic glycogen and lipofuscin. The overall activities of these enzymes in clam hemocytes, as estimated by the number of reactive sites, were unrelated to one another. Using consecutive double-staining techniques, individual cells were also found to vary in their enzyme content. These findings emphasize the biochemical individuality of circulating hemocytes and the variations noted probably reflect differences in number and composition of lysosomes.
The internal defense mechanism ofHelix pomatia discriminates between different types of foreign cells as demonstrated by determinations of their clearance rates. The rate of elimination is not dependent on the size of foreign cells but on their molecular surface properties. Circulating hemocytes are not involved in the first phase of the clearance event, which is characterized by an accumulation of nonself cells in the digestive gland, kidney and foot muscle ofHelix. Light microscopic studies of these organs reveal nonself cells to be attached to the membrane of cells lining hemolymph sinuses. The attachment of certain types of foreign cells is apparently mediated by opsonins as their clearance depends on the opsonin level of the hemolymph, whereas others are cleared without involvement of opsonizing molecules. Membrane bound molecules of the latter type of nonself cells seem to directly interact with carbohydrate-specific combining sites on the membranes of cells of the sinus walls, as their binding can be inhibited by N-acetyl-galactosamine, and N-acetyl-glucosamine.
The second phase of clearance apparently involves the attraction of circulating hemocytes by organtrapped foreign cells. The number of hemocytes in circulation decreases significantly, whereafter a rising percentage of hemocytes containing foreign cells can be observed in the circulation.
The effects of residing in a polycyclic aromatic hydrocarbon (PAH) contaminated environment on the cytometric characteristics of hemocytes from the American oyster Crassostrea virginica (collected from the Rappahannock River, Virginia, USA in Spring 1991) were analyzed using a multichannel Coulter counter (10000 hemocytes oyster-1). The percentage and relative volume of small-sized hemocytes (>2.5 to 5.1 m) was higher (PP6.2 to 10 m) was observed in the HP-oysters. Maintaining the RR-oysters at the HP site for 8 wk induced statistically significant increases in the number and relative volume contribution of the >2.5 to 5.1 m hemocytes and sharp decreases in the occurrence and relative volume of >6.2 to 13 m cells (P2.5 to 5.1 m) showed a significant decrease over time as compared to the baseline values. On the other hand, the relative number of >6.2 to 13 m hemocytes increased sharply within 8 wk (P
Five subpopulations of Crassostrea virginica hemocytes have been separated by employing a Percoll discontinuous density gradient. As reported previously (T. C. Cheng, J. W. Huang, H. Karadogan, L. R. Renwrantz, T. P. Yoshino, J. Invertebr. Pathol.36, 35–40, 1980), cells comprising subpopulation 2 are hyalinocytes. Quantitative determinations of the activity levels of two lysosomal hydrolases, acid phosphatase and lysozyme, have revealed that granulocytes comprising subpopulation 4 show the highest levels, followed by granulocytes comprising subpopulation 3. This and an earlier report of several differences between members of subpopulations of oyster hemocytes suggest functional differences.
The morphology, tinctorial properties, ultrastructure and some functions of bivalve haemocytes are reviewed in relation to the simple division of these cells into granular and agranular haemocytes, as suggested by Cheng. Whereas granular haemocytes (granulocytes) form a distinct group, agranular haemocytes are heterogeneous in appearance and ultrastructure. Three types of agranular haemocytes are identified; blast-like cells, basophilic macrophage-like cells, and hyalinocytes. Also the early stages of granulocyte development, and spent granulocytes, may be agranular. The distribution of blast-like cells suggests haematopoiesis may be widespread in connective tissue, with further development of haemocytes in the haemolymph. Consequently, the haemocytes of bivalve haemolymph are less differentiated than vertebrate leucocytes, and their composition may vary greatly between individuals. Not all types occur in each bivalve species; scallops lack granulocytes, and the hyalinocyte is a poorly defined cell type in several groups. There is evidence of functional heterogeneity in granulocytes and macrophage-like cells, and the functions of haemocyte types cannot be reliably extrapolated between species. Brown cells (rhogocytes) are regarded as part of the urinary system overlapping in tissue distribution and some functions.
Specimens of Biomphalaria glabrata, NIH albino strain, challenged with 2 μl of heat-killed Bacillus megaterium at a concentration of 9 × 109 bacteria/ml, showed significant elevation of serum aminopeptidase activity level at 2 hr postchallenge when compared with the serum activity levels of the enzyme in snails of the other experimental groups at four time intervals postchallenge. It was found that challenge with a heavy dosage of heat-killed bacteria did not cause a shift in the highest level of enzyme activity from 2 hr postchallenge. Also, the activity of aminopeptidase was higher at 2 hr postchallenge with a greater dosage of heat-killed bacteria.
Today, mollusc farming represents the fourth part of the mundial aquaculture production. These results have been permitted by the improvement of farming techniques commercially important but also by advances in the biology of a species. Valuable reproductive studies have been carried out in s.a. hatcheries on triploids and tetraploids animals. Since the last decade, advances in the knowledge of metabolism and growth mechanisms has helped the improvement of the brood stock. Nevertheless, the mundial expansion of bivalve farming increases the outbreak of strong epizooties. Investigations allow a valuable knowledge on the scope of molluscan defence mechanisms against pathogen agents. The results are discussed in relation to a possible selection for bivalve farming.
Circulating leucocytes of the mussel, Mytilus edulis, were studied by electron microscopy. Based on morphological criteria, the leucocytes were classified as agranular and granular leucocytes, dependent upon the presence or absence of specific granules in their cytoplasm. Furthermore, the existing literature is being critically revised, and it is suggested that agranular and granular leucocytes might belong to the same cell line.
Pericardial hemolymph was obtained from American Oysters (Crassostrea virginica) and the hemocytes characterized by flow cytometry. The cells were found to have a broad unimodal size distribution with a median diameter of 7 micrometers. Total protein measured by flow cytometric fluorescence of dansylated cells also revealed a broad unimodal distribution similar to that obtained for size. The proportion of hemocytes in each stage of the cell cycle was measured using DNA-specific DAPI fluorescence. Histograms showed a single peak representing the G0/G1 population. There was no evidence of S or G2+M phases of the cell cycle, nor was polyploidy seen. The forward and orthogonal light scatter of fixed hemocytes showed no evidence of sub-populations on the basis of cytoplasmic granularity. Thus, in terms of these parameters, oyster hemocytes appear to represent a single population exhibiting graded cellular differences.
It has been demonstrated that hemocytes collected from the American oyster, Crassostrea virginica, 2 hr postchallenge with live Bacillus megaterium are significantly less chemotactic to this bacterium. Since chemotaxis between hemocytes and non-self materials is an integral part of the cellular internal defense mechanism in mollusks, this finding may be important in our understanding of reduced cellular reactions in prechallenged animals.
Glutaraldehyde-fixed hemocytes of Crassostreu t,irginiccr were subjected to differential centrifugation
on a 5, 10, 15, and 25% discontinuous sucrose gradient. Five subpopulations of cells were
separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation
2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation
4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes
and aggregates of small cells. By using several plant and animal lectins, it was ascertained
that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the
albumin glands of Helix pomutirr and Cepcrea nemoralis while those of subpopulation 2
were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying
the Con A-peroxidase cytochemical technique, it was determined that approximately 20%
of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18%
of the large cells comprising subpopulation 5 possess such sites. These results suggest that the
subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may
be further subdivided by differences in specific surface binding sites.
The blood cells of Acteon tomatilis (L.) were examined
by light microscope to provide descriptions of
cell types, their size distributions, "in vitro" reactions,
and cytochemical properties. The haemolymph contains
hyalinocytes (diameter 6.0 sd 0.8 fan) which
represent about 20% of the total cell population, and
stain acidophilic. Haemocytes with basophilic granules
(4-19 fan in diameter) represent about 80% of
the total. Some of these granulocytes (about 15%)
contain large acidophilic globules. Comparable globule-
containing cells have been described from the
digestive glands of other opisthobranch molluscs, and
their biological role is discussed.e&sd
hyalinocytes (diameter 6.0 sd 0.8 fan) which
represent about 20% of the total cell population, and
stain acidophilic. Haemocytes with basophilic granules
(4-19 fan in diameter) represent about 80% of
the total. Some of these granulocytes (about 15%)
contain large acidophilic globules. Comparable globule-
containing cells have been described from the
digestive glands of other opisthobranch molluscs, and
their biological role is discussed.e&sd
Reported variability in numbers and relative proportions of hemocytes in marine bivalves may be related to environmental conditions and laboratory method differences. An automated identification assay, flow cytometry, removes much laboratory bias, but its usefulness is limited because the putative cell types in delineated subpopulations have never been confirmed. The present study was designed to: (1) confirm the identity of oyster hemocyte subpopulations described by flow cytometry, and (2) use flow cytometry in an experimental analysis of potential causes of variation. Light-scatter flow cytometry consistently differentiated three subpopulations in oysters from two mid-Atlantic (USA) sites. Cell sorting and microscopy identified them as granular, small granular, and agranular (hyalinocytes and apparently degranulated) hemocytes. Subpopulation proportions estimated by microscopy and by flow cytometry were significantly correlated (r(2) = 0.27 to 0.50). In a 4-week laboratory experiment, neither temperature (12 vs. 22 degrees C) nor food (fed vs. not fed) had a statistically significant effect on total or differential counts, or on hemocyte viability. Most of the variability was attributable to individual differences and was very similar to that reported for vertebrates. We hypothesize that variability in molluscan hemocytes may be more immediately linked to individual metabolic condition than to ambient changes.
Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology
Hemocytes of Crassostrea virginica were video recorded and tracked to determine their locomotive rates and to assign these rates to Wright-stained morphological variants. From 24 oysters examined in January, February, March, and May, 1571 hemocytes were video recorded, identified, and their rate of locomotion (ROL) measured. Granulocytes (three types) and agranulocytes (one lymphoid and three nonlymphoid types) were recognized. Focusing on 15 oysters in March and May, 20,318 hemocytes were counted from duplicate slides to verify the classification and to show that predominant hemocytes vary greatly between samples and among individual oysters, yet population differences can be detected. Measured rates of locomotion indicate that the granulocyte subpopulation moved significantly faster (3.3 microns/min) than the agranulocyte subpopulation (0.7 microns/min) because most (81%) agranulocytes were not mobile. Of the mobile hemocytes, granulocytes were also significantly faster (4.8 microns/min vs. 3.5 microns/min, P less than 0.0001), and basophilic granulocytes (BASOs) were the most active and abundant cell type. Examination of monthly percentages of cells and ROL indicates, however, that granulocyte dominance and ROL are not invariable. Granulocyte percentages of more than 60% in January, February, and March decreased to 32% in May, and BASO dominance was reduced to 15%. Further, percentages of mobile granulocytes decreased from greater than 65% in January, February, and March to 50% in May. ROL for all cells decreased from greater than 2.3 microns/min in these months to 1.0 microns/min in May. The fewer mobile hemocytes tracked in May had significantly (P less than .05) lower average ROL (4.0 microns/min) than those in January and March (4.7 microns/min each). Agranulocytes increased in May due to an increase in nonlymphoid cells.
The number and types of hemocytes in four size groups of the clam species Sunetta scripta and Villorita cyprinoides var. cochinensis, and leukocytosis in the 38- to 40-mm-size-group clams of both species subsequent to challenge with Vibrio alginolyticus at a concentration of 1 x 10(8) cells/0.02 ml of sterile 2% saline was investigated. In S. scripta, the mean total hemocyte count in the 42- to 44-mm size group was significantly lower than that of the three other size groups but there was no significant variation in total cell counts in the four size groups of V. cyprinoides var. cochinensis. Only two types of hemocytes, granulocytes and agranulocytes, occur and the percentage of agranulocytes was roughly half of that of granulocytes. The data on the effects of sham injection and Vibrio injection suggest that there is significant leukocytosis early in both clam species as a result of sham injection; the bacterial challenge produces significant leukocytosis in V. cyprinoides var. cochinensis both early (6 and 12 hr) and later (48,96 and 120 hr), but only at 48 hr in S. scripta; and in both clam species there is significant increase in total cell counts in Vibrio-injected ones than in untampered controls at various time intervals.
Fluorescein-labeled concanavalin A (F-Con A) was employed as a membrane probe to compare the surface glycosyl components associated with glass-adherent circulating hemocytes from a schistosome-susceptible (PR albino) and schistosome-refractory (10-R2) stock of snails Biomphalaria glabrata. Under hemolymph-free conditions, granular hemocytes (granulocytes), composed of two morphologically-distinct cell populations (rounded and spread hemocytes) bound F-Con A via specific carbohydrate surface receptors. Con A-binding induced in rounded hemocytes a redistribution (patching and capping) of lectin-receptor complexes. Receptor redistribution in both snail stocks was characterized by a high capping frequency (80–90% of adherent rounded cells) and temperature dependency. Differences in the response of albino and 10-R2 snail hemocytes to differing F-Con A concentrations, however, suggest that cells may possess subtle differences in physiology or function.
Vibrio vulnificus is a naturally occurring marine bacterium that causes invasive disease of immunocompromised humans following the consumption of raw oysters. It is a component of the natural microbiota of Gulf Coast estuaries and has been found to inhabit tissues of oysters, Crassostrea virginica (Gmelin 1791). The interaction of V. vulnificus with oyster host defenses has not been reported in detail. We examined the interaction of V. vulnificus with phagocytic oyster hemocytes as a function of time, temperature, bacterial concentration, pretreatment with hemolymph, and V. vulnificus translucent and opaque colony morphotypes. Within these experimental parameters, the results showed that the association of V. vulnificus with hemocytes increased with time, temperature, and initial V. vulnificus/hemocyte ratio. Pretreatment of V. vulnificus with serum or an increased serum concentration did not enhance V. vulnificus-hemocyte associations, a result suggesting the absence of opsonic activity. More than 50% of hemocytes bound the translucent, avirulent morphotype, whereas 10 to 20% were associated with the opaque, virulent form, a result indicating that the degree of encapsulation was related to resistance to phagocytosis, as previously described for mammalian phagocytes. Understanding these cellular interactions may, in part, explain the persistence of V. vulnificus in oyster tissues and the ecology of V. vulnificus in estuarine environments.
Molluscs bivalves have been widely used as bioindicators to monitor contamination levels in coastal waters. In addition, many studies have attempted to analyze bivalve organs, considered pollutant-targets, to understand the bio-accumulation process and to characterize the effects of pollutants on the organisms. Here we analyzed the effects of mercury exposure on flat oyster hemocytes. Optical and electronic microscope procedures were used to characterize hemocyte morphology. In addition, cell solutions treated with acridine orange were analyzed by flow cytometry and laser scanning cytometry in order to evaluate the variations of cytoplasmic granules (red fluorescence, ARF) and cell size (green fluorescence, AGF) of hemocyte populations over time. Light and electron microscopical studies enabled us to differentiate four hemocyte subpopulations, agranulocytes (Types I and II) and granulocytes (Types I and II). Slight morphological differences were observed between control and Hg-exposed cells only in granulocytes exposed to Hg for 30 days, where condensed chromatin and partially lysed cytoplasmic regions were detected. Flow and laser scanning cytometry studies allowed us to differentiate three hemocyte populations, agranulocytes (R1) and granulocytes (R2 and R3). The exposure time to Hg increased the average red fluorescence (ARF) of agranulocytes and small granulocytes, while there was no change in large granulocytes, which showed a loss of membrane integrity. In control oysters, the three hemocyte populations showed an increase of ARF after 19 days of exposure although initial values were restored after 30 days. The average green fluorescence (AGF) was more stable than the ARF throughout the experiment. In Hg-exposed oysters, the values of AGF of agranulocytes showed an increase at half Hg-exposure period while the AGF values of large granulocytes decreased throughout the experiment, confirming the instability of these types of cells. The relative percentage of small granulocytes and granulocytes showed time variations in both control and exposed oysters. However, the values of small granulocytes remained constant during the whole experiment. The fact that there were only changes in agranulocytes and large granulocytes suggested a possible relationship between these two types of cells. In a quantitative study, we found a significant linear relationship between the agranulocytes and large granulocytes.
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