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Demonstration of Lysosomal Enzymes in Hemocytes of Mercenaria mercenaria (Mollusca: Bivalvia)
Abstract
Seven hydrolytic enzymes (acid phosphatase, beta- glucuronidase, beta-N-acetylglucosaminidase, acid-beta-galactosidase, and A-, B-, and C-esterases were demonstrated by cytochemical techniques in hemocytes of Mercenaria mercenaria prior to and following in vitro exposure to the dinoflagellate Isochrysis galbana. All of the hydrolases were localized in blunt granules; acid phosphatase, beta-glucuronidase, and the esterases also were found in dot-like cytoplasmic granules. Acid-beta-galactosidase, beta-N-acetyl‐ glucosaminidase, and A-, B-, and C-esterases were observed within the phagosomes, while acid phosphatase and beta-glucuronidase were present only in the granules adjacent to these organelles. Following phagocytosis of algae, elevated levels of enzyme activity were demonstrated.
... A I'aide de techniques cytochimiques couplées à la microscopie optique, plusieurs auteurs ont mis en évidence différentes hydrolases lysosomales au niveau de diverses inclusions cytoplasmiques. Ainsi des estérases non spécifiques, des B glucuronidases et des phosphatases acides et alcalines ont été révélées dans les hémocytes de M. mercenaia (Moore et Gelder, 1985), de C. viryinica (Feng et Feng, 1971) et de M. arenaria (Huffman et Tripp, 1982). Ces deux demiers auteurs notent d'importantes variabilités dactivité enzymatique entre les cellules de la même lignée. ...
... Les Moore et Gelder (1985) modifiée. La classe 1 comprend les hémocytes dont des granules positifs occupent O à 25 7o de la surface cellulaire, dans la classe 4, fes granules occupent 75 à 100 olo de la surface. ...
... En effet, des phosphatases acides et des estérases non spécifiques ont été révélées dans les granules des granulocytes. Ces résultats concordent avec ceux de plusieurs auteurs ctrez différentes espèces de bivafves (Moore et Lowe, 1977;Moore et Gelder, 1985;Gelder et Moore' 1986)' ...
La capacité bioindicatrice des mollusques bivalves vis-à-vis des métaux est actuellement reconnue et utilisée dans le cadre du biomonitoring. La compréhension de cette capacité nécessitait une étude plus fondamentale des voies d'intoxication, de transport et d'élimination des métaux. Une étude anatomique, cytologique et microanalytique des hémocytes, de la glande péricardiale et des reins a montré le rôle des granulocytes dans le transport puis le rôle de détoxication de la glande péricardiale et des reins. Le système lysosomal joue un rôle important. La nature du métal modifie la répartition au sein des organes. Le plomb est bioconcentré en association avec P, S, Ca dans les concrétions des cellules du rein distal. La microanalyse de rayons x a été, à ce titre performante. Une adaptation éventuelle à la présence de micropolluants métalliques a été recherchée par numération des hémocytes, suivi de l'activité enzymatique et des capacités de phagocytose. Il y a tendance à l'augmentation du nombre des hémocytes ; augmentation de l'activité phosphatasique acide mais pas de modification significative de l'activité de phagocytose. Les réponses cytochimiques et biochimiques des hémocytes en présence de métaux peuvent être proposées comme méthodes d'évaluation précoce (7 jours) des effets de la contamination des eaux par les métaux ; le niveau de bioaccumulation étant évalué par la méthode des transplantations seulement après trois semaines
... m.56982; Table 2), which is known to catalyze the hydrolysis of galactosides into monosaccharides, was also found to be significantly higher in the dorsal as compared to the ventral tract. β-galactosidases are produced by hemocytes (Moore and Gelder, 1985) as well as the secreting cells (i.e. apocrine cells) located in the epithelium of the digestive tract (e.g. ...
... esophagus, intestine) of mollusks (Martin et al., 2011). They actively participate in the intracellular digestion of microbes after phagocytosis (Moore and Gelder, 1985). This enzyme could contribute to the early digestion of microbes although its source in the dorsal tract is unknown but might be related to a possible higher abundance of specific secretory cells along the lateral ordinary filaments and/or principal filaments of the gill plicae. ...
In the oyster Crassostrea virginica , the organization of the gill allows bidirectional particle transport where a dorsal gill tract directs particles meant to be ingested while a ventral tract collects particles intended to be rejected as pseudofeces. Previous studies showed that the transport of particles in both tracts is mediated by mucus. Consequently, we hypothesized that the nature and/or the quantity of mucosal proteins present in each tract is likely different. Using endoscopy-aided micro-sampling of mucus from each tract followed by multidimensional protein identification technologies, and in situ hybridization, a high spatial resolution mapping of the oyster gill proteome was generated. Results showed the presence in gill mucus of a wide range of molecules involved in non-self recognition and interactions with microbes. Mucus composition was different between the two tracts, with mucus from the ventral tract shown to be rich in mucin-like proteins, providing an explanation of its high viscosity, while mucus from the dorsal tract was found to be enriched in mannose-binding proteins, known to be involved in food particle binding and selection. Overall, this study generated high-resolution proteomes for C. virginica gill mucus and demonstrated that the contrasting functions of the two pathways present on oyster gills are associated with significant differences in their protein makeup.
... On the one hand, hemocytes are abundant in lysosomal hydrolases relative to metal transport and detoxication, 70 and their enzymatic activity was reported to be significantly elevated following phagocytosis. 71 On the other hand, in invertebrate tissues, granules larger than 1 μm were formed by the aggregation or fusion of immature and smaller granules with a lysosomal nature. 16,66 Consistently, the high abundance of 12 C 14 N − and 32 S − in these Ag-rich granules ( Figure 5A−C) was indicative of the preservation of enzymatic properties 66 and the metabolic products of lysosomal proteins (e.g., Ag-bound MT). ...
... 16,66 Consistently, the high abundance of 12 C 14 N − and 32 S − in these Ag-rich granules ( Figure 5A−C) was indicative of the preservation of enzymatic properties 66 and the metabolic products of lysosomal proteins (e.g., Ag-bound MT). 16 One previous study reported that more kinds of enzymes were detected in blunt granules than in dot-like granules, 71 which somehow agreed with the lower abundance of 12 C 14 N − in type 2 granulocytes ( Figure 5E−G) compared to type 1 granulocytes. As described in Figure 6, the higher Pearson coefficient between 109 Ag and 107 Ag (R = 0.949, p < 0.01) in type 1 granules demonstrated complete degradation of 109 AgNPs (transformed into Ag(I)) by lysosomal enzymes, whereas the weaker Pearson correlation (R = 0.471, p < 0.01) between 109 Ag and 107 Ag in type 2 granules indicated different forms of Ag distribution in the cytosol. ...
The extensive application of silver nanoparticles (AgNPs) requires a full examination of their biological impacts, especially in aquatic systems where AgNPs are likely to end up. Despite numerous toxicity studies from molecular to individual levels, it is still a daunting challenge to achieve in situ subcellular imaging of Ag and to determine the sites of AgNP interaction with organelles or macromolecules simultaneously. Here, by coupling high-resolution nanoscale secondary ion mass spectrometry elemental mapping with scanning electron microscopy ultrastructural characterization, we successfully visualized the subcellular localization and the potential toxicity effects of AgNPs in the oyster gill filaments. The stable isotope tracing method was also adopted to investigate the respective uptake and transport mechanisms of differently labeled 109AgNPs and 107Ag+ ions. 109Ag hotspots were colocalized with endosomes or lysosomes, proving an endocytosis-based entry of AgNPs which passed through the barrier of oyster gill epithelium. These 109Ag hotspots showed a strong colocalization with 32S-. For the first time, we provided visualized evidence of AgNP-induced autophagy in the oyster gill cells. We further identified two categories of hemocytes (blood cells) and illustrated their roles in AgNP transport and sequestration. The integration of morphological and functional aspects of Ag subcellular distribution in different target cells suggested that oysters were equipped with a specialized endolysosomal (epithelial cells) or phagolysosomal system (hemocytes) in regulating the cellular process of AgNPs, during which the lysosome was the most involved organelle and sulfur was the most relevant macronutrient element. This study highlighted not only the intracellular but also the intercellular AgNP translocation and transformation, providing important subcellular imaging of silver and reliable methodology regarding bio-nano interactions in natural environments.
... In this study, the lysosomal content was very low at 0 and 5 salinities, and with long-exposure time, a further decrease in the content was evident which could be linked to mortality of hemocytes (Gagnaire et al., 2006a(Gagnaire et al., , 2006b, eventually thinning out the ability of an organism to fight against pathogens. Moore and Gelder (1985) demonstrated an increase in the activity of hydrolytic enzymes or lysozyme including acid phosphatase, betaglucuronidase, beta-N-acetylglucosaminidase, acid-betagalactosidase after phagocytosis of the dinoflagellate Isochrysis galbana. These enzymes are secreted during phagocytosis (Cheng et al., 1975) and are involved in the degradation of glycoprotein complexes thus proving their role in destruction of phagocytosed organisms (Moore and Gelder, 1985). ...
... Moore and Gelder (1985) demonstrated an increase in the activity of hydrolytic enzymes or lysozyme including acid phosphatase, betaglucuronidase, beta-N-acetylglucosaminidase, acid-betagalactosidase after phagocytosis of the dinoflagellate Isochrysis galbana. These enzymes are secreted during phagocytosis (Cheng et al., 1975) and are involved in the degradation of glycoprotein complexes thus proving their role in destruction of phagocytosed organisms (Moore and Gelder, 1985). ...
Paphia malabarica is a predominant and commercially important bivalve in India, persistently challenged by wavering salinity in a monsoon-influenced estuary. To examine the organism's immunological response under such a condition we challenged P. malabarica with different salinities (0, 5, 15, 25 and 35) for varied periods using a two-way experimental approach (in vitro and in vivo). This is the first study to report the response of P. malabarica hemocytes to salinity stress from a monsoon-influenced estuary on the southwest coast of India. Evaluation of total hemocytes count, mortality, lysosomal content, reactive oxygen species production, phagocytic and esterase activity was carried out using flow cytometric analysis. In both the experimental conditions, hemocyte parameters were significantly compromised at lower salinities (0 and 5) with an evident immuno-salinity tolerance range of 15–35. The damaging impact of 0 and 5 salinities on hemocyte function intensified with a longer exposure period, indicating that prolonged exposure to low salinity could be detrimental to bivalve wellness if they are pushed beyond their tolerance range which is usually observed during the monsoon. Further studies should focus on the interactive effect of salinity tagged with different stressors influencing biology of P. malabarica.
... No correlation was found between the phagocytic index employed and the other immunological measures of hydrogen peroxide and lysozyme production. Moore and Gelder (1985) showed that Mercemria mercenaria which demonstrated increased phagocytosis, also had increased haemolymph enzyme activity. It was expected that a similar phenomenon would occur in the present study, as contact between bacteria and haemocytes is required for both respiratory burst activity (producing hydrogen peroxide) and for phagocytosis. ...
p>The aim of this study was to provide a comprehensive set of quantitative and qualitative baseline responses at physiological, metabolical and immunological levels, in the Pacific oyster Crassostrea gigas (Thunberg), the European flat oyster Ostrea eduli (L.), and the Manila clam Tapes philippinarum (Adams and Reeve). The energetics of these species were compared across a matrix of temperature and salinity conditions. Field trials examined the effect of exposure of three O. edulis populations to infection by the protozoan parasite Bonamia ostreae , and enzyme electrophoresis investigated the genetic basis for any differences. Changes in immunocompetence were monitored from field samples and with controlled Vibrio anguillarum bacterial challenges. Haemolymph and haemocytic responses were recorded.
Filtration rate had the most significant effect on scope for growth (SFG) indices measured in all species. C. gigas showed a much wider range of filtration rates than O. edulis and consequently had much higher SFG. Optimum environmental conditions for C.gigas occurred at 20-25<sup>o</sup>C and 19-25�, compared with 20<sup>o</sup>C and 33� for O. edulis , and 15-20<sup>o</sup>C at 33� in T. philippinarum . Separate winter and summer physiological behaviour was detected in C. gigas and O. edulis , with the change occurring at 15<sup>o</sup>C and 10-12<sup>o</sup>C respectively. Body condition indices were inversely proportional to SFG and were probably related to the reproductive cycle. Temperature was shown to have the most significant influence on energetic factors, with salinity having little effect.
Field trials investigating Bonamia effects in three O. edulis populations found a significant, inverse size relationship with most of the physiological measurements.</p
... Mercenaria mercenaria Mytilus edulis Mytilus galloprovincialis Moore and Gelder, 1985Pipe, 1990Carballal et al., 1997a Phosphatase acide ...
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.
... La présence extracellulaire des enzymes hydrolytiques lysosomales résulterait d'une dégranulation des granulocytes durant la phagocytose . Les activités lysosomales ont largement été étudiées chez les mollusques bivalves marins (Moore & Gelder, 1985 ;Beckmann et al., 1992 ;Toreilles et al., 1997 ;. ...
Le travail de thèse s’inscrit dans la problématique très actuelle des mortalités massives de naissain et de juvéniles d’huîtres creuses, C. gigas, et des questionnements autour de l’implication des pesticides dans ce phénomène. La première partie de la thèse a été consacrée à l’étude des effets de pesticides sur les capacités hémocytaires de l’huître creuse (in vitro et in vivo). L’effet immuno-modulateur des xénobiotiques sélectionnés (seul ou en mélange) a été exploré principalement au travers de la cytométrie en flux. La deuxième partie de la thèse a concerné l’étude des effets d’un mélange de pesticides sélectionnés sur le virus OsHV-1 et l’infection qu’il induit chez l’huître creuse. Les effets des pesticides sur le virus lui-même ont été évalués. Les expériences réalisées n'ont pas permis de mettre en évidence, dans les conditions testées, de dégradation des particules virales en présence des polluants. D’autre part, leurs effets sur les huîtres elles-mêmes ont été explorés après traitement des animaux avec un mélange de pesticides lors d’essais de pathologie expérimentale. Il a ainsi pu être montré que des huîtres préalablement contaminées par un mélange de pesticides à des concentrations retrouvées dans l’environnement semblaient être plus sensibles à l’infection virale. La troisième partie de la thèse a concerné l’étude de l’autophagie chez l’huître creuse, C. gigas. La publication du génome complet de cette espèce en 2012 a ouvert de nouvelles possibilités pour étudier l'immunité innée. L’étude de l’autophagie réalisée pour la première fois chez l’huître creuse a consisté lors d’une première étape en la recherche in silico de gènes impliqués dans cette voie et des protéines correspondantes par western blotting. Puis, le rôle de ce processus important dans l’immunité innée a été exploré au travers d’essais de reproduction d’infections en présence ou non de modulateurs de l’autophagie. Les résultats obtenus semblent montrer que l’autophagie soit un processus important pouvant être impliqué dans les capacités de défense de l’huître creuse vis-à-vis d’infections virales et bactériennes.
... Haemolymph cells of several species of molluscs have been characterized on the basis of enzymatic properties (Bayne et al., 1979; Granath and Yoshino, 1983). The role of lysosomes in molluscan phagocytes involves degradation of phagocytized material, as synthesis of lysosomal enzymes occurs after stimulation with certain exogenous agents (Moore and Gelder, 1985; Carballal et al., 1997). It was observed that b-glucuronidase, a widespread lysosomal hydrolase, is signi®cantly inhibited after TBT exposure (Fig. 1c). ...
The main purpose of this research is to detect and select bioindicators which may be helpful for monitoring and controlling coastal ecosystem quality with special attention to the risk of poisoning by toxic organotin compounds. A few species of marine invertebrates have been chosen, i.e., tunicates, molluscs and echinoderms, to test their reliability as indicators. The immunotoxicity test applied here is based on the analysis of the effects on in vitro yeast phagocytosis by haemocytes. In addition, the embryotoxicity test examines the effects of these compounds on various embryonic developmental stages beginning from post-fertilisation. The obtained results suggest that the effects of these environmental pollutants: i) are concentration-dependent, ii) are strictly related to their lipophilic properties, iii) influence cell calcium homeostasis, and iv) inhibit mitochondrial oxidative phosphorylation.
... La présence extracellulaire des enzymes hydrolytiques lysosomales résulterait d'une dégranulation des granulocytes durant la phagocytose . Les activités lysosomales ont largement été étudiées chez les mollusques bivalves marins (Moore & Gelder, 1985 ;Beckmann et al., 1992 ;Toreilles et al., 1997, Xue & Renault, 2000Da Silva et al., 2008). Les enzymes hydrolytiques lysosomales sont composées principalement de la β-glucuronidase, la phosphatase acide (ACP) et la phosphatase alcaline (ALP), les aminopeptidases, les estérases, l'amylase, la lipase, la β-galactosidase et le lysozyme. ...
The history of the French oyster production highlights the fragility of this production against overexploitation and disease outbreaks. In particular, the production of flat oyster, Ostrea edulis, has decreased following the emergence of two parasitic diseases including bonamiosis. The means to fight against bonamiosis are relatively limited. They are mainly based on oyster health surveillance to limit the spread of the disease. However, the use of predictive models of disease progression in infected area would help to improve stock management and minimize the impact pathogens. Moreover the development of resistant animals could help to revive this production. These different approaches require appropriate diagnostic tools, a good knowledge of the life cycle of the pathogen, and the interactions between the parasite and its host. In this context, the main objective of the phD work is to understand the interactions between the flat oyster Ostrea edulis and the parasite Bonamia ostreae, and particularly the molecular basis of the resistance to the parasite. In a first step, a subtractive cDNA bank allowed the identification of ESTs differentially expressed in haemocytes in response to the parasite. Expression of some genes, among which a galectin, was measured by Real Time PCR in the context of in vitro infections. In addition, the cellular response was investigated by flow cytometry and the infection was checked by microscopy. These experiments showed a multiplication of the parasite inside haemocytes associated with a decreased of esterases and of the production of ROS. In a second step, a comparative approach was carried out between a population of oysters resistant to bonamiosis and a natural population. Results suggest that modulation of apopotosis and decrease of phagocytosis could be involved in mechanisms related to resistance to bonamiosis. This work is the first study on the response of haemocytes of flat oysters to an infection with the parasite Bonamia ostreae at the cellular and molecular levels.
... There are reports that these enzymes viz., acid phosphatase, phenol oxidase and lysozyme have their origin from hemocytes. The granulocytes secrete these enzymes into the hemolymph when there is an infection in Mercenaria mercenaria (Moore and Gelder, 1985) and C. virginica (Cheng, 1990). In the present study, there was an increase in the granulocytes in the hemolymph and a corresponding increase of enzymes in the serum. ...
Juveniles of Crassostrea madrasensis (mean weight 85.5 ± 2.3 g) were exposed to live cells of Vibrio alginolyticus (1.2 x 10 6 cells g -1) by intramuscular injection. Hemolymph samples were collected at different time intervals to study the modulations in the cellular and humoral factors. There was an increase in total hemocyte count, percentage granulocytes, serum protein, serum acid phosphatase, serum phenol oxidase and serum lysozyme in response to bacterial challenge upto three to five days post-injection. A decrease in the ability of hemocytes to phagocytose yeast cells was also noted. The hemolymph parameters of the test group became similar to that of control animals within two weeks of exposure to live bacterial cells.
... 4). The significant defense role temperature of 25 'C, which favors parasite developof lysosomal enzymes in bivalve molluscs has also ment, may be one of the reasons why 100 % of the been documented (e.g. McDade & Tripp 1967a. b, challenged oysters are infected at 25 "C with the Cheng 1978, Cheng 1979, Huffrnann & Tripp 1982, est disease intensity. Moore & Gelder 1985), and reviewed and dicussed by Cheng (1983a, b, c) and Chu (1988). L concentrations in oysters also decreased with increased salinity (Chu et The ability to agglutinate latex beads was greater in 40 - z ...
... A number of lysosomal enzymes were de-214 Dis Aquat Org 30: 209-215,1997 tected in Ruditapes decussatus haemocytes and the presence of acid phosphatase in the granules suggests that these granules are lysosomes (Lopez et al. in press). The presence of lysosomal enzymes in bivalve mollusc haemocytes was cited by several authors (Moore & Gelder 1985, Feng 1988, Beckrnann et al. 1992. The absence of oxidative metabolism coupled to phagocytosis in R. decussatus haemocytes was previously reported (L6pez et al. 1994). ...
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.
... Acid phosphatases (ACP) (E.C.3.1.3.2) were demonstrated by the azo-dye coupling method (Diagnostic kit 386, Sigma Chemical Co.) using naphthol AS-B1 phosphate as substrate and Fast Garnet GBC as coupler (Goldberg & Barka 1962). The change in the presence and relative amounts of ACP following the exposure to metals was assessed according to the method of Moore & Gelder (1985) modified for the present study. To indicate the relative degree of reactivity, 200 hemocytes were examined on each slide and assigned to 1 of 4 categories: class 1 (0 to 25% of the endoplasm filled with positive granules), class 2 (25 to 50% of the endoplasm filled with positive granules), class 3 (50 to 75 % of the endoplasm filled with positive granules) and class 4 (75 to 100% of the endoplasm filled with positive granules). ...
This study examines the structural changes of the lysosomal system of the hemocytes of the zebra mussel Dreissena polymorpha experimentally exposed to lead and zinc. A cytochemical technique which demonstrated acid phosphatase activity as a lysosomal marker was used on blood cell monolayers. The results indicate that the effects of both metals on hemocytic lysosomes were variable and that no marked linear relationship between lysosomal changes and metal concentration and exposure time was observed. Hemocytes of exposed zebra mussels exhibited enlarged and/or more numerous lysosomes. The use of the lysosomal changes in the hemocytes as a biomarker of contaminant exposure is discussed.
... These enzymes have also been demonstrated in other bivalves. Acid phosphatase has been identi¢ed in the haemocytes of M. edulis (Moore & Lowe 1977 ), Mytilus californianus (Bayne et al.1979 ), Biomphalaria glabrata (Granath & Yoshino1983), Mercenaria mercenaria (Moore & Gelder 1985), Lymnaea luteola (Jyothirmayi & Rao1988), C. virginica (Cheng1989; Cheng & Downs 1998), Viviparous ater (Franchini & Ottaviani 1990), Sunetta scripta and Villorita cyprinoides var cochinensis (Suresh & Mohandas 1990b), Mya arenaria (Beckmann, Morse & Moore 1992 ) and Ruditapes decussatus (Lopez, Carballal, Azevedo & Villalba 1997). Acid phosphatase and peroxidase play an important role in aggregation and release of the antibacterial agents as reported in Tridacna crocea by Nakayama et al. (1997). ...
The haemocytes of the Indian edible oyster Crassostrea madrasensis were characterized using light and electron microscopy. The light microscopic study was conducted by staining a monolayer of the haemocytes with Geimsa. Cells without granules and with a large nucleus occupying much of the cytoplasmic area were grouped as hyalinocytes. Those with lesser amounts of basophilic cytoplasmic granules were characterized as semigranulocytes and those with large amounts of a mixture of acidophilic and basophilic granules were termed as granulocytes. Ultrastructural studies also revealed the presence of three types of haemocytes. Scanning electron microscopic studies were used to study the spreading behaviour of the haemocytes. Cytochemical studies revealed the presence of acidphosphatase, peroxidase and prophenol oxidase in the cells.
... Haemolymph cells of several species of molluscs have been characterized on the basis of enzymatic properties (Bayne et al., 1979; Granath and Yoshino, 1983). The role of lysosomes in molluscan phagocytes involves degradation of phagocytized material, as synthesis of lysosomal enzymes occurs after stimulation with certain exogenous agents (Moore and Gelder, 1985; Carballal et al., 1997). It was observed that b-glucuronidase, a widespread lysosomal hydrolase, is signi®cantly inhibited after TBT exposure (Fig. 1c). ...
The aim of this investigation was to quantify the effects of tributyltin (TBT) on the immune reactivity of haemocytes from the cultivated clam Tapes philippinarum (Adams and Reeve, 1850) using a series of in vitro bioassays. It is known that TBT has adverse effects on cellular immune functions like mobility, phagocytosis and lysosomal enzyme activity. As defining TBT-sensitive immunologic biomarkers in sentinel organisms is important in the field of ecotoxicology, the authors propose three indexes, amoebocytic (A.I.), phagocytic (P.I.), and lysosomal activity (L.A.I.), as sensitive and useful biomarkers to assess environmental risks due to TBT contamination.
... The occurrence of acid phosphatase activity, especially in the GR of P. perna, suggested that at least some of its granules correspond to a lysosomal system and function in the intracellular degradation of ingested foreign particles. The presence of a variety of lysosomal enzymes and other degrading factors has already been demonstrated in the granules of di#erent bivalve species (Cheng & Rodrick, 1975;Hu#man & Tripp, 1982;Yoshino & Cheng, 1976;Moore & Gelder, 1985;Pipe, 1990;Carballal et al., 1997c;Pipe et al., 1997). Both cell types of P. perna, but especially the GR, were capable of phagocytosing the yeast S. cerevisiae and zymosan particles in vitro. ...
The aim of this study was to establish the normal pattern of some haemato-immunological parameters in the mussel Perna perna to serve as a reference for further studies using this species as a biological indicator of environmental contaminants. We investigated the morphology and cytochemistry of the circulating haemocytes of P. perna , their role in phagocytosis and superoxide anion production, their numbers and composition, the agglutinin concentration in the plasma and the presence of phenoloxidase activity. Two blood cell types, hyalinocytes (HY) and granulocytes (GR), were found in the haemolymph of P. perna . HY were agranular cells with a basophilic cytoplasm, whereas GR contained abundant eosinophilic granules. HY comprised approximately 60% of the blood cell types in the circulation. Both haemocyte types, but especially the GR were actively phagocytic and contained acid phosphatase. After stimulation with zymosan, the blood cells were able to reduce NBT to formazan, suggesting the generation of the superoxide anion. Glycogen was not histochemically detectable in either haemocyte type. The number of cells in circulation (THC) did not significantly vary with sex or size of the mussels (3,145·5 and 3,668·5 cells/mm3in males and females respectively). The mussel plasma had an agglutinating activity against different mammalian erythrocytes. A phenoloxidase activity was not encountered either in the haemocyte fraction or in the mussel plasma, even after induction with the protease trypsin or non-self molecules, such as LPS from Gram-negative bacteria and β-1, 3-glucans from fungi.
... The hydrolytic effect of different lysosomal enzymes is one of the most important mechanisms involved in degradation of pathogenic organisms both inside and outside the hemocytes (Cheng, 1978(Cheng, , 1983. As a result, enzymatic activities in hemolymph have been studied as one of the immune capacity indicators in many bivalve species (Cheng, 1976(Cheng, , 1988Moore and Gelder, 1985;Gelder and Moore, 1986;Cheng and Downs, 1988;Chu and La Peyre, 1989;Beckmann et al., 1992;Hine and Wesney, 1994a;Toreilles et al., 1997). However, certain pathogens are able to survive enzymatic damage mediated by bivalve hemocytes. ...
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.
... The latter, is also enhanced by the ultrastructural localization of BSD in the dense bodies of the palps. Dense bodies that are found in various molluscan cell types are regarded as lysosomes (Moore and Gelder, 1985; Dimitriadis and Liosi, 1992; Pal and Cajaraville, 1994) and have already been reported as BSD accumulating sites in the digestive gland and the gills of M. galloprovincialis (Domouhtsidou and Dimitriadis, 2000). In the present study, metal localization in the palps is further enhanced by the results of X-ray microanalysis, performed after focusing the electron beam on the BSD, according to which Hg is traced on the autometallographical deposits. ...
The intracellular localization of heavy metals using autometallography (AMG) and X-ray microanalysis was studied in the palps, the digestive gland and the gills of mussels Mytilus galloprovincialis, after an experimental exposure to 0.1 mg l(-1) of Hg and 0.1 mg l(-1) of Pb, for 30 and 60 days. In the examined tissues, autometallographical black silver deposits (BSD) were localized mainly in the residual bodies and heterolysosomes of the digestive cells, as well as in the dense bodies of the epithelial cells. Metal deposition after Hg exposure was much more abundant compared to Pb exposure. Using X-ray microanalysis, Hg was traced on the BSD in all examined tissues, while Pb was not traced in these deposits. The results are discussed in comparison to previous results on long-term exposure to the same metals; in addition, the palps are introduced as a new metal storing organ and, finally, the use of X-ray microanalysis under a scanning electron microscope in order to enhance the specificity of AMG is suggested.
... They also contain hydrolytic enzymes and produce reactive oxygen 3 species (ROS), which play a key role in pathogen degradation (13)(14)(15). They have been used as immune capacity indicators in many bivalve species (16)(17)(18)(19). ...
The Pacific oyster, Crassostrea gigas, is extensively cultivated and represents an important economic activity. Oysters are reared in estuarine areas, subjected to various biotic and abiotic factors. One of the limiting factors in aquaculture is mortality outbreaks, which may limit oyster production, and the causes of these outbreaks are not completely understood. In this context, the effects of temperature and salinity on Pacific oyster, C. gigas, haemocytes, were studied. Haemocytes are the invertebrate blood cells and thus have been shown to be involved in defence mechanisms. Flow cytometry was used for monitoring several haemocyte parameters. An increase of temperature induced an increase of haemocyte mortality, in both in vitro and in vivo experiments. Temperature modulated aminopeptidase activity. An in vitro decrease of salinity was associated with cell mortality. During the course of in vivo experiments, an increase of phagocytic activity was reported at 15 per thousand and 50 per thousand. Environmental physical parameters may modulate haemocyte activities.
... Lysosomes appear to be a more generalised target of toxic stress induced by PAHs and PCBs. Lysosomal enzymes are considered to play an important role in invertebrate defence reactions involving bacterial destruction (Moore and Gelder, 1985; Moore et al., 1978). Their functional impairment may affect host resistance to a pathogen infection (Anderson, 1981). ...
The shellfish industry is an important economic activity in France, occurring mostly in estuarine zones subject to pollution due to anthropogenic activities. The harmful effects of pollutants on species inhabiting these estuarine zones are not well known. Among marine species, bivalve mollusks--particularly Pacific oyster, Crassostrea gigas--may serve a model of interest. The species is sedentary and filter-feeding, which favors bioaccumulation of pollutants in their tissues. Oysters may be suitable for studies on disturbance by pollutants of physiological activities, among which defense mechanisms are poorly documented in bivalves. In this study, effects of pollutants on hemocyte functions were monitored in Pacific oyster, C. gigas. Hemocytes were exposed in vitro to selected pollutants. The strategy for investigating the effects of pollutants on hemocyte functions is based on several biomarkers, which is more relevant than that of published papers based on single-endpoint experiments. Pollutants belonging to the most important groups of xenobiotics (PAHs, PCBs, and pesticides) were selected and their effect on hemocyte activities was analyzed using flow cytometry. Twenty-three pollutants were tested and eight of them showed significant modulation of hemocyte activities. PAHs and PCB 77 induced a decrease of hemocyte activity after an incubation periods of 4 and 24 h at 200 micro mol/L. Three pesticides (2,4D, paraoxon, and chlorothalonil) modulated hemocyte activities. A mixture of eight pesticides also decreased phagocytotic activity. This study is one of the first to investigate the effects of so many pollutants on hemocyte functions at the same time and therefore allows a real comparison of different pollutant effects.
The variations in hemolymph parameters in marine bivalves were monitored in both laboratory and field after exposure of animals to different metallic and organic pollutants. In the laboratory, experimental modification of cytometric parameters produced physiological changes int he hemolymph. Exposure of oysters to sub=lethal doses of metal ions, cadmium and copper resulted in an increase in total hemocyte counts (THC), a phenomenon often observed under pathological conditions, and in important shifts in differential hemocyte counts (DHC). Spontaneous hemocyte aggregation (SHA), which acts to maintain the integrity of the body, was altered in oysters and mussels following in vitro contact of the hemocytes with metal ions or with the biocide tributyltin (TBT). these effects on SHA were observed to be dependent on the applied concentration of the particular micropollutant. In the field, the measurement of THC, DHC and SHa in transplanted mussels revealed responses at the most exposed sites (mouth of the Elorn, Bay of Daoulas, harbour area). The serum of wild oysters and clams from exposed sites had increased levels of cellular peptidase reflecting sub-cellular modifications such as lysosome destabilisation. the modifications in hemolymphatic parameters, demonstrated in this study, are likely to have reduced immune system performance, and therefore are considered to indicate a state of immunomodulation. These changes in the immune system in bivalves in polluted areas, relative to those at control sites, indicate that measurement of hemolymphatic parameters in sentinel species should be strongly considered in monitoring of marine ecosystem under human impact.
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.
It has been 100 years since the publication of the landmark work of Kellog. Although much has been l e a r n e d about the anatomy and histology of Mercenaria mercenaria since that seminal work, we have a long way to travel on that tortuous pathway known as research. A case in point: although we now know much about the cytology, fine understand the life cycle very well and can spawn animals upon demand throughout the year independent of latitude; yet we have no knowledge concerning the fine structure and functions of follicle and nutritive cells of gonadal acini. In the past 15 years, however, substantial strides have been made in renal cytology, fine structure and general function largely due to the work of M.R Morse and her students. It is expected that investigators will direct concentrated studies of this type to all organ systems and cell types. A Mercenaria genome project should not be in the too distant future. A little more than 50 years ago, Thurlow Nelson stated that the eastern oyster, Crassostrea virginica, was the best known marine animal in the world; I expect that with continued and expanded research on thiscomplex, hearty and commercially impo mercenaria may well become the marine animal about which we have the most scientific information.
Environmental chemico-physical factors, pathogens, and biological interactions constantly affect organism physiology and behavior. Invertebrates, including bivalve mollusks do not possess acquired immunity. Their defense mechanisms rely on an innate, non-adaptive immune system employing circulating cells and a large variety of molecular effectors. The mechanisms underlying host defense depend on the presence of functional proteins in appropriate quantities, within a crucial time window. These proteins are encoded by genes whose transcription is tightly coordinated by complex programs of gene expression. Currently available advanced techniques allow the evaluation of this gene expression, expanding our understanding of the behavior and function of cells and tissues under varying conditions. In particular, DNA microarray technology enables measurement of a large predetermined set of known genes or sequences. Expressed sequence tag sequencing from redundant, normalized, subtractive hybridization libraries is a robust method for sampling the protein encoding genes that are expressed within a tissue. The elimination of microorganisms by defense cells is a dynamic process that involves integrating synthesis of granule proteins during differentiation, migration onto sites of infection, phagocytosis and killing of microorganisms,
modulation of their effector cells, and finally apoptosis. Understanding how this complex biological process is regulated can best be addressed using a systems biology approach to the study of organisms and populations in order to more effectively decipher the continuous challenge between two genomes, i.e., evolving host-pathogen interactions.
Lysosomal, tissue and cellular alterations of the gill, the palp and the intestine epithelium of the mussel Mytilus galloprovincialis (L.) collected from six stations along a closed estuarine system (Thermaikos Gulf, northern Greece) were monitored in May, August and December. Semi-quantitative evaluation of histopathological alterations was performed under the light microscope, while certain of the latter alterations were confirmed electron microscopically. In addition, morphometrical evaluation of the volume density of the lysosomes under the electron microscope was conducted. The observed alterations, as the detachment of gill epithelial cells from the basement membranes and the dilated extracellular spaces formed either between the lateral plasma membranes or between the infoldings of the basal plasma membrane in the palps and the intestine are possibly related to the degree of pollution at the examined stations. The particularly expanded extracellular spaces formed by basal plasma membrane infoldings in the intestine in spring may be attributable to red tide algal infection. On the other hand, differences in morphometric parameters of lysosomal structures indicated no direct relationships with pollution levels.
Large numbers of the soft-shelled clam Mya arenaria from New Bedford Harbor, Massachusetts, exhibit a blood cell disease referred to as hematopoietic neoplasia (HN). Diseased hemocytes differ morphologically from normal hemocytes by their high nucleocytoplasmic ratio. The phagocytic activity, one of the internal defense mechanisms, was compared in the two populations. Surface receptors, essential for recognition during phagocytosis, were demonstrated for both populations using Con A staining. Adherence and uptake were measured after in vitro incubation with yeast. Examination of hemocytes with scanning electron microscopy showed that yeast cells adhered only to the surface of normal hemocytes, and not to diseased hemocytes. Quantitative analysis of adherence and uptake of yeast by hemocytes showed that diseased hemocytes were unable to adhere and ingest yeast. This is thought to be caused by differences in cytoskeletal organization. Finally, the activity of four lysosomal enzymes was used to evaluate intracellular degradation in normal and diseased hemocytes. Histochemical analyses showed diseased hemocytes to have higher than normal acid phosphatase, nonspecific esterases and β-glucuronidase activity. These results indicate that enzyme activity cannot be directly correlated to intracellular degradation. However, it is possible that stress on the lysosomal system may activate certain enzymes.
The pericardial gland-kidney complex forms the major excretory system in the bivalve mollusc, Mya arenaria. Through electron microscopy, a reabsorptive and a secretory kidney cell type have been identified. The kidney cells of individuals collected from polluted sediments (New Bedford, Massachusetts), show qualitatively higher numbers of granules when compared to those of animals collected from a nonpolluted site (Orleans, Massachusetts). Otherwise, no difference in kidney cell architecture is discernible between the two populations. In animals from the Orleans population, two types of circulating hemocytes have been identified through electron microscopy: an agranulocyte and a granulocyte. The postulated role of the hemocytes in removal of pollutant is supported by the finding of a significantly higher number of granulocytes in New Bedford specimens. In addition, the granulocytes in these animals showed much larger inclusions. This study suggests that pollution-exposed Mya arenaria show quantitative and qualitative differences in the granulocytes at the electron microscope level.
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.
Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology
The haemocytes of Dreissena polymorpha were examined by light and electron microscopy. On the basis of cytology and cytochemistry, three main haemocyte types were identified' agranular haemocytes or hyalinocytes, granular haemocytes represented by the neutrophihc granulocytes and the large basophilic haemocytes (LBH). These cell types correspond to the haemocyte classification scheme commonly established for bivalve molluscs: the granular haemocytes (granulocytes) and the nongranular haemocytes (hyalinocytes) Hydrolytic enzyme activities, such as acid phosphatase and non-specific esterases, were demonstrated within the cytoplasmic inclusions of the neutrophilic granulocytes and the LBH β glucuronidase activity was also detected in the cytoplasnuc granules and vacuoles of the LBH. Ultrastructural and phosphatase reactivity was revealed within the matrix of the cytoplasmic granules. The presence of a range of hydrolytic enzymes, including hydrolases and glycosidases in the granules, suggests that these organelles belong to the lysosomal system X-ray microanalysis showed the presence of P, S, Si, Mg, Ca and heavy metals such as Cu, Zn, Pb and Fe in the matrix of the granules of the haemocytes in a natural population of zebra mussels. The occurrence of trace metals sequestrated in the lysosomes suggests the involvement of the granular haemocytes in metal accumulation, transport and detoxication mechanisms.
Calcium-binding phosphoprotein particles are the most abundant extracellular proteins in the hemolymph of heterodont bivalves, and granular hemocytes are the most abundant cells in the same fluid. In this study, the hemocytes of Rangia cuneata were examined ultrastructurally and probed with anti-phosphoprotein IgG to demonstrate that the granulocytes are a probable source of the hemolymph phosphoprotein. The granulocyte cytoplasm is laden with large vesicles containing an amorphous homogenous matrix and variable numbers of electron-dense particles; the latter are ultrastructurally similar to the extracellular phosphoprotein. The vesicle particles and matrix are related forms of the hemolymph phosphoprotein as evidenced by heavy gold labeling when Lowicryl sections were sequentially treated with rabbit-anti-phosphoprotein IgG and colloidal gold-anti-rabbit IgG. The vesicles may be the loci for posttranslational phosphorylation and eventual secretion of the calcium-binding phosphoprotein, or alternatively the vesicles may be digestive structures which degrade internalized phosphoprotein.
The aim of the present study was to isolate and characterize digestive gland cells of the bivalve mollusc Mytilus galloprovincialis Lmk. The digestive gland of bivalves is a complex organ composed of digestive and connective tissues but it is also invaded by the reproductive tissue as gametogenesis proceeds. The digestive tissue is comprised of stomach and intestinal epithelial cells, ciliated and non-ciliated duct cells, digestive cells and basophilic cells. the last two cell types are found lining the epithelium of the blind-ending digestive tubules and are the main focus of this study. Two different approaches were assayed for cell isolation, i.e., explant culture techniques and mechanical plus enzymatical digestion techniques. Cell viability was tested by trypan blue exclusion, neutral red uptake and ultrastructural analysis. The explant cultures were often contaminated with bacteria and spermatozoa and, moreover, cells migrating out of the explants possibly corresponded to hemocytes and not to digestive tissue cells. Mechanical plus enzymatical digestion with collagenase was concluded to be the method of choice for digestive cell isolation, with a percentage of about 30% of isolated cells corresponding to digestive cells. The ultrastructure of digestive cells isolated with the latter procedure closely resembled that of mussel digestive cells in vivo.
The intracellular localization of heavy metals using autometallography (AUM) was studied in the gills and the digestive gland of the common mussel Mytilus galloprovincialis, after an experimental exposure to 0.1 mg L(-1) of Hg, 0.1 mg L(-1) of Ag, 0.1 mg L(-1) of Pb, and 0.1 mg L(-1) of Cu for 98 days. In the gills, autometallographical black silver deposits (BSDs) were localized in the dense bodies observed in the apical and in the basal part of the cells. Among metals, Hg presented the highest accumulation, followed by Ag, Pb, and Cu. BSDs were more prominent in the abfrontal part of the gill filament in the case of Hg exposure and in the frontal part in the case of Ag and Pb exposure. In the digestive gland, Hg and Ag were localized in the heterolysosomes and the residual bodies of the digestive cells, as well as in the dense bodies of the basophilic cells. The heavy metal exposure also affected the gross morphology of the examined tissues and resulted in the fusion of residual bodies in the digestive cells, the fragmentation or vacuolization of the rough endoplasmic reticulum, and the increase in the number of granules in the basophilic cells. In the gills, fusion of the gill filaments was also noted.
We assayed European flat oyster, Ostrea edulis, hemocyte parameters, circulating and tissue-infiltrating hemocyte densities, circulating hemocyte type distribution and lysosomal enzyme contents, to possibly relate these hematological parameters to Bonamia ostreae infection. Circulating hemocyte densities were not statistically different between infected and uninfected oysters. In contrast, the number of tissue-infiltrating hemocytes increased with infection intensity suggesting a recruitment process at the site of infection and a possibility for cells to migrate from circulatory system to connective tissues. Lysosomal enzymes were localized mainly in granulocytes both infected and uninfected, and mean of alpha-naphtyl butyrate esterase activity decreased with increasing B. ostreae infection level. The main response observed was a change in hemocyte type distribution between uninfected and infected oysters and greater tissue-infiltrating hemocytes with increased infections. These results suggest that the decrease of circulating granulocytes, and, consequently of some cell enzyme activities may be related with B. ostreae infection.
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.
Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, beta-galactosidase, beta-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.
In the previous paper we presented findings which indicated that enzyme heterogeneity exists among PMN leukocyte granules. From histochemical staining of bone marrow smears, we obtained evidence that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appeared to be restricted to one of the two types. Clear results were obtained with alkaline phosphatase, but those with a number of other enzymes were suggestive rather than conclusive. Since the approach used previously was indirect, it was of interest to localize the enzymes directly in the granules. Toward this end, we carried out cytochemical procedures for five enzymes on normal rabbit bone marrow cells which had been fixed and incubated in suspension. The localization of reaction product in the granules was determined by electron microscopy. In accordance with the results obtained on smears, azurophil granules were found to contain peroxidase and three lysosomal enzymes: acid phosphatase, arylsulfatase, and 5'-nucleotidase; specific granules were found to contain alkaline phosphate. Specific granules also contained small amounts of phosphatasic activity at acid pH. Another finding was that enzyme activity could not be demonstrated in mature granules with metal salt methods (all except peroxidase); reaction product was seen only in immature granules. The findings confirm and extend those obtained previously, indicating that azurophil granules correspond to lysosomes whereas specific granules represent a different secretory product.
Since the pioneering work on structure and function of heteroglycans compiled in the classical books edited by A. Gottschalk in 19721, there have been several promising developments in glycoconjugate research, as reviewed in this article. In Part 1, contributed by A. Kobata, current knowledge on heteroglycan structures is presented and representative examples taken from higher organisms are given. Part 2, written by J. F. G. Vliegenthart and J. P. Kamerling, covers the most important achievements in methodology: procedures to obtain pure glycans and to analyze their structures. Part 3, contributed by J. Paulson, is devoted to biosynthesis of glycans now describable as pathways since several of the glycosyltransferases have been isolated and analyzed for specificity. In Part 4, contributed by E. Buddecke, current knowledge on functional roles of glycans is presented. It will become apparent that the prerequisite for valid work either in biosynthetic or functional context depends on solid structural information. This is particularly true whenever glycosyltransferase reaction products are being analyzed, or glycans involved in biological functions are investigated. Although in past years, a great deal of important knowledge has been gathered by use of crude glycosidase or glycosyltransferase activities (a notable example is found in reference 2), one may now postulate that glycans implicated in biological reactions should be thoroughly analyzed. This review may familiarize 'newcomers' with the field of glycoconjugate research with special emphasis on glycoprotein glycans. Glycolipids are not included in this article as they have recently been reviewed by S. I. Hakomori3. The reader is also referred to several excellent monographs4,5 and the Proceedings of the Glycoconjugate Symposia held biannually6-8.
How the author was initiated to the study of cellular immunity in invertebrates, especially molluscs, by the late Leslie A. Stauber is recalled. Phagocytosis is the principal mechanism by which molluscs defend themselves against invading nonself materials. The most active phagocytic hemocytes are the granulocytes. These cells are attracted to at least certain species of bacteria by chemotaxis. The attractants are proteins with molecular weights of approximately 10,000. Both microtubules and microfilaments are involved in migration of granulocytes to foreign substances. It is now known that molluscan granulocytes challenged with certain foreign substances undergo hypersynthesis of lysosomal enzymes and these are subsequently released into serum and are effective in degrading certain microorganisms and possibly helminths. In addition to extracellular degradation of foreign materials, there is an efficient intracellular degradation system associated with phagocytosis.
Based on ultrastructure, there are four types of hemocytes or hemocyte-like cells in the heart of the oyster Crassostrea virginica. Types I, II, and III are agranular or slightly granular, and type IV is granular. Type I cells are small (4.0 µm diameter) and have few organelles. Type II cells are round (6.5-7.5 µm diameter), have a moderately dense cytoplasm, and often contain a juxtanuclear body consisting of a deposit of dense material surrounded by mitochondria and dense, membrane-bound bodies. Juxtanuclear bodies usually are related to pairs of confronting cisternae of rough endoplasmic reticulum (RER). Some type II cells contain numerous foci of multiple confronting cisternae of RER and lipofuscin-like bodies. Type III cells are oval (8.5 x 6.0 µm), have an eccentric nucleus, numerous dense-cored vesicles, and abundant RER cisternae. Type III cells appear to be phagocytic. These cells also contain paracrystalline inclusions in the perinuclear space and in RER cisternae. Some cells containing such inclusions are broad, flattened, and closely related to the myocardial trabeculae. These cells have numerous pseudopodia, multiple confronting cisternae and heterogenous bodies, and an eccentric nucleus. The form of these cells and their close association with the myocardial trabeculae suggest that they are not circulating. Type IV cells are typical oyster granulocytes that have an eccentric nucleus, numerous clear-centered granules, and large cytoplasmic deposits of glycogen. Some granules have a PAS-positive content. Developmental or degenerative forms of type IV cells were not observed. Type IV cells were not phagocytic in this study.
Hemocytes of the hard clam, Mercenaria mercenaria, were observed to phagocytose Isochrysis galbana and several other species of unicellular algae, as well as Congo red-stained yeast cells. The “blunt” cytoplasmic granules received degraded materials from the phagosomes containing the algae but not those enclosing a yeast cell. Transfer of the degradation product(s) was traced by observing the fluorescence emission and by microspectrofluorimetric analysis of the phagocytosed material. The blunt granules also participated in the hemocyte's intracellular processing of vital dyes and endotoxin. It is suggested that the blunt granules are involved in the containment and/or further degradation of foreign materials taken up by the hemocytes.
The hemocytes of Mytilus californianus are of three types: small and large basophils and large granular acidophils. The basophils contain lysosomal enzymes and phagocytose colloidal carbon. Agglutinins for yeast and human A Rh+ve erythrocytes are present in plasma, but are not needed for effective phagocytosis; in vitro both acidophilic and basophilic hemocytes rapidly phagocytose these particles. Plasma proteins, analyzed electrophoretically, are under strong homeostatic control. When Mya arenaria mantle is placed orthotopically on M. californianus mantle, the implant is invaded by host hemocytes in a manner consistent with that described in other published reports on molluscan graft rejection. Steady state is achieved by 26 days postimplant. Second- and third-set implants are rejected more rapidly than are first-set implants, but this is not a specific response. Third-set implants elicit a host cellular response that is more localized than the response to first-set implants. These data do not permit conclusions with respect to memory in these molluscan immune responses, but do imply a qualitative “improvement” in this quasi-immune response of M. californianus.
Soft shell clams () are commonly found in coastal waters of both the eastern and western United States. These invertebrates, which have an open circulatory system, may develop neoplasms of the haemolymph which ultimately kill the host. In this study we have 1) recorded the prevalence of hematopoietic neoplasms (HN) in within a 50 mile radius of Woods Hole, Massachusetts and 2) utilized cells from one HN bearing clam to generate a series of monoclonal antibodies. Our data show that determinants are expressed on HN cells which are not detected on normal clam haemocytes, suggesting separate ontogenetic pathways of cell differentiation.
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.
Lavaged alveolar macrophages of mice were examined by light and electron microscopy primarily using cytochemical reactions for lysosomal enzymes. Among several methods tried, the Reed and Bennett procedure for N-acetyl-β-glucosaminidase (J. Histochem. Cytochem., 23:752–757 (1975)) was preferred for use in smears and the Gomori acid phosphatase method for ultrastructural study. Prior to examination, some mice were exposed to an aerosol of finely divided iron oxide for 1–3 hours at particle concentrations of 80–290 mg/m3, others breathing clean air provided macrophages for comparison. Both exposed and unexposed macrophage populations were composed of a similar range of subtypes differing in cytological appearance and enzymic activity. The exposed populations tended to contain a higher proportion of strongly reactive cells, notably of a kind possessing many elongate lysosomes; consequently, it appeared that many cells in these populations were substantially engaged in synthesizing lysosomes. Based on this material, the cycle of production was reconstructed. This is divisible into five natural stages, defined primarily by the intracellular distribution of lysosomal enzyme activity and to a lesser extent by other cytological criteria. Commencing in immature cells, where the predominant localization of acid phosphatase activity in the Golgi apparatus and a few rounded lysosomes (Stage 1), the cycle continues through a period dominated by extensive growth of the granular endoplasmic reticulum (Stage 2). With the appearance of a crop of elongate or lumbricoid lysosomes, the predominate locus of acid phosphatase activity shifts from the Golgi apparatus to these bodies (Stage 3). Thereafter, they often are seen together with an increased number of large rounded lysosomes (Stage 4), but in other cells only the latter are to be found (Stage 5). This lysosomal cycle operates in cells showing other signs of an aroused metabolism, and it appears not to be closely tied to the mitotic cycle. The nascent lumbricoid lysosomes seem to be formed at some distance from the Golgi apparatus, wheras the lysosomes produced by more quiescent cells often lie in close relationship to that organelle.
Intracytoplasmic interactions were examined in iron oxideexposed and -unexposed alveolar macrophages of mice by electron microscopy and ultrastructural cytochemistry. Submicronic-sized particles are taken up by microendocytosis into 800-Å tubules that sometimes open up into a large vacuole located midway between the plasmalemma and the Golgi complex and sometimes lead directly to that organelle, which is reactive for acid phosphatase while cells are only modestly engaged in lysosomal synthesis and then serves as the main locus for formation of heterolysosomes. In more activated cells, nascent lumbricoid lysosomes are budded directly off branches of the granular reticulum; in consequence, much of the newly synthesized acid phosphatase bypasses the Golgi apparatus, and heterolysosomes are formed throughout a widened expanse of cytoplasm. The lumbricoids exist either as free elements or as a labyrinthian network of lumbricoid units, and the two forms appear interconvertible. They are short lived in contrast to larger rounded lysosomes more commonly present, many of which are heterolysosomes as judged by their content of ingested particles. Lumbricoids readily fuse with exoplasmic structures; indeed, in some lumbricoid-producing cells terminal segments of the reticulum may share this propetry to a degree, since they resemble transitional elements when extended along large phagosomes. Microendosomes can be taken up into lumbriciods, but if the endosome is larger, lumbricoids will adhere to its surface and become part of the digestive vacuole. A labyrinth is formed to envelop large phagosomes or regions of cytoplasm marked for autophagy. The interior is invaded by branches and digestion ensues. In active macrophages the Golgi apparatus is enlarged. The centrioles organize an array of microtubules about themselves, and individual Golgi stacks are turned outward to give the organelle better access to phagosomes and lysosomes. In less active cells the microtubules disappear, and the stacks reform in a circle about the diplosome. Alveolar macrophages therefore make a variety of lysosomes. Depending on cellular activity, one kind or another predominates. Few of them fall cleanly into “primary” and “secondary” classes, and in their interactions with other cellular structures they are influenced by mechanisms operating on microscopic as well as molecular scales.
Lumbricillus lineatus selectively ingests masses of organic and inorganic interstitial particles from a sand-clay substratum in the upper littoral zone. Particle-masses are ingested, passed along the esophagus and into the anterior intestine where the pH becomes acid. A- and C-esterases, acid -galactosidase, acid phosphatase and -N-acetylglucosaminidase are present in the epithelium, while the rotating food masses are surrounded by a membrane of sulphated, acid glycoprotein. These enzymes, with the exception of acid phosphatase and the addition of aminopeptidase M, are also present in the epithelia of the mid and posterior intestinal regions where the pH is alkaline. The cells in the ventral wall of the mid intestinal region contain high concentrations of alkaline phosphatase, acid -galactosidase and -N-acetylglucosaminidase. The food consists of absorbed organics and bacteria with absorption and intracellular digestion occurring along the intestine, particularly in the mid ventral region.
ABSTRACT The binding and redistribution of Con A surface membrane
determinants was investigated in two Crassostrea virginica hemocyte subpopulations
(designated LS and SS) by employing a direct Con A-peroxidase
coupling method in conjunction with light and electron microscopical cytochemistry.
At 4°C small, adherent hemocytes exposed to Con A and horseradish
peroxidase (HPO) produced a uniform staining pattern on hemocyte surface
membranes. Polar redistribution or capping of Con A receptors was observed in
22.2% of LS and 11.0% of SS hemocytes when the incubating temperature was
increased from 4 to 21°C. Receptor patching, observed with equal frequency
(-15%) in both cell subpopulations, also exhibited a temperature dependency.
Capped hemocytes were readily identified by the polar concentration of darkstaining
HPO reaction product along the marginal surface of each cell. Receptor
patching was observed as multiple aggregates of reaction staining on blood
cell surface membranes. Cap formation in SS hemocytes was inhibited by
sodium azide (-N3) at each concentration of inhibitor tested. LS hemocyte capping
was inhibited only at the highest -N, concentration (5 x M). Cells of
both subpopulations were unable to completely reverse the effects of -N3
poisoning following inhibitor removal.
The hemocytes of the hard clam M. mercenaria were of three types: an agranulocyte, a small, and a large granulocyte. The agranulocyte, with only a thin periphery of cytoplasm surrounding the nucleus, had no visible cytoplasmic granules in living preparations but did exhibit a few centers of nonspecific esterase activity. This cell type represented 2% of the hemocyte population. The small granulocyte possessed four distinct granule types and comprised 61% of the total cell population. Large granulocytes accounted fro 37% of all hemocytes. While they contained the same four granule types identified in the small granulocyte, only one-third the total number were present. The nucleus of all three hemocyte types appeared morphologically similar. The four types of granules observed were a blunt, dot-like, a refractile and a filamentous granule. Blunt granules were identified as mitochondria, based on their ability to reduce Janus Green B to diethyl safranin, the presence of NADH dehydrogenase activity and boundary staining with Sudan black B. Dot-like granules were identified as lysosomes on the basis of neutral red staining, localization of acid phosphatase and nonspecific esterase activity and staining with Sudan black B. Refractile granules were demonstrated to be membrane-bound, lipid-filled structures that reacted positively with Sudan black B and Oil red O, respectively; these granules act as lipid storage centers. Nuclear similarity of the three cell types suggest that these cells might represent different stages of maturity, rather than three distinct cell lines. This was also indicated by the similar yet graded cytochemical reactions and the varying degree of motility and phagocytic activity demonstrated by hemocyte types.
Hemocytes of Mytilus edulis were examined cytologically and cytochemically. On the basis of structure, staining reactions, and phagocytic behavior, they were divided into two main groups: basophilic hemocytes and eosinophilic granular hemocytes (granulocytes). The basophilic cells were further divided into small lymphocytes and larger phagocytic macrophages reactive for lysosomal hydrolases. Mitosis was observed in granulocytes and in small lymphoid cells, believed to be the stem cells for the basophilic cell line. A few cells appeared to be intermediate between lymphocytes and small granulocytes. Macrophages were the main cell type involved in the clearance of injected carbon particles. However, granulocytes did show some phagocytic activity. Brown cells displaying apparent amoebocytic behavior were found to contain Fe3+ and Pb2+ in cytoplasmic inclusions, some of which were also reactive for β-glucuronidase and glucosaminidase. These cells appear to have a separate origin from the hemocytes.
1.1. The activities of lysozyme, acid and alkaline phosphatases, β-glucuronidase, amylase, lipase, glutamic-oxalacetic transaminase, and glutamic-pyruvic transaminase in the whole hemolymph and 4000 g pellets and supernatants of Crassostrea virginica and Mercenaria mercenaria were assayed. All of these enzymes, except for amylase, occurred in whole hemolymph as well as in the fractions of both species of molluscs.2.2. Amylase only occurred in the whole hemolymph and serum of C. virginica. Since this mollusc possesses a crystalline style, the amylase is believed to have originated from this structure.3.3. It is postulated that the lysosomal enzymes detected in the serum of both species of molluscs had been released from certain hemolymph cells and may play a role in destroying certain invading organisms.
Acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase) activity within electron-opaque, membrane-bound vesicles of Mercenaria mercenaria granulocytes has been localized by employing cytochemistry at the light and electron microscope levels. These vesicles can now be considered lysosomes. They presumably function, at least in part, as storage organelles for acid hydrolases, and are therefore analogous to the granules in mammalian polymorphonuclear and monocytic leucocytes. Lysosomes containing acid phosphatase are probably the sources of this enzyme found in cellular and serum fractions of the hemolymph of M. mercenaria, although the mechanism for enzyme release remains uncertain.
The fine structure of oyster leucocytes resembles to a great extent, that of typical eucaryotic cells. Organelles which have been described for the first time in this report are light granules, dense granules, protocentriole and X structure. Light microscopy reveals two morphological types of oyster leucocytes: agranular and granular. Based upon nuclear morphology and cytoplasmic compositions revealed in electron microscopy, at least three types of agranular and one type of granular cells are recognized.
In the Giemsa-stained preparations, granular leucocytes exhibit three distinct types of cytoplasmic granules: refractile, dark blue, and pink, which presumably correspond to light granules Type A, B, and C seen in the electron micrographs. A granular leucocyte may contain one or more types of granules. Cytochemical investigations show that oyster leucocytes contain at least three hydrolytic enzymes: non-specific esterases, acid, and alkaline phosphatase. The latter two enzymes constitute 63% of the enzyme activity detected. These intracellular enzymes may be associated with the light granules and/or lysosome-like bodies.
It is also demonstrated that the granular leucocyte population is significantly higher (P<0.001) in the oysters experimentally infected with Bacillus mycoides (72.19±4.71%) as contrasted with that of the controls (37.18±4.48%).
Leucocytes in progressive stages of degeneration are also described.
A chemical (phenol) stressor induces changes in phagocytic ability in the hard clam, Mercenaria mercenaria; cytoplasmic disorganization and selective lysis of hemocytes are observed. In invertebrates, infectious agents are primarily controlled by nonspecific cellular defense mechanisms and stressors may alter the host-parasite complex with pathological consequences.