Role of divalent cations in plasma opsonin-dependent and-independent erythrophagocytosis by hemocytes of the Asian clam,Corbicula fluminea

The in vitro phagocytosis-promoting properties of hemolymph from the freshwater clam, Corbicula fluminea, are described. Hemocytes were capable of phagocytosing aldehyde-fixed erythrocytes (RBCs) of seven vertebrate species with equal facility, but only in the presence of homologous clam plasma. The plasma factors mediating erythrophagocytosis were heat sensitive. Pretreatment (opsonizing) of target RBCs with plasma also resulted in enhancement of hemocyte particle uptake in the absence of plasma. Opsonin-dependent phagocytosis required the presence of divalent cations, especially calcium, although not in free ionic form. Evidence suggests that the plasma opsonin may normally exist as a divalent cation-macromolecular complex since opsonizing activity was retained after dialysis against Tris-buffered saline (TBS), but was lost following TBS/EDTA or TBS/EGTA dialysis. We also have identified an opsonin-independent phagocytosis mechanism in which Corbicula hemocytes are able to ingest nonopsonized RBCs in the absence of homologous plasma. Extracellular calcium or magnesium in the incubation medium is needed for particle uptake, although the direct binding of free ions to the target RBC surface does not appear to be mediating enhanced phagocytosis. From the present data, it is concluded that hemocyte recognition of aldehyde-fixed RBCs can be accomplished by either of two mechanisms: (1) by the coating of cells with plasma factors capable of triggering the phagocytic process (opsonization) or (2) by a plasma opsonin-independent mechanism in which extracellular divalent cations (e.g., Ca²⁺ or Mg²⁺) in the incubation buffer stimulate uptake of nonopsonized RBCs. The factors regulating in vitro erythrophagocytosis by clam hemocytes are considered to be analogous to those involved in nonimmune opsonin-dependent and -independent phagocytosis in mammalian macrophages.     

Quantification of cytotoxic hemocytes of Mytilus edulis using a cytotoxicity assay in agar

When a thin layer of agar containing a mixture of erythrocytes and Mytilus hemocytes is prepared on slides, the occurrence of plaques of lysed target cells can be observed around a limited number of hemocytes. These hemocytes remain completely intact cells and are viable as evidenced by their ability to phagocytose target cells and/or to form pseudopods. The number of hemocytes releasing cytotoxic molecules has been shown to vary greatly between different animals. The same holds true for the total number of circulating hemocytes, although no correlation exists between the number of hemocytes in the circulation and the percentage of cytotoxic blood cells.     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

Phagocytosis in invertebrates: Studies on the hemocytes of the clam Tridacna maxima

The two types of cells found in the hemolymph of the clam Tridacna maxima have been examined in vitro by light microscopy, and their morphological charcteristics described and illustrated. It was shown that the hemocytes, which exhibited rapidly spreading cytoplasm and extensive ruffled membrane activity, were able to phagocytose carbon particles experimentally introduced into the animals. Attention is drawn to the part that the phagocytic hemocytes of invertebrates may have to play in putative host defense mechanisms and physiological waste removal processes.     

Immunophenotyping of Mya arenaria neoplastic hemocytes using propidium iodide and a specific monoclonal antibody by flow cytometry

Disseminated neoplasia (DN) is a disorder referred to as hemic neoplasia (HN) in the soft-shell clam Mya arenaria. Traditionally, diagnosis is performed by hematocytology or histology. The intensity of the disease is generally given as the percentage of transformed neoplastic cells out of total number of hemocytes. Flow cytometry techniques have found a field of application in diagnosis of HN with analysis of ploidy. Hemocytes of the soft-shell clams with HN display tetraploid DNA content, as shown by propidium iodide staining. This feature makes difficult HN diagnosis in the soft-shell clam, especially for early stages of the condition, since the percentage of normal circulating cells undergoing mitosis, which also are tetraploid, remains unknown in molluscs. Use of specific monoclonal antibodies in a flow cytometry assay was foreseen as a way to overcome the difficulty. The purpose of this study was to develop a double staining protocol using propidium iodide for hemocyte cycle analysis and the MAb 1E10 for staining of HN cells. Our results showed a correlation between tetraploid and MAb 1E10-stained hemocytes in a single clam with moderate HN. This protocol offers some potential for further investigation of this cell disorder. However, a validation step will be necessary to confirm our preliminary results.     

Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: Changes in cell structure, numbers and adherence

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24 h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p < 0.01) than with 7SHRW. The cell adherence was markedly diminished (p < 0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p < 0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.     

Morula-like cells in photo-symbiotic clams harboring zooxanthellae

Symbiosis is observed between zooxanthellae, symbiotic dinoflagellates, and giant clams and related clams which belong to the families Tridacnidae and Cardiidae. We have previously shown that a photo-symbiotic clam Tridacna crocea has three types of hemocytes, the eosinophilic granular hemocyte with phagocytic activity, the agranular cell with electron lucent granules, and the morula-like cell with large (ca. 2 mum in diameter) colorless granules. The function of the morula-like cell is not clear, but it has not been reported in any other bivalves except photo-symbiotic clams T. crocea and Tridacna maxima. In order to clarify whether it is specific to photo-symbiotic clams or not, we studied hemocytes in the photo-symbiotic clams Tridacna derasa (Tridacnidae), Hippopus hippopus (Tridacnidae) and Corculum cardissa (Cardiidae), and a closely related non-symbiotic clam Fulvia mutica (Cardiidae). The eosinophilic granular hemocytes and the agranular cells were found in all of the clams examined. However, the morula-like cells which were packed with many large electron dense granules (ca. 2 mum in diameter), were observed only in the photo-symbiotic clams. In F. mutica, a closely related non-symbiotic clam, this type of hemocyte was not found. Instead a hemocyte with vacuoles and a few large granules containing peroxidase activity was observed. The large granules of F. mutica varied in size from ca. 1-9 mum in diameter. Present data suggests that the presence of morula-like cells is restricted to photo-symbiotic clams and that the hemocytes associated with the morula-like cells may have some functional relationship to symbiosis with zooxanthellae.     

Cells released in vitro from the embryonic yolk sac of the stick insect carausius morosus (BR.) (PHASMATODEA : HETERONEMIIDAE) may include embryonic hemocytes

The embryonic yolk sac and the adult dorsal vessel of the stick insect Carausius morosus (Br.) (Phasmatodea : Heteronemiidae) were shown to release a number of cells that appear morphologically similar to circulating adult hemocytes. Like adult hemocytes, these cells reacted positively when tested for both phenoloxidase activity and a monoclonal antibody specifically raised against a vitellin polypeptide. Based on this evidence, it is suggested that yolk sac-released cells behave as potential embryonic hemocytes. A model is thus proposed whereby the yolk sac might host a number of hemopoietic stem cells on their way to the dorsal vessel, and in so doing, it may temporally act as an embryonic hemopoietic organ.     

Involvement of nitric oxide in the in vitro interaction between Manila clam, Ruditapes philippinarum, hemocytes and the bacterium Vibrio tapetis

The Manila clam, Ruditapes philippinarum can become infected by the bacterium Vibrio tapetis which causing the Brown Ring Disease along North European Atlantic coasts. Variations in clam immune parameters have been reported in clam challenged with V. tapetis but no studies have been done on Nitric Oxide (NO) production. NO is a toxic agent to pathogens produced mostly by immune cells such as hemocytes in invertebrates. In this study, we demonstrated that NO production in hemolymph and extrapallial fluid of clams is dose dependent and increases with incubation time with V. tapetis. Moreover, the augmentation of NO production seems to be directly correlated to cell rounding and to the loss of pseudopods-forming capacity of hemocytes during the infection process.     

Characterization of a bacterial agglutinin in the hemolymph of the hard clam, Mercenaria mercenaria

The hemolymph of the hard clam, Mercenaria mercenaria, was found to agglutinate nonspecifically 4 of the 30 bacteria tested and a marine alga. The agglutinin is a protein (or a conjugated protein) because it is: (1) precipitated by trichloroacetic acid and ammonium sulfate; (2) inactivated by extraction with chloroform, but not with toluene or xylene; and (3) inactivated by chymotrypsin and protease, but not by deoxyribonuclease. Electrophoretic analysis shows that the agglutinin is composed of subunits each with a molecular weight of approximately 21,000. Calcium ions are required for the activity of the agglutinin and contribute to the heat stability of the molecule. Several saccharides, which may constitute a portion of the bacterial agglutinin receptors, were capable of partially inhibiting agglutination. In vitro studies using clam hemocytes showed that the phagocytosis of a marine bacterium, designated as RS-005, was enhanced by the presence of hemolymph. Adsorption of hemolymph samples with RS-005 bacteria removed the agglutinin activity for all types of cells tested and also abolished the opsonic effect.     

Histopathology of Aerococcus viridans var. homari infection (Gaffkemia) in the lobster, Homarus americanus, and a comparison with histological reactions to a gram-negative species, Pseudomonas perolens

Histological response of lobsters to injection of Aerococcus viridans var. homari, cause of gaffkemia, was followed over a 14-day period. Salient features in infected lobsters, Homarus americanus, were: aggregations of hemocytes occurring in hemal spaces throughout the tissues and increasing in number and size with time; the early phagocytosis of bacteria by the system of fixed phagocytes (FPs) present in hemal spaces of the hepatopancreas; and premature release of differentiating hemocytes from the hemopoietic tissue, so that by 14 days that tissue consisted mainly of large stem cells. Mass release of differentiating hemocytes presumably occurred to replace hemocytes lost from the circulation by their incorporation into aggregations or by lysis of individual cells ruptured through the pressure of phagocytized bacteria that were multiplying in them. Bacteria and their remains were present in FPs at 2 days but not visible in single or aggregated hemocytes until 6 days, when free bacteria were also present in the hemolymph. By 6 days, all bacteria, whether phagocytized or free, appeared normal and were surrounded by nonstaining halos that extended well beyond the stainable capsular material. As predicted earlier in physiological studies, gaffkemia is a nontoxic, noninvasive bacteremia. There was hemal stasis and consequent injury in the antennal gland due to free and aggregated hemocytes that occluded hemal spaces of that organ, but other tissues and organs appeared normal except for depletion of glycogen. Aggregations of hemocytes were present in lobsters 2 and 12 days after injection of a nonpathogenic, Gram-negative bacterium, Pseudomonas perolens. Unlike the case with gaffkemia, necrotic hemocytes were common in the aggregations, presumably in response to damage by endotoxin. A further difference was that aggregations were common in the heart of P. perolens-injected lobsters but rare in the heart of gaffkemic lobsters. Bacteria were not seen in hemolymph, hemocytes, or other cells of P. perolens-injected lobsters.     

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage. HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.     

First characterisation of the populations and immune-related activities of hemocytes from two edible gastropod species,the disk abalone,Haliotis discus discus and the spiny top shell,Turbo cornutus

The disk abalone Haliotis discus discus and the spiny top shell Turbo cornutus are edible gastropod species of high economic value, mainly in Asia. Mortality outbreaks and variations in worldwide stock abundance have been reported and suggested to be associated, at least in part, with pathogenic infections. Ecology, biology and immunology of both species are currently not well documented. The characterisation of the immune systems of these species is necessary to further assess the responses of H. discus discus and T. cornutus to environmental, chemical and disease stresses. In the present study, we investigated the morphology and immune-related activities of hemocytes in both species using light microscopy and flow cytometry. Two types of hemocytes were identified in the disk abalone hemolymph, blast-like cells and hyalinocytes; whereas four main hemocyte types were distinguished in the spiny top shell, blast-like cells, type I and II hyalinocytes, and granulocytes. Flow cytometric analysis also revealed differences between cell types in immune-related activities. Three subsets of hemocytes, defined by differing lysosomal characteristics, were observed in the hemolymph of the spiny top shell, and only one in the disk abalone. Phagocytic activity was higher in H. discus discus hemocytes than in T. cornutus hemocytes, and the kinetics of PMA-stimulated oxidative activity was different between hemocytes of the disk abalone and the spiny top shell. Finally our results suggest for the first time a predominant mitochondrial origin of oxidative activity in gastropod hemocytes.     

Expression of biomineralisation genes in tissues and cultured cells of the abalone Haliotis tuberculata

Mollusc shell biomineralisation involves a variety of organic macromolecules (matrix proteins and enzymes) that control calcium carbonate (CaCO₃) deposition, growth of crystals, the selection of polymorph, and the microstructure of the shell. Since the mantle and the hemocytes play an important role in the control of shell formation, primary cell cultures have been developed to study the expression of three biomineralisation genes recently identified in the abalone Haliotis tuberculata: a matrix protein, Lustrin A, and two carbonic anhydrase enzymes. Mantle cells and hemocytes were successfully maintained in primary cultures and were evaluated for their viability and proliferation over time using a semi-automated assay (XTT). PCR and densitometric analysis were used to semi-quantify the gene expression and compare the level of expression in native tissues and cultured cells. The results demonstrated that the three genes of interest were being expressed in abalone tissues, with expression highest in the mantle and much lower in the hemocytes and the gills. Biomineralisation genes were also expressed significantly in mantle cells, confirming that primary cultures of target tissues are suitable models for in vitro investigation of matrix protein secretion.     

Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations

Maintenance of hemocyte populations is critical for both development and immune responses. In insects, the maintenance of hemocyte populations is regulated by mitotic division of circulating hemocytes and by discharge from hematopoietic organs. We found cell clusters in the hemolymph of Mamestra brassicae larvae that are composed of small, spherical cells. Microscopic observations revealed that the cells in these clusters are similar to immature or precursor cells present in hematopoietic organs. The results of bromodeoxyuridine (BrdU) incorporation experiments demonstrate that these cells are mitotically active. Furthermore, these cells maintain their immature state and proliferate until late in the last larval instar. The results of in vitro experiments showed that most of the cells changed their morphology to one consistent with plasmatocytes or granulocytes, and that the change was promoted by addition of larval hemolymph to the culture medium, in particular when hemolymph was collected at a prepupal stage. Taken together, our results suggested that cells in clusters may be an additional source of hemocytes during larval development.     

Tiger shrimp (Penaeus monodon) penaeidin possesses cytokine features to promote integrin-mediated granulocyte and semi-granulocyte adhesion

Recent studies indicated that antimicrobial peptides (AMPs) play multiple roles in both innate and adaptive immune functions. The penaeidin of tiger shrimp Penaeus monodon shows an antimicrobial activity against Gram-positive bacteria and filamentous fungi. To study immunomodulation functions of the penaeidin, we transfected shrimp hemocytes in primary culture with penaeidin-specific small interfering RNA (siRNA-3) and observed a concomitant 20% reduction in adhesive hemocytes compared with mock-transfected cells. The addition of biosynthesized or chemically synthesized penaeidin or penaeidin proline-rich domain (PRD) to the culture medium of penaeidin knock-down hemocytes led to a full recovery in the number of adhesive hemocytes. The effect of penaeidin knock-down on the expression of tiger shrimp cell adhesion-associated molecules was examined using real-time Q-PCR. Results demonstrated 91% and 64% decreases in the expression of integrin-β and collagen, respectively, and a 396% increase in the expression of collagenase. The addition of chemically synthesized penaeidin after penaeidin knock-down hemocytes normalized the expression of these genes. The addition of the integrin-β ligand competitor RGDS to mock-transfected hemocytes decreased the number of adhesive hemocytes similar to penaeidin knock-down. In conclusion, penaeidin possesses an integrin-β-mediated cytokine feature that promotes shrimp granulocyte and semi-granulocyte adhesion. This is the first report about functional shrimp cytokine.