A quantitative study of phagocytosis by hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria

Fresh hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria were exposed to known concentrations of Bacillus megaterium, Escherichia coli, and Staphylococcus aureus in vitro and it was ascertained that all four types of cells of C. virginica and all three types of M. mercenaria became associated with the bacteria. Association is defined as either the first, i.e., contact and adherence, or second, i.e., engulfment, phase of phagocytosis. However, when the surfaces of each type of cell, as well as the percentages of each type in whole hemolymph, from both species of molluscs are taken into consideration, it is concluded that the granulocytes are the most important from the standpoint of phagocytosis.When hemocytes of M. mercenaria were exposed to Bacillus megaterium at 4°, 22°, and 37°C, it was found that the association indices were higher at the latter two temperatures. It is postulated, because of the results of Feng and Feng (1974), that nonself materials adhere with less frequency at 4°C and hence are not phagocytosed at this lower temperature.     

Light and electron microscopy of the leucocytes of Crassostrea virginica (Mollusca: Pelecypoda)

Summary 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.Contribution No. 71 from Marine Research Laboratory, University of Connecticut.The initial phase of this investigation was carried out at the Department of Zoology, Rutgers, The State University, New Brunswick, New Jersey, and supported by Public Health Service Research Grant AI-00781 from the National Institute of Allergy and Infectious Diseases of the National Institute of Health, awarded to Dr. L. A. Stauber. Supported by a grant from the University of Connecticut Research Foundation and Faculty Summer Fellowship to S. Y. Feng.     

Release of lysozyme from hemolymph cells of Mercenaria mercenaria during phagocytosis

Activity of the lysosomal enzyme, lysozyme, has been quantitatively determined in the serum and cells of the hemolymph of Mercenaria mercenaria which had been exposed to known quantities of Bacillus megaterium and also in the serum and cells of hemolymph which had not been exposed to bacteria. The results indicate that the level of enzyme activity is greater in serum of hemolymph that had been exposed to B. megaterium and concurrently, there is an equivalent decrease in the level of activity in the cells. This evidence indicates that the amount of lysozyme released from cells into serum is enhanced during phagocytosis of the bacteria.It has also been demonstrated that the release of lysozyme from cells occurs during the process of phagocytosis and is not a delayed phenomenon.Enzyme release by secondary phagosomes is reflected morphologically by what is commonly referred to as degranulation. This process does not involve the rupture of the plasma membrane of the hemolymph cells since biochemical studies have revealed that there is no release of the cytoplasmic enzyme, lactate dehydrogenase.     

Chemotactic attraction of Crassostrea virginica hemolymph cells to Staphylococcus lactus.

The nuclear polyhedrosis virus (NPV) of Porthetria dispar was isolated and purified through a two-step zonal centrifugation procedure. The LD₅₀ of the purified NPV was determined by a dose-response assay. Quantitative analyses were made of whole polyhedra and of separated fractions of polyhedral protein, virus rods, and denatured material, i.e., the pellet obtained from low speed centrifugation of dissolved polyhedra, to determine the protein, DNA, and “RNA” (orcinol-positive material) present in this NPV. Approximately one-half the “RNA” was present in the denatured material. Trace elements were also determined, and four, Fe, Mg, Cu, and Zn, of the ten assayed were present in the polyhedral protein fraction, while only Mg and Zn were in the virus rod fraction.     

Agglutinins in the hemolymph of the hard clam, Mercenaria mercenaria.

Lectins in the serum of the clam Mercenaria mercenaria agglutinate some red blood cells, bacteria, and yeast. The interaction of these substances with particles is affected by sugars, ions, temperature, and alteration of particle surfaces. Lectins are not needed for phagocytosis of foreign particles in vitro. In M. mercenaria these recognition molecules do not enhance defense mechanisms.     

Kinetic properties of lysozyme from the hemolymph of Crassostrea virginica

Lysozyme activity has been demonstrated in both the supernatant and pellet fractions of whole hemolymph of the American oyster, Crassostrea virginica, subjected to centrifugation at 4000 and 10,000 × g. In each case the enzyme activity is greater in the supernatant than in the pellet.The lytic activity of the molluscan lysozyme on Micrococcus lysodeikticus, like that of egg-white lysozyme, is salt dependent, is relatively heat stable, and is very sensitive to changes in ionic concentration. The optimal pH of the molluscan enzyme, however, ranges from 5.0 to 5.5, depending on the buffer employed.When tested against a number of bacteria, the oyster lysozyme has been found to be active against not only M. lysodeikticus but also Bacillus subtilis, B. megaterium, Escherichia coli, Gaffkya tetragena, Salmonella pullorum, and Shigella sonnei, although it is less active against the last four mentioned. It is not active against Staphylococcus aureus.It is postulated that the lysozyme in the serum of C. virginica has its origin in cytoplasmic phagosomes of granulocytes and is released when these organelles become ruptured.     

Interactive effects of elevated temperature and CO2 levels on metabolism and oxidative stress in two common marine bivalves (Crassostrea virginica and Mercenaria mercenaria)

Marine bivalves such as the hard shell clams Mercenaria mercenaria and eastern oysters Crassostrea virginica are affected by multiple stressors, including fluctuations in temperature and CO₂ levels in estuaries, and these stresses are expected to be exacerbated by ongoing global climate change. Hypercapnia (elevated CO₂ levels) and temperature stress can affect survival, growth and development of marine bivalves, but the cellular mechanisms of these effects are not yet fully understood. In this study, we investigated whether oxidative stress is implicated in cellular responses to elevated temperature and CO₂ levels in marine bivalves. We measured the whole-organism standard metabolic rate (SMR), total antioxidant capacity (TAOC), and levels of oxidative stress biomarkers in the muscle tissues of clams and oysters exposed to different temperatures (22 and 27 °C) and CO₂ levels (the present day conditions of ~ 400 ppm CO₂ and 800 ppm CO₂ predicted by a consensus business-as-usual IPCC emission scenario for the year 2100). SMR was significantly higher and the antioxidant capacity was lower in oysters than in clams. Aerobic metabolism was largely temperature-independent in these two species in the studied temperature range (22–27 °C). However, the combined exposure to elevated temperature and hypercapnia led to elevated SMR in clams indicating elevated costs of basal maintenance. No persistent oxidative stress signal (measured by the levels of protein carbonyls, and protein conjugates with malondialdehyde and 4-hydroxynonenal) was observed during the long-term exposure to moderate warming (+ 5 °C) and hypercapnia (~ 800 ppm CO₂). This indicates that long-term exposure to moderately elevated CO₂ and temperature minimally affects the cellular redox status in these bivalve species and that the earlier observed negative physiological effects of elevated CO₂ and temperature must be explained by other cellular mechanisms.     

Experimentally induced elevation of aminopeptidase activity in hemolymph cells of the American oyster,Crassostrea virginica

The levels of aminopeptidase activity in the serum and hemolymph cells of the American oyster, Crassostrea virginica, have been determined to be 2.89 ± 2.25 and 0.62 ± 0.39 Sigma units/ml, respectively. Low levels of aminopeptidase activity have been demonstrated cytochemically within cytoplasmic granules, i.e., secondary phagosomes, of circulating hemolymph cells. Exposure of whole hemolymph to heat-killed Bacillus megaterium or to sterile sea water results in a significant increase in cellular aminopeptidase activity, with the level of activity being the highest in cells that had been exposed to bacteria. The level of aminopeptidase activity in serum is unaltered in similarly challenged whole hemolymph. It is concluded that aminopeptidase synthesized in cells during phagocytosis or as a result of stimulation by exposure to sea water is not released into the serum but is retained intracellularly. Our studies suggest that it is the intracellular and not the serum aminopeptidases whith are of primary importance in the degradation of B. megaterium.     

Assessment of the northern distribution range of selected Perkinsus species in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) with the use of PCR-based detection assays

Perkinsus species are protistan parasites of molluscs. In Chesapeake Bay, Perkinsus marinus, Perkinsus chesapeaki, and Perkinsus andrewsi are sympatric, infecting oysters and clams. Although P. marinus is a pathogen for Crassostrea virginica, it remains unknown whether P. andrewsi and P. chesapeaki are equally pathogenic. Perkinsus species have been reported in C. virginica as far north as Maine, sometimes associated with high prevalence, but low mortality. Thus, we hypothesized that, in addition to P. marinus, Perkinsus species with little or no pathogenicity for C. virginica may be present. Accordingly, we investigated the distribution of Perkinsus species in C. virginica and Mercenaria mercenaria, collected from Maine to Virginia, by applying PCR-based assays specific for P. marinus, P. andrewsi, and a Perkinsus sp. isolated from M. mercenaria. DNA samples of M. mercenaria possessed potent PCR inhibitory activity, which was overcome by the addition of 1 mg/ml BSA and 5% (v/v) DMSO to the PCR reaction mixture. All 3 Perkinsus species were found in both host species throughout the study area. Interestingly, the prevalence of P. marinus in M. mercenaria was significantly lower than in C. virginica, suggesting that M. mercenaria is not an optimal host for P. marinus.     

Hemolymph cells of the bivalve mollusc Mercenaria mercenaria: an electron microscopical study.

The hemolymph cells of Mercenaria mercenaria were studied with the transmission electron microscope. Three morphological types of cells, granulocytes, hyalinocytes, and fibrocytes, are distinguishable and their fine structural characteristics are described. However, as a result of analyzing the fine structural features of the so-called fibrocytes of M. mercenaria, i.e., the inclusion of large aggregates of glycogen granules in their cytoplasm and the occurrence of primary phagosomes enclosing partially degraded exogenous material and digestive lamellae, it is suggested that fibrocytes are actually granulocytes which are at the terminal phase of their physiologic cycle relative to the degradation of phagocytized nonself materials. The cytoplasmic granules of M. mercenaria granulocytes are structurally different from those of Crassostrea virginica in that they are delimited by a unit membrane, rather than by a complex wall, and include a homogenously electron-dense material. Lipidlike droplets are reported from both granulocytes and hyalinocytes of M. mercenaria for the first time.     

Rickettsiae in gill epithelial cells of the hard clam, Mercenaria mercenaria

Rickettsiae are found in the gill epithelium of the hard clam, Mercenaria mercenaria. The procaryotes occur free in the cytoplasm of the epithelial cells at the tip of the filament and in the more proximal cells that support the lateral J cilia. The fine structure of the organisms, showing rippled cell walls, is typical of the rickettsiae. The increasing size of the inclusion representing late phase growth often culminates in lysis of the host cell. Masses (Gram-negative, Feulgen-positive) in ova, similar to those observed in the gill epithelium, suggest that transovarian transmission may occur.     

Direct plating procedure for enumerating Vibrio vulnificus in oysters (Crassostrea virginica).

A procedure for enumerating and identifying Vibrio vulnificus in oysters was developed and evaluated. This method consists of growth on a direct plating medium (VVE medium) for isolating the organism from shellfish tissues, followed by biochemical tests for differentiating and identifying presumptively positive isolates. Densities of V. vulnificus are reliably obtained in 2 to 4 days, and as few as 10 culturable cells per 100 g can be identified. The procedure was evaluated by using a DNA probe technique specific for the cytotoxin-hemolysin gene of V. vulnificus and gas chromatographic analysis of the fatty acid contents of positive isolates. Only 3.2 and 0.4% of the isolates gave false-positive and false-negative results, respectively. The average level of recovery on VVE medium for 33 strains, including both clinical and environmental isolates, was 92% of the level of recovery obtained with brain heart infusion agar supplemented with 1% NaCl. The densities of V. vulnificus in oyster homogenates and individual oysters harvested from gulf and Atlantic coastal waters revealed that seasonally high levels occurred. The VVE medium procedure facilitated enumeration of this pathogen in molluscan shellfish and had a distinct advantage over the widely used most-probable-number procedure for V. vulnificus enumeration, which requires 5 to 7 days and often gives improbable and imprecise results.     

Uptake and clearance of Vibrio vulnificus from Gulf coast oysters (Crassostrea virginica).

Oysters collected in late winter, when they were free of Vibrio vulnificus, were exposed in the organism in the laboratory. The oysters effectively concentrated the bacteria from seawater, but when the inoculum was removed, the bacteria were rapidly cleared from the oyster tissues. These results suggest that V. vulnificus may be found in oysters as a result of filtration of the bacteria from seawater rather than active multiplication of the bacteria in the oysters.     

Effect of gamma irradiation on shelf life and bacterial and viral loads in hard-shelled clams (Mercenaria mercenaria).

The feasibility of using 60Co gamma irradiation to inactivate total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, and F-coliphage in hard-shelled clams, Mercenaria mercenaria, was investigated. The results of three trials indicated average D10 values of 1.32 kGy for total coliforms, 1.39 kGy for fecal coliforms, 1.54 kGy for E. coli, 2.71 kGy for C. perfringens, and 13.50 kGy for F-coliphage. Irradiation doses of > 0.5 kGy were significantly lethal to the shellfish.     

Oyster-MSX interactions: Alterations in hemolymph enzyme activities in Crassostrea virginica during the course of Minchinia nelsoni disease development

Hemolymph enzyme activities were investigated during the course of Minchinia nelsoni (MSX) disease development in Crassostrea virginica. Aspartate aminotransferase, alanine aminotransferase, and phosphohexose isomerase activities increase significantly during the gill lesion stage of MSX disease. Enzyme activities are not significantly elevated during the general infection stage of MSX disease. The alteration of hemolymph enzyme activity is discussed with respect to host metabolism and possible humoral defense mechanisms.     

Isolation and partial characterization of a cadmium-binding protein from the American oyster (Crassostrea virginica).

American oysters (Crassostrea virginica) were exposed to 0.1 ppm cadmium for 0--15 days in a flowing seawater system and then placed into clean flowing seawater for 24 h prior to sacrifice. Whole oysters were homogenized and a cadmium-binding protein isolated and purified by a process of centrifugation, heat-treatment, Sephadex G-75 chromatography, DEAE cellulose chromatography and disc gel electrophoresis. A highly anionic protein which is not present in control oysters was found to be present in cadmium-exposed animals after 3 days of treatment and to increase in concentration at succeeding time points. The protein does not extensively bind zinc or copper. Amino acid analysis of the purified protein disclosed an amino acid composition characterized by a high percentage of dicarboxylic amino acids and relatively little cysteine.