Oyster Hemolymph Research Articles

Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures

Hemolymph and soft tissues of Pacific oysters (Crassostrea gigas) kept in sand-filtered seawater at temperatures between 1 and 8 degrees C were normally found to contain bacteria, with viable counts (CFU) in hemolymph in the range 1.4 x 10 to 5.6 x 10 bacteria per ml. Pseudomonas, Alteromonas, Vibrio, and Aeromonas organisms dominated, with a smaller variety of morphologically different unidentified strains. Hemolymph and soft tissues of horse mussels (Modiolus modiolus), locally collected from a 6- to 10-m depth in the sea at temperatures between 4 and 6 degrees C, also contained bacteria. The CFU in horse mussel hemolymph was of the same magnitude as that in oysters (mean, 2.6 x 10), and the bacterial flora was dominated by Pseudomonas (61.3%), Vibrio (27.0%), and Aeromonas (11.7%) organisms. In soft tissues of horse mussels, a mean CFU of 2.9 x 10 bacteria per g was found, with Vibrio (38.5%), Pseudomonas (33.0%), and Aeromonas (28.5%) constituting the major genera. After the challenge of oysters in seawater at 4 degrees C to the psychrotrophic fish pathogen Vibrio salmonicida (strains NCIMB 2245 from Scotland and TEO 84001 from Norway) and a commensal Aeromonas sp. isolated from oysters, the viable count in hemolymph increased 1,000-fold to about 10 bacteria per ml. In soft tissues, about a 1,000-fold increase in CFU to 6 x 10 was observed. V. salmonicida NCIMB 2245 invaded hemolymph and soft tissues after 14 days and dominated these compartments after 41 days, whereas strain TEO 84001 did not invade soft tissues to the same extent. Challenge with V. salmonicida NCIMB 2245 resulted in 100% mortality, whereas about 50% of the oysters survived challenge with the Norwegian strain, TEO 84001. The commensal Aeromonas sp. invaded hemolymph and soft tissues and caused 100% mortality. Oyster hemolymph contained agglutinins for Vibrio anguillarum but not for V. salmonicida, whereas we did not find agglutinins for either of these bacteria in horse mussels. Agglutinins for horse and human erythrocytes were found in hemolymph from both animals. We found no differences in agglutinin titers in oysters from different Norwegian locations, and long-term challenge with bacteria in seawater did not result in changes of agglutinin activity. These studies demonstrate that bacteria exist in hemolymph and soft tissues of marine bivalves at temperatures below 8 degrees C. Increased bacterial numbers in seawater at 4 degrees C result in augmented invasion of bacteria in hemolymph and soft tissues. V. salmonicida, a bacterium pathogenic for fish at low temperatures, invades bivalve hemolymph and soft tissues, and thus bivalves may serve as a reservoir for pathogens of fish at low seawater temperatures.

In Vitro Propagation of the Protozoan Perkinsus Marinus, A Pathogen of the Eastern Oyster, Crassostrea Virginica

ABSTRACT. Perkinsus marinus, a pathogen of eastern oysters (Crassostrea virginica), has been successfully propagated in vitro. Cultures of the parasite were initiated from heart fragments of an infected oyster. the cultured protozoan (designated Parkinsus‐1) was similar in morphology at both the light and transmission electron microscopy levels to histozoic stages of P. marinus in naturally infected oysters. In addition, cultured cells incubated in fluid thioglycollate medium produced enlarged cells (prezoosporangia) that stained blue‐black in Lugol's solution, a response characteristic to Perkinsus spp. and used in routine diagnosis. Polyclonal antibodies raised against P. marinus prezoosporangia reacted positively to Perkinsus‐1. Finally, the cultured cells infected susceptible oysters and reisolation of Perkinsus‐1 cells was possible from the hearts of experimentally infected oysters. the culture medium contained most of the known constituents of cell‐free hemolymph of oysters. the success achieved in culturing P. marinus will allow further investigations aimed at reducing mortalities caused by this important oyster pathogen and at addressing many unanswered questions about its biology and pathobiology.

Effect of repeated sampling on haemolymph pH, PO 2 and haemocyte activity in the Pacific oyster, Crassostrea gigas (Thunberg)

The effects of repeat bleeding via a novel cannulation technique, on the physiology of the Pacific oyster, Crassostrea gigas (Thunberg), were compared to control animals from which haemolymph was sampled by syringe and needle. The effects of surgery and cannulation were characterized by an initial alkalosis (up to 8 h) in the haemolymph of cannulated animals followed by an acidosis of up to 12 h; which occurred in both cannulated and control oysters. A decline in partial pressure of oxygen of oyster haemolymph, observed in both cannulated and control oysters, was directly related to shell closure following handling. Furthermore, a haemocyte concentration observed at 4 h followed by haemocyte dilution at 12 h post-surgery, suggested a handling-related stress response in both cannulated and control oysters. A heightened phagocytic activity of haemocytes at 4 h also supported the occurrence of handling-related stress response in all animals. The cannulation technique described here provides the investigator with a convenient tool for chronic, and repeated sampling of haemolymph with a transient disturbance to the animal.

Agglutinin activity in pacific oyster ( Crassostrea gigas) hemolymph following in vivo Vibrio anguillarum challenge

Hemolymph from the Pacific oyster ( Crassostrea gigas) contains lectins that agglutinate horse (Gigalin E) and human (Gigalin H) erythrocytes. The gigalins also agglutinate bacteria, including Vibrio anguillarum, and were adsorbed from oyster hemolymph at different temperatures by living, heat-killed, and freeze-dried V. anguillarum cells. Baseline activities of the two gigalins were established by measuring their activities in oyster hemolymph over a period of 4 years. A normal distribution of Gigalin H activity (mean titer 139) was found, whereas the distribution of Gigalin E activity in the same samples was skew (mean titer 512). No covariance was observed between the two agglutinin activities. Increased lectin activity above this baseline was found in oysters exposed for varying time intervals to V. anguillarum at different seasons and temperatures over a period of 2 years. Such exposure resulted in an increase in activity (titer) of four- to nine-fold for Gigalin E and three- to seven-fold for Gigalin H when compared with controls, and in augmentation in the hemolymph of a protein with the same electrophoretic mobility as affinity-purified oyster lectins (gigalins). Challenge with either living or heat-killed bacteria resulted in a significant increase of Gigalin E activity, whereas results for Gigalin H were variable. Oysters challenged with bacteria were observed to filter normally with open shells during the experiments. Also, no increase was found in hemolymph calcium that could indicate anoxia following bacterial challenge (0.49 ± 0.004 mg mL -1) compared to unexposed oysters (0.50 ± 0.001 mg mL -1). Increase in the concentration of free amino acids in oyster hemolymph was observed following exposure to bacteria (15.05 mM) and anaerobiosis (13.51 mM) compared to controls (9.06 mM), and changes (in mol %) of individual amino acids differed considerably between hemolymph from animals challenged with bacteria and animals kept anaerobic. The augmented lectin activity in oyster hemolymph, following in vivo exposure to increased bacteria in the seawater, suggests their involvement in enhancing bacterial clearance and defense in the oyster.

A direct monoclonal antibody sandwich immunoassay for detection of Bonamia ostreae (Ascetospora) in hemolymph samples of the flat oyster Ostrea edulis (Mollusca Bivalvia)

Currently recognition of molluscan diseases relies heavily upon light microscopic examination of stained histological sections of molluscan tissues Such histopathological methods are time consuming and relatively inefficient In the study of ongoing epizootics Alternatively, more effluent investigative tools are needed to accelerate disease recognition and quantification Such methods are needed to prevent control and eradicate molluscan diseases This paper reports the development of a direct sandwich enzyme-lmmunosorbent assay (ELISA) for the ascetosporan intrahemocytic parasite Bonamiao ostreae of the flat oyster Ostrea edulis. Bonamiasis is a commercially important epizootic disease of oysters A Bonamia-specific monoclonal antibody is employed in the assay The assay is performed In antibody-coated microplates employing standardized reagent and 50 µl oyster hemolymph samples This procedure was developed into a commercial product, available as a kit Based upon clinical studies, there was a 90 O/o agreement In test results when ELISAs were compared to the reference histopathologic light microscopic examination for the parasites

A study of steady state and kinetic regulation of chloride ion and osmotic pressure in hemolymph of oysters,Crassostrea virginica, exposed to tri-n-butyltin

Two sets of experiments were made to determine if bis(tri-n-butyl)tin oxide (TBT) [IUPAC: hexabutyldistannoxane] influenced osmotic pressure or chloride ion concentration in hemolymph of adult American oysters, Crassostrea virginica. In the first set, oysters were acclimated to 5, 25 or 40%;, then exposed to bis(tri-n-butyl)tin oxide dissolved in seawater (0.5, 1.0 or 2.0 micrograms/L) for 11 days. Measurements of the total osmotic pressure and chloride ion concentration in hemolymph indicated that mean values of neither was significantly affected under steady state conditions. Oysters exposed to TBT exhibited a higher variation around the mean value for both total osmotic pressure and chloride ion concentration. There was significant mortality of oysters exposed to TBT in 25 and 40, but not in 5%. In a second experiment, adult oysters were acclimated to 25%, and simultaneously exposed to the TBT concentrations listed above for 10 days. Then, subgroups of oysters were abruptly moved to 5 or 40% and the time-course of adjustment of hemolymph osmotic pressure and chloride ion concentration was measured. During the first 25 hr, there was little adjustment to 5% in controls or TBT exposed oysters; apparently, they remained closed most or all of the time. In marked contrast, hemolymph of oysters moved to higher salinities rapidly readjusted. Exposure to TBT had a delaying effect on the schedule of the increase in hemolymph osmotic pressure and chloride ion concentration. Results of these experiments show that TBT has an indirect effect on osmotic pressure and chloride ion concentration adjustment in oysters; it does not appear to act strongly as an anionophore to influence anion transfer across epithelia.

Invertebrate red blood cell carbonic anhydrase

This is the first report documenting the presence of carbonic anhydrase (CA) for any invertebrate red cells. CA activity was measured in plasma, hemolysates of blood cells, and in hemolymph of selected species of invertebrates. Annelid red blood cells (RBC) and sipunculid pink blood cells both possessed significant levels of CA activity. Molluscan RBC, on the other hand, lacked CA activity. The distribution appears to have fallen along phylogenetic lines, with CA being present only in blood cells of the two more closely related groups. However, the presence of extracellular CA was confirmed in oyster hemolymph. Oyster hemolymph CA showed a similar affinity (Ki) for the sulfonamide inhibitors acetazolamide and ethoxzolamide, as did the vertebrate RBC CA II isozyme, supporting the idea that this isozyme could be the ancestral form of the enzyme.

Seasonal and environmental variation in protein and carbohydrate levels in the hemolymph from American oysters ( Crassostrea virginica gmelin)

1. 1. Carbohydrate and protein levels in cell-free hemolymph of Crassostrea virginica exhibited a pronounced seasonal cycle, possibly linked to the oyster's reproductive cycle. 2. 2. There were persistent differences in the hemolymph carbohydrate and protein levels between oysters collected from different localities. 3. 3. The influence of laboratory changes in temperature and salinity, alone and in combination, on the oyster's hemolymph carbohydrate and protein concentration varied according to the season and site of collection. 4. 4. Hemolymph protein and carbohydrate levels in oysters starved for 7 days did not differ from those in fed animals.

Serum creatinine concentration and renal function in rheumatic diseases

The long-term evolutionary interaction between the host and symbiotic microbes determines their cooperative relationship. It is well known that the symbiotic microbes have evolved various mechanisms to either benefit or exploit the mammalian host immune system to maintain homeostasis. However, the strategies employed by the symbiotic microbes to overcome host immune responses in invertebrates are still not clear. In the current study, the hemolymph microbes in oyster <i>Crassostrea gigas</i> were found to be able to directly bind an oyster Ig superfamily member (IgSF) (designated as <i>Cg</i>IgIT) to inhibit the immune responses of hemocytes. The mRNA transcripts of <i>Cg</i>IgIT in hemocytes increased significantly after the stimulation with hemolymph microbes. <i>Cg</i>IgIT was found to be located on the hemocyte membrane and it was able to directly bind the hemolymph microbes and polysaccharides via its three Ig domains and recruited the protein tyrosine phosphatase <i>Cg</i>SHP2 through its ITIM. The recruited <i>Cg</i>SHP2 inhibited the activities of <i>Cg</i>ERK, <i>Cg</i>P38 and <i>Cg</i>JNK proteins to reduce the productions of dual oxidase 2 (<i>Cg</i>Duox2) and defensin 2 (<i>Cg</i>Def2), which eventually protected the hemolymph microbes from <i>Cg</i>Duox2/<i>Cg</i>Def2-mediated elimination. Collectively, the results suggest that the oyster IgIT-SHP2 signaling pathway can recognize bacteria capable of residing in oyster hemolymph and inhibit innate immune responses, which contributes to the maintenance, colonization, and survival of hemolymph microbes.

In Vitro Maintenance of Amoebocytes from the American Oyster (Crassostrea virginica)

Explant and monolayer cell cultures were initiated from oyster heart and embryonic tissue and maintained for periods of a few days to 6 mo depending on the type of tissue and the culture medium. A pH of 7.0–7.3 and a temperature of 20 °C were optimum. Vertebrate cell culture media prepared in a marine saline and supplemented with fetal bovine serum and protein digests provided a suitable basal medium. Supplementation of the basal medium with oyster hemolymph or extracts of oyster tissue markedly prolonged cell maintenance. Explant cultures of heart tissue with the subsequent outward migration of individual cells were most easily initiated and maintained for periods up to 6 mo. Although several cell types were observed, actively motile, granular amoebocytes predominated. No mitotic cells were observed even following exposure to a variety of mitogens. Cultures initiated from disaggregated larvae did yield actively dividing cells. Key words: oyster, cell culture, amoebocytes

Vivo clearance of enteric bacteria from the hemolymph of the hard clam and the American oyster

American oysters, Crassostrea virginica, and hard clams, Mercenaria mercenaria, were experimentally contaminated with Escherichia coli, Salmonella typhimurium, and Shigella flexneri either by intracardial injection or via the natural route of ingestion. Bacterial inactivation in the hemolymph was monitored for 72 h after exposure to these enteric pathogens at 20 and 6 degrees C. At 6 degrees C, both mean bacterial uptake by ingestion and subsequent clearance was singificantly lower that at 20 degrees C. However, substantial bacterial clearance from the hemolymph occurred for both shellfish at each temperature. At 20 degrees C, viable bacteria were no longer detectable after 24 h in hemolymph of either clams or oysters after exposure to contaminated water containing 4 x 10(3) bacteria per ml.

High Performance Liquid Chromatographic Separation and Fluorescent Measurement of Taurine, a Key Amino Acid

Abstract A rapid, sensitive method for the analysis of taurine in oyster hemolymph (blood) has been developed. Highly fluorescent, substituted isoindoles formed by the reaction of taurine and other amino acids with o-phthaldialdehyde/ethanethiol reagent were separated on a reverse phase, high performance liquid chromatographic column. It was necessary to carefully control the concentration of the sodium ion in the phosphate buffer in order to maintain an adequate resolution of both taurine and tyrosine from β-alanine and arginine. Calibration plots of the fluorescent taurine derivative were linear over 2.5 orders of magnitude with a limit of detection of 0.10 nanomoles per ml of oyster hemolymph.

Effects of Salinity and Selected Petroleum Hydrocarbons on the Osmotic and Chloride Regulation of the American Oyster, Crassostrea virginica

Previous articleNext article No AccessEffects of Salinity and Selected Petroleum Hydrocarbons on the Osmotic and Chloride Regulation of the American Oyster, Crassostrea virginicaRoger D. Anderson, and Jack W. AndersonRoger D. Anderson Search for more articles by this author , and Jack W. Anderson Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by Volume 48, Number 4Oct., 1975 Article DOIhttps://doi.org/10.1086/physzool.48.4.30155667 Views: 4Total views on this site Citations: 13Citations are reported from Crossref Journal History This article was published in Physiological Zoology (1928-1998), which is continued by Physiological and Biochemical Zoology (1999-present). PDF download Crossref reports the following articles citing this article:Edward Olmedo, Michael Smith Development of an IoT Water Quality Monitoring System for Data-Based Aquaculture Siting, (Mar 2021): 1–8.https://doi.org/10.1109/SoutheastCon45413.2021.9401906Olaf Heilmayer, Julian Digialleonardo, Lianfen Qian, Guritno Roesijadi Stress tolerance of a subtropical Crassostrea virginica population to the combined effects of temperature and salinity, Estuarine, Coastal and Shelf Science 79, no.11 (Aug 2008): 179–185.https://doi.org/10.1016/j.ecss.2008.03.022C.T Tirard, R.M Grossfeld, J.F Levine, S Kennedy-Stoskopf Effect of Osmotic Shock on Protein Synthesis of Oyster Hemocytes In Vitro, Comparative Biochemistry and Physiology Part A: Physiology 116, no.11 (Jan 1997): 43–49.https://doi.org/10.1016/S0300-9629(96)00115-6Elizabeth Bokman, Roy B. Laughlin A study of steady state and kinetic regulation of chloride ion and osmotic pressure in hemolymph of oysters,Crassostrea virginica, exposed to tri-n-butyltin, Archives of Environmental Contamination and Toxicology 18, no.66 (Nov 1989): 832–838.https://doi.org/10.1007/BF01160297J. A. Nell, P. R. Dunkley Effects of temperature, nutritional factors and salinity on the uptake of L-methionine by the Sydney rock oyster Saccostrea commercialis, Marine Biology 80, no.33 (Jan 1984): 335–339.https://doi.org/10.1007/BF00392829R.C. Dean, A.A. Paparo Effects of changes of salinity and calcium concentration on ctenidial ciliary activity in the oyster Crassostrea virginica (Gmelin), Comparative Biochemistry and Physiology Part A: Physiology 74, no.33 (Jan 1983): 587–594.https://doi.org/10.1016/0300-9629(83)90552-2Bruce M. S. Mahoney, George S. Noyes Effects of petroleum on feeding and mortality of the American oyster, Archives of Environmental Contamination and Toxicology 11, no.55 (Sep 1982): 527–531.https://doi.org/10.1007/BF01056358Bruce M. Mahoney, Harold H. Haskin The effects of petroleum hydrocarbons on the growth of phytoplankton recognised as food forms for the eastern oyster, Crassostrea virginica gmelin, Environmental Pollution Series A, Ecological and Biological 22, no.22 (Jun 1980): 123–132.https://doi.org/10.1016/0143-1471(80)90188-9Lynn M. Amende, Sidney K. Pierce Cellular volume regulation in salinity stressed molluscs: The response ofNoetia ponderosa (Arcidae) red blood cells to osmotic variation, Journal of Comparative Physiology ? B 138, no.44 (Jan 1980): 283–289.https://doi.org/10.1007/BF00691562Susan R. Zell, Mark H. Bamford, Herbert Hidu Cryopreservation of spermatozoa of the American oyster, Crassostrea virginica Gmelin, Cryobiology 16, no.55 (Oct 1979): 448–460.https://doi.org/10.1016/0011-2240(79)90059-2J.W. Anderson AN ASSESSMENT OF KNOWLEDGE CONCERNING THE FATE AND EFFECTS OF PETROLEUM HYDROCARBONS IN THE MARINE ENVIRONMENT, (Jan 1979): 3–21.https://doi.org/10.1016/B978-0-12-718260-5.50005-0J.W. Anderson RESPONSES TO SUBLETHAL LEVELS OF PETROLEUM HYDROCARBONS: ARE THEY SENSITIVE INDICATORS AND DO THEY CORRELATE WITH TISSUE CONTAMINATION?, (Jan 1977): 95–114.https://doi.org/10.1016/B978-0-08-021613-3.50014-0FREDERICK G. JOHNSON SUBLETHAL BIOLOGICAL EFFECTS OF PETROLEUM HYDROCARBON EXPOSURES: BACTERIA, ALGAE, AND INVERTEBRATES, (Jan 1977): 271–318.https://doi.org/10.1016/B978-0-12-466902-4.50011-4

Hemolymph-free amino acids and related nitrogenous compounds of Crassostrea virginica infected with Bucephalus sp. and Minchinia nelsoni

1. 1. Seventeen free amino acids and related amines were detected in the normal oyster hemolymph. Free taurine, alanine, glycine and serine were found in high concentrations. Phosphoethanolamine, β-alanine, ornithine, phosphoserine, arginine, lysine and glutamic acid were present in moderate amounts, while leucine, isoleucine, γ-aminobutyric acid, aspartic acid, threonine and methionine existed in low and trace quantities. 2. 2. The total concentration as well as composition of normal oyster hemolymph-free ami amino acids and amines varied with seasons and geographic locations at which the oysters were maintained. 3. 3. In general the depletion of β-alanine, ornithine, arginine, lysine, leucine and isoleucine was accompanied by concomitant increases in free taurine, aspartic acid, glutamic acid, threonine and phosphoserine in the infected oyster hemolymph.

Physical and Chemical Characterization of an Oyster Hemagglutinin

Abstract The natural hemagglutinin of the oyster Crassostrea virginica was examined for possible structural relationship with vertebrate immunoglobulins. The hemagglutinin activity was associated with a heterogeneous group of rapidly sedimenting molecules. The major active component in oyster hemolymph has a sedimentation coefficient of 33.4 S, but minor components with lower sedimentation coefficients are also present. The molecules can be dissociated into subunits consisting of single polypeptide chains having an approximate molecular weight of 20,000. Complete dissociation could be observed in 5 m guanidine-HCl indicating noncovalent linkage of the subunit. Amino acid composition of the subunit was distinguished by a relatively high histidine content and the absence of lysine. The sequence of the first 2 residues from the NH2 terminus of the subunit was found to be Thr-Ala. Mannose, galactose, and glucosamine were present with a total carbohydrate content of 13%. These data do not demonstrate obvious structural similarities to mammalian immunoglobulins.

Lysozyme in the hemolymph of the oyster, Crassostrea virginica

Oyster hemolymph lyses suspensions of some gram-positive bacteria and degrades cell walls isolated from Micrococcus lysodeikticus with the release of amino and reducing sugars. This indicates that the bacteriolytic substance in oyster hemolymph is lysozyme.

Mechanism of agglutination of red blood cells by oyster hemolymph

Hemolymph from normal oysters ( Crassostrea virginica) agglutinates the red blood cells of many vertebrate species. Inhibition tests indicate that the reactive site on human cells is the ABO blood group hapten; chemically different sites serve as points of attachment in other species. The reactive protein molecule requires calcium to agglutinate human cells but does not require calcium to agglutinate cells of nonhuman species. In the absence of calcium, hemagglutinin is heat-labile and is degraded to protein subunits with loss of biological activity. Presumably the intact complex molecule has the proper spatial orientation to attach to specific sites on red cells and to bind them in a lattice formation that results in agglutination.

Hemagglutinins from oyster hemolymph.

Oyster hemolymph hemagglutinin agglutinated fish, rabbit, and human, red blood cells, was active over a wide pH range (optimum pH 7.5), and was heat-labile. Dialysis caused a greater reduction in the activity in the aged hemolymph than it did in the fresh hemolymph. Ultracentrifugation removed 90% of the protein; the remaining 11% retained all the activity. Gel filtration revealed two distinct hemagglutinins associated with two main protein peaks. Fractionation according to solubility gave seven different protein fractions, but only the salt-soluble and a very minor water-soluble Fraction possessed hemagglutinating activity. Agar-gel diffusion tests on the protein fractions showed a complexity of patterns which was partially resolved by immunoelectrophoretic techniques.