Cryopreservation of oyster (Crassostrea gigas) embryos

Several critical variables associated with successful cryopreservation of oyster embryos (Crassostrea gigas) were examined. These were 1) embryo developmental stage, 2) kind and concentration of cryoprotectant, 3) equilibration time, and 4) freezing rate. The percentage of survival was scored as the number of recovered embryos that swam actively 12 h after thawing and had developed into veliger stage. The oyster embryos became increasingly susceptible to the cryoprotectants as the concentration was increased and the equilibration time was lengthened. The stage of development appears to be a critical factor for survival of oyster embryos, with trochophore stage embryos more resistant than morula and gastrula stages embryos to cryoprotectant exposure and having better surviving after freezing. The optimum cryoprotectant concentration for the trochophore embryos differed markedly from the morula stage. Cryopreservation of fertilized eggs (2 to 8 cells) was unsuccessful. Varying degrees of success were achieved using gastrula- and trochophore-stage embryos. Maximum survival was obtained when trochophore embryos incubated in 10% propylene glycerol-artificial sea water were cooled at -2.5 degrees C/min to -30 degrees C and were then directly placed into liquid nitrogen. The results showed a clear effect of the stage of development on survival.     

Identification of bacterial chemoattractants for oyster (Crassostrea virginica) hemocytes

The cytoplasmic and cell wall components of the Gram-positive bacterium Bacillus megaterium and the cytoplasmic and cell envelope components of the Gram-negative bacterium Escherichia coli were assayed for chemotactic activity for the hemocytes of Crassostrea virginica. The cellular components were separated by differential centrifugation and gel filtration was used to determine the approximate molecular weights of the chemoattractant molecules. Active fractions were assayed for glycoproteins and lipoproteins. As a result, it is known that hemocytes are chemotactically attracted to proteins of approximately 10,000 daltons which are associated with the cell wall of B. megaterium and the cell envelope of E. coli.     

Esterification of vertebrate-type steroids in the Eastern oyster (Crassostrea virginica)

Characteristics of acyl-coenzyme A (acyl-CoA):steroid acyltransferase from the digestive gland of the oyster Crassostrea virginica were determined by using estradiol (E2) and dehydroepiandrosterone (DHEA) as substrates. The apparent Km and Vmax values for esterification of E2 with the six fatty acid acyl-CoAs tested (C20:4, C18:2, C18:1, C16:1, C18:0, and C16:0) were in the range of 9-17 microM E2 and 35-74 pmol/min/mg protein, respectively. Kinetic parameters for esterification of DHEA (Km: 45-120 microM; Vmax: 30-182 pmol/min/mg protein) showed a lower affinity of the enzyme for this steroid. Formation of endogenous fatty acid esters of steroids by microsomes of digestive gland and gonads incubated in the presence of ATP and CoA was assessed, and at least seven E2 fatty acid esters and five DHEA fatty acid esters were observed. Some peaks eluted at the same retention times as palmitoleoyl-, linoleoyl-, oleoyl/palmitoyl-, and stearoyl-E2; and palmitoleoyl-, oleoyl/palmitoyl-, and stearoyl-DHEA. The same endogenous esters, although in different proportions, were produced by gonadal microsomes. The kinetic parameters for both E2 (Km: 10 microM; Vmax: 38 pmol/min/mg protein) and DHEA (Km: 61 microM; Vmax: 60 pmol/min/mg protein) were similar to those obtained in the digestive gland. Kinetic parameters obtained are similar to those observed in mammals; thus, fatty acid esterification of sex steroids appears to be a well-conserved conjugation pathway during evolution.     

Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches.

Metallothioneins are typically low relative molecular mass (6000-7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-X(n)-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan alphabeta-domain structure. Oyster metallothioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of alphabeta-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the alpha-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four alpha-domains and that lack a beta-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evolutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family.     

Chronic infections of Haplosporidium nelsoni (MSX) in the oyster Crassostrea virginica

Oysters inhabiting areas enzootic for the parasite Haplosporidium nelsoni (MSX) are exposed to infective particles each summer, and it is often difficult to distinguish newly acquired lesions from older infections. To study long-term parasitism without the complication of new infections, MSX-infected oysters were moved from Delaware Bay to a disease-free area on the New Jersey coast. Because infections seen after the transfer were acquired in Delaware Bay during a known infective period, it was possible to determine how long oysters remained infected and how they were affected by chronic parasitism. Chronic infections displayed the same seasonal cycle (high levels in winter and late spring, low levels in summer and early spring) that occurs in enzootic areas with annual reinfection. Within individual oysters, chronic MSX became localized, relapsed into general infections, and then became localized again in a sequence that was probably controlled by temperature. Some experimental oysters survived with MSX for at least 4 years. Hemocytosis persisted in these oysters, and their poor condition suggested that chronically infected individuals would have lowered resistance to additional stress.     

A lectin on the hemocyte membrane of the oyster (Crassostrea virginica)

Using antisera produced against a serum lectin we have shown by employing immunocytofluorescence that hemocytes from the oyster, Crassostrea virginica, possess a lectin which is situated on the external surface of the cell membrane. The antisera block the binding of hemocyte microsomes to protease-treated vertebrate erythrocytes, thus confirming that the hemocyte membrane lectin is serologically related to the serum lectin. The major serum lectin has an apparent mass of 34,000. Flow cytometry has revealed that the distribution of the surface lectin on hemocytes represents a heterogeneous expression on a population basis, but no discrete cell subpopulations can be identified.     

Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica)

Lysozyme was purified from the plasma of eastern oysters (Crassostrea virginica) using a combination of ion exchange and gel filtration chromatographies. The molecular mass of purified lysozyme was estimated at 18.4 kDa by SDS-PAGE, and its isoelectric point was greater than 10. Mass spectrometric analysis of the purified enzyme revealed a high-sequence homology with i-type lysozymes. No similarity was found however between the N-terminal sequence of oyster plasma lysozyme and N-terminal sequences of other i-type lysozymes, suggesting that the N-terminal sequences of the i-type lysozymes may vary to a greater extent between species than reported in earlier studies. The optimal ionic strength, pH, cation concentrations, sea salt concentrations, and temperature for activity of the purified lysozyme were determined, as well as its temperature and pH stability. Purified oyster plasma lysozyme inhibited the growth of Gram-positive bacteria (e.g., Lactococcus garvieae, Enterococcus sp.) and Gram-negative bacteria (e.g., Escherichia coli, Vibrio vulnificus). This is a first report of a lysozyme purified from an oyster species and from the plasma of a bivalve mollusc.     

Studies on the gill ciliation of the American oyster Crassostrea virginica (Gmelin)

Latero-frontal, para-latero-frontal, and frontal ciliary tracts on the gill filaments of Crassostrea virginica (Gmelin) were studied with light microscopy and scanning electron microscopy. Latero-frontal cirri are complex structures composed of varying numbers of paired cilia. The multiple pairs of cilia which constitute a single cirrus are closely appressed for a portion of their length; they then branch laterally from the central axis in a plume-like fashion. Latero-frontal cirri of adjacent gill filaments create a filtration sieve which should be capable of retaining particles smaller than 1 μm in diameter. Para-latero-frontal cilia are short, closely spaced cilia arranged as a staggered row along the frontal side of each tract of latero-frontal cirri. Latero-frontal cirri and para-latero-frontal cilia occur on ordinary, principal, and transitional gill filaments. Frontal ciliary tracts of ordinary filaments are divided into a central, ventrally directed coarse tract, flanked on either side by a dorsally directed fine ciliary tract. The coarse tract is covered by cirri which are comprised of five to eight cilia, while the fine frontal tracts are made up of individually functioning cilia. The frontal ciliary tracts of principal and transitional filaments bear only dorsally directed fine cilia. The unique direction of effective beat of the coarse frontal cirri of ordinary filaments, in combination with the action of fine frontal cilia and the strategic location of mucus producing cells, is used to describe a possible mechanism for the sorting of filtered particles.     

Occurrence of symbiotic turbellarians in the oyster,Crassostrea virginica

Crassostrea virginica was collected from several locations where it is cultured, both along the Northumberland Strait of New Brunswick and Malpeque Bay on the coast of Prince Edward Island. The oysters were found with two turbellarians on their gills. Urastoma cyprinae (Graff) was found in the oysters mostly during the warmer months of the year in numbers averaging as high as 50 worms per host (N = 50) and with as much as 78% of the host population infected (N = 100). Paravortex gemellipara (Linton) was also found during warmer months, but much less frequently or abundantly.Both male and female oysters were found to have U. cyprinae. No eggs or recent young of U. cyprinae were found in hosts; female-mature individuals of P. gemellipara with young were found from June through August.     

The effects of some selected bacteria on embryos and larvae of the American oyster,Crassostrea virginica

An investigation was conducted to determine the effects of selected bacteria on healthy embryos and larvae of the American oyster, Crassostrea virginica. A total of 156 bacterial types were isolated from moribund larvae and particulate matter collected with a 36 μm Nitex screen. Most of the isolates were nonpigmented motile, gram-negative rods sensitive to chloramphenicol and neomycin, and were classified as Pseudomonas and Vibrio.Three oyster developmental stages were exposed to selected bacteria: embryos in the first 48 hr of development, 2-day-old larvae, and presetting, 10-day-old larvae. Twenty of the bacterial strains tested caused gross morphological abnormalities, decreased growth, and/or increased mortality for one or more of the three developmental stages. Neither culture filtrate nor heat-killed bacteria produced these effects.     

Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas).

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.     

Epizootiology and pathology of juvenile oyster disease in the Eastern oyster, Crassostrea virginica.

Juvenile Oyster Disease (JOD) causes mortalities of small cultured oysters, Crassostrea virginica. The present study was an intensive epizootiological and pathological investigation of JOD in eight sequentially deployed cohorts at sites on Long Island, New York. JOD symptoms and mortalities began in all groups at about the same time. Lesions on the mantle were detected histologically about 1 week before the principal symptom, a conchiolin deposit on the inner shell, appeared. Mortality began about 1 week later and reached 60-90% in oysters <25 mm. Mantle lesions were highly correlated with subsequent conchiolin-deposit prevalence and with total mortality. Larger juveniles (25-40 mm) were affected by the disease and produced conchiolin deposits, but mortalities did not exceed 30%. Mortalities were consistently related to size, but not necessarily to age or length of "exposure" in the field. There was no indication that JOD was linked to a particular broodstock or hatchery. Wild spat deployed at experimental sites showed JOD symptoms before the hatchery-produced groups did and cohorts maintained inside a hatchery experienced essentially no JOD. Histological examination of cohorts experiencing high mortalities failed to reveal an obvious etiological agent, but showed a disease pattern similar to that described for other bivalve diseases with a bacterial etiology. Similarities and differences between this and other studies of JOD suggest that one or more bacterial species is responsible for JOD, but that a trigger, probably temperature, is also involved and may vary from site to site.     

Genetic linkage map of the eastern oyster Crassostrea virginica Gmelin

Amplified fragment length polymorphisms (AFLPs), along with some microsatellite and Type I markers, were used for linkage analysis in Crassostrea virginica Gmelin, the eastern oyster. Seventeen AFLP primer combinations were selected for linkage analysis with two parents and their 81 progeny. The 17 primer combinations produced 396 polymorphic markers, and 282 of them were segregating in the two parents. Chi-square analysis indicated that 259 (91.8%) markers segregated in Mendelian ratio, while the other 23 (8.2%) showed significant (P < 0.05) segregation distortion, primarily for homozygote deficiency and probably due to deleterious recessive genes. Moderately dense linkage maps were constructed using 158 and 133 segregating markers (including a few microsatellite and Type I markers) from male and female parents, respectively. The male framework map consisted of 114 markers in 12 linkage groups, covering 647 cM. The female map had 84 markers in 12 linkage groups with a length of 904 cM. The estimated genome length was 858 cM for the male map and 1296 cM for the female map. The observed genome coverage was 84% for the male and female map when all linked markers were considered. Genetic maps observed in this study are longer than the cytogenetic map, possibly because of low marker density.     

Interindividual variation of malate dehydrogenase activity in the oyster Crassostrea virginica

Intertidal environments are dynamic, stressful niches and variation in physiological parameters may determine distribution and survival of individuals in a population. We demonstrated that mitochondria of the oyster Crassostrea virginica oxidize malate more readily than other Krebs cycle intermediates and investigated the level of interindividual variability in oyster malate dehydrogenase (MDH) activity and total protein content in muscle tissues. Both MDH activity and total protein evidenced a high level of interindividual variation in heart and adductor among a sample of more than 50 oysters. Normalization to total DNA failed to explain the variation in either MDH activity or protein content of phasic adductor and explained less than 40% of the variation in heart. This range of MDH titers defines a continuum of biochemical phenotypes important for understanding the relative selection forces operative on metabolic pathways within the muscles of the oyster.