Purification of a novel arthropod defensin from the American oyster, Crassostrea virginica

An antimicrobial peptide was purified from acidified gill extract of a bivalve mollusk, the American oyster (Crassostrea virginica), by preparative acid-urea--polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. The 4265.0 Da peptide had 38 amino acids, including 6 cysteines. It showed strongest activity against Gram-positive bacteria (Lactococcus lactis subsp. lactis and Staphylococcus aureus; minimum effective concentrations [MECs] 2.4 and 3.0 microg/ml, respectively) but also had significant activity against Gram-negative bacteria (Escherichia coli D31 and Vibrio parahemolyticus; MECs 7.6 and 15.0 microg/ml, respectively). Comparison of the amino acid sequence with those of other known antimicrobial peptides revealed that the novel peptide had high sequence homology to arthropod defensins, including those from other bivalves, the mussels Mytilus edulis and Mytilus galloprovincialis. This is the first antimicrobial peptide to be isolated from any oyster species and we have named it American oyster defensin (AOD).     

Cryopreservation of spermatozoa of the American oyster, Crassostrea virginica Gmelin

Spermatozoa of the American oyster Crassostrea virginica were frozen to ?196 °C in concentrated Hanks' salt solution (2.6×) containing 8% dimethyl sulfoxide. About 0.2 ml of spermatozoa that were stored in liquid nitrogen for 68 days fertilized 91% of 65,600 eggs compared to fresh spermatozoa, which fertilized 92% of approximately the same number of eggs from the same females. Larvae resulting from fertilization of fresh eggs with 39-day-old cryopreserved spermatozoa appeared normal after 11 days.     

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.     

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.     

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.     

Eight PCR primers to amplify EST‐linked microsatellites in the Eastern oyster,Crassostrea virginica genome

Twenty‐four microsatellite repeat sequences were identified by screening a total of 4446 eastern oyster, Crassostrea virginica, expressed sequence tags. Polymerase chain reaction primers were designed to amplify 12 of these loci. After optimizing reaction parameters, eight loci showed high variability with consistent amplification that could be scored unambiguously. Ninety two C. virginica from the James River, VA, were genotyped at these loci. Number of alleles per locus ranged from 11 to 53, expected and observed heterozygosities ranged from 0.69 to 0.97, and from 0.30 to 0.99, respectively. Discrepancies between expected and observed heterozygosities were common and likely caused by null alleles.     

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.     

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.     

Short‐term heat stress impairs testicular functions in the American oyster,Crassostrea virginica: Molecular mechanisms and induction of oxidative stress and apoptosis in spermatogenic cells

Global temperature is increasing due to anthropogenic activities. Abnormal temperature has devastating effects on growth, reproduction, and development of aquatic organisms. In this study, we examined the effects of short‐term exposure to elevated temperatures (28 and 32°C for 1 week) on testicular functions, heat shock protein‐70 (HSP70), dinitrophenyl protein (DNP, a biomarker of reactive oxygen species [ROS]), and nitrotyrosine protein (NTP, an indicator of reactive nitrogen species [RNS]) expressions, protein carbonyl (PC, a measure of ROS) contents, nitrates/nitrites (NOx, a metabolite of nitric oxide) levels, extrapallial fluid (EPF) conditions, and cellular apoptosis in American oyster (Crassostrea virginica). Higher temperatures significantly decreased (~26%) sperm production in oysters compared with controls (24°C). HSP70, NTP, and DNP expressions were increased after heat exposure, consistent with increased EPF pH, and cellular apoptosis. The enhanced apoptosis in spermatogenic cells is associated with increased caspase 3/7 activity, PC contents, and NOx levels in testicular tissues. Together these results suggest that elevated temperature drastically increases oxidative stress and cellular apoptosis which in turn leads to decreased testicular functions in oysters. To the best of our knowledge, this study reports the first findings on the impacts of elevated temperatures on testicular functions in oysters.     

Cytometric investigations on hemocytes of the American oyster, Crassostrea virginica

Pericardial hemolymph was obtained from American Oysters (Crassostrea virginica) and the hemocytes characterized by flow cytometry. The cells were found to have a broad unimodal size distribution with a median diameter of 7 micrometers. Total protein measured by flow cytometric fluorescence of dansylated cells also revealed a broad unimodal distribution similar to that obtained for size. The proportion of hemocytes in each stage of the cell cycle was measured using DNA-specific DAPI fluorescence. Histograms showed a single peak representing the G(0)/G(1) population. There was no evidence of S or G(2)+M phases of the cell cycle, nor was polyploidy seen. The forward and orthogonal light scatter of fixed hemocytes showed no evidence of sub-populations on the basis of cytoplasmic granularity. Thus, in terms of these parameters, oyster hemocytes appear to represent a single population exhibiting graded cellular differences.     

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.     

The Effects of Inorganic Phosphates on Trochophore Larvae of the Oyster,Crassostrea virginica

Summary

Trochophore larvae were exposed to monosodium orthophosphate (MSOP), sodium hexametaphosphate (SHMP), sodium tripolyphosphate (STPP), and tetrasodium pyrophosphate (TSPP) at concentrations of 15 ppm and 150 ppm phosphate for 24, 48, and 72 hr. All four compounds caused morphological developmental changes, increased mortality, and inhibition of shell growth at both 15 ppm and 150 ppm. The rank order of effectiveness of the compounds was determined from percentage abnormal larvae, percentage survival, and from shell measurements. The order was MSOP>SHMP>STPP≥TSPP for abnormality and mortality and TSPP>STPP>MSOP>SHMP for shell growth inhibition. The relative effect of the individual compounds in causing mortality varied with the time of exposure. Orthophosphate retarded growth rates of the two valves to different degrees.     

Differences in heat shock protein 70 expression during larval and early spat development in the Eastern oyster, Crassostrea virginica (Gmelin, 1791)

For a variety of species, changes in the expression of heat shock proteins (HSP) have been linked to key developmental changes, i.e., gametogenesis, embryogenesis, and metamorphosis. Many marine invertebrates are known to have a biphasic life cycle where pelagic larvae go through settlement and metamorphosis as they transition to the benthic life stage. A series of experiments were run to examine the expression of heat shock protein 70 (HSP 70) during larval and early spat (initial benthic phase) development in the Eastern oyster, Crassostrea virginica. In addition, the impact of thermal stress on HSP 70 expression during these early stages was studied. C. virginica larvae and spat expressed three HSP 70 isoforms, two constitutive, HSC 77 and HSC 72, and one inducible, HSP 69. We found differences in the expression of both the constitutive and inducible forms of HSP 70 among larval and early juvenile stages and in response to thermal stress. Low expression of HSP 69 during early larval and spat development may be associated with the susceptibility of these stages to environmental stress. Although developmental regulation of HSP 70 expression has been widely recognized, changes in its expression during settlement and metamorphosis of marine invertebrates are still unknown. The results of the current study demonstrated a reduction of HSP 70 expression during settlement and metamorphosis in the Eastern oyster, C. virginica.     

Settlement and Survival of the Oyster Crassostrea virginica on Created Oyster Reef Habitats in Chesapeake Bay

Efforts to restore the Eastern oyster (Crassostrea virginica) reef habitats in Chesapeake Bay typically begin with the placement of hard substrata to form three‐dimensional mounds on the seabed to serve as a base for oyster recruitment and growth. A shortage of oyster shell for creating large‐scale reefs has led to widespread use of other materials such as Surf clamshell (Spisula solidissima), as a substitute for oyster shell. Oyster recruitment, survival, and growth were monitored on intertidal reefs constructed from oyster and Surf clamshell near Fisherman’s Island, Virginia, U.S.A. and on a subtidal Surf clamshell reef in York River, Virginia, U.S.A. At the intertidal reefs, oyster larvae settlement occurred at similar levels on both substrate types throughout the monitoring period but higher levels of post‐settlement mortality occurred on clamshell reefs. The oyster shell reef supported greater oyster growth and survival and offered the highest degree of structural complexity. On the subtidal clamshell reef, the quality of the substrate varied with reef elevation. Large shell fragments and intact valves were scattered around the reef base, whereas small, tightly packed shell fragments paved the crest and flank of the reef mound. Oysters were more abundant and larger at the base of this reef and less abundant and smaller on the reef crest. The availability of interstitial space and appropriate settlement surfaces is hypothesized to account for the observed differences in oyster abundance across the reef systems. Patterns observed emphasize the importance of appropriate substrate selection for restoration activities to enhance natural recovery where an underlying habitat structure is destroyed.     

Improved attachment and spreading in primary cell cultures of the eastern oyster, Crassostrea virginica

At present, establishment of a cell line from bivalve molluscs has been unsuccessful, and in vitro work is limited to primary cell cultures. We sought to improve attachment and spreading of cells of the eastern oyster, Crassostrea virginica, to aid primary cultures and to assist development of a bivalve cell line. Our objectives were to examine the effects of substrate on ventricle cell viability, attachment, and spreading by testing of collagen I, collagen IV, fibronectin, laminin, poly-D-lysine, and two types of uncoated tissue culture plates (Falcon and Corning). Experiments were conducted by incubating cells with the various substrates for 24 h and 5 d. An assay with a tetrazolium compound (MTS) was used to estimate cell numbers based on metabolic activity. Although differences in MTS assay values for substrate effect on cell viability were detected at 24 h and at 5 d (P > 0.0001), these were attributed to variations in metabolic activity due to different levels of attachment and spreading among treatments. Differences among treatments were detected in attachment and spreading at 24 h and 5 d (for all, P > 0.0001). At 24 h, poly-D-lysine induced the highest levels of attachment and spreading; no other factor performed better than the uncoated Falcon substrate, and collagen I performed most poorly. At 5 d, poly-D-lysine and the uncoated Corning substrate induced significantly higher levels of attachment and spreading than did the uncoated Falcons substrate, and collagen I performed most poorly. From these results, poly-D-lysine best promoted cell attachment and spreading. Fibronectin (at 24 h) and laminin (at 5 d) warrant further study. Along with improvements in medium composition, future work should involve screening of other attachment factors and combinations of factors, including those of bivalve origin.