Sublethal effects of oil-contaminated sediment to early life stages of the Eastern oyster, Crassostrea virginica.

The explosion of the Deepwater Horizon (DWH) oil drilling rig resulted in the release of crude oil into the Gulf of Mexico. This event coincided with the spawning season of the Eastern oyster, Crassostrea virginica. Although oil bound to sediments constitutes an important source of polycyclic aromatic hydrocarbon (PAH) exposure to benthic organisms, toxicity of sediment-associated DWH oil has not been investigated in any bivalve species. Here, we evaluated the sublethal effects of acute exposure of gametes, embryos and veliger larvae of the Eastern oyster to different concentrations of unfiltered elutriates of sediment contaminated with DWH oil. Our results suggest that gametes, embryos and veliger larvae are harmed by exposure to unfiltered elutriates of contaminated sediment. Effective concentrations for fertilization inhibition were 40.6 µg tPAH50 L-1 and 173.2 µg tPAH50 L-1 for EC201h and EC501h values, respectively. Embryo exposure resulted in dose-dependent abnormalities (EC20 and EC50 values were 77.7 µg tPAH50 L-1 and 151 µg tPAH50 L-1, respectively) and reduction in shell growth (EC2024h value of 1180 µg tPAH50 L-1). Development and growth of veliger larvae were less sensitive to sediment-associated PAHs compared to embryos. Fertilization success and abnormality of larvae exposed as embryos were the most sensitive endpoints for assessing the toxicity of oil-contaminated sediment. Bulk of measured polycyclic aromatic hydrocarbons were sediment-bound and caused toxic effects at lower tPAH50 concentrations than high energy water accommodated fractions (HEWAF) preparations from the same DWH oil. This study suggests risk assessments would benefit from further study of suspended contaminated sediment.

Evaluation of toxicity of Deepwater Horizon slick oil on spat of the oyster Crassostrea virginica.

The 2010 explosion of the Deepwater Horizon (DWH) oil rig generated the largest marine oil spill in US history with millions of barrels of crude oil released in the Gulf of Mexico (GoM). The eastern oyster, Crassostrea virginica, is an ecologically and economically important species in the northern GoM. Due to its biological characteristics (sessile, filter feeding), juvenile oysters may have been affected. This study investigated the effects of surface-collected DWH oil prepared as high-energy water-accommodated fraction (HEWAF) on the survival of 2-month-old oyster spat, and evaluated the potential impacts of HEWAF on particle clearance rate and spat tissue. Exposure of oysters to a range of oil/HEWAF (0-7-66-147-908-3450 µg tPAH50 (sum of 50 polycyclic aromatic hydrocarbons) L-1) resulted in non-dose-dependent mortalities and reduced clearance rates of algal food (Tisochrysis lutea). A morphometric study of the digestive tubules (DGTs) indicated a dose-dependent response to oil exposure on lumen dilation, on epithelium thinning of the DGT, and a significant change in DGT synchrony (LOEC = 66 µg tPAH50 L-1). This finding suggests that structural changes occurred in the digestive gland of exposed oysters most likely due to an oil-related stress. In addition, histological observations showed that tissues in contact with HEWAF (gills, palp, connective tissue, digestive gland) were adversely impacted at ≥ 7 µg tPAH50 L-1, and exhibited pathological symptoms typical of an inflammatory response (e.g., hemocyte diapedesis and infiltration, syncytia, epithelium sloughing).

Perkinsus marinus, a protozoan parasite of the eastern oyster, has a requirement for dietary sterols.

Perkinsus marinus, a protozoan parasite of the eastern oyster, Crassostrea virginica, causes high mortality in its host along the Atlantic and Gulf coasts of North America. P. marinus meronts cultured in vitro in medium containing complete lipid supplement (cod liver oil, cholesterol and alpha tocopherol acetate in detergent) are able to synthesize a wide variety of lipids, yet cultures cannot be maintained in lipid-free medium. To determine P. marinus lipid requirements meronts were inoculated into media containing different combinations of lipid components in detergent. Treatments included complete lipid supplement (positive control), detergent only (negative control), cholesterol in detergent, alpha tocopherol acetate in detergent and cholesterol+alpha tocopherol acetate in detergent. Meronts proliferated in the positive control medium and media containing cholesterol or cholesterol+alpha tocopherol acetate, but failed to proliferate in the negative control medium and the medium containing just alpha tocopherol acetate. Gas chromatography analysis of P. marinus meronts grown in medium with added (13)C sodium acetate (0.5 mg mL(-1)) revealed the presence of fatty acids containing (13)C, but the only sterol present was cholesterol containing no (13)C. These results suggest that P. marinus cannot synthesize sterols and must sequester them from its host.

Viability, infectivity and fatty acid synthetic activity of Perkinsus marinus meront cells incubated in estuarine and artificial seawater.

We investigated the viability and fatty acid synthetic activity of in vitro cultured Perkinsus marinus (Dermo) in lipid-free medium and estuarine water, and the infectivity of P. marinus maintained in artificial seawater (ASW). Viability and fatty acid synthetic activity in 7 d old P. marinus meronts maintained in lipid-free medium and estuarine water were tested. The infectivity of meronts incubated in ASW was examined by first incubating P. marinus meronts in ASW for 2, 3 or 7 d, and then inoculating viable ASW-incubated meronts into the shell cavity of individual oysters Crassostrea virginica. P. marinus infection prevalence and intensity in oysters were determined 9 wk post-inoculation. Heavy mortality occurred in meronts maintained in estuarine water, a drop from an initial value of 100% viable to 7.8 and 6.1% after 3 and 14 d incubation, respectively. Viability was 85 and 67% in meronts maintained in lipid-free medium for 3 and 24 d, respectively. Meronts kept in lipid-free medium for 14 d retained their ability to synthesize fatty acids. Viable meronts incubated in ASW remained infective for up to 7 d. The infection prevalences were 85, 48 and 100%, in the treatments inoculated with viable meronts that were incubated in ASW for 2, 3 and 7 d, respectively. Infection prevalence in the group inoculated with viable meronts immediately after they were transferred to ASW ranged from 61 to 85%. Our results suggest that in nature meronts can survive for at least 14 d outside the host. Viable meronts are not only infective, but are also able to replicate and retain their fatty acid synthetic ability for 7 d.

Assessment of the cell viability of cultured Perkinsus marinus (Perkinsea), a parasitic protozoan of the Eastern oyster, Crassostrea virginica, using SYBRgreen-propidium iodide double staining and flow cytometry.

A flow cytometry (FCM) assay using SYBRgreen and propidium iodide double staining was tested to assess viability and morphological parameters of Perkinsus marinus under different cold- and heat-shock treatments and at different growth phases. P. marinus meront cells, cultivated at 28 degrees C, were incubated in triplicate for 30 min at -80 degrees C, -20 degrees C, 5 degrees C, and 20 degrees C for cold-shock treatments and at 32 degrees C, 36 degrees C, 40 degrees C, 44 degrees C, 48 degrees C, 52 degrees C, and 60 degrees C for heat-shock treatments. A slight and significant decrease in percentage of viable cells (PVC), from 93.6% to 92.7%, was observed at -20 degrees C and the lowest PVC was obtained at -80 degrees C (54.0%). After 30 min of heat shocks at 40 degrees C and 44 degrees C, PVC decreased slightly but significantly compared to cells maintained at 28 degrees C. When cells were heat shocked at 48 degrees C, 52 degrees C, and 60 degrees C heavy mortality occurred and PVC decreased to 33.8%, 8.0%, and 3.4%, respectively. No change in cell complexity and size was noted until cells were heat shocked at >or=44 degrees C. High cell mortality was detected at stationary phase of P. marinus cell culture. Cell viability dropped below 40% in 28-day-old cultures and ranged 11-25% in 38 to 47-day-old cultures. Results suggest that FCM could be a useful tool for determining viability of cultured P. marinus cells.

Effects of triclosan on growth, viability and fatty acid synthesis of the oyster protozoan parasite Perkinsus marinus.

Perkinsus marinus, a protozoan parasite of the Eastern oyster Crassostrea virginica, has severely impacted oyster populations from the Mid-Atlantic region to the Gulf of Mexico coast of North America for more than 30 yr. Although a chemotherapeutic treatment to reduce or eliminate P. marinus from infected oysters would be useful for research and hatchery operations, an effective and practical drug treatment does not currently exist. In this study, the antimicrobial drug triclosan 5-chloro-2-(2,4 dichlorophenoxy) phenol, a specific inhibitor of Fab1 (enoyl-acyl-carrier-protein reductase), an enzyme in the Type II class of fatty acid synthetases, was tested for its effects on viability, proliferation and fatty acid synthesis of in vitro-cultured P. marinus meronts. Treatment of P. marinus meront cell cultures with concentrations of > or = 2 microM triclosan at 28 degrees C (a temperature favorable for parasite proliferation) for up to 6 d stopped proliferation of the parasite. Treatment at > or = 5 microM at 28 degrees C greatly reduced the viability and fatty acid synthesis of meront cells. Oyster hemocytes treated with > or = 20 microM triclosan exhibited no significant (p < 0.05) reduction in viability relative to controls for up to 24 h at 13 degrees C. P. marinus meronts exposed to > or = 2 microM triclosan for 24 h at 13 degrees C exhibited significantly (p < 0.05) lower viability relative to controls. Exposure of P. marinus meronts to triclosan concentrations of > or = 20 microM resulted in > 50% mortality of P. marinus cells after 24 h. These results suggest that triclosan may be effective in treating P. marinus-infected oysters.

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.

Arachidonic acid synthetic pathways of the oyster protozoan parasite, Perkinsus marinus: evidence for usage of a delta-8 pathway.

The meront stage of the oyster protozoan parasite, Perkinsus marinus, is capable of synthesizing saturated and unsaturated fatty acids including the essential fatty acid, arachidonic acid [20:4(n-6)]. Eukaryotes employ either delta-6 (Delta-6) or delta-8 (Delta-8) desaturase pathway or both to synthesize arachidonic acid. To elucidate the arachidonic acid synthetic pathways in P. marinus, meronts were incubated with deuterium-labeled precursors [18:1(n-9)-d6, 18:2(n-6)-d4, 18:3(n-3)-d4, and 20:3(n-3)-d8]. The lipids were extracted, converted to fatty acid methyl esters, and analyzed using gas chromatography/mass spectrometry and gas chromatography/flame ionization detection. Deuterium-labeled 18:2(n-6), 20:2(n-6), 20:3(n-6), and 20:4(n-6) were detected in meront lipids after 1-, 3-, 5-, and 10-day incubation with 18:1(n-9)-d6. Deuterium-labeled 20:2(n-6), 20:3(n-6) and 20:4(n-6) were found in lipids from meronts after incubation with 18:2(n-6)-d4 methyl ester. No labeled 18:3(n-6) was detected in either incubation. Apparently, when incubated with 18:1(n-9)-d6, the parasite first desaturated 18:1(n-9)-d6 to 18:2(n-6)-d6 by Delta-12 desaturase, then to 20:2(n-6)-d6 by elongation, and ultimately desaturated to 20:3(n-6)-d6 and 20:4(n-6)-d6 using the sequential Delta-8 and Delta-5 desaturation. Similarly, when incubated with 18:2(n-6)-d4, P. marinus converted the 18:2(n-6)-d4 to 20:2(n-6)-d4 by elongation and 20:2(n-6)-d4 to 20:3(n-6)-d4 by Delta-8 desaturase then by Delta-5 desaturase to 20:4(n-6)-d4. These results provide evidence that P. marinus employed the Delta-8 rather Delta-6 pathway for arachidonic acid synthesis. Additional support for the presence of a Delta-8 pathway was the demonstrated ability of the parasite to metabolize 18:3(n-3)-d4 to 20:3(n-3)-d4 and 20:4(n-3)-d4, and 20:3(n-3)-d8 to 20:4(n-3)-d6 and 20:5(n-3)-d6 using the sequential position-specific Delta-8 and Delta-5 desaturases.

Relationship between PCB accumulation and reproductive output in conditioned oysters Crassostrea virginica fed a contaminated algal diet.

Because of their resistance to environmental degradation, polychlorinated biphenyls (PCBs) are among the most widespread environmental contaminants. PCBs have high bioaccumulation potential and may affect a number of biological/physiological processes including disruption of the endocrine system function, lipid metabolism and reproduction. The objective of this study was to test whether conditioning sexually immature oysters with PCB-contaminated algal diets affects their subsequent reproductive success. Sexually immature oysters were conditioned in individual containers and fed daily with 0.7 g algal paste containing 0, 0.35 or 3.5 microg PCBs for up to 76 days. The impact of sediment load on PCB accumulation in oysters was also tested by exposure of a subset of oysters to clay particles. Oysters in different treatments were sampled 56 days after conditioning with PCB-contaminated algal diets to determine uptake and distribution of PCBs in gonad, digestive gland, mantle, gill and muscle, and the presence of gametes. Tissues from oysters exposed to PCBs alone for 56 days were also analyzed for lipid and fatty acid composition. Following 61 and 76 days of PCB exposure, remaining oysters from all treatments were induced to spawn via thermal stimulation. Non-spawned oysters were stripped to determine if sexual products were present. Oysters exposed to PCBs alone and PCBs plus clay particles showed similar trends in PCB accumulation, but concentrations were generally lower in the latter. PCB accumulation in oysters increased with an increase in algal-associated PCB concentrations, varied with organ types and was correlated with lipid content. The highest PCB concentration was in the gonad and the lowest in gill and muscle. PCB-153, -138/158, -118, -90/101 and -149 were the dominant congeners in all tissue compartments, except the muscle where PCB-28/31 was the dominant congener pair. PCB exposure appeared to impair both lipid metabolism and reproductive success. Although PCB exposure produced only slight changes in the lipid class composition in the oysters, decreases in phospholipids were observed in gonad, muscle and mantle of oysters exposed to 3.5 microg PCBs daily for 56 days. After 56 days of conditioning with PCB-sorbed algal paste, no well-developed mature eggs were observed in any of the oysters examined for the presence of sexual products. No significant difference was noted in reproductive success (production of spawned females and males) between sediment-treated and non-treated groups after 76 days of PCB exposure compared to controls, PCB-exposed oysters produced fewer spawned females, but no dose-dependent relationship was observed.

Perkinsus marinus, a protozoan parasite of the Eastern oyster (Crassostrea virginica): effects of temperature on the uptake and metabolism of fluorescent lipid analogs and lipase activities.

The effects of temperature on the uptake and metabolism of fluorescent labeled palmitic acid (FLC16) and phosphatidylcholine (FLPC) and lipase activities in the oyster protozoan parasite, Perkinsus marinus, meront stage were tested at 10, 18, and 28 degrees C. Temperature significantly affected not only the uptake, assimilation, and metabolism of both FLC16 and FLPC in P. marinus, but also its triacylglycerol (TAG) lipase activities. The incorporation of both FLC16 and FLPC increased with temperature and paralleled the increase in the amount of total fatty acids in P. marinus meront cultures. The incorporation of FLC16 was higher than FLPC at all temperatures. The percentage of FLC16 metabolized to TAG was significantly higher at higher temperatures. Trace amounts of incorporated FLC16 were detected in monoacylglycerol (MAG) and PC at 18 and 28 degrees C. P. marinus meronts metabolized FLPC to TAG, diacylglycerol (DAG), monoacylglycerol (MAG), free fatty acids (FFA), phosphatidylethanolamine (PE), and cardiolipin (CL). The conversion of FLPC to TAG and PE was highest at 28 degrees C. The relative proportions of individual fatty acids and total saturated, monounsaturated and polyunsaturated fatty acids changed with temperatures. While total saturated fatty acids (SAFAs) increased with temperature, total monounsaturated fatty acids (MUFAs) decreased with temperature. Total polyunsaturated fatty acids (PUFAs) increased from 28 to 18 degrees C. The findings of increase of total SAFAs and decrease of total MUFAs with the increase of temperatures and upward shift of total PUFAs from 28 to 18 degrees C suggest that, as in other organisms, P. marinus is capable of adapting to changes in environmental temperatures by modifying its lipid metabolism. Generally, higher lipase activities were noted at higher cultivation temperatures. Both TAG lipase and phospholipase activities were detected in P. marinus cells and their extra cellular products (ECP), but phospholipase activities in both the cell pellets and ECP were very low. Also, lipase activities were much lower in ECP than in the cells. The observations of low metabolism, bioconversion of incorporated fluorescent lipid analogs and lipase activities at low temperatures are consistent with the low in vitro growth rate and low infectivity of P. marinus at low temperatures.

Heat-shock protein (HSP70) response in the eastern oyster, Crassostrea virginica, exposed to PAHs sorbed to suspended artificial clay particles and to suspended field contaminated sediments.

Sediments are a potentially significant source of pollutants, containing not only organic contaminants but heavy metals as well. The heat shock protein response (HSP70 family) in the eastern oyster exposed to suspended clay particles spiked with polynuclear aromatic hydrocarbons (PAHs) and to suspended field contaminated sediments (SFCS) was investigated. In experiment 1, oysters were exposed to 1.0, 1.5 or 2.0 g suspended clay particles with concentrations of 65.6, 159.0 and 242 micro g PAHs per g of wet clay particles, respectively, and sampled after 40 days. Controls were exposed to 0, 1.0, 1.5, and 2.0 g suspended unspiked clay particles. In experiment 2, oysters were exposed to 0, 1.0, 1.5, and 2.0 g SFCS and the HSP70 expression was determined after 5, 10, 20 and 40 days exposure. Oysters exposed to suspended clay particles spiked with PAHs showed a significant increase in HSP70 levels, while oysters exposed to 1.0, 1.5 or 2.0 g suspended unspiked clay particles did not show changes (P=0.78) in HSP70 levels compared to the group receiving 0 g clay particles. Exposure to the SFCS resulted in a significant increase in HSP70 as a function of exposure (P<0.001) and treatment (P=0.006). The response, however, was not dose dependent. Compared to the control group (0 g SFCS), groups exposed to 1.0, 1.5 and 2.0 g SFCS reached significantly higher levels in HSP70 at 40 days of exposure, with those exposed to 2.0 g SFCS expressing the highest levels. The HSP70 expression for each treatment showed fluctuations at various time intervals. No mortalities were recorded during the exposure experiments. The major contaminants in the SFCS were PAHs, heavy metals and polychlorinated biphenyls (PCBs). These results reveal that exposure to PAHs sorbed to clay particles and to SFCS induced a HSP70 response in the eastern oyster.

Phospholipid biosynthesis in the oyster protozoan parasite, Perkinsus marinus.

Perkinsus marinus is a protozoan parasite that causes high mortality in its commercially and ecologically important host, the Eastern oyster Crassostrea virginica. In order to understand the host-parasite relationship in lipid metabolism, the ability of P. marinus to synthesize phospholipids from polar headgroup precursors was investigated. Pulse/chase experiments were conducted using radiolabled serine, choline, ethanolamine and inositol. Timecourse incubations revealed that in vitro cultured P. marinus meronts can utilize the cytidine diphosphate-diacylglycerol (CDP-DAG) pathway to synthesize phosphatidylinositol (PI) from inositol and phosphatidylserine (PS) from serine. Serine label was also incorporated into phosphatidylethanolamine (PE), phosphatidylcholine (PC) and lysophosphatidylcholine (LPC). Incubations of P. marinus cells with increasing concentrations of radiolabeled serine resulted in more radioactivity recovered in neutral lipids than in polar lipids at the highest substrate concentration tested (344 microM). This suggests that excess serine label was being utilized for fatty acid synthesis and stored as triacylglycerols. Additional incubations were conducted with radiolabeled choline and ethanolamine at concentrations equimolar to the highest serine concentration tested. Ethanolamine label was also incorporated into PE, PS, PC and LPC. Choline label was incorporated into PC. These results suggest the presence of three pathways for de novo synthesis of phospholipids in P. marinus: CDP-choline, CDP-ethanolamine and CDP-DAG. At equivalent substrate concentrations (344 microM) the highest incorporation of labeled substrate into total phospholipids was with serine followed by ethanolamine and choline, respectively. P. marinus phospholipid biosynthetic capabilities appear to be similar to those of Plasmodium and Trypanosoma species.

Cellular responses and disease expression in oysters (Crassostrea virginica) exposed to suspended field contaminated sediments.

Exposure of oysters to water soluble fractions derived from field-contaminated sediments (FCS) containing predominantly lower molecular weight organic aromatic compounds, has been previously demonstrated to enhance pre-existing infections caused by the protozoan parasite, Perkinsus marinus (Dermo), and the prevalence of experimentally induced infections. To further explore the role of pollution on the onset and progression of disease, effects of suspended FCS from an estuarine creek in Virginia, USA, dominated by higher molecular weight polycyclic aromatic hydrocarbons (PAHs) on cellular responses and Dermo disease expression in oysters (Crassostrea virginica) were examined. Sediments were collected from a PAH polluted estuarine creek in Virginia, USA. To test effects on cellular response, oysters from Maine were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 70.2, 105, or 140 microg PAHs, respectively) for 5, 10, 20, and 40 days. Hemocyte activities and plasma lipid, protein and lactate dehydrogenase (LDH) levels were then measured. Exposure stimulated neutral red uptake, MTT reduction, and 3H-leucine incorporation in oyster hemocytes at various exposure times, but did not affect the plasma protein, lipid and LDH levels. To test effects on Dermo expression, oysters from a Dermo enzootic area, with an initial estimated infection prevalence of 39%, were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 75.0, 113, or 150 microg PAHs, respectively) for 30 days. Exposure enhanced disease expression in oysters. However, no significant change was noted in any measured cellular or humoral parameters.

De novo arachidonic acid synthesis in Perkinsus marinus, a protozoan parasite of the eastern oyster Crassostrea virginica.

The capability of synthesizing fatty acids de novo in the meront stage of the oyster protozoan parasite, Perkinsus marinus, was investigated employing stable-isotope-labeled precursors (1,2 13C-acetate and palmitic-d(31) acid). Fatty acid methyl esters derived from 1,2 13C-acetate and palmitic-d(31) acid were analyzed using gas chromatography/mass spectrometry and gas chromatography/flame ionization detection. Results revealed that in vitro cultured P. marinus meronts utilized 13C-acetate to synthesize a range of saturated and unsaturated fatty acids. The saturated fatty acids 14:0, 16:0, 18:0, 20:0, 22:0, 24:0 and the unsaturated fatty acids, 18:1(n-9), 18:2(n-6), 20:1(n-9), 20:2(n-6), 20:2(n-9), 20:3(n-6), 20:4(n-6) were found to contain 13C, after 7, 14, and 21 days incubation with the precursor. This indicates that meronts can synthesize fatty acid de novo using acetate as a substrate. Meronts efficiently elongated 16:0-d(31) to 18:0, 20:0, 22:0, 24:0, but desaturation activity was limited, after 7 and 14 days cultivation. Only a small quantity of 18:1-d(29) was detected. This suggests that meronts cannot directly convert exogenous palmitic acid or its products of elongation to unsaturated counterparts. The ability to synthesize 20:4(n-6) from acetate is particularly interesting. No parasitic protozoan has been reported to be capable of synthesizing long chain essential fatty acids, such as 20:4(n-6) de novo. Future study will be directed to determine whether the observed in vitro activities indeed reflect the in vivo activities, when meronts are associated with the host.