Interactions between Crassostrea virginica larvae and Deepwater Horizon oil: Toxic effects via dietary exposure.

The Deepwater Horizon (DWH) disaster released crude oil in the Gulf of Mexico for 87 days, overlapping with the reproductive season and recruitment of the oyster Crassostrea virginica. The pelagic larval life stages of C. virginica are particularly vulnerable to contaminants such as polycyclic aromatic hydrocarbons (PAHs) and oil droplets. Based on their lipophilic properties, PAHs and oil droplets can adsorb onto phytoplankton and filter-feeding C. virginica larvae may be exposed to these contaminants bound to suspended sediment, adsorbed onto algal and other particles, or in solution. This study examined the effects of exposure of C. virginica larvae to algae mixed with DWH oil. In a 14-day laboratory exposure, 5 day-old C. virginica larvae were exposed to Tisochrysis lutea mixed with four concentrations of unfiltered DWH oil (HEWAF) in a static renewal system. Larval growth, feeding capacity, abnormality and mortality were monitored throughout the exposure. Total PAH (n = 50) content of the water medium, in which larvae were grown, were quantified by GC/MS-SIM. Oil droplets were observed bound to algae, resulting in particles in the size-range of food ingested by oyster larvae (1-30 µm). After 14 days of exposure, larval growth and survival were negatively affected at concentrations of tPAH50 as low as 1.6 µg L-1. GC/MS-SIM analysis of the exposure medium confirmed that certain PAHs were also adsorbed by T. lutea and taken up by oyster larvae via ingestion of oil droplets and/or contaminated algae. Long-term exposure to chronic levels of PAH (1.6-78 µg tPAH50 L-1) was shown to negatively affect larval survival. This study demonstrates that dietary exposure of oyster larvae to DWH oil is a realistic route of crude oil toxicity and may have serious implications on the planktonic community and the food chain.

Sensitivity of eastern oyster (Crassostrea virginica) spermatozoa and oocytes to dispersed oil: Cellular responses and impacts on fertilization and embryogenesis.

The 2010 Deepwater Horizon (DWH) oil spill released millions of barrels of oil and dispersant into the Gulf of Mexico. The timing of the spill coincided with the spawning season of Crassostrea virginica. Consequently, gametes released in the water were likely exposed to oil and dispersant. This study aimed to (i) evaluate the cellular effects of acute exposure of spermatozoa and oocytes to surface slick oil, dispersed mechanically (HEWAF) and chemically (CEWAF), using flow-cytometric (FCM) analyses, and (ii) determine whether the observed cellular effects relate to impairments of fertilization and embryogenesis of gametes exposed to the same concentrations of CEWAF and HEWAF. Following a 30-min exposure, the number of spermatozoa and their viability were reduced due to a physical action of oil droplets (HEWAF) and a toxic action of CEWAF respectively. Additionally, reactive oxygen species (ROS) production in exposed oocytes tended to increase with increasing oil concentrations suggesting that exposure to dispersed oil resulted in an oxidative stress. The decrease in fertilization success (1-h), larval survival (24-h) and increase in abnormalities (6-h and 24-h) may be partly related to altered cellular characteristics. FCM assays are a good predictor of sublethal effects especially on fertilization success. These data suggest that oil/dispersant are cytotoxic to gametes, which may affect negatively the reproduction success and early development of oysters.

Lethal and sub-lethal effects of Deepwater Horizon slick oil and dispersant on oyster (Crassostrea virginica) larvae.

In April 2010, crude oil was spilled from the Deepwater Horizon (DWH) oil platform for 87 days, coincident with the spawning season and recruitment of the oyster, Crassostrea virginica, in the Gulf of Mexico. Impacts of acute exposures to surface-collected DWH oil (HEWAF), dispersed oil (CEWAF) and dispersant alone (Corexit 9500A(®)) on planktonic larval stages of C. virginica (veliger, umbo and pediveliger) were tested in the laboratory. Exposures to HEWAF, CEWAF and dispersant were toxic to larvae impairing growth, settlement success and ultimately survival. Larval growth and settlement were reduced at concentrations of tPAH50 ranging from 1.7 to 106 µg L(-1) for HEWAF and 1.1-35 µg L(-1) for CEWAF, concentrations well within the range of water sampled during the DWH oil spill. Sublethal effects induced by oil and dispersant could have significant ecological implications on oyster populations and on the whole estuarine ecosystem.

Impacts of Deepwater Horizon oil and associated dispersant on early development of the Eastern oyster Crassostrea virginica.

The explosion of the Deepwater Horizon (DWH) oil platform resulted in large amounts of crude oil and dispersant Corexit 9500A® released into the Gulf of Mexico and coincided with the spawning season of the oyster, Crassostrea virginica. The effects of exposing gametes and embryos of C. virginica to dispersant alone (Corexit), mechanically (HEWAF) and chemically dispersed (CEWAF) DWH oil were evaluated. Fertilization success and the morphological development, growth, and survival of larvae were assessed. Gamete exposure reduced fertilization (HEWAF: EC201h=1650µg tPAH50L(-1); CEWAF: EC201h=19.4µg tPAH50L(-1); Corexit: EC201h=6.9mgL(-1)). CEWAF and Corexit showed a similar toxicity on early life stages at equivalent nominal concentrations. Oysters exposed from gametes to CEWAF and Corexit experienced more deleterious effects than oysters exposed from embryos. Results suggest the presence of oil and dispersant during oyster spawning season may interfere with larval development and subsequent recruitment.

Uptake and interconversion of fluorescent lipid analogs in the protozoan parasite, Perkinsus marinus, of the oyster, Crassostrea virginica.

Uptake, distribution, and interconversion of fluorescent lipid analogs (phosphatidylcholine, PC; cholesteryl ester, CHE; phosphatidylethanolamine, PE; palmitic acid, C16; sphingomyelin, SM) by the two life stages, meront and prezoosporangium, of the oyster protozoan parasite, Perkinsus marinus, were investigated. Class composition of these two life stages and lipid contents in meront cells were also examined. Both meronts and prezoosporangia incorporated and modified fluorescent lipids from the medium, but their metabolic modes differ to some extent. Results revealed that among the tested analogs, neutral lipid components (CHE and C16) were incorporated to a greater degree than the phospholipids (PC, PE, and SM). HPLC analysis of meront lipids showed that while the majority of the incorporated PC, CHE, and PE remained as parent compounds, most of the incorporated C16 was in triacylglycerol (TAG) and SM was in ceramide and free fatty acids. The cellular distribution of fluorescent labels varied with lipid analogs and the extent of their metabolism by the parasite. Fluorescence distribution was primarily in cytoplasmic lipid droplets of both life stages after 24 h incubation with PC. After 24 h incubation with SM, fluorescence appeared in the membrane and cytosol. Total lipid contents in meront cultures increased during proliferation and TAG accounted for most of the increased total lipids. Since total lipid content per meront cell did not increase until the day of culture termination, the lipid increase in the meront culture was mainly a result of increased cell numbers. Both life stages contain relatively high levels of phospholipids, 53.8% in 8-day-old meronts and 39.4% in prezoosporangia. PC was the predominant phospholipid.

Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica).

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.

Acid phosphatase activity in Perkinsus marinus, the protistan parasite of the American oyster, Crassostrea virginica.

The effect of temperature (4, 12, 20, and 28 C) and osmolality (400, 570, and 840 mOsm/kg) on extracellular acid phosphatase (AP) secretion in vitro, and ultrastructural localization of AP activity in the parasite were investigated. The extracellular AP secretion by Perkinsus marinus was cell density dependent (P < 0.001). Increasing culture temperatures resulted in increased P. marinus proliferation concomitant with AP secretion (P < 0.0001). AP secretion was similar in P. marinus cultured at 400 and 570 mOsm/kg media, but higher than P. marinus cultured at 870 mOsm/kg media. Results of the ultrastructural study revealed that intense AP activity was in the nucleus of the parasite. Based on its distribution in the nucleus, AP may be playing a role in events leading to cell cycle regulation.

Suppression of chemiluminescence of eastern oyster (Crassostrea virginica) hemocytes by the protozoan parasite Perkinsus marinus.

Experiments were conducted to determine the ability of the protistan parasite, Perkinsus marinus, to inhibit chemiluminescence of hemocytes from the eastern oyster, Crassostrea virginica. Luminol-enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen intermediates (ROI) generated by oyster hemocytes using zymosan as a stimulant. To determine whether P. marinus suppresses ROI evoked from zymosan-stimulated hemocytes, live or heat killed P. marinus in filtered estuarine water (YRW) (salinity = 20 ppt) were added to (1) zymosan-stimulated hemocytes after CL reached its peak, or (2) hemocytes at the same time as zymosan, and reduction of CL responses were recorded. In both tests, controls received only estuarine water. Live P. marinus meronts significantly suppressed ROI production by zymosan-stimulated hemocytes. The suppression of ROI production was dose dependent. Suppression of ROI production from zymosan-stimulated hemocytes by heat killed P. marinus was significantly less than by live P. marinus. Similarly, CL of hemocytes was reduced, though not significantly when hemocytes were exposed to YRW preincubated with P. marinus. When P. marinus meronts were used as a stimulant, no CL response was elicited. Results of this study suggest that P. marinus cells are able to suppress ROI release from oyster hemocytes, thus evading this component of the host's defense.