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.

Heat shock protein (hsp70) expression and thermal tolerance in sublethally heat-shocked eastern oysters Crassostrea virginica infected with the parasite Perkinsus marinus.

To investigate whether sublethal heat shock protects Perkinsus marinus (Dermo)-infected oysters Crassostrea virginica from lethal heat stress, and the effects of P. marinus infection on sublethal heat shock response, oysters were first experimentally challenged with P. marinus. Then, when infections in oysters progressed to moderate levels (parasite burden = 10(4) to 10(5) cells g(-1) wet tissue weight), oysters were treated with a sublethal heat shock at 40 degrees C for 1 h (heat shock + Dermo challenge). Other treatment groups included heat-shocked, unchallenged (non-P. marinus challenged) oysters and non-heat-shocked, P. marinus-challenged and -unchallenged oysters. Thermal tolerance was compared among these treatments by administering a lethal heat treatment at 44 degrees C for 1 h, 7 d after sublethal heat shock. Sublethal heat shock enhanced survival to lethal heat treatment in both P. marinus-challenged and -unchallenged oysters. Although levels of hsp70 isoforms (hsp69 and hsp72) did not vary significantly by heat shock or infection with P. marinus, responses due to these treatments were apparent when comparing hsp70 levels within infected and uninfected oysters. Infection enhanced expression of hsp69, regardless of whether oysters were heat shocked or not. In uninfected oysters, hsp72 increased due to heat shock 2 and 7 d post heat shock. Overall, this study demonstrates that heat shock can improve survival in oysters, even in oysters infected with P. marinus. Expression of hsp70 varied among isoforms after sublethal and lethal heat shocks and in infected and uninfected oysters. The heat shock response was not negatively affected by P. marinus infection.

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.

Oxidative pyrolysis and postpyrolytic derivatization techniques for the total analysis of maillard model systems: investigation of control parameters of maillard reaction pathways.

Factors that regulate various pathways of Maillard reaction leading to aroma, color, or carcinogen generation have not been identified, due to the difficulties associated with analyzing complex reaction mixtures. In particular, the role played by oxidation in directing aromagenic, chromogenic, or carcinogenic pathways is not well understood. In order to overcome the analytical difficulties, novel Py-GC/MS-based methodologies were developed to analyze volatile and nonvolatile residues of Maillard reaction products generated from the same model system under air or helium atmosphere. The analysis of nonvolatiles was achieved through a postpyrolytic in situ derivatization technique using hexamethyldisilazane, and pyrolysis under air was achieved through modification of the GC equipped with sample concentration trap to allow gas stream switching and subsequent isolation of the pyrolysis chamber from the analytical stream. In this approach label incorporation from the starting materials can be observed in both volatile and nonoxidative conditions for mechanistic studies. In addition, monitoring of redox potentials, oxygen consumption, and color generation of relevant model systems over time were also carried out at different temperatures. The data collected have indicated that perturbation in the redox potential of Maillard model systems by external (oxidizing conditions) or internal (formation of reductones) factors can alter the balance among the four critically important groups of precursors: alpha-dicarbonyl, alpha-hydroxycarbonyl, 2-aminocarbonyls, and 2-(amino acid)-carbonyl compounds and hence control the relative importance of aromagenic versus chromogenic pathways.

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.

Effects of palm carotenoids in rat hepatic cytochrome P450-mediated benzo(a)pyrene metabolism.

Using benzo(a)pyrene (BaP) metabolism as a probe for chemical carcinogenesis, in vitro and in vivo effects of palm-oil carotenoid [beta-carotene (BC), alpha-carotene (AC), or canthaxanthin (CTX)] on BaP metabolism in the rat hepatic cytochrome P450-mediated monooxygenase system were studied. Apparent Michaelis-Menten constants (Km) for formation of the precursor carcinogen, 7,8-dihydrodiol BaP, were found to be 14.4 (BC), 1.74 (AC), and 0.7 (CTX) mumol/L. The order of anticarcinogenic strength established in this study was BC much greater than AC greater than CTX. Increased formation of the detoxification intermediate, 3-hydroxy BaP, with increased carotenoid concentration was observed. The order of detoxification strength was BC greater than AC = CTX. The presence of carotenoids in vivo inhibited BaP metabolism. Using 9,10-dihydrodiol BaP as an indicator for inhibition, the order of the antioxidative activity was palm oil (with carotenoids) greater than BC greater than CTX greater than palm oil (without carotenoids). BC and AC may selectively modify the rat-liver microsomal enzymes, thus providing a biochemical mechanism for the inhibitory effect of palm carotenoids on chemical carcinogenesis.

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.

In vitro interaction of Perkinsus marinus merozoites with eastern and Pacific oyster hemocytes.

This study compared hemocyte responses of eastern and Pacific oysters to Perkinsus marinus, in vitro. Except for the percentage of hemocytes associated with P. marinus there was little or no significant difference between eastern and Pacific oysters with regard to their hemocytic response to P. marinus. In phagocytosis assays, merozoites were bound to all hemocyte types but in unequal proportions, unlike zymosan which was found predominantly associated with granulocytes. The number of merozoites enlarging in Ray's fluid thioglycollate medium after incubation with hemocytes in plasma for one day was significantly lower than after incubation in plasma alone in both oyster species. Electron microscopy or merozoites indicated that the parasites were rapidly phagocytosed and that some of the merozoites showed signs of degeneration in less than 12 h. The results suggest that limited intracellular killing of P. marinus had occurred, but was probably not mediated by oxygen metabolites, since no increase in chemiluminescence was observed when hemocytes of either eastern or Pacific oysters were exposed to merozoites.

Spinal cord proliferative sparganosis in Taiwan: a case report.

A 43-year-old woman suffered from low back pain and bilateral footdrop. A cisternal myelogram unexpected revealed multiple filing defects in the spinal canal extending from the lower cervical region to the caudal equina. Diagnostic exploration revealed numerous cystic organisms adhering to the spinal cord and nerve roots. Histopathological examination showed these organisms to be proliferative sparganum cestode larvae. Although these cestode larval infections have been reported a dozen times in humans from various parts of the world, this is probably the first reported case of spinal cord infection.

Lipid class composition of the protozoan Perkinsus marinus, an oyster parasite, and its metabolism of a fluorescent phosphatidylcholine analog.

Perkinsus marinus is one of two important protozoan parasites of the eastern oyster, Crassostrea virginica. The other is Haplosporidium nelsoni. Lipids extracted from 7-d-old in vitro cultured P. marinus meronts, incubated with fluorescent-labeled phosphatidylcholine (FL PC) and nonincubated P. marinus meronts, were analyzed by a high-performance liquid chromatography (HPLC) system equipped with a diol phase column, in combination with thin-layer chromatography coupled with a flame-ionization detector (TLC/FID), and high-performance thin-layer chromatography (HPTLC). Various polar and neutral lipid classes were separated by HPLC using a two-gradient solvent system. Five polar lipid classes--phosphatidylcholine (PC), phosphatidylethanolamine (PE), cardiolipin (CL), sphingomyelin (SM), and phosphatidylserine (PS)--were identified from P. marinus extracts. Four neutral lipid classes--triacylglycerol (TAG), steryl ester (SE), cholesterol (CHO), and fatty alcohol--were distinguished. TLC/FID analysis of meront lipids showed that the weight percentages of PC, PE, CL, SM, PS/PI, TAG, SE, and CHO were 21, 10.7, 4, 2.3, 4.3, 48.7, 7.8, and 1.2%, respectively. HPLC and HPTLC analyses revealed the presence of two SM and PS isomers in P. marinus extracts. Perkinsus marinus effectively incorporated FL PC acquired from the medium and metabolized it to various components (i.e., free fatty acid, monoacylglycerol, diacylglycerol, TAG, PE, and CL). Uptake and interconversion of FL PC in P. marinus meronts increased with time. After 48 h the total uptake of fluorescence (FL) was 28.9% of the FL PC added to the medium, and 43% of the incorporated FL resided in TAG.

Brain SPECT imaging with 99Tcm-hexamethylpropyleneamine oxime in the early detection of cerebral infarction: comparison with transmission computed tomography.

A new cerebral blood flow agent, 99Tcm-hexamethylpropyleneamine oxime (HM-PAO), was evaluated for early detection of acute cerebral infarction in conjunction with the transmission computed tomographic (CT) studies. The data from 22 cases were analysed. Results reveal that 99Tcm-HM-PAO enables the early detection of acute cerebral infarction prior to CT with rather proper depiction of the extent of physiological abnormality in the majority of patients. This promising result together with the lack of logistical problems will make 99Tcm-HM-PAO a useful and practical agent worldwide for diagnosing and managing acute cerebral infarction.

The effect of xanthine derivatives on red blood cells: microelectrophoretic studies.

The effect of xanthine derivatives on the variation of surface charges of red blood cells has been investigated. Results of mobility curves showed that the derivatives increase the surface charges on the cells, whereas there is little effect on the surface charges of liposomes.

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.

The effect of PCBs on glycogen reserves in the eastern oyster Crassostrea virginica.

Recent declines in Chesapeake Bay oyster populations have been attributed to disease, and reduced water quality from pollution. The stress associated with pollutant exposure may reduce energy available for growth and reproduction. Polychlorinated biphenyls (PCBs) are lipophilic contaminants that may potentially affect mobilization of lipid reserves, increasing reliance on glycogen stores, which could otherwise be utilized to supply energy for gametogenesis. Thus, PCBs may indirectly affect glycogen stores in oysters in a deleterious manner. To test for this effect, reproductively inactive oysters were exposed to PCBs by feeding individuals 0.7 g of algal paste containing 0, 0.35, or 3.5 micrograms PCBs daily for 8 weeks. Additionally, a group of oysters was exposed to PCBs (0, 0.35, and 3.5 micrograms) plus 0.3 g of non-toxic artificial sediment to examine interactive effects of sediment particles and PCBs. Adductor muscle, mantle, and gonadal tissues were analyzed for glycogen content. Results suggest that glycogen content is reduced in the adductor muscle with increasing PCB exposure, but there are no effects of PCBs in the mantle and gonadal tissues.

PCB uptake and accumulation by oysters (Crassostrea virginica) exposed via a contaminated algal diet.

Reproductively active oysters were fed daily with 0.2 g algal paste containing 0, 0.1, and 1.0 microgram polychlorinated biphenyls (PCBs) (1:1:1 mixture of Aroclor 1242, 1254 and 1260) for either 15 or 30 days, and accumulation of PCBs in different organ tissues and eggs assessed. The effects of PCB exposure on lipid content, lipid class and fatty acid composition were also evaluated. PCBs were accumulated by the oysters and transferred to the eggs. PCB accumulation in oysters was dose, time and tissue dependent. Mean PCB contents were 3150, 1970, and 250 ng/g dry wt., respectively, in the visceral mass, gills + mantle and muscle of oysters fed algal paste containing 1.0 microgram PCBs for 30 days. The PCBs in the eggs from the same oysters reached 671 ng PCBs/g dry wt. Feeding oysters with PCB-sorbed algal paste for 30 days significantly increased phospholipid and free fatty acid contents in gills + mantle tissue compared to the same tissues in the undosed control.

Effects of PCBs sorbed to algal paste and sediments on the stress protein response (HSP70 family) in the eastern oyster, Crassostrea virginica.

This study examined the stress protein response (HSP70 family) of reproductively inactive oysters fed 0.7 g algal paste containing 0, 0.35 and 3.5 micrograms polychlorinated biphenyls (PCBs) daily. A second set of treatment groups investigated the combined effect of PCBs and sediments (0.3 g sediments daily per oyster) on HSP70 response. After 8 weeks of PCB exposure, oyster tissues (mantle and gill) were sampled and analyzed for HSP70. Preliminary results did not show a significant effect in HSP70 response in oysters fed PCB sorbed to algal paste, albeit PCBs accumulated up to 1342 ng/g dry weight in the mantle, and up to 180 ng/g dry weight in gill tissues. However, the addition of sediments caused a significant increase in HSP70 levels of gills and mantle, although the mantle was less sensitive to the sediments.

Common carotid artery dissection diagnosed by ultrasonic image: report of a case.

Carotid artery dissection is not uncommon and usually starts at the internal carotid artery. It rarely begins in the common carotid artery. It may cause symptoms of cerebral vascular insufficiency such as stroke and transient ischemic attacks. The case reported here is a 38-year-old man with an acute onset of left hemiparesis 2 hours after drinking three bottles of beer. Computed tomographic scan showed cerebral infarction in the territory of the right middle and anterior cerebral arteries. Ultrasonic imagings showed an intimal flap, starting at the bifurcation of the right common carotid artery, indicative of arterial dissection. Angiography confirmed the diagnosis and the arterial dissection appeared to extend into and terminate at the internal carotid artery.

Acquired but reversible loss of erythrocyte complement receptor 1 (CR1, CD35) and its longitudinal alteration in patients with severe acute respiratory syndrome.

This longitudinal study investigates the change of erythrocyte complement receptor (E-CR1) expression in patients with severe acute respiratory syndrome (SARS). Circulating E-CR1 expression was semiquantified by flow cytometric analyses in 54 SARS patients and in 212 healthy individuals as a control. Since E-CR1 expression is influenced by the genetic polymorphisms in the CR1 gene, a major genetic polymorphism located within intron 27 of the CR1 gene was simultaneously analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The results showed that the expression level of E-CR1 (referred to as net fluorescence intensity values, NFI) was statistically correlated with the relevant genetic genotypes among the Chinese population including the healthy individuals (NFI: 5.14 +/- 0.82, 3.57 +/- 0.66 and 2.67 +/- 0.32 for HH, HL and LL genotypes, respectively) and SARS patients (NFI: 3.52 +/- 0.91 and 2.63 +/- 0.70 for HH and HL genotypes, respectively). Interestingly, the expression density of E-CR1 was found to fall significantly during the initiation and progressive phases (weeks 1 and 2 after the disease onset) and gradually returned close to normal through their whole convalescent phase (beginning from weeks 2 or 3 to weeks 7 or 8) in SARS patients irrespective CR1 genotype. In conclusion, our findings, at least, suggest that E-CR1 is likely involved in immune pathogenesis of SARS disease.

Effects of field-contaminated sediments and related water soluble components on haemocyte function and Perkinsus marinus susceptibility and expression in oysters.

This paper reviews and discusses our recent findings on the effects of contaminated sediments (CSs) and related water-soluble fractions (WSFs) on haemocyte function/activity and the onset and progression of an infectious disease caused by the protozoan parasite, Perkinsus marinus (Dermo) in the eastern oyster, Crassostrea virginica. Sediments used to generate WSFs and sediments used for the whole CS exposure experiments were collected in different areas of the southern branch of the Elizabeth River, a heavily polluted sub-estuary of the Chesapeake Bay, USA. The WSFs were dominated by low molecular weight polycyclic aromatic hydrocarbons (PAHs). The CSs used for whole CS exposure experiment had elevated concentrations of high molecular weight PAHs. Polychlorinated biphenyls (PCBs) and metals were also present in the CSs. No PCBs were detected in the WSFs. In vitro exposure of haemocytes to WSFs derived from CSs reduced to haemocytes' chemotaxic, phagocytic, and chemiluminescent responses to some extent. Exposure of oysters to suspended CSs stimulated neutral red uptake, mitochondrial dehydrogenase production and (3)H-leucine incorporation in haemocytes. Exposure of oysters to 0, 15, 30% WSFs increased the oysters' susceptibility to laboratory-induced infection caused by P. marinus. Exposure of oysters to 15, and 30% dilutions of WSFs for 33 days or to 1.0, 1.5, and 2.0g CSs for 30 days significantly elevated the expression/progression of latent P. marinus infection in oysters in a dose-dependent manner.