Fu-Lin E. Chu

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 hosts defense.

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.

Haemocytic and humoral activities of eastern and Pacific oysters following challenge by the protozoan Perkinsus marinus

Abstract Eastern oysters ( Crassostrea virginica ) and Pacific oysters ( Crassostrea gigas ) were exposed to the protozoan parasite Perkinsus marinus . Pacific oysters were less susceptible to P. marinus infection than eastern oysters. The concentration of circulating haemocytes, percentage of granulocytes and plasma haemagglutinin titre increased in Pacific oysters exposed to P. marinus as compared to unexposed Pacific oysters. Similar increases were not observed in eastern oysters exposed to P. marinus . The percentage of granulocytes, percentage of phagocytic haemocytes, number of zymosan per phagocytic haemocytes and plasma haemagglutinin titre were greater in Pacific oysters than in eastern oysters heavily infected with P. marinus . However, the concentration of circulating haemocytes and the concentrations of plasma protein and lysozyme were lower in Pacific oysters. The data suggest that Pacific oysters may offer a less favourable environment for the development of P. marinus as compared to eastern oysters for at least two possible reasons; i.e. the elevated cellular and humoral activities may degrade the parasite more effectively, and/or the lower plasma protein levels may limit parasite growth.

Host-parasite interactions: Marine bivalve molluscs and protozoan parasites, Perkinsus species.

This review assesses and examines the work conducted to date concerning host and parasite interactions between marine bivalve molluscs and protozoan parasites, belonging to Perkinsus species. The review focuses on two well-studied host-parasite interaction models: the two clam species, Ruditapes philippinarum and R. decussatus, and the parasite Perkinsus olseni, and the eastern oyster, Crassostrea virginica, and the parasite Perkinsus marinus. Cellular and humoral defense responses of the host in combating parasitic infection, the mechanisms (e.g., antioxidant enzymes, extracellular products) employed by the parasite in evading host defenses as well as the role of environmental factors in modulating the host-parasite interactions are described.

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.

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.

Lipid and fatty acid composition of striped bass (Morone saxatilis) larvae during development

Abstract Changes in lipid class, fatty acid composition, protein, and dry and wet weights of fertilized eggs and developing larvae of striped bass ( Morone saxatilis ) fed with the live food, Artemia , were investigated. A decrease of wet and dry weights and moisture was observed at the beginning of the larval stage. Larvae regained the original moisture level, and wet and dry weights increased steadily after feeding. Total lipids decreased from 190 μg/egg in fertilized eggs to 151 μg/egg during hatching and increased after feeding. When total lipid contents were expressed as a percentage of larval dry weight, a decline of lipid did not occur until after feeding. Total protein, on the other hand, increased right after feeding, but there was some variation between days. Polar lipids increased significantly from 20 μg/egg at the egg stage to 199 μg/larva at 26 days post-hatching (DPH), 2 days before the onset of metamorphosis, while neutral lipids declined from 175 μg/egg to 80 μg/larva during the same time period. Wax/steryl esters decreased from 150 μg/egg in fertilized eggs to 32 μg/larva at 26 DPH. Triacylglycerols dropped from 21 μg/egg to 15 μg/larva before feeding and increased gradually after feeding. In contrast, the level of cholesterol increased 2–3-fold. There was a significant increase of phospholipids, particularly phosphatidylcholine in larvae after feeding. The fatty acid composition of fish larvae was significantly influenced by the diet, Artemia . There was an indication of catabolism of endogenous eicosapentaenoic and docosahexaenoic acids during metamorphosis.

Ontogenetic changes of lipolytic enzymes in striped bass (Morone saxatilis)

Abstract Ontogenetic changes of triacylglycerol hydrolase (TAGH), wax ester hydrolase (WEH) and phospholipase A2 (PLA2) activities were determined in the fertilized eggs and premetamorphosed larvae of striped bass and the larval food Artemia using radioassays. Maximum activities of the three enzymes were observed at a substrate concentration of 500 μM and in the presence of 5 mM Na-taurocholate. PLA2 showed the major activity in the eggs, followed by TAGH and WEH. TAGH became the dominant lipolytic activity after feeding was initiated at 7 days post-hatching (DPH). The proportions of TAGH:PLA2: WEH in the fertilized eggs were 1.1:1.6:1.0. In 20 DPH fed larvae, however, the proportion changed to 6.3:1.9:1.0. In unfed larvae, the enzyme activities were significantly lower compared to feeding larvae (P TAGH > WEH. The contribution of dietary enzymes to the total lipolytic processes is estimated to be less than 6.5% of the total enzyme activity throughout the development. The results of this study indicate that first feeding striped bass larvae have the capacity to digest 47% of their daily lipid ingestion.

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.

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

Abstract. 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°C, were incubated in triplicate for 30 min at −80°C, −20°C, 5°C, and 20°C for cold‐shock treatments and at 32°C, 36°C, 40°C, 44°C, 48°C, 52°C, and 60°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°C and the lowest PVC was obtained at −80°C (54.0%). After 30 min of heat shocks at 40°C and 44°C, PVC decreased slightly but significantly compared to cells maintained at 28°C. When cells were heat shocked at 48°C, 52°C, and 60°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≥44°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.