Leah M. Oliver

A survey of oysters Crassostrea virginica from Tampa Bay, Florida: associations of internal defense measurements with contaminant burdens

Oysters from 16 sites in Tampa Bay, Florida, were collected during a 6-week period in winter 1993 and analyzed for both biological characteristics and tissue chemical concentrations. Using previous sediment contamination and toxicity data, oyster tissues from the selected sites were expected to exhibit a wide range in both quantity and type of chemicals. Chemical analysis showed tissue concentrations at some of these sites to be greater than national averages, as reported by the National Status and Trends Mussel Watch Program, for total PAH, total PCB, total chlordanes, DDT, Cu, Pb and Zn. Measures of oyster internal defense, including hemocyte density, rate of locomotion and superoxide generation, varied significantly among sites and were generally higher at sites with higher tissue concentrations of xenobiotic chemicals. Potential associations between oyster defense characteristics and accumulated chemical contaminants, either singly or in chemical classes, were explored using correlation analysis and a composited ranking procedure. Positive relationships were found for hemocyte characteristics with certain trace metal (Cu, Sn and Zn) and PAH analytes, whereas negative relationships were found with certain PCB and pesticide analytes. Heightened defenses in contaminated conditions may reflect a hemocyte process for sequestration and detoxification of environmental contaminants. Oysters from four of the 16 sites were additionally collected in June and September 1993 and site-related differences did not closely parallel those obtained in winter. Seasonal environmental factors may have altered contaminant-related differences among sites.

Relationships between tissue contaminants and defense-related characteristics of oysters (Crassostrea virginica) from five Florida bays.

Evidence linking bivalve defense responses with pollutant exposure is increasing. Contaminant effects on immune or defense responses could influence the ability of an organism to resist infectious disease. This study explored relationships between xenobiotic chemicals accumulated in oyster (Crassostrea virginica) tissue and various measures of putative oyster internal defense activities and physiological condition. Defense-related and physiological measurements were made on individual oysters collected from 22 sites at five Florida bays and pooled oyster tissue from each site was analyzed for polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and certain pesticides. Chemical concentrations, physiological condition, and hemocyte and hemolymph characteristics varied across bays and among sites within a bay. Within-bay comparisons showed that sites with high oyster defense-related activities often had accompanying high tissue concentrations of one or more classes of xenobiotic chemicals. Correlation analysis performed across bays demonstrated significant positive relationships between most defense-related characteristics and at least one contaminant, including various PAH, PCB and trace metal analytes. In combination with other recent studies, these results strengthen the hypothesis that certain xenobiotic chemicals may be associated with elevated oyster hemocyte activities, even though the ultimate influence on disease resistance remains unknown.

Appraisal of prospective bivalve immunomarkers.

Worldwide concern over threats to natural resources and public health has led to increased efforts to monitor and assess environmental conditions. This has stimulated the need for development and application of select biological and ecological measurements, or indicators, that are responsive to environmental stress. Measures of bivalve mollusc defence activities, such as haemocyte density, phagocytic activity, locomotion and production of cytotoxic molecules; and haemolymph constituents, such as agglutinins and lysozyme, have potential as indicators and appear to be responsive to xenobiotic chemical insults in the aquatic environment. However, basic research on the relevance of these measurements in inferring resistance to disease or enhanced survival is currently insufficient, reducing their value as potential biomarkers to address environmental objectives. In addition, variation in defence activities caused by seasonal temperature and reproductive cycling, salinity changes, nutritional status, diseases ...Worldwide concern over threats to natural resources and public health has led to increased efforts to monitor and assess environmental conditions. This has stimulated the need for development and application of select biological and ecological measurements, or indicators, that are responsive to environmental stress. Measures of bivalve mollusc defence activities, such as haemocyte density, phagocytic activity, locomotion and production of cytotoxic molecules; and haemolymph constituents, such as agglutinins and lysozyme, have potential as indicators and appear to be responsive to xenobiotic chemical insults in the aquatic environment. However, basic research on the relevance of these measurements in inferring resistance to disease or enhanced survival is currently insufficient, reducing their value as potential biomarkers to address environmental objectives. In addition, variation in defence activities caused by seasonal temperature and reproductive cycling, salinity changes, nutritional status, diseases and parasites, and genetic stocks is high and may limit applicability of bivalve defence-related measurements as indicators. This review examines these sources of variability and their possible implications for interpreting changes in bivalve defence activity as an indicator of stress. Examples of contaminant-induced changes in bivalve defence functions are described.

A rapid tetrazolium dye reduction assay to assess the bactericidal activity of oyster (Crassostrea virginica) hemocytes against Vibrio parahaemolyticus

Abstract An assay was developed to assess the ability of oyster, Crassostrea virginica , hemocytes to kill the human pathogenic bacterium, Vibrio parahaemolyticus (ATCC 17802). Bacterial killing was estimated colorimetrically by the enzymatic reduction of a tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium (MTS), and phenylmethasulfazone (PMS). The assay proposed here provides an indicator of immunocompetence of oysters against V. parahaemolyticus . The assay involved: (1) exposure of plasma-free oyster hemocytes to a streptomycin (SM)-resistant mutant strain of V. parahaemolyticus in a 96-well plate for 3 h at 17°C in SM-augmented sea water; (2) growout of surviving bacteria in nutrient broth for 2 h at 37°C; (3) addition of MTS and PMS; and (4) measurement of MTS/PMS reduction product (formazan) at 490 nm using a microplate reader. Advantages of this assay include the absence of radio-isotopes used in some killing assays and requirement of low volumes of plasma and numbers of hemocytes. In addition, we demonstrated greater precision than traditional, plate counting methods for bacterial estimation. This technique has the potential to evaluate oyster capacity to eliminate microbial agents and to assess effects of environmental changes and pollutant stress on defense capabilities of oysters.

Greater hemocyte bactericidal activity in oysters (Crassostrea virginica) from a relatively contaminated site in Pensacola Bay, Florida

Bivalve mollusks such as Crassostrea virginica inhabiting polluted estuaries and coastal areas may bioaccumulate high concentrations of contaminants without apparent ill effects. However, changes in putative internal defense activities have been associated with contaminant accumulation in both experimental and long-term field exposures. In an effort to elucidate these relationships, 40 oysters were collected from Bayou Chico (BC) and East Bay (EB) in Pensacola Bay, FL, two estuaries known to differ in the type and magnitude of chemical contaminants present. Oyster tissue concentrations of metals, tri- and dibutyltin (TBT, DBT), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were measured in individual oysters, as were hemocyte counts (HCs), hemocyte bacterial killing indices (KI), serum lysozyme (LYS) and serum protein (PRO) levels. Average HC, KI, LYS and PRO were significantly higher in BC oysters, which also had significantly higher tissue concentrations of total trace metals, butyltins (BTs), PAHs, PCBs, pesticides, and Mn, Cu, Zn and Sn. EB oysters had low organic contaminant levels and no detectable BTs, but significantly higher concentrations of Al, Cr, Fe, Ag, Cd, and Hg. Simple correlation analysis between specific defense measurements and specific chemical analytes showed specific positive relationships that corroborated previous findings in other FL estuaries. Canonical correlation analysis was used to examine relationships between defense measurements and tissue metals using linearly combined sets of variables. Results were also consistent with previous findings-the highest possible canonical correlation was positive: r=0.864, P<0.0019 among canonical variables composed of HC, KI and LYS for defense, and Fe, Cu, Ag, Cd, Sb, Sn, Ni, Pb and Hg for metals.

Stimulation of defense factors for oysters deployed to contaminated sites in Pensacola Bay, Florida.

A positive association between chemical contaminants and defense factors has been established for eastern oysters (Crassostrea virginica) from Florida, but it is unknown whether such factors can be stimulated through short-term exposure to contaminants in the field. Hatchery oysters were deployed at two contaminated sites and one reference site near Pensacola, Florida, during spring and summer in 1998. Putative defense measurements, notably hemocyte count and bactericidal activity, were significantly elevated after 12-week deployment during summer at the most contaminated site. This site exhibited a dramatic increase in chemical concentrations in oyster tissue relative to both the initial concentrations in hatchery oysters and to oysters deployed at the reference site. Hemocyte activity was not stimulated after 16-week deployment of hatchery oysters in spring, despite similar increases in tissue chemical concentrations, so defense activation by short-term exposure may covary with other unmeasured environmental or physiological parameters. Using the converse approach, Pensacola Bay oysters were collected from two contaminated sites and deployed at the reference site for 16 weeks during spring. Results from this converse deployment were ambiguous; serum lysozyme concentrations were reduced for oysters transplanted from both sites, but hemocyte activities were not significantly changed. The principal outcome from this study was the demonstration of enhanced defense activities for oysters upon short-term summer deployment at a contaminated site.

Contrasting responses of coral reef fauna and foraminiferal assemblages to human influence in La Parguera, Puerto Rico

Coral reef biota including stony corals, sponges, gorgonians, fish, benthic macroinvertebrates and foraminifera were surveyed in coastal waters near La Parguera, in southwestern Puerto Rico. The goal was to evaluate sensitivity of coral reef biological indicators to human disturbance. Proxies for human disturbance were measured as distance to town (DTT) and rankings of a low-level sediment contamination gradient analyzed from a previous study. Contaminant rank and DTT showed that percent mud, stony coral taxa richness, reef rugosity, and numbers of invertebrates and sponges were higher at sites closer to human disturbance, but a foraminiferal assemblage index was significantly lower at sites with higher proxies for human disturbance. Fish indicators showed no significant relationships with human activity, but associations between fish community measures and certain measures of stony corals, gorgonians and sponges were found. Contrasting responses between foraminifera and reef organisms may be due to greater exposure and sensitivity of foraminifera to sediment contaminants.

Assessing land use, sedimentation, and water quality stressors as predictors of coral reef condition in St. Thomas, U.S. Virgin Islands

Coral reef condition on the south shore of St. Thomas, U.S. Virgin Islands, was assessed at various distances from Charlotte Amalie, the most densely populated city on the island. Human influence in the area includes industrial activity, wastewater discharge, cruise ship docks, and impervious surfaces throughout the watershed. Anthropogenic activity was characterized using a landscape development intensity (LDI) index, sedimentation threat (ST) estimates, and water quality (WQ) impairments in the near-coastal zone. Total three-dimensional coral cover, reef rugosity, and coral diversity had significant negative coefficients for LDI index, as did densities of dominant species Orbicella annularis, Orbicella franksi, Montastraea cavernosa, Orbicella faveolata, and Porites porites. However, overall stony coral colony density was not significantly correlated with stressors. Positive relationships between reef rugosity and ST, between coral diversity and ST, and between coral diversity and WQ were unexpected because these stressors are generally thought to negatively influence coral growth and health. Sponge density was greater with higher disturbance indicators (ST and WQ), consistent with reports of greater resistance by sponges to degraded water quality compared to stony corals. The highest FoRAM (Foraminifera in Reef Assessment and Monitoring) indices indicating good water quality were found offshore from the main island and outside the harbor. Negative associations between stony coral metrics and LDI index have been reported elsewhere in the Caribbean and highlight LDI index potential as a spatial tool to characterize land-based anthropogenic stressor gradients relevant to coral reefs. Fewer relationships were found with an integrated stressor index but with similar trends in response direction.