Product Citations and Abstracts

Worldwide concern over threats to natural resources and public health has led to increased efforts to monitor and assess environmental condition. This has stimulated the need for development and application of select biological and ecological measurements, or indictors, that are responsive to environmental stress. Measures of bivalve mollusk defense activities, such as hemocyte density, phagocytic activity, locomotion and production of cytotoxic molecules, and hemolymph 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 defense 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 defense-related measurements as indicators. This review examines these sources of variability and their possible implications of interpreting changes in bivalve defense activity as an indicator of stress. Examples of contaminant-induced changes in bivalve defense functions are described.

Oliver, Leah M., William S. Fisher, James T. Winstead, Becky L. Hemmer and Edward R. Long. 2001. Relationships Between Tissue Contaminants and Defense-Related Characteristics of Oysters (Crassostrea virginica) from Five Florida Bays. Aquat. Toxicol. 55(3-4):203-222. (ERL,GB 1106).

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 reamins unknown.

Oliver, Leah M., William S. Fisher, Aswani K. Volety and Ziad Malaeb. 2003. Greater Hemocyte Bactericidal Activity in Oysters (Crassostrea virginica) from a Relatively Contaminated Site in Pensacola Bay, Florida. Aquat. Toxicol. 64(4):363-373. (ERL,GB 1162).

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 di-butyltim (TBT, DBT), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were measured in individual oysters, as were hemocyte counts (HC), 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 correlations was positive: r = .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.

Oliver, Leah M. and William S. Fisher. 1995. Comparative Form and Function of Oyster Crassostrea virginica Hemocytes from Chesapeake Bay (Virginia) and Apalachicola Bay (Florida). EPA/600/J-95/461. Dis. Aquat. Org. 22(3):217-225. (ERL,GB 855).

Oysters Crassostrea virginica from Chesapeake Bay, Virginia, and Apalachicola Bay, Florida, USA, were collected in March and October 1992 to investigate possible differences in defense-related hemocyte activities between individuals from geographically separate populations. In March, hemolymph drawn from Chesapeake Bay oysters contained an average of 1.08 x 106 hemocytes ml-1 hemolymph, significantly lower than the average 1.63 x 106 hemocytes ml-1 hemolymph obtained from Apalachicola Bay oysters. Hemocyte number did not differ significantly in the October comparison. At both times of year, Chesapeake Bay oyster hemolymph samples contained significantly greater proportions of granular hemocytes compared to Apalachicola Bay hemolymph samples. Hemocyte samples from Chesapeake Bay oysters demonstrated a higher percentage of mobile hemocytes and greater particle binding ability than Apalachicola Bay oyster hemocytes when tested in March, but the reverse was found in the October experiments. Chesapeake Bay oyster hemocytes produced significantly more superoxide anion as measured by nitroblue tetrazolium reduction than did Apalachicola Bay oyster hemocytes in both March and October. Oyster hemolymph levels of the protozoan parasite Perkinsus marinus did not differ significantly between the 2 sites at either time of year. These results demonstrate the importance of background studies to characterize site-specific differences in oyster hemocyte defense-related functions.

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)-2H-tet razolium (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.

Genthner, Fred J., Aswani K. Volety, Leah M. Oliver and William S. Fisher. 1999. Factors Influencing In Vitro Killing of Bacteria by Hemocytes of the Eastern Oyster (Crassostrea virginica). Appl. Environ. Microbiol. 65(7):3015-3020. (ERL,GB 1063).

A tetrazolium dye reduction assay was used to study factors governing 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 transluclent (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 winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificusto 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 theV. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than 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.

Fisher, William S., Leah M. Oliver, James T. Winstead and Edward R. Long. 2000. Survey of Oysters Crassostrea virginica from Tampa Bay, Florida: Associations of Internal Defense Measurements with Contaminant Burdens. Aquat. Toxicol. 51(1):115-138. (ERL,GB 1090).

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 characteristics, 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.

Fisher, William S., Leah M. Oliver, James T. Winstead and Aswani K. Volety. 2003. Stimulation of Defense Factors for Oysters Deployed to Contaminated Sites in Pensacola Bay, Florida. EPA/600/J-03/405. Aquat. Toxicol. 64(4):375-391. (ERL,GB 1161).

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 and one reference site near Pensacola, Florida, during spring and summer 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 week 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.

Fisher, William S. and Leah M. Oliver. 1996. Whole-Oyster Procedure for Diagnosis of Perkinsus marinus Disease Using Ray's Fluid Thioglycollate Culture Medium. J. Shellfish Res. 15(1):109-117. (ERL,GB 919).

Diagnosis of Perkinsus marinus disease of eastern oysters Crassostrea virginica has been routinely accomplished by incubating oyster tissues in a fluid thioglycollate medium described by Ray in the early 1950s. At least three modifications of the technique are available with applications to different diagnostic needs. Of these, the whole-oyster technique is potentially the most valuable modification because it includes all oyster tissues and does not rely on subjective estimates of intensity. A variety of protocols and approaches were examined in an attempt to develop a standardized procedure for quantitative whole-oyster diagnosis that optimizes sensitivity, specificity, precision and accuracy. A recommended procedure, with possible variations, is presented here with the expectation that its presentation will foster further refinement and improvement. We conclude that the recommended whole-oyster diagnostic technique is capable of providing reliable quantifications of prevalence and intensity and has great potential for examining correlations of total P. marinus body burdens with measurements of oyster biology and for evaluating or calibrating other diagnostic techniques.

Fisher, William S., Leah M. Oliver and Patrice Edwards. 1996. Hematologic and Serologic Variability of Eastern Oysters from Apalachicola Bay, Florida. J. Shellfish Res. 15(3):555-564. (ERL,GB 971).

Eastern oysters (Crassostrea virginica) were collected monthly from two sites approximately 15 km apart in Apalachicola Bay, Florida during a one-year period. Hematological and serological measurements were made on hemolymph withdrawn from the adductor muscle. The two sites experienced nearly identical temperature patterns during the study period, but salinity and other physical factors fluctuated. Significant differences attributable to sampling date were found for circulating hemocyte density, phagocytic activity and superoxide anion (O2-) producing ability, and for serum protein, lysozyme, and agglutinating activity, using data from both sites combined. This variability was most likely related to temperature or temperature-influenced reproductive cycling. Oyster hemocyte locomotion did not vary significantly with time over the study period nor were significant differences found between sites. Significant differences between site means (combined for all dates) were found for O2-, protein and lysozyme and significant date * site interactions were found for phagocytosis, agglutination and lysozyme, indicating that local conditions, such as salinity fluctuations, influenced these measurements. An accurate description of variability in oyster defensive functions will require more frequent sampling and a better understanding of local environmental influences.

Fisher, William S., James T. Winstead, Leah M. Oliver, H. Lee Edmiston and George O. Bailey. 1996. Physiologic Variability of Eastern Oysters from Apalachicola Bay, Florida. J. Shellfish Res. 15(3):543-553. (ERL,GB 972).

Eastern oysters, Crassostrea virginica, were collected monthly during a one-year period from two study sites in Apalachicola Bay, Florida, and several measurements were made of their physiological condition. Continuous and intermittent temperature measurements at both sites showed highly coincident ambient temperature regimes. Salinity measurements however, were erratic and varied dramatically between sites. Oyster gonad size and gametogenic condition were highly synchronous at both sites, supporting the concept of temperature-driven reproductive cycles. Other measurements, including condition index, wet:dry tissue weight ratio, digestive tubule condition and vesicular connective tissue condition, showed significant variability due to sampling month, but also differed due to site and/or to interaction between date and site, indicating that local effects influenced oyster physiology. Temperature control over condition index and wet:dry tissue weight seems apparent, but it is not known whether the changes resulted directly from temperature or from temperature-driven reproductive and metabolic cycles. A significant difference between site means at specific dates was observed for digestive tubule condition and may relate to short-term salinity differences. Other physiological variations could not be attributed to any of the physical conditions monitored (temperature, salinity, pH and dissolved oxygen). Variability of oyster physiological measurements inherent at different sites and seasons must be well understood to properly interpret them in the context of biological indicators of environmental condition.

Fisher, William S., Leah M. Oliver, John E. Rogers and Debbie L. Santavy. 2007. Effects of Global Change on Coral Reef Ecosystems. Chapter 12. In: A Summary of NHEERL Ecological Research on Global Climate Change. Edited by Peter A. Beedlow and David T. Tingey. EPA/600/R-05/007. U.S. Environmental Protection Agency, Office of Research and Development, Washington, DC 20460. Pp. 12-1 - 12-13. (ERL,GB X1098).