Assessment of the Role of Reactive Oxygen Species in Innate Immunity of the Eastern Oyster, Crassostrea virginica

EPA Grant Number: U915015
Title: Assessment of the Role of Reactive Oxygen Species in Innate Immunity of the Eastern Oyster, Crassostrea virginica
Investigators: Bramble, Lisa A.
Institution: University of Maryland - College Park
EPA Project Officer: Lee, Sonja
Project Period: January 1, 1996 through December 31, 1999
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1996) RFA Text |  Recipients Lists
Research Category: Fellowship - Pathology , Academic Fellowships , Health Effects


The objective of this research project was to test the hypothesis that reactive oxygen species (ROS) generated by hemocytes of the bivalve Crassostrea virginica (eastern oyster) are involved in the bactericidal activity mediated by phagocytic cells. Demonstration of ROS production by hemocytes of a variety of bivalve species has led to the hypothesis that ROS function as microbicidal agents in these organisms, as is the case in mammalian innate immunity.


Stimulation of superoxide anion (O2-) and hydrogen peroxide (H2O2) production by eastern oyster hemocytes upon exposure to the bacterium Listonella anguillarum was assessed using lucigenin- and luminol-enhanced chemiluminescence (CL), respectively. Hemocytes produced significantly elevated levels of O2- in response to heat-killed L. anguillarum, but not to viable bacteria. Stimulation of hemocyte-derived luminol CL was not observed when oyster immunocytes were exposed to either live or dead L. anguillarum. Results suggest that the bacterial antioxidant enzyme catalase may suppress hermocyte H2O2 production.

Morone saxatilis (striped bass) phagocytes were used as a positive comparative model in this research project. When assayed under identical conditions, the ROS response of striped bass macrophages to zymosan was two orders of magnitude greater than the oyster hemocyte response. In contrast to the oyster hemocytes, viable L. anguillarum stimulated striped bass phagocyte ROS production. It is suggested that the comparatively low levels of ROS generated by eastern oyster hemocytes are unable to exceed bacterial antioxidant capability, such that a CL response is not elicited.

In a subsequent study, the effect of ROS inhibition on phagocyte bactericidal activity was evaluated. Elimination of O2- production by the NADPH oxidase inhibitor diphenyleneiodonim reduced bactericidal activity of striped bass phagocytes towards Bacillus megaterium and Pseudomonas fluorescens by 50 percent to 100 percent. In contrast, abrogation of the ability of oyster hemocytes to produce ROS had no effect on their ability to kill B. megaterium and reduced bactericidal activity towards P. fluorescens by only 4 percent.

These data suggest that eastern oyster hemocytes do not produce sufficient levels of ROS to serve as cytotoxic effector molecules. It is suggested that future research evaluate the possibility that ROS produced by C. virginica hemocytes function as immunoregulatory molecules.

Supplemental Keywords:

fellowship, reactive oxygen species, ROS, eastern oyster, Crassostrea virginica, phagocytes, oyster hemocytes.

Progress and Final Reports:

  • 1996
  • 1997
  • 1998
  • Final