Reactive oxygen species (ROS) production by amoebocytes of Asterias rubens (Echinodermata)

An adapted peroxidase, luminol-enhanced chemiluminescence method in an EDTA-free, Ca++-containing medium is described and used to characterise reactive oxygen species (ROS) production by starfish immunocytes using a standard microplate reader luminometer. ROS production was stimulated by direct interaction of immunocytes with bacteria or bacterial wall components, but not by the soluble stimulant PMA nor the lectin concanavalin A. Produced ROS detected by this method are apparently superoxide anions, hydrogen peroxide and peroxynitrite. Comparison with other chemiluminescence methods indicates that the described method is the only one to detect the stimulation of starfish immunocytes by the Gram-positive bacteria, Micrococcus luteus, a fact that questions previous reports indicating a lack of stimulation by pathogens. The adapted method provides a rapid determination of the overall ROS production, which is suitable for both disease control and immunotoxicological studies in echinoderms.