Fluorometric measurement of oxidative burst in lobster hemocytes and inhibiting effect of pathogenic bacteria and hypoxia

The stimulation of hemocytes during phagocytosis leads to the generation of a series of oxygen radicals known as reactive oxygen species (ROS). Among these, hydrogen peroxide plays an important microbicidal role by directly killing microorganisms or by serving as an intermediate for other antimicrobial radicals. In this study, we adapted a technique using 2′,7′- dichlorofluorescein-diacetate (DCFH-DA) to measure H₂O₂ production in lobster hemocytes. After oxidation by hydrogen peroxide, this molecule produces a fluorescent product that can be easily detected. The respiratory burst was successfully activated in lobster hemocytes by the addition of zymosan particles, but not with phorbol myristate acetate. After optimization, we used the technique to investigate the effect of different bacterial strains, including lobster pathogens, on the oxidative burst. Results demonstrate that Aureococcus viridans, a bacterial pathogen that is able to survive phagocytosis by lobster hemocytes, quenches ROS production. The comparison of ROS production in lobsters collected from field sites submitted to different levels of dissolved oxygen suggests that this technique provides a good indicator of lobster physiological status and immunocompetency.