Immunostimulation of tiger shrimp (Penaeus monodon) hemocytes for generation of microbicidal substances: analysis of reactive oxygen species.

NBT staining was used to determine the presence of superoxide anions (O2-) produced by tiger shrimp (Penaeus monodon) hemocytes attached to a coverslip. When cells were treated with beta-glucan, blue granules were observed in 41% of studied hemocyte cytoplasm. For zymosan-treated, PMA-treated, and control cells, the percentages of hemocytes showing similar blue granules were 31, 9, and 5%, respectively. A comparison of stimulative effects on 15 hemocyte suspensions, each collected from a single tiger shrimp, showed that beta-glucan had the strongest effect on intracellular O2- generation, followed by zymosan and PMA (2.5, 2, and 1.3 times greater than the O2- generated by the control group, respectively). After oxidizing phenol red to measure the amounts of hydrogen peroxide (H2O2) produced by the hemocytes, we found that beta-glucan had the strongest stimulative effect (12.2 nmol/mg protein), followed by zymosan and PMA (7.2 and 2.6 nmol/mg, respectively). However, a luminol-enhanced chemiluminescence analysis of hypochlorites (OCl-) produced by the experimental hemocytes showed that neither zymosan nor beta-glucan had a stimulative effect on OCl- production. However, following PMA stimulation, hemocyte chemiluminescence was detected although only at 1.7 mV. Using H2O2 as substrate and guaiacol as an electron acceptor, the enzyme activity of crude enzyme extract derived from broken hemocytes was analyzed; enzyme activity similar to that of human myeloperoxidase (MPO) (0.104 U/mg protein) was observed. The data showed that only PMA had any stimulative effect on MPO-like enzyme activity (2.23 times that of the control group); zymosan and beta-glucan did not have any observable effects on this specific enzyme activity. This is the first documented demonstration of a respiratory burst in shrimp hemocytes.