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

Abstract

NBT staining was used to determine the presence of superoxide anions (O − 2) produced by tiger shrimp ( Penaeus monodon) hemocytes attached to a coverslip. When cells were treated with β-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 β-glucan had the strongest effect on intracellular O − 2 generation, followed by zymosan and PMA (2.5, 2, and 1.3 times greater than the O − 2 generated by the control group, respectively). After oxidizing phenol red to measure the amounts of hydrogen peroxide (H 2O 2) produced by the hemocytes, we found that β-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 β-glucan had a stimulative effect on OCl − production. However, following PMA stimulation, hemocyte chemiluminescence was detected although only at 1.7 mV. Using H 2O 2 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 β-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.