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Measurement of reactive oxygen intermediate production in haemocytes of the penaeid shrimp, Penaeus vannamei

Munoz, M., Cedeno, R., Rodriguez, J., Knaap, W.P.W. van der., Mialhe, E., Bachere, E.
Aquaculture 2000 no.1/3 pp. 89-107
Vibrio, quantitative analysis, Litopenaeus vannamei, assays, bacteria, shrimp culture, propiconazole, pathogens, pollutants, nitroblue tetrazolium, anions, oxides, culture media, mortality, hemocytes
A spectrophotometric nitroblue tetrazolium (NBT) reduction assay was used to demonstrate the production of superoxide anions (O2-) by haemocytes of the white shrimp Penaeus vannamei. It was found that haemocytes, without receiving an experimental stimulant, showed a rather high background activity. Therefore, optimal parameters (number of haemocytes, type of incubation medium, type and concentration of stimulants) were first established, in order to obtain a reliable and reproducible quantitative assay. With this optimized assay, and using specific inhibitors, it was shown that it is indeed the production of O2- that was measured. Activities varied strongly among individual shrimp specimens. Live bacteria, among those Vibrio strains, induced O2- production in the haemocytes, in a dose-dependent manner. Whereas Vibrio anguillarum and a probiotic strain of V. alginolyticus evoked clear reactions, a pathogenic strain of V. harveyi failed to elicit O2- production in haemocytes. It is discussed that this may explain this strain's capability of evading the host's oxidative microbicidal activity, which would be a virulence factor in these bacteria. Heat-killed bacteria hardly induced NBT reduction in the cells. The fungicide propiconazole or Tilt, found as a pollutant in the aquatic environment where the shrimp are reared, was tested for its effect on NBT reduction by the haemocytes. In haemocytes that did not receive an experimental stimulant, Tilt induced the reduction of NBT in a dose-dependent manner. In experimentally stimulated haemocytes, however, Tilt strongly reduced the reaction upon the stimulant PMA. Probable explanations for these seemingly controversial effects of Tilt are discussed, as are possible consequences of this sort of pollutants for shrimp aquaculture. This easy to perform and relatively cheap and simple quantitative assay for measuring the activity of an oxidative microbicidal mechanism in shrimp haemocytes, appears quite reliable and may therefore prove to be a valuable tool for monitoring shrimp health and immunologic status.