A rapid method to quantify total haemocyte count of Penaeus monodon using ATP analysis

Decapod crustaceans generally have 3 major categories of blood cells (haemocytes) - hyaline cells, semi-granular cells and granular cells - that can be distinguished by morphological features and have distinct functions(1)). Mean total haemocyte counts (THCs) for penaeid shrimp range from 20 to 40 x 10(6) cells/mL(1,2)) Molting, development, reproductive status, nutritional condition and disease have been shown to influence haemocyte abundance(1,2)). Infections of penaeid shrimp with fungus(3)) or virus(4)) result in a profound haemocytopenia. The THC declines almost 50-75% during these infections. Granulocytes are preferentially lost until late in the course of the infection(3)). Monitoring of THC, together with other diagnostic indices, is critical in diagnosing the progress of an infection. Traditionally, haemocyte counting is conducted with the aid of a haemocytometer under a light microscope. Quantification of the THC is often laborious and time-consuming especially when a series of examinations are to be performed within a limited time. Flow cytometry has been used to study haemocytes during the course of the molting for Penaeus japonicus(5)) and P. monodon(2)), the results are however not satisfactory. The luminometric assay for ATP is a rapid means of measuring all living organisms present in a sample. Cell counting based on ATP analysis has been successfully used in quantifying the amount of bacteria present in industrial and drinking water, foodstuffs(6)), pharmaceuticals, and cosmetics(7)) Clinical applications includes determining the presence of bacteria in body fluid and assessing the effect of antibiotics on microbial growths(8)). We hereinafter report the results of a study in which the THC story was followed using the ATP method in the course of a white spot syndrome virus (WSSV) infection in P. monodon.