Hemolymph (100 µL) was collected from both treated and control groups and centrifuged at 800 g for 5 mins (Model GS-15R, Rotor No. F2402; Beckman, Fullerton, CA, USA). After centrifugation, the supernatant was discarded, the hemocytes washed three times with Hank’s buffered salt solution and then stained with NBT solution (0.3%, 100 µL) for 30 mins at 37°C. The staining reaction was terminated by removing the NBT solution and adding absolute methanol. After three washings with 70% methanol, the hemocytes were air dried and 120 µL of 2-M KOH and 140 µL of DMSO added to dissolve cytoplasmic formazan. The optical density of the dissolved formazan was

read at 630 nm. Alkaline and acid phosphatase activities assays were performed according to the methods described by Gestal

et al. [23]. Briefly, ALP and ACP were measured using p-nitro phenyl phosphate Atezolizumab 16 mM as a standard substrate. Glycine NaOH buffer and sodium acetate buffer were used for ALP and ACP assays, respectively. VX-809 chemical structure Mixtures containing 0.2 mL of the substrate and 50 µL of hemolymph were incubated for 30 min at 37°C. Released p-nitrophenol in the resulting supernatants was measured at 410 nm and the amount calculated from the standard curve. One-way ANOVA followed by Tukey’s test was performed to identify significant differences among experimental groups at each sampling time using Statistical Analysis Software (SAS Institute, Cary, NC, USA). For statistically significant differences, an α value of < 0.05 (P < 0.05) was required. Linear regression analysis (comparison

between biochemical and immune variables and salinity of WSSV-challenged hemolymph of F. indicus) was performed to analyze WSSV infection and the influence of each salinity concentration. The unchallenged control F. indicus kept in 25 g/L survived. Mortality began at 24 hrs in the challenged shrimp kept in 5 and 35 g/L. Over AZD9291 order 24–96 hrs, the cumulative mortality of F. indicus maintained in 5 and 35 g/L was significantly higher than that of shrimp kept in 25 and 15 g/L (P < 0.05). At 72 hrs pi, the cumulative mortality of challenged F. indicus maintained in 25 g/L was the lowest among the experimental groups, whereas the cumulative mortality of the challenged F. indicus transferred to 5 g/L was the highest among the four treatments. No mortality was recorded in any of the unchallenged groups during the experimental period. In WSSV challenged animals, mortality increased in parallel with sampling time. For all salinity concentrations except for 25 g/L salinity, the mortality rates ranged from 63.3 ± 3.3% (15 g/L) to 83.3 ± 3.3% (5 g/L). From the start of the experiment (24th hour), animals exposed to 5 g/L salinity had a mortality of 53.3 ± 3.3%. However, animals at 25 g/L showed a comparatively lower mortality rate after infection with WSSV (Table 1). Total hemolymph protein concentration increased significantly at 48 and 72 hrs pi (P < 0.