In addition we used a polyclonal antibody against penaeidin (25) and a commercial immunohistochemistry kit (DiagXotics) to detect WSSV. We present evidence of the role of the three hemocyte subpopulations SGH, LGH, and HH, as well as peneidins and α2-macroglobulin in immune processes that occur in the LO. Immunostaining of other shrimp tissues with antibodies against

hemocytes is described. Hemocyte subpopulations in tissue sections were studied in animals from a full-sib family of L. vannamei shrimp. After induced infection with WSSV performed per os, these animals exhibited strong hemocyte infiltration and spheroids formation by histological observations (24). Briefly, WSSV inoculates were prepared following the protocol of Melena et al. (26). Gills were collected from shrimps with severe WSD lesions, as detected by histology, and were Bortezomib supplier homogenized in buffer TN (Tris HCL 20 mM, NaCl 0,4 M, pH 7.2). After centrifugation at 1200 g for 5 min, the supernatant was recovered and filtered through a 0.45 μm membrane and stored at –80°C until needed. 3 g shrimp were infected by intramuscular injection with 50 μL of the viral solution in the second abdominal segment. After 48 hr, moribund shrimp were removed and stored at –80°C. Severity of infection was verified by histology. These shrimp were

used as infected material in the induced infection. Eighty animals from the full-sib family of L. vannamei shrimp distributed in 16 tanks were starved one day before infection. The infected material was given twice a day at a total dose of 8% of the biomass. A water exchange of 100% PRKD3 was performed 3 hr after each application Akt activator of the infected material. Animals were sampled before infection and 24, 48 and 72 hr after infection (20 animals per sample) for histopathology analysis. Davidson’s AFA (alcohol, formalin, glacial acetic acid) fixative was used to preserve samples. Shrimp tissue was processed according to procedures outlined in Bell and Lightner (27). Sections were cut into 5 μm slices and stained with Mayer Bennet hematoxylin and eosin (H&E).

Tissues were carefully examined paying special attention to LO traits (WSSV lesions, hemocyte presence, spheroids formation). Immunohistochemistry of hemocyte subpopulations was performed on 20 animals (five per sample) following Destoumieux et al. (25) procedures. Briefly, the tissue sections were fixed on positively charged slides (Fisher Scientific, Loughborough, UK), and after rehidratation, they were incubated for 20 min at room temperature in TBS (100 mM Tris, 150 mM NaCl, pH 7.5), and then for 1 hr in the same solution supplemented with 0,5 (w:v) skim milk. One hour of incubation at 25°C was further performed with the specific antibodies (see below). Alkaline phosphatase-labeled anti-rabbit IgG or anti-mouse IgG (depending on the specific antibody) were used according to manufacturer’s instructions (Sigma-Aldrich, St.