2c). Some individual cells were recognized by 41B12 MAB in the stromal matrix of LO tubules, but a well defined labeling of exocyted α2-macroglobulin was detected in the external stromal matrix and in the fibrous material of outer tubule walls of LO (Fig. 3a). Vesicles inside the LOS were

immunostained by MABs 41B12 (Fig. 3b,c), 40E10 (Fig. 2a) and antipeneidin polyclonal antibody (Fig. 2c). No signal was detected in the LOS with the MAB 40E2. Other tissues labeled with the antibodies used in this study were: hematopoietic tissue (MABs 41B12, 40E10 and 40E2), podocytes of the antennal gland (40E10 MAB) (Fig. 4a), and phagocytic reserve heart cells (41B12 MAB) (Fig. 4b). A strong signal for 41B12 MAB was detected in the connective tissue BGJ398 in vitro of

the esophagus, stomach and infiltrating hemocytes in the hepatopancreas. 40E2 MAB immunostaining was detected mainly in hemocytes located in the connective tissue of the oral region (mandible, labrum and paragnatha). Although antibodies have been used as reagents for characterizing immune cells in the LO of shrimp (8,22), the panel of four antibodies against hemocytes used in this study, offer a new insight into the hemocyte interactions in the LO of WSSV infected shrimp. Our work shows the presence of SGH in the stromal matrix of LO. Winotaphan et al. (22) and van de Braak (23) stated that LO constitutes a site of hemocyte differentiation from undifferentiated HH into GH and SGH. In a previous study Rodríguez et al. (15) and Glutamate dehydrogenase Bachère et al. Everolimus cell line (17) reported that the MAB 40E10 recognized HH and SGH in hemocyte subpopulations separated by a percoll gradient. However, immunogold assays showed that 40E10 MAB labeled only SGH and not HH containing cytoplasmic glycoprotein deposits and/or striated granules (16) (Fig. 5a). These previous

findings suggested that SGH are present in circulation as a heterogeneous group of cells, possibly at different differentiation states of varying size and density. Our results support conclusions drawn by van de Braak et al. (23) and Whinotaphan et al. (22), that the stromal matrix of LO is the tissue in which SGH differentiation takes place. However, these findings also suggest that undifferentiated SGH and HH are two different cell groups. α2-macroglobulin is an evolutionarily conserved element of the innate immune system whose best characterized function is the clearance of active proteases of tissue fluids (for a review see Armstrong, 28). Proteases can act as virulence factors of a diverse array of pathogens (28). The MAB 41B12 recognizes α2-macroglobulin, and using inmunogold assay Perazollo et al. (18) determined its sub cellular localization in granules of LGH of F. paulensis, while Rodríguez (16), using the same MAB and the same technique detected α2-macroglobulin in striated vesicles of HH of M. japonicus (Fig. 5b).