Acidification of the phagosome in Crassostrea virginica hemocytes following engulfment of zymosan

Phagocytic hemocytes are responsible for engulfing and internally degrading foreign organisms within the hemolymph and tissue of the eastern oyster, C rassostrea virginica. Since rapid acidification of the phagosome lumen is t ypically essential for activation of hydrolytic and reactive oxygen interme diate (ROI) producing enzymes in vertebrate cells, we measured phagosomal p H in oyster hemocytes by using the emission fluorescence of two fluorescent probes, rhodamine and Oregon Green 488 (OG 488), conjugated to zymosan to determine whether oyster hemocyte phagosomes become acidified after phagocy tosis of zymosan. The average pH of 1079 phagosomes within 277 hemocytes 1 h after phagocytosis of zymosan was 3.9 +/- 0.03. Observations of 141 hemoc ytes with internalized zymosan by light microscopy revealed that, over a 60 -min time period, 51% of highly granular hemocytes became partially granula r, and 29% became agranular. In addition, 83% of partially granular hemocyt es containing zymosan at time = 0 became agranular within 60 min. A compari son revealed that the phagosomes of agranular hemocytes were much more acid ic (pH 3.1 +/- 0.02) than those of highly granular hemocytes (4.9 +/- 0.02; P < 0.05). These values are significantly lower than most reported in the literature for blood cells from metazoan organisms.