Osmotic tolerance and volume regulation in in vitro cultures of the oysterpathogen Perkinsus marinus

Growth rate. cell size, osmotic tolerance, and volume regulation were exami ned in cells of Perkinsus marinus cultured in media of osmolalities ranging from 168 to 737 mOsm (6.5-27.0 ppt). Cells cultured at the low osmolalitie s of 168 and 256 mOsm (6.5 and 9.7 ppt) began log phase growth 4 days posts ubculture, whereas cells cultured at the higher osmolalities 341, 433, and 737 mOsm (12.7. 16.0, and 27.0 ppt) began log phase growth 2 days postsubcu lture. During log phase growth, cells from the higher osmolalities 341, 433 , and 737 mOsm had shorter doubling times than cells from the lower osmolal ities 168 and 256 mOsm. During both log and stationary phase growth, the me an cell diameter of cells cultured at 168 mOsm was significantly greater th an cells cultured at 341 and 737 mOsm; the mean diameters of cells cultured at 341 and 737 mOsm did not differ significantly from each other. P. marin us cells cultured in various osmolalities were exposed to artificial seawat er treatments of 56-672 mOsm (2.5-24.7 ppt). After the hypoosmotic treatmen t of 56 mOsm, cells that had been cultured in medium of low osmolality, 168 mOsm, showed only 41% mortality whereas the cells from the 341-, 433-, and 737-mOsm culture groups experienced 100% mortality. During the hyperosmoti c shock, all of the groups exhibited mortalities of less than 10%. In P. ma rinus cells cultured in medium of 737 mOsm and then placed in a 50% dilutio n, cell diameter increased 13% which was a volume increase of 44.5%, but ce lls returned to baseline size (size before osmotic shock) within 5 minutes. P. marinus cells cultured at low osmolalities can withstand both hypo- and hyperosmotic stress and use volume-regulatory mechanisms during hypoosmoti c stress. Results suggest that transferring infected oysters to low salinit y will result in strains of P. marinus acclimated to low salinity that will be able to withstand periodic events of extremely low salinity.