Visualisation of the symbiosome membrane surrounding cnidarian algal cells

dVirtually nothing is known of the role of the cnidarian symbiosome, primarily due to the difficulty in visualising its membrane. We used the fluorescent dye MDY-64 to stain symbiosome membranes surrounding algae of the anthozoan Zoanthus robustus. MDY-64 did not stain cultured symbiotic dinoflagellates, confirming that this dye binds to a membrane of host cell origin. Another fluorescent dye, amino-chloromethylcoumarin (CMAC) stained the cytoplasm of both endoderm cells and algal cells from the zoanthid, a coral and an anemone. By drawing a suspension of endoderm cells from Z. robustus back and forth (5-7 times) through a hypodermic needle, we obtained approximately 73% of the algae in intact symbiosomes, with only 6% of the algae remaining in intact endoderm cells, and 21% free of both endoderm cell and symbiosome. About 15 additional passages of the cells through the needle removed the symbiosome membranes, leaving approximately 85% of the algae free of all host cell material. Use of detergents to remove the endoderm cell plasma membrane damaged both the symbiosome and algal membranes. Transmission electron microscopy showed variable numbers of membranes surrounding the algae. The ability to isolate dinoflagellate cells with and without symbiosome membranes will allow studies of the role of this membrane.