Ciliates from ancient permafrost: Assessment of cold resistance of the resting cysts

There is evidence that resting cysts of soil ciliates and numerous taxa of other protists can survive in permafrost for thousands of years at subzero temperatures; however, our knowledge about mechanisms of long term cryobiosis remains incomplete. In order to better understand the means by which ancient cysts survive, we investigated resistance to cyclical supercooling stress of resting cysts of the soil ciliate Colpoda steinii (Colpodida, Ciliophora). Three clonal strains were used for comparison, isolated from Siberian tundra soil, ancient Holocene (5-7,000 y) and late Pleistocene (32-35,000 y) permafrost sediments. To determine the viability of the ancient and contemporary ciliate cysts we improved and validated a cultivation-independent method of vital fluorescent staining with a combination of two nucleic acid binding dyes, acridine orange and propidium iodide. The viability of Colpoda steinii cysts during low-temperature experiments was measured using both the proposed vital fluorescent staining method and standard germination test. Our results indicate that the dual-fluorescence technique is a more accurate, rapid, and efficient method for estimating cyst viability. We found that cysts of ancient ciliates display lower tolerance to the impact of cyclical cold compared to cysts of contemporary ciliates from Siberian permafrost affected soils.