Survival of tardigrades (Hypsibius exemplaris) to subzero temperatures depends on exposure intensity, duration, and ice-nucleation—as shown by large-scale mortality dye-based assays

Abstract Tardigrades are an emerging model system for understanding a diversity of environmental stress responses, yet few studies describe the physiology of cold tolerance in hydrated, active tardigrades. Here, we develop methods to screen tardigrades for survival in a high-throughput manner, to investigate the impacts of several key environmental conditions on survival. The visualization of dye uptake (SYTOX Green) in hydrated, cold-exposed Hypsibius exemplaris allows us to quickly and accurately quantify the survival of thousands of animals, under a range of ecologically-relevant low temperatures, exposure times, conditions, and thermal acclimations. As a proof-of-concept, we show that SYTOX Green uptake more accurately predicts 2-week survival outcomes of tardigrades post-cold exposure, compared to previous methods of scoring survival (locomotion). We show that hydrated, active tardigrades survive mild cold exposures of - 10°C at high rates of ∼98%. Survival of tardigrades to exposures of -15°C depends on environmental freezing in pure mineral water, and survival decreased exponentially with exposure time at -20°C (to 45% after 24 hours; with freezing occurring at nearly all -20°C timepoints). To investigate the role of environmental ice-formation on tardigrade survival vs. temperature, we incubated unacclimated tardigrades with ice-nucleating bacteria—which initiate environmental freezing at higher temperatures (-1.8 to 3.8°C). Surprisingly, we found a significant increase in survival of tardigrades frozen at -20°C (p-value = 0.0152) with the addition of Pseudomonas syringae compared to non-inoculated controls, as well as observing high-survival of tardigrades in ice-nucleated samples exposed to -10°C and -15°C. This indicates the species’ tolerance to environmental ice formation and exposure to our lowest temperature (-20°C), under certain conditions of controlled environmental ice formation. A 3-week acclimation of tardigrades to mild cold (1°C and 4°C) in constant darkness did not significantly improve survival after acute exposure to low temperature, but acclimating animals to 15°C did. Overall, we find that H. exemplaris—an emerging tardigrade model species—has a range of cold tolerance capabilities, dependent on time, temperature, environmental ice-formation, and culturing conditions. This work offers a framework with new tools for performing large-scale physiological assays in numerous species, establishing tardigrades as a tractable and uniquely informative model system in comparative physiology and the study of environmental stress. Key Findings - Active, hydrated tardigrades—with no prior acclimation to cold—have high survival of temperatures above -15°C, even in response to prolonged exposures. - Below -15°C, tardigrade survival declines exponentially with increasing exposure time. - Incubating tardigrades with ice-nucleating bacteria significantly improves survival after cold exposure, illustrating the importance of ice-formation dynamics and environmental microbes. - A 3-week acclimation of tardigrades to mild cold (1°C & 4°C) does not significantly improve survival to low temperature, while acclimation to 15°C (vs the standard culture condition of 20°C) does. - Uptake of the dye SYTOX Green is a more accurate metric of tardigrade mortality in response to cold exposure, compared to the traditional method of scoring lack of locomotion during recovery. Competing Interest Statement The authors have declared no competing interest.