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

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{degrees}C at high rates of [~]98%. Survival of tardigrades to exposures of -15{degrees}C depends on environmental freezing in pure mineral water, and survival decreased exponentially with exposure time at -20{degrees}C (to 45% after 24 hours; with freezing occurring at nearly all -20{degrees}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{degrees}C). Surprisingly, we found a significant increase in survival of tardigrades frozen at -20{degrees}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{degrees}C and -15{degrees}C. This indicates the species tolerance to environmental ice formation and exposure to our lowest temperature (-20{degrees}C), under certain conditions of controlled environmental ice formation. A 3-week acclimation of tardigrades to mild cold (1{degrees}C and 4{degrees}C) in constant darkness did not significantly improve survival after acute exposure to low temperature, but acclimating animals to 15{degrees}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{degrees}C, even in response to prolonged exposures. - Below -15{degrees}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{degrees}C & 4{degrees}C) does not significantly improve survival to low temperature, while acclimation to 15{degrees}C (vs the standard culture condition of 20{degrees}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.