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
Authors: Lyons Ana M , Roberts Kevin T , Williams Caroline M 
Published: March 2024

AbstractTardigrades 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-exposedHypsibius exemplarisallows 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 ofPseudomonas syringaecompared 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 thatH. 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.