UC Berkeley

Tardigrades are microscopic, aquatic animals that are an emerging model system for studying physiological responses to a wide range of environmental stressors. Past research has explored tardigrades’ extreme tolerance to desiccation and radiation, but little is known about the phylum’s tolerance to subzero temperatures, especially under more ecologically relevant conditions. Here, I use Hypsibius exemplaris—a lab-friendly cosmopolitan species, with a publicly available genome—as a model species to characterize the physiological response of active, hydrated tardigrades to low temperatures ranging from 1°C to -20°C. I develop methods to characterize numerous cold tolerance phenotypes in tardigrades, through multiple lenses: high-accuracy measurement of survival, plasticity of chill coma, and comparative proteomics and ice-affinity purification of cold-treated tardigrades (aimed at identifying underlying molecular mechanisms of cold tolerance and putative tardigrade-specific ice-binding proteins).

In Chapter 1, I develop new methods to score mortality of cold-exposed tardigrades using the live/dead cell-marker SYTOX Green, to improve the accuracy of survival-based assays. I determined that visualization of SYTOX Green uptake was a more accurate and more specific method of determining mortality in tardigrades, compared to the traditional methods of scoring survival based on locomotion. This new method for determining survival allows for higher throughput of scoring tardigrade phenotypes and provides differentiation of live, dead, and injured tardigrades (showing localized regions of damage in the tardigrade body-plan, as they first experience cellular death). Using this new assay, I monitored survival of hydrated mixed-staged adult H. exemplaris over a range of acute exposure times (2-120 hours) and subzero temperatures (-10°C, -15°C, and -20°C), in order to dissect the impacts of time, temperature, and their interaction on cold tolerance. Tardigrades exposed to -10°C showed little SYTOX Green uptake and high survival across all timepoints, and samples supercooled rather than froze. Survival of tardigrades exposed to -15°C was highly variable, and depended on the water-state of the cold exposed samples (liquid or frozen). When exposed to -20°C, tardigrade survival declined exponentially with increasing exposure time and nearly all samples froze. Although I predicted that freezing may drive tardigrade mortality based on these results, I surprisingly found that tardigrades incubated with the ice-nucleating bacteria Pseudomonas syringae had significantly improved survival after cold exposure, illustrating the importance of ice-formation dynamics and environmental microbes. Lastly, a 3-week thermal acclimation of tardigrades to mild cold (1°C & 4°C) did not significantly improve survival to low temperature, while acclimation to 15°C (vs the standard culture condition of 20°C) did. This work suggests that H. exemplaris is sensitive to ecologically relevant cold and assays developed here will be useful for exploring the thermal physiology of additional tardigrade species. More so, evidence collected here suggests that this tardigrade species is capable of surviving environmental freezing, under certain ecologically relevant conditions.

The goal of Chapter 2 was to explore cold tolerance phenotypes of tardigrades at low but non-freezing temperatures. Although no such phenotype was documented in past tardigrade literature, I predicted that tardigrades would experience a state of reversible paralysis known as chill coma, with exposure to sufficiently low temperatures and exposure times. Chill coma is often a non-lethal phenotype commonly seen in insects, but it nonetheless can impact the ecology and overall fitness of an organism living in winter conditions, as it prevents individuals from feeding, reproducing, and evading predators. Chill coma has been demonstrated to be a plastic phenotype in response to a variety of environmental conditions, as seen in insects. I predicted that the amount of time required for tardigrades to recover from chill coma (known as chill coma recovery time or CCRT) would vary with low temperature and exposure time. By analyzing video recordings of adult H. exemplaris recovering from cold-exposure, I observed that tardigrades experience a cold-induced state of reversible paralysis indicative of chill coma after 12h at 1°C. The impact of longer mild cold exposures depended on temperature: above -1°C, recovery times decreased with increasing duration of exposure, indicating that beneficial acclimation may have occurred. Below -1°C to -4°C, recovery times increased with increasing exposure duration, suggesting that sublethal damage may have accumulated. I also observed that chill coma recovery time was plastic depending on photoperiod acclimation. Tardigrades that were acclimated to shorter-day photoperiods (winter-like) for 2 weeks had significantly faster CCRTs than tardigrades acclimated to longer-day photoperiods (summer-like). In summary, this chapter characterizes a new cold tolerance phenotype in tardigrades, as well as its plasticity. Determining the underlying mechanisms that control the chill coma plasticity in tardigrades will further illustrate similarities or differences between the insect and tardigrade nervous system, in future work.

The goal of Chapter 3 was to characterize how the tardigrade proteome responds to cold exposure, as well as to discover and functionally verify candidate proteins that help tardigrades perturb freeze damage—especially novel candidate ice-binding proteins. Here, I used comparative proteomics to broadly determine how the protein repertoire shifts in H. exemplaris due to ecologically relevant cold conditions. Cohorts of 1000+ adult tardigrades were exposed to a cold treatment down to -10°C (or a control treatment of 20°C), and proteins were extracted from whole-animals via optimized sample preparation methods for data-independent acquisition mass spectrometry (DIA-MS). In a parallel set of samples, I performed a specialized ice-affinity purification method (IAP) and DIA-MS to enrich for and identify proteins that bind to ice. I predicted that tardigrades utilize a wide range of biochemical mechanisms related to cold tolerance, as seen in insects, as well as IBPs or other cold tolerance proteins that are unique to the phylum, with novel structures or functions. Gene ontology (GO) enrichment analysis of proteins are significantly more abundant after cold illustrated changes in neuron and neuromuscular function, calcium ion balance, cell division processes (such as apoptosis), and glucose metabolism. GO enrichment of proteins less abundant after cold treatment suggested changes in lipid metabolism, cuticle production, development, and redox. Ice-affinity purification and a bioinformatics pipeline identified a ranked list of novel candidate ice-binding proteins that tardigrades may utilize to survive freezing. This study is the first comparative proteomics study exploring the impacts of cold stress, in any eukaryotic animal, and results provide a framework for what molecular mechanisms tardigrades may use to survive cold.

This dissertation fills a gap in knowledge about the fundamental physiology and molecular mechanisms that an especially cold-hardy microscopic organism (tardigrades, specifically Hypsibius exemplaris) uses to survive subzero temperatures. Development of new phenotypic assays allowed for the rigorous characterization of the plasticity of tardigrade survival and chill coma, in response to time, temperature, and their interaction. Despite tardigrades’ reputation for being virtually indestructible, I determined numerous time and temperature-dependent conditions that result in increased mortality and reduced performance (CCRT), that may impact tardigrade ecology and winter-time behavior. Interestingly, I also determined that tardigrade’s survival to environmental freezing improves drastically when animals are exposed to ice-nucleating microbes, which may be found in their natural environments. Finally, I determined a number of molecular mechanisms that may underlie cold tolerance in tardigrades, as illustrated by the first-ever comparative proteomics study of any animal in response to cold, along with generating a ranked list of novel, tardigrade-specific candidate ice-binding proteins. Overall, I hope that the tools and findings of this work will help establish tardigrades as an increasingly important and tractable model system, in the field of comparative physiology and beyond.