Physical and Mechanistic Characterization of Tardigrade Cryptobiotic States in Response to Environmental Stressors

Tardigrades, known more commonly as ‘water bears,’ are semi‐microscopic invertebrates that possess a remarkable ability to withstand extreme stress. This stress tolerance is inferred via cryptobiosis, a collection of quiescent states enabling long‐term survival to extreme stressors through temporary metabolic suspension and upregulation of protective biomolecules. While cryptobiotic abilities span taxa across all kingdoms, the tardigrades ability to survive desiccation, freezing, oxygen starvation, fluctuating osmotic pressure, and ionizing radiation (using anhydrobiosis, cryobiosis, anoxybiosis, osmobiosis, and irradiation‐induced dormancy, respectively) is matched by none.Tardigrade cryptobioses, particularly those that form tuns, seem to occur via two distinct but connected processes, one of which facilitates and/or initiates muscular contraction and one of which expels internal water stores. However, the mechanisms through which these cryptobioses are initiated, maintained, and regulated are still unknown. Additionally, while research has shown distinctions between cryobiosis and anhydrobiosis, it is unclear to what extent tun‐forming cryptobioses (i.e. anhydrobiosis and osmobiosis) are distinctly regulated. Furthermore, while anhydrobiosis and cryobiosis have been well characterized, other forms of cryptobiosis have not been empirically explored.Our work has focused on charactering the physiological and metabolic distinctions between cryptobioses in Hypsibius exemplaris,a model tardigrade species. We have mapped all known cryptobiotic states through the implementation of confocal microscopy and biochemical characterization. Together we have shown structural and metabolic distinctions between tun‐forming and non‐tun cryptobiotic states. These quantitative measurements provide insight into mechanisms corresponding to protective physiological changes and represent the first known empirical analysis of the five cryptobiotic states.