Evidence that local viscosity and NOX-dependent ROS increases render the tardigrade H. exemplaris resilient to...

Biological phase changes provoked by stress, such as vitrification or gel-sol transitions, enable many organisms, including extremotolerant tardigrades, to enter quiescent states and survive extreme environmental conditions. Protein-driven phase transitions are hypothesized to produce large-scale changes in intracellular viscosity, allowing tardigrades to survive extreme stresses such as desiccation. We report that the tardigrade Hypsibius exemplaris undergoes both large-scale and local increases in intracellular viscosity following exposure to anoxic and hyperosmotic stress. Such dramatic shifts in cellular viscosity would be expected to enhance cellular resilience to physical force. Indeed, we found that tardigrades can survive, behave normally, and reproduce after exposure to the highest simulated hypergravity (HG) achievable in an ultracentrifuge (one million times Earth's gravity). In contrast, Caenorhabditis elegans, a similarly sized animal, does not survive these extreme forces owing to loss of cellular integrity. Remarkably, tardigrades frozen during exposure to extreme hypergravitational force show minimal disruption of fine cellular ultrastructure and little evidence of stratification of cellular components whose density varies by nearly a factor of two. Further, exposure to anoxia, hyperosmotic stress, and HG all result in a large increase in reactive oxygen species (ROS), which is required for survival under these extreme environments. Inhibition of NADPH oxidase (NOX) suppresses survival both to HG and hyperosmotic stress. Our findings suggest that intracellular viscosity changes in response to multiple extreme stresses may underlie the resilience of these animals to extraordinary physical stress, and that survival in or recovery from these states relies on ROS signaling via NADPH oxidase.