Mitochondria-Derived Reactive Oxygen Species Regulation of Tardigrade Osmobiosis Revealed by Proteomics of Hypsibius exemplaris

Tardigrades are microscopic invertebrates renowned for their ability to survive extreme environmental stress such as radiation, extreme temperatures, and desiccation. Yet, the biochemical mechanisms they utilize to survive these extremes are poorly understood. Herein, we implement proteomics to investigate the biomolecular underpinnings of tardigrade osmobiosis - a survival state in response to osmotic pressure. Using two solutes, sucrose and NaCl, we reveal that de novo gene expression is not required for osmobiosis induction. While sucrose and NaCl induce slightly different proteomic effects, both solutes lead to an increased abundance or oxidation of proteins involved in ER or mitochondrial activity. Further, we investigate the role of mitochondrial reactive oxygen species (ROS) in tardigrade osmobiosis and demonstrate that inhibition of the alternative oxidase (AOX) within the mitochondrial respiratory chain (MRC) increases the rate of osmobiosis formation with both sucrose and NaCl. Subsequent electron paramagnetic resonance (EPR) spectroscopy reveals an increased rate of ROS formation in osmobiotes with AOX inhibited. Finally, inhibition of mitochondrial ROS significantly decreases the rate of osmobiote formation suggesting a regulation of osmobiosis through MRC-derived ROS. In sum, this work suggests mitochondrial-ROS signaling is necessary for tardigrade osmobiosis and further clarifies the biochemical mechanisms contributing to tardigrade extremotolerance.