O'Dell, H. M. | bioRxiv Science

bioRxiv2024

Fluorescence Shadow Imaging of Hypsibius exemplaris Reveals Morphological Differences Between Sucrose- and CaCl2-Induced Osmobiotes

Tardigrades are renowned for their ability to survive a wide array of environmental stressors. In particular, tardigrades can curl in on themselves while losing a significant proportion of their internal water content to form a structure referred to as a tun. In surviving varying conditions, tardigrades undergo distinct morphological transformations that could indicate different underlying mechanisms of stress sensing and tolerance specific to the stress condition. Presently, methods to effectively distinguish between morphological transformations, including between tuns induced by different stress conditions, are lacking. Herein, a new approach for discriminating between tardigrade morphological states is developed and utilized to compare sucrose- and CaCl2-induced tuns. A novel method of shadow imaging with confocal laser scanning microscopy enabled production of three-dimensional renderings of tardigrades in various physiological states resulting in tardigrade volume measurements. Combining these measurements with qualitative morphological analysis using scanning electron microscopy revealed that sucrose- and CaCl2-induced tuns have distinct morphologies, including differences in the amount of water expelled during tun formation. Further, varying the concentration of the applied stressor did not affect the amount of water lost, pointing towards water expulsion being a controlled process that is adapted to the specific stressors. Summary StatementA novel method for volume measurements of biological specimens was developed and paired with scanning electron microscopy to allow for morphological comparisons between tardigrade osmobiotes.

physiology↗

bioRxiv2023

Chemobiosis reveals tardigrade tun formation is dependent on reversible cysteine oxidation

Tardigrades, commonly known as waterbears, are eight-legged microscopic invertebrates renowned for their ability to withstand extreme stressors, including high osmotic pressure, freezing temperatures, and complete desiccation. Limb retraction and expelling their internal water stores results in the tun state, greatly increasing their ability to survive. Emergence from the tun state and/or activity regain follows stress removal, where resumption of life cycle occurs as if stasis never occurred. However, the mechanism(s) through which tardigrades initiate tun formation is yet to be uncovered. Herein, we use chemobiosis to demonstrate that tardigrade tun formation is mediated by reactive oxygen species (ROS). We further reveal that tuns are dependent on reversible cysteine oxidation, and that this reversible cysteine oxidation is facilitated by the release of intracellular reactive oxygen species (ROS). We provide the first empirical evidence of chemobiosis and map the initiation and survival of tardigrades via osmobiosis, chemobiosis, and cryobiosis. In vivo electron paramagnetic spectrometry suggests an intracellular release of reactive oxygen species following stress induction; when this release is quenched through the application of exogenous antioxidants, the tardigrades can no longer survive osmotic stress. Together, this work suggests a conserved dependence of reversible cysteine oxidation across distinct tardigrade cryptobioses.

biochemistry↗