Abstract 1608 Tardigrade Fluorescence Shadow Imaging Reveals Morphological Differences Between Osmobiotes

Tardigrades are capable of surviving a vast array of environmental stressors, including extreme temperatures, extreme pressures, desiccation, high concentrations of osmolytes, and exposure to chemical toxicants. The principal mechanism of tardigrades' tolerance to these stressors is their entrance into cryptobioses, states of near metabolic cessation that usually coincide with the upregulation of bioprotectants as well as distinct morphological transformations. One such transformation is the formation of a 'tun' which is achieved by tardigrades expelling large proportions of their internal water content and curling in on themselves to form a compact structure. Tardigrades are known to form tuns in response to desiccation, high concentrations of osmolytes (both ionic and non-ionic), and chemical toxicants. While all of these stress conditions are "tun-forming", it is unclear how similar the changes undergone are because methods to effectively distinguish between them are presently lacking. Thus, the central aim of this work was to remedy this situation. A new approach for discriminating between tardigrade morphological states was developed and utilized to compare sucrose- and CaCl2-induced tuns, both of which are putative osmobiotes. A novel method of shadow imaging with confocal laser scanning microscopy enabled production of three-dimensional renderings of tardigrades in various physiological states, allowing for 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. Varying the concentration of the applied stressor did not affect the amount of water expelled, indicating that water expulsion during tun formation is not primarily driven by osmosis. Overall, the data presented here point towards tardigrades having unique, active responses to two putative osmotic stressors. This research was supported by the National Science Foundation, the North Carolina Space Grant, and the NASA West Virginia Space Grant Consortium.