Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through a comprehensive search using a desolvating agent, trifluoroethanol (TFE), we identified 336 proteins, collectively dubbed “TFE-Dependent ReversiblY condensing Proteins (T-DRYPs).” Unexpectedly, we rediscovered CAHS proteins as highly enriched in T-DRYPs, 3 of which were major components of T-DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. Furthermore, CAHS proteins increased cell stiffness in a hyperosmotic stress-dependent manner and counteract the cell shrinkage caused by osmotic pressure, and even improved the survival against hyperosmotic stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeleton-like proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and could contribute to the exceptional physical stability in a dehydrated state.

Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective cytoplasmic-abundant heat-soluble (CAHS) proteins which are essential for the anhydrobiotic survival of tardigrades. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through comprehensive analysis, we identified 336 such proteins, collectively dubbed "dehydration-induced reversibly condensing proteins (DRPs)". Unexpectedly, we rediscovered CAHS proteins as highly enriched in DRPs, 3 of which were major components of DRPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro, which increases the mechanical strength of cell-like microdroplets. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeletal proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and contribute to the exceptional stability of dehydrated tardigrades.

Considering recent developments in gene manipulation methods for honey bees, establishing simple, robust, and indoor assay systems which can analyze behavioral components in detail is important for the rise of honey bee behavioral genetics. We focused on the antennal movements of the honey bee, which are used for not only multimodal sensory perception but also interactions between individuals. We developed an experimental system for analyzing the antennal responses (ARs) of the honey bee using DeepLabCut, a markerless posture-tracking tool using deep learning. The tracking of antennal movements during the presentation of vertical (downward and upward) motion stimuli using DeepLabCut successfully detected the ARs reported in the previous studies, where bees tilted their antennae in the direction opposite to the motion stimuli. In addition, we successfully detected ARs in response to horizontal (forward and backward) motion stimuli. An investigation of the developmental maturation of honey bee ARs showed that ARs to motion stimuli were not detected in bees immediately after emergence but became detectable through post-emergence development in an experience-independent manner. Furthermore, unsupervised clustering analysis using multidimensional data created by processing tracking data using DeepLabCut classified antennal movements into different clusters, suggesting that data-driven behavioral classification can apply to AR paradigms. These results reveal novel AR to visual stimuli and developmental maturation of ARs and suggest the efficacy of data-driven analysis for behavioral classification in behavioral studies of the honey bee.