(D, E) Effects of translation inhibitor (250 M cycloheximide) on recovery rates at 1 h (D) and 24 h (E) after rehydration. (B, D, F, H, J) after rehydration. N = 4 unless otherwise stated; 20 tardigrades each. Statistically significant differences among samples were determined by the Tukey-Kramer test (*, gene expression is required for successful transition to anhydrobiosis in this tardigrade. We then screened 81 chemicals and identified 5 chemicals that significantly impaired anhydrobiotic survival after severe desiccation, in contrast to little or no effect on survival after high humidity exposure only. In particular, cantharidic acid, a selective inhibitor of protein phosphatase (PP) 1 and PP2A, exhibited the most profound inhibitory effects. Another PP1/PP2A inhibitor, okadaic acid, also significantly and specifically impaired anhydrobiotic survival, suggesting that PP1/PP2A activity plays an important role for anhydrobiosis in this species. This is, to our knowledge, the first report of the required activities of signaling molecules for desiccation tolerance in tardigrades. The identified inhibitory chemicals could provide novel clues to elucidate the regulatory mechanisms underlying anhydrobiosis in tardigrades. Introduction For terrestrial organisms, desiccation is one of the most commonly encountered environmental stresses. To avoid deleterious water loss, most animals escape from a desiccated OSI-420 environment using their mobility, and retain their body water by the proper intake of water and by preventing surface water evaporation [1,2]. In contrast, some small animals, whose mobility is limited and whose large surface/volume ratio enhances evaporation, have adapted to tolerate a loss of body water in order to withstand a desiccated environment [3]. When encountering desiccation, these animals drop water and enter a metabolically inactive dehydrated state referred to as anhydrobiosis, and resume their metabolic activity upon rehydration. Tardigrades are tiny animals comprising the phylum Tardigrada, in which more than 1000 species have been reported [4]. All tardigrades are principally aquatic and require surrounding water to grow and reproduce, though some species have anhydrobiotic abilities. When desiccated, anhydrobiotic tardigrades contract their bodies longitudinally with the loss of body water, to form a compact shape called a tun, and are able to tolerate almost complete dehydration [5]. For successful transition to anhydrobiosis, many anhydrobiotic animals require pre-exposure to high humidity conditions, called preconditioning, prior to severe dehydration [6C9]. During preconditioning, animals are thought to sense environmental desiccation and prepare for upcoming severe dehydration. Some anhydrobiotic animals, such as the sleeping chironomid, can tolerate desiccation at 23% RH or above after preconditioning at 98% RH for 4 days [21], and their desiccation OSI-420 tolerance largely depends on two genes, osm11 and osm9, OSI-420 which are expressed in head neurons and required for osmotic avoidance, suggesting that certain head neurons participate in their desiccation tolerance [22]. Therefore, the regulatory mechanisms of desiccation tolerance likely vary among animal species. Tardigrades accumulate only small amounts of trehalose upon desiccation [13], and an anhydrobiotic tardigrade, is an anhydrobiotic tardigrade which requires longer preconditioning in a high humidity condition to acquire tolerance against severe desiccation [6]. This implies the presence of regulatory mechanisms to induce anhydrobiosis in this species in response to preconditioning. is easy to maintain in the laboratory, and the strain is established [23] and used for expressed sequence tag and genomic projects, providing plenty of genetic information (http://www.ncbi.nlm.nih.gov/nucest/?term=hypsibius+dujardini). Therefore, this species is suitable for molecular dissection of the regulatory mechanisms of anhydrobiosis in tardigrades. Here, we used a chemical genetic approach and suggested that gene expression is required for entering anhydrobiosis in was purchased from Sciento (UK) and maintained HAS3 at 18C. Tardigrades were reared on 1.2% agar plates overlaid with volvic water containing sp. (Sciento, UK) as food. Water and food were replaced once or twice a week. Chemicals -Amanitin, cycloheximide, J-8, and cantharidic acid were purchased from Enzo Life Sciences (USA). Triptolide was purchased from MedChem Express (USA). 3,4-Methylenedioxy–nitrostyrene (MNS), 2-aminoethyl diphenylborinate (2-APB), and okadaic acid were OSI-420 purchased from Santa Cruz Biotechnologies (USA). The 81 chemicals used for the screening were provided by the Drug Discovery Initiative, The University of Tokyo (Japan) and are listed.