Tiny animals known as tardigrades use a combination of DNA repair machinery and a novel protein to mend their genome after intense ionizing radiation.

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 mechanisms of stress sensing and tolerance specific to the stress condition. Methods to effectively distinguish between morphological transformations, including between tuns induced by different stress conditions, are lacking. Herein, an approach for discriminating between tardigrade morphological states is developed and utilized to compare sucrose- and CaCl2-induced tuns, using the model species Hypsibius exemplaris. A novel approach of shadow imaging with confocal laser scanning microscopy enabled production of three-dimensional renderings of Hys. exemplaris in various physiological states resulting in 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 by Hys. exemplaris being a controlled process that is adapted to the specific stressors.

Tardigrades can survive remarkable doses of ionizing radiation, up to about 1,000 times the lethal dose for humans. How they do so is incompletely understood. We found that the tardigrade Hypsibius exemplaris suffers DNA damage upon gamma irradiation, but the damage is repaired. We show that this species has a specific and robust response to ionizing radiation: irradiation induces a rapid upregulation of many DNA repair genes. This upregulation is unexpectedly extreme-making some DNA repair transcripts among the most abundant transcripts in the animal. By expressing tardigrade genes in bacteria, we validate that increased expression of some repair genes can suffice to increase radiation tolerance. We show that at least one such gene is important in vivo for tardigrade radiation tolerance. We hypothesize that the tardigrades\' ability to sense ionizing radiation and massively upregulate specific DNA repair pathway genes may represent an evolved solution for maintaining DNA integrity.

Increasing temperature influences the habitats of various organisms, including microscopic invertebrates. To gain insight into temperature-dependent changes in tardigrades, we isolated storage cells exposed to various temperatures and conducted biochemical and ultrastructural analysis in active and tun-state Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa, & Roszkowska, 2020. The abundance of heat shock proteins (HSPs) and ultrastructure of the storage cells were examined at different temperatures (20 °C, 30 °C, 35 °C, 37 °C, 40 °C, and 42 °C) in storage cells isolated from active specimens of Pam. experimentalis. In the active animals, upon increase in external temperature, we observed an increase in the levels of HSPs (HSP27, HSP60, and HSP70). Furthermore, the number of ultrastructural changes in storage cells increased with increasing temperature. Cellular organelles, such as mitochondria and the rough endoplasmic reticulum, gradually degenerated. At 42 °C, cell death occurred by necrosis. Apart from the higher electron density of the karyoplasm and the accumulation of electron-dense material in some mitochondria (at 42 °C), almost no changes were observed in the ultrastructure of tun storage cells exposed to different temperatures. We concluded that desiccated (tun-state) are resistant to high temperatures, but not active tardigrades (survival rates of tuns after 24 h of rehydration: 93.3% at 20 °C, 60.0% at 35 °C, 33.3% at 37 °C, 33.3% at 40 °C, and 20.0% at 42 °C).

A small number of conserved signaling pathways regulate development of most animals, yet we do not know where these pathways are deployed in most embryos. This includes tardigrades, a phylum with a unique body shape. We examined expression patterns of components of the BMP and FGF signaling pathways during embryonic segmentation and mesoderm development of the tardigrade Hypsibius exemplaris. Among the patterns examined, we found that an FGF ligand gene is expressed in ectodermal segment posteriors and an FGF receptor gene is expressed in underlying endomesodermal pouches, suggesting possible FGF signaling between these developing germ layers. We found that a BMP ligand gene is expressed in lateral ectoderm and dorsolateral bands along segment posteriors, while the BMP antagonist Sog gene is expressed in lateral ectoderm and also in a subset of endomesodermal cells, suggesting a possible role of BMP signaling in dorsal-ventral patterning of lateral ectoderm. In combination with known roles of these pathways during development of common model systems, we developed hypotheses for how the BMP and FGF pathways might regulate embryo segmentation and mesoderm formation of the tardigrade H. exemplaris. These results identify the expression patterns of genes from two conserved signaling pathways for the first time in the tardigrade phylum.

Encystment is a natural process that involves cyst formation, and at least some species of tardigrades can form cysts. However, the encystment process and cyst structure among tardigrades are still poorly understood. Despite some aspects of the encysted animals\' systems organisation being examined in the past, the morphology and structure of the nervous system have never been thoroughly investigated. This study covers anatomical, histological and morphological details and proposes physiological aspects of the nervous system in encysted Thulinius ruffoi up to 11 months duration in encystment. This is the first record of the nervous system organisation in a species belonging to the family Doryphoribiidae. The cyst formation results in morphological changes in the nervous system. It comprises central and peripheral elements, which may be observable even after many months since the cyst formation. Based on the nervous system\'s organisation in cysts, there is no sign that histolysis is a part of encystment.

Tardigrades are renowned for their ability to enter the extremotolerant state of latent life known as cryptobiosis. While it is widely accepted that cryptobiosis can be induced by freezing (cryobiosis) and by desiccation (anhydrobiosis), the latter involving formation of a so-called tun, the exact mechanisms underlying the state-as well as the significance of other cryptobiosis inducing factors-remain ambiguous. Here, we focus on osmotic and chemical stress tolerance in the marine tidal tardigrade Echiniscoides sigismundi. We show that E. sigismundi enters the tun state following exposure to saturated seawater and upon exposure to locality seawater containing the mitochondrial uncoupler DNP. The latter experiments provide evidence of osmobiosis and chemobiosis, i.e., cryptobiosis induced by high levels of osmolytes and toxicants, respectively. A small decrease in survival was observed following simultaneous exposure to DNP and saturated seawater indicating that the tardigrades may not be entirely ametabolic while in the osmobiotic tun. The tardigrades easily handle exposure to ultrapure water, but hypo-osmotic shock impairs tun formation and when exposed to ultrapure water the tardigrades do not tolerate DNP, indicating that tolerance towards dilute solutions involves energy-consuming processes. We discuss our data in relation to earlier and more contemporary studies on cryptobiosis and we argue that osmobiosis should be defined as a state of cryptobiosis induced by high external osmotic pressure. Our investigation supports the hypothesis that the mechanisms underlying osmobiosis and anhydrobiosis are overlapping and that osmobiosis likely represents the evolutionary forerunner of cryptobiosis forms that involve body water deprivation.

Even for tardigrades, often called the toughest animals on Earth, a hypomagnetic field (HMF) is an extreme environment. However, studies on the effect of HMF on tardigrades and other invertebrates are scarce. Mitochondria play an important role in an organism\'s response to extreme conditions. The effect of HMF on the mitochondrial inner membrane potential (Δψ), a well-known marker of mitochondria functionality, shows that mitochondria are very sensitive to HMF. To measure the HMF effect on Paramacrobiotus experimentalis, we calculated the tardigrade survival rate and Δψ level after HMF treatments of different durations. We also estimated the relationship between the age and sex of the tardigrade and the HMF effect. We observed age- and sex-related differences in Δψ and found that Δψ changes after HMF treatment were dependent on its duration as well as the animal\'s age and sex. Furthermore, active P. experimentalis individuals displayed a high survival rate after HMF treatment. The data may contribute to the understanding of tardigrade aging and their resistance to extreme conditions including HMF, which in turn may be useful for future space explorations.

Introduction: Tardigrades are small aquatic invertebrates with well documented tolerance to several environmental stresses, including desiccation, low temperature, and radiation, and an ability to survive long periods in a cryptobiotic state under arrested metabolism. Many tardigrade populations live in habitats where temporary exposure to hypoxia is expected, e.g., benthic layers or substrates that regularly undergo desiccation, but tolerance to hypoxia has so far not been thoroughly investigated in tardigrades. Method: We studied the response to exposure for hypoxia (<1 ppm) during 1-24 h in two tardigrade species, Richtersius cf. coronifer and Hypsibius exemplaris. The animals were exposed to hypoxia in their hydrated active state. Results: Survival was high in both species after the shortest exposures to hypoxia but tended to decline with longer exposures, with almost complete failure to recover after 24 h in hypoxia. R. cf. coronifer tended to be more tolerant than H. exemplaris. When oxygen level was gradually reduced from 8 to 1 ppm, behavioral responses in terms of irregular body movements were first observed at 3-4 ppm. Discussion: The study shows that both limno-terrestrial and freshwater tardigrades are able to recover after exposure to severe hypoxia, but only exposure for relatively short periods of time. It also indicates that tardigrade species have different sensitivity and response patterns to exposure to hypoxia. These results will hopefully encourage more studies on how tardigrades are affected by and respond to hypoxic conditions.

The cosolvent 2,2,2-trifluoroethanol (TFE) is often used to mimic protein desiccation. We assessed the effects of TFE on cytosolic abundant heat soluble protein D (CAHS D) from tardigrades. CAHS D is a member of a unique protein class that is necessary and sufficient for tardigrades to survive desiccation. We find that the response of CAHS D to TFE depends on the concentration of both species. Dilute CAHS D remains soluble and, like most proteins exposed to TFE, gains α-helix. More concentrated solutions of CAHS D in TFE accumulate β-sheet, driving both gel formation and aggregation. At even higher TFE and CAHS D concentrations, samples phase separate without aggregation or increases in helix. Our observations show the importance of considering protein concentration when using TFE.