Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR.

Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, methods for genetically manipulating tardigrades have long been desired. Despite our prior success in somatic cell gene editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions that led to the generation of gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRISPR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. This establishment of a straightforward method for generating homozygous knockout/knock-in individuals not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades.

Cambrian lobopodians shed light on the origin of the tardigrade body plan.

Phylum Tardigrada (water bears), well known for their cryptobiosis, includes small invertebrates with four paired limbs and is divided into two classes: Eutardigrada and Heterotardigrada. The evolutionary origin of Tardigrada is known to lie within the lobopodians, which are extinct soft-bodied worms with lobopodous limbs mostly discovered at sites of exceptionally well-preserved fossils. Contrary to their closest relatives, onychophorans and euarthropods, the origin of morphological characters of tardigrades remains unclear, and detailed comparison with the lobopodians has not been well explored. Here, we present detailed morphological comparison between tardigrades and Cambrian lobopodians, with a phylogenetic analysis encompassing most of the lobopodians and three panarthropod phyla. The results indicate that the ancestral tardigrades likely had a Cambrian lobopodian-like morphology and shared most recent ancestry with the luolishaniids. Internal relationships within Tardigrada indicate that the ancestral tardigrade had a vermiform body shape without segmental plates, but possessed cuticular structures surrounding the mouth opening, and lobopodous legs terminating with claws, but without digits. This finding is in contrast to the long-standing stygarctid-like ancestor hypothesis. The highly compact and miniaturized body plan of tardigrades evolved after the tardigrade lineage diverged from an ancient shared ancestor with the luolishaniids.

In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada.

Water is essential for life, but anhydrobiotic tardigrades can survive almost complete dehydration. Anhydrobiosis has been a biological enigma for more than a century with respect to how organisms sustain life without water, but the few choices of genetic toolkits available in tardigrade research have been a challenging circumstance. Here, we report the development of an in vivo expression system for tardigrades. This transient transgenic technique is based on a plasmid vector (TardiVec) with promoters that originated from an anhydrobiotic tardigrade Ramazzottius varieornatus. It enables the introduction of GFP-fused proteins and genetically encoded indicators such as the Ca2+ indicator GCaMP into tardigrade cells; consequently, the dynamics of proteins and cells in tardigrades may be observed by fluorescence live imaging. This system is applicable for several tardigrades in the class Eutardigrada: the promoters of anhydrobiosis-related genes showed tissue-specific expression in this work. Surprisingly, promoters functioned similarly between multiple species, even for species with different modes of expression of anhydrobiosis-related genes, such as Hypsibius exemplaris, in which these genes are highly induced upon facing desiccation, and Thulinius ruffoi, which lacks anhydrobiotic capability. These results suggest that the highly dynamic expression changes in desiccation-induced species are regulated in trans. Tissue-specific expression of tardigrade-unique unstructured proteins also suggests differing anhydrobiosis machinery depending on the cell types. We believe that tardigrade transgenic technology opens up various experimental possibilities in tardigrade research, especially to explore anhydrobiosis mechanisms.

Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel.

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 Use Intrinsically Disordered Proteins to Survive Desiccation.

Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance.

Conserved and specific gene expression patterns in the embryonic development of tardigrades.

Tardigrades, commonly known as water bears, are enigmatic organisms characterized by their remarkable resilience to extreme environments despite their simple and compact body structure. To date, there is still much to understand about their evolutionary and developmental features contributing to their special body plan and abilities. This research provides preliminary insights on the conserved and specific gene expression patterns during embryonic development of water bears, focusing on the species Hypsibius exemplaris. The developmental dynamic expression analysis of the genes with various evolutionary age grades indicated that the mid-conserved stage of H. exemplaris corresponds to the period of ganglia and midgut development, with the late embryonic stage showing a transition from non-conserved to conserved state. Additionally, a comparison with Drosophila melanogaster highlighted the absence of certain pathway nodes in development-related pathways, such as Maml and Hairless, which are respectively the transcriptional co-activator and co-repressor of NOTCH regulated genes. We also employed Weighted Gene Co-expression Network Analysis (WGCNA) to investigate the expression patterns of tardigrade-specific genes during embryo development. Our findings indicated that the module containing the highest proportion of tardigrade-specific genes (TSGs) exhibits high expression levels before the mid-conserved stage, potentially playing a role in glutathione and lipid metabolism. These functions may be associated to the ecdysone synthesis and storage cell formation, which is unique to tardigrades.

Ecology explains anhydrobiotic performance across tardigrades, but the shared evolutionary history matters more.

Desiccation stress is lethal to most animals. However, some microinvertebrate groups have evolved coping strategies, such as the ability to undergo anhydrobiosis (i.e. survival despite the loss of almost all body water). Tardigrades are one such group, where the molecular mechanisms of anhydrobiosis have been more thoroughly studied. Despite the ecological, evolutionary and biotechnological importance of anhydrobiosis, little is known about its inter- and intra-specific variability nor its relationship with natural habitat conditions or phylogenetic history. We developed a new index-anhydrobiotic recovery index (ARI)-to evaluate the anhydrobiotic performance of tardigrade populations from the family Macrobiotidae. Moreover, we compared the explanatory role of habitat humidity and phylogenetic history on this trait using a variance partitioning approach. We found that ARI is correlated with both microhabitat humidity and yearly rainfall, but it is mostly driven by phylogenetic niche conservatism (i.e. a high portion of ARI variation is explained by phylogeny alone). Finally, we showed that anhydrobiotic performance is highly variable, even between closely related species, and that their response to local ecological conditions is tightly linked to their phylogenetic history. This study not only presents key insights into an emerging model system, but also provides a new methodological approach for wider scale studies of the ecological and evolutionary implications of anhydrobiosis.

Morphological and molecular identification of freshwater eutardigrade Dactylobiotus parthenogeneticus (Bertolani, 1982) in the Greater Zab River of Kurdistan Region - Iraq.

Dactylobiotus parthenogeneticus is one of the widespread species of tardigrade all over the world. Tardigrades of this species were collected from the Greater Zab River in Erbil City-Iraq by filtering water of the river through a plankton net with a mesh of 45 µm pore. The samples were mounted on a slide with a cover slip and examined under the microscope to determine morphological characteristics and measurements. Based on these characters the species identified to be D. parthenogeneticus. To support this diagnosis, DNA barcoding techniques were applied to do molecular analysis and sequencing on the cytochrome oxidase subunit I (COI) gene. The sequence was subjected to the GenBank database of NCBI and recorded with the accession number PP140905. The result of the sequencing and molecular analysis of the cytochrome oxidase subunit I (COI) gene confirmed to be the same species diagnosed by relying upon morphological characters. This study represents one of the pioneer researches and documents on tardigrades and found D. parthenogeneticus for the first time in the Greater Zab River in Kurdistan, North of Iraq. Tardigrades play a magnificent role in different trophic levels and can be utilized as an indicator of ecosystem health.

Transcriptome analysis of the tardigrade Hypsibius exemplaris exposed to the DNA-damaging agent bleomycin.

Tardigrades are microscopic animals that are renowned for their capabilities of tolerating near-complete desiccation by entering an ametabolic state called anhydrobiosis. However, many species also show high tolerance against radiation in the active state as well, suggesting cross-tolerance via the anhydrobiosis mechanism. Previous studies utilized indirect DNA damaging agents to identify core components of the cross-tolerance machinery in species with high anhydrobiosis capacities. However, it was difficult to distinguish whether transcriptomic changes were specific to DNA damage or mutual with anhydrobiosis. To this end, we performed transcriptome analysis on bleomycin-exposed Hypsibius exemplaris. We observed induction of several tardigrade-specific gene families, including a previously identified novel anti-oxidative stress family, which may be a core component of the cross-tolerance mechanism. We also identified enrichment of the tryptophan metabolism pathway, for which metabolomic analysis suggested engagement of this pathway in stress tolerance. These results provide several candidates for the core component of cross-tolerance, as well as possible anhydrobiosis machinery.

Antioxidant Defense in the Toughest Animals on the Earth: Its Contribution to the Extreme Resistance of Tardigrades.

Tardigrades are unique among animals in their resistance to dehydration, mainly due to anhydrobiosis and tun formation. They are also very resistant to high-energy radiation, low and high temperatures, low and high pressure, and various chemical agents, Interestingly, they are resistant to ionizing radiation both in the hydrated and dehydrated states to a similar extent. They are able to survive in the cosmic space. Apparently, many mechanisms contribute to the resistance of tardigrades to harmful factors, including the presence of trehalose (though not common to all tardigrades), heat shock proteins, late embryogenesis-abundant proteins, tardigrade-unique proteins, DNA repair proteins, proteins directly protecting DNA (Dsup and TDR1), and efficient antioxidant system. Antioxidant enzymes and small-molecular-weight antioxidants are an important element in the tardigrade resistance. The levels and activities of many antioxidant proteins is elevated by anhydrobiosis and UV radiation; one explanation for their induction during dehydration is provided by the theory of "preparation for oxidative stress", which occurs during rehydration. Genes coding for some antioxidant proteins are expanded in tardigrades; some genes (especially those coding for catalases) were hypothesized to be of bacterial origin, acquired by horizontal gene transfer. An interesting antioxidant protein found in tardigrades is the new Mn-dependent peroxidase.

Two new tardigrade genera from New Zealand's Southern Alp glaciers display morphological stasis and parallel evolution.

Tardigrada is an invertebrate phylum that often constitutes a dominant micrometazoan group on glaciers worldwide. We investigated tardigrades residing in surface ice above the equilibrium line altitude (ELA) on three temperate glaciers of New Zealand's Southern Alps. Morphological, morphometric and multilocus DNA analyses (CO1, 18S rRNA, 28S rRNA, ITS-2) revealed two new genera comprising four species, of which two are formally described here: Kopakaius gen. nov. nicolae sp. nov. and Kararehius gen. nov. gregorii sp. nov. The former is represented by three genetically distinct phyletic lineages akin to species. According to CO1, Kopakaius gen. nov. nicolae sp. nov. inhabits Whataroa Glacier only while the remaining two Kopakaius species occur on Fox and Franz Joseph Glaciers, suggesting low dispersal capabilities. Although morphological characteristics of the new genera could indicate affinity with the subfamily Itaquasconinae, phylogenetic analysis placed them confidently in the subfamily Diphasconinae. Kopakaius gen. nov. lack placoids in the pharynx similar with some Itaquasconinae, whereas dark pigmentation and claw shape aligns them with the glacier-obligate genus, Cryobiotus (subfamily Hypsibiinae), which is an example of parallel evolution. The second genus, Kararehius gen nov. could be classified as Adropion-like (subfamily Itaquasconinae), but differs greatly by genetics (placed in the subfamily Diphasconinae) as well as morphology (e.g., lack of septulum), exemplify deep stasis in Hypsibiidae. Our results suggest that glacier fragmentation during the Pleistocene triggered tardigrade speciation, making it a suitable model for studies on allopatric divergence in glacier meiofauna.

The unbearable uncertainty of panarthropod relationships.

Panarthropoda, the clade comprising the phyla Onychophora, Tardigrada and Euarthropoda, encompasses the largest majority of animal biodiversity. The relationships among the phyla are contested and resolution is key to understanding the evolutionary assembly of panarthropod bodyplans. Molecular phylogenetic analyses generally support monophyly of Onychophora and Euarthropoda to the exclusion of Tardigrada (Lobopodia hypothesis), which is also supported by some analyses of morphological data. However, analyses of morphological data have also been interpreted to support monophyly of Tardigrada and Euarthropoda to the exclusion of Onychophora (Tactopoda hypothesis). Support has also been found for a clade of Onychophora and Tardigrada that excludes Euarthropoda (Protarthropoda hypothesis). Here we show, using a diversity of phylogenetic inference methods, that morphological datasets cannot discriminate statistically between the Lobopodia, Tactopoda and Protarthropoda hypotheses. Since the relationships among the living clades of panarthropod phyla cannot be discriminated based on morphological data, we call into question the accuracy of morphology-based phylogenies of Panarthropoda that include fossil species and the evolutionary hypotheses based upon them.

Proteomics Reveals How the Tardigrade Damage Suppressor Protein Teaches Transfected Human Cells to Survive UV-C Stress.

The genome sequencing of the tardigrade Ramazzottius varieornatus revealed a unique nucleosome-binding protein named damage suppressor (Dsup), which was discovered to be crucial for the extraordinary abilities of tardigrades in surviving extreme stresses, such as UV. Evidence in Dsup-transfected human cells suggests that Dsup mediates an overall response in DNA damage signaling, DNA repair, and cell cycle regulation, resulting in an acquired resistance to stress. Given these promising outcomes, our study attempts to provide a wider comprehension of the molecular mechanisms modulated by Dsup in human cells and to explore the Dsup-activated molecular pathways under stress. We performed a differential proteomic analysis of Dsup-transfected and control human cells under basal conditions and at 24 h recovery after exposure to UV-C. We demonstrate via enrichment and network analyses, for the first time, that even in the absence of external stimuli, and more significantly, after stress, Dsup activates mechanisms involved with the unfolded protein response, the mRNA processing and stability, cytoplasmic stress granules, the DNA damage response, and the telomere maintenance. In conclusion, our results shed new light on Dsup-mediated protective mechanisms and increases our knowledge of the molecular machineries of extraordinary protection against UV-C stress.

Time-series transcriptomic screening of factors contributing to the cross-tolerance to UV radiation and anhydrobiosis in tardigrades.

BACKGROUND: Tardigrades are microscopic animals that are capable of tolerating extreme environments by entering a desiccated state of suspended animation known as anhydrobiosis. While antioxidative stress proteins, antiapoptotic pathways and tardigrade-specific intrinsically disordered proteins have been implicated in the anhydrobiotic machinery, conservation of these mechanisms is not universal within the phylum Tardigrada, suggesting the existence of overlooked components. RESULTS: Here, we show that a novel Mn-dependent peroxidase is an important factor in tardigrade anhydrobiosis. Through time-series transcriptome analysis of Ramazzottius varieornatus specimens exposed to ultraviolet light and comparison with anhydrobiosis entry, we first identified several novel gene families without similarity to existing sequences that are induced rapidly after stress exposure. Among these, a single gene family with multiple orthologs that is highly conserved within the phylum Tardigrada and enhances oxidative stress tolerance when expressed in human cells was identified. Crystallographic study of this protein suggested Zn or Mn binding at the active site, and we further confirmed that this protein has Mn-dependent peroxidase activity in vitro. CONCLUSIONS: Our results demonstrated novel mechanisms for coping with oxidative stress that may be a fundamental mechanism of anhydrobiosis in tardigrades. Furthermore, localization of these sets of proteins mainly in the Golgi apparatus suggests an indispensable role of the Golgi stress response in desiccation tolerance.

Evolution of DNA Methylation Across Ecdysozoa.

DNA methylation is a crucial, abundant mechanism of gene regulation in vertebrates. It is less prevalent in many other metazoan organisms and completely absent in some key model species, such as Drosophila melanogaster and Caenorhabditis elegans. We report here a comprehensive study of the presence and absence of DNA methyltransferases (DNMTs) in 138 Ecdysozoa, covering Arthropoda, Nematoda, Priapulida, Onychophora, and Tardigrada. Three of these phyla have not been investigated for the presence of DNA methylation before. We observe that the loss of individual DNMTs independently occurred multiple times across ecdysozoan phyla. We computationally predict the presence of DNA methylation based on CpG rates in coding sequences using an implementation of Gaussian Mixture Modeling, MethMod. Integrating both analysis we predict two previously unknown losses of DNA methylation in Ecdysozoa, one within Chelicerata (Mesostigmata) and one in Tardigrada. In the early-branching Ecdysozoa Priapulus caudatus, we predict the presence of a full set of DNMTs and the presence of DNA methylation. We are therefore showing a very diverse and independent evolution of DNA methylation in different ecdysozoan phyla spanning a phylogenetic range of more than 700 million years.

Application of CRISPR/Cas9 system and the preferred no-indel end-joining repair in tardigrades.

Tardigrades are small aquatic animals known for the tolerant ability against various extreme stresses. Recent studies identified several tardigrade-unique proteins as protective factors of biomolecules from extreme stresses. Due to the limitation of the technique available in tardigrades, the function of these protective molecules has largely been studied utilizing the systems of in vitro and the heterologous expression in other organisms. Although RNAi is feasible in tardigrades, their effects are variable and not always sufficient. To analyze the functions of the tardigrade protective proteins, in vivo genetic manipulations have been desired. In this study, we used a tardigrade Hypsibius exemplaris as a model whose genome is available, and developed the delivery method of Cas9 ribonucleoproteins (RNPs) to adult tardigrade cells. Cas9 RNPs containing two kinds of crRNAs were injected to the body cavity of adult tardigrades and subjected to the subsequent electroporation to facilitate the incorporation of RNPs to the cells. Using this delivery method, we detected the deletion of the intervening region between two crRNAs from the genome. Intriguingly, all examined joining sites exhibited no incorporation of insertions/deletions (indels), suggesting that no-indel end-joining is dominant repair system in this tardigrade. We also detected similar removal of the intervening region even in the tardigrades injected with Cas9 RNPs without electroporation and in this case the no-indel end-joining is detected in still dominant but not all examined joining sites. This study provides the development of the delivery method of Cas9 RNPs to tardigrade cells and our data also suggested that simultaneous application of more than two crRNAs/gRNAs are recommended to disrupt the target gene by CRISPR/Cas9 system to avoid scarless repair in the tardigrade.

Molecular phylogenetics, speciation, and long distance dispersal in tardigrade evolution: A case study of the genus Milnesium.

Microorganisms (sensu lato, i.e., including micrometazoans) are thought to have cosmopolitan geographic distributions due to their theoretically unlimited dispersal capabilities, a consequence of their tiny size, population dynamics, and resistant forms. However, several molecular studies of microorganisms have identified biogeographic patterns indicating cryptic speciation and/or weak species definitions. Using a multi-locus approach with the genus Milnesium (Tardigrada), we aimed to determine the genetic structure of populations worldwide and the effects of long distance dispersal (LDD) on genetic connectivity and relationships across the six continents. Our results on this micrometazoan's genetic structure and LDD at global and micro-local scales indicate contrasting patterns not easily explained by a unique or simple phenomenon. Overall, we report three key findings: (i) confirmation of long distance dispersal for tardigrades, (ii) populations with globally-shared or endemic micro-local haplotypes, and (iii) a supported genetic structure instead of the homogeneous genetic distribution hypothesized for microorganisms with LDD capabilities. Moreover, incongruences between our morphological and molecular results suggest that species delimitation within the genus Milnesium could be problematic due to homoplasy. Duality found for Milnesium populations at the global scale, namely, a molecular phylogenetic structure mixed with widely distributed haplotypes (but without any apparent biogeographic structure), is similar to patterns observed for some unicellular, prokaryotic and eukaryotic, microorganisms. Factors influencing these patterns are discussed within an evolutionary framework.

An ancient, Antarctic-specific species complex: large divergences between multiple Antarctic lineages of the tardigrade genus Mesobiotus.

Antarctica has been isolated and progressively glaciated for over 30 million years, with only approximately 0.3 % of its area currently ice-free and capable of supporting terrestrial ecosystems. As a result, invertebrate populations have become isolated and fragmented, in some cases leading to speciation. Terrestrial invertebrate species currently found in Antarctica often show multi-million year, and even Gondwanan, heritage, with little evidence of recent colonisation. Mesobiotus is a globally distributed tardigrade genus. It has commonly been divided into two "groups", referred to as harmsworthi and furciger, with both groups currently considered cosmopolitan, with global reports including from both the Arctic and the Antarctic. However, some authors considered that Meb. furciger, as originally described, may represent an Antarctic-specific lineage. Using collections of tardigrades from across the Antarctic continent and publicly available sequences obtained from online databases, we use mitochondrial and nuclear ribosomal sequence data to clarify the relationships of Antarctic Mesobiotus species. Our analyses show that all Antarctic members belong to a single lineage, evolving separately from non-Antarctic representatives. Within this Antarctic lineage there are further deep divisions among geographic regions of the continent, consistent with the presence of a species complex. Based on our data confirming the deep divisions between this Antarctic lineage, which includes representatives of both groups, we recommend that the use of furciger and harmsworthi group terminology is now abandoned, as it leads to systematic and biogeographical confusion.

Extreme freeze-tolerance in cryophilic tardigrades relies on controlled ice formation but does not involve significant change in transcription.

Subzero temperatures are among the most significant factors defining the distribution of organisms, yet, certain taxa have evolved to overcome this barrier. The microscopic tardigrades are among the most freeze-tolerant animals, with selected species reported to survive milli-Kelvin temperatures. Here, we estimate survival of fully hydrated eutardigrades of the species Ramazzottius varieornatus following exposures to -20 °C and  -80 °C as well as -196 °C with or without initial cooling to -80 °C. The tardigrades easily survive these temperatures, yet with a significant decrease in viability following rapid cooling by direct exposure to -196 °C. Hence, post-freeze recovery of R. varieornatus seems to rely on cooling rate and thus controlled ice formation. Cryophilic organisms are renowned for having cold-active enzymes that secure appropriate reaction rates at low temperatures. Hence, extreme freeze-tolerance in R. varieornatus could potentially involve syntheses of cryoprotectants and de novo transcription. We therefore generated a reference transcriptome for this cryophilic R. varieornatus population and explored for differential gene expression patterns following cooling to -80 °C as compared to active 5 °C controls. Specifically, we tested for fast transcription potentially occurring within 25 min of cooling from room temperature to a supercooling point of ca. -20 °C, at which the tardigrades presumably freeze and enter into the ametabolic state of cryobiosis. Our analyses revealed no evidence for differential gene expression. We, therefore, conclude that extreme freeze-tolerance in R. varieornatus relies on controlled extracellular freezing with any freeze-tolerance related genes being constitutively expressed.

Differential expression profiling of heat stressed tardigrades reveals major shift in the transcriptome.

Tardigrades are renowned for their extreme stress tolerance, which includes the ability to endure complete desiccation, high levels of radiation and very low sub-zero temperatures. Nevertheless, tardigrades appear to be vulnerable to high temperatures and thus the potential effects of global warming. Here, we provide the first analysis of transcriptome data obtained from heat stressed specimens of the eutardigrade Ramazzottius varieornatus, with the aim of providing new insights into the molecular processes affected by high temperatures. Specifically, we compare RNA-seq datasets obtained from active, heat-exposed (35 °C) tardigrades to that of active controls kept at 5 °C. Our data reveal a surprising shift in transcription, involving 9634 differentially expressed transcripts, corresponding to >35% of the transcriptome. The latter data are in striking contrast to the hitherto observed constitutive expression underlying tardigrade extreme stress tolerance and entrance into the latent state of life, known as cryptobiosis. Thus, when examining the molecular response, heat-stress appears to be more stressful for R. varieornatus than extreme conditions, such as desiccation or freezing. A gene ontology analysis reveals that the heat stress response involves a change in transcription and presumably translation, including an adjustment of metabolism, and, putatively, preparation for encystment and subsequent diapause. Among the differentially expressed transcripts we find heat-shock proteins as well as the eutardigrade specific proteins (CAHS, SAHS, MAHS, RvLEAM, and Dsup). The latter proteins thus seem to contribute to a general stress response, and may not be directly related to cryptobiosis.