Ralph O. Schill

Tardigrades survive exposure to space in low Earth orbit

Summary Vacuum (imposing extreme dehydration) and solar/galactic cosmic radiation prevent survival of most organisms in space [1]. Only anhydrobiotic organisms, which have evolved adaptations to survive more or less complete desiccation, have a potential to survive space vacuum, and few organisms can stand the unfiltered solar radiation in space. Tardigrades, commonly known as water-bears, are among the most desiccation and radiation-tolerant animals and have been shown to survive extreme levels of ionizing radiation [2–4]. Here, we show that tardigrades are also able to survive space vacuum without loss in survival, and that some specimens even recovered after combined exposure to space vacuum and solar radiation. These results add the first animal to the exclusive and short list of organisms that have survived such exposure.

Trehalose and anhydrobiosis in tardigrades – evidence for divergence in responses to dehydration

To withstand desiccation, many invertebrates such as rotifers, nematodes and tardigrades enter a state known as anhydrobiosis, which is thought to require accumulation of compatible osmolytes, such as the non‐reducing disaccharide trehalose to protect against dehydration damage. The trehalose levels of eight tardigrade species comprising Heterotardigrada and Eutardigrada were observed in five different states of hydration and dehydration. Although many species accumulate trehalose during dehydration, the data revealed significant differences between the species. Although trehalose accumulation was found in species of the order Parachela (Eutardigrada), it was not possible to detect any trehalose in the species Milnesium tardigradum and no change in the trehalose level has been observed in any species of Heterotardigrada so far investigated. These results expand our current understanding of anhydrobiosis in tardigrades and, for the first time, demonstrate the accumulation of trehalose in developing tardigrade embryos, which have been shown to have a high level of desiccation tolerance.

Stress gene (hsp70) sequences and quantitative expression in Milnesium tardigradum (Tardigrada) during active and cryptobiotic stages

SUMMARY The eutardigrade Milnesium tardigradum can undergo cryptobiosis, i.e. entry into a reversible ametabolic stage induced by dehydration, cooling and, probably, osmotic and anoxic stress. For the first time in tardigrades, we described partial sequences of three heat-shock protein (hsp70 family) genes and examined gene expression on the way from an active to a cryptobiotic and back to an active stage again. Results showed different patterns of gene expression in the hsp70 isoforms. All three isoforms seem to be true heat-shock proteins since transcription could be clearly enhanced by temperature elevation. Isoform 1 and, at a lower level, isoform 3 do not seem to have a specific function for cryptobiosis. By contrast, transcription of isoform 2 is significantly induced in the transitional stage between the active and the cryptobiotic stage, resulting in a comparatively high mRNA copy number also during cryptobiosis. This pattern of induction implies that isoform 2 is the most relevant hsp70 gene for M. tardigradum individuals entering the cryptobiotic stage.

A transcriptome approach to ecdysozoan phylogeny.

The monophyly of Ecdysozoa, which comprise molting phyla, has received strong support from several lines of evidence. However, the internal relationships of Ecdysozoa are still contended. We generated expressed sequence tags from a priapulid (penis worm), a kinorhynch (mud dragon), a tardigrade (water bear) and five chelicerate taxa by 454 transcriptome sequencing. A multigene alignment was assembled from 63 taxa, which comprised after matrix optimization 24,249 amino acid positions with high data density (2.6% gaps, 19.1% missing data). Phylogenetic analyses employing various models support the monophyly of Ecdysozoa. A clade combining Priapulida and Kinorhyncha (i.e. Scalidophora) was recovered as the earliest branch among Ecdysozoa. We conclude that Cycloneuralia, a taxon erected to combine Priapulida, Kinorhyncha and Nematoda (and others), are paraphyletic. Rather Arthropoda (including Onychophora) are allied with Nematoda and Tardigrada. Within Arthropoda, we found strong support for most clades, including monophyletic Mandibulata and Pancrustacea. The phylogeny within the Euchelicerata remained largely unresolved. There is conflicting evidence on the position of tardigrades: While Bayesian and maximum likelihood analyses of only slowly evolving genes recovered Tardigrada as a sister group to Arthropoda, analyses of the full data set, and of subsets containing genes evolving at fast and intermediate rates identified a clade of Tardigrada and Nematoda. Notably, the latter topology is also supported by the analyses of indel patterns.

Freeze tolerance, supercooling points and ice formation: comparative studies on the subzero temperature survival of limno-terrestrial tardigrades

SUMMARY Many limno-terrestrial tardigrades live in unstable habitats where they experience extreme environmental conditions such as drought, heat and subzero temperatures. Although their stress tolerance is often related only to the anhydrobiotic state, tardigrades can also be exposed to great daily temperature fluctuations without dehydration. Survival of subzero temperatures in an active state requires either the ability to tolerate the freezing of body water or mechanisms to decrease the freezing point. Considering freeze tolerance in tardigrades as a general feature, we studied the survival rate of nine tardigrade species originating from polar, temperate and tropical regions by cooling them at rates of 9, 7, 5, 3 and 1°C h–1 down to –30°C then returning them to room temperature at 10°C h–1. The resulting moderate survival after fast and slow cooling rates and low survival after intermediate cooling rates may indicate the influence of a physical effect during fast cooling and the possibility that they are able to synthesize cryoprotectants during slow cooling. Differential scanning calorimetry of starved, fed and cold acclimatized individuals showed no intraspecific significant differences in supercooling points and ice formation. Although this might suggest that metabolic and biochemical preparation are non-essential prior to subzero temperature exposure, the increased survival rate with slower cooling rates gives evidence that tardigrades still use some kind of mechanism to protect their cellular structure from freezing injury without influencing the freezing temperature. These results expand our current understanding of freeze tolerance in tardigrades and will lead to a better understanding of their ability to survive subzero temperature conditions.

DNA damage in storage cells of anhydrobiotic tardigrades.

In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 +/- 1.98% DNA in tail, compared to 0.44 +/- 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 +/- 6.41% of DNA was found in the comet tail. After ten months, 23.66 +/- 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation.

Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades

BackgroundTardigrades represent an animal phylum with extraordinary resistance to environmental stress.ResultsTo gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (Milnesium tardigradum, Hypsibius dujardini, Echiniscus testudo, Tulinus stephaniae, Richtersius coronifer) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer http://waterbear.bioapps.biozentrum.uni-wuerzburg.de. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data.ConclusionDifferent protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences.

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

Background Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Principal Findings Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. Conclusions The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.

Selective neuronal staining in tardigrades and onychophorans provides insights into the evolution of segmental ganglia in panarthropods

BackgroundAlthough molecular analyses have contributed to a better resolution of the animal tree of life, the phylogenetic position of tardigrades (water bears) is still controversial, as they have been united alternatively with nematodes, arthropods, onychophorans (velvet worms), or onychophorans plus arthropods. Depending on the hypothesis favoured, segmental ganglia in tardigrades and arthropods might either have evolved independently, or they might well be homologous, suggesting that they were either lost in onychophorans or are a synapomorphy of tardigrades and arthropods. To evaluate these alternatives, we analysed the organisation of the nervous system in three tardigrade species using antisera directed against tyrosinated and acetylated tubulin, the amine transmitter serotonin, and the invertebrate neuropeptides FMRFamide, allatostatin and perisulfakinin. In addition, we performed retrograde staining of nerves in the onychophoran Euperipatoides rowelli in order to compare the serial locations of motor neurons within the nervous system relative to the appendages they serve in arthropods, tardigrades and onychophorans.ResultsContrary to a previous report from a Macrobiotus species, our immunocytochemical and electron microscopic data revealed contralateral fibres and bundles of neurites in each trunk ganglion of three tardigrade species, including Macrobiotus cf. harmsworthi, Paramacrobiotus richtersi and Hypsibius dujardini. Moreover, we identified additional, extra-ganglionic commissures in the interpedal regions bridging the paired longitudinal connectives. Within the ganglia we found serially repeated sets of serotonin- and RFamid-like immunoreactive neurons. Furthermore, our data show that the trunk ganglia of tardigrades, which include the somata of motor neurons, are shifted anteriorly with respect to each corresponding leg pair, whereas no such shift is evident in the arrangement of motor neurons in the onychophoran nerve cords.ConclusionsTaken together, these data reveal three major correspondences between the segmental ganglia of tardigrades and arthropods, including (i) contralateral projections and commissures in each ganglion, (ii) segmentally repeated sets of immunoreactive neurons, and (iii) an anteriorly shifted (parasegmental) position of ganglia. These correspondences support the homology of segmental ganglia in tardigrades and arthropods, suggesting that these structures were either lost in Onychophora or, alternatively, evolved in the tardigrade/arthropod lineage.

Stress response in tardigrades: differential gene expression of molecular chaperones

Semi-terrestrial tardigrades exhibit a remarkable tolerance to desiccation by entering a state called anhydrobiosis. In this state, they show a strong resistance against several kinds of physical extremes. Because of the probable importance of stress proteins during the phases of dehydration and rehydration, the relative abundance of transcripts coding for two α-crystallin heat-shock proteins (Mt-sHsp17.2 and Mt-sHsp19.5), as well for the heat-shock proteins Mt-sHsp10, Mt-Hsp60, Mt-Hsp70 and Mt-Hsp90, were analysed in active and anhydrobiotic tardigrades of the species Milnesium tardigradum. They were also analysed in the transitional stage (I) of dehydration, the transitional stage (II) of rehydration and in heat-shocked specimens. A variable pattern of expression was detected, with most candidates being downregulated. Gene transcripts of one Mt-hsp70 isoform in the transitional stage I and Mt-hsp90 in the anhydrobiotic stage were significantly upregulated. A high gene expression (778.6-fold) was found for the small α-crystallin heat-shock protein gene Mt-sHsp17.2 after heat shock. We discuss the limited role of the stress-gene expression in the transitional stages between the active and anhydrobiotic tardigrades and other mechanisms which allow tardigrades to survive desiccation.