Comparative genomics of the tardigrades <i>Hypsibius dujardini</i> and <i>Ramazzottius varieornatus</i>

Tardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Panarthropoda by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda

RNA sequencing data for gamma radiation response in the extremotolerant tardigrade Ramazzottius varieornatus

Tardigrades are microscopic animals of which terrestrial species are capable of tolerating extreme environments by entering a desiccated ametabolic state known as anhydrobiosis. Intriguingly, they survive high dosage gamma rays (>4,000 Gy), possibly through a mechanism known as cross-tolerance. We hypothesized that anhydrobiosis genes are also regulated during cross-tolerance, thus we submitted Ramazzottius varieornatus to 500 Gy 60Co gamma-ray and conducted time-course low-input RNA-Seq. The gene expression was quantified with RSEM and differential expression was determined with DEseq2. Differentially expressed genes were submitted to gene ontology enrichment analysis with GOStat. The transcriptome dynamically shifted nine hours post-exposure

Analysis of DNA Repair and Protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after Exposure to UVC Radiation

Tardigrades inhabiting terrestrial environments exhibit extraordinary resistance to ionizing radiation and UV radiation although little is known about the mechanisms underlying the resistance. We found that the terrestrial tardigrade Ramazzottius varieornatus is able to tolerate massive doses of UVC irradiation by both being protected from forming UVCinduced thymine dimers in DNA in a desiccated, anhydrobiotic state as well as repairing the dimers that do form in the hydrated animals. In R. varieornatus accumulation of thymine dimers in DNA induced by irradiation with 2.5 kJ/m² of UVC radiation disappeared 18 h after the exposure when the animals were exposed to fluorescent light but not in the dark. Much higher UV radiation tolerance was observed in desiccated anhydrobiotic R. varieornatus compared to hydrated specimens of this species. On the other hand, the freshwater tardigrade species Hypsibius dujardini that was used as control, showed much weaker tolerance to UVC radiation than R. varieornatus, and it did not contain a putative phrA gene sequence. The anhydrobiotes of R. varieornatus accumulated much less UVC-induced thymine dimers in DNA than hydrated one. It suggests that anhydrobiosis efficiently avoids DNA damage accumulation in R. varieornatus and confers better UV radiation tolerance on this species. Thus we propose that UV radiation tolerance in tardigrades is due to the both high capacities of DNA damage repair and DNA protection, a two-pronged survival strategy

Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells

<div><p>Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called “anhydrobiosis”. Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, <i>Ramazzottius varieornatus</i>, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from <i>Ramazzottius varieornatus</i>. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble <i>in vitro</i>, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.</p></div

Protein structure and mitochondria-targeting potential of RvLEAM.

<p>(a) Schematic representation of RvLEAM protein structure with hydropathy plot. Pink box indicates Mitochondria Targeting Peptide (MTP) at the N-terminus and the nine green boxes indicate LEA motifs found in RvLEAM protein. The six yellow bars in the hydropathy plot indicate predicted helix regions (H1–6) (b) Alignment of LEA motif sequences of RvLEAM with consensus. Greek characters in the consensus sequence indicate the following: Ψ, basic residue; Ω, acidic or amide residue; Φ, aliphatic residue; X, non-conserved residue. Residues consistent with the consensus are highlighted in deep green, and amino acids at non-conserved positions are shown in light green. Sequence logo created from nine LEA motifs of RvLEAM is shown below the alignment. (c) Amphipathic distribution of amino acids in predicted helical region of repeating LEA motifs 3 and 4. Amino acids are indicated as hydrophilic (pink), negatively-charged (red), positively-charged (blue), and hydrophobic (yellow) residues. (d) Subcellular localization of RvLEAM-GFP fusion protein (top) and GFP alone (bottom) in human HEp-2 cells. GFP signals (left). Mitochondria stained with Mito-Tracker (centre). Merged images (right).</p

Heat-soluble property of RvLEAM protein.

<p>Heat-solubility of RvLEAM protein is shown using either RvLEAM-induced bacterial lysate (a) or purified RvLEAM protein (b). ‘S’ indicates starting sample before heat treatment. After heat treatment, the proteins were separated into a soluble fraction (HS) and insoluble precipitate (HP). Arrows indicate RvLEAM protein.</p

Subcellular localization of a novel mitochondrial heat-soluble MAHS protein.

<p>(a) Subcellular localization of MAHS-GFP fusion protein in human HEp-2 cells. (b) Heat-solubility of MAHS protein using bacterial lysate (left) or purified protein (right). Starting samples before heat treatment (S), soluble fraction after heat treatment (HS), and insoluble precipitate (HP) are shown. Red arrows indicate MAHS protein. (c) MAHS protein structure with hydrophathy plot. Mitochondria Targeting Peptide and conserved MAHS motif are indicated by the pink box and red box, respectively. Yellow and blue bars in the hydropathy plot indicate predicted helix regions (H1–5) and strand regions (S1–7), respectively. (d) Sequence alignment of conserved MAHS motifs among three tardigrade species, <i>R. varieornatus</i>, <i>H. dujardini</i> (Hd), and <i>M. tardigradum</i> (Mt). Pink and yellow indicate hydrophilic and hydrophobic residues, respectively (d) Amphipathic nature of predicted helix in MAHS motif. Amino acids are coloured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118272#pone.0118272.g001" target="_blank">Fig. 1c</a>.</p

Effects of RvLEAM and MAHS proteins on metabolic activity of human cells under hyperosmotic stress.

<p>Human HEp-2 cells (black), and HEp-2 cells expressing RvLEAM (light grey) or MAHS (dark grey) were exposed to medium supplemented with defined concentrations of sucrose for 48 h. After replacement of the medium with serum-free isotonic medium, cell metabolic activities were analysed using PrestoBlue reagents. Metabolic activities at each sucrose concentration are shown as a percentage of the mean activity observed in the cells at 0 mM sucrose (n = 3, ±SEM). Statistical significance against untransfected HEp-2 cells was determined by <i>Dunnett</i>’s test (*, <i>P</i><0.05; **, <i>P</i><0.01). <i>P</i>-values are shown above asterisks where statistically significant.</p

Mitochondrial localization of RvLEAM protein in tardigrade embryo.

<p>Tardigrade embryos were collected with eggshells and immunohistochemical analysis was performed using 10-μm frozen sections. Images show a representative section that was simultaneously reacted with anti-RvLEAM antiserum (a, d) and anti-ATP5A (mitochondrial ATP synthase) antibody (b, e). (c, f) Merged images. (d-f) Magnified images corresponding to the white boxes in (a-c).</p