Tardigrade species used in this study include H. dujardini and P. richtersi, and were obtained and cultured as described below.

Method Details

Tardigrade Culture and Collection Gabriel et al., 2007 Gabriel W.N.

McNuff R.

Patel S.K.

Gregory T.R.

Jeck W.R.

Jones C.D.

Goldstein B. The tardigrade Hypsibius dujardini, a new model for studying the evolution of development. H. dujardini specimens were obtained originally from Sciento and were cultured in glass petri-dishes filled with spring water (Deer Park) and fed unicellular Chlorococcum sp. algae similar to (). P. richtersi was extracted from hazel leaf litter collected at Formigine (Northern Italy; N 44°34.253’, E 10°50.892′, 80 m a.s.l.). Dry leaf litter was stored at −80°C until specimen collection. To isolate P. richtersi, leaf litter was sprinkled with tap water for 15 min, and then submerged in water for 30 min. Active P. richtersi specimens were then extracted by sieves (250 μm and 37 μm mesh) under running water, and animals were isolated via direct microscopic observation. M. tardigradum short reads were downloaded from NCBI (Accessions SRA: SRX426237-SRX426240).

H. dujardini RNA Extraction and Library Preparation For RNaseq experiments three biological replicates were used for each condition: wet, drying, or frozen. To isolate RNA from desiccating specimens, 400 μL of Trizol was used to wash specimens from dishes into a 1.5 mL Eppendorf tube. For frozen and wet specimens, excess liquid was removed from pelleted animals and 400 μL of Trizol was added directly to the tubes. Plastic pestles were placed in tubes and the tubes dipped into liquid nitrogen. The frozen samples were ground with pestles and allowed to thaw. Five rounds of freeze-thaw homogenization were conducted. An additional 100 μL of Trizol was used to wash the pestles. Chloroform (100 μl) was mixed with each sample. Tubes were capped, shaken for 20 s, and allowed to sit at room temperature for 3 min. Samples were then centrifuged at 10,000g for 18 min at 4°C. The clear top layer was removed to a fresh tube and an equal volume of 100% ethanol was added. Samples were then processed using QIAGEN’s RNeasy Mini Kit (QIAGEN, Cat# 74104) according to manufacturer’s instructions. RNA samples were used for library construction using the Illumina mRNA TruSeq v2 kit.

P. richtersi RNA Extraction and Library Preparation We isolated RNA from biological replicates of P. richtersi specimens (three wet replicates and two dry replicates) by methods to those used for H. dujardini. RNA was extracted using the Epicenter MasterPure RNA Purification kit (Cat# MCR85102). RNA samples were used for library construction using the Illumina mRNA TruSeq v2 kit.

Transcriptome Sequencing, Assembly and Differential Expression Analysis Haas et al., 2013 Haas B.J.

Papanicolaou A.

Yassour M.

Grabherr M.

Blood P.D.

Bowden J.

Couger M.B.

Eccles D.

Li B.

Lieber M.

et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Li and Dewey, 2011 Li B.

Dewey C.N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. Robinson et al., 2010 Robinson M.D.

McCarthy D.J.

Smyth G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. RNaseq libraries were multiplexed and sequenced on the Illumina HighSeq 2000 platform. Raw transcriptome reads for M. tardigradum were obtained from NCBI’s SRA database (Accessions SRA: SRX426237-SRX426240). Pooled reads (H. dujardini – wet + drying + frozen; P. richtersi – wet + dry; M. tardigradum - Accessions SRA: SRX426237-SRX426240) were used for de novo assembly of transcripts using the program Trinity (). Read mapping was performed for each RNaseq library using RSEM () against the appropriate reference transcriptome, followed by normalization. For M. tardigradum, differential expression analysis was performed comparing active (SRA: SRX426237) and inactive (SRA: SRX426238) read counts. For H. dujardini and P. richtersi a transcript/gene was considered ‘expressed’ if it had a sum across all sequencing libraries of mapped read counts of 100 or more. Mapped read counts were used to perform differential expression for expressed genes using the program edgeR (). A transcript was deemed differentially expressed (enriched) if it had both a p value and a false discovery rate of < 0.05.

Protein Expression and Purification E. coli codon optimized gBlocks encoding tardigrade CAHS proteins were synthesized (Integrated DNA Technologies) and cloned into the pET28b expression vector. BL21star (DE3) E. coli were transformed with pET28b + CAHS plasmids. A single bacterial colony was used to inoculate 10 mL of Lennox broth (LB, 10 g/L, tryptone, 5 g/L yeast extract, 5 g/L NaCl) supplemented with 60 μg/mL of kanamycin. The culture was shaken at 37°C overnight (New Brunswick Scientific Innova I26, 225 rpm). Three of these cultures were used to inoculate 1 L of supplemented M9 media (50 mM Na 2 HPO 4 , 20 mM KH 2 PO 4 , 9 mM NaCl, 4 g/L glucose, 1 g/L 15NH 4 Cl, 0.1 mM CaCl 2 2 mM MgSO 4 , 10 mg/L thiamine, 10 mg/L biotin, and 60 μg/mL of kanamycin). The 1 L cultures were shaken at 37°C until the optical density at 600 nm reached 0.5. IPTG (1 mM final concentration) was then added to induce expression. After 4 hr, the cells were pelleted at 1,000g at 10°C for 30 min. The cell pellets were stored at −20°C. Pellets were resuspended in 12.5 mL of 50 mM HEPES, 50 mM NaCl (pH 8.0) supplemented with half a Roche cOmplete EDTA-free protease inhibitor tablet (Sigma-Aldrich Cat. #4693159001). Cells were then lysed by heat shock at 95°C for 15 min. Lysates were cooled at room temperature for 30 min. Insoluble components were removed by centrifugation at 20,000g and 10°C for 30 min. 2 (final concentration 2 mM) was added to the heat soluble fraction before digestion with 1250 units of Benzonase (Sigma-Aldrich) at 37°C for 1 hr. Benzonase was then inactivated by heating to 95°C. After cooling to room temperature, the lysate was sterile filtered using a 0.45 μm filter and transferred to 10,000 MWCO dialysis tubing. Samples were dialyzed against 50 mM sodium phosphate (pH 7.0) overnight followed by dialysis against three changes of 17 MΩ cm-1 H 2 O for at least 3 hr each. The dialysate was again filtered before being flash frozen in CO 2 (s)/ethanol and lyophilized for 48 hr (Labconco FreeZone). Purity was determined by SDS-PAGE, DNA electrophoresis, and an ethidium bromide fluorescence assay. Alpha-synuclein and ubiquitin were purified according to Smith et al. (2015) Smith A.E.

Zhou L.Z.

Pielak G.J. Hydrogen exchange of disordered proteins in Escherichia coli. Wang et al. (2012) Wang Y.

Sarkar M.

Smith A.E.

Krois A.S.

Pielak G.J. Macromolecular Crowding and Protein Stability. MgCl(final concentration 2 mM) was added to the heat soluble fraction before digestion with 1250 units of Benzonase (Sigma-Aldrich) at 37°C for 1 hr. Benzonase was then inactivated by heating to 95°C. After cooling to room temperature, the lysate was sterile filtered using a 0.45 μm filter and transferred to 10,000 MWCO dialysis tubing. Samples were dialyzed against 50 mM sodium phosphate (pH 7.0) overnight followed by dialysis against three changes of 17 MΩ cmO for at least 3 hr each. The dialysate was again filtered before being flash frozen in CO(s)/ethanol and lyophilized for 48 hr (Labconco FreeZone). Purity was determined by SDS-PAGE, DNA electrophoresis, and an ethidium bromide fluorescence assay. Alpha-synuclein and ubiquitin were purified according toand, respectively.

NMR Purified CAHS proteins were dissolved at 10 g/L in 50 mM sodium phosphate (pH 7.0), 90:10 (vol/vol) H 2 O:D 2 O by boiling and then centrifuged at 14,000g for 10 min to remove undissolved material. 15N-1H HSQC spectra were acquired at 298 K on an 850 MHz Bruker Avance III spectrometer equipped with a TCI cryoprobe. Sweep widths were 11,000 Hz and 3,500 Hz in the 1H and 15N dimensions, respectively. Each spectrum comprised 256 increments of 24 scans per increment. One-dimensional spectra were taken 20 min after sample preparation using a 1H sweep width of 13,500 Hz and comprised 128 scans. Each pair of H 2 O/D 2 O spectra was normalized using the methyl resonances at 0.8 ppm. Purified ubiquitin (2 mM) was resuspended in 50 mM sodium phosphate (pH 7.0), 95:5 (vol/vol) H 2 O:D 2 O and centrifuged at 20,000g for 5 min to remove undissolved material. 15N-1H HSQC spectra were acquired at 298 K on the 850 MHz spectrometer. Sweep widths were 14,000 Hz and 3,500 Hz in the 1H and 15N dimensions, respectively. Each spectrum comprised 256 increments of 4 scans per increment. One-dimensional spectra were taken 20 min after sample preparation using a 1H sweep width of 14,000 Hz and comprised 128 scans. Each one-dimensional spectrum was normalized using the methyl resonance at −0.15 ppm, and all spectra are referenced to DSS. Purified α-synuclein (0.1 mM) was resuspended in 50 mM sodium phosphate (pH 7.0), 95:5 (vol/vol) H 2 O:D 2 O and centrifuged at 20,000g for 5 min to remove undissolved material. 15N-1H HSQC spectra were acquired at 298 K on the 850 MHz spectrometer. Sweep widths were 14,000 Hz and 3,500 Hz in the 1H and 15N dimensions, respectively. Each spectrum comprised 256 increments of 4 scans per increment. One-dimensional spectra were taken 20 min after sample preparation using a 1H sweep width of 14,000 Hz and comprised 128 scans. Each one dimensional spectrum was normalized using the methyl resonance at 1 ppm, and all spectra are referenced to DSS.

Trehalose Phosphatase Identification Levin et al., 2016 Levin M.

Anavy L.

Cole A.G.

Winter E.

Mostov N.

Khair S.

Senderovich N.

Kovalev E.

Silver D.H.

Feder M.

et al. The mid-developmental transition and the evolution of animal body plans. Bemm et al., 2016 Bemm F.

Weiß C.L.

Schultz J.

Förster F. Genome of a tardigrade: Horizontal gene transfer or bacterial contamination?. Boothby et al., 2015 Boothby T.C.

Tenlen J.R.

Smith F.W.

Wang J.R.

Patanella K.A.

Nishimura E.O.

Tintori S.C.

Li Q.

Jones C.D.

Yandell M.

et al. Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade. Koutsovoulos et al., 2016 Koutsovoulos G.

Kumar S.

Laetsch D.R.

Stevens L.

Daub J.

Conlon C.

Maroon H.

Thomas F.

Aboobaker A.A.

Blaxter M. No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini. In animals the final step in trehalose production is mediated by the enzyme trehalose phosphatase (EC 3.1.3.12). C. elegans tps-1&2 (trehalose-6-phosphates) as well as gob-1 (trehalose phosphatase) and the D. melanogaster Tps1 (a trehalose-6-phosphate/trehalose phosphatase fusion) protein sequences were obtained from http://www.wormbase.org/#012-34-5 and http://www.flybase.org , respectively, and used as query sequences for BLAST analysis against a database derived from our transcriptome assembly with an E-value cutoff of 1E-10. No transcript (from our or the Yanai lab’s transcriptome ()) or predicted sequences from genome assemblies () aligned to gob-1 with an E-value less than or equal to 1E-10. Several H. dujardini predicted protein sequences aligned to tps-1, tps-2, Tps1 – but these sequences were not expressed in either hydrated or desiccated specimens and lacked a conserved trehalose phosphatase and HAD domain, which are found in both C. elegans and D. melanogaster trehalose phosphatases. Additionally, these sequences reciprocally BLAST to C. elegans tps genes not gob-1. Together, the detection of low levels of trehalose (at best) in some tardigrade species, coupled with the lack of expression of genes involved in trehalose anabolism and the absence of trehalose phosphatases suggests that H. dujardini does not rely on trehalose to survive desiccation.

CEGMA Analysis Parra et al., 2007 Parra G.

Bradnam K.

Korf I. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. The percent of core eukaryotic genes contained within our assembly was determined as a means of assessing the completeness of the transcriptome using CEGMA () with default settings. Identification of TDP-encoding Transcripts Transcript sequences were used as BLASTx queries and searched against NCBI’s non-redundant protein database. Reciprocal best BLAST was performed with an E-value cutoff of 1E-10.

RNA Interference Tenlen et al., 2013 Tenlen J.R.

McCaskill S.

Goldstein B. RNA interference can be used to disrupt gene function in tardigrades. Tenlen et al., 2013 Tenlen J.R.

McCaskill S.

Goldstein B. RNA interference can be used to disrupt gene function in tardigrades. Double stranded RNA (dsRNA) was made and microinjections performed with slight modification of a published protocol (). dsRNAs were diluted to a concentration of 1 μg/μl in nuclease-free water. Specimens were not sedated with levamisole as previously described () to reduce the number of factors potentially influencing survival. Injected specimens were transferred to 30 mm plastic dishes filled with fresh spring water and left overnight. The next day, specimens were either left in spring water with fresh food added (control), desiccated, or frozen. For each RNAi treatment and stress condition three individual trials were performed, with ten tardigrades injected per trial.

H. dujardini Desiccation Forney and Brandl, 1992 Forney C.F.

Brandl D.G. Control of Humidity in Small Controlled environment Chambers using Glycerol-Water Solutions. After injection (RNAi studies) or directly from larger cultures used for RNaseq, H. dujardini specimens were transferred to 35 mm plastic petri dishes filled with fresh spring water without algal food. Specimens were starved for 24 hr. Melted 2% agar (300 μl) was used to evenly coat the lid of 35 mm dishes and excess agar removed. After solidification, tardigrades were transferred to the center of coated lids. Using a mouth pipette, excess water was removed and lids were placed in humidified chambers. The relative humidity (95% for slow drying and 70% for quick drying) of each chamber was established using a mixture of glycerol and water () and monitored using a hygrometer. Tardigrades were dried overnight, enough time for tun formation to occur, and then removed and exposed to laboratory conditions (∼35% relative humidity) for 24 hr to allow for further desiccation. Rehydration was achieved by pipetting 1.5 mL of spring water into dishes. Rehydrated samples were left for 2 hr before observation and quantification of survival. Coordinated movement was used to score survival.

P. richtersi Desiccation 2 or 1 cm2) with mineral water (9 μl or 30 μl, respectively) and exposing them initially to 80% relative humidity (RH) and 18°C for 4 hr, then to 50% RH at 18°C for 4 hr in a climatically controlled chamber, and finally to 0%–3% RH at room temperature for 12 hr ( Altiero et al., 2011 Altiero T.

Guidetti R.

Caselli V.

Cesari M.

Rebecchi L. Ultraviolet radiation tolerance in hydrated and desiccated eutardigrades. P. richtersi specimens were desiccated by placing each group of animals on a Whatman filter paper (25 mmor 1 cm) with mineral water (9 μl or 30 μl, respectively) and exposing them initially to 80% relative humidity (RH) and 18°C for 4 hr, then to 50% RH at 18°C for 4 hr in a climatically controlled chamber, and finally to 0%–3% RH at room temperature for 12 hr (). At the end of this treatment animals exhibit the typical tun shape.

H. dujardini Freezing After injection (RNAi studies) or directly from larger cultures (RNaseq), H. dujardini specimens were transferred to 35 mm plastic petri dishes filled with fresh spring water without algal food. Specimens were starved for 24 hr. Specimens were then transferred to 1.5 mL microcentrifuge tubes, and the volume of spring water adjusted to 1 mL. The tubes were centrifuged briefly to move specimens to the bottom and then placed in a styrofoam box at −80°C for 24 hr. For RNAi studies, thawing was achieved by moving tubes to ambient laboratory conditions (∼20°C) for 2 hr. Following thawing the contents of each tube were transferred to a new 35 mm dish for observation and quantification of survival. Coordinated movement was used to score for survival. For RNaseq, thawing was accelerated by warming the specimens by hand and then rapidly moving on to RNA extraction.

Bacterial Heterologous Expression and Desiccation Survival Assay Cloning and transformation of bacteria was performed as described above. For expression, 10 mL cultures were grown overnight. The following day an aliquot of overnight culture was added to fresh culture media at a ratio of 1:200. Cultures were grown to log phase (OD 600 0.4-0.8). Expression of CAHS genes was then induced with 1 mM IPTG and the cultures grown for an additional 4 hr. Optical densities were measured again and approximately 108 cells were transferred to 1.5 mL microcentrifuge tubes and spun at 4,000g for 20 min. Excess culture media was removed, and cells were washed with water and re-pelleted. Water was quickly removed with a pipette and pellets were dried overnight in a SpeedVac (Savant SpeedVac SC100). The tubes, caps open, were transferred to a sealed desiccator filled with Drierite (Sigma-Aldrich, Cat. #238937) for 1 week. Rehydration and pellet dispersal was achieved by adding 1 mL of culture media to dry pellets and vortexing for 10 min. Cells were then transferred to kanamycin plates and grown overnight at 37°C. The following day colonies were counted and survival reported as colony forming units/108 cells plated.

Yeast Heterologous Expression and Desiccation Survival Assay The strain MAT α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 nth1::G418R can1::P TDH3 -AGT1 was used. This strain is a haploid alpha strain, with the nth1 trehalase gene deleted and replaced with G418 and with the AGT1 trehalose transporter under a constitutive highly expressed TDH3 promoter. Tardigrade CAHS coding sequences were cloned into the p413-GPD plasmid. Tardigrade genes were under the same TDH3 promoter on CEN plasmids, with histidine selection. Standard yeast propagation and transformation procedures were used. Strains were grown in selective, synthetic complete, media (2% glucose without histidine). Cultures were grown to logarithmic phase from an overnight culture by incubation overnight at 30°C. Cultures were re-diluted to an OD 600 of ∼0.05 and allowed to reach mid-log phase (OD 600 0.4 - 0.6). Desiccation tolerance assays were performed as follows. Approximately 107 cells were withdrawn from liquid cultures, washed twice in water and brought to a final volume of 1 ml. Undesiccated controls were plated for colony counting. Aliquots (200 μl) were then transferred to a 96-well tissue culture plate (Becton Dickinson, 353075), centrifuged and most of the water removed without disturbing the cell pellet. Cells were desiccated in a 23°C incubator with a constant 60% RH, with the lid raised, for 48 hr. Samples were resuspended in water and plated for colony counting.

Lactate Dehydrogenase Enzyme Activity Assay Goyal et al. (2005) Goyal K.

Walton L.J.

Tunnacliffe A. LEA proteins prevent protein aggregation due to water stress. Assays for lactate dehydrogenase (LDH) activity were largely based on the procedure outlined by. LDH from rabbit muscle (Roche) was diluted to 0.1 g/L in 100 μL of 25 mM Tris/HCl (pH 7) containing various concentrations of CAHS D, CAHS G, BSA (Sigma), or D-trehalose (Aldrich Chemical Company). Half of each sample was stored at 4°C while the other half was dehydrated in an EZ-2 Personal Evaporator (Genevac) for 16 hr on the aqueous setting with the heat lamp off. Dehydrated samples were re-dissolved in 250 μL of water, control samples were diluted with 200 μL of water, and all samples were kept on ice until their activity was measured. To determine the enzyme activity of LDH, 10 μL of LDH/excipient solution was added to 990 μL of 100mM sodium phosphate (pH 6), 100 μM NADH and 2mM pyruvate. The absorbance at 340 nm was measured every 0.1 s for 1 min in a Cary Series UV-Vis Spectrophotometer (Agilent Technologies). The percent activity was determined by comparing the initial, linear reaction rate of each dehydrated and rehydrated sample compared to its corresponding unstressed control. Experiments were performed in triplicate. For LDH assays, protein expression was performed as described above, except that 10 mL of overnight cultures were used to inoculate 1 L of LB. Differential Scanning Calorimetry For dried tardigrades, CAHS proteins, and modified yeast strains, samples (6-9 mg) were hermetically sealed in Tzero aluminum pans (TA Instruments) and accessed using a Discovery Series differential scanning calorimeter (TA Instruments). Samples were equilibrated at −20°C, and then ramped to 250°C at a rate of 5°C /min. The temperature profile was cycled once.