Osmotic stress tolerance and transcriptomic response of Ramazzottius varieornatus (Eutardigrada: Ramazzottiidae) following tun formation

To thrive in harsh environments, tardigrades have evolved the ability to enter the quiescent state of cryptobiosis, often characterized by transition into a so-called ‘tun’. Here, we investigate osmobiosis, a substate of cryptobiosis induced by rising osmolyte concentrations. We follow the behaviour and morphology of Ramazzottius varieornatus during transfer from freshwater conditions into 3 Osmol/kg sucrose solution. The tardigrades easily survive the extreme change in external osmolality. During gradual exposure, they initiate tun formation at 0.2 Osmol/kg, with most specimens fully contracted into a tun at 0.5 Osmol/kg. The first transcriptomic profiling of osmobiotic tuns in comparison to active tardigrades reveals a modest shift, with 16% of the 3322 differentially expressed transcripts having a |log 2 fold change| > 1. A gene ontology enrichment analysis shows enrichment within protein homeostasis and neurohormonal signalling, with a ubiquitin-conjugating enzyme and neurotransmitter receptor transcripts being down-and upregulated, respectively. A putative Hsp70 is upregulated, whereas transcripts related to eutardigrade-specific proteins, antioxidant defence and DNA repair show minor fold changes. Among putative membrane transporters, a monocarboxylate and two amino acid transporters are downregulated. Our findings are in agreement with previous studies in Ramazzottius indicating that cryptobiosis and tun formation involve no change or modest change in transcription.


INTRODUCTION
Cryptobiosis (latent life) is defined as the state of an organism where metabolism comes to a reversible standstill or is hardly measurable (Keilin 1959).Various clades of life use cryptobiosis as a survival strategy in adverse environmental conditions (Clegg 2001).How cryptobiosis evolved and how living organisms endure the extreme stress and reversible shutdown of metabolism associated with the state are long-standing questions.
Phylum Tardigrada comprises two major evolutionary lineages, eutardigrades and heterotardigrades, both of which contain cryptobiotic and non-cryptobiotic species (Møbjerg et al. 2011, Jørgensen andMøbjerg 2015).Tardigrades are meiofaunal organisms that need a film of water to be in their active feeding and reproducing state (Rebecchi et al. 2020, Møbjerg andNeves 2021).These microscopic organisms are, nevertheless, found worldwide and thrive in habitats spanning ecological realms, including extreme habitats such as the deep sea, hot springs, polar regions and deserts (Nelson et al. 2018).In order to survive in extreme or changing environments tardigrades rely on a range of survival strategies, including cryptobiosis (Møbjerg and Neves 2021).Tardigrades can stay in the cryptobiotic state for decades and finally return to life when environmental conditions become more favourable ( Jørgensen et al. 2007, Hengherr et al. 2008a, Tsujimoto et al. 2016, Roszkowska et al. 2020).
Notably, tardigrades can lose > 95% of their water (Crowe 1972), reducing body volume by ≤ 90% (Halberg et al. 2013a), while contracting into the anhydrobiotic tun state.ATP-driven muscular contractions are a requisite for tun formation (Halberg et al. 2013a).Specifically, the musculature facilitates the longitudinal contraction of the trunk, a retraction of the legs and head, and a rearrangement of internal organs.During dessication, as water leaves the animal and metabolism eventually ceases, muscle protein filaments are likely to lock in a rigor state, providing a vital stabilizing role, ensuring the three-dimensional structure of the ATP-depleted tun (Møbjerg and Neves 2021).Hence, actin and myosin filaments provide a vital stabilizing framework at the whole-animal level of the desiccated ametabolic tun state.Furthermore, intensive research focusing on anhydrobiosis has suggested that a variety of compounds participate in desiccation tolerance at the cell and organelle levels (Hibshman et al. 2020, Møbjerg andNeves 2021).These include trehalose, heat shock proteins (HSPs), cold shock proteins, late embryogenesis abundant (LEA) proteins, and tardigrade-specific proteins, such as cytoplasmic abundant heat-soluble (CAHS), mitochondrial abundant heat-soluble (MAHS) and secretory abundant heat-soluble (SAHS) proteins, LEA proteins located in the mitochondria (RvLEAM) and the damage suppressor (Dsup) (Hengherr et al. 2008b, Yamaguchi et al. 2012, Tanaka et al. 2015, Hashimoto et al. 2016, Boothby et al. 2017, Chavez et al. 2019, Kamilari et al. 2019, Nguyen et al. 2022).Notably, different tardigrade taxa and lineages exhibit unique molecular adaptations, and the tardigrade-specific proteins (i.e.CAHS, MAHS, SAHS, RvLEAM and Dsup) seem to be present across eutardigrades but are missing in heterotardigrades (Kamilari et al. 2019, Murai et al. 2021).All tardigrades seem to have a comprehensive number of genes encoding proteins involved in antioxidant defence (Kamilari et al. 2019).
Here, we investigate the phenomenon osmobiosis, which is likely to be the most ancient form of the cryptobiosis substates that involve water loss, i.e. osmobiosis, anhydrobiosis and cryobiosis (Hygum et al. 2016, Møbjerg and Neves 2021, Møbjerg et al. 2022).Specifically, phylum Tardigrada is likely to have a marine origin, and the ability to form osmobiotic tuns (i.e.tuns induced by high external osmolyte concentrations) is present among all extant tardigrade lineages, indicating that the tun is an ancient and homologous trait that evolved in the sea ( Jørgensen andMøbjerg 2015, Hygum et al. 2016).Hence, from an evolutionary perspective, it is especially interesting to investigate osmobiosis, because this state is likely to represent the forerunner of anhydrobiosis and cryobiosis.Marine tidal tardigrades are highly tolerant of both gradual and acute osmotic stress induced by high-concentration NaCl solutions, if necessary entering osmobiosis in the course of seconds (Hygum et al. 2016, Sørensen-Hygum et al. 2018).Limnoterrestrial species only seem to tolerate extreme external salt concentrations when given time to acclimate (Wright et al. 1992, Heidemann et al. 2016).These species, however, readily form tuns and survive acute exposures to high concentrations of non-ionic osmolytes (Heidemann et al. 2016).
Here, we investigate osmotic stress tolerance and osmobiotic tun formation in the limnoterrestrial eutardigrade Ramazzottius varieornatus (Eutardigrada: Ramazzottiidae).Specifically, we report the behavioural and morphological response of the tardigrades to extreme changes in external osmolality following gradual or acute exposure to 3 Osmol/kg sucrose solution.With the aim of providing new insights into the molecular processes involved in cryptobiosis, we also provide the first transcriptomic profiling of tardigrades following acute exposure to a highosmolality solution.

Tardigrade cultivation and handling
Approximately 1760 specimens of R. varieornatus were isolated from a sediment sample collected in February 2018 from a Danish roof gutter (55°56′36.5″N12°30′00.9″E)and subsequently stored at ~−20°C (see Neves et al. 2020).Specifically, two smaller portions of this sample were thawed in September and November 2021, respectively, diluted in ultrapure water (Millipore Milli-Q Reference, Merck, Darmstadt, Germany) and kept in glass containers at ~5°C to prepare cultures (Neves et al. 2020).Small amounts of sediment from these cultures were transferred to Petri dishes before each experiment, and tardigrades were subsequently isolated from the sediment under a stereomicroscope and transferred to embryo dishes using handpulled Pasteur pipettes.

Osmotic stress tolerance assay
To investigate osmotic stress tolerance in R. varieornatus, we prepared sucrose solutions of ~100, ~200, ~500, ~1000, ~2000 and ~3000 mOsmol/kg, measuring osmolality by vapour pressure osmometry (VAPRO model 5600).In an initial gradual exposure experiment, solutions were prepared by serial dilution of sucrose in moderately hard reconstituted water (MHRW adapted from Khanna et al. 1997: 96 mg NaHCO 3 , 60 mg CaSO 4 •2H 2 O, 123 mg MgSO 4 •7H 2 O and 4 mg KCl dissolved in 1 L of ultrapure water).The measured osmolalities of MHRW and the six sucrose solutions were as follows (mean ± SE; N = 3-5): 5 ± 0, 107 ± 1, 205 ± 1, 506 ± 2, 1005 ± 3, 2006 ± 7 and 3001 ± 4 mOsmol/kg, respectively.Groups of tardigrades (N = 5 × 20 specimens) and small amounts of sediment were transferred to a six-well plate filled with 1 mL MHRW and left at ~5°C for 1 h.Gradual exposures were conducted by chronologically replacing the medium in the well plate with 1 mL of the series of sucrose solutions.Efforts were made to ensure that as much medium as possible was removed between steps, but without exposing the tardigrades to desiccation.Hence, the osmolality was stepped up from 5 ± 0 mOsmol/kg (MHRW) to ~100, ~200, ~500, ~1000, ~2000 and, finally, ~3000 mOsmol/kg, with a 1 h gap between each step.The specimens were subsequently kept at 3 Osmol/kg for 14 h, before being returned to MHRW in a reverse stepwise manner.The tardigrades were finally left in MHRW for 48 h.The response of single specimens was assessed under a stereomicroscope at every transition of osmolality and compared with that of the control groups (N = 5 × 20 specimens) kept in MHRW for the entire experimental period, i.e. 72 h.
In a subsequent second experimental series, tardigrades were challenged by direct transfer into 3 Osmol/kg sucrose solutions (measured osmolality: 3051 ± 25 mOsmol/kg, mean ± SE; N = 3) prepared by adding sucrose to ultrapure water.Groups of tardigrades (N = 5 × ~20 specimens) and small amounts of sediment were initially transferred to a six-well plate and exposed to ultrapure water for 1 h and subsequently (at t = 0 h) to 3 Osmol/kg directly.The specimens were kept for 24 h in 3 Osmol/kg before being retransferred to ultrapure water with sediment for 48 h.Control groups (N = 5 × ~20 specimens) were kept on sediment in ultrapure water for 72 h.
Every specimen in each of the experimental and control series was monitored under a stereomicroscope for activity and behaviour/morphology at specific time points.A given specimen was considered active if it displayed a well-coordinated movement of the legs (A + ) or any other signs of life (A), e.g.clinging to the sediment or responding to a gentle tactile stimulus.Alternatively, the specimens were marked as inactive.This category included specimens that had entered an osmobiotic tun (T), but also non-contracted inactive specimens (I) (for scoring details, see Supporting Information, Tables S1 and S2).Activity in proportion was calculated by dividing the number of active specimens by the total number of specimens in each group (Heidemann et al. 2016, Sørensen-Hygum et al. 2018).Statistical comparisons were conducted in RStudio v.4.1.1 (2021-08-10) using the prop.test() function.To illustrate the change in activity over experimental time, boxplots were made with the ggplot2 package.
Light micrographs were taken with an Olympus DP27 digital microscope camera mounted on an Olympus BX53 compound microscope using the Olympus cellSens Standard software.The final assemblage of figures was conducted in CorelDRAW v.2021.5.
Total RNA extraction, mRNA library construction, and sequencing Tardigrades were pooled (N = 6 × ~200-320 specimens) and transferred to BeadBug microtubes (Benchmark Scientific, Sayreville, NJ, USA) containing 1 mL of either ultrapure water (control) or a 3 Osmol/kg (measured osmolality: 3031 ± 33 mOsmol/kg, mean ± SE; N = 5) sucrose solution (osmobiotic tuns).The tardigrades were subsequently kept in these solutions for 24 h at ~5°C before total RNA was extracted using the RNeasy Plus Universal Mini Kit (Qiagen, Hilden, Germany).Extractions included a homogenization step with circonia/silica beads at 50 Hz for 2 min (Neves et al. 2022), followed by standard extraction steps according to the kit protocol.The six RNA samples were sent to BGI Europe A/S (Copenhagen, Denmark) for library construction and sequencing.Initially, the RNA samples were evaluated for quantity and quality using an Agilent 4200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA) (Table 1), after which mRNA was oligo dT selected and reverse transcribed.The obtained cDNA was ligated to adaptors, amplified, separated into single strands, and cyclized for DNA nanoball sequencing on the DNBseq-G400 platform.The paired-end reads (100 bp read length) were trimmed of adapters The quantity and quality of RNA extracts were determined by BGI (Agilent 4200 TapeStation).RIN, RNA integrity number; Q20(%), percentage of reads with a quality higher than Q20, representing a sequencing error rate of 1%.Data provided by BGI.  1).Note that there are no tuns at t = 72 h (both active and inactive specimens are fully distended at this time point; see Supporting Information, Table S1).and low-quality sequences by BGI Europe A/S, resulting in a total of ~273 million clean reads, with an average read number per sample of ~45.6 million, corresponding to ~22.8 million read pairs per sample (Table 1).Finally, we double-checked the RNA-seq datasets using FastQC (Ewels et al. 2016).

Differential expression analyses
Clean reads obtained from the six RNA-seq datasets (i.e. three samples exposed to ultrapure water and three samples exposed to 3 Osmol/kg) were quasi-mapped to a reference transcriptome (ENA PRJEB47629; see Møbjerg et al. 2022) using Salmon v.1.10 (Patro et al. 2017).We used the transcriptome assembly obtained from the Danish R. varieornatus population, because this population is different from the Japanese population used to generate the genome available from public databases (i.e. the Japanese R. varieornatus population is a different species).Transcript abundance (i.e. the number of read counts for each transcript) was quantified using Salmon, and transcripts expressed in more than half of the six samples were retained for further analyses (19 267 of 19 456 transcripts).An initial principal components analysis revealed a clear batch effect (Supporting Information, Fig. S1), probably originating from size (and thus age) differences among specimens in different batches.Specifically, the sample thawed in November 2021 contained mostly small (probably juvenile) tardigrades.Consequently, the dataset was subjected to batch effect correction using ComBat-seq (Zhang et al. 2020).Differential expression analyses were conducted on the corrected dataset using three methods: Limma (Ritchie et al. 2015), EdgeR (Robinson et al. 2009) and DESeq2 (Love et al. 2014).Specifically, log 2 fold changes (log 2 FC) were determined by comparing transcript abundance between tardigrades exposed to high osmolality and active control tardigrades using a false discovery rate (FDR) or adjusted P-value of ≤ 0.05.A given transcript was considered for subsequent analyses only if identified as differentially expressed by all three methods.

Gene ontology enrichment analysis
A gene ontology (GO) enrichment analysis was performed on the differentially expressed transcripts congruently identified by Limma, EdgeR and DESeq2 using topGO v.2.46.0 (Alexa and Rahnenfuhrer 2021).Statistical significance was tested with the topgoFisher algorithm ('weighth01') using a significance cut-off of P ≤ 0.05.Fold enrichment was calculated as the number of significant differentially expressed transcripts divided by the total number of annotated transcripts within a given GO term.
In the subsequent acute exposure experiment (Fig. 3; Supporting Information, Table S2), we tested the response of tardigrades to direct transfers from ultrapure water containing small amounts of sediment to 3 Osmol/kg sucrose solutions.The tardigrades generally thrived in clean water containing small amounts of substrate from the sample locality (Fig. 3B).Upon transfer to 3 Osmol/kg, the tardigrades immediately entered the osmobiotic tun state (Fig. 1C), and they remained in this state for 24 h (Fig. 3A, t = 24 h), until return into ultrapure water, with 65 ± 8.5% of the specimens having regained activity 1 h after the return (Fig. 3A, t = 25 h).Almost all specimens (95 ± 0.2%) were active at the final assessment (Fig. 3A; t = 72 h), i.e. the activity was not significantly different from that of the control group [99 ± 1.0% active; Fig. 3B; t = 72 h; χ 2 (1) = 1.51,P = 0.22].

Transcriptomic profiling of the osmobiotic tun
In order to elucidate molecular mechanisms involved in osmobiotic tun formation, we continued our investigation by comparing transcriptomes obtained from osmobiotic tuns and active tardigrades.Specifically, we sequenced six RNA samples, including three RNA-seq replicates from tuns and active specimens, respectively (Table 1).Following a batch-effect adjustment, the three differential expression analyses (Limma, EdgeR and DESeq2) identified 3322 (1684 up-and 1638 downregulated) transcripts (Fig. 4), accounting for 12.3% of the 27 054 Unigenes in the reference transcriptome.Most of these transcripts had a minor fold change.Specifically, 521 (15.7%) of the 3322 transcripts identified across the three methods had a |log 2 FC| > 1.Of the 3346 transcripts identified by DESeq2, 537 (16.0%) had a |log 2 FC| > 1 (Fig. 5).Table 2 shows the top 10 most up-and downregulated transcripts with a predicted function.The Supporting Information (Table S3) includes a list of highly up-and downregulated transcripts without annotation.A GO enrichment analysis revealed 12 significantly enriched GO molecular function categories (Table 3) (Supporting Information, Fig. S2; Tables S4-S15).Six of these categories were associated with various processes involved in protein homeostasis (Table 3; GO:0003735, GO:0003743, GO:0004579, GO:0004813, GO:0004571 and GO:0051082).Notably, a ubiquitin-conjugating enzyme was among the most downregulated transcripts in the tuns (Table 2).Three significantly enriched GO terms were associated with neurohormonal signalling (Table 3; GO:0004715, GO:0070594 and GO:0005484), and several transcripts related to neuronal function were found among the most up-and downregulated transcripts (Table 2), including a γ-aminobutyric acid (GABA) receptor (upregulated), a glutamate receptor (upregulated) and extended synaptotagmins (downregulated).The enriched GO terms also involved transcripts of various metabolic enzymes (Table 3; GO:0004471 and GO:0005506), with a cytochrome P450 (CYP) transcript being downregulated (Unigene6627_Ro-001, log 2 FC = −1.04).Lastly, the significantly enriched GO term chitin binding (Table 3; GO:0008061) covered transcripts related to several classes of proteins, including mucins, a di-Nacetylchitobiase and chitinase-like proteins, which were all represented by minor fold changes (|log 2 FC| < 1).An overview of selected differentially expressed transcripts encoding proteins previously proposed to play a role in tardigrade stress tolerance is given in Table 4 (see also Supporting Information, Tables S16-S20).Specifically, Table 4 includes 176 transcripts related to HSPs and co-chaperones, eutardigrade-specific proteins and proteins involved in antioxidant defence, DNA repair and membrane transport.The vast majority of these transcripts (i.e.172 transcripts) had minor fold changes (|log 2 FC| < 1), including all transcripts related to eutardigrade-specific proteins (CAHS, MAHS, SAHS and Dsup), antioxidant defence and DNA repair.In addition, most transcripts related to HSPs and co-chaperones had minor fold changes, except for the upregulation of a putative Hsp70 (log 2 FC = 1.70; see Supporting Information, Table S16).Among the 117 transcripts related to membrane transport proteins, including various channels, cotransporters and ATPases, two putative amino acid transporters and a monocarboxylate transporter were highly downregulated (log 2 FC = −1.49to −1.08; see Supporting Information, Table S20).A putative unorthodox aquaporin (AQP11) was the only aquaporin found to be differentially expressed in the osmobiotic tuns, albeit with a minor fold change (log 2 FC = −0.47;see Supporting Information, Table S20).

DISCUSSION
Our data confirm that Ramazzottius species are highly tolerant of extreme changes in external non-ionic osmolyte concentrations (Heidemann et al. 2016).Our gradual exposure experiments show that tun formation in R. varieornatus is initiated at an external osmolality of 0.2 Osmol/kg.For most specimens, full tun formation is acquired at an external osmolality of 0.5 Osmol/kg.We hypothesize that the tardigrades, by an unknown mechanism, sense the decrease in external water potential, which triggers the behavioural response associated with tun formation.This is supported by the apparent upregulation of transcripts related to GABA and glutamate receptors (Table 2).Our data also indicate that the tun state is acquired as external osmolality approaches that of the body fluids, assuming that R. varieornatus has a body fluid osmolality in the range 0.4-0.5 Osmol/kg, as shown for other limnoterrestrial parachelan eutardigrades (Halberg et al. 2013b).Thus, instead of spending energy on hyper-regulating body fluids, as seen in tardigrades in an active state (reviewed by Møbjerg et al. 2018), R. varieornatus contracts into a tun and ceases normal body activity.The acute exposure experiment also revealed that R. varieornatus easily survives direct transfer into 3 Osmol/kg sucrose solution, emphasizing that limnoterrestrial tardigrades do not require any acclimation when exposed to high concentrations of non-ionic osmolytes (Heidemann et al. 2016).
Notably, we provide the first molecular investigation into osmobiotic tun formation by comparing RNA-seq datasets between tuns and active tardigrades.Among the 3322 differentially expressed transcripts, 521 transcripts (15.7%) display a |log 2 FC| > 1, indicating that osmobiotic tun formation is associated with minor changes in transcription.This observation is in line with previous investigations on anhydrobiotic tun formation in Ramazzottius following slow desiccation (Yoshida et al. 2017).As a comparison, little to no transcriptomic change is found in Ramazzottius following rapid desiccation (Hashimoto et al. 2016, Yoshida et al. 2017) and freezing (Møbjerg et al. 2022).As noted above, we see an increase in the transcription of selected neurotransmitter receptors.Interestingly, our results support previous investigations indicating that Hsp70 might play a role in the prevention of protein unfolding and aggregation during dehydration, a common stressor of anhydrobiosis and osmobiosis (Reuner et al. 2010, Schokraie et al. 2011, Møbjerg and Neves 2021).Our data indicate that a ubiquitin-conjugating enzyme is among the most downregulated transcripts in the tuns.Downregulations also involve a CYP transcript and transcripts of putative amino acid and monocarboxylate transporters.An obvious task for future studies is to investigate in more detail when metabolism comes to standstill and when it restarts following osmobiosis and anhydrobiosis, e.g. by measuring oxygen uptake (Pedersen et al. 2020(Pedersen et al. , 2021)).
In summary, our data emphasize that Ramazzottius species are highly tolerant of extreme changes in external non-ionic osmolyte concentrations and that such changes induce osmobiotic tun formation.We also present the first differential expression analysis of osmobiotic tuns, providing a foundation for future investigations into the molecular mechanisms associated with tun formation.

Figure 3 .
Figure3.Activity of Ramazzottius varieornatus after direct transfer to 3 Osmol/kg and subsequent direct return to ultrapure water (A) and during a 72 h exposure to ultrapure water containing small amounts of sediment (B).Tardigrades were pooled into five groups, each containing ~20 specimens.Observed data points (filled circles) represent the proportion of active tardigrades in each group.Horizontal lines illustrate medians, boxes interquartile ranges and whiskers 1.5 × interquartile ranges.Insets illustrate the habitus of the tardigrades at specific time points (cf.Fig.1).
the DESeq2 analysis.BLASTX best hit results of each transcript against NCBI (GB) and the UniProtKB/Swiss-Prot database, and BLASTP best hit results against NCBI (GB) are provided, with the percentage of identity (I), e-value (Eval) and query coverage (QC) within parentheses when available.

Table 1 .
Summary data for RNA samples generated from active control specimens and osmobiotic tuns of Ramazzottius varieornatus.

Table 3 .
Significantly enriched gene ontology (GO) terms as identified by TopGO and listed according to the P-value from topgoFisher (algorithm = 'weighth01').

Table 4 .
Selected differentially expressed transcripts encoding proteins previously proposed to be involved in tardigrade stress tolerance.