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A novel nematode species from the Siberian permafrost shares adaptive mechanisms for cryptobiotic survival with C. elegans dauer larva [1]

['Anastasia Shatilovich', 'Institute Of Physicochemical', 'Biological Problems In Soil Science Ras', 'Pushchino', 'Zoological Institute Ras', 'St. Petersburg', 'Vamshidhar R. Gade', 'Max Planck Institute For Molecular Cell Biology', 'Genetics', 'Dresden']

Date: 2023-08

Abstract Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways that enable the organism to survive for prolonged periods. Recently, nematode individuals have been reanimated from Siberian permafrost after remaining in cryptobiosis. Preliminary analysis indicates that these nematodes belong to the genera Panagrolaimus and Plectus. Here, we present precise radiocarbon dating indicating that the Panagrolaimus individuals have remained in cryptobiosis since the late Pleistocene (~46,000 years). Phylogenetic inference based on our genome assembly and a detailed morphological analysis demonstrate that they belong to an undescribed species, which we named Panagrolaimus kolymaensis. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis in P. kolymaensis and in C. elegans is partly orthologous. We show that biochemical mechanisms employed by these two species to survive desiccation and freezing under laboratory conditions are similar. Our experimental evidence also reveals that C. elegans dauer larvae can remain viable for longer periods in suspended animation than previously reported. Altogether, our findings demonstrate that nematodes evolved mechanisms potentially allowing them to suspend life over geological time scales.

Author summary Survival in extreme environments for prolonged periods is a challenge that only a few organisms, are capable of. It is not well understood, which molecular and biochemical pathways are utilized by such cryptobiotic organisms, and how long they might suspend life. Here, we show that a soil nematode Panagrolaimus kolymaensis, suspended life for 46,000 years in the Siberian permafrost. Through comparative analysis, we find that P. kolymaensis and model organism C. elegans utilize similar adaptive mechanisms to survive harsh environmental conditions for prolonged periods. Our findings here are important for the understanding of evolutionary processes because generation times could be stretched from days to millennia, and long-term survival of individuals of species can lead to the refoundation of otherwise extinct lineages.

Citation: Shatilovich A, Gade VR, Pippel M, Hoffmeyer TT, Tchesunov AV, Stevens L, et al. (2023) A novel nematode species from the Siberian permafrost shares adaptive mechanisms for cryptobiotic survival with C. elegans dauer larva. PLoS Genet 19(7): e1010798. https://doi.org/10.1371/journal.pgen.1010798 Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, UNITED STATES Received: February 17, 2023; Accepted: May 24, 2023; Published: July 27, 2023 Copyright: © 2023 Shatilovich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All the data files are available at https://doi.org/10.5281/zenodo.6590382. Funding: This work was supported by the Russian Foundation for Basic Research (19-29-05003-mk) to AS and ER. VRG and TVK acknowledge the financial support from the Volkswagen Foundation (Life research grant 92847). PHS and TTH are supported by a DFG ENP grant to PHS (DFG project 434028868). GMH is funded by a UCD Ad Astra Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Organisms from diverse taxonomic groups can survive extreme environmental conditions, such as the complete absence of water or oxygen, high temperature, freezing, or extreme salinity. The survival strategies of such organisms include a state known as suspended animation or cryptobiosis, in which they reduce metabolism to an undetectable level [1]. Spectacular examples of long-term cryptobiosis include a Bacillus spore that was preserved in the abdomen of bees buried in amber for 25 to 40 million years [2], and a 1000 to 1500 years-old Lotus seed, found in an ancient lake, that was subsequently able to germinate [3]. Metazoans such as tardigrades, rotifers, and nematodes are also known for remaining in cryptobiosis for prolonged periods [4,5]. The longest records of cryptobiosis in nematodes are reported for the Antarctic species Plectus murrayi [6] (25.5 years in moss frozen at -20°C), and Tylenchus polyhypnus [7] (39 years desiccated in an herbarium specimen). Intensive research during the last decade has demonstrated that permafrost (perennially frozen sediments) are unique ecosystems preserving life forms at sub-zero temperatures over thousands of years [8,9,10,11]. Permafrost remains are an exceptional source for discovering a wide variety of unicellular and multicellular living organisms surviving in cryptobiosis for prolonged periods [1,12,13]. The Siberian permafrost is a unique repository for preserving organisms in sub-zero temperatures for millions of years. Expeditions in the past decade have resulted in the revival of several organisms across various taxa from the Siberian permafrost [14,15,16,17]. The possibility to exploit permafrost as a source for reanimating multicellular animals was recognized as early as 1936. A viable Cladocera crustacean, Chydorus sphaericus, preserved in the Transbaikalian permafrost for several thousand years [18,19], was discovered by P. N. Kapterev, who worked at the scientific station Skovorodino as a GULAG prisoner. Unfortunately, this observation remained unnoticed for many decades. We recently reanimated soil nematodes that were preserved in Siberian permafrost for potentially thousands of years, and initial morphological observations provisionally described them as belonging to the genera Panagrolaimus and Plectus. Previous studies demonstrated several species of Panagrolaimus can undergo cryptobiosis in the form of anhydrobiosis (through desiccation) and cryobiosis (through freezing) [20,21,22,23,24]. In various nematodes, entry into anhydrobiosis is often accompanied by a preparatory phase of exposure to mild desiccation, known as preconditioning [22,25]. This induces a specific re-modelling of the transcriptome, the proteome, and metabolic pathways that enhances survival ability [26,27,28]. Some panagrolaimids possess adaptive mechanisms for rapid desiccation where most of the cellular water is lost, while others possess freezing tolerance without loss of water at sub-zero temperatures by inhibiting the growth and recrystallisation of ice crystals [22]. Here, we present a high-quality genome assembly, detailed morphological phylogenetic analysis, and define a novel species, Panagrolaimus kolymaensis. Precise radiocarbon dating indicates that P. kolymaensis remained in cryptobiosis for about 46,000 years, since the late Pleistocene. Making use of the model organism C. elegans, we demonstrate that C. elegans dauer larvae and Panagrolaimus kolymaensis utilize comparable molecular mechanisms to survive extreme desiccation and freezing, i.e. upregulation of trehalose biosynthesis and gluconeogenesis.

Discussion The new nematode species from permafrost can now be placed into the genus Panagrolaimus [40], which contains several described parthenogenetic and gonochoristic species [34,41]. Many Panagrolaimus display adaptation to survival in harsh environments [22] and the genus includes the Antarctic species P. davidi [23]. The genus Panagrolaimus is exceptional in its morphological uniformity even among nematode species that are hard to classify based on morphology in general. Thus, species designation via microscopic (including SEM) analysis is unreliable, which is further complicated by the absence of males in parthenogenetic species. Males have an important diagnostic feature such as spicules and pericloacal papillae, females differ from one species to another mainly by morphometrics, where interspecies differences (absolute measures and ratios) might be subtle. Our specimens are similar based on absolute sizes and ratios to females of the bisexual species Panagrolaimus detritophagus [42]. The only non-overlapping morphometric character is index “b” (body length: pharynx length): 5.6–6.8 in P. kolymaensis versus 4.4–5.1 in P. detritophagus. Consequently, we turned to phylogenomic methods under the phylogenetic species concept to place the species on the tree. This showed that this species is an outgroup to other known Panagrolaimus species, raising the possibility of a second independent evolution of parthenogenesis in the genus, in contrast to previous findings [34,35,41]. Alternatively, the hybrid origin of parthenogenetic Panagrolaimus could influence the phylogenetic positioning of strains, raising the possibility that the new species is a true sister to the other parthenogenetic strains. To fully resolve the phylogenetic positioning further, extensive sampling, and genome sequencing of Panagrolaimus species is needed. We found P. kolymaensis to be triploid and thus a hybrid origin is possible, as seen in other parthenogenetic Pangrolaimus [34]. The highly contiguous genome of P. kolymaensis will allow for analyses of this trait in comparison to other Panagrolaimus species currently being genome sequenced. Our results provide a deeper insight into the homology of molecular and biochemical mechanisms between C. elegans and P. kolymaensis, which are not only taxonomically but also ecologically distinct. C. elegans can mostly be found in rotting fruits and plants in temperate regions [43,44], while Panagrolaimus species are globally distributed and prevalent in leaf litter and soil [41], including in harsh environments [22]. We show through orthology analysis that the well-studied molecular pathways used by C. elegans larvae to enter the dauer state, such as insulin [45,46] (DAF-11, DAF-2 & DAF-16), TGF-β [47] (DAF-7), steroid [48] (DAF-9, DAF-12) are present in the genome of the P. kolymaensis (S4C Fig). The presence of homologous genes in two species does not necessarily demonstrate their functionality in both. Therefore, further functional analyses are needed to study molecular pathways in detail. Trehalose accumulation (Fig 4B) and depletion of triacylglycerols (S5A and S5B Fig) ensure the functionality of the trehalose biosynthesis pathway and utilization of glyoxylate shunt during desiccation in P. kolymaensis. Without the activity of the enzyme TPS-2 and glyoxylate shunt, it is unfeasible to synthesize trehalose in nematodes. We do not eliminate the possibility of other biochemical features that might contribute to the desiccation survival ability of P. kolymaensis, but with regards to trehalose biosynthesis and the glyoxylate shunt, our data suggest that the molecular tool kit is partially orthologous. In our future studies, we intend to perform RNAi-based experiments to infer the concrete mechanisms. Our results hint at convergence or parallelism in the molecular mechanisms organizing dauer formation and cryptobiosis. As mentioned above, preconditioning enhances the survival of P. kolymaensis by rendering them desiccation tolerant. We previously reported that preconditioning elevates trehalose biosynthesis in C. elegans dauer larvae and the elevated trehalose renders desiccation tolerance by protecting the cellular membranes [25]. It is not surprising that P. kolymaensis upregulates trehalose, however the magnitude of trehalose elevation is higher than C. elegans dauer larvae. This indicates that central regulators (DAF-16, DAF-12) of trehalose upregulation may differentially regulate tps-2 in P. kolymaensis [38,49,50]. Although P. kolymaensis utilizes the glyoxylate shunt and gluconeogenesis to upregulate trehalose levels, it is intriguing to observe that they accumulate substantial levels of trehalose-6-phosphate. Further investigation of this observation using RNAi or inhibitor-based experiments will provide insights into molecular mechanisms of metabolic regulation in P. kolymaensis upon preconditioning. Our findings for the first time demonstrate that C. elegans dauer larvae possess an inherent ability to survive freezing for prolonged periods if they undergo anhydrobiosis. It is tempting to speculate that undergoing anhydrobiosis might be a survival strategy of C. elegans to survive the seasonal changes in nature. In summary, our findings indicate that by adapting to survive cryptobiotic state for short time frames in environments like permafrost, some nematode species gained the potential for individual worms to remain in the state for geological timeframes. This raises the question of whether there is an upper limit to the length of time an individual can remain in the cryptobiotic state. Long timespans may be limited only by drastic changes to the environment such as strong fluctuations in ambient temperature, natural radioactivity, or other abiotic factors. These findings have implications for our understanding of evolutionary processes, as generation times may be stretched from days to millennia, and long-term survival of individuals of species can lead to the refoundation of otherwise extinct lineages. This is particularly interesting in the case of parthenogenetic species, as each individual can find a new population without the need for mate finding, i.e. evading the cost of sex. Finally, understanding the precise mechanisms of long-term cryptobiosis and cues that lead to successful revivals can inform new methods for long term storage of cells and tissues.

Methods Materials and C. elegans strains [1-14C] -acetate (sodium salt) was purchased from Hartmann Analytic (Braunschweig, Germany). All other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany). The Caenorhabditis Genetic Centre (CGC) which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) provided daf-2(e1370) and E. coli NA22 strains. Genomic DNA isolation from P. kolymaensis nematodes After isolation (S1 Text), to ensure our strain P. kolymaensis (Pn2-1) can adapt to different laboratories, we grew them for multiple generations. The strain was in culture for several generations during genomic DNA isolation and was frozen after genomic DNA isolation was performed. P. kolymaensis nematodes (isofemale strain Pn2-1) were grown on several plates of NGM agar plated with E. coli NA22 bacteria at 20°C. Worms were collected from the plates, washed with water at least three to five times by centrifugation at 1000 g to remove any residual bacteria and any debris. The worm pellet was dissolved in 5 volumes of worm lysis buffer (0.1M Tris-HCl pH = 8.5, 0.1M NaCl, 50mM EDTA pH = 8.0) and distributed in 1.5 ml of microcentrifuge tubes. These tubes are incubated at -80°C for 20 minutes. 100 μl of Proteinase ‘K’ (20 mg/ml) was added to each tube and they are incubated at 60°C overnight. 625 μl of cold GTC buffer (4M Guanidinium Thiocynate, 25mM Sodium citrate, 0.5% (v\v) N-lauroylsarcosine, 7%(v/v) Beta Mercaptoethanol) was added to the tube, incubated on ice 30 min, and mixed by inverting every 10 min. 1 volume of phenol–chloroform-isoamyl alcohol (pH = 8) was added to the lysate and mixed by inverting the tube 10–15 times. Tubes were centrifuged for 5 min at 10,000 g at 4°C to separate the phases. The upper aqueous phase was carefully collected into a fresh tube. One volume of fresh chloroform was added and mixed by inverting the tubes for 10–15 times and centrifuged for 5 min at 10,000 g at 4°C to separate the phases. One volume of cold 5 M NaCl was added, mixed by inverting the tubes and incubated on ice for 15 min. After incubation these tubes were centrifuged for 15 min at 12,000–16,000 g at 4°C. The supernatant containing the nucleic acids were slowly transferred into a fresh tube. One volume of isopropanol was added to the tube, inverted few times, and incubated on ice for 30 minutes. After incubation, the tubes were centrifuged at 3000 g for 30–45 min at 25°C and the supernatant was discarded without disturbing the pellet. The pellet was washed twice with 1 ml of 70% ethanol, tubes were centrifuged at 3000 g for 5 min and supernatant was discarded and incubated at 37°C for 10–15 min to dry the pellet. The pellet was resuspended carefully in TE buffer. The quality of the genomic DNA was analyzed with pulse field gel electrophoresis. Genome sequencing and assembly The long insert library was prepared as recommended by Pacific Biosciences according to the ‘Procedure & Checklist-Preparing gDNA Libraries Using the SMRTbellExpress Template Preparation Kit 2.0’ protocol. In summary, RNAse treated HMW gDNA was sheared to 20 kb fragments on the MegaRuptordevice (Diagenode) and 10 μg sheared gDNA was used for library preparation. The PacBio SMRTbell library was size selected in two fractions (9-13kb, > 13kb) using the BluePippin device with cassette definition of 0.75% DF MarkerS1 3–10 kb Improved Recovery. The second fraction of the size-selected library was loaded with 95 pM on plate on a Sequel SMRT cell (8M). Sequel polymerase 2.0 was used in combination with the v2 PacBio sequencing primer and the Sequel sequencing kit 2.0EA, with a runtime of 30 hours. We created PacBio CCS reads from the subreads.bam file using PacBio’s ccs command linetool (version4.2.0), outputting 8.5Gb of high-quality CCS reads (HiFi reads N50 of 14.4 kb). HiCanu (version 2.2) [51] was used to create the contig assembly. Blobtools [52] (version 1.1.1) was used to identify and remove bacterial contigs. The final triploid contig assembly consists of 856 contigs has a N50 of 3.82 Mb and a size of 266Mb. The mitochondrial genome was created with the mitoHifi pipeline (version 2, https://github.com/marcelauliano/MitoHiFi based on the assembled contigs and the closely related reference mitochondrial genome of Panagrellus redivivus (strain: PS2298/MT8872, ENAaccession: AP017464). The mitoHifi pipeline identified 49 mitochondrial contigs ranging from 13-32Kb. The final annotated circular mitochondrial genome has a length of 17467 bp. To identify pseudohaplotypes in the P. kolymaensis genome assembly, we selected the longest isoform of each predicted protein-coding gene in our assembly and in the C. elegans genome (downloaded from WormBase Parasite, release WBPS15) using AGAT (version 0.4.0) and clustered them into orthologous groups (OGs) using OrthoFinder (version 2.5.2). We identified OGs that contained three Panagrolaimus sequences (i.e. groups that were present as single-copy in all three pseudohaplotypes) and used these to identify trios of multi-megabase size contigs derived from the three pseudohaplotypes. Synteny between the three pseudohaplotypes was visualized using Circos to plot the positions of each homeolog (version 0.69–8). Genome annotation RepeatModeler 1.0.8 (http://www.repeatmasker.org/) was used with parameter ‘-engine ncbi’ to create a library of repeat families which was used with RepeatMasker 4.0.9 to soft-mask the Panagrolaimus genome. To annotate genes, we cross mapped protein models from an existing Panagrolaimus as external evidence in the Augustus based pipeline. The completeness of our predictions was evaluated using BUSCO on the gVolante web interface. Orthology analysis We conducted a gene orthology analysis using genomic data from P. kolymaensis, the Plectid nematode species from the permafrost, as well as genomic data from WormBase Parasite (https://parasite.wormbase.org; accessed 17/12/2020): Caenorhabditis elegans, Diploscapter coronatus, Diploscapter pachys, Halicephalobus mephisto, Panagrellus redivivus, Panagrolaimus davidi, Panagrolaimus sp. ES5, Panagrolaimus sp. PS1159, Panagrolaimus superbus, Plectus sambesii, and Propanagrolaimus sp. JU765. For plectids, genomic resources are scarce. We therefore added transcriptome data of Plectus murrayi, Anaplectus granulosus, Neocamacolaimus parasiticus, and Stephanolaimus elegans, with the latter three transcriptomes kindly provided by Dr. Oleksandr Holovachov (Swedish museum of natural history). Transcriptomes for Anaplectus granulosus, and Neocamacolaimus parasiticus have been published and are readily available [53,54]. All three transcriptomes were assembled de novo with Trinity [55]. The exact procedures are described in the respective publications [53,54]. The Stephanolaimus elegans transcriptome was assembled using the same methodologies as Neocamacolaimus parasiticus. The Plectus murrayi transcriptome was built from raw reads deposited at NCBI (https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos2/sra-pub-run-13/SRR6827978/SRR6827978.1; accessed 22.12.2020) and assembled using Galaxy Trinity version 2.9.1 [55,56]. All default options were used including in silico normalization of reads before assembly. Transdecoder (conda version 5.5.0) [57] was used to translate to amino acid sequence. Identical reads were removed with cd-hit version 4.8.1 [58,59], with shorter isoforms removed using the Trinity get_longest_isoform_seq_per_trinity_gene.pl command [57] (Trinity conda version 2.8.5; Anaconda Software Distribution, Conda, Version 4.9.2, Anaconda, Nov. 2020). Amino acid translations of the longest isoforms were extracted with AGAT (Dainat, https://www.doi.org/10.5281/zenodo.3552717) from genome assembly FASTA files and genome annotation GFF3 files using the ‘agat_convert_sp_gxf2gxf.pl’, ‘agat_sp_keep_longest_isoform.pl’ and ‘agat_sp_extract_sequences.pl’ scripts, respectively. All FASTA headers were modified to allow for simple species assignment of each sequence in subsequent analysis. Orthology analysis was conducted with OrthoFinder v. 2.5.1 [60,61] using default settings. For genes of interest, we constructed alignments with MAFFT v. 7.475 [62] using the localpair and maxiterate (1000) functions. Spurious sequences and areas that were not well aligned were removed with Trimal v. 1.4.rev22 [63] (procedure stated in S1 Orthology analysis below each phylogeny). We then ran phylogenetic analysis with Iqtree2 v. 2.0.6 [64], with -bb 1000 option, testing the model for each analysis (models eventually used stated in S1 Orthology analysis). PFAM domains were explored using Interproscan v. 5.50–84.0 [65]. The phylogenies were visualized with Dendroscope 3.7.6 [66] and figures were created with Inkscape (https://inkscape.org). Most of our analysis was performed on the HPC RRZK CHEOPS of the Regional Computing Centre (RRZK) of the University of Cologne. Phylogenomics Sequences of 18S and 28S genes from 44 taxa across the Propanagrolaimus, Panagrolaimus, Panagrellus and Halicephalobus genera (all listed in S1 Text were aligned (MAFFT L-INS-I v7.475) [62], concatenated [67]and used to infer a species tree using maximum likelihood via (IQTREE) [68] and partitioned by best-fit models of sequence evolution for both [69]. Nodal support was determined using 1000 bootstrap pseudoreplicates. A further 60 genes from 101 taxa (all listed in S1 Text) were used to confirm the taxonomic position using the supermatrix concatenation methods outlined above. Given the limitations of differential gene sampling, we expanded our phylogenomic analyses to include a coalescence approach using 12,295 ML gene trees inferred for orthogroups containing the target animal. Instances of multiple genes per species per group were treated as paralogs/orthologs and analysed using ASTRAL-Pro [70]. Given the number of copies of genes per orthogroup, we explored whether auto or alloploidy was the source of extra genes observed using the gene-tree reconciliation approach implemented in GRAMPA (Gene-tree Reconciliation Algorithm with MUL (Multi labelled)-trees for Polyploid Analysis) [71]. All gene trees rooted at the midpoint and the final ASTRAL-pro species tree were used as inputs, with the most parsimonious result analyzed further. Desiccation survival assay C. elegans dauer larvae desiccation assays were performed as described in [25]. P. kolymaensis desiccation assays were performed similarly as described in [25] with mixed population (Mixture of all larval stages and adults) of the nematodes. Exposure of nematodes to extreme environments C. elegans dauer larvae or mixed population (Mixture of all larval stages and adults) of P. kolymaensis nematodes were preconditioned and desiccated as described in [28], then transferred to elevated temperature of 34°C, freezing (-80°C) and anoxia. Anoxic environment was generated in a desiccation chamber at 60%RH by flushing the Nitrogen gas into the chamber. The concentration of oxygen inside the chamber was monitored. After each timepoint they were rehydrated with 500 μl of water for 2–3 hours. Rehydrated worms were transferred to NGM agar plates with E. Coli NA22 as food. Survivors were counted after overnight incubation at 15°C. Each experiment was performed on two different days with at least two technical replicates. Trehalose quantification from nematode lysates Trehalose measurements were performed as described in previous reports [27]. Radiolabeling, metabolite extraction and 2D-TLC The above-mentioned procedures were performed according to previous reports [27,28]. Identification of trehalose-6-phosphate from TLC plates Normalized aqueous fractions from the non-preconditioned and preconditioned samples were separated by high performance thin layer chromatography (HPTLC), using 1-propanol-methanol-ammonia (32%)-water (28:8:7:7 v/v/v/v) as first, dried for 15 min and 1-butanol-acetone-glacial acetic acid–water (35:35:7:23 v/v/v/v) second dimension respectively. Using the trehalose as a standard on both dimensions of the TLC, the regions of interest were scrapped out from the TLCs. The scraped-out silica was extracted with 10 ml of 50% methanol twice. The fractions were combined, dried under vacuum and dissolved in 100 μl of MS mix solution containing 4:2:1 (Isopropanol:Methanol:Chloroform) with 7.5 mM ammonium formate. Mass spectrometric analysis was performed on a Q Exactive instrument (Thermo Fischer Scientific, Bremen, DE) equipped with a robotic nanoflow ion source TriVersa NanoMate (Advion BioSciences, Ithaca, USA) using nanoelectrospray chips with a diameter of 4.1 μm. The ion source was controlled by the Chipsoſt 8.3.1 soſtware (Advion BioSciences). Ionization voltage was + 0.96 kV in negative mode; backpressure was set at 1.25 psi. The temperature of the ion transfer capillary was 200°C; S-lens RF level was set to 50%. FT MS spectra were acquired within the range of m/z 50–750 at the mass resolution of R m/z 200 = 140000; automated gain control (AGC) of 3×106 and with the maximal injection time of 3000 ms. FT MS/MS spectra were acquired within the range of m/z 50–750 at the mass resolution of R m/z 200 = 140000; automated gain control (AGC) of 3×104 and with a maximal injection time of 30 s. Triacylglycerols measurement from P. kolymaensis lysates Non-preconditioned and preconditioned pellets were lysed in 200 μl of isopropanol with 0.5 mm Zircornium beads twice for 15 min. The lysates were centrifuged at 1300 g for 5 min. The supernatant was carefully collected without any debris, 20 μl of the lysate was used for protein estimation. Normalization was performed according to soluble protein levels, supernatant volumes corresponding to 50–100 μg of proteins were dried in the desiccator. 700 μl of IS ((10:3 (Methyl tert-butyl ether: ethanol)) mix (warmed to room temperature) was added to dried samples and left on the shaker for 1 hour. The samples were centrifuged at 1400 rpm and 4°C. 140 μl of water was added and left on the shaker for 15 min. These samples were centrifuged at 13400 rpm for 15 min. The upper organic fraction was collected and transferred to 1.5 ml glass vial and left for drying in the desiccator. The dried samples were reconstituted in a volume of 300 μl of 4:2:1 (Isopropanol:Methanol:Chloroform). Volume corresponding to 1 μg was used for injection. LC-MS/MS analysis was performed on a high-performance liquid chromatography system (Agilent 1200 HPLC) coupled to a Xevo G2-S QTof (Waters). The samples were resolved on a reverse phase C18 column (Cortecs C18 2.7um from Waters) with 50:50:0.1:1% (Water:Methanol:Formicacid:1MAmmoniumformate) and 25:85:0.1:1% (Acetonitrile:Isopropanol:Formic acid:1M Ammonium formate) as mobile phase. The following gradient program was used: Eluent B from 0% to 100% within 12 min; 100% from 12 min to 17min; 0% from 17 min to 25 min. The flow rate was set at 0.3 ml/min. The samples were normalised according to the total protein concentration and the worm numbers. TAG 50:00:00 was used as internal standard. Skylinesoftware (https://skyline.ms/project/home/software/Skyline/begin.view) was used to analyse the raw data. TAGs were extracted from Lipidmaps (https://www.lipidmaps.org/) database.

Acknowledgments VG is thankful to Andrej Shevchenko, members of the Volkswagen grant and Kurzchalia lab for helpful discussions and the core facilities of MPI-CBG for assistance. We are grateful to Dr. S. Gubin for field study and sampling, our colleagues in Soil Cryology Lab, Pushchino and North-East Scientific Station in Chersky, Republic of Sakha (Yakutia) for their help and cooperation. The authors thank Long Read Team of the DRESDEN-concept Genome Center, DFG NGS Competence Center, part of the Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden and MPI-CBG. We thank https://www.copernicus.eu/en for the base map in Fig 1A. The authors are thankful to Richard Roy and Jens Bast for critically reading the manuscript. We thank Iain Pattern for suggestions on writing the manuscript.

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