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

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.

Fig 1. Study site.

(A) Location of the Duvanny Yar outcrop on the Kolyma River, northeastern Siberia, Russia. https://climate.copernicus.eu/sites/default/files/inline-images/C3S_indicator_sea_ice_sidebar_figS2_branded.png) (https://climate.copernicus.eu/data-protection-and-privacy-statement) (B) View of the upper part of outcrop composed of ice wedges and permafrost silty deposits. (C) Lithostratigraphic scheme of deposits, showing location of studied rodent borrow (red circle). (D) fossil rodent burrow with herbaceous litter and seeds buried in permafrost deposits; m a.r.l. = meters above river level.

Fig 2. General morphology of P. kolymaensis, female.

Scanning electron pictures (A, C), light microscopy photographs (E, F) and graphic presentations (B, D, G) of holotype: A, B) entire body, C, D) anterior ends, E) anterior body, F) perivulvar body region, G) tail. Abbreviations: l.f.–lateral field, ov–ovary, pro–procorpus of the pharynx, t.b.–terminal bulb of the pharynx, u–uterus with eggs, v–vulva, v.p.–ventral pore. Scale bars: A, D, E, F, G– 20 μm, B– 100 μm, C– 2 μm.

Fig 3. Genome assembly and phylogenomics reveals that the newly discovered P. kolymaensis species is triploid.

A) Kmer spectra of the P. kolymaensis PacBio HiFi data. Kmers of length 19 were counted using Jellyfish. B) Circos plot showing the triploid structure of the P. kolymaensis genome. Lines represent the position of 6,715 homeologs in eight contigs that comprise 39.9 Mb (15%) of the assembly. Homeologs were identified by clustering protein-coding genes into orthogroups using OrthoFinder and selecting groups containing three sequences. Contig IDs and scale is shown. C) Inferred species tree for all taxa. The maximum likelihood tree inferred using a concatenated supermatrix (18S and 28S genes) with bootstrap support values is displayed. All genera are represented as monophyletic clades. P. kolymaensis is highlighted in red and basal to all other Panagrolaimus taxa. Internal nodes, where all subsequent branches represent identical sequences, are displayed with a black star. D) P. kolymaensis possesses C. elegans gene orthologs to enzymes required for TCA cycle, glyoxylate shunt, glycolysis, gluconeogenesis, trehalose synthesis, and polyamine synthesis. Black filled circles: Ortholog presence suggested by orthogroup clustering, phylogenetic analysis, and domain architecture. White filled circles: No ortholog found via current analysis. Coloured filled circles: Presence of P. kolymaensis gene(s), related to several C. elegans genes (all genes of same colour) that are all co-orthologous to that gene (those genes). Label: C. elegans enzyme names and orthogroup that contains that gene according to our orthogroup clustering.

Fig 4. C. elegans dauer larvae and P. kolymaensis might utilize similar mechanisms to survive cryptobiosis.

A) Survival rate of P. kolymaensis nematodes to desiccation and freezing (-80°C). Error bars indicate standard error of mean of two independent experiments with two technical replicates performed on two different days. Statistical comparison was performed using unpaired t-test with Welch correction. n.s. p > 0.05, ****p < 0.0001. For desiccation (non-preconditioned) n = 289, freezing (non-preconditioned) n = 675, desiccation (preconditioned at 98%RH) n = 953 and freezing (preconditioned at 98%RH) n = 1295. B) P. kolymaensis nematodes and daf-2(e1370) dauer larvae upregulate trehalose levels upon preconditioning at 98%RH. Error bars indicate standard error of mean of two independent experiments with three replicates performed on two different days. Statistical comparison was performed using two-way ANOVA with Holm-Sidak’s multiple comparison test, ****p < 0.0001. C-D) 2D-thin layer chromatography of ¹⁴C-acetate labelled metabolites from P. kolymaensis that were non-preconditioned and preconditioned at 98%RH. Enumerated spots indicate trehalose (1), glucose (2), glutamate (3), glutamine (4), serine/glycine (5) and phenylalanine (6). Representative images from at least two independent experiments performed on two different days. E) Desiccated daf-2 (e1370) dauer larvae survive to freezing (-80°C) for an extremely long period. Error bars indicate standard error of mean of two independent experiments with two technical replicates performed on two different days. F) Brood size of desiccated dauer larvae exposed to freezing remain like that non- desiccated dauer larvae. Average brood size is the mean of seven dauer larvae per each condition. Statistical comparison was performed by using non-parametric Kolmogorov-smirnov test n.s p>0.05.