Figures
Abstract
Tardigrades can survive hostile environments such as desiccation by adopting a state of anhydrobiosis. Numerous tardigrade species have been described thus far, and recent genome and transcriptome analyses revealed that several distinct strategies were employed to cope with harsh environments depending on the evolutionary lineages. Detailed analyses at the cellular and subcellular levels are essential to complete these data. In this work, we analyzed a tardigrade species that can withstand rapid dehydration, Ramazzottius varieornatus. Surprisingly, we noted an absence of the anhydrobiotic-specific extracellular structure previously described for the Hypsibius exemplaris species. Both Ramazzottius varieornatus and Hypsibius exemplaris belong to the same evolutionary class of Eutardigrada. Nevertheless, our observations reveal discrepancies in the anhydrobiotic structures correlated with the variation in the anhydrobiotic mechanisms.
Citation: Galas S, Le Goff E, Cazevieille C, Tanaka A, Cuq P, Baghdiguian S, et al. (2024) A comparative ultrastructure study of the tardigrade Ramazzottius varieornatus in the hydrated state, after desiccation and during the process of rehydration. PLoS ONE 19(6): e0302552. https://doi.org/10.1371/journal.pone.0302552
Editor: Marcos Rubal García, UMinho CBMA: Universidade do Minho Centro de Biologia Molecular e Ambiental, PORTUGAL
Received: September 7, 2023; Accepted: April 7, 2024; Published: June 6, 2024
Copyright: © 2024 Galas 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 relevant data are within the manuscript.
Funding: This research was supported by the funding CNRS “Défi Origines 2018” – Project GigaTardi (grant no. 265880 – Giga18). A part of the present study was supported by the funding UM I-Site « Soutien à la recherche 2023 - programme d'excellence I-SITE », Volet « Recherche exploratoire » Université de Montpellier - France 2030 (grant no. PE23PR01-RC02-DSUPTOWORM).
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Tardigrades are tiny metazoan animals that range in size from approximately 0.1–1.2 mm and have four pairs of legs [1]. They can be called “water bears” because of their appearance and “moss piglets” because of where they can be found. Nearly 1500 tardigrades species have thus far been described [2], which are distributed from the depths of the oceans to the highest mountain peaks [3]. The worldwide distribution of tardigrade species can be either endemic or cosmopolitan [4–8], and their transport by birds or snails has recently been suggested [9–11].
As earlier as their discovery in the 18th century [12], tardigrades have demonstrated an ability to adopt a latent state due to a shortage of water, which is called anhydrobiosis. These tardigrades, rather terrestrial species, can enter an anhydrobiosis state in response to desiccation to form an anhydrobiote, allowing the organism to wait for the return of water [13, 14]. Thus, by reaching nearly complete desiccation, tardigrades can survive for many years as anhydrobiotes [15–18]. During the course of desiccation, tardigrades contract and retract their whole body to assume a characteristic “tun”-shaped anhydrobiote structure. Tardigrades can then lose up to 97% of their bound and free body water content [19].
Other invertebrates, such as rotifers, nematodes and dipteran larvae [12, 20–23], can enter anhydrobiosis and some of them are also tolerant to other extreme physical stresses. For example, the nematode Panagrolaimus superbus displays tolerance to ultra-low temperature (-196°C), X-radiations (500Gy) or ultracentrifugation (400,000xg) [24] and bdelloids rotifers are able to withstand high doses of ionizing radiation, up to 1000 Gy [25, 26]. Likewise, Adineta vaga is known for its resistance to X-ray, protons and Fe ions [27]. However the particularity of the tardigrade is that it resists a more extensive set of stresses. Indeed, tardigrades are resistant to temperatures ranging from -272 to +150°C [28, 29], very high pressures (up to 7.5 Gpa) equivalent to that at a depth of up to 180 km from the Earth’s surface [30], radiation at levels up to 5000 Gy [31–33] and exposure to solar radiation at a low Earth orbit in a space vacuum during a ten-day space flight [34]. These characteristics make them an emerging model for space biology [35].
To date, the genomes of four tardigrade species are available [36]. The genomes of two Eutardigrada species: Ramazzottius varieornatus (Ram. varieornatus) and Hypsibius exemplaris (Hys. exemplaris) [37–39], enabled the identification of gene products involved in anhydrobiosis. For instance, the Dsup (damage suppressor) gene was identified in Ram. Varieornatus and was suggested to protect both human and plant cells from gamma ray irradiation [31, 38, 40] as well as human cultured cells from oxidation by free radicals [31, 38]. The molecular capacity of the Dsup gene products to protect nucleosomes from direct oxidation by hydroxyl radicals was thereafter evidenced by an in vitro assay [41].
While Ram. varieornatus is tolerant to a rapid desiccation process (minutes), Hys. exemplaris can undergo effective anhydrobiosis after only an obligate preconditioning period (hours) [14, 36, 42].
Accordingly, both species show contrasting gene expression in response to desiccation [36, 43]. Thus, while Ram. varieornatus is believed to express anhydrobiosis involved genes constitutively, Hys. exemplaris requires a de novo expression induction of orthologs genes [36]. To date, tardigrade species belonging to the Heterotardigrada class seem to lack bona fide Dsup orthologs [41].
It has been shown [44] that tardigrades belonging to the Eutardigrada class, such as Ram. varieornatus and Hys. exemplaris, also possess genes [38, 45, 46] encoding proteins that are involved in resistance to desiccation stress, such as the cytosolic abundant heat-soluble (CAHS), secretory abundant heat-soluble (SAHS), late embryogenesis abundant mitochondrial (RvLEAM), and mitochondrial abundant heat-soluble (MAHS) proteins. These intrinsically disordered proteins (IDPs) are involved in the maintenance of cellular structures during desiccation processes [38, 46–51].
Species of the Eutardigrada possess specific genes involved in stress resistance that differ from those of Heterotardigrada, but some discrepancies have also been reported among eutardigrade species. For example, Ram. varieornatus possesses a trehalose-6-phosphate synthase gene, while Hys. exemplaris does not [43]. Trehalose-6-phosphate synthase can produce the nonreducing sugar trehalose [52], which has been proposed to play a role in mediating desiccation tolerance in some organisms, such as Caenorhabditis elegans (Maupas, 1899) [53], Saccharomyces cerevisiae (Meyen, 1838) [54] and chironomids, by vitrifying their cellular contents. However, other desiccation-tolerant invertebrates, such as rotifers, do not require this sugar [55–58] and the presence of the trehalose is still unclear in tardigrades [59–63].
To date, few reports have attempted to describe the ultrastructures of anhydrobiotic organisms [64–66]. Halberg et al. [67] described the tun morphology of the Richtersius coronifer with an emphasis on muscular organization, while Czernekova et al. [68, 69] investigated the internal morphologies of dehydrated organs, tissues and cells in the same species. Poprawa et al. [14] characterized ultrastructure of storage cells in tuns of Hys. exemplaris.
In a previous report [70], we used electron microscopy to compare hydrated specimens and anhydrobiotic tuns of Hys. exemplaris. We highlighted deep modifications occurring up to the subcellular level in the anhydrobiote and during the course of exit from anhydrobiosis. We also uncovered the materialization of an anhydrobiote-specific and reversible extracellular structure [70].
In the present study, we studied the structures and ultrastructures of the cells and organelles of anhydrobiotic Ram. varieornatus specimens by electron microscopy and compared them to the ultrastructures of active hydrated specimens. Finally, we compared strategies used by the Eutardigrada species Ram. varieornatus and Hys. exemplaris to resist anhydrobiosis.
2. Materials and methods
2.1 Materials
The Yokozuna-1 strain of the extremotolerant Ram. varieornatus (Bertolani and Kinchin, 1993) [71], (Eutardigrada, Hypsibiidae), provided by Takekazu Kunieda (University of Tokyo), was used for all experiments. Tardigrades were cultured as previously described [29]. They were fed with the unicellular algae Chlorella vulgaris (Beijerinck, 1890) [72] (strain A60) on 2% Bacto agar plates prepared with Volvic water and incubated at 20°C under constant dark conditions. Algae were purchased from the Sciento Company (Manchester, UK).
2.2 Desiccation protocol
Twenty specimens in a drop of water were placed on a filter paper inside Petri dish, which were left at room temperature (20–22°C) and relative humidity (RH) (between 30–36%) for one hour. To confirm good dehydration, the dessication process of the specimens was monitored by direct observation under a stereomicroscope in order to assure that the tardigrades underwent a proper anhydrobiosis process and formed tuns. We therefore control size reduction and the total absence of movement. The anhydrobionts were stored at 20°C and at room RH (between 30–36%) in an incubator for one week before analysis.
2.3 Rehydration protocol
To rehydrate the desiccated Ram. varieornatus after one week of dehydration, Volvic water droplets were added to the filters. Tardigrades were maintained in water at room temperature (20–22°C) and prepared for TEM after 5 and 15 minutes of contact with liquid. Specimens of Ram. varieornatus start to move around 5 minutes after rehydration.
2.4 Transmission electron microscopy
According to Richaud et al. [70], samples were fixed in 2.5% glutaraldehyde in PHEM buffer (1X, pH 7.4) overnight at 4°C, rinsed in PHEM buffer and postfixed in 0.5% osmic acid for 2 hours in the dark at room temperature. After two rinses in PHEM buffer, samples were dehydrated in a graded series of ethanol (30–100%) and embedded in EmBed 812 using an automated microwave tissue processor for electronic microscopy (Leica EM AMW). Ultrathin sections (70 nm; Leica-Reichert Ultracut E) were collected from different levels of each block, counterstained with 1.5% uranyl acetate in 70% ethanol and lead citrate and observed using a Tecnai F20 transmission electron microscope at 200 kV at the CoMET MRI facilities (INM, Montpellier, France). For TEM, five tardigrades were analyzed for each condition: tuns, rehydrated for 5 minutes, rehydrated for 15 minutes and hydrated.
2.5 Tardigrade size
We measured the sizes of hydrated and tun tardigrades from the tip of the head to the extreme end of the body without legs. Measurements from DIC images obtained using a Zeiss LSM880 Fast Airyscan confocal microscope at the DBS-Optique MRI facilities (Montpellier, France) were determined with ImageJ software. For each condition, five specimens were measured.
2.6 Number of nuclei
We counted the number of nuclei in hydrated and tuned animals. Tardigrades were fixed for 30 min with 4% paraformaldehyde (PFA) in mineral water (Volvic) and then permeabilized for 20 min at room temperature with 0.2% Triton X-100 in PBS buffer (Amresco, Inc.). Fixed and permeabilized tardigrades were labeled with DAPI (Euromedex 1050-A) at 1 μg/mL in PBS for 30 min at room temperature. Finally, the specimens were rinsed several times with PBS and mounted in Dako. Counts from DAPI z-stack stained images obtained using a Zeiss LSM880 Fast Airyscan confocal microscope at the DBS-Optique MRI facilities (Montpellier, France) were determined with ImageJ software. For each condition, hydrated and dehydrated tardigrades, the number of nuclei was counted in five animals, i.e. 10 tardigrades in total.
2.7 Mitochondrial size
We measured the mitochondrial sizes under every condition: tuns, rehydrated for 5 minutes, rehydrated for 15 minutes and hydrated. For each condition, mitochondria were observed in each cell types and in the same proportions to avoid sampling bias. The sizes of 150, 110, 136 and 140 mitochondria were measured in each group, respectively, using ImageJ software. Mitochondria were measured in cross-sections through and through on the longer side.
2.8 Statistical analysis
According to Richaud et al. [70], we used XLSTAT software (Addinsoft, New York, NY, USA) to compare mitochondrial sizes among animals that were dehydrated, rehydrated for 5 or 15 minutes and hydrated and to compare the body sizes and numbers of nuclei between hydrated and dehydrated animals.
3. Results
3.1. Comparison of hydrated and anhydrobiotic Ramazzottius varieornatus
3.1.1. Cell compaction of anhydrobiotic tardigrades.
We first used confocal laser microscopy with differential interference contrast (DIC) to obtain a global view of the external morphologies of hydrated specimens versus anhydrobiotic tuns. In parallel, we used the DNA-specific dye DAPI to enable nuclei counting. Fig 1A and 1C shows representative images of the characteristic contraction in anhydrobiotic tun compared to hydrated specimen. Length measurements of hydrated specimens revealed an average size of 153 +/- 17 μm, while the anhydrobiotic tuns showed an average size of 104 +/- 29 μm (Fig 1E), revealing a size reduction of 32% (Fig 1E). However, staining the nuclei of both the hydrated (Fig 1B) and anhydrobiotic groups (Fig 1D) with DAPI revealed a nonsignificant t difference in the total cell counts (Fig 1F).
varieornatus: (a-d) Confocal microscopy images with DIC (a and c) and DAPI (b and d) staining. (e-f) Statistics on body sizes and nucleus numbers. (e) Error bars indicate the standard deviation and the star indicates a significant difference (Kolmogorov-Smirnov test, p = 0.048; α = 0.05). (f) Error bars indicate the standard deviation.
3.1.2. Comparative analysis of cell structure and ultrastructure.
Hydrated individuals showed a large space between cells, named extracellular space (ecs) (Fig 2A). Sizes of this space throughout the body of the tardigrade were not similar. Epidermal cells, bordered by the cuticle, were clearly visible together with numerous pigmented vesicles (Fig 2C). Muscle cells possessed long fibers with long dark mitochondria (Fig 2E), and digestive cells appeared with long villosities (Fig 2G). Additionally, numerous lipid droplets were observed inside the cells regardless of the cell type.
(a, b) Overview of a body part. (c, d) Ultrastructure of epidermal cells. (e, f) Ultrastructure of muscle cells. (g, h) Ultrastructure of digestive cells. (i, j) Extracellular space overview. Cut: cuticle, DC: digestive cell, EC: epidermal cell, MC: muscle cell, m: mitochondria, mv: microvilli, p: pigment, ecs: extracellular space.
We also observed ecs (Fig 2B) features in the anhydrobiotic tun group (Fig 2B). We observed a space between the epidermal cells and the cuticle (Fig 2D). The global structure of this cell type was not affected by dehydration (Fig 2D) except a cell compaction. Indeed, epidermal cells appear to be thinner in tuns (Fig 2D). This may be due to dehydration-induced compaction. Conversely, muscular fibers were less discernible in the anhydrobiotic tun group than in the hydrated group because of compaction (Fig 2F). Similar to the hydrated tardigrades, the digestive cells of the anhydrobiotic group exhibited long villosities (Fig 2H), and lipid droplets were still present. No apoptotic cells were observed in any of the specimens.
In both the hydrated specimens and anhydrobiotic tun groups, dot-like structures were observable in the ecs (Fig 2I and 2J).
The hydrated specimens and anhydrobiotic groups showed comparable organelle structures, with numerous mitochondria being observed in both groups. Moreover, the mitochondrial structures were comparable (Fig 3A and 3C vs 3B and 3D) and not degraded. Surprisingly, the mitochondrial cristae in the anhydrobiotic tun group were comparable to those in hydrated animals (Fig 3C and 3D); however, a statistically significant size reduction of 24% (Fig 3E) was observed in mitochondria of the anhydrobiotic tun group compared with the hydrated specimens group. We observed many mitochondria with atypical shapes in the anhydrobiotic tun group (Fig 3B and 3D) that were not observable in the hydrated specimen group (Fig 3A and 3C).
(a-d) Transmission electron microscopy images. (e) Mean mitochondrial size. The error bars indicate the standard deviation, and the stars indicate significant differences (Student’s t-test, α = 0.05). Table 1 shows the complete statistical results. (f, g) Histogram of the mitochondrial size frequencies of hydrated tardigrades (f) and desiccated tardigrades (g). m: mitochondria.
Fig 3F and 3G shows the size distribution frequencies of up to 140 and 150 mitochondria measured in both the hydrated and anhydrobiotic tun groups respectively.
3.2. Temporal change in anhydrobiotic tuns during rehydration
To better understand the functional structures of stress-resistant anhydrobiotic tuns, we assessed the ultrastructural changes in the anhydrobiotic tun group over the course of rehydration. Because anhydrobiotic tuns take only a few minutes (10–20 minutes) to wakeup (size recovery and detectable movements) from dehydration, they were dehydrated for one week, rehydrated for 5 and 15 minutes and then assessed by TEM.
3.2.1. Rehydration of anhydrobiotic tuns for 5 minutes.
After 5 minutes of rehydration, Ram. varieornatus specimens begin to move and become active. We observed a size evolution that fell between that of the hydrated and anhydrobiotic specimens. Following this observation, we again noticed persistence of the anhydrobiotic state with decoupling between the epidermal cells and the cuticle (Fig 4A). Moreover, the global structure of the epidermal cells containing the already described vesicles was maintained (Fig 4A versus Fig 2C and 2D), and muscle cells exhibited a normal structure with long contractile fibers (Fig 4C). Gut cells also showed a normal ultrastructure compared to those of the hydrated group (Fig 4E versus Fig 2G and 2H). The mitochondrial size was intermediate between those in the anhydrobiotic and hydrated groups of tardigrades (Fig 5B and 5E). Mitochondrial size differences between the anhydrobiotic and hydrated groups were evident based on their size frequency distributions (S1 Fig). More than 110 mitochondria were assessed, and their intermediate sizes were also confirmed by statistical analysis (Table 1). We observed a higher concentration of mitochondria around muscle fibers. Previously described lipid droplets were still observable inside the cells (data not shown) as were the dot-like structures in the es (Fig 4G).
(a, b) Ultrastructure of epidermal cells. (c, d) Ultrastructure of muscle cells. (e, f) Ultrastructure of digestive cells. (g, h) Extracellular space overview. cut: cuticle, DC: digestive cell, EC: epidermal cell, MC: muscle cell, m: mitochondria, mv: microvilli, p: pigment, ecs: extracellular space.
(a–d) Transmission electron microscopy images. Scale bar a-d = 500 nm. (e) Mean mitochondrial sizes. The error bars indicate the standard error of the mean. <*> indicates a significant difference at p < 0.05 (Student’s t-test, α = 0.05). <***> indicates a significant difference at p < 0.0001 (Student’s t-test, α = 0.05). See Table 1 for all of the statistical results. m: mitochondria.
3.2.2. Rehydration of anhydrobiotic tuns for 15 minutes.
Fifteen minutes after rehydration, Ram. varieornatus have a normal activity (mobility, food consumption, movements) and are fully active. The tardigrade size was already comparable to that of the hydrated group. In agreement with this observation, the epidermal cells recovered contiguously with the cuticle (Fig 4B), and the muscle cells appeared as classical long fibers, like in the hydrated group (Fig 4D versus Figs 2E, 2F and 4E). In addition, the digestive cells showed a normal structure compared with that in the hydrated group (Fig 4F versus Figs 2G, 2H and 4E). Moreover, the mitochondrial sizes were equivalent to those in the hydrated control group (Fig 5C and 5E). This was shown by evaluating the size frequency distribution of up to 136 mitochondria (S1 Fig) and confirmed by statistical analysis (Table 1). Finally, the lipid droplets, previously described in other conditions were still present, as were the dot-like structures in the ecs (Fig 4H).
4. Discussion
Ramazzottius varieornatus can cope with rapid dehydration and is known to be one of the most resilient to desiccation among the limno-terrestrial tardigrades [36, 38]. However, no information on internal reorganization during anhydrobiosis is available.
We have previously reported [70] that upon desiccation, Hys. exemplaris shows active secretory cells that are closely related to a specific and reversible extracellular structure surrounding each cell. This specific extracellular structure and the accompanying secretory cells disappear during rehydration, implying their direct association with resistance to dehydration stress. However, Hys. exemplaris is more sensitive to desiccation than Ram. varieornatus [38, 43, 44] and thus requires preconditioning steps to achieve successful anhydrobiosis [42, 47, 73].
Fig 6 summarizes their ultrastructural divergences during the dehydration process and anhydrobiote formation. Contrary to Hys. exemplaris [70], no secretory cells with a dense network of endocytoplasmic reticulum were found in Ram. varieornatus.
Abstract design: Laurence Meslin CNRS, ISEM 2023.
The presence of active secretory cells during the formation of the Hys. exemplaris tuns was suggested to be associated with the production of a specific extracellular structure surrounding each cell [70]. In agreement with this observation, we were unable to detect this specific extracellular structure in the tuns (Fig 2) of the Ram. varieornatus. Moreover, ecs is always present in Ram. varieornatus, anhydrobionts and hydrated specimens, and its pre-existence could explain the high capacity of Ram. varieornatus to resist anhydrobiosis stress, its speed to reach the tun stage without preconditioning and its speed of return from anhydrobiotic state with an active form.
The shape of mitochondria in Ram. varieornatus and Hys. exemplaris tuns cells represents another divergent ultrastructural characteristic between the two species (Fig 5). Compared to those in the hydrated groups, the anhydrobiotic mitochondria of Hys. exemplaris exhibited a reduced size (15%) and a decreased cristae size [70], while those of Ram. varieornatus showed a slightly greater size reduction (24%) but comparable cristae (Fig 5A versus 5D). This cristae size difference between Ram. varieornatus and Hys. exemplaris may explain the respiration reactivation and faster anhydrobiotic exit of Ram. varieornatus anhydrobionts.
In the present study, we have shown that the internal ultrastructures of individual Ram. varieornatus anhydrobionts are slightly different from those in active hydrated individuals, which contrasts with a previous report showing the neosynthesis of a specific extracellular structure associated with deep internal ultrastructural modifications in anhydrobiotic Hys. exemplaris individuals compared to hydrated individuals [70].
It is possible that the removal of the specific extracellular structure from desiccated Hys. exemplaris during the anhydrobiosis exit may slow the entire rehydration process, while Ram. varieornatus, lacking a detectable equivalent anhydrobiosis-specific ultrastructure, may not be influenced by the same way during the rehydration.
In summary, the desiccation process of Ram. varieornatus does not appear to be equivalent to that of Hys. exemplaris. These differences may at least partially explain the significant differences in desiccation resistance between both species.
Supporting information
S1 Fig. Distribution of mitochondrial size frequency depending on the tardigrade status.
https://doi.org/10.1371/journal.pone.0302552.s001
(TIF)
Acknowledgments
We thank the Montpellier MRI-RIO Imaging platform (MRI-DBS Optique and MRI-CoMET) (Montpellier, France).
References
- 1.
Kinchin I. The biology of tardigrades. London: Portland Press; 1994.
- 2. Degma P, Guidetti R. Actual checklist of Tardigrada species. 2023.
- 3. Nelson DR. Current status of the tardigrada: evolution and ecology. Integr Comp Biol. 2002;42: 652–659. pmid:21708761
- 4. Gąsiorek P, Vončina K, Michalczyk Ł. Echiniscus testudo (Doyère, 1840) in New Zealand: anthropogenic dispersal or evidence for the "Everything is Everywhere" hypothesis? New Zealand Journal of Zoology. 2019;46: 174–181.
- 5. Kaczmarek Ł, Mioduchowska M, Kačarević U, Kubska K, Parnikoza I, Gołdyn B, et al. New records of antarctic tardigrada with comments on interpopulation variability of the Paramacrobiotus fairbanksi Schill, Förster, Dandekar and Wolf, 2010. Diversity. 2020;12: 108.
- 6. Kayastha P, Stec D, Sługocki Ł, Gawlak M, Mioduchowska M, Kaczmarek Ł. Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Sci Rep. 2023;13: 2196. pmid:36750641
- 7.
McInnes S, Pugh P. Tardigrade Biogeography. In Water bears: the biology of tardigrades, in: Schill R. O., Springer International Publishing; 2018. pp. 115–129. https://doi.org/10.1007/978-3-319-95702-9_4
- 8. Morek W, Surmacz B, López-López A, Michalczyk Ł. "Everything is not everywhere": time-calibrated phylogeography of the genus Milnesium (Tardigrada). Mol Ecol. 2021;30: 3590–3609. pmid:33966339
- 9. Mogle MJ, Kimball SA, Miller WR, McKown RD. Evidence of avian-mediated long distance dispersal in American tardigrades, PeerJ. 2018;6: e5035. pmid:30018851
- 10. Robertson MW, Russo NJ, McInnes SJ, Goffinet B, Jiménez JE. Potential dispersal of tardigrades by birds through endozoochory: evidence from sub-antarctic white-bellied seedsnipe (Attagis malouinus), Polar Biol. 2020;43: 899–902.
- 11. Książkiewicz Z, Roszkowska M. Experimental evidence for snails dispersing tardigrades based on Milnesium inceptum and Cepaea nemoralis species. Sci Rep. 2022;12: 4421. pmid:35422107
- 12. Spallanzani L. Opuscoli di Fisica Animale e Vege Tipografica, Modena. 1776: 203–285.
- 13. Kaczmarek Ł, Roszkowska M, Fontaneto D, Jezierska M, Pietrzak B, Wieczorek R, et al. Staying young and fit? Ontogenetic and phylogenetic consequences of animal anhydrobiosis. Journal of Zoology. 2019;309: 1–11.
- 14. Poprawa I, Bartylak T, Kulpla A, Erdmann W, Roszkowska M, Chajec Ł, et al. Verification of Hypsibius exemplaris Gąsiorek et al., 2018 (Eutardigrada; Hypsibiidae) application in anhydrobiosis research. PLOS ONE. 2022;17: e0261485. pmid:35303010
- 15. Goldstein B, Blaxter M. Tardigrades. Curr Biol. 2002;12: R475. pmid:12176341
- 16. Roszkowska M, Kmita H, Kaczmarek Ł. Long-term anhydrobiosis in two taxa of moss dwelling Eutardigrada (Tardigrada) desiccated for 12 and 15 years, respectively. The European Zoological Journal. 2020;87: 642–647.
- 17. Tsujimoto M, Imura S, Kanda H. Recovery and reproduction of an antarctic tardigrade retrieved from a moss sample frozen for over 30 years. Cryobiology. 2016;72: 78–81. pmid:26724522
- 18. Wełnicz W, Grohme MA, Kaczmarek L, Schill RO, Frohme M. Anhydrobiosis in tardigrades-the last decade. J Insect Physiol. 2011;57: 577–583. pmid:21440551
- 19. Crowe JH. Anhydrobiosis: an unsolved problem. The American Naturalist. 1971;105: 563–573.
- 20. Bertolani R, Guidetti R, Jönsson IK, Altiero T, Boschini D, Rebecchi L. Experiences with dormancy in tardigrades. Journal of Limnology. 2004;63: 16–25.
- 21. Crowe J. The physiology of cryptobiosis in Tardigrades, in: Memorie Dell’Istituto Italiano Di Idrobiologia, Dott. Marco de Marchi Verbania Pallanza. 1975. pp. 37–59.
- 22. Doyère LMF. Mémoire sur les Tardigrades. Sur la faculté que possèdent les tardigrades, les rotifères, les anguillules des toits, et quelques autres animalcules, de revenir à la vie après avoir été complètement desséchés. Ann Sci Nat. 1842;18: 5–35.
- 23. Rebecchi L, Altiero T, Guidetti R. Anhydrobiosis: the extreme limit of desiccation tolerance. Invertebrate Survival Journal. 2007.
- 24. Souza TD, Carli GD, Pereira T. Survival potential of the anhydrobiotic nematode Panagrolaimus superbus submitted to extreme abiotic stresses. Invertebrate Survival Journal. 2017;14: 85–93.
- 25. Gladyshev E, Meselson M. Extreme resistance of bdelloid rotifers to ionizing radiation. PNAS. 2008;105 (13): 5139–5144. pmid:18362355
- 26. Hespeels B, Fontaneto D, Cornet V et al. Back to the roots, desiccation and radiation resistances are ancestral characters in bdelloid rotifers. BMC Biol. 2023;21: 72. pmid:37024917
- 27. Hespeels B, Penninckx S, Cornet V et al. Iron Ladies–How Desiccated Asexual Rotifer Adineta vaga Deal With X-Rays and Heavy Ions? Front. Microbiol. 2020;11: 1792. pmid:32849408
- 28. Hengherr S, Worland MR, Reuner A, Brümmer F, Schill RO. High-temperature tolerance in anhydrobiotic tardigrades is limited by glass transition. Physiol Biochem Zool. 2009;82: 749–755. pmid:19732016
- 29. Horikawa D, Kunieda T, Abe W, Watanabe M, Nakahara Y, Yukuhiro F, S et al. Establishment of a rearing system of the extremotolerant tardigrade Ramazzottius varieornatus: a new model animal for astrobiology. Astrobiology. 2008;8: 549–56. pmid:18554084
- 30. Ono F, Mori Y, Sougawa M, Takarabe K, Hada Y, Nishihira N, et al. Effect of very high pressure on life of plants and animals. J. Phys.: Conf. Ser. 2012;377: 012053.
- 31. Hashimoto T, Kunieda T. DNA protection protein, a novel mechanism of radiation tolerance: Lessons from tardigrades. Life (Basel). 2017;7: 26. pmid:28617314
- 32. Jönsson KI. Radiation tolerance in tardigrades: Current knowledge and potential applications in medicine. Cancers (Basel). 2019;11: 1333. pmid:31505739
- 33. Jönsson KI, Harms-Ringdahl M, Torudd J. Radiation tolerance in the eutardigrade Richtersius coronifer. Int J Radiat Biol. 2005;81: 649–656. pmid:16368643
- 34. Jönsson KI, Rabbow E, Schill RO, Harms-Ringdahl M, Rettberg P. Tardigrades survive exposure to space in low Earth orbit. Current Biology. 2008;18: R729–R731. pmid:18786368
- 35. Guidetti R, Rizzo AM, Altiero T, Rebecchi L. What can we learn from the toughest animals of the Earth? Water bears (tardigrades) as multicellular model organisms in order to perform scientific preparations for lunar exploration. Planetary and Space Science. 2012;74: 97–102.
- 36. Yoshida Y, Tanaka S. Deciphering the biological enigma—Genomic evolution underlying anhydrobiosis in the phylum Tardigrada and the Chironomid Polypedilum vanderplanki. Insects. 2022;13: 557. pmid:35735894
- 37. Arakawa K, Yoshida Y, Tomita M. Genome sequencing of a single tardigrade Hypsibius dujardini individual. Sci Data. 2016;3: 160063. pmid:27529330
- 38. Hashimoto T, Horikawa DD, Saito Y, Kuwahara H, Kozuka-Hata H, Shin-I T, et al. Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nat Commun. 2016;7: 12808. pmid:27649274
- 39. Koutsovoulos G, Kumar S, Laetsch DR, Stevens L, Daub J, Conlon C, et al. No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini. Proceedings of the National Academy of Sciences. 2016;113: 5053–5058. pmid:27035985
- 40. Kirke J, Jin XL, Zhang XH. Expression of a tardigrade Dsup gene enhances genome protection in plants. Mol Biotechnol. 2020;62: 563–571. pmid:32955680
- 41. Chavez C, Cruz-Becerra G, Fei J, Kassavetis GA, Kadonaga JT. The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals. ELife. 2019;8: e47682. pmid:31571581
- 42. Kondo K, Kubo T, Kunieda T. Suggested involvement of PP1/PP2A activity and de novo gene expression in anhydrobiotic survival in a tardigrade, Hypsibius dujardini, by chemical genetic approach. PLOS ONE. 2015;10: e0144803. pmid:26690982
- 43. Yoshida Y, Koutsovoulos G, Laetsch DR, Stevens L, Kumar S, Horikawa DD, et al. Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus. PLOS Biology. 2017;15: e2002266. pmid:28749982
- 44. Kamilari M, Jørgensen A, Schiøtt M, Møbjerg N. Comparative transcriptomics suggest unique molecular adaptations within tardigrade lineages. BMC Genomics. 2019;20: 607. pmid:31340759
- 45. Tanaka S, Tanaka J, Miwa Y, Horikawa DD, Katayama T, Arakawa K, et al. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic tardigrade improve osmotic tolerance of human cells. PLOS ONE. 2015;10: e0118272. pmid:25675104
- 46. Yamaguchi A, Tanaka S, Yamaguchi S, Kuwahara H, Takamura C, Imajoh-Ohmi S, et al. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade. PLOS One. 2012;7: e44209. pmid:22937162
- 47. Boothby TC, Tapia H, Brozena AH, Piszkiewicz S, Smith AE, Giovannini I, et al. Tardigrades use intrinsically disordered proteins to survive desiccation. Mol Cell. 2017;65: 975–984.e5. pmid:28306513
- 48. Kasianchuk N, Rzymski P, Kaczmarek Ł. The biomedical potential of tardigrade proteins: A review. Biomedicine & Pharmacotherapy. 2023;158: 114063. pmid:36495665
- 49. Krakowiak M, Bartylak T, Kmita H, Kaczmarek Ł, Nawrot R. Tardigrade proteins: molecular tools in the phenomenon of anhydrobiosis. Zoological Journal of the Linnean Society. 2024;200: 269–281.
- 50. Tanaka A, Nakano T, Watanabe K, Masuda K, Honda G, Kamata S, et al. Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel. PLOS Biology. 2022;20: e3001780. pmid:36067153
- 51. Tanaka S, Aoki K, Arakawa K. In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada. Proceedings of the National Academy of Sciences. 2023;120: e2216739120. pmid:36693101
- 52. Yoshida Y, Nowell R, Arakawa K, Blaxter M. Horizontal gene transfer in Metazoa: Examples and methods. 2019: 203–226.
- 53. Maupas E. La mue et l’enkystement chez les nématodes. Arch. Zool. Exp. Gen. 1899;7: 563–628.
- 54. Meyen J. Jahresbericht uber die Resultate der Arbeiten im Felde der physiologischen Botanik von dem Jahre 1837. Arch. Naturgesch. 1838;4: 1–186.
- 55. Erkut C, Penkov S, Khesbak H, Vorkel D, Verbavatz JM, Fahmy K, Kurzchalia TV. Trehalose renders the dauer larva of Caenorhabditis elegans resistant to extreme desiccation. Current Biology. 2011;21: 1331–1336. pmid:21782434
- 56. Lapinski J, Tunnacliffe A. Anhydrobiosis without trehalose in bdelloid rotifers. FEBS Lett. 2003;553: 387–390. pmid:14572656
- 57. Sakurai M, Furuki T, Akao KI, Tanaka D, Nakahara Y, Kikawada T, et al. Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplanki. Proc Natl Acad Sci U S A. 2008;105: 5093–5098. pmid:18362351
- 58. Tapia H, Koshland DE. Trehalose is a versatile and long-lived chaperone for desiccation tolerance. Curr Biol. 2014;24: 2758–2766. pmid:25456447
- 59. Cesari M, Altiero T, Rebecchi L. Identification of the trehalose-6-phosphate synthase (tps) gene in desiccation tolerant and intolerant tardigrades. Italian Journal of Zoology. 2012;79.
- 60. Guidetti R, Altiero T, Rebecchi L. On dormancy strategies in tardigrades. J Insect Physiol. 2011;57: 567–576. pmid:21402076
- 61. Hengherr S, Heyer AG, Köhler HR, Schill RO. Trehalose and anhydrobiosis in tardigrades-evidence for divergence in responses to dehydration. FEBS J. 2008;275: 281–288. pmid:18070104
- 62. Jönsson KI, Persson O. Trehalose in three species of desiccation tolerant tardigrades. The Open Zoology Journal. 2010;3: 1–5.
- 63. Westh P, Ramløv H. Trehalose accumulation in the tardigrade Adorybiotus coronifer during anhydrobiosis. Journal of Experimental Zoology. Part A: Ecological Genetics and Physiology (Print Edition). 1991;258: 303–311.
- 64. Bird AF, Buttrose MS. Ultrastructural changes in the nematode Anguina tritici associated with anhydrobiosis. J Ultrastruct Res. 1974;48: 177–189. pmid:4843794
- 65. Dickson MR, Mercer EH. Fine structural changes accompanying desiccation in Philodina roseola (Rotifera). J. Microsc. 1967;6: 331–348.
- 66. Morris JE. Dehydrated cysts of Artemia salina prepared for electron microscopy by totally anhydrous techniques. J Ultrastruct Res. 1968;25: 64–72. pmid:4179173
- 67. Halberg KA, Jørgensen A, Møbjerg N. Desiccation tolerance in the tardigrade Richtersius coronifer relies on muscle mediated structural reorganization. PLOS ONE. 2013;8: e85091. pmid:24391987
- 68. Czerneková M, Jönsson KI, Chajec L, Student S, Poprawa I. The structure of the desiccated Richtersius coronifer (Richters, 1903). Protoplasma. 2017;254: 1367–1377. pmid:27677802
- 69. Czerneková M, Janelt K, Student S, Jönsson KI, Poprawa I. A comparative ultrastructure study of storage cells in the eutardigrade Richtersius coronifer in the hydrated state and after desiccation and heating stress. PLOS ONE. 2018;13: e0201430. pmid:30096140
- 70. Richaud M, Le Goff E, Cazevielle C, Ono F, Mori Y, Saini NL, et al. Ultrastructural analysis of the dehydrated tardigrade Hypsibius exemplaris unveils an anhydrobiotic-specific architecture. Sci Rep. 2020;10: 4324. pmid:32152342
- 71. Bertolani R, kinchin IM. A new species of Ramazzottius (Tardigrada, Hypsibiidae) in a rain gutter sediment from England. Zool. J. Linn. Soc. 1993;109: 327–333.
- 72. Beijerinck MW. Culturversuche mit Zoochlorellen, Lichenengonidien und anderen niederen Algen. Botanische Zeitung. 1890;47: 725–739, 741–754, 757–768, 781–785.
- 73. Wright JC. Desiccation tolerance and water-retentive mechanisms in tardigrades. Journal of Experimental Biology. 1989;142: 267–292.