research on water bears

January 17, 2024

Cute Little Tardigrades Are Basically Indestructible, and Scientists Just Figured Out One Reason Why

Tardigrades are microscopic animals that can survive a host of conditions that are too extreme to ever occur on Earth—and scientists want to learn their secrets

By Meghan Bartels

This tardigrade, imaged by scanning electron microscope, is less than 0.1 millimeter across.

Steve Gschmeissner/Science Photo Library

Tiny tardigrades have three claims to fame: their charmingly pudgy appearance, their delightful common names (water bear and moss piglet), and their stunning resilience in the face of threats such as the vacuum of space and temperatures near absolute zero. “They’re masters of protecting themselves,” says Derrick Kolling, a chemist at Marshall University.

Now Kolling and his colleagues have identified a key mechanism contributing to tardigrades’ toughness : a kind of molecular switch that triggers a hardy dormant state. It’s just one piece of the complex system the minuscule creatures use to survive harsh circumstances, but the researchers hope the new work, published in the journal PLOS ONE , will encourage further investigations. “It’s opened up a whole huge repertoire of experiments we can now pursue,” says study co-author Leslie Hicks, a chemist at the University of North Carolina at Chapel Hill.

The research began when, on a whim, Kolling put a tardigrade into a machine that detects “free radicals,” or atoms and molecules that contain unpaired electrons. An animal’s normal metabolic processes, as well as environmental stressors such as smoke and other pollutants, can create free radicals inside cells, so he thought it was likely that tardigrades would also produce such molecules.

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When they accumulate, free radicals—most notably, reactive forms of oxygen—snatch electrons from their surroundings to achieve stability in a process known as oxidation. This reaction can damage cells and compounds within the body. But in small quantities, free radicals can also act as signaling molecules, Hicks says, and her laboratory studies show that they affect a cell’s behavior when they glom on to and pop off of a variety of proteins.

When Kolling told Hicks about seeing free radicals in a tardigrade, she wondered whether these molecules could have anything to do with the animal’s hardiness. The team devised several experiments to temporarily expose water bears to stress-inducing, free-radical-producing conditions, such as high levels of salt, sugar and hydrogen peroxide. Under these forms of stress, tardigrades curl up into a temporary, protective state of dormancy called a tun. The researchers monitored the conditions in which the tardigrades hunkered down and found that free radicals did seem to induce the tun state, although the mechanism was not yet clear.

Hicks studies signaling interactions between free radicals and the amino acid cysteine, and she decided to test whether that molecule could play a part in tun formation. So she and her colleagues introduced the tardigrades to different kinds of molecules known to block cysteine oxidation. Under stressful conditions, with cysteine unavailable to the free radicals being produced, the tardigrades couldn’t form tuns—pointing to cysteine oxidation as a required mechanism.

Kazuharu Arakawa, a biologist who studies tardigrades at Keio University in Japan, says the new work aligns with previous research; an earlier study showed the role of oxidation in a midge known for withstanding total desiccation (the process of drying out). The similarities suggest the mechanism may be a common trigger for tuns and other forms of hardy dormancy, a phenomenon that scientists call cryptobiosis.

But water bears still hold many mysteries. When they enter the tun state, they temporarily shut down their metabolism—a feat that even cysteine oxidation can’t explain, says Hans Ramløv, a comparative animal physiologist at Roskilde University in Denmark. “There is no single study to date that explains it,” he says. “In my opinion, we are far, far from understanding that.”

Kolling and Hicks agree that there’s much more research to be done to understand how free radicals work in tardigrades. The resilient tun state isn’t the only tactic water bears use to survive environmental stress, and the team plans to scrutinize other strategies. The researchers also aim to study various species of tardigrades (they examined only Hypsibius exemplaris ), and they expect to find that cysteine oxidation is widely used among the animals.

Hicks hopes that in the long run the work can inform investigations of aging and space travel, which both involve free-radical damage to vital cellular machinery such as DNA and proteins.

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Why NASA sent tiny water bears into space

A new experiment aboard the International Space Station ( ISS ) is studying tardigrades , tiny creatures also known as water bears because of the way they look under a microscope. Tardigrades can tolerate the hottest and coldest environments on Earth, and can survive decades without water. The new experiment – called Cell Science-04 – aims to identify the genes involved in water bears’ abiliy to survive and adapt to high-stress environments, including the one astronauts experience in space. NASA said scientists hope the findings can help guide research into protecting humans from the stresses of long-duration space travel.

The tardigrades arrived at the ISS on June 5, 2021, via the SpaceX Dragon cargo spacecraft. But the Cell Science-04 water bears aren’t the first tardigrades to visit space. In 2007, a European research team sent 3,000 living tardigrades into Earth orbit for 12 days on the outside of a FOTON-M3 rocket ( 68% of them survived .). This time, the water bears will live onboard, inside special science hardware that lets scientists carry out long-term studies of cultures of cells, tissues, and microscopic animals in space by allowing real-time, remote monitoring, and control over the tardigrades’ living conditions.

Microscopic water bears to help protect astronaut health

Tardigrades possess superpowers when it comes to surviving really harsh conditions . They can live for up to 60 years, can survive for up to 30 years without food or water, and are able to endure temperature extremes of up to 150 degrees Celsius (300 degrees F), the deep sea and even the microgravity and elevated radiation levels of space. Water bears survive extreme conditions by going into a state of suspended animation. Essentially, water bears can dry up and survive for years without water, carried around on the wind. When they do come into contact with water, they revive and get on with their lives as though nothing happened.

The Cell Science-04 experiment will help reveal how tardigrades do it.

University of Wyoming biologist Thomas Boothby is principal investigator of the experiment. Boothby said in a statement :

We want to see what ‘tricks’ they use to survive when they arrive in space, and, over time, what tricks their offspring use. Are they the same or do they change across generations? We just don’t know what to expect.

Boothby said that one option in the tardigrade bag of tricks could be producing tons more antioxidants to combat harmful changes in the body caused by increased radiation exposure in space. He said :

We have seen them do this in response to radiation on Earth, and we think the ways tardigrades have evolved to withstand extreme environments on this planet may also be what protects them against the stresses of spaceflight.

NASA said in a statement :

The research team will look at what happens with tardigrade genes in space. Knowing which ones turn on or off in response to short-term and long-term spaceflight will help researchers identify specific ways tardigrades use to survive in this stressful environment. If one solution they have is to turn up the dial on antioxidant production, for example, genes involved in that process should be affected. Checking which genes are also activated or deactivated by other stresses will help pinpoint the genes that respond exclusively to spaceflight. Cell Science-04 will then test which are truly required for tardigrade adaptation and survival in this high-stress environment.

In the long run, NASA said, revealing what makes tardigrades so tolerant could lead to ways of protecting biological material, such as food and medicine, from extreme temperatures, drying out and radiation exposure, which will be invaluable for long-duration deep-space exploration missions.

Bottom line: A new experiment aboard the ISS is studying tardigrades – aka water bears – to better understand how they tolerate extreme environments, including the one astronauts experience in space.

Eleanor Imster

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New Research

Water Bears Are the Master DNA Thieves of the Animal World

Foreign genes from bacteria, fungi and plants may have bestowed these animals with their ability to tolerate boiling, freezing and the vacuum of space

Rachel Nuwer

Tardigrades are animals that thrive in extremes. Also known as water bears or moss piglets, the aquatic, microscopic invertebrates can survive freezing and boiling temperatures as well as the harsh conditions of outer space. A dried-out tardigrade can be reanimated just by adding water—even decades later. They’re found on every continent including Antarctica, and they live in environments ranging from the deepest ocean trenches to the hottest deserts to the tops of the Himalaya.

Now scientists have discovered that tardigrades possess yet another extreme claim to fame: Their genome contains the most foreign DNA of any animal species known.

Rather than inheriting all of their genes from their ancestors, tardigrades get a whopping one-sixth of their genetic makeup from unrelated plants, bacteria, fungi and archaeans, researchers report today in PNAS . The bizarre mashup highlights the fact that species can take shape in much less linear ways that commonly imagined.

“When most people think of the diversity of life and flow of genetic information, they picture a tree with big branches generating smaller ones, but without any connection between the limbs,” says study leader Thomas Boothby , a Life Sciences Research Foundation postdoctoral fellow at the University of North Carolina, Chapel Hill. “We’re beginning to realize that instead of the tree of life, it might be more appropriate to think of the web of life.”

Boothby turned to the tardigrade genome in the hopes of uncovering the most basic underpinnings of the creatures’ extreme survival strategies. To catalog every gene, he and his colleagues first extracted and sequenced many short chunks of DNA from thousands of tardigrades. Using a computer program, they stitched those sequences back together to produce the code in its entirety.

“When we did that, we initially saw that there were a lot of genes that looked like they didn’t come from animals,” Boothby says. “Our gut reaction was that we messed something up and must have contaminated our sample.”

To double check, the team turned to the polymerase chain reaction, a method that amplifies targeted regions of genetic material only if they match with specific primers. In this case, they wanted to see if they could amplify animal and bacterial genes as single units, which would only be possible if they were physically linked within the same genome. “We did that for over 100 genes, with 98-percent success,” Boothby says.

Convinced their reading of the genome was correct, the team then reconstructed the evolutionary ancestry of specific gene sequences. This confirmed that what looked like foreign genes actually were just that, rather than look-a-likes developed by tardigrades themselves.

“The results told us pretty unambiguously that genes that look foreign really are coming from non-animals,” Boothby says.  

All told, the tardigrade genes are made of 17.5 percent foreign material. Most of those strange genes have bacterial origins—thousands of species are represented within the tardigrade’s genetic makeup. Many of the genes are known or suspected to play roles in stress tolerance for their original owners.

“I think the findings are extremely surprising,” says Andrew Roger , a biologist at Dalhousie University in Canada. That an animal could acquire such a large proportion of its genes from foreign sources is “amazing and unprecedented.”

In some cases, foreign genes have actually replaced tardigrade ones, while in others, tardigrades kept their own versions but incorporated single or multiple copies from one or several bacteria species. “We speculate that this wasn’t a one-time event, but probably was ongoing and may still be happening today,” Boothby says. 

Researchers have known for years that bacteria and other microbes can engage in horizontal gene transfer—the swapping of genetic material between unrelated species. But only recently have scientists begun to realize that this method of genetic development can also occur in animals.

Compared to tardigrades, other animals’ genomes, including humans, contain very little foreign material. Until now, rotifers—another microscopic aquatic animal—held the record at 8 to 9 percent. For tardigrades and rotifers, the heavy dose of foreign genes likely plays a significant role in bestowing them with superior survival skills.

“If they can acquire DNA from organisms already living in stressful environments, they may be able to pick up some of the same tricks,” Boothby says. But precisely how tardigrades managed to cobble together so much foreign genetic material remains unknown.

Boothby and his colleagues suspect that the animals’ ability to dry out and reanimate might play a role. When tardigrades desiccate, their genomes fragment. After life-giving liquid restores them, the membranes surrounding their cells remain leaky for a while, and as the cells quickly work to repair their own genomes, they may accidentally work in some DNA from the environment.

“This paper confirms the importance of the study of the whole genome, here applied to an unusual but very interesting and often-neglected animal model,” says Roberto Bertolani , an evolutionary zoologist at the University of Modena and Reggio Emilia in Italy.

“One interesting point that the authors make is the possible relationship between desiccation, membrane leakiness and DNA breakages that may predispose these animals to incorporate and integrate many foreign genes.”

For now that’s just a hypothesis, so Boothby plans to investigate this and other lingering questions. His work with this extreme creature could even give humans a better shot at survival: Studying tardigrade genes may one day aid development of pharmaceuticals and vaccines that no longer have to be kept on ice and instead can be dried out and reanimated on the spot in a rural clinic or crisis zone. 

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Rachel Nuwer | | READ MORE

Rachel Nuwer is a freelance science writer based in Brooklyn.

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• Published: 20 September 2016

Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein

• Takuma Hashimoto 1   na1 ,
• Daiki D. Horikawa 1 , 2 , 3   na1 ,
• Yuki Saito 1 ,
• Hirokazu Kuwahara 1 , 4 ,
• Hiroko Kozuka-Hata 5 ,
• Tadasu Shin-I 6 ,
• Yohei Minakuchi 7 ,
• Kazuko Ohishi 6 ,
• Ayuko Motoyama 7 ,
• Tomoyuki Aizu 7 ,
• Atsushi Enomoto 8 ,
• Koyuki Kondo 1 ,
• Sae Tanaka 1 ,
• Yuichiro Hara 9 ,
• Shigeyuki Koshikawa 10 , 11 ,
• Hiroshi Sagara 5 ,
• Toru Miura 10 ,
• Shin-ichi Yokobori 12 ,
• Kiyoshi Miyagawa 8 ,
• Yutaka Suzuki 13 ,
• Takeo Kubo 1 ,
• Masaaki Oyama 5 ,
• Yuji Kohara 6 ,
• Asao Fujiyama 7 , 14 ,
• Kazuharu Arakawa 3 ,
• Toshiaki Katayama 15 ,
• Atsushi Toyoda 7 &
• Takekazu Kunieda 1  

Nature Communications volume  7 , Article number:  12808 ( 2016 ) Cite this article

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• Animal physiology
• DNA-binding proteins
• DNA damage and repair

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus , one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼ 40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.

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Introduction.

Tardigrades, also known as water bears, are tiny aquatic animals having four pairs of legs 1 . More than 1,000 species have been reported from various habitats such as marine, fresh water or limno-terrestrial environments. All tardigrades require surrounding water to grow and reproduce, but some species—typically those living in the limno-terrestrial environments—have the ability to tolerate almost complete dehydration. When encountering desiccation, tolerant tardigrades lose body water and enter a contracted dehydrated state called anhydrobiosis, which is a reversible ametabolic state. The dehydrated tardigrades withstand a wide range of physical extremes that normally disallow the survival of most organisms, such as extreme temperatures (from −273 °C 2 to nearly 100 °C 3 , 4 ), high pressure (7.5 GPa) 5 , immersion in organic solvent 4 , 6 , exposure to high dose of irradiation 7 , 8 and even direct exposure to open space 9 . Although such unusual tolerance of some tardigrades has long fascinated researchers, the molecular mechanisms enabling such exceptional tolerance have remained largely unknown.

Recently, a finding was reported by a research group at the University of North Carolina (UNC) claiming the presence of extensive horizontal gene transfer (HGT) in a tardigrade genome (17.5% of genes have foreign origin) as a potential basis of tolerant ability, based on their own draft genome assembly of a freshwater tardigrade, Hypsibius dujardini (N50=15.9 kb; hereafter referred to as the UNC assembly) 10 . In contrast, another research group offered a counterargument, suggesting that a substantial portion of the UNC assembly were derived from contaminating microorganisms 11 . There is also a significant discrepancy between the estimated genome size of the species (80–110 Mbp) 11 and the span of the UNC assembly (212.3 Mb), which could be explained by the presence of contaminating sequences at least partially. It is controversial whether extensive HGT is real or an inaccurate interpretation of contaminating sequences. Contaminating sequences substantially affect genome analyses, leading to misinterpretation of the gene repertoire in the target organisms, as well as poor assembly or even chimeric misassembly. Metagenomic approaches could be used to identify putative contaminating sequences based on sequence similarity to phylogenetically distant taxa 11 , but possible misidentification and erroneous elimination from the assembly may lead to a biased representation of the gene repertoire for the target organism. A bona fide tardigrade genome sequence largely free from contamination is therefore needed.

The possible contribution of foreign genes was discussed in the presumed tolerant ability of the sequenced species, H. dujardini 10 . However, freshwater tardigrades, including H. dujardini , are among the least tolerant members of the phylum Tardigrada and H. dujardini cannot withstand exposure to low humidity conditions without a long pre-exposure to high-humidity conditions 12 , 13 . Furthermore, no data have been reported for their tolerability against extreme stress in a dehydrated state, although they exhibit some tolerance to radiation in a hydrated state 14 . The controversial extensive HGT was thoroughly examined in the poorly tolerant H.dujardini , but no other gene repertoire analysis has been reported for tardigrades. Therefore, the genomic basis for the exceptional tolerance of tardigrades remains to be elucidated.

To this end, we conducted a precise genome analysis using one of the most stress-tolerant tardigrade species, R. varieornatus , which tolerates direct exposure to low-humidity conditions and withstands various extremes in the dehydrated state 4 , 15 . We determined a high-quality genome sequence largely free from contamination that allows us to precisely analyse the gene repertoire, such as the proportion of HGT, and characteristic gene expansion or deletion. We also analysed the gene expression profiles during dehydration and rehydration. Furthermore, we focused on the abundantly expressed tardigrade-unique genes and present evidence for the relevance of tardigrade-unique proteins to tolerability, based on our investigation of the effect of a novel tardigrade-unique DNA-associating protein on DNA protection and radiotolerance in human cultured cells.

High-quality genome sequence of extremotolerant tardigrade

R. varieornatus is an extremotolerant tardigrade species, which becomes almost completely dehydrated on desiccation ( Fig. 1a,b ) and withstands various physical extremes 4 . The genome sequence of R. varieornatus was determined by using a combination of the Sanger and Illumina technologies ( Supplementary Table 1 ). To minimize microbial contamination we cleansed egg surfaces with diluted hypochlorite and before sampling the tardigrades were starved and treated with antibiotics for 2 days. After the removal of short scaffolds (<1 kb) and mitochondrial sequences, we obtained the assembly spanning 56.0 Mbp (301 scaffolds). Coverage analysis (160 × Illumina sequencing) revealed that 199 scaffolds (99.7% in span) had considerable coverage (>40), whereas 102 scaffolds had exceptionally low coverage (<1; Supplementary Fig. 1 and Supplementary Data 1 ). We considered these 102 scaffolds (153 kb in span) as derived from contaminating organisms and excluded them from our assembly. As a result, our final assembly spans 55.8 Mbp (199 scaffolds; N50=4.74 Mbp; N90=1.3 Mbp; Supplementary Table 2 ). The span is highly concordant with the genome size estimated by DNA staining in the tardigrade cells ( ∼ 55 Mbp; Supplementary Fig. 2 ), suggesting sufficiency of our assembly span and no significant inflation by contaminated organisms. We also constructed a full-length complementary DNA library from dehydrated tardigrades and determined paired-end sequences. BLAST search of these Expression Sequence Tag (EST) data against our genome assembly revealed 70,674 of 70,819 sequences (99.8%) were successfully mapped ( E -value<10 65 ). The completeness of our assembly was also supported by high coverage (95.6%) in essential eukaryotic genes assessed by Core Eukaryotic Genes Mapping Approach 16 ( Supplementary Table 2 ) and the very low duplication rate in Core Eukaryotic Genes Mapping Approach (1.13) indicated that our assembly was largely free from inflation by contaminating organisms. We generated gene models based on our messenger RNA-sequencing (RNA-seq) data for six states (two embryonic stages and four states of adults during dehydration and rehydration) and merged them with ab initio gene models, to produce the comprehensive gene set, containing 19,521 protein-coding genes. The genome of this species was highly compact and, correspondingly, the mean length of coding sequences (1,062 bp), exons (234 bp) and introns (402 bp) were fairly short and genes were densely distributed with short inter-coding sequence distances (mean 1,099 bp; Supplementary Table 3 ).

( a , b ) Scanning electron microscopy images of the extremotolerant tardigrade, R. varieornatus , in the hydrated condition ( a ) and in the dehydrated state ( b ), which is resistant to various physical extremes. Scale bars, 100 μm. ( c ) Classification of the gene repertoire of R. varieornatus , according to their putative taxonomic origins and distribution of best-matched taxa in putative HGT genes.

No extensive HGT in R. varieornatus genome

To evaluate the significance of HGT in the tardigrade gene repertoire, we first performed BLAST search against the non-redundant database of National Centre for Biotechnology Information. Among the 19,521 tardigrade proteins, 10,957 proteins (56.1%) had similar proteins below the threshold ( E -value≤10 −5 ) used to estimate HGT in rotifers 17 . The vast majority exhibited the best similarity with metazoan proteins and were thus classified as metazoan origin (10,249 proteins; 52.5% of total proteins; Fig. 1c ). We examined putative HGT based on HGT indices that were calculated by subtracting the best bit score of the metazoan hit from that of the non-metazoan hit in BLAST searches, as used in previous reports 10 , 17 . Only 234 proteins (1.2%) had HGT scores higher than the previously defined threshold (≥30) 10 , 17 and were classified as putative HGT genes ( Fig. 1c and Supplementary Data 2 ). Of 234 putative HGT genes, 226 genes were encoded in the scaffolds containing metazoan-origin genes and all 234 putative HGT genes were supported by substantial coverage of genomic reads ( Supplementary Fig. 3 and Supplementary Data 2 ), suggesting that these putative HGT genes were encoded in the tardigrade genome rather than mis-incorporated minor contaminating sequences. In our evaluation of genome assembly, we excluded 102 scaffolds due to the extremely low coverage as a sign of possible contamination origins. To examine the impact of this exclusion on the estimated HGT proportion, we applied the same gene prediction on the excluded scaffolds and found 152 additional protein-coding genes. Of these 152 genes, 129 exhibited high HGT indices (≥30) and were classified as putative HGT genes. Even taking into account these genes, the proportion of putative HGT genes was still only 1.8% ( Supplementary Table 2 ). In any case, the proportion of HGT in our genome was much lower than those reported for the UNC assembly of H. dujardini (17.5%) 10 . In addition to the HGT proportion, we also found a striking contrast in putative taxonomic origins of HGT genes. In the UNC assembly, most (>90%) of the putative HGT genes were presumed to be of bacterial origin. In contrast, more than half (65%) of the putative HGT genes have probable eukaryotic origins in our assembly, mainly fungal origin ( Fig. 1c ).

Our transcriptome analyses revealed that 138 of 234 putative HGT genes were certainly transcribed (fragments per kilobase of exon per million mapped fragments ≥5) and were considered as functional ( Supplementary Data 2 ). These functional HGT genes included several tolerance-related genes, for example, catalases. Catalase is an antioxidant enzyme that decomposes hydrogen peroxide, which is hazardous to the organism, and antioxidant enzymes are presumed important to counteract oxidative stress during desiccation 18 . In our assembly, we found three catalases and one putative pseudo-gene. All of them had high HGT scores and contained an extra domain at the carboxy terminus compared with other metazoan catalases ( Supplementary Fig. 4 ). This structure resembles those of bacterial clade II catalases. Catalases are classified into three sub-groups, termed clade I, II and III, and all other metazoan catalases are classified as clade III 19 . Phylogenetic analyses confirmed the classification of tardigrade catalases as clade II ( Supplementary Fig. 5 ).

Expansion of stress-related genes in the tardigrade genome

Comparison of the gene repertoire with other metazoans revealed characteristic expansion of several stress-related gene families such as superoxide dismutases (SODs) and MRE11 ( Supplementary Fig. 6 and Supplementary Data 3 ). Sixteen SODs were found in our assembly, whereas less than ten SODs are found in most metazoans. SOD is a detoxifying enzyme of superoxide radicals, a type of reactive oxygen species (ROS) 18 . As desiccation induces oxidative stress, expanded SODs could contribute to better tolerance against desiccation 18 . MRE11, another expanded gene family, plays important roles in repair processes of DNA double-strand breaks (DSBs) 20 . Four MRE11 genes were found in our assembly, whereas most animals possess only one copy. DNA in tardigrade cells undergo DSBs during long preservation in a dehydrated state and expanded MRE11 might be beneficial for efficient repairing damaged DNA. In the UNC assembly of H. dujardini , expansions by HGT were reported for several other DNA repair genes such as Ku , umuC , Ada and recA ( Rad51 ) 10 . We observed no significant expansion or sign of HGT for those genes in R. varieornatus ( Supplementary Data 3 ). Furthermore, all MRE11 genes in R. varieornatus were suggested to be of metazoan origin ( Supplementary Data 2 ). Thus, the expansion of MRE11 was likely to be due to gene duplication events during evolution to this lineage, rather than acquisition from other non-metazoan organisms through HGT. We also detected the expansion of some other gene families, for example, guanylate cyclases ( Supplementary Fig. 6 ). Their relation to tardigrade physiology is, however, currently elusive.

Selective loss of peroxisomal oxidative pathway

We also evaluated whether some metabolic pathways had been lost in our tardigrade genome. To assess this, we mapped genes found in model organisms but missing in our tardigrade genome to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways 21 . Statistical analysis revealed the significant gene loss in the peroxisomal pathway (corrected P -value=0.007, Fisher’s exact test; Supplementary Data 4 ). Many oxidative enzymes including those in the conserved β-oxidation pathway and several peroxisome biogenesis factors were missing ( Supplementary Figs 7 and 8 ). β-Oxidation is a major catabolic pathway of fatty acids, normally catalysed by two sets of enzymes, one in the mitochondria and the other in the peroxisome 22 . All members of the peroxisomal set were missing, whereas a complete set of mitochondrial enzymes was present ( Supplementary Data 5 ), suggesting actual gene loss in the peroxisomal β-oxidation process rather than insufficient genome sequencing.

Selective loss of stress responsive pathways

In addition to the KEGG pathways, we searched for the non-curated gene networks lost in the tardigrade genome by connecting putative lost genes using the protein–protein interaction database, STRING 23 . We found that eight lost genes had an interconnected network in the highly conserved stress-responsive signalling pathways ( Fig. 2 and Supplementary Data 6 ). Three of these genes, HIF1A , PHD and VHL , are central components to regulate response to hypoxia 24 . REDD1 is a downstream target of HIF1A 25 , as well as a downstream target induced by p53 on genotoxic stress 26 . REDD1 activates the TSC1/TSC2 complex, leading to downregulation of mammalian target of rapamycin complex 1 (mTORC1) activity 25 . The other lost gene, Sestrin , is also a downstream gene of p53 connecting genotoxic stress to mTOR signalling 27 . As TSC1/TSC2 is activated by oxidative stress 28 , the tardigrade lacks the signalling components connecting various stresses such as hypoxia, genotoxic stress and oxidative stress, to downregulation of mTORC1. In contrast, all other signalling components are present for regulation of mTORC1, depending on physiologic demands such as energy deprivation sensing 29 and amino acid sensing 30 .

Gene networks involved in the regulation of mTORC1 activity. Magenta indicates genes absent in the tardigrade genome and green indicates retained genes. The interconnected eight genes mediating environmental stress stimuli to downregulate mTORC1 were selectively lost, whereas all components involved in sensing and mediating physiologic demands were present.

Constitutive abundant expression of tardigrade-unique genes

We examined gene expression profiles during dehydration and rehydration using mRNA sequencing and comparative analyses detected only minor differences ( Supplementary Data 2 ), suggesting that the tardigrade can enter a dehydrated state without significant transcriptional regulation. This finding is consistent with the fact that this tardigrade, R. varieornatus , tolerates rapid desiccation by direct exposure to low humidity conditions. We speculated that putative protective proteins are constitutively expressed. During inspection of abundantly expressed genes, we noticed that many abundantly expressed genes are classified as tardigrade-unique genes that exhibited no or low similarity to non-tardigrade proteins ( Supplementary Fig. 9 ).

These abundantly expressed proteins included previously identified tardigrade-unique heat-soluble proteins, CAHS and SAHS, both of which maintain solubility even after heat treatment and are proposed to be involved in the protection of biomolecules during desiccation 31 , 32 . We found significant expansion of these tardigrade-unique protein families, as 16 CAHS genes and 13 SAHS genes in our assembly, whereas no counterparts were found in other phyla, except 3 SAHS genes with low similarity to several metazoan fatty acid-binding proteins. In accordance with the identification of CAHS and SAHS proteins as predominant proteins in the heat-soluble proteome of the tardigrade, our transcriptome data confirmed the abundant expression of these family members in the adult stage as well as in embryonic stages, although dominantly expressed members differed depending on the stage ( Supplementary Data 2 ). We found a reasonable number of genes unique to the species or the phylum (8,023 genes; 41.1% of the gene repertoire; Fig. 1c ). Abundantly expressed unique genes might be good candidates involved in the tolerability of the tardigrade.

Identification of a tardigrade-unique DNA-associated protein

R. varieornatus exhibits extraordinary tolerance against high-dose radiation 4 . Considering DNA as a major target of radiation damage, we hypothesized that tardigrade-unique proteins associate with DNA to protect and/or to effectively repair DNA in the tardigrade. To explore this possibility, we isolated the chromatin fraction from the tardigrade and used tandem mass spectrometry to identify the proteins contained in the bands selective to the chromatin fraction ( Supplementary Fig. 10 ). Among the identified proteins ( Supplementary Tables 4 and 5 ), we examined subcellular localization of putative nuclear proteins by expressing them as green fluorescent protein (GFP)-fused proteins in Drosophila Schneider 2 (S2) cells. Only one protein, termed Damage suppressor (Dsup), co-localized with nuclear DNA ( Supplementary Fig. 11 ) and similar co-localization was also observed in human cultured HEK 293T cells ( Fig. 3a ). Our transcriptome data revealed abundant expression of Dsup in an early embryonic stage (within the top 100 abundantly expressed genes; Supplementary Data 2 ), which is consistent because nuclear DNA extensively replicates in the embryonic stage. To verify the localization of Dsup protein in tardigrade cells, we performed immunohistochemistry with frozen sections of tardigrade embryos. In almost all tardigrade cells expressing Dsup, Dsup proteins co-localized with nuclear DNA ( Supplementary Fig. 12 ).

( a ) Subcellular localization of Dsup-GFP fusion proteins transiently expressed in HEK293T cells. Nuclear DNA was visualized by Hoechst 33342. Scale bars, 10 μm. ( b ) Mobility shift of DNA by bacterially expressed Dsup protein in a dose-dependent manner (10, 50, 75 or 100 ng). Black arrowhead indicates the predicted size of the probe DNA (3 kbp, 10 ng). Red arrowhead indicates the position of the extremely slowly migrating DNA in the presence of Dsup protein. A similar extensive mobility shift was observed with histone H1.

Dsup protein showed no sequence similarity to any proteins or motifs in BLASTP and InterProScan searches. In silico prediction revealed a putative long α-helical region in the middle and a putative nuclear localization signal at the C terminus ( Supplementary Fig. 13 ). Dsup protein is highly basic (pI=10.55), especially in the C-terminal region, suggesting its potential association with DNA through electrostatic interactions. Mutational analyses using variously truncated Dsup proteins fused with GFP revealed that the C-terminal region (Dsup-C) is required and sufficient for co-localization with nuclear DNA ( Supplementary Fig. 14a–c ). Expression of Dsup-C induced an abnormally aggregated distribution of nuclear DNA, whereas full-length Dsup-expressing cells had an almost normal distribution of nuclear DNA, similar to that in control cells ( Supplementary Figs 14a and 15 ).

To examine the affinity of Dsup protein to DNA, we performed a gel-shift assay using bacterially expressed Dsup protein in vitro . Pre-incubation with purified Dsup protein significantly retarded the migration of linearized plasmid DNA in a dose-dependent manner ( Fig. 3b ), suggesting that Dsup protein has certain affinity to DNA in vitro . When Dsup protein was mixed with DNA at a 10:1 (wt:wt) ratio, the migration of DNA was almost completely inhibited. This retarded mobility of DNA could be due to formation of huge DNA–Dsup protein complexes and/or neutralization of the negative charge of DNA. These results suggested the physical affinity of Dsup protein to DNA molecules, although physiological specificity and mode of interaction between Dsup protein and DNA remain elusive. A similar drastic band shift was observed with the ubiquitous chromatin protein histone H1 (ref. 33 ). Dsup protein required the higher protein:DNA ratio for a complete band shift compared with histone H1, suggesting relatively weak affinity to DNA of Dsup than histone H1. Dsup protein lacking the C-terminal region (DsupΔC) completely lost the ability to shift the DNA mobility and Dsup-C alone was sufficient to shift the DNA band ( Supplementary Fig. 14d ). These findings indicated that the C-terminal region of Dsup is responsible for association with DNA as well as for co-localization with nuclear DNA.

Dsup protein suppresses DNA damage in human cultured cells

We hypothesized that the association of Dsup proteins with nuclear DNA might help to protect DNA from irradiation stress. To examine this possibility, we established a HEK293 cell line stably expressing Dsup under the control of the constitutive CAG promoter. Co-localization of Dsup protein with nuclear DNA was confirmed by immunocytochemistry in the established line ( Supplementary Fig. 16a ). X-ray irradiation induces various types of DNA damage, including DNA breaks, mainly single-strand breaks (SSBs). To examine the effect of Dsup on X-ray-induced DNA breaks, Dsup-expressing cells and untransfected HEK293 cells were exposed to 10 Gy X-ray irradiation. After irradiation, the cells were exposed to an alkaline condition (pH>13) to denature the damaged DNA and dissociated single-strand DNA fragments were analysed in single-cell electrophoresis (alkaline comet assay). The short fragmented DNA migrated to more distant location from the nuclei (comet tail region) and thus the proportion of DNA in the comet tail was considered an indicator of DNA breaks. In irradiated Dsup-expressing cells, the proportion of tail DNA was only 16%—less than half of that in the untransfected HEK293 cells (33%; Fig. 4a ). This finding suggested that Dsup protein suppressed X-ray-induced SSBs in human cultured cells. There are two modes for X-ray to induce SSBs: the direct absorption of X-ray energy into the DNA (direct effects) and through attack by ROS generated from water molecules activated by X-ray energy (indirect effects) 34 . We, therefore, examined the effect of Dsup protein on DNA SSBs generation by ROS. Exposure to hydrogen peroxide induced severe fragmentation of DNA (71% of total DNA in tail) in control HEK293 cells. In contrast, DNA fragmentation in Dsup-expressing cells was substantially suppressed to only 18% of total DNA in the tail ( Fig. 4b ), indicating that Dsup protein was able to protect DNA from ROS as well as X-rays. Pretreatment with the antioxidant, N -acetyl- L -cysteine (NAC) also substantially suppressed peroxide-induced SSBs. The combination of NAC and Dsup led to even greater suppression, although the suppression induced by their combination was less than the sum of those in each condition individually, suggesting that NAC and Dsup at least partially share the same suppression mechanism, most probably counteracting oxidative stress.

( a ) The effects of Dsup on SSBs by 10 Gy X-ray irradiation in alkaline comet assays. The irradiated cells were immediately subjected to the assay. Representative images are shown for each condition. In the pseudo-coloured images in the inset, red to blue circles indicate nuclear DNA and magenta indicates fragmented DNA in tail. DNA fragmentation was assessed by the proportion of DNA detected in the tail region (% of DNA in Comet Tail). At least 281 comets were analysed for each condition. ** P <0.01 and *** P <0.001 (Welch’s t -test: non-irradiated, t -value=−3.199, P -value=0.0015; irradiated: t -value=8.599, P -value<1.0 E −15). ( b ) The effects of Dsup on SSBs caused by hydrogen peroxide (H 2 O 2 ) treatment in alkaline comet assays. Cells were treated with 100 μM H 2 O 2 for 30 min at 4 °C, to induce DNA damage with or without pretreatment with 10 mM NAC as an antioxidant for 30 min. At least 203 comets were analysed for each condition. *** P <0.001 (Tukey–Kramer’s test). ( c ) The effects of Dsup on DSBs by 5 Gy X-ray irradiation in neutral comet assays. Three hundred comets were analysed for each condition. ** P <0.01 and *** P <0.001 (Welch’s t -test: non-irradiated, t -value=2.758, P -value=0.0060; irradiated; t -value=7.406, P -value=4.7 E −13). Values represent mean±s.d. in all panels. Scale bars, 100 μm.

Besides SSBs, high-dose X-ray irradiation also induces DSBs, which are much more hazardous for organisms due to their difficult repair. We next examined the effect of Dsup protein on DSBs using a neutral comet assay, in which DNA fragmentation was analysed without dissociating in a neutral condition. The proportion of fragmented DNA was ∼ 40% reduced in Dsup-expressing cells compared with that in the untransfected cells ( Fig. 4c ). These findings together suggest that Dsup protein suppressed X-ray-induced DNA DSBs and SSBs.

We further verified the suppression of DNA breaks by Dsup proteins using another DSB quantification method. In irradiated cells, histone H2AX around DSBs becomes phosphorylated within an hour 35 , referred to as γ-H2AX, and γ-H2AX can be used as an indicator of DSBs. We visualized γ-H2AX by immunofluorescence and counted the number of foci per nucleus at 1 h after irradiation. For this experiment, we irradiated cells with a relatively lower dose (1 Gy) of X-ray to avoid overlap of neighbouring foci and minimize counting errors. The Dsup-expressing cells exhibited an ∼ 40% reduced number of γ-H2AX foci compared with untransfected cells ( Fig. 5a ). We further established Dsup knockdown cells by transfecting a small hairpin RNA (shRNA) expression construct in Dsup-expressing cells. Dsup expression was successfully reduced by 77% in the knockdown cells ( Fig. 5b ). The reduction of DNA damage completely disappeared by Dsup knockdown ( Fig. 5c,d ). These findings indicated that Dsup protein is responsible for suppressing DNA damage in irradiated human cultured cells. When using a stable line expressing mutant Dsup protein, DsupΔC, which lacks the C-terminal DNA-associating region ( Supplementary Fig. 16b ), we detected no reduction of DNA fragmentation in the alkaline comet assays ( Supplementary Fig. 17a ), suggesting that the association with DNA is prerequisite for Dsup protein to protect DNA from X-ray. This view was further supported by the impaired suppression of the γ-H2AX foci in the DsupΔC-expressing cells ( Supplementary Fig. 17b ).

( a ) Distribution of the numbers of γ-H2AX foci per nucleus is shown. Each dot represents an individual nucleus of a HEK293 cell (Control) or a Dsup-expressing cell (Dsup) under non-irradiated and irradiated conditions. *** P <0.001; NS, not significant (Welch’s t -test). ( b ) Significant decrease of Dsup transcript in shRNA-introduced cells (Dsup+shDsup) compared with that in untreated Dsup-expressing cells (Dsup shRNA(−)). n =3. Values represent mean±s.e.m. *** P <0.001 (Student’s t -test). ( c ) Quantitative comparison of γ-H2AX foci number among untransfected HEK293 cells (Control), Dsup-expressing cells (Dsup) and Dsup-knockdown cells (Dsup+shDsup) under non-irradiated and 1 Gy X-ray irradiated conditions. At least 70 cells were analysed for each condition. Values represent mean±s.d. ** P <0.01; NS indicates not significant (Tukey–Kramer’s test). ( d ) Representative images detecting γ-H2AX foci in each condition. Fluorescent images were converted to binary images for automatic counting of foci. Scale bar, 10 μm.

Dsup improves viability of irradiated human cultured cells

To test whether DNA protection by Dsup protein could also improve cellular survival after irradiation, we measured the cell viability after irradiation. In general, 3–7 Gy of X-ray induces severe DNA damage in mammalian cells, leading to loss of proliferative ability 36 . Accordingly, we irradiated cells with 4 Gy X-ray at 1 day post seeding (dps), which was the minimum dose enough to suppress proliferation of untransfected HEK293 cells in our condition. After irradiation, cell proliferation was examined at 24 h intervals for 8 days using PrestoBlue Cell Viability reagent, which measures the total reducing power of the cell culture 37 . Dsup-expressing cells exhibited slightly better cell viability after irradiation compared with those of untransfected HEK293 cells ( Supplementary Fig. 18a–c ). At 4 days after the cell viability analysis (12 dps), we noticed a drastic difference between Dsup-expressing cells and untransfected cells under phase-contrast microscopy ( Supplementary Fig. 18d ). Almost all irradiated untransfected cells had an abnormal round shape and were mostly detached from the culture dish, typical characteristics of dead cells. In contrast, many irradiated Dsup-expressing cells had a normal morphology and attached to the culture dishes, suggesting that these cells retained the characteristics of live adherent cells and perhaps even had proliferative ability.

To confirm their proliferative ability, we examined the temporal change in cell numbers over a longer period after irradiation with 4 Gy of X-ray. Even under non-irradiated conditions, Dsup-expressing cells proliferated slightly faster than the untransfected cells, whereas Dsup-knockdown cells exhibited similar proliferation to that of untransfected cells ( Fig. 6b ). At 10–12 dps, the cell numbers became nearly saturated. Under irradiated conditions, almost all untransfected cells detached from the culture dish and had an abnormal round shape ( Fig. 6a ). In contrast, some of the irradiated Dsup-expressing cells attached to the culture dish with an apparently normal morphology and such cells increased over time ( Fig. 6a ). Cell counting analyses confirmed these observations. At 8 dps, the number of irradiated untransfected cells was almost unchanged from that at the seeding and further decreased at 10 and 12 dps ( Fig. 6b ). In contrast, the number of Dsup-expressing cells increased even at 8 dps compared with that at the seeding and drastically increased at 10 and 12 dps ( Fig. 6b ), suggesting that at least some fraction of irradiated Dsup-expressing cells retained proliferative ability. Growth rates at 8–12 dps were comparable to those of non-irradiated Dsup-expressing cells. In Dsup-knockdown cells, the improvements in cell viability and proliferative ability were completely abolished and their phenotypes were similar to those of untransfected HEK293 cells ( Fig. 6 ). These findings suggested that Dsup protein confers increased radiotolerance to human cultured cells. Cells expressing a Dsup mutant lacking the DNA-associating domain (DsupΔC) exhibited impaired improvement of radiotolerance compared with those expressing full-length Dsup protein ( Supplementary Fig. 19 ), suggesting that DNA targeting is important for full improvement of the radiotolerance by Dsup. As radiosensitivity of mammalian cells is affected by the cell cycle 38 , we compared the cell cycle distribution between Dsup-expressing cells and untransfected cells using flow cytometry. However, no significant differences were detected ( Supplementary Fig. 20 ), suggesting that the improved radiotolerance conferred by Dsup protein was not due to alterations of the cell cycle.

( a ) Representative microscopic images with phase contrast at 8, 10 and 12 dps, of untransfected HEK293 cells (Control), Dsup-expressing cells (Dsup) and Dsup -knockdown cells (Dsup+shDsup) irradiated with 4 Gy X-ray at 1 dps. Scale bar, 200 μm. ( b ) Comparison of growth curves of untransfected cells (Control), Dsup-expressing cells (Dsup) and Dsup-knockdown cells (Dsup+shDsup) in non-irradiated and irradiated conditions. Values represent mean±s.d.

The genome sequence of R. varieornatus determined in this study is the first example of an extremotolerant tardigrade genome. All examined data, including congruence of the assembly span with the estimated genome size, and high coverage of EST and a core eukaryotic gene set, support the completeness of the determined genome sequence. The clear separation of minor contaminating scaffolds based on coverage and the consistent GC proportion and coverage of the scaffolds in the final assembly suggested that our assembly is largely free from contamination. The quality of our assembly is two orders of magnitude better (N50 ∼ 4.7 Mb) than those of the two draft genomes of the freshwater tardigrade, H. dujardini (N50 ∼ 15.9 or 50.5 kb) and thus could be useful as a reference genome of the phylum.

The estimated HGT proportion (1.2%) in our final assembly is one order of magnitude lower than that in the controversial UNC assembly of H. dujardini (17.5% HGT) 10 . We did not exclude any sequences from the assembly based on sequence similarity to foreign organisms (for example, bacteria) and thus there was no preferential removal of HGT genes and no bias to underestimate the HGT proportion. The HGT index is a useful indicator for the possibility of HGT, but is not a sufficient criterion to guarantee true HGT. Indeed, previous phylogenetic analyses validated only an average of 55% of the genes with a high HGT index (≥30) as foreign origin 39 . Thus, our estimated HGT proportion (1.2%) was rather overestimated. The number of putative HGT genes (234) in our assembly is in the range of those in nematodes (129–241) estimated with the same criterion 39 and we therefore concluded that R. varieornatus contains only a moderate number of HGT genes. Extensive HGT is thus not a common feature in the phylum Tardigrada and is also not correlated with extremotolerance, because R. varieornatus has superior tolerability compared with H. dujardini without extensive HGT.

As desiccation causes severe oxidative stress 18 , desiccation-tolerant animals should have the ability to mitigate this type of stress. Multiple gene repertoire traits in the tardigrade genome suggested enhanced tolerability against oxidative stress, such as characteristic expansion of antioxidative enzymes, SODs and acquisition of bacterial-origin catalases (clade II). Bacterial clade II catalases exhibit greater resistance to denaturing conditions, such as high temperature or 7 M urea than metazoan clade III catalases 40 and, thus, tardigrade clade II catalase might be active even in hyperosmotic conditions during dehydration/rehydration and contribute to desiccation tolerance. Loss of peroxisomal oxidative enzymes including those in β-oxidation could be another strategy to adapt to oxidative stress. In peroxisomal β-oxidation, acyl-CoA oxidases catalyse the initial conversion of acyl-CoA and produce hydrogen peroxide as a side product. On the other hand, in mitochondria, similar conversion is catalysed by acyl-CoA dehydrogenases, which produce FADH 2 instead of hydrogen peroxide ( Supplementary Fig. 7 ). Thus, the lack of peroxisomal β-oxidation pathway probably leads to decreased hydrogen peroxide production during fatty acid metabolism. Decreased production of hydrogen peroxide would help the animal by preserving antioxidant capacity to combat oxidative stress during desiccation. Hydrogen peroxide produced in metazoan peroxisomes is normally decomposed by the resident enzyme, catalase. The putative decrease of hydrogen peroxide is consistent with the loss of typical metazoan catalases (clade III) in the tardigrade genome.

Although the stress-responsive pathway is widely used to adapt to various environmental stresses, the decoded tardigrade genome is unexpectedly missing signalling pathways that mediate stress stimuli to inactivate mTORC1, probably leading to degradation of damaged cellular components by autophagy 41 . We speculate that the tardigrade avoids excessive destruction of cellular components after severe stress by suppressing autophagy induction and this might be beneficial to resume cellular activity by using partially damaged biomolecules after rehydration. These findings suggest that the tardigrade is insensitive to environmental stress, at least with respect to autophagy induction.

Minor changes in gene expression profiles during dehydration and rehydration suggested constitutive expression of tolerance-related genes in R. varieornatus . Some tardigrade-unique genes, including putative protective proteins CAHS and SAHS, were abundantly and constitutively expressed, and could be candidates involved in desiccation tolerance. Dsup protein is a prominent example of tardigrade-unique abundant proteins involved in tolerability and is, to our knowledge, the first DNA-associating protein demonstrated to protect DNA and improve the radiotolerance of cultured animal cells. Although Dsup improved radiotolerance of HEK293 cells, cultured cell lines including HEK293 cells are potentially pre-adapted to oxidative environments in an artificial culture system and Dsup might enhance radiotolerance in conjunction with the partial adaptation of cultured cell lines.

We detected ∼ 40 foci of γ-H2AX, a relatively high number, in HEK 293 cells at 1 h after 1 Gy irradiation on glass coverslips. Glass materials are reported to enhance irradiation effects approximately twofold by generating the secondary electrons 42 . Taking this effect into account, the detected number of γ-H2AX in our assay is in good accordance with previous reports in which 20 ∼ 30 DSBs were detected after 1 Gy irradiation 43 , 44 .

In our comet assays, Dsup-expressing cells were irradiated on ice or treated with hydrogen peroxide at 4 °C and immediately subjected to electrophoresis, suggesting that DNA fragmentation was detected before significant DNA repair. In the γ-H2AX foci assay as well, we detected γ-H2AX foci at 1 h after irradiation when enough γ-H2AX has accumulated to be detected in human cells and the accumulation of γ-H2AX is normally retained for at least several hours 35 , 45 . Thus, we concluded that the reduced number of DNA breaks in Dsup-expressing cells was due to the suppression of DNA breaks, rather than facilitation of DNA repair processes, which is proposed in some other radiotolerant animals, such as the sleeping chironomid or rotifers 46 , 47 ( Supplementary Fig. 21 ). In some desiccation-tolerant animals, protective molecules, such as trehalose, are thought to play important roles in the protection of biomolecules against dehydration stress. Dsup could be a DNA-targeted protectant in the tardigrade, although this finding would not exclude the possibility of the presence of an effective DNA repair system, for example, expanded MRE11s could contribute to facilitation of DNA repair.

Although association of proteins with DNA is potentially beneficial to physically shield DNA from environmental stress, including ROS, it could interfere with DNA replication and transcription. Indeed, overexpression of several DNA-binding proteins, such as a bacterial histone-like nucleoid-structuring protein or a small acid-soluble spore protein associated with spore DNA of Bacillus subtilis , causes severe condensation of DNA and loss of cell viability 48 , 49 . The C-terminal region of Dsup alone similarly induced an abnormal aggregation of DNA and we were unable to establish stably expressing cell lines, likely to be due to cytotoxic effects. The apparent lack of such negative effects in full-length Dsup-expressing cells suggests that the amino-terminal and middle regions play important roles to relieve the adverse effects induced by association of Dsup-C to DNA (for example, possible heterochromatinization and/or interference on transcription and replication). Dsup protein affords DNA protection without impairing cell viability and is quite suitable for future application to confer the tolerance to other animal cells.

Improvement of radiotolerance by Dsup suggests that unique proteins in the tardigrades confer exceptional tolerance to harsh environmental stresses. Dsup-expressing human cultured cells exhibited better tolerance to 4 Gy of X-ray irradiation, whereas R. varieornatus exhibited far superior tolerance against high-dose irradiation, such as 4,000 Gy of He-ion beam in adults, and a lower, but still significant, dose of irradiation in mitotically active embryos (LD 50 ∼ 500 Gy) 50 . There may be additional factors besides Dsup in the tardigrade genome that contribute to the exceptional tolerance. The genome sequence and gene repertoire of the extremotolerant tardigrade revealed in this study provide a treasury of genes to improve or augment the tolerant ability in stress-sensitive animal cells.

Experimental animals

The YOKOZUNA-1 strain of the extremotolerant tardigrade R. varieornatus was used for all experiments. The strain was established from a single individual 4 to minimize genetic variance. The tardigrades were reared on water-layered agar plates by feeding them alga, Chlorella vulgaris (Chlorella Industry, Japan), at 22 °C 4 with additional hygienic treatment using hypochlorite.

Genome size determination

The genome size of the animal was determined by flow cytometry 51 . Briefly, ∼ 100 starved adult tardigrades were collected and homogenized in Galbraith buffer (pH 7.2) 52 using a Kontes Dounce tissue grinder. Dissociated cells were obtained by filtration through a CellTrics disposable filter (30 μm pore size; Partec) and stained with 50 μg ml −1 propidium iodide. The DNA content in each cell was analysed using a FACSCanto flow cytometer and FACSDiva software (BD Biosciences).

The genome size was also estimated by Feulgen densitometry method 53 . Adult tardigrades were squashed on a slide glass. After air drying and fixation, the slide was hydrolysed in 5.0 N HCl and stained using Schiff reagent. The density of Feulgen stain was measured using image analysis software, FMBIO Analysis (Hitachi Software, Tokyo, Japan). In total, 119 cells from 10 animals were examined. Drosophila melanogaster was used as a reference.

Genome DNA extraction

After 2 days starvation and antibiotics treatment, tardigrades were extensively cleansed and genomic DNA was extracted using a Blood and Cell Culture DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. Eluted DNA solution was supplemented with DNA carrier, Ethachinmate (Nippon Gene) and precipitated by ethanol. In total, ∼ 15,000 individuals were subjected to genomic analyses including whole genome shotgun (WGS), fosmid and Illumina sequencing.

Fosmid library construction and sequencing

The fosmid library (GRVF) was constructed from sheared genomic DNA and pKS300 cloning vector. After in vitro packaging using Gigapack III Gold Packaging Extract (Agilent Technology), the phage particles were transfected to Escherichia coli XL1-BLUE. Fosmid clone DNA from each 96-well plate was prepared by the standard alkaline lysis method (Kurabo PI-1100). End sequencing of 30,336 fosmid clones was performed using a BigDye terminator kit version 3 and the ABI 3730xl DNA Analyzers (Applied Biosystems).

WGS sequencing and assembly

The genome sequence of R. varieornatus was determined by using a combination of the Sanger and Illumina technologies. First, a WGS library with an average insert size of 3.5 kb was constructed. End sequencing of 489,216 clones was then performed using the ABI 3730xl DNA Analyzers (Applied Biosystems). After quality and vector clipping, the WGS and fosmid end sequence data were assembled by the PCAP.REP assembler (version 06/07/05). Gap closing and re-sequencing of low-quality regions were performed by a combination of primer walking and direct sequencing of fosmid/WGS clones and PCR products. Complete sequences of four fosmid clones were generated by the shotgun sequencing method. Second, Illumina-sequencing libraries were prepared using a Paired End DNA Sample Prep kit and a Mate Pair Library Prep kit. Paired-end (240 and 480 bp) and mate-pair (4.8, 5.8 and 7.3 kb) libraries run on the Genome Analyzer IIx sequencers (Illumina). After the pre-processing steps, de novo assembly was performed using the SOAPdenovo version 1.3. The contig sequences (8,086 contigs, total bases: 49,160,052 bp) were then incorporated into the Sanger-based assembly sequence, to close the gaps and resolve the problematic regions.

RNA sequencing

After 2 days starvation, extensively washed tardigrades were used. Dehydrated tardigrades were obtained by exposing the washed tardigrades to 33.8% relative humidity on a nylon mesh and filter paper. Rehydrated tardigrades were collected at 80 min and 3 h after rehydration. Total RNA was extracted using TRIzol reagent (Invitrogen). Embryos were collected at 2-day intervals after egg laying as 0–2 days and 3–4 days, and extensively washed. Six sequencing libraries were constructed from four adult samples during anhydrobiosis (hydrated, dehydrated and rehydrated at 80 min and 3 h) and two embryonic samples using a mRNA-Seq Sample Prep kit (Illumina). The sequencing was performed using the Genome Analyzer IIx and HiSeq2000 sequencers (Illumina).

Full-length cDNA library construction and EST sequencing

Total RNA was extracted from dehydrated tardigrades using TRIzol reagents and a full-length cDNA library (cYOK) was constructed by the oligo-capping method 54 . DNA template for each clone was amplified from the bacterial culture in a glycerol stock 384-well plate using a TempliPhi DNA amplification kit (GE Healthcare). EST sequencing of 38,400 cDNA clones was performed using the ABI 3730xl capillary sequencers (Applied Biosystems).

Prediction of protein-coding genes

For ab initio prediction of protein-coding genes, primary training data were created by: (1) predicting the longest open reading frames (ORFs) longer than 300 bp from the cDNA sequences; (2) screening translated ORF sequences with BLASTP 55 e -value< e −50 match against UniRef90 (ref. 56 ); (3) mapping the screened translated ORF sequences against the genome sequence using exonerate programme 57 . Thus, using the derived training data, genes were predicted from the genome sequence using SNAP 58 for initial bootstrap learning. Using the gene prediction of SNAP in the longest seven scaffolds, gene model is further trained and the final predictions were made using GlimmerHMM 59 . In parallel, RNA-seq reads were mapped to the genome sequence using TopHat software 60 and gene model was generated using cufflinks 61 . To dissociate artificial fusions of adjacent coding sequences, non-overlapping ORFs were extracted. The transcriptome-based gene model was merged with the ab initio gene model to produce the comprehensive gene set.

Annotation of genes

For non-coding RNAs, transfer RNAs were predicted using the combination of Aragorn v.1.2.28 (ref. 62 ) and tRNAscan-SE 1.23 (ref. 63 ), and rRNAs were predicted using RNAmmer v.1.2 (ref. 64 ). For functional annotation of protein-coding genes, sequence similarities were searched in Swiss-Prot knowledgebase 56 using BLASTP 55 with e -value< e −25, domains were searched in Conserved Domains Database 65 using RPS-BLAST with e -value< e −5 and orthologous groups were searched using KEGG Automatic Annotation Server 66 with bidirectional best hit method. Gene Ontology terms were obtained from the best Swiss-Prot match.

Gene expansion and lost pathway analysis

Putative orthologues in other metazoans were assigned for all tardigrade proteins based on a reciprocal BLAST search to reference protein sequences obtained from the UniProt proteome database 56 . Gene numbers were compared between the tardigrade and other metazoans. For detection of lost pathways, we assigned KEGG orthology identifiers to all tardigrade proteins and two well-established model invertebrates ( D. melanogaster and Caenorhabditis elegans ) using the KEGG Automatic Annotation Server programme 66 . We took the proteins conserved in both model invertebrates as a background and evaluated the statistical significance of missing genes in the tardigrade genome for each KEGG pathway using the KOBAS programme 67 . To find lost gene networks in addition to curated KEGG pathways, putative lost genes were inter-connected using the STRING database (cutoff score 0.9) 23 . The gene networks containing high number of putative lost genes were inspected manually.

Assessment of taxonomic origin of predicted proteins

BLASTP search was performed to retrieve similar proteins from the National Centre for Biotechnology Information non-redundant database for each tardigrade protein and the taxonomy information was retrieved based on their GeneInfo Identifiers. Tardigrade proteins were excluded from the retrieved list. When no similar proteins were retrieved with the defined threshold, the query proteins were classified as ‘no similarity’ ( E -value>10 −3 ) or ‘low similarity’ ( E -value>10 −5 ). The proteins exhibiting the best score with metazoan proteins were classified as ‘metazoan origin’. For rest proteins, HGT indices were calculated to assess possible foreign origin, by subtracting the bit score of the best metazoan hit from that of the best non-metazoan hit as defined previously 17 . We allowed metazoan hits with an E -value threshold of 10. The proteins with high HGT index (≥30) were classified as ‘putative HGT proteins’. The threshold value was determined as 30 in the previous work 17 . Those with a lower HGT index were classified as ‘Indeterminate’.

Protein identification

Chromatin fraction was separated by partial disruption and differential centrifugation 68 . Five hundred tardigrades were homogenized with a Dounce tissue grinder (Radnoti, RD440910; with 30–40 μm clearance) on ice in Buffer A (10 mM HEPES-HCl pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol and Complete EDTA-free protease inhibitor cocktail (Roche)). To solubilize the cell membrane, Triton X-100 (Wako) was added to final 0.1% and incubated for 8 min on ice. The nuclear fraction was precipitated by low-speed centrifugation (4 min, 1,300  g , 4 °C) and washed twice with buffer A. The nuclear fraction was lysed by hypotonic shock in Buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM dithiothreitol and Complete EDTA-free protease inhibitor cocktail (Roche)) for 30 min on ice. After centrifugation (4 min, 1,700  g , 4 °C), insoluble chromatin was obtained as a precipitate and the supernatant was recovered as the nuclear soluble fraction. The chromatin fraction was washed two more times with Buffer B. Each fraction was analysed by SDS–PAGE and proteins were visualized using a Silver Quest Staining Kit (Invitrogen). Selective bands (B1 and B2) were excised and treated with trypsin. The fragmented peptides were analysed by nano liquid chromatography–electrospray ionization–quadrupole time of flight–tandem mass spectrometry. Proteins were identified using MASCOT software (Matrix Science; P <0.01; Mascot score cutoff was 37). Detected peptides are shown in Supplementary Table 5 .

Subcellular localization analysis of GFP fusion protein

For expression of GFP-fused full-length Dsup protein, the coding sequence of Dsup was amplified and inserted into Asp718 and BamHI sites of pAcGFP1-N1 (Clontech). HEK293T cells were transiently transfected with the expression construct using X-tremeGENE 9 reagent (Roche). After 24 h, the cells were stained with Hoechst 33342 (Lonza) to visualize nuclear DNA. Fluorescent signals were observed under a confocal microscope (LSM710, Carl Zeiss).

Immunohistochemistry

Anti-Dsup antibody was raised and affinity-purified against bacterially expressed Dsup protein. For immunohistochemistry on frozen sections, tardigrade embryos within 3 days after egg-laying were fixed with 4% paraformaldehyde at room temperature (RT) for 15 min and were embedded in Agarose-LGT (Nacalai Tesque) 32 . The embedded gels were incubated in sucrose series, 15 and 30% overnight each at 4 °C and embedded in O.C.T. Compound (Sakura Finetek Japan). Cryosections (10–14 μm thickness) were prepared using a cryostat (Leica CM1850, Leica). After three washes with 0.1% Tween 20 in Tris-buffered saline, the sections were blocked with 2% goat serum for 1 h and reacted with the affinity-purified anti-Dsup antibody (at 1/200 dilution) overnight at 4 °C and then with Alexa Fluor 488 anti-Rabbit IgG (Molecular Probes, A-11008, at 1/1,000 dilution) for 45 min at RT. Nuclear DNA was counterstained with 4′,6-diamidino-2-phenylindole (Invitrogen). Fluorescent signals were observed using a confocal microscope (LSM710, Carl Zeiss).

In silico analysis based on the Dsup protein sequence

Secondary structures were predicted by the CLC main workbench 6.9.1 (CLC Bio). The nuclear localization signal was predicted using the cNLS Mapper ( http://nls-mapper.iab.keio.ac.jp/ ). A hydrophobicity plot was generated by ProtScale ( http://web.expasy.org/protscale/ ) with the Kyte and Doolittle model 69 . A protein charge plot was generated using EMBOSS 70 . Subcellular localizations were predicted by WoLF PSORT 71 and TargetP 72 .

DNA electrophoretic mobility shift assay

The protein–DNA association was examined by a gel-shift assay 73 . Recombinant Dsup protein was produced as follows. The coding sequence of Dsup was amplified and inserted into NdeI and XbaI sites of pCold-I vector (TaKaRa), which contains the 6xHis tag at the N terminus. The construct was transformed to BL21 (DE3) cells and protein production was induced with isopropyl β- D -1-thiogalactopyranoside and cold treatment according to the manufacturer’s protocol. Recombinant Dsup protein was purified with Ni-NTA His-Bind Superflow (Novagen) in denaturing conditions using 8 M Urea and dialysed in PBS using a Micro-Dialyzer (Nippon Genetics). PBS was prepared from ten times concentrated stock solution (Wako, 163-25265). As a DNA probe, pBluescript II plasmid DNA was linearized by digestion with HindIII and subjected to the assay. Purified recombinant Dsup proteins (10, 50, 75 or 100 ng) were incubated with purified linearized pBluescript DNA (10 ng) in PBS for 20 min at RT. Purified histone H1 protein (bovine) was purchased from Upstate and was used as a positive control. After the incubation, the samples were mixed with gel loading dye (10 mM Tris-HCl pH 8.0, 1 mg ml −1 bromophenol blue, 20% glycerol) and were electrophoresed in a 0.5% agarose gel in Tris-borate-EDTA (TBE) buffer. DNA was stained with SYBR Green I and visualized by a transilluminator (ATTO).

We obtained HEK293 cells (RCB1637) and HEK293T cells (RCB2202) from RIKEN BioResource Center (BRC). The identity of these cell lines was validated by short tandem repeat profiling and all cell lines were negative for mycoplasma contamination (RIKEN BRC). The cells were maintained in Dulbecco’s modified essential medium (Nacalai Tesque) containing 10% fetal bovine serum (Corning). A Dsup expression vector was constructed by inserting the coding sequence of Dsup into KpnI and NotI sites of pCXN2KS, a modified pCAGGS vector 74 . The expression construct was transfected to HEK293 cells using X-tremeGENE 9 DNA Transfection Reagent (Roche) and stably transfected cells were selected by 700 μg ml −1 G418 (Calbiochem) treatment for 3 weeks. We observed many cells with an abnormal morphology (for example, giant cells or elongated form) and those cells could not be maintained. Clonal cell populations were obtained by limiting dilution and Dsup expression was examined by western blotting analysis and immunohistochemistry. Clones showing non-nuclear localization of Dsup protein immunoreactivity were discarded. The clone expressing the highest level of Dsup protein with nuclear localization was chosen. The target sequence for the shRNA was designed based on the online analysis software siDirect 75 and BLOCK-iT RNAi Designer ( http://rnaidesigner.lifetechnologies.com/rnaiexpress/ ) as 5′-GAA CGT AAC CGT TAC CAA AGG-3′. To construct a vector expressing shRNA, oligonucleotides encoding the stem-loop shRNA sequence were synthesized and inserted into the AgeI-EcoRI site of pLKO.1 puro 76 : the inserted sequence was 5′-ACC GGT GAA CGT AAC CGT TAC CAA AGG TTC AAG AGA CCT TTG GTA ACG GTT ACG TTC TTT TTG AAT TC-3′. The shRNA expression construct was transfected to the Dsup-expressing stable cell line. After selection by 2 μg ml −1 puromycin (Sigma) treatment, cell cloning was performed as described above.

Comet assay

A comet assay was performed using the CometAssay Kit (Trevigen) under alkaline or neutral conditions essentially according to the manufacturer’s protocol. Briefly, cells were irradiated on ice using an X-ray generator, the Pantak HF 350 (Shimadzu) operating at 200 kV–20 mA with a filter of 0.5 mm Cu and 1 mm Al at a fixed dose rate of 1.73 Gy min −1 . We selected irradiation doses that increased the proportion of tail DNA to a 30–50% of total DNA to clearly visualize the irradiation-dependent increase of DNA damage without catastrophic fragmentation (10 and 5 Gy were used for alkaline or neutral conditions, respectively). The irradiated cells were immediately trypsinized and collected as a cell suspension. Cell suspensions were mixed with molten agarose and solidified as a thin layer on slide glasses by chilling at 4 °C for 30 min. For alkaline comet assays, the slide glasses were soaked for 1 h in manufacturer’s lysis solution (Trevigen) at 4 °C for 1 h to lyse the cells and then immersed in alkaline solution (200 mM NaOH, 1 mM EDTA pH>13) for 1 h at RT, in the dark and electrophoresed in freshly prepared alkaline solution at 25 V and 4 °C for 1 h. For neutral comet assay, the cell-mounted slide glasses were soaked in the lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 1% sarcosinate and 0.01% Triton X-100) at 4 °C 77 and then washed in TBE buffer for 30 min and electrophoresed in freshly prepared TBE buffer at 25 V and 4 °C for 1 h. After electrophoresis, the comets were visualized by staining with SYBR Green I and captured with an Imager Z1 (Carl Zeiss). DNA fragmentation was quantified for at least 120 comets per condition using CASP software 78 .

Hydrogen peroxide treatment

Cells were treated with 100 μM hydrogen peroxide (H 2 O 2 ) at 4 °C for 30 min. Half of the cells were pretreated with an antioxidant, 10 mM NAC (Sigma) for 30 min before the hydrogen peroxide treatment. DNA damage was evaluated by the alkaline comet assay with the electrophoresis at 25 V at 4 °C for 30 min, immediately after the treatment. At least 302 comets were analysed for each condition.

γH2AX foci detection

The cultured cells on Chambered Coverglass (Thermo Scientific) were irradiated with 1 Gy of X-ray using the Pantak HF 350 X-ray generator (Shimadzu). One hour after X-ray irradiation, the cultured cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 15 min. The cells were blocked with 10% goat serum for 1 h and reacted with the anti-phospho-histone H2A.X (Ser139) antibody clone JBW301 (Merck Millipore, 05-636, at 1/800 dilution) for 1 h and then with Alexa Fluor 488 anti-mouse IgG (Molecular Probes, A-11001, at 1/500 dilution) for 45 min. Nuclear DNA was counterstained with 4′,6-diamidino-2-phenylindole (Invitrogen). All reactions and procedures were essentially performed at RT. Fluorescent signals were observed by confocal microscopy (LSM710, Carl Zeiss). The depth-coded projections were captured as stacks of ten optical sections of z-series at 1-μm intervals and converted to binarized images by ImageJ version 1.47. The threshold value for image conversion was manually adjusted until a visual best fit between the original and converted images was observed ( Supplementary Fig. 22 ). The numbers of γ-H2AX foci were counted using the ImageJ software 79 .

Quantification of Dsup transcript by realtime reverse transcriptase–PCR

Total RNA was extracted from cell pellet using the RNeasy mini kit following the manufacturer’s instructions (Qiagen) and reverse-transcribed using PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa). Dsup expression was quantified by real-time PCR using LightCycler 480 Instrument II (Roche) and knockdown efficiency was calculated. Human β-actin was used as an internal control. Sequences for primer sets were as follows: Dsup : forward 5′-TCC ACA GAA CCC TCT TCC AC-3′ and reverse 5′-TCT TGA CAA TGG CAG CTG AG-3′. β-actin : forward 5′-TGA GCG CGG CTA CAG CT-3′ and reverse 5′-TCC TTA ATG TCA CGC ACG ATT T-3′.

Cell cycle analysis

Cell cycle analysis was performed using flow cytometry based on DNA content and incorporation of 5-bromo-2-deoxyuridine (BrdU) 80 . BrdU (Sigma) was added to cell cultures at 10 μM at 37 °C for 1 h. After pulse labelling, the cells were collected as a cell suspension by trypsinization. Cells were fixed with 90% ice-cold ethanol with gentle vortexing and incubated on ice for 1 h. Cells were rinsed in PBS and further incubated with 2 N HCl/0.5% Triton X-100 at RT for 30 min. After that, cells were suspended in 0.1 M sodium tetraborate for 30 min. Cells were incubated with 1/50 diluted anti-BrdU mouse IgG (555627, BD Pharmingen) at RT for 1 h and reacted with 1/500 diluted Alexa Fluor 488 anti-mouse IgG (Molecular Probes, A-11001) for 30 min after two washes with PBS. Cells were finally incubated with PBS containing 10 μg ml −1 RNase (Sigma) and 5 μg ml −1 propidium iodide (Dojindo) at RT for 30 min in the dark and then filtered through 77-μm nylon mesh to remove cell clusters. Cells were analysed by flow cytometry using BD FACSVerse (BD Bioscience). At least 10,000 events were collected and data were analysed using FlowJo software (Tree Star Inc.).

Cell count and measurement of cell viability

Cells were seeded in poly- L -lysine-coated 24-well plates (Iwaki) at a density of 1,000 cells per well. After 24-h incubation (1 dps), the cells were irradiated with 4 Gy of X-ray using the Pantak HF 350 X-ray generator. With 24 h intervals, the cells were incubated with PrestoBlue Cell Viability Reagent (Invitrogen) for 2 h and the fluorescence was measured using a microplate fluorometer, the Spectra max Gemini EM (Molecular Devices). To count the cell number, the cells were washed gently with PBS and treated with trypsin, then recovered as a cell suspension at 8, 10 and 12 dps. The numbers of cells in the suspensions were counted using an automatic cell counter, the Z1 Particle Counter (Beckman Coulter). We examined three wells for each condition.

Statistical analysis

The effects of Dsup or its derivatives in alkaline/neutral comet assays, γ-H2AX assays and cell viability assays were evaluated by statistical tests. For pairwise comparisons, two-tailed Student’s t -test or two-tailed Welch’s t -test was used depending on the equality of variance between samples determined by F-test (significance level=0.05). For comparisons among three or more samples, Tukey–Kramer’s test was used to evaluate the differences between all possible comparison pairs. All statistical measures and tests of the comet assays, γ-H2AX assays and cell viability assays are provided in Supplementary Tables 6–15 .

Data availability

All sequence data were deposited to DDBJ/GenBank/EMBL under the accession numbers: (i) BDGG01000001–BDGG01000199 for the nuclear genome scaffolds, (ii) AP017609 for the assembled mitochondrial genome, (iii) FT955276–FT997721 for the GRVF end sequences, (iv) AP013349–AP013352 for the complete sequences of fosmid clones, (v) HY377478–HY448296 for the EST sequences of full-length cDNA clones and (vi) 2343876328–2344039843, 2343537664–2343876048 and 2343264041–2343530383 for WGS trace data. All Illumina sequence reads were deposited to the DDBJ Sequence Read Archive (DRA) under accession numbers (i) DRA001119 for WGS and (ii) DRA001120 for RNA-seq. The sequence of Dsup has been submitted to DDBJ with the accession number, LC050827. The corresponding Bioprojects were deposited to DDBJ/GenBank/EMBL under the accession numbers: PRJDB5011 (Umbrella), PRJDB4588 (Genome assembly), PRJDB1451 (genome short reads), PRJDB2359 and PRJDB2360 (RNA-seq data). The genome browser and the relevant databases are available at http://kumamushi.org/ .

Additional information

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Acknowledgements

We thank Professor Hiroshi Mitani and Associate Professor Yoshikazu Kuwahara for valuable suggestions regarding the comet assay, and Professor Jun-ichi Miyazaki for generous gift of the pCXN2 vector. We are also grateful to Yuriko Hasebe and Nobuhiro Kido for technical assistance in gene model construction, and to the staff of Comparative Genomics Laboratory at NIG for supporting genome sequencing. We thank Yumiko Ishii for her assistance with the cell cycle analysis and Noriko Eto of the OPEN FACILITY of Hokkaido University Sousei Hall with the genome size determination using FACSCanto flow cytometer. This work was supported by JSPS/MEXT KAKENHI Grant Numbers 25281016, 16H02951, 16H01632, 20017010, 16064101 and 221S0002. T.H. received a Grant-in-Aid for JSPS Fellows (No. 25-1805) from the Japan Society for the Promotion of Science.

Author information

Takuma Hashimoto and Daiki D. Horikawa: These authors contributed equally to this work

Authors and Affiliations

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan

Takuma Hashimoto, Daiki D. Horikawa, Yuki Saito, Hirokazu Kuwahara, Koyuki Kondo, Sae Tanaka, Takeo Kubo & Takekazu Kunieda

Graduate School of Environmental Earth Science, Hokkaido University, Kita 8, Nishi 5, Kita-ku, Sapporo, 060-0810, Hokkaido, Japan

Daiki D. Horikawa

Institute for Advanced Biosciences, Keio University, Mizukami 246-2, Kakuganji, Tsuruoka, 997-0052, Yamagata, Japan

Daiki D. Horikawa & Kazuharu Arakawa

Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, 152-8550, Tokyo, Japan

Hirokazu Kuwahara

Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, 108-8639, Tokyo, Japan

Hiroko Kozuka-Hata, Hiroshi Sagara & Masaaki Oyama

Genome Biology Laboratory, National Institute of Genetics, 1111 Yata, Mishima, 411-8540, Shizuoka, Japan

Tadasu Shin-I, Kazuko Ohishi & Yuji Kohara

Comparative Genomics Laboratory, National Institute of Genetics, 1111 Yata, Mishima, 411-8540, Shizuoka, Japan

Yohei Minakuchi, Ayuko Motoyama, Tomoyuki Aizu, Asao Fujiyama & Atsushi Toyoda

Laboratory of Molecular Radiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan

Atsushi Enomoto & Kiyoshi Miyagawa

Phyloinformatics Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minami, Chuo-ku, Kobe, 650-0047, Hyogo, Japan

Yuichiro Hara

Laboratory of Ecological Genetics, Graduate School of Environmental Science, Hokkaido University, Kita 10, Nishi 5, Kita-ku, Sapporo, 060-0810, Hokkaido, Japan

Shigeyuki Koshikawa & Toru Miura

The Hakubi Center for Advanced Research and Graduate School of Science, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, 606-8502, Kyoto, Japan

Shigeyuki Koshikawa

Department of Applied Life Sciences, Laboratory of Extremophiles, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, 192-0392, Tokyo, Japan

Shin-ichi Yokobori

Department of Computational Biology and Medical Science, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, 277-8562, Chiba, Japan

Yutaka Suzuki

Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies), 1111 Yata, Mishima, 411-8540, Shizuoka, Japan

Asao Fujiyama

Database Center for Life Science, 178-4-4 Wakashiba, Chiba, 277-0871, Kashiwa, Japan

Toshiaki Katayama

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Contributions

D.D.H., H.K. and T. Kunieda prepared tardigrades and nucleic acids. The shotgun library and the fosmid library were constructed by K.O., A.M., T.A., A.T. and A.F. Sanger sequencing was performed by K.O., A.M., T.A., A.T., A.F. and Y.K., and assembled by T.S.-I., Y.M., A.F., Y.K. and A.T. The full-length cDNA library was constructed by Y. Suzuki. Illumina sequencing was performed by T.A., A.T., A.F. and Y. Suzuki. D.D.H., S.K. and T.M. determined the genome size by dye staining. S.T. and H.S. captured SEM images. K.A. and T. Kunieda constructed gene model and gene annotation was performed by K.A. Gene repertoires were analysed by Y.H., T.H., S.T., K.K., T. Kubo and T. Kunieda. S.Y. analysed mitochondrial genome. T. Katayama built the genome browser and databases. Y. Saito isolated the chromatin proteins and mass spectrometry was performed by H.K.-H. and M.O. T.H. performed all functional analyses of Dsup protein and radiation analyses were performed by T.H., A.E. and K.M. T.H. and T. Kunieda wrote the manuscript.

Corresponding authors

Correspondence to Atsushi Toyoda or Takekazu Kunieda .

Ethics declarations

Competing interests.

T. Kunieda and T.H. declare competing financial interests, as a part of the work described in this publication has been applied for a patent (Japanese patent application number 2015-032209). All other authors declare no competing financial interests.

Supplementary information

Supplementary information.

Supplementary Figures 1-22, Supplementary Tables 1-15 and Supplementary Methods (PDF 19995 kb)

Supplementary Data 1

Summary of assembled scaffolds (XLSX 80 kb)

Supplementary Data 2

Annotations and expression profiles of the final gene model of R. varieornatus (XLSX 5680 kb)

Supplementary Data 3

Selective expansion in stress-related gene family (XLSX 74 kb)

Supplementary Data 4

Pathway enrichment analysis of lost genes (XLSX 56 kb)

Supplementary Data 5

Selective loss of peroxisomal oxidative pathway (XLSX 70 kb)

Supplementary Data 6

Selective loss of stress-responsive mTORC1 regulatory pathway (XLSX 59 kb)

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Hashimoto, T., Horikawa, D., Saito, Y. et al. Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nat Commun 7 , 12808 (2016). https://doi.org/10.1038/ncomms12808

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Water bears in space.

Thomas Boothby, assistant professor for the Department of Molecular Biology at the University of Wyoming, teaches us about tardigrades, more commonly known as water bears, that are headed up to the International Space Station for a scientific study to learn how these extremophiles adapt to microgravity. HWHAP Episode 197

Listen to the Podcast

If you’re fascinated by the idea of humans traveling through space and curious about how that all works, you’ve come to the right place.

“Houston We Have a Podcast” is the official podcast of the NASA Johnson Space Center from Houston, Texas, home for NASA’s astronauts and Mission Control Center. Listen to the brightest minds of America’s space agency – astronauts, engineers, scientists and program leaders – discuss exciting topics in engineering, science and technology, sharing their personal stories and expertise on every aspect of human spaceflight. Learn more about how the work being done will help send humans forward to the Moon and on to Mars in the Artemis program.

On Episode 197, Thomas Boothby, assistant professor for the Department of Molecular Biology at the University of Wyoming, teaches us about tardigrades, more commonly known as water bears, that are headed up to the International Space Station for a scientific study to learn how these extremophiles adapt to microgravity. This episode was recorded on May 6, 2021.

Gary Jordon (Host): Houston, we have a podcast. Welcome to the official podcast of the NASA Johnson Space Center, Episode197, “Water Bears in Space.” I’m Gary Jordan and I’ll be your host today. On this podcast we bring in the experts, scientists, engineers, and astronauts, all to let you know, what’s going on in the world of human spaceflight. Water bears are about to head to the International Space Station. If you’re not familiar with water bears, or tardigrades, they are super-tiny animals that are best known for their ability to survive in some of the harshest conditions: extreme heat, extreme cold, bottom of the ocean, near volcanoes, highly radioactive environments, and even the vacuum of space. Exactly how they survive in these conditions is something that Dr. Thomas Boothby has been studying for years. Thomas is an assistant professor at the University of Wyoming Department of Molecular Biology, and he’s taking his research to the International Space Station as the principal investigator for Cell Science-04, which is, you guessed it, sending water bears to space to study how they adapt to microgravity. I got a chance to chat with Thomas about water bears and this investigation that will be making its way to the space station aboard the SpaceX Dragon on the upcoming CRS-22 mission, so let’s get right to it. Enjoy.

Host: Dr. Thomas Boothby, thanks for coming on Houston We Have a Podcast today.

Thomas Boothby: Absolutely, my pleasure to be here.

Host: Hey, this mission that’s going to be carrying your experiment to the International Space Station is right around the corner, about to launch. How are you feeling in anticipation of this, of this launch coming up?

Thomas Boothby: Me, personally, I’m extremely excited. We’ve been working on this since 2015, so a lot of hard work and time has gone into this, and really exciting that the launch is right around the corner.

Host: Well, let’s get right into it, Thomas. We’re going to be talking about water bears today and I got to say, I am a huge fan. I discovered them like a, I think it was back, man, it was a couple years ago. “Animal Planet” did this, did this show called “The Most Extreme,” and they did one on like the most extreme survivors, and that’s where they just went deep into the survivability of a water bear. And I was like absolutely fascinated, I could not believe what these things were capable of surviving in. So, let’s just start there, understanding water bears, tardigrades, because that will sort of help us transition into this specific science investigation that’s going to space station. So, let’s start with Tardigrades 101, Thomas, take us through what these things are.

Thomas Boothby: Well, so, tardigrades, or water bears as they’re sometimes commonly referred to as, are a group of microscopic animals that are capable of surviving some of the harshest conditions that we know of. So, despite being these like teeny, tiny, little microscopic organisms that you need a microscope to see, they’re extremely robust, so they can survive a number of extremes that we typically think of as being restrictive to life. So, some examples of sort of extreme environments or conditions that tardigrades can survive include being dried out to the point where they essentially have no water left inside their body or cells; they can be frozen down to just above absolute zero, they can be heated up, in some cases, past the boiling point of water, they can survive thousands of times as much radiation as you or I could; they can go days or weeks with little or no oxygen, and maybe the sort of most remarkable feat that they’ve been shown to perform is that they can actually survive in the vacuum of outer space. They’re the only animal that we know of that can, that can do this, so they’re really, they’re really quite amazing and unique.

Thomas Boothby: So, if I were to, if you were to have a picture of a tardigrade, how would you, how would you describe sort of what they look like?

Thomas Boothby: Yeah, so, what I tell people is think about the little, like gummy bear candy, and imagine that, but with eight legs instead of four. They look like these kind of chubby, little eight-legged gummy bears. Yeah, I think, you know, they’re pretty adorable. If people have seen pictures of them, they’re pretty charismatic, but that’s usually how I describe them.

Host: Yeah, and they’re, I mean the pictures I’ve seen of them, they kind of look clear, right?

Thomas Boothby: Yeah, so depending on what kind of microscope you’re using to look at them, if you’re using like a light microscope, many tardigrades are transparent, so you can, you can see through them. Others aren’t, so different species of tardigrades actually, like morphologically, like how they look, is pretty distinct. You have some that, yeah, as you said, there’s kind of clear. You have others that almost look like they have like armored plates on their backs; they look like little tanks, and those are a little bit harder to see through, but yeah, there’s actually quite a bit of a sort of a morphological diversity within the group of animals.

Host: So, in the beginning of this chat you talked about all of these different, very extreme conditions that the water bears can survive in. So, if I were to go hiking around planet Earth, where could I find them?

Thomas Boothby: So, tardigrades have actually been found almost any, anywhere and everywhere that folks have looked for them. They’ve been found, you know, on the tops of mountains, like in the Himalayas, in the deep ocean, in mud volcanoes, tropical rainforests, in Antarctica, but amazingly, if you just go in your backyard, they’re probably living back there.

Host: Oh, wow, I didn’t realize they were, they were so widespread.

Thomas Boothby: Absolutely.

Host: So, really, when it comes to the extreme stuff, right, I guess, my backyard, I wouldn’t really consider that very extreme, but let’s just say, you know, like near a volcano or in like a very high-pressure environment, you know, what are they doing? Are they just sort of swimming around or do they go into some sort of process to help them survive these extreme conditions?

Thomas Boothby: Yeah, so one of, one of the tardigrade’s sort of greatest tricks is this ability to go into an ametabolic state, so a state where, essentially, they shut down all the sort of life processes that are going on inside of them. And when they do this, they pull their eight little legs and head inside their cuticle, that’s sort of like their exoskeleton that surrounds their body. And they curl up in this little ball-like structure known as a tun. So, have you ever seen like one of these little like roly-poly bugs?

Host: Yeah.

Thomas Boothby: They’re kind of like a tiny, little, microscopic version of that, where they curl up in this little ball, they shut down all their life processes that are going on and, you know, for all intents and purposes, you know, they sort of, it’s almost like they’re dead, but they’re in this state, they’re extremely resilient, and they’re able to ride out the, the sort of rough, harsh, extreme conditions. So, you know, if that’s a desiccating environment where water is being lost, you know, they’ll curl up in this little ball-like structure, dry out, and then, you know, when water returns to the environment, they uncurl. They come out of this ball-like structure and within an hour or so, you’ll see them scrambling around, eating, reproducing like nothing happened to them.

Host: Unbelievable. I’m sure you’ve been studying this for a long time, so, you know, you talk about when water is reintroduced to environment, or they’re, you know, they’re in a better environment where they can get out of this tun. How long have you seen some of these water bears in this state, before they return back to, you know, kind of frolicking through, and eating, and reproducing and all that?

Thomas Boothby: Yeah, so, tardigrades are able to enter this ametabolic state, and many species are extremely stable and viable in that, in that state. So, kind of an average would be about a decade or so in this, in this ametabolic state, but there are reports that tardigrades have been shown to survive, you know, for like over a hundred years; these were experiments going into herbariums, where they had preserved plant material, and people gathered tardigrades off that preserved plant material, which, you know, was documented and cataloged, when it was gathered and preserved. And they’ve been able to purportedly revive tardigrades that are, you know, hundreds of years old.

Host: Unbelievable. Now, I mean, it’s, this is a very unique trait for an animal. You know not everyone, not every—every one —every animal can do this. So, what is it about the tardigrade? What unique quality do they possess to be able to do this sort of thing?

Thomas Boothby: Well, that’s a really excellent question, and that’s something that my lab and other labs are trying to uncover. We’ve found some hints and clues, but certainly there’s a lot more to learn. But one of the really interesting features of tardigrades that we found was, that when they start to dry out or enter these sorts of extreme environments, they start to produce a very special class of protein. And this is actually a type of protein that is unique to tardigrades. So, so no other organisms that we’ve, that we’ve looked at possess similar, similar proteins. And what these proteins do is something very interesting: they build up in their concentration. So, the tardigrades just start making more and more and more of these things. And what these proteins seem to do is they make the environment inside the tardigrade, so like inside the tardigrade cells, really, really viscous. So, imagine, you know, as opposed to water, which is very liquid, imagine it more like becoming like honey, where it’s very sort of gooey and viscous. And what this sort of increased viscosity does is it slows down all the bad things that are happening. So, you know, parts of cells start to break down, or unfold, or fuse together, normally, when a cell is drying out, but in this sort of super-viscous environment all those things are still happening, they’re just happening very, very slowly. And when this super, sort of super-viscous environment gets even drier, it, what it does is it forms a glass, so like glass in a windowpane, and this is really important because glass has a very different molecular makeup than say something like a crystal. So, if tardigrades made something that filled their cells with crystals, that would be really bad because crystals are very sharp and pointy, they can puncture cells or crush, you know, sensitive material inside of the tardigrade cells, but these glasses are much smoother and sort of more amorphous, and they’re actually able to encapsulate these sensitive molecules inside of tardigrade cells, and actually preserve them within this sort of glass-like matrix or structure. And what’s really amazing is, when water returns to the system, when you rehydrate a tardigrade, that glassy material just kind of melts away, and it goes back into solution, it dissolves into the, into the water, and it releases all those sensitive molecules that were stabilized inside of it back into the tardigrade cell, where they can perform their normal biological functions.

Host: Thomas, this is, this is amazing. I mean, I, my next question I feel like is a genuine one, but I feel like just everything you’ve just described sort of answers it for me, just how interesting this is, but what got you interested in this fascinating world of researching tardigrades?

Thomas Boothby: Yeah, that’s a great question. So, so besides tardigrades just being, you know, at least to me, like really fascinating, you know, wanting to understand kind of the, the outliers in biology, right, like, you know, tardigrade biology is quite unique, and in my opinion, understudied, and so, you know, just from a purely, from a place of pure intellectual curiosity, understanding how these little creatures are able to do something that, you know, for us, would be so sort of mind-bogglingly impossible to achieve is, was really, really of interest to me. And then, beyond sort of the fundamental biology of tardigrades, I was really attracted to studying them because of some of the potential applications, you know: what we could do in terms of taking the fundamental biological findings that we made studying tardigrades, and sort of the promise of applying that knowledge to trying to solve real world problems was really, really sort of attractive to me.

Host: So, tell me about, you’re, you’re at the University of Wyoming, right? So, you sort of went and described a little bit more about this protein, and you mentioned that you’re still doing a lot more research to figure out exactly what’s going on to allow the tardigrade to have this sort of unique process. So, tell me about some of your research that you’re doing over there.

Thomas Boothby: Absolutely. So, we’ve got, we’ve got quite a bit of sort of diverse research going on here. On sort of the fundamental biological side, we’re really interested in understanding how these tardigrade proteins are working. So, like, what are the building blocks that make up these proteins that make them so special and so protective? We’re also really interested in understanding whether or not these proteins and other tricks that tardigrades use to, say, survive when they dry out, we’re really interested to know if those are the same tricks that tardigrades use when they’re faced with other extremes, like freezing, for example. So, do tardigrades have sort of one, one way of surviving many different extremes or do they have many different tricks for surviving all these different extremes? And then on the more applied side, we’re really interested in how we can take that knowledge and adapt it to addressing real world problems, like stabilizing pharmaceuticals, or developing crops that are more resistant to extreme environments, so that’s kind of our research, in a nutshell.

Host: So I imagine, you mentioned there are tardigrades all over the world and you want to understand more about the, some of these different processes, or at least when they hibernate, or I guess, go into this, you said, I forget the exact state, something about metabolism, but essentially, into this state, and in this tun, do you get to travel to some of those locations as part of your research, like to, you know, volcanoes, or to whatever, deep sea, and understand, like pressure, or are you bringing them to the lab and doing everything in the, at your university?

Thomas Boothby: Yeah, so, so a little bit of both. So, a couple years ago, as part of a NSF (National Science Foundation) training, training grant, I was able to go down to Antarctica, and we were finding tardigrades down there, along with doing some experiments. One of the reasons that our lab located to Wyoming was to be closer to some of these extreme environments that we study organisms from. So, Wyoming has a number of really diverse extreme environments. You know, people typically think of, you know, sort of the hot springs out in Yellowstone, but then there’s also Wyoming’s Red Desert, which is an immense, high-elevation desert, so, so very cold and very dry, with sort of Martian-like environments. And then, of course, you have the Bighorn Mountains, the Snowy Range Mountains, so, you know, we kind of have all different types of environments out here in Wyoming where we go in and collect organisms from.

Host: That’s pretty cool. Yeah, you got to enjoy those kinds of trips then, you got to enjoy the harsh environments.

Thomas Boothby: Absolutely. Yeah, it helps, it helps to be a little bit tough if you want to go and study these little critters out there.

Host: Well, look, the space station is only 250 miles from Wyoming, you just got to go straight up. So, how did it happen where you were looking at all these different extreme environments and you thought, ah, you know, where we should go is the space station?

Thomas Boothby: Yeah, so, you know, how that kind of came about was, I was just really curious in this observation that tardigrades actually survive a number of extremes that they would never have been exposed to, so it’s kind of this perplexing question of, you know, how could an organism evolve to tolerate a condition that it, it didn’t evolve in? And spaceflight and space environments are probably some of the sort of most foreign or alien environments that you can think of for an organism that evolved on Earth. And so, there have been some space studies using tardigrades before. In particular there was a, there was a Russian capsule that went up in 2007, which actually exposed tardigrades to the vacuum of space, and they were left out there for about ten days in low-Earth orbit, and they were shown to be viable after that exposure. There was another mission involving some Italian scientists, which showed that tardigrades could survive and reproduce without any negative effects during spaceflight. And so, yeah, I got really interested in trying to understand how, right, not just, can they do this, but how are they able to do this? And so, that’s really, kind of the, kind of main driving scientific question for Cell Science-04 mission, is understanding how tardigrades adapt to being exposed to outer space, or to space conditions, rather. And then under those prolonged spaceflight conditions, how do they change and adapt after that initial exposure, you know, say over multiple generations?

Host: So, let’s get into it, let’s get into the experiment that’s going on the International Space Station, you called it Cell Science-04. So, what’s the, what’s this experiment that’s going up?

Thomas Boothby: Yeah, so what we’re really interested in doing is looking at what the initial changes in gene expression, so, so how tardigrades are adapting to spaceflight environments, is initially, and then how that changes over multiple generations. So, essentially, what we’re going to be doing is sending tardigrades up from the Kennedy Space Center to the ISS, and we’re going to basically have two different pools of tardigrades. One pool is going to be our sort of founding generation, where after a week of being in space, we’re going to preserve them in a special chemical preservative, but then the second pool we’re going to let culture, and grow, and reproduce for two months, and that’ll represent about four generations of tardigrades. So, they’ll have time to reproduce, and their offspring will reproduce, and so on, and so forth, for four generations. And then we’re going to preserve those multigenerational tardigrades. And when we get these preserved tardigrades back to our lab here in Wyoming, what we do is we extract a certain molecule called RNA, and this is kind of an intermediate molecule between the tardigrade’s DNA, their genes, and the final products that those genes are sort of the blueprint to make. And so, by looking at these molecules that we can extract, we can tell what changes in gene expression tardigrades are inducing when they’re exposed to space initially, and when they’re exposed to spaceflight conditions over the long term. And our hope is that by understanding how these tough little organisms are able to survive spaceflight conditions, that this will give us hints and clues into, you know, how we might safeguard astronauts during prolonged space missions.

Host: See, that’s going to be a big deal, especially for NASA’s plans to go to the Moon and Mars, just one extra step to help out in that process, very, very fascinating stuff.

Host: I’m curious to hear about how you’ve been preparing to get this experiment going. You know, you, I guess had to start with the initial process of figuring out how to get the tardigrades into space, but what’s been the process from the initial concept, to getting everything packed, and basically, ready to go on a rocket?

Thomas Boothby: Yeah. So, so initially, kind of the biggest consideration was just trying to figure out how we’re going to grow these little animals in space. So, in the lab, we normally grow them in these sorts of big glass petri dishes filled with a liquid medium, but in space that wouldn’t work so much because the, in microgravity the liquid media that the tardigrades grow in would just sort of float away. So, yeah, initially, it was validating a bioculture system that had been developed by some NASA engineers and adapted to this project. And then, it was really just going through a number of sort of dry runs and seeing, you know, in ground-based experiments, how our experimental plan for the actual flight experiment went. You know, it was a lot of optimizing things that don’t sound very exciting, like how fast a pump moves water through the system, or how much oxygen we need to deliver to the media. But, you know, all that sort of nitty-gritty detail has been worked out and we’re now, actually, this, just this week, in the process of prepping our samples that will go up to the ISS, so that basically involves loading the tardigrades into syringes that will be frozen and can be stored frozen and delivered to the ISS in this sort of inactive state. And then along with that we’re packaging up a lot of the food that the tardigrades eat because, over multiple generations, they’re going to need to be fed a couple times to stay healthy. So, the species of tardigrade that we use, they eat unicellular algae, so little, little algal cells, so we’re also getting those loaded into syringes, and ready to be sent up to the space station.

Host: So, actually, running the experiment on station, it sounds like this, whatever setup you have is going to be installed on a facility on space station, and every once in a while, is it going to require astronaut interaction to go ahead and use this syringe and feed the water bears, over generations?

Thomas Boothby: Absolutely. So, yup, when the, when our samples get up to the space station, they’ll be in syringes, and the astronauts will need to thaw out the tardigrades to sort of reactivate them, and then inject them into this bioculture system. Once they’re in the bioculture system, it’s pretty hands-off. We have telemetry, so we can sort of monitor the temperature and the flow rates and everything, inside the bioculture system. But then, yeah, you’re exactly right, at sort of two-week intervals an astronaut is going to need to attach an algal syringe to the bioculture system and inject fresh algae into it for the, for the tardigrades to eat, but, and then, and then at the end of the experiment they’re going to need to essentially sort of dismantle a portion of that bioculture system, which will be frozen and stored until we can get it back at our lab in Wyoming. So, there will definitely be some astronaut interaction with this, with this experiment, but there are also sort of large portions that are, that are automated.

Host: Yeah. Honestly, it sounds pretty easy in terms of the astronaut crew time needed, just, you know, feed it, it sounds like not even that often. You said once every two weeks was the feeding schedule?

Thomas Boothby: Yup.

Host: Yeah, see, it’s not even that bad. When it comes to measuring though, you said you’re going to, it is going to return, that’s part of the plan is it returns back to Earth, you go to the lab, and you have a number of things that you’re going to be looking at. Is there anything on orbit that will be, that you have in terms of measuring tools? It sounds like you have the, in the facility, you have the ability to control climate and watch all data coming in, but are you going to be doing any data gathering from the facility while it’s in orbit?

Thomas Boothby: So, the only sort of measurements that we’re making on orbit are environmental measurements. So, you know, what the, what the environment that the bioculture system is in. And we’re going to be using that telemetry, so, you know, the temperature, and whatnot, to replicate those experiments back here on Earth, and so we call those our near-synchronous ground controls. So we’re basically going to be doing the exact same experiment, but here on Earth, almost in real time with the, with the flight mission. But yeah, all the, all the sort of biological data that will be gathered is going to be done once we get the samples back from the space station, then we’ll process them here, here in the lab in Wyoming.

Host: So, you said — you said we, right? So, it’s not just, it’s not just you sending the water bears up and looking at everything. It sounds like you got, you got a decent support team that’s helping to bring all of this together.

Thomas Boothby: Yeah, absolutely. And yeah, it’s really great that you bring that up because, you know, I’m just one person in a team of really amazing folks. So, here, in the lab, in Wyoming, specifically, we have Cherie Hesgrove and Ryan Bettcher are working on this project. But then, on the NASA and the KBR side of things, we have a lot of people who really, sort of deserve some credit. So, specifically, Medaya Torres is our CS-04 mission scientist, Natayla Dvorochkin is our contract support scientist, and then on the KBR side there’s a bunch of people that I’d just like to acknowledge really quickly: Daniel Nolan, Kevin Sims, Oscar Roque, Christina Lim, Crystal Kumar, Kris Vogelsong, Brandon Schmitt, and Jamie Bales, Susan Markey, Meghan Feldman. And then on the NASA engineering side, Peter Zell and David Pletcher. And I’m sure I’m forgetting some other folks, but it’s been a, it’s been a huge team and group effort to get to this point, and yeah, I think it’s worth taking a couple minutes just to acknowledge all these folks.

Host: Absolutely. Yeah, and that’s part of the whole deal, right, is it’s not just, you know, it’s not just, “hey, Thomas, let’s get your experiment on the International Space Station,” it does really take a team, not only to get it up there, but to do all the work, to monitor it, make sure it’s working fine, and then of course, what you, what you’re all anticipating is when you get the water bears back from space into the lab in Wyoming and you get to conduct some fascinating research from that, from that group of water bears that went up there.

Host: Yeah. Now, one of the things I’m thinking of, Thomas, is, you know, there’s, well, I think what we’re all anticipating is when you’re starting that research, you know, what are the potential applications that you are thinking of in terms of maybe something we can learn, that we can bring back to benefit us here on Earth, or something that we can use to further space exploration. What are some of the things that you’re looking at that might have potential benefits to this experiment?

Thomas Boothby: Yeah. Well, definitely, part of the sort of stated goal of this mission is, you know, to start to build a foundation for developing therapies or countermeasures that might better safeguard astronauts in the future during prolonged space missions. So, you know, as I sort of mentioned before, spaceflight can be a really challenging sort of environment for organisms, including humans, who have evolved to the conditions on Earth. So, in space, you have much less gravity, you’re in microgravity, and you’re also exposed to a lot more radiation. So, for humans who spend a lot of time in space, you know, there can be detrimental effects to being in these environments. And so, one of the things we’re really keen to do is understand, you know, how are tardigrades surviving and reproducing in these environments, and can we learn anything about the tricks that they’re using that might be adapted to safeguarding astronauts. So, for example, if we see that tardigrades, when exposed to sort of this increased radiation in space, which produces a lot of reactive oxygen species, which are these sort of damaging chemical moieties that are really bad for cells, if tardigrades are producing a lot of reactive oxygen species scavengers, which basically kind of negate those negative effects, then that might be something that we would consider either through, you know, like a dietary supplement, or something like that, providing astronauts with increased antioxidants or reactive oxygen species scavengers. That would just help them stay healthier in space for longer.

Host: See, this makes me think about this experiment and this, like you said, you want to, you want to set a foundation, right, that’s what you were talking about whenever you were thinking of potential application. And I think that’s a very exciting thing to say because what makes me, it makes me think that this is scalable, right? You can continue the research, maybe, maybe bringing next cell science investigations up to the International Space Station. And you were just mentioning the radiation environment, which in low-Earth orbit is a little bit different from say, the Moon. And with the Artemis program, with NASA returning to the Moon, there are potential, there are potential options to have investigations like this, where you can study water bears in an even higher radiation environment and gather even more unique data. So, it, to me it sounds like this is something that you can continue for a while.

Thomas Boothby: Absolutely, we hope so. I think, you know, there’s a lot more to learn about tardigrades and a lot of, you know, continuing potential benefits to society.

Host: And that’s a, that’s such a big deal and it’s all happening on board the International Space Station coming here real soon. So, Dr. Thomas Boothby, thank you again for coming on Houston We Have a Podcast. And really, best of luck to you and your team as you gear up for this launch of a, on a SpaceX Cargo Dragon to the International Space Station. Best of luck to you as your, as your journey just begins for Cell Science-04.

Thomas Boothby: Great. Thanks very much.

Host: Hey, thanks for sticking around. I hope you learned something about water bears and you’re as excited as I am for this launch of CRS-22. You can watch these water bears launch from Florida, travel to the International Space Station. Just check out our website NASA.gov/ntv has the latest on our TV schedule when you can see the launch live. If you like this podcast, we are one of several NASA podcasts across the entire agency; you can check all of them out at NASA.gov/podcasts . We, Houston We Have a Podcast, are on the Johnson Space Center pages of Facebook, and Twitter, and Instagram. So, if you want to talk to us, just use the hashtag #AskNASA on your favorite platform, you can submit an idea or ask us a question, just make sure to mention it’s for us at Houston We Have a Podcast. This episode was recorded on May 6, 2021. Thanks to Alex Perryman, Pat Ryan, Norah Moran, Belinda Pulido, Jennifer Hernandez, Rachel Barry, and the International Space Station Program Research Office for helping to set us up with Thomas. And, of course, thanks again to Dr. Thomas Boothby for taking the time to come on the show. Give us a rating and feedback on whatever platform you’re listening to us on and tell us what you think of our podcast. We’ll be back next week.

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The Tardigrade: Practically Invisible, Indestructible ‘Water Bears’

By Cornelia Dean

• Sept. 7, 2015

When scientists at the American Museum of Natural History mounted an exhibit about creatures that survive under conditions few others can tolerate, they did not have to go far to find the show’s mascot.

“We just got them from Central Park,” said Mark Siddall , a curator of the show, Life at the Limits . “Scoop up some moss, and you’ll find them.”

He was talking about tardigrades , tiny creatures that live just about everywhere: in moss and lichens, but also in bubbling hot springs, Antarctic ice, deep-sea trenches and Himalayan mountaintops. They have even survived the extreme cold and radiation of outer space.

Typically taupe-ish and somewhat translucent, and a sixteenth of an inch or so long, they are variously described as resembling minuscule hippopotamuses (if hippos had giant snouts and eight legs, each with several claws), mites or, most commonly, bears. Many people call them “water bears” or “bears of the moss.” (The word “tardigrade” is from the Latin for “slow walker” and pronounced TAR-dee-grade.)

Once an object of interest only among zoological specialists, tardigrades now are generating widespread enthusiasm. Admirers have produced artwork and children’s books about them, and have even organized the International Society of Tardigrade Hunters “to advance the study of tardigrade (water bear) biology while engaging and collaborating with the public.”

According to the society, formed this year at the University of North Carolina at Chapel Hill, people can find tardigrades if they gather some lichen or moss, especially on a damp day, put it in a shallow dish of water, and “agitate” it a bit. Debris will settle to the bottom of the dish, and tardigrades will probably be prowling in it.

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Survivalists at the Micro Level: The Amazing World of Tardigrades

Welcome to the incredible microcosm of survivalists – tardigrades. Known as water bears, these microscopic marvels defy the limits of endurance in the most challenging environments. From the deep sea to towering mountains, tardigrades showcase unparalleled adaptability. In this exploration, we unravel the secrets behind their resilience, cryptobiosis mastery, and survival in space, unveiling the amazing world of tardigrades as true champions at the micro level.

Microscopic Wonders

Tardigrades, affectionately termed water bears, are exceptional microscopic organisms, epitomizing extraordinary resilience. Despite their diminutive size, these creatures exhibit an unparalleled ability to withstand extreme conditions, navigating environments where others falter. From the depths of the ocean to the highest mountain peaks, tardigrades showcase a tenacity that defies their minuscule stature. Colloquially celebrated as water bears, these microscopic wonders capture the essence of survival at the micro level, embodying an awe-inspiring ability to endure and thrive in the face of challenges that surpass the limits of larger organisms.

Diverse Habitats

Tardigrades, commonly known as water bears, emerge as masters of adaptation, thriving across a spectrum of diverse habitats that span the Earth’s extremes. From the lightless depths of the deep sea to the lofty heights of mountainous terrains, these microscopic marvels showcase an unparalleled adaptability to the harshest conditions nature offers.

In the ocean’s abyssal plains, tardigrades navigate the crushing pressures, demonstrating a resilience that sets them apart. Scaling mountainous landscapes, they endure oxygen-thin air and temperature extremes. Tardigrades are equally at home in mosses, lichens, and leaf litter, showcasing their ability to persist in microenvironments that pose challenges to most life forms.

The astounding adaptability of tardigrades across such diverse ecosystems reflects their prowess in conquering extremes. This adaptability not only fuels their survival but also positions them as extraordinary indicators of life’s potential in environments deemed inhospitable to many. As we explore their presence in various habitats, we gain a profound appreciation for the resilience and versatility that define these microcosmic survivalists.

Resilience Unveiled

Tardigrades, colloquially known as water bears, reveal a profound mystery in the realm of resilience, defying conventional limits and thriving where few organisms dare to venture. This microscopic wonder unfolds a tale of unparalleled endurance, showcasing the secrets behind their ability to withstand extreme conditions. Whether subjected to harsh temperatures, intense pressures, or the vacuum of space, tardigrades stand resilient, unraveling nature’s secrets of survival at a scale that challenges our understanding. The water bear’s resilience unveils a microcosmic marvel, inviting us to explore the extraordinary mechanisms that enable them to triumph in the face of adversity.

Cryptobiosis Mastery

Tardigrades, the microscopic marvels often referred to as water bears, wield an extraordinary survival strategy – cryptobiosis. In this remarkable state, they adeptly shut down their metabolism, a feat that enables them to endure the harshest conditions, including desiccation. This microcosmic mastery allows water bears to suspend their biological activities, essentially entering a state of suspended animation. Cryptobiosis emerges as the key to their resilience, unveiling a fascinating adaptation that empowers tardigrades to navigate extreme environments and persevere in the face of challenges that would spell doom for many other life forms.

Space Survivors:

Embark on a cosmic journey with tardigrades, the resolute water bears, as they venture into the inhospitable realms of outer space and emerge as extraordinary space survivors. In pioneering experiments, these microscopic marvels have defied the odds, enduring the vacuum and radiation of space aboard spacecraft. Strapped to the exterior of satellites and shuttles, tardigrades have withstood the harsh conditions, showcasing an incredible resilience that challenges our perceptions of life’s fragility beyond Earth. As space travelers, they offer insights into the potential for life to endure in extraterrestrial environments, inspiring awe and curiosity about the microorganisms that defy the cosmic challenges of the void.

Biological Adaptability: 

Tardigrades, endearingly known as water bears, unveil a stunning display of biological adaptability, demonstrating an unparalleled ability to persist in diverse and hostile environments. These diminutive creatures navigate extremes with remarkable ease, showcasing a versatility that defies conventional limits. From the depths of the ocean to the peaks of mountains, tardigrades embody an exceptional adaptability that allows them to thrive where others struggle. Their ability to confront and conquer varied challenges reflects a microcosmic triumph of biological resilience, offering a testament to nature’s ingenuity in creating life forms capable of flourishing in the most contrasting landscapes.

Anhydrobiosis Feat:

Delve into the extraordinary phenomenon of anhydrobiosis, a captivating survival strategy mastered by tardigrades, the resilient water bears. In this remarkable process, these microscopic marvels can shed almost all body water content, entering a desiccated state, only to spring back to life upon rehydration. Anhydrobiosis allows tardigrades to withstand extreme dehydration, navigating environments where water scarcity would typically be lethal. Witnessing this feat of desiccation tolerance unveils a microcosmic spectacle of adaptation, where tardigrades seemingly defy the constraints of conventional life, showcasing a remarkable ability to suspend life processes and revive when conditions once again become conducive.

Scientific Marvels:

Tardigrades, often affectionately called water bears, emerge as true scientific marvels, unravelling mysteries that captivate researchers and broaden our understanding of extremophiles and life’s potential in extreme environments. These microscopic organisms, with their unparalleled resilience, become invaluable subjects of study, providing critical insights into the boundaries of life on Earth and beyond.

As extremophiles, tardigrades redefine our understanding of where life can thrive. Their ability to endure extreme temperatures, pressures, and even the vacuum of space positions them as extraordinary contributors to astrobiology and the exploration of extraterrestrial life.

Studying tardigrades not only expands our knowledge of these microscopic marvels but also fuels scientific curiosity about the adaptability of life in the cosmos. In unlocking the secrets held by water bears, scientists glimpse into the intricacies of survival, resilience, and the potential for life to persist in the most challenging corners of the universe. Tardigrades stand at the intersection of scientific fascination, offering a gateway to exploring the frontiers of life’s tenacity and adaptability in environments previously thought uninhabitable.

Microbial Survivors:

Acknowledging the microscopic wonders known as tardigrades reveals an extraordinary narrative of tiny survivors contributing significantly to our understanding of microbial survival strategies. These water bears, though minuscule, play a pivotal role in unraveling the intricacies of how microorganisms persist in diverse and often challenging environments.

Tardigrades, with their resilience to extreme conditions, act as ambassadors for microbial life, showcasing adaptability that transcends the limitations of size. By navigating the abyssal depths of the sea, scaling towering mountains, and even braving the rigors of outer space, these microbial survivors become invaluable subjects in the exploration of microbial survival tactics.

Understanding the strategies employed by tardigrades sheds light on the broader realm of microbial life. Their ability to withstand desiccation, endure radiation, and thrive in various ecosystems underscores the versatility of microbial survival. As we acknowledge the significance of tardigrades, we gain insights into the fundamental mechanisms that govern microbial resilience, fostering a deeper appreciation for the myriad ways these tiny survivors contribute to the intricate tapestry of life on our planet and, potentially, beyond.

Conclusion 

In delving into the amazing world of tardigrades, the microscopic survivalists, we unveil a testament to nature’s ingenuity and the resilience of life at the micro level. From the depths of the ocean to the vastness of outer space, these water bears defy conventional limits, showcasing unparalleled adaptability and survival strategies.

Tardigrades, with their mastery of cryptobiosis, endurance in space, and ability to navigate extreme environments, stand as extraordinary ambassadors of microbial resilience. Their significance extends beyond their diminutive size, offering valuable insights into extremophiles and challenging our understanding of where life can thrive.

As we conclude this exploration, we acknowledge the exceptional role tardigrades play in advancing scientific knowledge. Their microcosmic triumphs inspire awe and curiosity, inviting us to ponder the broader implications for life’s potential in the universe. Tardigrades, the unsung heroes of microscopic survival, continue to illuminate the wonders of life at the smallest scales, leaving an indelible mark on the scientific landscape.

• A: Tardigrades, colloquially known as water bears, are microscopic, water-dwelling animals known for their resilience.
• A: Tardigrades inhabit diverse environments, including mosses, lichens, leaf litter, deep-sea environments, and even outer space.
• A: Tardigrades earned the nickname “water bears” due to their lumbering movement reminiscent of a bear’s walk.
• A: Cryptobiosis is a state where tardigrades suspend their metabolism, enabling them to survive extreme conditions like desiccation.
• A: Yes, tardigrades have demonstrated the ability to survive the vacuum and radiation of outer space.
• A: Tardigrades have been discovered in a wide range of environments, from the deep sea to high mountain peaks.

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Frontiers for Young Minds

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Water Bears—The Most Extreme Animals on The Planet (And in Space!)

Can you imagine that there is an eight-legged bear that tolerates colder temperatures than the polar bears do in the Arctic? Can you imagine that this bear is able to grow older than the grizzly bears in North America? And can you imagine that this bear grows by molting, like spiders or snakes? These so-called water bears, scientifically named tardigrades, are the most extreme animals on our planet. They not only survive in ice, but also in boiling water. Moreover, they can stop breathing for long periods and they have even traveled to outer space, surviving without an astronaut’s suit. Since water bears can withstand the harshest conditions on earth and beyond, they may teach us how we can protect ourselves from extreme environmental conditions.

Are Water Bears True Bears?

What are water bears? Are they really bears? This question is easy to answer: no, the only thing that water bears and bears have in common is the fact that both are animals. The shape of a water bear slightly resembles that of true bears, such as the polar bear or the grizzly, but they are most closely related to the huge group called the arthropods , which includes insects, spiders, millipedes, and crabs. However, you cannot see a water bear with the naked eye, because these animals are very tiny. They usually grow to <1 mm ( Figure 1 ). Water bears were discovered more than 200 years ago [ 1 ]. The German pastor and biologist Johann Goeze initially named them “little water bears,” because of their size and their preference for wet living spaces.

• Figure 1 - Water bears, also called tardigrades, are extremely small compared to other animals.
• This image of a water bear was taken with a scanning electron microscope. The water bear micrograph by Bob Goldstein and Vicky Madden ( https://en.wikipedia.org/wiki/Tardigrade#/media/File:Waterbear.jpg ). Photographs of the grasshopper and the cat by S. Elleuche.

Water bears love wet or at least humid environments where they can remain covered by a layer of water. They are among the most successful lifeforms known and are widely distributed all over our planet. We can observe water bears in all oceans, rivers, seas, and lakes, and in wetlands , but they are mainly found in mosses or swamps. Water bears have even conquered the highest mountains, rainforests, and Antarctica. Many different types of water bears have been found and described. They even conquered Hollywood, where you may have encountered water bears in the Marvel superhero movies “Ant-Man” and “Ant-Man and the Wasp,” when Scott Lang disappears into the quantum realm.

Water bears have a strange shape—they are of stout build with four pairs of short and stubby legs, ending with four to eight claws, and they appear to lumber along as they move ( Figure 1 ). The first three pairs of legs are used for moving, while the water bears use the last pair of legs to hang on to the surface on which they walk. Even with so many legs, water bears usually do not walk but instead passively slide, using the flow of water or wind. The way they move is also reflected by their scientific name: tardigrades . Tardigradum means “slow walker,” and this name was given to water bears by Loredano Spallanzani, a former Italian biologist, due to the slow and sedate behavior of these animals, which might look like laziness.

How Do Water Bears Grow?

Just like almost any other creature on our planet, water bears must eat food and breathe air to generate the energy needed for their cells to divide and their bodies to grow. In contrast to true bears, water bears are just too tiny to eat salmon or seals. Honey is also not on their menu. Nevertheless, water bears basically eat everything. While they mainly prefer vegetarian foods like plants and algae, they will also eat microscopic animals.

Unlike most other animals, the bodies of water bears are created following a specific plan. Every type of adult water bear even has exactly the same number of cells. Their cells are continuously dividing, but the water bear is covered by a non-growing and non-flexible sheath, or protective outer covering. As soon as the sheath becomes too tight, water bears will shed the sheath in a process called molting , similar to spiders and snakes. Although both humans and water bears need oxygen to survive, water bears do not breathe the way we do. In fact, they do not even possess respiratory organs like lungs. Water bears take up air through the surfaces of their bodies, just like insects. Water bears can even stop breathing and eating for some time, similar to the process of hibernation that allows other animals, such as polar bears, to slow down their bodily processes to survive the winter months. However, water bears are even more impressive, because not only can they sleep for a couple of months, but they can also become extremely old and thrive in the most extreme places on earth.

What Are the Most Extreme Living Spaces for Water Bears?

Water bears are the most extreme animals that we know—they basically tolerate almost every extreme condition that we can think of. They can survive in the Arctic alongside polar bears, or in Antarctica, where penguins feel at home ( Figure 2A ). Water bears even survive in the laboratory at temperatures below −200°C, which is more than twice as cold as the coldest temperature that was ever observed in nature. Under such extreme conditions, the water bears enter a stage that resembles death. During this death-like resting stage, called dormancy , water bears stop all functions that usually define life: they stop breathing, they stop moving and growing, and they even stop digesting their last meal [ 2 ]. Depending on how long they are in dormancy, it can take several hours to wake them up. Some water bears have even been seen to last for a century in dormancy.

• Figure 2 - (A) Water bears can survive in extremely cold habitats, like the icy Himalaya mountains, and at temperatures as low as −150°C.
• (B) Water bears can survive in extremely hot habitats, like the hottest deserts, and at temperatures as high as 100°C. (C) Water bears can even survive in the vacuum of space!

On the other end of the temperature scale, there are microbes that can grow at temperatures around 120°C. These heat-loving microbes are called extremophiles [ 3 , 4 ]. Water bears do not love the extreme heat, but not only can water bears survive in the desert, they can even tolerate temperatures around 150°C ( Figure 2B )—temperatures that would kill most extremophiles. Even more impressive is the fact that water bears can be repeatedly heated up and frozen without dying. These abilities have allowed water bears to become unrivaled in their success over the course of evolution. More than 1,000 different types of water bears are known, with the oldest species dating back more than 500 million years.

Water bears do not only survive the coldest cold or the hottest heat without food and without air to breathe, but they can also go without water and they are resistant to radiation. Since those extreme conditions exist in space, scientists asked themselves whether water bears might even be able to travel in space ( Figure 2C ). Scientists knew that the high pressure present in the deep sea could be tolerated by water bears, but in space there is a vacuum, with lower pressure compared to earth. Nevertheless, several species of water bears were sent into space and all of them returned home in healthy condition. Moreover, more than 1,000 water bears in dormancy were crash-landed on the moon as passengers of a spacecraft in 2019. It is expected that most of these robust animals have survived the crash and could be revived by water and oxygen in the future.

Could Water Bears Be Used to Help Humans?

For a long time, scientists have been trying to understand the water bears’ resistance to radiation. Although radiation in the form of X-rays can be used by doctors to examine broken bones, radiation can also cause the destruction of the body’s instruction manual. This instruction manual is called the genome , and it is similar in every living organism on earth, including water bears. There must be a reason for the immense resistance to radiation seen in water bears, which is more than 1,000 times higher than humans’ resistance.

One part of the genome of water bears has recently been identified and reproduced in a laboratory [ 5 ]. When this factor was added to human cells grown in the same laboratory, the human cells tolerated more intense radiation than did human cells without the water bear factor. These early experiments may lead to future applications of water bear factors that could not only be used to protect the human cells against radiation, but possibly also to stabilize drugs or to increase the resistance of crop plants to environmental conditions like drought.

What We Have Learned From Water Bears

So, now you can see that those little water bears are quite different from the bears we know well. We have learned from these animals that they not only tolerate the most extreme conditions on our planet, they are even capable to survive in Space. Because of these unique properties, water bears are fascinating and among the most interesting model organisms for us to further study.

Arthropods : ↑ This group of animals is characterized by the outer skeleton and includes insects, spiders, millipedes, and crabs.

Wetland : ↑ A living space for multiple organisms that is temporary or permanently flooded by water and inhabited by aquatic plants.

Tardigrade : ↑ A scientific nomenclature for a group of animals that are also known as water bears or moss piglets.

Molting : ↑ Some animals, such as water bears, insects, spiders and snakes do not grow continuously. They have to replace their outer sheath when it became too tight.

Dormancy : ↑ Death-like resting stage during which each kind of activity such as growth or ingestion is temporarily stopped.

Extremophiles : ↑ Microorganisms that love to live in the most extreme environments on the planet. Water bears are no true extremophiles because, although they can tolerate extreme conditions, they do not prefer such environments.

Genome : ↑ A kind of construction plan that is included in every living cell in all organisms (Bacteria, Fungi, Plants, Animals etc.), which determines the look and composition of most cellular compon.

Conflict of Interest

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The author thanks Sylvia Wiese and Jan Friesen for critically reading the manuscript.

[1] ↑ Jönsson, K. I. 2019. Radiation tolerance in tardigrades: current knowledge and potential applications in medicine. Cancers 11:1333. doi: 10.3390/cancers11091333

[2] ↑ Fontaneto, D. 2019. Long-distance passive dispersal in microscopic aquatic animals. Mov. Ecol . 7:10. doi: 10.1186/s40462-019-0155-7

[3] ↑ Elleuche, S., Schröder, C., Stahlberg, N., and Antranikian, G. 2017. “Boiling water is not too hot for us!”—preferred living spaces of heat-loving microbes. Front. Young Minds . 5:1. doi: 10.3389/frym.2017.00001

[4] ↑ Schröder, C., Burkhardt, C., Antranikian, G. 2020. What we learn from extremophiles. ChemTexts 6:8. doi: 10.1007/s40828-020-0103-6

[5] ↑ Hashimoto, T., Horikawa, D. D., Saito, Y., Kuwahara, H., Kozuka-Hata, H., Shin, I. T., et al. 2016. Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nat. Commun . 7:12808. doi: 10.1038/ncomms12808

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Study determines microscopic water bears will be Earth’s last survivors

In Animals , Marine Science , Research News , Science & Nature , Space , Spotlight / 19 July 2017

By Megan Watzke

The world’s most indestructible species, the tardigrade, an eight-legged micro-animal, also known as the water bear, will survive until the Sun dies, according to a new Harvard-Smithsonian Center for Astrophysics and Oxford University collaboration.

The world’s most indestructible species, the tardigrade, an eight-legged micro-animal, also known as the water bear, will survive until the Sun dies, according to a new Harvard-Smithsonian Center for Astrophysics and Oxford University collaboration.

The new study published in Scientific Reports, has shown that the tiny creatures will survive the risk of extinction from all astrophysical catastrophes, and be around for at least 10 billion years–far longer than the human race.

Although much attention has been given to the cataclysmic impact that an astrophysical event would have on human life, very little has been published around what it would take to kill the tardigrade, and wipe out life on this planet. The research implies that life on Earth in general, will extend as long as the Sun keeps shining. It also reveals that once life emerges, it is surprisingly resilient and difficult to destroy, opening the possibility of life on other planets.

Tardigrades are the toughest, most resilient form of life on earth, able to survive for up to 30 years without food or water, and endure temperature extremes of up to 150 degrees Celsius, the deep sea and even the frozen vacuum of space. The water-dwelling micro animal can live for up to 60 years, and grow to a maximum size of 0.5 mm, best seen under a microscope.

Researchers from the Universities of Oxford and the Harvard-Smithsonian Center for Astrophysics, have found that these life forms will likely survive all astrophysical calamities, such as an asteroid, since they will never be strong enough to boil off the world’s oceans.

Three potential events were considered as part of the research, including; large asteroid impact, and exploding stars in the form of supernovas or gamma-ray bursts.

Asteroids:   There are only a dozen known asteroids and dwarf planets with enough mass to boil the oceans, these include Vesta and Pluto, however none of these objects will intersect the Earth’s orbit and pose no threat to tardigrades.

Supernova:  In order to boil the oceans an exploding star would need to be 0.14 light-years away. The closest star to the Sun is four light years away and the probability of a massive star exploding close enough to Earth to kill all forms of life on it, within the Sun’s lifetime, is negligible.

Gamma-Ray bursts:  Gamma-ray bursts are brighter and rarer than supernovae. Much like supernovas, gamma-ray bursts are too far away from earth to be considered a viable threat. To be able to boil the world’s oceans the burst would need to be no more than 40 light-years away, and the likelihood of a burst occurring so close is again, minor.

“Without our technology protecting us, humans are a very sensitive species. Subtle changes in our environment impact us dramatically. There are many more resilient species’ on earth. Life on this planet can continue long after humans are gone,” says Rafael Alves Batista, co-author and post-doctoral research associate in the Department of Physics at Oxford University. “Tardigrades are as close to indestructible as it gets on Earth, but it is possible that there are other resilient species examples elsewhere in the universe. In this context there is a real case for looking for life on Mars and in other areas of the solar system in general. If tardigrades are Earth’s most resilient species, who knows what else is out there.”

David Sloan, co-author and post-doctoral research associate in the Department of Physics at Oxford University, said: “To our surprise we found that although nearby supernovae or large asteroid impacts would be catastrophic for people, tardigrades could be unaffected. Therefore it seems that life, once it gets going, is hard to wipe out entirely. Huge numbers of species, or even entire genera may become extinct, but life as a whole will go on.”

In highlighting the resilience of life in general, the research broadens the scope of life beyond Earth, within and outside of this solar system. Abraham Loeb, co-author and chair of the Astronomy Department at Harvard University, said: “It is difficult to eliminate all forms of life from a habitable planet. Organisms with similar tolerances to radiation and temperature as tardigrades could survive long-term below the surface in these conditions. The subsurface oceans that are believed to exist on Europa and Enceladus, would have conditions similar to the deep oceans of Earth where tardigrades are found, volcanic vents providing heat in an environment devoid of light. The discovery of extremophiles in such locations would be a significant step forward in bracketing the range of conditions for life to exist on planets around other stars.”

A paper detailing this work appeared on July 14, 2017 in Scientific Reports , an open, online journal from the publishers of Nature.

Related posts:

Tags: asteroids , astronomy , astrophysics , Center for Astrophysics | Harvard & Smithsonian , extinction , Smithsonian Astrophysical Observatory

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What is a tardigrade?

Tardigrades are invertebrates belonging to the phylum Tardigrada. They are related to arthropods (e.g., crustaceans and insects ) and nematodes (i.e., roundworms). Also known as water bears, tardigrades are known for their appearance and their ability to survive in extreme environments.

Tardigrades can survive extreme conditions by going into a “tun” state, in which their body dries out and their metabolism drops to as little as 0.01 percent of its normal rate. When conditions return to normal, the tardigrade revives itself. A tardigrade can stay in a tun state for decades.

Research shows that tardigrades can be killed by exposure to hot water for an extended period of time. One study showed that one hour of exposure to water at 82.7 °C (180.9 °F) can kill a tardigrade in its “tun” state, where it goes into suspended animation and becomes hard to destroy.

Where do tardigrades live?

Tardigrades can be found in moist habitats, such as damp moss or underwater sediment. Tardigrades exist all over the world because of their ability to withstand extreme conditions.

tardigrade , (phylum Tardigrada), any of more than 1,100 species of free-living tiny invertebrates belonging to the phylum Tardigrada. They are considered to be close relatives of arthropods (e.g., insects , crustaceans ). Tardigrades are mostly about 1 mm (0.04 inch) or less in size. They live in a variety of habitats worldwide: in damp moss , on flowering plants , in sand , in fresh water, and in the sea. In adapting to this wide range of external conditions, a large number of genera and species have evolved.

Tardigrades have a well-developed head region and a short body composed of four fused segments, with each segment bearing a pair of short, stout, unjointed limbs generally terminated by several sharp claws . The animals have no known specialized organs of circulation or respiration; the tardigrade’s body cavity ( hemocoel ) is filled with fluid that transports blood and oxygen (the latter of which diffuses through the animal’s integument and is stored in cells within the hemocoel). The alimentary canal traverses the body from end to end. Most plant-eating tardigrades feed by piercing individual plant cells with their stylets (spearlike structures near the mouth) and then sucking out the cell contents. A few tardigrades are predatory carnivores. Tardigrades may reproduce sexually or through asexual reproduction (by means of parthenogenesis or through self-fertilization [ hermaphroditism ]). Eggs are discharged either into the posterior end of the alimentary canal or directly to the exterior through an opening in front of the anus .

The most remarkable feature of the tardigrades is their ability to withstand extremely low temperatures and desiccation (extreme drying). Under unfavourable conditions, they go into a state of suspended animation called the “ tun” state—in which the body dries out and appears as a lifeless ball (or tun). In this state their metabolism may decline to as little as 0.01 percent of its normal rate. Tardigrades can survive as tuns for years, or even decades, to wait out dry conditions. In addition, specimens kept for eight days in a vacuum , transferred for three days into helium gas at room temperature , and then exposed for several hours to a temperature of −272 °C (−458 °F) came to life again when they were brought to normal room temperature. Sixty percent of specimens kept for 21 months in liquid air at a temperature of −190 °C (−310 °F) also revived. Tardigrades are easily distributed by wind and water while in the tun state.

What are tardigrades and why are they nearly indestructible?

Tardigrades are near-microscopic animals that can survive some of the most extreme conditions on Earth, including freezing temperatures, crushing pressures, and even the vacuum of space.

What makes tardigrades so indestructible?

How big are tardigrades, where do tardigrades live, what extremes can tardigrades survive, how do tardigrades enter a 'tun' state, what do tardigrades eat, how do tardigrades reproduce, tardigrade anatomy, are tardigrades endangered.

Tardigrades, often called water bears or moss piglets, are near-microscopic aquatic animals with plump, segmented bodies and flattened heads. They have eight legs, each tipped with four to eight claws or digits, and somewhat resemble the hookah-smoking caterpillar from "Alice in Wonderland." Though tardigrades are disarmingly cute, they are also nearly indestructible and can even survive in outer space.

Tardigrades were discovered in 1773 by the German zoologist Johann August Ephraim Goeze, who dubbed them "little water bear." Three years later, Italian biologist Lazzaro Spallanzani named the group "Tardigrada," or "slow stepper," for their toddling gait, according to the Science Education Resource Center at Carleton College (SERC). There are currently about 1,300 known tardigrade species within the Tardigrada phylum (a classification category) according to the  Integrated Taxonomic Information System (ITIS), a resource for species names and classifications created by a partnership of U.S. federal agencies.

Tardiges have an unusual strategy for surviving harsh conditions: They enter an almost death-like state called cryptobiosis, expelling more than 95% of the water from their bodies, retracting their heads and legs and curling into a dehydrated tun. 

By the 1970s, scientists determined that different forms of cryptobiosis in tardigrades could be caused by four environmental triggers: desiccation, freezing, lack of oxygen and excess salt, reported a 2020 study published in the journal Scientific Reports .

During cryptobiosis, a tardigrade's metabolic activity drops to as little as 0.01% of normal levels. Its cells are protected from damage by water-soluble proteins that are unique to tardigrades, known as tardigrade disordered proteins, or TDPs. When tardigrades expel their body's water, TDP molecules form a tough, glasslike cocoon around cells. This keeps cellular material safe while the tardigrade is a tun and enables it to reanimate in water when conditions are more hospitable, according to a 2017 study published in the journal Molecular Cell . 

"Tardigrades are fascinating little beasties," Sandra McInnes , a tardigrade researcher with the British Antarctic Survey, who has been studying species that occur in the frozen snowscapes of Antarctica since 1980, previously told Live Science. " Tardigrades have this ability to cope with extreme environments by shutting down their metabolism. This ability to cope with drying out or freezing is what gives them their durability in the Antarctic."

Sandra McInnes is a tardigrade researcher with the British Antarctic Survey. She also is an associate editor dedicated to tardigrade-related manuscripts at the journal Zootaxa.

However, tardigrades do have a fatal weakness: They wilt under heat, which could be a problem as climate change increases temperatures. A 2020 study published in the journal  Scientific Reports found that tardigrades in water temperatures of about 100 degrees Fahrenheit (37.8 degrees Celsius) can die in just one day.

" Tardigrades are definitely not the almost-indestructible organism as advertised in so many popular science websites," study co-author Ricardo C. Neves , a postdoctoral scientist in biology at the University of Copenhagen, previously told Live Science.

Water bears can range from 0.002 to 0.05 inches (0.05 to 1.2 millimeters) long, but they usually don't get any bigger than 0.04 inches (1 mm) long, according to the World Tardigrada Database .

A tardigrade's body typically consists of only 1,000 cells, according to an article published in the journal Arthropod Structure and Development in 2019. In comparison, the human body is made up of many trillions of cells.

As their name implies, water bears live just about anywhere there's liquid water, inhabiting the ocean, freshwater lakes and rivers, and the water film that coats terrestrial mosses and lichens. 

They can survive a wide range of environments: from altitudes of over 19,600 feet (6,000 meters) in the Himalayan mountain range to ocean depths more than 15,000 feet (4,700 m) below the surface, according to the University of Michigan's Animal Diversity Web (ADW). 

Related: Tardigrades probably see in black and white 

Not all tardigrades live in extreme environments, but water bears are known for surviving extreme conditions that would kill most other forms of life, by transforming into a dehydrated ball known as a tun. 

Researchers have found that tardigrades in a tun state can withstand temperatures as low as minus 328 degrees Fahrenheit (minus 200 degrees Celsius) and hotter than 300 degrees F (148.9 C), Smithsonian magazine reported. They can also survive exposure to radiation, boiling liquids, and up to six times the pressure of the deepest part of the ocean, according to the Science Education Resource Center at Carleton College in Minnesota. 

A 2008 study published in the journal Current Biology revealed that some species of tardigrades — when dehydrated — could weather a 10-day trip into low-Earth orbit, and return to Earth unharmed by solar ultraviolet radiation and the vacuum of space. 

More recently, desiccated tardigrades have been shot from a high-speed gun , traveling nearly 3,000 feet per second (900 meters per second) and surviving a crushing impact of about 1.14 gigapascals of pressure. 

Their findings , however, suggest that several thousands of tun-state tardigrades that were carried on the Israeli lunar mission Beresheet would not have survived after the lander crashed on the moon , on April 11, 2019. The shock pressure of the metal lander hitting the moon would've been "well above" the limits tardigrades could survive. "We can confirm they didn't survive," Alejandra Traspas-Muina , who led the research as a PhD student at the Queen Mary University of London, told Science magazine .

While scientists have long known that to survive extremes, tardigrades enter their tun state through a metamorphosis known as anhydrobiosis, exactly how they do this has been a longstanding mystery. In a study published in the journal PLOS One in 2024, researchers discovered the molecular underpinnings that allow them to enter their near-invincible state . 

The team exposed the tardigrade species Hypsibius exemplaris to a series of life-threatening conditions, such as dangerous levels of hydrogen peroxide and temperatures of minus 112 degrees Fahrenheit (minus 80 degrees C) and measured the chemical environment inside the tardigrades' cells. 

They found tardigrades produce free radicals — oxygen atoms with an additional unpaired electron that emerge in animal cells during a phase known as oxidative stress. In most animlas, this is harmful, but in tardigrades, free radicals react with the amino acid cysteine to transform them into their tun state. When they inhibited the cysteine oxidation process, the tardigrades were incapable of entering the tun state. 

Most tardigrades suck fluids from cells in plants, algae and fungus, puncturing cell walls with needlelike stylets in their mouths and hoovering up the liquid inside. 

However, some species can consume entire living organisms, such as rotifers, nematodes and even other tardigrades, according to Illinois Wesleyan University's Species Distribution Project (SDP).

Reproduction in tardigrades may be sexual or asexual, depending on the species. For egg-layers, females produce up to 30 eggs at a time, and eggs may be fertilized either inside the female's body; in her shed cuticle after the male ejaculates his sperm there; or while attached to sand or substrate, according to ADW. Other tardigrade species are self-fertilizing hermaphrodites that reproduce through parthenogenesis — a process in which an embryo develops without external fertilization. 

Embryos typically are fully developed within 14 days of fertilization, though their development can last up to 90 days depending on environmental conditions such as dryness and temperature, according to ADW. Young tardigrades do not have a larval stage and resemble miniature adults upon hatching, though they usually have fewer claws and spines than fully-grown water bears do. The youngsters grow in several stages by molting their external cuticle "skin," and each molt can take five to ten days to complete.

Inside the tardigrade's tiny body, you won't find any bones, according to research published in the Proceedings of the National Academy of Sciences (PNAS) in August 2021. Instead, they are supported by a hydrostatic skeleton. This is a fluid-filled compartment called a hemolymph. Similarly to human blood, the hemolymph is filled with nutrients. 

Although they lack a spinal cord , tardigrades have a ventral nervous system , according to the book Forest Canopies , published in 2004. This sends signals between the tardigrade's brain and body and is the functional equivalent of a vertebrate's spinal cord. 

Water bears have a complete digestive system, but no circulatory or respiratory system. Instead, oxygen from the water enters their bodies through their cuticle walls. To aid circulation, they have muscles which contract to transport the nutrients in their hydrostatic skeleton.

Kingdom : Animalia 

Subkingdom : Bilateria 

Infrakingdom : Protostomia 

Superphylum : Ecdysozoa 

Phylum : Tardigrada

Source:  ITIS

Tardigrade tuns can be revived even after decades have passed. In 2016, scientists revived two tuns and a tardigrade egg that had been in cryptobiosis for more than 30 years, Live Science previously reported . Reanimation from even longer tun states might be possible. In 1948, a researcher in Italy purportedly revived a tun from a dried-out piece of moss that was over 120 years old, the BBC reported in 2015. However, no other researcher has since reanimated a tardigrade from a tun that old, according to the BBC.

And in some tardigrades, fluorescence could lend them protection against radiation by transforming UV rays into harmless blue light, Live Science previously reported . 

Tardigrades have not been evaluated by the International Union for Conservation of Nature, the global organization that monitors conservation status for animals and natural habitats; and they aren't on any other endangered list. In fact, tardigrades have survived all five mass extinctions on Earth since the group evolved about half a billion years ago, according to the University of Wisconsin Madison , and water bears could survive after humanity is long gone , researchers found.

Related: The 5 mass extinction events that shaped the history of Earth 

In 2017, scientists from Harvard and Oxford universities looked at the probabilities of certain astronomical events — Earth-pummeling asteroids, neighboring supernova blasts and gamma ray bursts, to name a few — that could take place over the next several billion years. Then, they evaluated the likelihood of those events wiping out Earth's hardiest species. While such disasters would likely eradicate humans, the researchers found that little tardigrades would survive most cosmic cataclysms, they reported in their study published in the journal Scientific Reports .

"To our surprise, we found that although nearby supernovas or large asteroid impacts would be catastrophic for people, tardigrades could be unaffected," David Sloan, a co-author of the study and a researcher at Oxford, said in a statement .

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Mindy Weisberger is an editor at Scholastic and a former Live Science channel editor and senior writer. She has reported on general science, covering climate change, paleontology, biology and space. Mindy studied film at Columbia University; prior to Live Science she produced, wrote and directed media for the American Museum of Natural History in New York City. Her videos about dinosaurs, astrophysics, biodiversity and evolution appear in museums and science centers worldwide, earning awards such as the CINE Golden Eagle and the Communicator Award of Excellence. Her writing has also appeared in Scientific American, The Washington Post and How It Works Magazine.  Her book "Rise of the Zombie Bugs: The Surprising Science of Parasitic Mind Control" will be published in spring 2025 by Johns Hopkins University Press.

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Behold The Mighty Water Bear

Water bears, aka tardigrades, can withstand boiling, freezing and the vacuum of space. Biologist Bob Goldstein, of University of North Carolina, Chapel Hill, studies these millimeter-long creatures to try to understand how organisms develop.

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Why Tardigrades Are So Badass: 7 Secrets of the Only Animal That Can Survive in Space

All hail the toughest organism on (and beyond) Earth: the weird and wonderful water bear.

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Tardigrades are one of the most fascinating creatures on Earth— and in space.

Now, we may finally understand the mechanism tiny water bears use to live in such harsh conditions, from deep-sea vents at the bottom of the ocean to the top of the Himalayas. Scientists already knew that tardigrades could survive extreme environments by entering what’s called a “tun” state for extended periods of time. Still, they didn’t understand how the eight-legged microscopic invertebrates could initiate that tun state in the first place.

In other words, they “use a molecular sensor that detects harmful conditions in their environment, telling them when to go dormant and when to resume normal life,” per a press statement .

We talked to leading researchers to learn a bit more about what makes these little water bears so amazing. Here are our seven favorite facts about tardigrades, according to the latest research.

✅ 2 More Things You Should Know About the Weird and Wonderful Tardigrade At Popular Mechanics , we love the water bear so much that we thought we’d share two more mini bonus facts about these resilient (and cute!) invertebrates. // Can tardigrades live forever? // Not exactly. Tardigrades, in their active state, can technically only live for a few months. However, in between those active periods, they enter the tun state. The phenomenon underlying this state is known as cryptobiosis , in which the water bear suspends its metabolism to 0.01 percent of its typical level. Species that undergo cryptobiosis can “live” in this state for more than 30 years . // Can tardigrades survive on Mars? // A 2019 study found that two species of water bear— Ramazzottius varieornatus and Hypsibius exemplaris— could survive simulated Martian conditions for up to 30 days through cryptobiosis.

1) Tardigrades are everywhere .

Tardigrades are a class of microscopic animals with eight limbs and strange, alien-like behavior. William Miller, a leading tardigrade researcher at Baker University, says they are remarkably abundant. Hundreds of species “are found across the seven continents; everywhere from the highest mountain to the lowest sea,” he says. “Many species of tardigrades live in water, but on land, you find them almost everywhere there’s moss or lichen.”

In 2007 , scientists discovered these microscopic critters can survive an extended stay in the cold, irradiated vacuum of outer space. A European team of researchers sent a group of living tardigrades to orbit the earth on the outside of a FOTON-M3 rocket for 10 days. When the water bears returned to Earth, the scientists discovered that 68 percent lived through the ordeal.

Although tardigrades are unique in their ability to survive in space, Miller insists there is no reason to believe they evolved for this reason or—as a misleading VICE documentary has implied—that they are of extraterrestrial origin. Rather, the tardigrade’s space-surviving ability is the result of a strange response they’ve evolved to overcome an earthly life-threatening problem: a water shortage.

Land-dwelling tardigrades can be found in some of the driest places on Earth. “I’ve collected living tardigrades from under a rock in the Sinai desert, in a part of the desert that hadn't had any record of rain for the previous 25 years,” Miller says. Yet these are technically aquatic creatures, and require a thin layer of water to do pretty much anything, including eating, having sex, or moving around. Without water, they’re about as lively as a beached dolphin.

💡 Did You Know? There are 11,000 species of tardigrades, according to Encyclopedia Britannica.

2) Tardigrades can pause their biological clock.

But land-dwelling tardigrades have evolved a bizarre solution to living through drought : When their environment dries up, so do they. Tardigrades will enter a state called desiccation, in which they shrivel up, losing all but around three percent of their body’s water and slowing their metabolism down to an astonishing 0.01 percent of its normal speed—a metabolic state known as cryptobiosis. In this state, the tardigrade just persists, doing nothing, until it’s inundated with water again. When that happens, the creature pops back to life like a re-wetted sponge and continues onward as if nothing had happened.

What’s even more astonishing is that tardigrades can survive being in this strange state for more than a decade. According to Miller, a few researchers believe some species of tardigrades might even be able to survive desiccation for up to a century. Yet the average lifespan of a (continuously hydrated) tardigrade is rarely longer than a few months.

“It sounds quite strange,” says Miller, “that even though these tardigrades only live for a few weeks or months, that lifetime can be stretched over many, many years.”

Similarly, tardigrades can also survive being frozen. A new study published in Journal of Zoology in September 2022 shows that tardigrades exposed to freezing temperatures entered cryptobiosis and lived longer than those who never entered this state during their lives.

Out of a total of 716 tardigrades, those that were periodically frozen became “sleeping beauties,” living around twice as long as the control group. The oldest tardigrade stuck around for 169 days, with 94 days at room temperature. In the control group, which stayed warm, the oldest tardigrade lived for 93 days.

“During inactive periods, the internal clock stops and only resumes running once the organism is reactivated,” explains zoologist Ralph Schill, one of the researchers. “So, tardigrades, which usually only live for a few months without periods of rest, can live for many years or even decades.”

3) Tardigrades probably can’t see in color.

Recent research from the Genome Biology and Evolution journal reports that the resilient little critters don’t have the same opsins (light-sensitive, photoreceptive proteins) as animals who use their eyes to see color. One of the tardigrade species ( Ramazzottius variornatus ) that was analyzed in this study didn’t have eyes at all but did have active opsins. Another species ( Hypsibius exemplaris ) did have eyes, but their opsins didn’t respond to light stimuli—a necessary feature for color vision . It’s technically still possible that tardigrades can see some color, but it’s more likely that they see things in black and white. Their eyes are very simple, after all. Further research is needed to determine how their vision works.

4) Tardigrades can survive the harshest atmospheres.

In its desiccated state, the tardigrade is ridiculously, almost absurdly resilient. Laboratory tests have shown that tardigrades can endure both an utter vacuum and intense pressures more than five times as punishing as those in the deepest ocean. Even temperatures up to 300 degrees Fahrenheit and as low as minus 458 degrees Fahrenheit (just above absolute zero) won’t spell out the creature’s doom.

But the exact source of its resilience is a mystery, says Emma Perry, a leading tardigrade researcher at Unity College in Maine. “In general, we know very little about how this species functions, especially when we’re talking about the molecular level.”

There are clues. Scientists have learned that when the tardigrade enters its desiccated state, “it replaces some of its cell contents with a sugar molecule called trehalose,” Perry says.

Researchers believe this trehalose molecule not only replaces water, but also in some cases can physically constrain the critter’s remaining water molecules, keeping them from rapidly expanding when faced with hot and cold temperatures. This is important, because expanding water molecules (like what happens when you get frostbite) can mean instant cellular death for most animals.

5) Even space radiation is no match for tardigrades.

Space is deadly, and not just because of the vacuum. Outside our protective atmosphere there is killer radiation caused by distant supernovae, our sun, and other sources. Space radiation comes in the form of harmful charged particles that can imbed in the body of animals, ripping apart molecules and damaging DNA faster than it can be repaired.

But here, too, the tardigrade seems oddly prepared for life in space. According to Peter Guida , the head of NASA’s space radiation laboratory, one of the biggest radiation concerns for astronauts (and space-bound tardigrades) is a set of molecules called reactive oxygen species. Ionizing radiation enters the body and bores into wayward molecules that contain oxygen. In simple terms, those newly irradiated molecules then troll through the body causing all sorts of harm.

Tardigrades, in their desiccated state, produce an abnormal amount of antioxidants (yes, these actually exist outside the health-food world), which effectively neutralize those roaming, evil reactive oxygen species. Partly because of this talent, tardigrades have been found to withstand higher radiation doses with far greater success than researchers would otherwise believe they should.

The reason that tardigrades would have evolved to survive high radiation doses is a mystery, too. However, Miller points to a leading theory: Perhaps tardigrades evolved to be swept up by the wind and survive in the earth’s atmosphere—which would explain not only their hardiness, but also why they’re found all over the world.

6) Still, tardigrades aren’t completely invincible.

There might be one thing tardigrades are not so well-equipped to handle: high temperatures over a prolonged period of time, per a study published in Scientific Reports in January 2020. The study revealed that this temperature-based Achilles’ heel also extends to when tardigrades are in their protective tun states.

Researchers studied Ramazzottius varieornatus, a species of tardigrade, in tun state and noted nearly 50 percent of the tardigrades exposed to 181 degrees Fahrenheit over the course of an hour perished. Active tardigrades—that is, those not in tun state—fared even worse.

These temperature experiments show that, given time, most tardigrades can adjust to intense temperature fluctuations: The tardigrades who had an hour to acclimate to intense heat faced higher mortality rates, compared to those who had a full 24 hours.

“Tardigrades can survive pressures that are comparable to those created when asteroids strike Earth, so a small crash like this is nothing to them,” Lukasz Kaczmarek, an expert on tardigrades, told The Guardian .

So what does this mean for us? If humans could replicate cryptobiosis in the way tardigrades do, we’d live far longer than the average life expectancy . According to Kaczmarek, when a tardigrade enters the tun state, it doesn’t age. It becomes dormant at one month old and can wake up years later and still biologically be the same age.

“It may be that we can use this in the future if we plan missions to different planets, because we will need to be young when we get there,” said Kaczmarek.

7) Some tardigrades lay spiked eggs.

The ever-mysterious, alien-like creatures have presented scientists with yet another quandary: What’s the deal with a newly discovered tardigrade species that can lay spiked eggs?

In a June 2020 paper published in Scientific Reports , scientists reveal that Dactylobiotus ovimutans , the new species, displayed a “range of eggshell morphologies” despite the fact that “the population was cultured under controlled laboratory condition.”

The researchers believe an “ epigenetic factor” could be causing the range of shapes and features seen on the D. ovimutans eggs. But the mystery still remains: Why has D. ovimutans turned to epigenetics (the activation/deactivation of genes that has no affect on an organism’s DNA sequence) when it comes to their offspring?

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Melting permafrost releasing toxic mercury into the Arctic, scientists say

The addition of heavy metals into the water system could impact the food chain.

Melting permafrost in the Arctic is releasing toxic mercury into the water system, potentially impacting the food chain, scientists say.

Arctic permafrost is melting at rapid rates, potentially putting the food chain and the communities who depend on it in "grave danger," according to researchers at the University of Southern California who studied the transport of sediment in the Yukon River in Alaska.

MORE: Arctic fossils indicate ice shelf is not as stable as previously thought, scientists say

As the Yukon River flows west across Alaska to the Bering Sea, the eroding permafrost along the way is adding sediment embedded with mercury into the water, a paper published Thursday in the journal Environmental Research Letters found. The mercury has likely been sequestered in the permafrost for millennia, the scientists said.

"There could be this giant mercury bomb in the Arctic waiting to explode," Josh West, professor of Earth sciences and environmental studies at USC Dornsife College of Letters, Arts and Sciences, said in a statement.

The researchers analyzed mercury in sediments in riverbanks and sandbars, tapping into deeper soil layers.

They also used remote sensing data from satellites to monitor how fast the Yukon River is changing course -- significant because it affects how much mercury-laden sediments are eroded from riverbanks and deposited along sandbars, according to the paper.

"The river can quickly mobilize large amounts of sediment containing mercury," said Isabel Smith, a doctoral candidate at USC Dornsife and co-author of the study, in a statement.

MORE: Climate change is altering the length of days on Earth, according to new research

The rivers are also reburying a considerable amount of mercury, leading the researchers to emphasize the importance of understanding both the erosion and reburial processes.

The addition of the toxic metals poses an environmental and health threat to at least 5 million people living in the Arctic, the researchers found.

Risk of contamination through drinking water is minimal, West said.

"We’re not facing a situation like Flint, Michigan," West said. "Most human exposure to mercury comes through diet."

But the long-term effects could be devastating, particularly for Arctic communities dependent on hunting and fishing, the researchers said.

The impact is expected to build over time as the metal accumulates in the food chain, especially through fish and game that humans consume.

"Decades of exposure, especially with increasing levels as more mercury is released, could take a huge toll on the environment and the health of those living in these areas," Smith said.

MORE: Prolonged ice-free periods putting Hudson Bay polar bear population at risk of extinction: Study

The Arctic is often considered the front line for climate change, with existing research pointing to a plethora of impacts melting at the North Pole will have on the rest of the planet.

The region is melting fasting than predicted , a paper published in the Proceedings of the National Academy of Sciences last year found. Days are getting longer as both poles melt, redistributing the mass of water that is contributing to sea level rise, a study published in PNAS in July found.

A recent study of fossils derived from beneath the Greenland ice shelf indicate that the region was once ice-free and that the ice sheet is not as stable as previously thought, according to a study published earlier this month in Nature.

Melting of the Greenland ice sheet could expose 400 million people to flooding risk, a paper published in Nature in 2019 found.

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Density, harvest rates, and growth of a reintroduced American black bear population

Less than 30% of all species reintroductions have been successful and it is important that factors associated with success or failure be identified. Officials experimentally translocated 14 adult female American black bears (Ursus americanus) from Great Smoky Mountains National Park, North Carolina and Tennessee, USA, to Big South Fork National River and Recreation Area in the Cumberland Plateau of Kentucky and Tennessee, USA, in 1996–1997. Since that time, the reintroduced bear population has continued to expand in size and range so our study objective was to use spatially explicit capture-recapture methods across a wide spatial extent to estimate bear population abundance and growth. We constructed 440 (223 in KY, 217 in TN) hair traps in our primary sampling area in 2019 arranged in clusters of 4–9 traps/cluster, which we augmented with data from 138 hair traps in a secondary sampling area in Tennessee collected in 2018. We extracted and genotyped DNA from hair samples to construct spatially explicit capture histories, using spatial covariates to model inhomogeneous densities. Population abundance estimates across our 36,035-km 2  study area were 411 males and 406 females excluding cubs. Based on an initial standing population of 18 adult and subadult bears, the mean annual growth rate (λ) from 1998 to 2019 was 1.199. The mean annual harvest rate in Kentucky from 2013 to 2019 was 5.1% and in Tennessee from 2014 to 2019 was 13.2%. Based on simulations, the hunting seasons reduced mean λ from 1.217 to 1.199, but growth was rapid despite harvest. Genetic diversity was retained, with similar expected heterozygosity as in the source population. The lack of conspecifics, highly productive habitat, and an initial age and sex distribution that was skewed toward the most fecund members of the population likely contributed to the rapid growth and high levels of gene retention in this bear population.

Citation Information

Related Content

Joseph clark, ph.d., supervisory research ecologist.

Disclaimer: Early release articles are not considered as final versions. Any changes will be reflected in the online version in the month the article is officially released.

Volume 30, Number 9—September 2024

Research Letter

Thelazia callipaeda eyeworms in american black bear, pennsylvania, usa, 2023.

Suggested citation for this article

We identified a Thelazia callipaeda eyeworm in an American black bear in Pennsylvania, USA, on the basis of its morphological features and molecular analysis. Our finding highlights emergence of a T. callipaeda worm sylvatic transmission cycle in the United States.

Thelaziosis is an emerging zoonotic disease caused by nematodes of the genus Thelazia (Spirurida, Thelazioidea). In the United States, 3 zoonotic species have been identified: Thelazia gulosa ( 1 ), T. californiensis ( 2 ), and most recently T. callipaeda ( 3 ). In Asia and Europe, T. callipaeda is considered the main agent of thelaziosis in humans, domestic animals, and wild animals ( 4 ). Over the past decade, the geographic distribution and prevalence of T. callipaeda infection has increased worldwide in scale and intensity ( 4 ). The first autochthonous case in the United States was reported in 2018 in a domestic dog ( Canis lupus familiaris ) from New York with a history of unilateral epiphora and blepharospasm. Since then, additional cases in domestic dogs and cats have been reported, predominately from the northeastern United States ( 3 , 5 ).

T. callipaeda eyeworms are found in the conjunctival sac and lacrimal duct of the definitive host. They are transmitted when a male zoophilic secretophagous Phortica variegata fly ingests first-stage larvae from the host’s lachrymal secretions. In the vector, the first-stage larvae develop to the infective third-stage larvae in the testes, migrate to the mouthparts, and are transferred to another host during subsequent feeding on lachrymal secretions ( 4 ).

The role of wildlife in the epidemiology and emergence of T. callipaeda eyeworms is not completely known. In Europe, cases of T. callipaeda eyeworm infection have been detected in a wide range of hosts, including wild carnivores, omnivores, and lagomorphs ( 6 , 7 ). Wild canids, particularly red foxes ( Vulpes vulpes ), seem to play a large role in maintaining the sylvatic cycle in thelaziosis-endemic areas of Europe ( 7 ). However, knowledge of the sylvatic transmission cycle of T. callipaeda eyeworms, along with their environmental and anthropogenic factors, remains limited. Considering the emergence of those zoonotic nematodes in non–thelaziosis-endemic areas and the need for more information about their ecology and epidemiology in the United States, we report a case of T. callipaeda eyeworm infection in an American black bear ( Ursus americanus ) and identify a new geographic location of transmission.

In November 2023, an adult, female American black bear was legally harvested in Coolbaugh Township, Monroe County, Pennsylvania. During processing of the bear for taxidermy preparation, multiple linear nematodes were observed behind the third eyelid. Nematodes were extracted and submitted for identification. Two additional harvested bears from Monroe and Pike Counties, Pennsylvania, were also reported to have similar ocular nematode infections, but specimens from those bears were not collected.

• Figure 1 . Morphologic features of adult female Thelazia callipaeda eyeworm isolated from an American black bear in Coolbaugh Township, Monroe County, Pennsylvania, USA, 2023. A) Anterior end showing the large, deep,...

We identified 9 female and 4 male adult nematodes from the bear as T. callipaeda on the basis of morphologic and morphometric features ( 8 ). The nematodes were characterized by the presence of a cup-shaped buccal capsule and cuticular transverse striations, as well as the location of the vulvar opening anterior to the esophageal-intestinal junction on the female worms ( Figure 1 ). Female nematodes were 1.16–1.46 cm long and 0.36–0.42 mm wide; male worms were 0.82–1.06 cm long and 0.31–0.42 mm wide. The number of transverse cuticular striations ranged from 160 to 400/mm in the cephalic, midbody, and caudal regions.

Figure 2 . Phylogenetic relationship of Thelazia callipaeda isolate from an American black bear in Coolbaugh Township, Monroe County, Pennsylvania, USA, 2023 (GenBank accession no. PP739308), and other species of ...

We extracted genomic DNA from a midbody fragment of a female adult worm and amplified, sequenced, and analyzed the partial cytochrome oxidase c subunit I ( cox 1) gene, as previously described ( 2 ). We generated a 623-bp cox 1 sequence (GenBank accession no. PP739308), which showed 99%–100% maximum identity with T . callipaeda sequences available in GenBank. Phylogenetic analysis was performed by using the maximum-likelihood method and confirmed the taxonomic identification of T . callipaeda . The isolate clustered with all previous isolates from domestic animals in North America and with some isolates from Europe ( Figure 2 ), indicating circulation of the newly introduced pathogen in wildlife habitats and transmission from domestic animals to wildlife.

The presence of adult T. callipaeda eyeworms in an American black bear suggests the establishment of a sylvatic transmission cycle in the United States and expansion of the number of definitive host species used by the zoonotic nematode. In the past decade, wild carnivores have been identified as primary definitive hosts associated with the sylvatic cycle in thelaziosis- endemic and non–thelaziosis-endemic areas of Europe and Asia ( 7 ). American black bears are the most widely distributed species of bear in North America, inhabiting diverse regions throughout Mexico, Canada, and the United States ( 9 ). Given the bears’ extensive geographic distribution and frequent and close interaction with humans and pets ( 10 ), thelaziosis in the black bear population raises concerns about the rapidly increasing incidence and geographic range of T. callipaeda eyeworms in the United States. Although further research into the extent to which black bears play a role in the maintenance of the sylvatic cycle and transmission of T. callipaeda eyeworms is needed, the presence of the zoonotic nematode in such a wide range of hosts implicates exposure and risk for transmission to threatened and endangered species and direct or indirect risk for transmission to humans and domestic animals.

Dr. Sobotyk is an assistant professor of clinical parasitology and director of the Clinical Parasitology Laboratory at the University of Pennsylvania, Philadelphia, PA. Her research focuses on zoonotic helminth infections in domestic and wild animals and improvement and development of diagnostic techniques for detecting parasitic infections of veterinary and public health relevance.

Acknowledgment

We thank the Pennsylvania Game Commission and Dillon Gruver for their continued support. We also acknowledge Shawn Lamparter’s Wildlife Design for recognition and prompting submission of the specimens.

• Bradbury  RS , Breen  KV , Bonura  EM , Hoyt  JW , Bishop  HS . Case report: conjunctival infestation with Thelazia gulosa : a novel agent of human thelaziasis in the United States. Am J Trop Med Hyg . 2018 ; 98 : 1171 – 4 . DOI PubMed Google Scholar
• Sobotyk  C , Foster  T , Callahan  RT , McLean  NJ , Verocai  GG . Zoonotic Thelazia californiensis in dogs from New Mexico, USA, and a review of North American cases in animals and humans. Vet Parasitol Reg Stud Reports . 2021 ; 24 : 100553 . DOI PubMed Google Scholar
• Schwartz  AB , Lejeune  M , Verocai  GG , Young  R , Schwartz  PH . Autochthonous Thelazia callipaeda infection in dog, New York, USA, 2020. Emerg Infect Dis . 2021 ; 27 : 1923 – 6 . DOI PubMed Google Scholar
• Otranto  D , Mendoza-Roldan  JA , Dantas-Torres  F . Thelazia callipaeda. Trends Parasitol . 2021 ; 37 : 263 – 4 . DOI PubMed Google Scholar
• Manoj  RRS , White  H , Young  R , Brown  CE , Wilcox  R , Otranto  D , et al. Emergence of thelaziosis caused by Thelazia callipaeda in dogs and cats, United States. Emerg Infect Dis . 2024 ; 30 : 591 – 4 . DOI PubMed Google Scholar
• Papadopoulos  E , Komnenou  A , Karamanlidis  AA , Bezerra-Santos  MA , Otranto  D . Zoonotic Thelazia callipaeda eyeworm in brown bears ( Ursus arctos ): A new host record in Europe. Transbound Emerg Dis . 2022 ; 69 : 235 – 9 . DOI PubMed Google Scholar
• Otranto  D , Dantas-Torres  F , Mallia  E , DiGeronimo  PM , Brianti  E , Testini  G , et al. Thelazia callipaeda (Spirurida, Thelaziidae) in wild animals: report of new host species and ecological implications. Vet Parasitol . 2009 ; 166 : 262 – 7 . DOI PubMed Google Scholar
• Otranto  D , Lia  RP , Traversa  D , Giannetto  S . Thelazia callipaeda (Spirurida, Thelaziidae) of carnivores and humans: morphological study by light and scanning electron microscopy. Parassitologia . 2003 ; 45 : 125 – 33 . PubMed Google Scholar
• Garshelis  DL , Scheick  BK , Doan-Crider  DL , Beecham  JJ , Obbard  ME . The American black Bear ( Ursus americanus ). The IUCN Red List of Threatened Species. Washington (DC): International Union for Conservation of Nature. 2016 :e.T41687A114251609.
• Di Salvo  AR , Chomel  BB . Zoonoses and potential zoonoses of bears. Zoonoses Public Health . 2020 ; 67 : 3 – 13 . DOI PubMed Google Scholar
• Figure 2 . Phylogenetic relationship of Thelazia callipaeda isolate from an American black bear in Coolbaugh Township, Monroe County, Pennsylvania, USA, 2023 (GenBank accession no. PP739308), and other species of Thelazia available...

Suggested citation for this article : Sobotyk C, Dietrich J, Verocai GG, Maxwell L, Niedringhaus K. Thelazia callipaeda eyeworms in American black bear, Pennsylvania, USA, 2023. Emerg Infect Dis. 2024 Sep [date cited]. https://doi.org/10.3201/eid3009.240679

DOI: 10.3201/eid3009.240679

Original Publication Date: August 14, 2024

Table of Contents – Volume 30, Number 9—September 2024

Please use the form below to submit correspondence to the authors or contact them at the following address:

Caroline. Sobotyk, University of Pennsylvania, School of Veterinary Medicine, Matthew J. Ryan Veterinary Hospital, Rm 4034, 3900 Delancey St, Philadelphia, PA 19104-6051, USA

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Bears Defensive Coordinator Likes Benefits in Using Starters

Gene chamberlain | aug 14, 2024.

• Chicago Bears

Bears coach Matt Eberflus didn't speak with reporters after Tuesday's practice and as a result it's still unclear whether the Bears will use starters against Cincinnati Saturday at Soldier Field.

He is not scheduled to talk to media again on Wednesday.

Bengals coach Zac Taylor already has said his starters won't play but Bears defensive coordinator Eric Washington did shed some light on the topic by saying Cincinnati's actions doesn't necessarily indicate what the Bears will do.

Just being completely objective, the Bears have 2 of the top 4 WRs in the division. Comparing each team’s WR1-3, the Bears have 3 of the top 4 in every comparison. The media needs to understand that ITS OKAY to give the Bears their flowers… pic.twitter.com/LN5mCPs2uL — Bartholomew Willijax (@B_Willijax) August 13, 2024

"We're on a ramp-up kind of mentality," Washington said. "We want to continue to see the defense, the guys we;re going to be counting and the guys that are competing to make the team. We want to continue to see them in live action and in different situations, so I think the experience of a preseason game is important.

"Obviously, coach Eberflus will make the final call on the specifics of that, but I think it's always valuable when we can get out and compete and we can kind of test the boundaries of our system and test the things that are really core values for us."

The critical thing is where the Bears are in preseason.

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"I think what matters is like I just mentioned, just where we are with our installation, where we are with our defensive process and where are we as it pertains to certain members of our team," Washington added.]

Eberflus has stressed throughout the offseason and also in camp how valuable the practice with the Bengals is, as opposed to the game and Washington agreed.

"Well, we just get a chance to work against another group as opposed to working against each other," Washington said. "We're going to have different looks. We're going to have a different skillset across the board.

I'm thrilled to have Hall of Famer Brian Urlacher on my show this week! His resilience led him to All-America honors & a legendary career with the Chicago Bears, leaving behind a legacy of outstanding achievement & leadership when he retired in 2012. Tune in on Thursday @ 6 PM… pic.twitter.com/sz8iHMbl7L — Lou Holtz (@CoachLouHoltz88) August 5, 2024

"It's just an opportunity for us to work against a different unit and to have to adjust on the fly without the benefit of going and game-planning and doing those sorts of things, just to really see our players respond and react with the things that are going to be presented to us."

The practice at least gives the defense a chance to face one of the best quarterbacks in the league, Cincinnati's Joe Burrow, as well as receivers Ja'Marr Chase and Tee Higgins.

"Well, we certainly adds a dimension to all of that, because of who he is and what he's capable of doing," Washington said. "It's an outstanding offense. I've had direct experience with that.

"It just gives us a chance to face an outstanding group, an outstanding offense, and we've been facing one our own offense for a couple weeks and now it gives us a chance to look at somebody else."

Here’s an another clip from my latest unboxing video. This is Jim McMahon’s 1st Sports Illustrated cover appearance from 1985 graded at 9.4. I will die on the hill that the 1985 Chicago Bears defense is still the greatest single season defensive performance in NFL history.… pic.twitter.com/tgkOQVf2Tq — CGC Sports Illustrated (@CGC_SI) August 6, 2024

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GENE CHAMBERLAIN

BearDigest.com publisher Gene Chamberlain has covered the Chicago Bears full time as a beat writer since 1994 and prior to this on a part-time basis for 10 years. He covered the Bears as a beat writer for Suburban Chicago Newspapers, the Daily Southtown, Copley News Service and has been a contributor for the Daily Herald, the Associated Press, Bear Report, CBS Sports.com and The Sporting News. He also has worked a prep sports writer for Tribune Newspapers and Sun-Times newspapers.

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