Everything you need (and want) to know about tardigrades

By now, most of us have heard of the extremely hardy (and cute) tardigrade, otherwise known as the water bear or moss piglet. These microscopic animals have been subjected to some torturous conditions in a bid to understand how (and why) they are able to survive in such harsh environments. A lot of questions remain unanswered, but the science of tardigrades has certainly come a long way in the last couple of decades – and their genomes are key to unravelling the mystery.

Getting to know you

“Strange is this little animal, because of its exceptional and strange morphology and because it closely resembles a bear en miniature. That is the reason why I decided to call it little water bear.”

– J.A.E. Goeze (Pastor at St. Blasii, Quedlinburg, Germany), 1773

It was in 1773 that German zoologist Johann August Ephraim Goeze first came across these miniature lichen-residing animals and named them “Kleiner Wasserbär,” or “little water bear” after their resoundingly cute and unmistakably bear-like appearance. Five years later, Italian scientist Lazzaro Spallanzani would name them “tardigrada” or “slow steppers” in recognition of their strangely sluggish gait. Tardigrades, as we now call them, have been a curiosity of biological research ever since.

But why is the science community so obsessed with them? Could it all be down to their charming appearance? Decide for yourselves…

There’s no denying these eight-legged segmented micro-animals are pretty cute. But the real allure of the tardigrade lies in its incredible hardiness. These small creatures have been subjected to all manner of supposedly unliveable conditions – from temperatures near absolute zero to crushing pressures and radiation that should easily kill them. They’ve even been thrown into the vacuum of space – all without batting an eyelid.

The question is – how do they survive such extreme conditions? To answer this, we’ll first need to get to know our little bear-like friends a bit better.

Going on a (water) bear hunt

One of the biggest appeals of the water bear is that they are around 0.5mm in size and are often found lurking in mosses and lichens. This means they can be viewed quite easily using a standard microscope, making them very accessible to students the world over.

If you want to cast your eyes over the moss piglet yourself, follow these easy steps:

Collect a clump of moss or lichen (dry or wet) and place in a shallow dish, such as a Petri dish.
Soak in water (preferably rainwater or distilled water) for 3-24 hours.
Remove and discard excess water from the dish.
Shake or squeeze the moss/lichen clumps over another transparent dish to collect trapped water.
Starting on a low objective lens, examine the water using a stereo microscope.
Use a micropipette to transfer tardigrades to a slide, which can be observed with a higher power under a compound microscope.

Body morphology

Tardigrades are short, plump, and covered in a tough cuticle (similar to that of grasshoppers and other insects) that they must shed to grow. They have four pairs of legs, with 4-6 claws on each foot. Their specialised mouthpart – called a bucco pharyngeal apparatus – allows them to suck the nutrients out of plants and other microorganisms.

Their tiny bodies contain no bones and are instead supported by a hydrostatic skeleton – a fluid-filled compartment known as a hemolymph. They have no spinal cord but do have a similar system in place – a ventral nervous system that sends signals between the brain and body.

The slow walkers

Besides their ability to live in harsh conditions, another strange thing about tardigrades is that most of their cousins – the other soft-bodied, microscopic animals – do not have legs.

To uncover more about the tardigrades’ unique locomotion at the small scale, researchers recorded footage of the Hypsibius exemplaris species moving. Unfortunately, you can’t use the usual techniques to force tardigrades along like you can other insects because they are so small – so there was a lot of footage for the researchers to watch! Eventually, they discovered that despite being separated by around 20 millions years of evolution and belonging to a completely different phylum, the tardigrade locomotion most closely resembles that of insects.

Finding their place on the tree of life

Tardigrades seem to straddle the divide between arthropods and nematodes, making their position on the tree of life much debated. Scientists tend to agree they are closely related to nematodes, but research has previously rejected this idea based on microRNA data showing this is a long branch attraction artifact.

All in all, 1,300 species have been identified in the phylum Tardigrada so far (though it’s difficult to put a true number on it because of difficulties differentiating species). The truth is this may only represent a small portion of their diversity. Despite the increased interest in these microscopic organisms in recent years, the diversity of tardigrades is not reflected in the genomic data available. This paucity of information means the debate around where they lie on the tree of life is very much still open.

The limits of tardigrade life

This is the fun part. Just how hardy is the tardigrade? This question has intrigued scientists the world over for many decades – and boy, they’ve had a good go at testing the tardigrade’s limits.

Tardigrades have been known to survive:

Low temperatures of 0.05 kelvins (-272.95 degrees Celsius or functional absolute zero)
High temperatures of 150 degrees Celsius
Pressures of 40,000 kilopascals
The burning ultraviolet radiation of space
Being shot from a high-speed gun (traveling at nearly 3,000 feet per second and the impact of 1.14 gigapascals of pressure)
Being stored in a freezer for 30 years

Despite these impressive stats, it is worth noting that tardigrades are not technically classed as “extremophiles” because they do not thrive in harsh conditions – they simply survive. There is also some confusion between the tardigrade’s sheer hardiness and its ability to live forever. In reality, tardigrades only have a lifespan of a few months – in the active state. This can (and is) interrupted by long periods where they are (to all intents and purposes) “dead.” But more on that later.

Nonetheless, these robust little creatures have been on Earth for about 600 million years – preceding the dinosaurs by about 400 million years. In fact, tardigrades have survived all five mass extinction events, and it is thought they could be around long after humanity has died out. This brings us onto the next intriguing thing about tardigrades…

To infinity, and beyond?

Amidst the many bold claims about the science-fiction-like applications of these hardy animals lies the idea that we might be able to use their genes to engineer people with the radiation tolerance to withstand space. Is there any truth behind this? Perhaps.

Putting their hardiness to one of the biggest tests, tardigrades have been shown to survive after exposure to outer space. In 2007, dehydrated tardigrades were taken into low Earth orbit as part of the FOTON-M3 mission. They were exposed to either the hard vacuum of outer space (and protected from UV radiation) or vacuum and solar UV radiation for 10 days straight. After their return to Earth, the water bears that were protected from UV were reanimated within 30 minutes of rehydration. Subsequent mortality was high, but many were able to produce viable embryos before they died.

In 2017, researchers from Oxford and Harvard Universities decided to test how resilient different forms of life were to potential astrophysical events – things like asteroids, supernova blasts and gamma ray bursts. Of course, humans stood no chance. But who stuck around? Tardigrades. They found that these little organisms could survive in most cases, pointing to the potential for them to survive on Earth long after humans have been wiped out.

In more recent experiments, researchers have tested whether the hardy little animals can survive violent impacts similar to those that might explain how life originated on Earth – or even how it could be sent to other planets. In April 2019, an Israeli spacecraft carrying thousands of tardigrade passengers crashed into the surface of the moon. At the time, there was a lot of speculation about whether the critters could have survived and if we’d accidentally repopulated this barren landscape.

Why does this matter? Not only does it give us a clue as to how one of the prevailing theories behind the origin of life on Earth could hold true – panspermia – but it could also give us an insight into how life might be sent to other planets.

To put an end to this debate, astrochemist Alejandra Traspas and astrophysicist Mark Burchell loaded the water bears into guns (lab-grade two-stage, light-gas guns) and fired them at sandbags. The tardigrades were then collected, given water, and watched for reanimation. The results showed that tardigrades can withstand impacts up to 900 meters per second and shock pressures of 1.14 gigapascals. Higher speeds than this, and the bears turned to mush. This sounds promising, but meteorites tend to crash into other planets at higher shock pressures than what the tardigrades could survive. The general consensus is that the Israeli tardigrades would have died on impact, but some meteorite crashes could have impacts much lower than this, leaving the story somewhat open-ended. It certainly makes for the beginnings of a great sci-fi tale.

The how and the why: Tardigrade survival mechanisms

To understand how tardigrades are able to withstand such extreme conditions, we first have to understand why they developed these unique abilities in the first place. The answer lies in their evolutionary history.

Just like all other major animal groups, tardigrades first appeared in the sea. Millions of years later, animals started to venture onto land. But there was one major problem – the lack of water. Other groups developed unique adaptions such as waterproof skin (like humans) which keeps the water in our bodies. Tardigrades went down a different route – they developed the tun state.

Dead or alive: The tardigrade tun state

Though tardigrades can be found in moss and lichens, they are still – as per their evolutionary history – truly an aquatic animal. They require a film of water to surround their bodies to allow them to take in oxygen and expel carbon dioxide. Without this, they start to dry out, stop metabolising, and curl up into a desiccated form known as a “tun.” The incredible thing is that at the faintest whiff of water, they immediately bounce back into their original metabolising state.

**Figure 1: Tardigrades have evolved a suite of survival tactics to escape the vagaries of their localized and vulnerable environments.** Anoxybiosis and encystment, described in the upper part of this figure, are responses one might see in a variety of organisms. The bottom half of the chart shows three states of cryptobiosis, in which metabolism is suspended—an act usually diagnostic of death. Cryobiosis occurs in response to freezing, and anhydrobiosis in response to drying. During the latter, an organism surrenders its internal water to become a desiccated pellet. Both result in the formation of a durable shrunken state called a *tun*. More rarely, a tun is created to resist osmotic assault, which requires water. In the tun state, tardigrades can survive for many years, impervious to extremes far beyond those encountered in their natural environments. **Source:** American Scientist. **Illustration by Tom Dunne.**

The process whereby an organism temporarily suspends its metabolism is known as cryptobiosis. In this state, tardigrades completely slow down their metabolism to almost undetectable levels – less than 0.01% of normal. Their levels of water also drop to around 1%. They are one of only a few groups of species capable of undergoing cryptobiosis, and they can remain in this half-dead state for more than 30 years. It is in this tun state that the tardigrades are able to withstand some of the harshest conditions known to man. The ability to form tuns is preserved among all species of Tardigrada, so it is likely an ancient and homologous trait. As can be seen in Figure 1, there are other states they can enter in order to survive. Encystment is one example which involves the animal creating layers of cuticle in a much slower process compared to the tun state.

Anhydrobiosis is the specific ametabolic state which allows tardigrades to survive without water for long periods. They’ve been known to be resuscitated after 9 years in this state, though they could potentially be capable of surviving much longer. In the half-dead tun state, their body water content can decrease to between one and 3 percent.

What do we mean by “half-dead?” Well, according to all traditional definitions of life, there must be some form or reproduction and metabolism involved. In their tun state, tardigrades are doing nothing that we would associate with being alive – they are not making or consuming ATP at all. They are indistinguishable from dead or inorganic matter. So, are they really alive? This is yet another debate we will have to leave to the experts.

A very special genome

The tardigrade genome varies in size from 50 bases to 44 million bases. In genome terms, this is relatively small – but sequencing is still no easy task. The inherent nature of the tardigrade – its microscopic scale – makes it incredibly difficult to study. Each tardigrade contains just 0.2 nanograms of DNA – ideally, you’d like a microgram or more for analysis. There have also been issues with trying to rear large numbers in the lab. What’s more, their DNA is locked within their tough exoskeleton, making it difficult to get at.

It’s only in recent years, with the advent of new and improved sequencing techniques, that scientists have been able to take a closer look at their genomes and get to grips with how they survive in certain conditions. But this was not without some controversy…

#Tardigate

The sequencing of the first draft tardigrade genome in 2015 led to some surprising findings that would eventually culminate in what was known as #Tardigate across the research community. What was so surprising? The authors of the paper found that one species of tardigrade – the Hypsibius dujardini – had acquired up to one sixth of its genes through the process of horizontal gene transfer (HGT).

HGT is the process through which genetic information is passed from one individual to another, outside of DNA transfer from parent to offspring. The transfer of antibiotic resistance genes between bacteria is one good example of this, and it is generally considered to occur at a much lower frequency in animals. HGT on the scale seen in the 2015 paper was unheard of in animals, and the finding led scientists around the world to conclude that they’d found the unique mechanism tardigrades used to hone their survival skills – by acquiring useful traits from bacteria, plants, funghi and archaea. It also challenged existing models around how life evolves, suggesting that animals can acquire new traits by borrowing genes from other species.

Alas, no matter how nice and simple this explanation seemed to be, it did not hold true. Just a few months after the initial draft genome was published, a second group published their own – this time, only 1-2% of the tardigrade genes seemed to have been gained through HGT. This frequency of HGT is much more in line with what we’d expect to see in animals.

So, what caused the difference? It’s likely to be a simple case of contamination. Due to the complexities around working with the small tardigrade genomes, bacteria can sometimes be sampled alongside tardigrade DNA. The 2015 study had seen that 18% of the tardigrade DNA they sampled was from “non-animal sources” and had suggested this was due to HGT. In reality, what they’d seen was simply contamination – indeed, several complete genomes of interesting bacteria could be found in the published “tardigrade” genome.

What a difference a protein makes

Initially, it was believed that the incredible hardiness of the tardigrade was down to trehalose – a sugar that some bacteria, plants, funghi and invertebrates use to survive extreme cold temperatures and a lack of water. However, a 2016 study from Takekazu Kunieda and his team from the University of Tokyo soon put paid to this notion.

After sequencing the R. varieornatus genome, the team decided to investigate any genes unique to the tardigrade. In particular, they focused on a gene that coded for a protein called damage suppressor protein (Dsup). This protein was found to associate with nuclear DNA, and the team guessed it may play a role in protecting the tardigrade from DNA damage.

To investigate this theory further, they engineered cultured human embryonic kidney cells to express Dsup. Compared with controls, these cells appeared to have higher survival rates when exposed to X-rays and they exhibited 40% less X-ray-induced DNA damage. The findings suggested tardigrade-unique proteins are integral to the organism’s ability to tolerate certain environments.

Alongside Dsup, the team also uncovered two other groups of unique proteins – cytoplasmic-abundant heat-soluble (CAHS) proteins and secretory-abundant heat-soluble (SAHS) proteins – that seemed to confer some survival tactics to tardigrades. All three proteins are classed as intrinsically disordered proteins (IDPs) – proteins that lack a single, well-defined 3D structure – and seemed to be highly expressed in response to desiccation. In recent years, the importance of IDPs has become more apparent, with some estimating that a third of the proteome may contain disordered sequences. These dry-tolerant proteins are intrinsically disordered in water, but once conditions dry out, they form a secondary structure that allows them to stabilize DNA, proteins and cell membranes.

It is thought that CAHS and SAHS may protect the water bears by associating with the phospholipid bilayers in membranes, helping to maintain their structure. In extremely dry conditions, CAHS proteins can transform the tardigrade cells from shapeless and flexible into a “bioglass” that holds the proteins and molecules together until they are rehydrated.

So, it seems researchers might have figured out how the hardy animals protect their DNA and proteins. But there are many other components and structures that make up an organism – and we still have no idea how they are able to protect these. Until a few years ago, most tardigrade research was curiosity-driven with little work being done to explore potential applications – but this is all likely to change. The hope is that uncovering the mechanisms that tardigrades use to protect their many different biological components will bring about new potential applications in the years to come.

From sci-fi to the real world: Tardigrade applications

Given their close relation to nematodes – like C. elegans – tardigrades have been proposed as an invaluable model organism for a range of scientific studies. They are physiologically simple, they have a fast-breeding cycle, and they have a precise, highly patterned development plan. Of course, their main (and most intriguing) use will be in understanding how biological materials can survive remarkably extreme conditions.

One medically important application of tardigrade research could be in vaccine development. As we are now all aware after the recent pandemic, vaccines must be kept at low temperatures. But maintaining the so-called “cold-chain” can throw up a number of potential issues and is also very costly. One alternative could be to use the inherent properties of intrinsically disordered proteins to allow vaccines to be stored at room temperature. In fact, Thomas Boothby (Molecular Biology Department at the University of Wyoming) is already working on tweaking some of these proteins so that they can be used in vaccines. “We have patents out on these things and have some partnerships,” he said. “If all goes well, hopefully we will see this technology out soon.” Their research has already proven that IDPs can protect protein-based pharmaceuticals (like biologics) around 10 times more efficiently than current FDA-approved protectants.

Of course, the cold-tolerant properties of tardigrade proteins are just one route for useful applications. In 2020, researchers stumbled across a new species of tardigrade that were capable of surviving intense doses of ultraviolet radiation. When they looked into the mechanism behind this, it seemed the animals were fluorescing – they were absorbing the shorter wavelengths of light and emitting longer ones, protecting their cells from radiation damage in the process. The researchers decided to go one step further and ground the tardigrades into a paste that was then applied to C. elegans worms. Remarkably, these worms were then able to tolerate much higher doses of UV radiation. The team hopes this could open the door to a type of tardigrade “sunscreen” that is used to protect humans from radiation exposure.

**Figure 3: *Paramacrobiotus* BLR strain shows tolerance to UV radiation and exhibits fluorescence under UV light.** (a) (i) Survival of *Paramacrobiotus* BLR strain after UV radiation exposure for 15 min (1 kJ m⁻²). *Hypsibius exemplaris* was used as control. (ii) Their comparable survival in the absence of UV radiation. (b) Fluorescence microscopy images of *Paramacrobiotus* BLR strain and *H. exemplaris* taken under identical microscope settings. (c) Fluorescence images of the lysates of *Paramacrobiotus* BLR strain and *H. exemplaris* under UV light (365 nm). (d) Images of hypopigmented *Paramacrobiotus* BLR strain showing reduced fluorescence under UV light compared with the pigmented ones. Images were taken under identical microscope settings. (e) Survival of hypopigmented *Paramacrobiotus* BLR strain after UV radiation exposure for 1 h. **Credit:** H Suma et al., Biology letters, 16, 10 (2020). DOI: **10.1098/rsbl.2020.0391**

The anti-desiccation properties of tardigrade-specific proteins could also come in handy – this time in food production. Yeast, bacteria, and crops could all be manipulated to express the genes for IDPs and potentially create drought-resistant versions – something of increased importance as the climate continues to warm.

As mentioned earlier, previous experiments have found that tardigrades can survive the hard vacuum of outer space, as well as strong radiation. Understandably, this has made people very excited about what this could mean for humans. Could their genes be used to help us populate other planets in the solar system? To test this further, the Cell Science-04 experiment aboard the International Space Station will try to identify the genes involved in their ability to survive and adapt to high-stress environments – like those that astronauts experience. NASA hopes this will help guide research into protecting humans from the stress of long-duration space travel.

Scientists have already inserted the gene for Dsup into human cells in the lab, with many of them able to survive levels of exposure to X-rays and peroxide chemicals that would usually kill normal cells. This research could also apply to crops – when Dsup was inserted into tobacco plants, it was able to protect the DNA from ethyl methanesulfonate and induce quicker growth. These plants were also more protected from UV radiation exposure.

Earlier this year, a team of researchers modified human cells to produce extremotolerance-associated proteins – like those from tardigrades. They found that the proteins conferred apoptosis protection to the cells, which could be useful in cancer research (specifically, in protecting cells from the damages of chemotherapy).

Another application of this research is in supporting the healing process – for example to prevent bleeding and cell death in traumatic injuries. Pamela Silver, a synthetic biologist at Harvard Medical Institute and the Harvard Wyss Institute, is looking into how IDPs can be used to stabilize traumatic injuries in combat zones. “The time from when one is wounded to when one gets to the hospital is a critical time,” said Silver. “In medicine, that window of time is called ‘the golden hour’ and we would like to extend it for as long as possible.”

Her team is also looking into other proteins – some 300 in fact – that could offer other medical applications. “For example, the exposure to hypoxia is medically relevant for conditions such as heart attack and stroke, and also potentially relevant to the mechanism by which these proteins work in tardigrades. We have been looking at the ability of these proteins to have an effect on low oxygen conditions and we have similarly found candidates.”

These applications are very exciting, and the reality is that the extreme hardiness of the tardigrade could be applied to an almost infinite number of challenges here on Earth. It is of course important to not get carried away with some of the more sci-fi-esque stories surrounding these little creatures – but who can blame us when they come in such a cute package?

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