The Arctic ground squirrel can survive even if its body temperature drops below the freezing point of water

Most animals hate the cold. When winter comes around many species burrow underground to hibernate or migrate to lower latitudes where conditions are warmer.

But a few strange creatures do the opposite. They actually embrace the freezing conditions.

We are still unravelling the mysteries of these amazing animals that freeze. For one species in particular, doing so could prove significant. Several scientists are trying to work out how the Arctic ground squirrel (Spermophilus parryii) became the only known warm-blooded mammal to be able to tolerate subzero body temperatures. Solving the mystery could hold the key to freezing human organs for transplant without damaging them.

It might even provide a boost for the controversial field of cryonics, in which human corpses are put into deep freeze in the hope that they can be returned to life with future medical advances.

Subzero temperatures are a problem for all living things because water expands as it freezes to become ice. “When water in an animal's cells freezes, the ice crystals that form expand and physically rupture the cell, causing death,” explains David Denlinger, an entomologist at Ohio State University in Columbus. To survive freezing temperatures, animals must find a way to prevent ice from forming inside their cells.

Cryoprotectants, much like the antifreeze you put into your car radiator, prevent ice forming by reducing the freezing point of water

The most famous freeze-tolerant species is probably the wood frog (Rana sylvatica), which can survive subarctic temperatures for weeks at a time.

“Freeze tolerant organisms use various "tricks" to limit the damage that occurs when ice forms within tissues,” explains Jon Costanzo from the Laboratory for Ecophysiological Cryobiology at Miami University in Ohio. “Our team was the first to examine the extreme freeze tolerance in wood frogs living in subarctic Alaska,” he says. “Those frogs all survived freezing to -14C!”

The frogs are able to cope with freezing temperatures by producing “cryoprotectants” – substances that prevent ice crystals from forming inside their cells.

Cryoprotectants, much like the antifreeze you put into your car radiator, prevent ice forming by reducing the freezing point of water.

“Cryoprotectants act by lowering the body's freezing point, preventing water freezing at temperatures well below 0C,” explains Denlinger. When a cryoprotectant chemical dissolves in water, it forms strong bonds with water molecules. Water molecules that are bonded to the cryoprotectant can no longer bond with other water molecules to form ice, meaning that the water can be cooled to subzero temperatures without freezing.

Wood frogs produce urea, glucose and glycogen to act as cryoprotectants in response to subzero temperatures, and researchers have now identified freeze-induced genes that are responsible for transporting glucose into cells.

The red flat bark beetle from Alaska can supercool its body fluids to -50C

Freezing vertebrates and insects also produce antifreeze proteins, which bind to ice crystals and prevent them growing. One compound, antifreeze glycolipid (AFGL) is used as a defence against freezing in organisms ranging from plants to beetles, and in 2014 scientists identified the same glycolipid as an antifreeze compound in the wood frog.

Most impressive among freezing animals may be the invertebrates. A host of arthropods from cockroaches to caterpillars can tolerate freezing temperatures for days at a time.

Many of these species use cryoprotectants and antifreeze proteins to protect their cells, and Denlinger says that body fluid freezing points (also called supercooling points) as low -25C are not uncommon.

One truly remarkable beetle, the red flat bark beetle (Cucujus clavipes puniceus) from Alaska, can supercool its body fluids to -50C. But there is huge variation between individuals – some can tolerate body temperatures as low as -100C.

By dehydrating themselves, Antarctic midges are able to survive temperatures as low as -20C

These deep-supercooling beetles have higher levels of antifreeze proteins and cryoprotectants like glycerol, which help them to minimise ice formation even at such extreme temperatures.

But these remarkable adaptations do not explain the freezing feats of the ground squirrel. Arctic ground squirrels don’t use cryoprotectants to protect their cells, and no antifreeze compounds have been identified in their blood.

“Mammals do not flood their bodies with cryoprotectants,” says Brian Barnes from the Institute of Arctic Biology at the University of Alaska Fairbanks, who has been studying the hibernation of the Arctic ground squirrel for over two decades. Something else is going on inside the squirrels’ bodies.

The Antarctic midge (Belgica antarctica) has come up with an alternative solution to cope with freezing temperatures. “The Antarctic midge does not make cryoprotectants nor antifreeze proteins,” says Denlinger. “Instead, it simply gets rid of its body water.”

If there’s no water, there can be no ice

Most insects can survive losing 20-30% of their water, but the Antarctic midge can lose up to 70% and still survive, he says. By dehydrating themselves, Antarctic midges are able to survive temperatures as low as -20C.

While frozen, their metabolism stops and they appear lifeless. “When it has lost so much water, it does not appear to be alive,” says Denlinger, “but when you add water it quickly becomes fully hydrated again and goes on its merry way.”

“The midge has the ability of cryoprotective dehydration," says Shin Goto, who studies animal physiology at the Osaka City University in Japan. He explains that when their habitat freezes, the surrounding ice draws water from the midges’ highly permeable bodies, preventing any ice from forming inside their delicate tissues.

Dehydration is an effective way to prevent ice formation – if there’s no water, there can be no ice. But no mammal can survive such extreme dehydration. Cryoprotective dehydration definitely doesn’t explain the mystery of the Antarctic ground squirrel’s super-cool stunt. 

Prevention isn’t the only way to deal with ice. Another trick used by wood frogs and other freeze-tolerant animals is to produce substances known as ice-nucleating agents, which actively encourage ice formation.

Although counter-intuitive, ice-nucleating compounds produced in the right place ensure that ice forms between cells, not inside them. This ‘extracellular ice’ is less harmful because its sharp crystals are kept away from delicate machinery inside the cell.

These frozen animals have evolved adaptations to survive a lack of oxygen

An ice nucleator could be almost anything: a speck of dust or even a bacterial cell. Inside freeze-tolerant organisms they are thought to be proteins or fats, although scientists are yet to isolate these natural ice-nucleators in the laboratory.

“Ice nucleators are molecular mimics of ice,” says Barnes. They have a similar structure to ice crystals, enabling them to act as a seed to start the ice-crystal formation process. Wood frog blood contains ice-nucleating proteins, and ice-nucleators have been identified in insects, molluscs and even plants.

When frozen, wood frogs and other freeze-tolerant vertebrates are forced to shut their systems down – they have no heartbeat or breathing. So as well as adapting to having ice inside their bodies, these frozen animals have evolved adaptations to survive a lack of oxygen, known as anoxia.

Painted turtle (Chrysemys picta marginata) hatchlings produce antioxidants and proteins that bind iron - part of the body’s usual response to a lack of oxygen – in reaction to freezing temperatures. A study published in 2015 confirmed a similar defence mechanism to anoxia in dehydrated Antarctic midges, whose larvae increase the expression of antioxidants when frozen.

The Arctic ground squirrel can survive body temperatures to -3C, but they do not freeze

But like cryoprotective dehydration, ice-nucleation is not a strategy that would work for ground squirrels. While invertebrates like the Antarctic midge, and cold-blooded animals like the wood frog, have evolved to tolerate some freezing inside their bodies, this just isn’t a possibility for mammals.

In fact, it is the very absence of ice-nucleators that turns out to be key to the squirrel’s unique abilities.

“Arctic ground squirrels cleanse their bodies of would be ice-nucleators before entering hibernation,” says Barnes. Although the exact mechanism remains a mystery, Barnes’ research suggests the ground squirrels may achieve this by producing masking agents, which neutralise ice-nucleators before ice has a chance to form around them.

“The Arctic ground squirrel can survive body temperatures to -3C, but they do not freeze”, he explains. “Instead body fluids within the arctic ground squirrel enter a ‘supercooled’ state”.

Without any effective nucleators to get the ice crystals started, water in the squirrel’s blood simply can’t freeze.

Whatever the strategy for tolerating subzero body temperatures, the strain of this physiological feat is evident. Most freeze-tolerant species can only pull off the trick once a year. When tested in spring, wood frogs were only able to tolerate temperatures down to -5C; still impressive, but nothing compared to their autumnal feats.

“We don't fully understand why freeze tolerance is so abruptly reduced in spring, but a limited capacity for cryoprotectant production is probably at issue,” explains Costanzo. “Both major cryoprotectants, glucose and urea, are at lower levels in spring frogs” – and these spring frogs also suffer more ice damage when artificially frozen.

Understanding these processes could help us develop new techniques to freeze human organs for storage prior to transplantation

This may be a common problem; one study found that four species of freeze-tolerant frog all showed the same loss of freeze-tolerance in spring, linked to a reduced ability to produce cryoprotectants.

In fact, most freeze-tolerant species benefit from at least a short period of acclimatisation to an oncoming freeze. Sudden drops in temperature can be bad news even for the hardiest of species.

The Isabella tiger moth (Pyrrharctia isabella) lives in the Arctic and produces caterpillars that can survive air temperatures down to -20C – supercooling their body fluids as low as -10C. For these caterpillars, 12 weeks of acclimatisation reduced the freezing point of their body fluids by nearly 2C. It gave them time to physiologically adapt by producing glycerol, proline and amino acids to act as cryoprotectants.

The Arctic ground squirrel is also far better at coping with the cold during the winter months. “Blood sampled from hibernating animals can supercool to much lower levels than blood sample from animals in summer,” says Barnes, although the exact mechanism remains a mystery.

He suspects it may be linked to larger scale seasonal changes in the squirrels. “We are currently studying how they time their annual cycles of hibernation.” He says these are determined by an internal “calendar” in the brain, which sets the timing of the physiological changes that adapt them for the winter months.

Across the animal kingdom, creatures living in the coldest parts of the world have developed adaptations to cope with freezing conditions. Understanding these processes could help us develop new techniques to freeze human organs for storage prior to transplantation. What’s more, learning how animals freeze may be the key to making human cryopreservation a reality.

If we are to cryopreserve human cells successfully, we need to understand how warm-blooded organisms like the squirrel cope

Currently, organs for transplant are chilled, but not frozen. This means they are viable for just a few hours, and so an organ that could save someone’s life can’t always reach the patient in time. Increasing the viability of transplanted organs by freezing them could revolutionise organ transplantation – and cold-tolerant animals like the arctic ground squirrel could hold the secrets to making this possible.

In fact, research into freeze-tolerance is already yielding results.

Antifreeze proteins identified in fish and insects are an obvious target for improving cryopreservation techniques, and in 2005 a team of researchers at the University of California, Berkeley and Sheba Medical Center in Israel, successfully used antifreeze proteins isolated from Antarctic fish to freeze and preserve rat hearts for 21 hours. These hearts were then successfully transplanted into recipient rats, where they continued to beat for at least 24 hours.

Tissue preservation companies are now investigating whether insect antifreeze proteins, which are more effective, could act as potential cryoprotectants for human organ preservation.

What we can learn about freezing humans from insects and frogs may be dwarfed by what the arctic ground squirrel can teach us, though. Freezing an insect or amphibian is one thing, but if we are to cryopreserve human cells successfully, we need to understand how warm-blooded organisms like the squirrel cope with subzero temperatures.

Even the most impressive animals can only cope with temperatures down to about -50C

If we could identify the ground squirrel’s technique for removing ice-nucleators, and apply that to humans, we might be able to supercool cells and organs without even a single crystal of ice forming inside.

Some also hope freezing technology may one day help us to preserve whole humans. In fact, cryonics methods are already beginning to be informed by our understanding of the protective mechanisms used by freeze tolerant animals.

For instance, some cryonics companies dehydrate bodies, replacing the blood with a solution of cryoprotectants such as glycerol and dimethyl sulfoxide, and finally deep-freezing them, in the hope that doing so won’t damage the human tissues or prevent reanimation in the future.

But even the most impressive animals can only cope with temperatures down to about -50C, nowhere near the deep-freezing temperatures used in whole body cryopreservation.

Shin Goto sees potential for research into insect cryoprotectants and anti-freeze proteins to improve cryopreservation of human cells, tissues and organs. But he says he doubts that we will ever be able to freeze and thaw whole humans. “A human is too large in size to freeze and thaw,” he says.

David Denlinger agrees, saying “I think that is pretty unlikely, but I do hold out hope for freezing and thawing human tissues.” He adds that cryoprotective dehydration as seen in the Antarctic midge has “tremendous implications for the field of organ storage”.

Learning how animals freeze may be the key to making human cryopreservation a reality

“If we truly understood how [the midge] does this, we could possibly adopt such mechanisms to store human tissues and organs,” he says.

Three decades of research into freeze-tolerant fish, frogs and insects has taught us a lot about how to survive subzero temperatures. But many mysteries remain.

The arctic ground squirrel, the only mammal to survive subzero body temperatures for extended periods of time, could offer our best chance of safely entering the world of subzero tissue preservation. It’s just about possible that, one day, a squirrel could save your life.

See a video of an Arctic ground squirrel in action