A water bear, Acutuncus antarcticus, descended from the egg frozen in 1983 called SB-3 shows off its latest meal of Chlorella algae inside its transparent stomach. Scale bar 200 micrometers. Fig. 1 from Tsujimoto et al. 2016.

Scientists seem to take a semi-sadistic pleasure in subjecting one of the cutest animals on Earth – the humble water bear – to some of the most extreme conditions imaginable. Water bears have been put through drought, cold, heat, pressure, radiation, even the vacuum of space, all in the name of science. That’s because the little animals, more formally called tardigrades ("slow steppers"), are some of the toughest animals on the planet, in spite of their petite size -- around half a millimeter -- and cuddle-me looks.

Waterbear.jpg
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Most of these extreme survival abilities likely link to their need to survive drying-out on the mosses on which they typically live (alternate water bear name: moss piglet). The mosses themselves possess a similar ability – called poikilohydry – uncommon among plants as well, but key to the environments in which both creatures live. The genetic machinery water bears need to repair themselves after dessication seems to work equally well after freezing, extreme heat, irradiation, or extravehicular activity.

Though many people had tested their ability to wake up and walk away after drying or freezing, few had tested whether they could actually go on to make little water bears after spending decades in the deep freeze.

Now, scientists from Japan have answered that burning question for us by thawing water bears collected in a moss sample in Antarctica and frozen during the Reagan administration – on November 6, 1983, to be precise, a time of Atari, Thriller, and skateboards. 31 years later, on May 7, 2014, they woke up to smartphones, Beyonce, and “hoverboards”, in their own tiny version of “Back to the Future” or “Futurama”. Perhaps luckily for them, the water bears remained blissfully unaware of any human cultural changes that took place during their long snooze, and went about doing what water bears do best: walking around on their eight bear claws, eating algae, and looking cute.

In the biology world, looking dead while you’re actually still alive is called “cryptobiosis”, and water bears are far from the only animals that can do it. Among the tiny animals, nematodes, also called roundworms, and fascinating little aquatic animals called rotifers can also do it.

But in the studies of such animals to date, most scientists have looked at animals that were dried out, not frozen. The record holder in this department are two young females and three larvae of the plant parasitic nematode Tylenchus polyhypnus, which survived 39 years without water – or, apparently, hope.

Further, most investigators sought simply whether the animals survived, and not the condition of the animals nor whether they were actually capable of reproducing after such tribulation. The longest such report was of two pairs of male and female nematodes that spent 8.7 years in a dried soil sample. The recovered females laid many eggs that hatched, producing fertile offspring.

In this study, by three scientists at the National Institute of Polar Research in Tokyo, Japan, and published in the journal Cryobiology in February, the water bears – an Antarctic species called Acutuncus antarcticus -- had been cooling their heels at -20°C for over 30 years.

They had been collected by Hiroshi Kanda in Yukidori Valley, Queen Maud Land, East Antarctica during the 24th Japanese Antarctic Research Expedition. He wrapped his samples in paper, put them in plastic bags, and stuck them in the Minus 20, probably with little idea what feats his samples would one day perform. One of those samples was thawed on May 7, 2014 at 3ºC for 24 hours. The sample was then put into a Petri dish and soaked in water for another 24 hours. Using tweezers, scientists picked apart the moss and extracted individual water bears with a pipette under the dissecting microscope.

The International Tardigrade Posture of Death is a fully extended body. Two of the tardigrades they pulled out had not assumed this position, and these two, which the scientists named Sleeping Beauty-1 and Sleeping Beauty-2 (SB-1 and SB-2 for short), were put into individual wells on a culture plate so they could be watched and fed. The scientists also found one egg, which they put into another well and dubbed SB-3.

In each well was tardigrade bedding in the form of 1.5% agar gel covered in 600 microliters of fine French mineral water (Volvic) and a gourmet selection of Chlorella algae. Each week the bedding and food was changed by moving the tardigrades to a new suite in another culture dish.

SB-1 was the first to show signs of life, twitching its fourth pair of legs on the first day after being rehydrated in a scene that surely must have recalled Mary Shelley. On day five, it twisted its body and slowly moved its first and second pairs of legs. On day 6, it tried to stand up, and by day 9, it managed to slowly crawl. Its appetite had returned by day 13, when it was finally able to swallow some food.

SB-1 had three eggs in its ovary. They began developing on day 21 and were laid on day 23. By day 45, SB-1 had laid 19 eggs in five separate batches, of which 14 hatched (74%).

SB-2 initially recovered similarly, but stopped eating after some tentative nibbling and died on day 20.

The egg SB-3 hatched on day 6. It laid its own first egg on day 14 and continued laying eggs until it was 32 days old, laying 15 total, of which 7 hatched (47%) – a much lower success rate than SB-1.

Previously, the longest any tardigrade was recorded to survive was only for 8 or 9 years after drying, not freezing. Previous authors have suggested 10 years may be the upper limit for dessicated tardigrades, because the exposure to air produces oxidative damage (similar to the way iron rusts in air) that is difficult to repair. Being frozen, on the other hand, may slow or eliminate these destructive chemical reactions and thus prolong survival, the authors of this study suggest.

That the water bears nevertheless incurred some damage from 30 years in the deep freeze is suggested by their prolonged recovery, and by the unfortunate demise of SB-2, they said. Other studies have suggested that the longer a nematode or water bear remains frozen, the longer it seems to take them to recover. The increasing recovery time is probably proportional to the cellular damage that accumulates over time in extremis.

The damage accrued over 30 years in cold storage may have also been the reason SB-1’s first egg required double the normal time to hatch, and SB-3 had lower reproductive success than usual after hatching from the egg in which it had been frozen.

The 30+ years of freezing survived by Sleeping Beauties 1 through 3 is a new record for cryptobiosis among tardigrades, exceeding the previous one by more than 20 years. But these tardigrades didn’t just survive. Two of them walked away from the ordeal and started families. It doesn’t get much tougher than that.

Reference

Tsujimoto, Megumu, Satoshi Imura, and Hiroshi Kanda. "Recovery and reproduction of an Antarctic tardigrade retrieved from a moss sample frozen for over 30 years." Cryobiology Volume 72, Issue 1, Pages 78–81 (February 2015).