Round Ice: Frozen in Time

 

The nematode P. kolymaensis

As the world heats up let’s turn to animals that cool down.

Roundworms, phylum Nematoda, are an unfamiliar group to most folks. Biology 101 students may remember them, though not necessarily fondly. These are one of the wormy phyla, long and skinny, but they are vastly different from the more well-known earthworms, which are in the phylum Annelida, segmented worms. 

A short aside, in the world of animals, there are many wormlike groups: round worms (Nematoda), segmented worms (Annelida), flatworms (Platyhelminthes), peanut worms (Sipuncula), ribbon worms (Nemertea), horsehair worms (Nematomorpha), Acanthocephala worms, and Priapulid worms, to name a few. Being worm-like is a successful body plan.

The Nematode species count is more than 40,000, but that number is far lower than the actual number of species on Earth; so many still to be discovered. They are abundant, found everywhere in large numbers. They are so abundant in soil and in us that a biologist quipped:

In short, if all the matter in the universe except the nematodes were swept away, our world would still be dimly recognizable, and if, as disembodied spirits, we could then investigate it, we should find its mountains, hills, vales, rivers, lakes and oceans represented by a thin film of nematodes. The location of towns would be decipherable, since for every massing of human beings there would be a corresponding massing of certain nematodes….”

Nathan Augustu Cobb, 1914. Nematodes and Their Relationships.

 

Yes, nematodes are everywhere, and that includes inside us: There are parasitic nematodes, heartworm, the scourge of pets and their owners, among them. Parasitic nematode hosts include humans and farm animals. Hookworms, pinworms, and the worm that causes trichinosis. Trichinosis, which usually infects humans due to them eating undercooked meat, is a fun disease that will cause diarrhea, vomiting, weakness, and may lead to heart inflammation. Yes, great fun.

Nematodes also include a well-studied lab animal, often called a model organism, Caenorhabditis elegans. From this worm, we (the royal we) have learned much. We have determined everything from the fate of cells as development proceeds (which dividing cell first becomes a nervous system cell, for example) to how dopamine leads to addiction, to aging of cells, to an understanding of apoptosis (programmed cell death). They are also used to screen drugs for toxicity. In short, these buggers have helped us tremendously.

Then…

In 2023, a team of them science types published a paper describing something almost impossible: A whole microscopic nematode that had been frozen solid in Siberian permafrost since the Pleistocene. The landscape was a bit different then; woolly mammoths roamed the tundra. That they found it was not the nearly impossible part; they took this worm and thawed it out, and it was… alive. Let this sink in for a minute. The approximate age of the worm is 46,000 years.

Melting permafrost. inset of nematode.

This species, named Panagrolaimus kolymaensis, when revived in the laboratory, not only survived, it reproduced. 

Note: the genus name refers to the type of pharynx structure Panagrolaimus, laimus = gullet, and the species epithet gives homage to the location discovered, the Kolyma River in northeast Russia.

As suggested above, with the quote, the phylum Nematoda is one of the most ecologically diverse on Earth, found in virtually every habitat from deep-sea sediments to garden soil to inside bodies. Let’s add one more evolutionary feat, they have also evolved an astonishing capacity to cheat death.

The survival strategy is a state of suspended animation so complete that metabolism becomes undetectable. No heartbeat (they have no heart anyway), no breathing (they have no lungs), no measurable biochemical activity. Their long-term survival depended on several integrated mechanisms. A key one is trehalose, a disaccharide sugar whose production is increased to 20-fold when the worm enters this suspended state, known as cryptobiosis. Trehalose coats cellular membranes and macromolecules, acting to prevent structural damage during freezing and desiccation. To produce this protective sugar in such quantities, the worms convert their stored fat reserves into sugar via a specialized metabolic pathway.

 

During the last 46 thousand year this Siberian permafrost has been a stable repository that maintained sub-zero temperatures; otherwise, the worm’s protective coating of sugar, would degrade and they would awaken or start to decompose.

 

C. elegans, mentioned above as a model study organism, shares some genes and pathways with the frozen Rip Van Winkle. Thus, this may be an old, evolutionarily conserved survival mechanism.

 

This ability to survive freezing is not observed just in nematodes. Surviving ice formation within body tissues has evolved across a wide sweep of animal life: insects, tardigrades, rotifers, intertidal mollusks, frogs, turtles, and salamanders all include members that routinely freeze and thaw. 

Animals generally employ three distinct cold-survival strategies: freeze avoidance, in which body fluids are supercooled to remain liquid below 0°C; freezing tolerance, in which ice formation in body tissues is survived; and cryoprotective dehydration, in which body water is lost entirely to prevent freezing. Freezing tolerance is a common strategy in cold-hardy insects from the Southern Hemisphere.

For freeze-tolerant animals, the key challenge is not preventing ice but controlling it. Most species limit supercooling deliberately, allowing ice to begin forming at relatively high subzero temperatures. A slow freeze gives the organism time to deploy its biochemical defenses before a catastrophic ice surge causes irreversible damage. The cardinal rule is that ice remains in extracellular spaces. If ice forms inside cells, membranes rupture, and the cell architecture collapses. Panagrolaimus nematodes have suspended (see what I did there) the cardinal rule and evolved the rare ability to survive ice formation inside their own cells, an intracellular freezing that would kill almost any other animal. 

Many of the cardinal rule following soil-dwelling species, earthworms, frogs, and hatchling turtles, exploit ice-binding proteins that prevent recrystallisation, stopping small ice crystals from merging into larger, tissue-shredding ones. The Alaskan wood frog and the beetle Upis ceramboides go further. These species produce a xylomannan-based antifreeze glycolipid that integrates into cell membranes, blocking ice from moving from extracellular fluid into the inner cell cytoplasm. 

Alaskan Wood Frog.

The Alaskan Wood Frog is at the pinnacle of vertebrate extremists. When a wood frog freezes, its heart stops. Its lungs stop. Blood circulation ceases entirely. The animal enters prolonged ischemia and hypoxia that would be fatal to virtually any mammal within minutes. The survival trick is a metabolic shutdown reducing energy expenditure to as low as 1% of normal resting levels. They actively silence energy-expensive genes, both DNA methylation and histone deacetylation, repress transcription during the frozen state. 

These frogs spend an average of 193 consecutive days frozen solid each winter, far longer than their southern counterparts. Their liver glycogen stores reach 3.5 times higher levels than in southern populations, fueling the massive glucose export upon freezing. The mitochondria reconfiguration, enhancing antioxidant defenses; this primes them to deal with the respiratory burst that occurs on thawing. Studying these mechanisms aids organ cryopreservation and biobanking research.  The lowly wood frog is a blueprint for preserving human tissue.

Considering extreme cold tolerance in animals is not complete without mentioning tardigrades, the winners of extreme living. The microscopic water bears famous for surviving conditions that would sterilize most spacecraft. Freeze-tolerant tardigrades can endure temperatures as low as −196°C through controlled extracellular ice formation and cellular dehydration. Their freeze-tolerance genes seem to be constantly active rather than modified, as seen in the wood frog. Like nematodes, tardigrades encase themselves, but they rely on proteins instead of the sugar trehalose.

Tardigrade

A 46,000-year-old worm, a frog that spends six months frozen solid, a water bear that could survive liquid nitrogen; all these critters have solved this fundamental problem: How to pause life completely and then start it again. Studying them is not only fascinating but could lead to connections of human health and cryopreservation. But, no, I don’t want to join the head of Walt Disney and be frozen.

Sorry about the Walt Disney jibe. It’s not actually true, he was cremated, but the rumor persist. There have been lots of people frozen after their death, but he was not among them. It’s an odd thing, and none of those people will be brought back to life, after all they were not first encased in trehalose sugar. 

 

Sources and Further Readings:

Boothby TC, Tapia H, Brozena AH, Piszkiewicz S, Smith AE, Giovannini I, Rebecchi L, Pielak GJ, Koshland D, & Goldstein B. 2017. Tardigrades use intrinsically disordered proteins to survive desiccation. Molecular Cell: 65(6): 975–984.e5.

Higuita ML, & Griffiths LG. 2022. Lessons from nature: Leveraging the freeze-tolerant wood frog as a model to improve organ cryopreservation and biobanking. Acta Biomaterialia 134: 144–159.

Larson DJ, Middle L, Vu H, Zhang W, Serianni AS, Duman J, & Barnes BM. 2014. Wood frog adaptations to overwintering in Alaska: New limits to freezing tolerance. The Journal of Experimental Biology 217(12): 2193–2200. 

Møbjerg A, Kodama M, Ramos-Madrigal J, Neves RC, Jørgensen A, Schiøtt M, Gilbert MTP, & Møbjerg N. 2022. Extreme freeze-tolerance in cryophilic tardigrades relies on controlled ice formation but does not involve significant change in transcription. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 271: 111245.

Morgan-Richards M, Marshall CJ, Biggs, PJ, & Trewick SA. 2023. Insect freeze-tolerance downunder: The microbial connection. Insects 14(1): 89. 

Shatilovich A, Gade VR, Pippel M, Hoffmeyer TT, Tchesunov A V, Stevens L, Winkler S, Hughes GM, Traikov S, Hiller M, Rivkina E, Schiffer PH, Myers EW, & Kurzchalia TV. 2023. A novel nematode species from the Siberian permafrost shares adaptive mechanisms for cryptobiotic survival with C. elegans dauer larva. PLoS Genetics 19(7):

Storey KB, & Storey JM. 2017. Molecular physiology of freeze tolerance in vertebrates. Physiological Reviews 97(2): 623–665. 

Wade S, Hadj-Moussa H, & Storey KB. 2023. mRNA m6A methylation in wood frog brain is maintained during freezing and anoxia. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology 339(3): 325–334. 

Wharton DA. 2012. Supercooling and freezing tolerant animals. In P. Wilson (Ed.), Supercooling (pp. 17–28). InTech. http://www.intechopen.com/books/supercooling/supercooling-and-freezing-tolerant-animals