Why Arctic Fish Don’t Freeze: Extreme Marine Life

Antifreeze proteins

Polar sea water usually hovers around its freezing point: −1.8 °C, (28.8 °F). Under sheets of ice, in the deep, dark water of the polar regions, you might expect to find a desolate ecosystem. Perhaps only extremophile microbes. This is not the case. Under the ice, fishes are thriving!

How is this possible? They’re cold-blooded (ectotherms), after all. Their bodies don’t produce their own heat, and fishes are theoretically temperature consistent with the seawater. A tropical fish would immediately freeze if dropped into Arctic waters since the temperature of the saline liquid is below the freezing point of the fresh water in the cells of the fish.

The mechanism that has allowed fishes to survive in the presence of structurally diverse antifreeze proteins in their tissues. These proteins possess the powerful ability to separate the melting and growth temperatures of ice. These proteins have evolved independently in different kinds of cold-adapted ectothermic animals, including insects and teleost fishes, where they protect against lethal freezing of the body fluids. The AF proteins stick to developing ice crystals and prevent them from growing enough to damage tissue.

Trypsin-Like Serine Proteases

Antifreeze proteins function very similarly to trypsin-like serine proteases, which “attack” or stick to biomolecules through various mechanisms. TLSP enzymes are responsible for protein hydrolysis in our digestive system, clotted blood, infection resistance, and egg fertilization.

The AFGP found in Antarctic fishes, in fact, evolved from a serine protease of the pancreas.² But not all AFGPs are the same. They have evolved independently many times and allowed those species that possess them to adaptively radiate throughout a freezing niche with little competition.

Antifreeze proteins come in many “flavors”...fungi, bacteria, insects, polar fishes and even plants utilize antifreeze proteins to prevent their tissues from crystallizing in freezing temperatures. This is a case of convergent evolution, meaning TLSPs are relatively evolutionarily malleable. Humans haven't evolved them because our genome has not experienced pressure to develop AFGPs - we are warm-blooded endotherms, and we generate our own heat.

AFGPs are part of a larger class of ice-binding proteins (IBPs) which are utilized by subzero tolerant organisms. These proteins include:

1. Antifreeze proteins (AFGPs) with high thermal hysteresis antifreeze activity

2. Low thermal hysteresis IBPs

3. Ice nucleating proteins (INPs)

Multiple structurally distinct IBPs have arisen even within related taxa. True antifreeze proteins (1) are found in freeze-avoiding organisms which will die if frozen. In contrast, less active proteins (2) are found in freeze tolerant species, which are able to survive being frozen.

Molecular Evolution

About 30 million years ago, the waters of the Antarctic region became very cold. The fossil record shows Antarctica's once great diversity of fish was reduced to a single suborder Notothenioid. Their secret to success? The divergence of the antifreeze protein. These fish survived and had the opportunity to radiate into their choice of diet and habitats. They grew enormously in population size, creating subpopulations.² More than 90 species of these fish exist today. This is a classic example of adaptive radiation in evolution, similar to Darwin’s 13 finch species on the Galapagos.

On the other side of the globe, Arctic fishes evolved antifreeze proteins too, but through a different mechanism. The stunning diversity and spatial distribution of AFGPs leads scientists to believe that the different types evolved recently in response to sea level glaciation 2 million years ago in the northern hemisphere, and 30 million years ago in Antarctica. This sort of independent development of similar adaptations is referred to as convergent evolution.

The reason that multiple types of protein do the same job and have the same name is that they are very similarly shaped when they are folded, and they act on different surfaces of the ice molecules. Ice is always composed of oxygen and hydrogen, but it has many different surfaces for binding.

Applications in technology

Scientists have identified many nuances of antifreeze proteins, and they have been very useful in developing a number of human technologies, like to create perfectly consistent ice cream! In order to bring these advances into medicine, an interesting question was required to be answered: why do antifreeze proteins lock onto the ice but not liquid water? 

“If AFPs bound as easily to liquid water as they do to ice, this lifesaving action could turn killer, as animals would quickly dehydrate,” says Matthew Blakeley at the Laue-Langevin Institute in Grenoble, France.¹

How do we ensure healthy tissue is not damaged by molecules similar to antifreeze proteins when they are sent into the human body to attack a tumor? The answer lies in the tertiary structure of proteins: their shape. The shape of a protein is determined by a few factors. Proteins are amino acid chains. Between 20 common amino acids, at least two are linked to create a polypeptide chain, or protein. Each amino acid building block has a particular chemical profile based on its elemental components and their configuration. The profile of two amino acids determines what reaction will take place when they are in proximity. Every protein folds to a particular, signature shape due to the interaction between its amino acid components.

Antifreeze proteins act as a structural lock and key, based on their shape and the shape of ice crystals. They are compatible with the spiky formation of ice crystals, not the loose structure of water. By nature, they will not bind to water, and not dehydrate the organism they belong to.

Connection to the Gulf of Maine

The Gulf of Maine is our home, and we have AFGPs to thank for the survival of our fishes! Some of our favorites - sculpin, sea raven, ocean pout, and the Atlantic tomcod use AFGPs to tolerate our cold waters.

Cheers to the beauty of molecular evolution!

References

1. Barras, Colin. (2011, April 13). Polar Animals' Antifreeze has a Spiky Secret. Retrieved from https://www.newscientist.com/article/mg21028083-300-polar-animals-antifreeze-has-a-spiky-secret/

2. Logsdon, J. M., & Doolittle, W. F. (1997). Origin of antifreeze protein genes: A cool tale in molecular evolution. Proceedings of the National Academy of Sciences of the United States of America, 94(8), 3485–3487.

How to: Dig Spaghetti Worms

Spaghetti Worm

U-shaped burrow and castings. The hole ('volcano') is where the mouth of the worm is. The castings pile is the other end of the worm! The middle of the worm is deepest underground, at the center of the U's curve.

Spaghetti worms (Amphitrite spp.) live in super slimy tube burrows. They have a U-shaped burrow with the mouth at one end and their anus at the other end. The appearance of the front end of the worm gives it its name. Looking at the head you will see a bunch of red and tan colored tentacles stretching out of its burrow. The reddish tentacles are used for respiration. The whitish or tan tentacles are used for feeding. 

The worm lives in a burrow for protection. Only the feeding tentacles come up above the bottom. They stretch out in a radius around the burrow and they pick up food particles, plankton, detritus - this is all reeled back into the mouth as a food source. Imagine a worm that is named for the red (tomato sauce) and the tan (pasta) tentacles.

Locating the worm

We find these worms on the mudflat by looking for their piles of poop (castings) that lie in a pile at the end of the U-shaped tube burrow. Another interesting thing about these worms is that they sometimes share their tube with 12-scaled worms. Now, scaled worms are typically found on rocky bottoms in the depressions under the stones. What the advantage is to be tucked down in a tight little tube with a spaghetti worm is beyond me.....it has to be for feeding or protection I would guess. If anyone would like a very interesting research project - this is wide open!

Care

The spaghetti worm is a very versatile lab specimen. You can keep them in a small tub of salt water without any aeration, in a fridge for weeks. Some of our customers use these as a food source for anemones and fish.

Spaghetti worms (Amphitrite spp.).

The front end of the worm toward the bottom of the photograph is tentacled.

Fisherman's Bucket

Hermits, hermits, hermits!  

Yesterday a local fisherman, Mike brought us in a pile of goodies fresh off his sea urchin drag boat. The weather had finally warmed up and the seas subsided enough for him to go out safely and try to make his living.

Besides the sea stars he brought in, I had asked him to keep an eye out for some hermit crabs and other small invertebrates. The hermits were HUGE - mostly flat - clawed variety, but there were some hairy hermits as well. Some of them were a bit stressed due to the anoxic conditions in the small pail and they had exited their shells. This was a bit concerning, at first - I wondered how they would do outside their shells. I placed them into my tank 'naked' for the evening and when I returned in the morning, they had all found suitable shells to move back into... 'shall I slip into something a bit more comfortable...?'.

The catch 

To my surprise, many of the hermits were egg-laden (gravid, berried?). You wouldn't be able to see this unless they had come out of their shells. The eggs look just like a lobster's egg mass.  The color was very black and the eggs a bit smaller though.

Hermit crab party.

Hairy hermit crab in my hand.

Hairy hermit crab with eggs (berried).

Flat clawed hermits.

Hermit crabs are one of the easier marine invertebrates to take care of in a tank. I feed mine bits of clam or squid when I have it. They will all gather around a lump of feed like cows at a trough, snipping and tearing bits to feed into their mouths. It is pretty neat to watch. As for the shell species that were represented - there were all the major mollusc snails we have here in Cobscook Bay. Waved whelk shells, moon snail shells, Stimpson's Coleus shells, and dogwinkle shells.  The moon snail shells are the most impressive. Since these snails get very large, the Hermits that inhabit them are big too.  

Sea grapes.

Sea peaches.

Toad crab.

Sea vase.

As for other species that came up in the fisherman's trawl, we found tunicates, other crabs, and worms.  There were several toad crabs, Hyas genus. The largest of these was about 8" across. The sea squirts in the pail were Sea Grapes (Molgula), Sea Vase (Ciona), Sea Potatoes (Boltenia), and Sea Peaches (Halocynthia). All of these were cemented to other tunicates in clusters or they attach to mussels, rocks, and other debris. Final inspection of the pail's contents revealed some fan worms, finger sponge, scaleworms and some tiny isopods - benthic creatures that had come up in the holdfasts of the tunicates and sponge. I dumped the remainder in our tanks. Hopefully, they will adapt to the smaller quarters and find their niche - it's a crab eat, worm eat, snail world in there!

Tidepool Tim