Notothenioids are fish capable of surviving in the sub-zero waters surrounding Antarctica. Equipped with antifreeze proteins for protection, they are benthic, living on the bottom of the sea, feeding on krill and other fish. Many are sit-and-wait predators, moving little with relatively low metabolic rates. A subgroup of the notothenioids, the icefish, are remarkable for being the only adult vertebrates to have lost the oxygen transporting protein haemoglobin. Christina Cheng and Kristin O'Brien tell Journal of Experimental Biology about these extraordinary creatures and how they survive in one of the planet's most inhospitable environments.
A giant Antarctic toothfish (Dissostichus mawsoni) from McMurdo Sound, the largest member of the Notothenioidei suborder. Photo credit: Paul Cziko.
Christina Cheng, can you tell us about the extraordinary notothenioids that live in the Southern Ocean?
C.C.: They are stenothermal, which means that they live within a very narrow temperature range. The most stenothermal fish are in the most southerly waters, such as McMurdo Sound (78°S), which is at the southern limit of marine life. Their habitats range from –2°C, which is the freezing temperature of seawater, up to 4°C along the relatively northerly Scotia Arc Islands, ∼57°S. –2°C to +4°C is a very narrow temperature range compared to the 10°C or more range in high-latitude northern oceans, and the fish succumb to heat at 6°C. They evolved in this very narrow range of temperatures without experiencing any major thermal fluctuations.
How did the notothenioids in the Southern Ocean become separated from other fish?
C.C.: About ∼300–275 million years ago all the continents were packed together in the supercontinent, Pangea, but about 200 million years ago, they started to drift apart. The last continent that detached from Antarctica was South America about 40 million years ago. Then the Antarctic plate started to move towards the south pole and there were no more surrounding landmasses. This allowed the unrestricted flow of water around the continent, resulting in the Antarctic circumpolar current about 36 million years ago. The Antarctic circumpolar current became larger until it became the most massive ocean current on earth today. It rotates clockwise and goes very deep, like a revolving curtain of water, forming a giant barrier that organisms cannot disperse across. So, the fauna in the Southern Ocean became highly endemic. The current also acts as a thermal barrier, so there is very little or no heat exchange between the water in the Southern Ocean and the warmer water to the north. When the global carbon dioxide drawdown occurred, about 34 million years ago, Antarctica began to become extremely cold and glaciated. Eventually the marine waters reached freezing temperatures, –2°C, with ice forming in the water column.
Cold temperatures are not the only danger to teleost (bony) fishes; ice in the water column is also a risk. The two together are a lethal combination. The blood of teleost fish is more dilute than seawater, so the fish's freezing point is much higher than –2°C, at about –1°C to –0.8°C. If there's no ice, fishes can undercool and remain unfrozen. But if there is ice and they run into an ice crystal, then ice will instantly nucleate in the body and they will die. The fishes either had to evolve a solution or become extinct, which was what happened to most of the fishes that were originally in the Southern Ocean. There is a fossil site near the tip of the Antarctic Peninsula that shows that about 40 million years ago the fauna was very cosmopolitan, including sharks, other elasmobranchs and bony fishes, which we see in the north. But those fossil fish all became extinct and only the notothenioids survived and became the dominant group because their ancestor evolved an antifreeze glycoprotein that allowed them to survive in freezing ice conditions.
If there is ice and they run into an ice crystal, then ice will instantly nucleate in the body and they will die
Which protein did the first antifreeze glycoprotein evolve from?
C.C.: The gene for the antifreeze glycoprotein came from a trypsinogen gene. Trypsinogen is the precursor of the protease trypsin, which is an enzyme that cuts up other proteins, but the current antifreeze glycoprotein protein sequence is very different from the modern trypsinogen sequence. The antifreeze protein has a very simple sequence – alanine-alanine-threonine, which gets repeated with the occasional substitution of a proline for an alanine, and there are sugars attached to the threonines in the protein. But the protein still uses the ancient trypsinogen signal peptide sequence, which tells the cells that this protein is for export. Antifreeze glycoproteins range in size from just 2,600 Da up to 50,000 Da.
How do antifreeze glycoproteins stop ice crystals from forming?
C.C.: The antifreeze proteins recognise ice that comes into the body of the fish from outside. Teleost fish drink water to replenish the body water that's constantly lost to the outside, but when they drink water they also ingest ice and they eat food that has ice associated with it, so it is unavoidable that external ice gets into the fish. The antifreeze then recognises ice crystals, because there is structural matching between the regularly spaced sugars along the length of the protein, which are about 9.3 Å apart, and the water molecules on a particular surface of the ice that have similar spacing. When antifreeze molecules bind to the face of ice crystals, water molecules can only join the ice crystal in between the absorbed antifreeze molecules. So instead of growing flat ice crystal planes, the crystal grows small highly curved fronts and at a critical radius it is thermodynamically unfavourable for new water molecules to join the crystal lattice, at which point ice growth stops and the body fluids stay liquid. In the more northerly latitudes, such as the Antarctic Peninsula and Scotia Arc Islands, the water warms up enough for these internalized ice crystals to melt, but the fish in the most southerly waters – including McMurdo Sound, where there is very little warming even in the summer – cannot melt these ice crystals. The fish are destined to carry them through the rest of their lives. We do see one organ that often carries ice and that is the spleen, because the spleen is a filtration organ for blood and so maybe the antifreeze-stabilized ice crystals in the blood are sequestered in it, so they don't get stuck in the fish's microcirculation and cause problems.
Where do the fish produce antifreeze proteins?
C.C.: Most of our plasma proteins are made in the liver and the antifreeze proteins produced by other polar fishes are made in the liver also. But after many years of work, we found that the notothenioids don't make antifreeze in the liver. Instead, the antifreeze glycoproteins are made in the pancreas and the anterior stomach mucosa. The pancreas secretes enzymes with the antifreeze into the small intestine and the stomach mucosa secretes into the stomach chamber. We think the reason for the pancreas and the stomach being the two major sites of antifreeze glycoprotein synthesis is that the gut fluid has to be protected from freezing, because the fish drink icy seawater, eat ice-studded food and there's a lot of liquid secretion in the intestine, such that fluid in the intestine is more dilute than sea water and at risk of freezing. So, it makes a lot of sense that antifreeze evolved from a pancreatic trypsinogen and is secreted as food comes in to protect the gastrointestinal fluid. The trouble is, how do these fish achieve such high levels of antifreeze in the blood? That's still an unresolved question. There's no direct anatomical connection between the pancreas and the general blood circulation. We also know that the antifreeze glycoproteins do not get degraded in the digestive tract because of the very high sugar component, which protects the antifreeze glycoproteins, and there is no known protease that cuts the antifreeze protein sequence. We have some evidence that the antifreeze is probably taken up by rectal cells and returned to the circulation, but that is a slow process and we still don't know if there are specific transporters that are involved. In any case, the antifreeze glycoproteins are plentiful in the blood circulation, as well as in all other extracellular compartments except for the eye fluid, endolymph and urine.
Antifreeze glycoproteins are made in the pancreas and the anterior stomach mucosa
Kristin O'Brien, how do icefish differ from the other notothenioids?
K.O’B.: Icefish don't have haemoglobin. They lost the ability to synthesize the protein somewhere between 2 and 8.5 million years ago. Probably the most striking characteristic of these animals is their milky white blood, which is a result of the fact that they do not express haemoglobin, which binds oxygen and delivers it to tissue. Perhaps you've had anaemia, which makes you feel really tired; these animals have a haematocrit (blood volume that's occupied by red blood cells) of zero, which is really astounding. Despite this, they survive and thrive. Today there are 16 species of Antarctic icefishes, 15 of which are found in the Southern Ocean surrounding Antarctica, and one species, Champsocephalus esox, is found off the coast of Chile and Patagonia in warmer waters. There are several modifications in their cardiovascular system that have enabled them to survive without haemoglobin. All icefish oxygen is carried within their circulatory system just dissolved in their blood plasma, which is the watery part of your blood. They have a very large blood volume, which increases the amount of oxygen that can be carried. They also have enlarged hearts, enabling them to pump this large volume of blood and to reduce the work of the heart pumping they have large diameter blood vessels, so they can circulate a fairly large volume of blood at a high rate to maintain oxygen delivery to the tissues. Also, these animals are not super active. They're mostly benthic fish – they are sea bottom huggers – they're fairly sedentary and they have a low metabolic rate, meaning they don't have a high demand for oxygen. Because they live in a very cold environment, there's much more oxygen in the Southern Ocean at –1.5°C, compared with a warmer ocean at 20°C, because the oxygen solubility in water increases as the temperature decreases. So, when the mutation that led to the loss of haemoglobin occurred in the ancestral fish, it's thought they survived because they lived in this very cold environment and there was plenty of oxygen already dissolved in their blood. In fact, Bruce Sidell (University of Maine, USA) once conducted an experiment lowering the haematocrit of a red-blooded notothenioid using a chemical that causes red blood cells to break open and they survived just fine. The loss of haemoglobin is considered a neutral mutation; it didn't impact the fitness or the ability of that animal to survive and reproduce. Also, there isn't a lot of competition with other species in the Southern Ocean, so the icefish probably don't have to work very hard to get the food that they need. But it's unlikely that they will be able to begin expressing haemoglobin again if they needed it, because the haemoglobin protein is composed of two subunits that are encoded by two different genes: the alpha- and beta-globin genes. Icefish completely lack the beta-globin gene and they only have a partial remnant of the alpha-globin.
The most striking characteristic of these animals is their milky white blood, which is a result of the fact that they do not express haemoglobin
Can you describe the icefish's natural environment for us?
K.O’B.: The continental shelf surrounding Antarctica is very deep, about 500 m, icy cold and there are limited intertidal regions for fish to colonise. They don't have swim bladders, so they mostly sit at the bottom. We know from fishing in the area that they are not found together in great numbers in one place. But in 2021, the research vessel Polarstern, was in the Weddell Sea and they made the absolutely amazing discovery of icefish nesting. They found over 60 million nests in a rocky area about 240 km2 and most were guarded by one individual, so perhaps icefish breed together or in common areas. The phenomenon likely has something to do with an oceanographic feature in the area that make it favourable for reproduction. It was a little bit warmer than surrounding areas, and the water was nutrient rich, so the researchers hypothesized that these features may make the area favourable for breeding.
What's the life expectancy of an icefish?
K.O’B.: It's estimated that they live somewhere between 12 and 20 years and they don't reproduce until they're somewhere between 3 and 10 years old, although this probably varies by species. That may be, in part, because it's energetically expensive to produce eggs and reproduce. These animals live in a very seasonal environment, food is mostly available in the summer when there's light for phytoplankton to grow, so given the energetic constraints, it may be that they have to be larger in order to have the capacity to reproduce.
What other physiological modifications have these fish made to be able to live at this cold environment?
K.O’B.: In response to cold temperature, membranes remodel to maintain fluidity. So, the fluidity of membranes in an Antarctic fish at –2°C is similar to that of a temperate fish swimming around at 20°C. They do that by increasing the proportion of unsaturated fatty acids in their membranes and also the cholesterol content in plasma membranes. My collaborator Elizabeth Crockett at Ohio University, USA, has measured membrane fluidity in icefish and red blooded notothenioid species and it seems that the membranes are more fluid in the icefish compared to red blooded notothenioids, which may be advantageous for improving oxygen delivery in the cells and in the tissues, because oxygen is more soluble in lipid compared to water. The ability of oxygen to diffuse in the membrane will be improved in a more unsaturated, more fluid, membrane.
The fluidity of membranes in an Antarctic fish at −2°C is similar to that of a temperate fish swimming around at 20°C
What other physiological mechanisms have icefish lost?
K.O’B.: Probably the best characterised example is that the icefish, and all of the Antarctic notothenioids, have lost the ability to increase levels of proteins called ‘heat shock proteins’. As temperature increases, the bonds that maintain protein structures weaken and so proteins begin to unfold. Heat shock proteins help proteins to refold into their proper conformation. Studies by Gretchen Hofman (University of California, Santa Barbara, USA) many years ago showed that Antarctic fish are unique because they've lost the ability to increase levels of these molecular chaperones in response to an increase in temperature. The loss of the heat shock response will make it challenging for all Antarctic notothenioids to withstand a warming climate, but icefish may be at a greater risk because they rely on oxygen dissolved in their blood plasma, which will decline as the ocean warms.
When I was working with Bruce [Sidell] we also discovered that icefish have really unusual mitochondria – they're much larger than those in red blooded species. But the density of the inner membrane, where you find the components that produce ATP, in each mitochondrion is lower compared to red blooded species, although the overall density of mitochondria in tissue is greater and therefore the total surface area of membranes is greater. Essentially, they've increased the membrane density within the cells. Oxygen is also more soluble in lipid than in the watery cytosol. Our hypothesis is that these lipid membranes serve as a highway through which oxygen can diffuse from a capillary to the enzyme cytochrome c oxidase, which is involved in producing ATP. In addition to lacking haemoglobin, six of the 16 species of icefish also lack the intracellular oxygen binding protein myoglobin in their hearts, so they have white hearts, which is really fascinating. Myoglobin has been lost multiple times during the radiation of teleost fishes, so it's not specific to Antarctic icefish, but it is a double whammy for icefish: they don't have haemoglobin, which reduces their blood oxygen carrying capacity and they don't have myoglobin, which stores oxygen in the heart and red muscle and enhances its diffusion to mitochondria. So again, to compensate in part for the loss of both haemoglobin and myoglobin, they've expanded the density of mitochondria and their cells.
The loss of the heat shock response will make it challenging for all Antarctic notothenioids to withstand a warming climate
What do you want to find out next about icefish?
K.O’B.: Right now, we're investigating their ability to withstand low oxygen – hypoxia – and we conducted experiments last spring in Antarctica with Yangfan Zhang, who's a postdoc at Harvard University. Our hypothesis is that notothenioids might have a lower capacity to withstand low oxygen compared with temperate fishes and that icefish that lack haemoglobin will be more sensitive to hypoxia than red blooded species. But if notothenioids are more hypoxia tolerant it could lead to the possibility that the ancestral notothenioid had some characteristics that allowed them to withstand low oxygen and that permitted the loss of haemoglobin.
Chi-Hing Christina Cheng works at the Department of Evolution, Ecology and Behavior, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
E-mail: [email protected]
Kristin O'Brien works at the Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK 99775, USA.
E-mail: [email protected]
Christina Cheng and Kristin O'Brien were interviewed by Kathryn Knight. The interviews have been edited and condensed with the interviewees' approval.