Chi-Hing (Christina) Cheng

Your research focuses on antifreeze proteins in fish. I'm curious how you became interested in this topic, and more specifically, how you got involved in working in Antarctica.

Well, just like in science, there was a lot of serendipity. I began working in Antarctica because I met Art DeVries, who later on became my husband. We were both in the same department – he was a new faculty member, and I was a grad student in a different lab. He gave me the opportunity to go to McMurdo as a field assistant. In that first season I was just totally awestruck by the environment and what people can do. And I like catching fish and cutting up fish and trying to understand their physiology in that very cold water. So, I knew I had to go back, and the rest is history.

In that first season I was just totally awestruck by the environment and what people can do. And I like catching fish and cutting up fish and trying to understand their physiology in that very cold water. So, I knew I had to go back, and the rest is history.

In terms of the antifreeze protein focus, Art discovered antifreeze in Antarctic fish, and he really started this whole new field of cold adaptation by the evolution of these macromolecules. Prior to this discovery, the conventional paradigm was that freezing point is lowered due to adding a lot of small solutes. Nobody had ever thought that large molecules, like proteins, could depress freezing point because they just don't contribute very much osmotic concentration because of their size. So, antifreeze proteins are revolutionary as a paradigm-changing concept.

Of course, working in Art’s lab, I had to work on antifreeze proteins. My first task at that time was purifying antifreeze protein so that we could figure out the protein sequence and how they work, and I also characterized other fishes that have different types of antifreeze protein than the Antarctic fish. I found that kind of work satisfying. Biochemistry really appeals to me, and I like to tinker at the lab bench and make things happen, get results.

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The “molecular revolution” happened a short time later. PCR – or polymerase chain reaction, which is a technique to amplify tiny amounts of DNA to huge quantities that could be easily studied in detail – was invented, and a lot of start-up companies were providing supplies for doing molecular biology. I wasn't trained in molecular biology. I essentially learned it on my own and then got into cloning and analyzing genes. The goal is to figure out where the genes for these novel antifreeze proteins come from and the mechanisms of how they evolved. That became my niche, and I've worked on it for many years since then.

It’s impressive how you took advantage of new technologies to answer these research questions. A follow-up question is, what was your research focus prior to meeting Art and being introduced to Antarctic fish?

...it’s really serendipitous that I got involved in Antarctic research.

I was actually doing my PhD research in a lab that worked on radiation biology, essentially looking at damages and repair after being exposed to radiation. That radiation lab was at one end of the hallway in the basement, and Art’s lab was at the other end. I think that is the “most romantic” way of meeting somebody, in the middle of a basement! Like I said, it’s really serendipitous that I got involved in Antarctic research.

Truly! When was that first field season at McMurdo that really changed your research focus?

That was 1984, which really dates me. I’ve been thinking about how many times I've gone, and in total I have deployed 19 times. Sixteen times to McMurdo and three times to Palmer Station.

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Wow! Can you expand on your initial impressions of Antarctica?

My first season down there, the first time I stepped on that continent and looked at the station, I was like, “Wow! How could this happen?”. I couldn't imagine the logistics and work that it took to put all of that infrastructure in this really remote place for science to happen. Being a new person bubbling with excitement for this new experience, I didn't know any better about how to do things. It was kind of bewildering, and I was a very shy person those days, so I was trying to figure out how to connect with the support people and how to do the science. It was very interesting, but a bit challenging. Even so, my impression was, “My goodness, this is just amazing! This is out of the world.”

When I first went there in the ‘80s, the whole place seemed like the wild wild west...

The station and the U.S. Antarctic Program have changed a lot over the years. When I first went there in the ‘80s, the whole place seemed like the wild wild west, if you will. It was a wild frontier and there was a lot of spontaneity. There were no people saying that you can't do this, you can't do that. There was a lot of freedom and spontaneity in trying to improvise science or do science on the fly; you could fabricate things. For example, if you needed a box to haul something out to the sea ice, you could find scrap wood and then build it yourself. These days, of course, you can't do that. You have to go through the chain of command. Back then, that freedom was really exhilarating. Plus, I felt that there was a lot more connection between the grantees and NSF. For example, the director at the time of my first deployments was Peter Wilkniss. He would come into the lab – the old Eklund Lab, which was later replaced by the Crary Lab – and he would check in on us and have a party or dance with us scientists. I don't think those things happen anymore.

The flip side of that freedom is the environmental impacts. Back in those days, people were free to throw stuff away and garbage was not handled well. There was this garbage dump where they put everything and then regularly just burned it, which polluted the environment. So, environmentally, the station was not in very good shape. And all of that is because the concept of stewardship for this pristine region was not a concept yet. It was in the late ‘80s when Greenpeace championed the World Park idea and set up camp in Cape Evans, not too far from McMurdo. That fostered action to clean up the place.

...being a basic scientist and having this opportunity and the funding support to go to the end of the world to be able to do really cool science – I'm really, really grateful for that.

And then, of course, over the years, there has been a lot more emphasis on safety and not doing any damage to the environment. So, there is a lot of training, a lot of rules, a lot of regulations that you have to comply with. That takes away some of the spontaneity of doing things because you have this tier of people supervising all of the structure. Then, as a scientist, you are at the other end of having to comply with the structure. I think it has built some distance between the grantees and NSF or OPP (Office of Polar Programs) personnel. These days, it is kind of difficult for new people to connect with OPP officers, even though they are actually quite open. You can call them, and you can write to them, but there isn't frequent spontaneous communication. That's kind of a barrier, especially with this COVID thing that has deferred a lot of funded programs.

But all in all, being a basic scientist and having this opportunity and the funding support to go to the end of the world to be able to do really cool science – I'm really, really grateful for that. I always keep that in mind, regardless of what other hassles come up along the way. I think that's important, and I think other people who have been funded to go to the Antarctic for years and years should be mindful of that gratitude.

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Yes, with the current situation, it’s important to remember that any Antarctic science happening at all is pretty remarkable.

Polar programs, after all, are human enterprises. Plus, there are environmental contingencies that affect what can be done and what cannot be done. I think people just need to be patient, on both sides, and more open communication would help alleviate a lot of second guessing.

Definitely. Speaking of research, are there any discoveries that you and Art have made that you’d like to highlight?

Polar fish biology is a huge field. Even antifreeze, which is one component of polar fish biology, is a big field in and of itself. There are many, many facets. Once you figure out that these proteins are useful in allowing these fish to avoid freezing to death, then you want to characterize the protein’s sequence, you want to figure out how it actually works to prevent that freezing. That then gets into a lot of structure-function studies and ice physics, because the antifreeze binds to ice crystals that get inside the fish and prevent them from nucleating the rest of the body fluids. So, there's a lot of physical and chemical studies. And then, of course, this whole area of where the antifreeze genes come from gets you into molecular and genomic evolution. I would say that our work is complementary. In the early days we caught fish together. These days, Art works a lot more on structure and function aspects. I work a lot more on molecular and genome biology. But together, our common goal is to understand how polar fish can thrive in that extreme environment.

I can highlight two discoveries from our work between the ‘80s and early 2000s. One of them is figuring out where those antifreeze genes in the Antarctic notothenioid fishes came from. The fish have this protein with sugars on it. It's called antifreeze glycoprotein – “glycol” referring to the sugar component. We call it AFGP for short. So, we caught fish, and I sequenced and analyzed the gene. The upshot is that it came from a protease gene called trypsin. Protease is a digestive enzyme that cuts up other proteins. When you eat, your protein has to be digested. Since trypsin cuts up other protein, it is not functionally related to the antifreeze protein that binds ice crystals, even though it is the evolutionary precursor.

After we figured out that AFGP evolved from trypsin, we wanted to know whether it's expressed where trypsin is expressed. Art helped a lot with sampling the digestive juices from pancreatic secretion, and then I ran the gel and all of that molecular stuff. We figured out that yes, antifreeze is also made in there along with the trypsin. But back to the evolution part, the AFGP gene came from a trypsin precursor, but the precursor has a very, very different sequence. The making of a new gene usually results from cells making a new copy of a gene by chance during DNA replication, which happens quite regularly. Then you have two redundant copies. One copy usually just goes away because its function is redundant. But if there's selection that can act on the new copy because it has a bit of another function that is beneficial to the organism, then that copy will remain. The sequence will change under that selective force over time to refine that new function.

But again, trypsin is very different from AFGP. There didn't seem to be any sort of secondary function that would be related to binding the ice crystal. We did a lot of molecular analyses. Back in those days, sequencing and decoding genes were not easy, so it took a while. What we found is that the coding sequence for the antifreeze function, the function of binding ice, actually came from a very simple nine nucleotide snippet of the DNA in the trypsin gene. Nine nucleotides code for three amino acids. It was those three amino acids specified by those nine nucleotides that became acted on by natural selection, and then it just duplicated many times. The AFGP peptide backbone is repeats of these three amino acids, and every third one has a sugar attached to it. That sugar is what binds to ice crystals.

I think that was one of the big discoveries. It’s used as an example in textbooks and people teach this stuff.

The gist of the story is that this initial nine nucleotide snippet was partly from an intron of the trypsin ancestor. Intron sequence is non-coding DNA. So, the AFGP functional coding sequence came from basically copying that partially non-coding snippet many times and generating this really large, life-saving adaptation. Back in those days when we described this new process, we didn't know what to call it. Since then, in the last 10 to 20 years, there's this whole field called de novo gene evolution. “De novo gene” means new genes that came from previously non-coding DNA sequences. This AFGP gene evolution in the Antarctic notothenioid fish to this day is known as the first example of de novo gene evolution. It’s a very concrete example, as well as an example that has very clear adaptive function and huge significance on the fitness of the species that evolved it. So, I think that was one of the big discoveries. It’s used as an example in textbooks and people teach this stuff.

Are there many other examples of these de novo genes?

There are a lot of de novo genes that have been detected since then in mammalian systems and model species like Drosophila. But the trouble there is that, while they can detect genes, they think are de novo genes, rarely do they know the origin, what the genes do, what is the selective force that drove them to become new genes, or what fitness consequence the new genes confer. And so, the field is expanding tremendously, and there are some instances where they actually were able to nail down the function. But by and large, most of the de novo genes that have been detected have no known function. So, the antifreeze system is really cool because we know what it does. We know how good it is for the animal. And we can even relate it to an environmental selection, which is the freezing of the ocean water. It's a neat story without many holes.

Cool, thank you for explaining the genetics so clearly. I'm a geologist and don't have any background in this field.

Actually, when we figured out how AFGP evolved, we also estimated when it evolved. We were interested in correlating the evolution to glaciation around the Southern Ocean. We estimated that the AFGP gene probably evolved from its trypsin ancestor about 7 to 15 million years ago, which is kind of in the ballpark of mid-Miocene cooling. We wrote that in the paper, and the marine geologists were like, “Hey, can we use molecular tools to answer some of our questions on the timing of these geological events?”.

What a great example of how interdisciplinary Antarctic research is! What is the second big discovery you’ve made?

It's not as revolutionary, but there is a very unusual physiology of the Antarctic notothenioid fish related to where the AFGP is being synthesized. There are many different species that have different types of antifreeze, and they are transported in the blood. Ice crystals get into the blood, somehow, and antifreeze sees them, binds to them, and stops them from growing. If they don't grow, then the rest of the blood and body fluids will remain liquid, and the fish doesn't freeze and die. Plasma proteins, meaning proteins circulating in the blood, are primarily made in the liver. So, many of the ice-binding proteins that circulate in the blood come from the liver. The reason why the liver is such a major source of plasma protein is that its anatomy allows it to directly put the proteins into the blood stream. There is a connection between all the blood sinusoids in the liver which converge to a blood vessel that leaves the liver and goes to the heart, where blood then gets distributed.

Early studies showed that AFGPs are made in the liver. Later on, when other antifreeze proteins were discovered in other fish species, both in the Southern Ocean as well as in the northern seas, we found synthesis of their respective type of antifreeze in the liver. Now in the Antarctic, I came along, equipped with these molecular techniques, and I could never find antifreeze mRNA expression in the liver of notothenioid fish. I spent at least two years figuring out whether it was my technique that was bad, but in the end, we found that it wasn't my technique. The notothenioid fish just don't make AFGPs in the liver. Instead, they make them in the pancreas.

We have multiple lines of evidence to show that. One of them is staining sections of these tissues and using antibodies to detect the protein. We've done it from embryo to new larvae and to adult tissue, and we never see any protein being made in the liver. But there’s a lot of booming signals in the pancreas. Somehow the Antarctic notothenioids are such an oddball, making AFGP in the pancreas. It is logical that the pancreas makes the antifreeze, because when food comes in and when the fish drinks seawater, in come ice crystals. Ice crystals get into the gut fluid, and the gut fluid is quite dilute because of the massive secretion of acidic (stomach) and alkaline (intestine) solutions during digestion. When gut fluid is more dilute with respect to the seawater outside, the ice crystal is going to just grow like gangbusters. And so, if the digestive enzymes and antifreeze are dumped into the gut when food and water comes in, then the antifreeze proteins will stop that nucleation, which makes a lot of sense. The caveat is that there's a lot of antifreeze moving around in the blood, and so how does the antifreeze that’s made in the pancreas reach the blood? There’s no direct anatomical connection between the pancreas and the blood vessels in the systemic circulation. All the secretions from the pancreas go through a pancreatic duct that empties into the small intestine. So, that opens up another whole area of figuring out how gastrointestinal AFGPs get reclaimed or recycled back into the circulation. We have some evidence that it might be taken up by some rectal cells where a lot of water is also reabsorbed. May be the antifreeze are taken up by those cells and then returned to the circulation.

...in this very isolated environment, animals can evolve very strange things, very different physiology than animals elsewhere.

So, that was the discovery of a weird physiological phenomenon. It shows you that in this very isolated environment, animals can evolve very strange things, very different physiology than animals elsewhere. After we found that the pancreas is the only place where Antarctic notothenioids make the AFGPs, we looked at all the other species that are known to make their type of antifreeze, and we also found that their pancreas also makes their antifreeze in addition to the liver. It makes sense for all of those other species. For the Antarctic notothenioids, that question of how antifreeze can get back to the circulation and build up to such high concentrations is still an open question.

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Very interesting. I have another question about your research, in light of the Antarctic being a really rapidly changing region. Has your work informed how these cold specialized fish can adapt to climate change?

That's a very important question, and a lot of people have been thinking about it. We don’t have direct answers to this question. If given enough time, any organism can gradually adapt to that changing environment and survive. The worry here is that climate change might be very rapid in the foreseeable future, and if the change is too drastic, then the animals will not have the plasticity to deal with it, and then they will just die. Many polar fish biologists have tried to tackle this question in different ways.

The first method was to look at how high a temperature the fish can tolerate without dying. Art actually did this very first study back in the 1960s, keeping the fish at warmer temperatures than the minus 1.9°C from McMurdo Sound. They found that the fish can only survive up to 6°C, and then they die off. That thermal tolerance is low and is fixed. If the climate change is very rapid, then you're looking at extinction.

The other, indirect, way of looking at the heat tolerance is to acutely warm them and see if they can mount a heat shock response. A heat shock response is essentially universal across all species – all life, actually – from bacteria to humans. The mechanism involves heat shock proteins, which are part of a whole family of proteins called molecular chaperones. One of their main functions is to stop denaturation of proteins. If the cell is under stress, whether it's heat stress or metal toxicity, the proteins in it will be under stress. When the proteins are stressed, then they will start to unwind. We call this denaturation. The proteins need a very unique structure for them to be functionally active. The chaperones themselves are very resistant to these stresses, so they recognize unfolding proteins and kind of hang on to them so that they do not completely go to hell, essentially. When that stress period passes, then there's a chance for the protein to refold to a functional shape. So, with these acute warming experiments, we’re trying to figure out what is the top end of their ability, the maximum temperature they can tolerate. This was first studied by another polar biologist, Gretchen Hofmann. They found that the Antarctic notothenioid fish actually could not mount this heat shock response. In other words, the heat shock proteins do not become what we call upregulated, meaning the proteins are not induced by heat to become made in a large quantity. This tells us that, again, if the ocean temperature goes up very rapidly, these Antarctic notothenioid fishes will not have the mechanism to deal with it. They will die.

Having said that, over evolutionary time – well, even now – you have a gradient of thermal niches going from the very cold, high latitude McMurdo Sound all the way up to the Scotia Arc Islands, which are much milder. When you test fish from McMurdo Sound versus fish that are in the Scotia Arc, or even the West Antarctic Peninsula, you see a gradual increase in the top thermal tolerance temperature. In addition to that, there are a number of species that have somehow escaped the polar front and over evolutionary time have established themselves in the southern coast of South America, some sub-Antarctic islands, New Zealand, et cetera. These are places where you never see any freezing temperatures. So, the fish obviously have been able to re-adapt to non-freezing environments even though their ancestry is so cold-specialized.

This drives home the point: if you give them enough time, they should be able to make it. One way to understand how they make it is to look at whole genomes of what we call secondarily temperate notothenioids and see what has changed that could allow them to readapt to a temperate environment, temperatures that will kill their Antarctic kins. We haven't been able to pull out good answers yet, but that's sort of the approach that I and other people are using. Hopefully we will have some answers later on, but right now it looks like they are probably quite vulnerable to rapid climate change.

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Thank you for that explanation. The different timescales are interesting to me, as a geologist. Shifting focus to a broader scope, can you talk a little bit about your role on the Polar Research Board (PRB) and how you're working to promote impactful polar research?

So, I'm really new to the PRB! I've been there only for a year or so, and I’m still trying to figure out the roles of the PRB members. My very first task in the first year was to brainstorm with a few other board members on a question that had been discussed in a meeting by the whole Board. That question pertains to impacts of climate change on coastal ecosystems. How are trends like melting glacier freshening of the coastal water going to impact the fauna? Is there a domino effect of that and changing water chemistry? How do we document these things?

This is especially important, I think, in the Arctic coastal regions. It is impacting the subsistence livelihood of the Indigenous people. There's a lot of emphasis on trying to understand what these impacts may be. And so, we brainstormed and came up with questions. Then we identified specialists and experts to come and give a presentation during an open session of a PRB meeting. It was a very good meeting. I think there were a lot of different perspectives represented, and experts shared data they had already generated as well as their prediction of what the tipping point may be. The idea was to have a focused discussion of a particular polar research area, and this was a very informative discussion.

...this is something that I am proud to have done for the Antarctic community.

One thing that I did prior to my current tenure in the PRB was serving as a committee member on the 2015 A Strategic Vision for NSF Investments in Antarctic and Southern Ocean Research. This study was initiated by the NSF Office of Polar Programs, who asked the Polar Research Board to conduct a study with this focus. We proposed three priority areas, one of them being polar genomics. At that time, the technology for whole genome sequencing was on the way up. It is still quite expensive, but it's a lot cheaper now than 30 years ago, when the human genome was being sequenced. We saw sequencing as a method for answering questions about the impacts of climate change on organisms across the board presently and in the past. Like I discussed earlier, we’re interested in organisms’ abilities to handle climate change. I did a lot of work, a lot of writing, and I got the committee to agree that genomics is an important initiative. Since then, NSF has funded quite a few investigators who use genome sequencing and genomic tools to interrogate biology. While not related to my current membership in PRB, this is something that I am proud to have done for the Antarctic community.

That is an impressive contribution to the community. For my last question, what advice would you give to early career scientists who want to become involved in Antarctic research?

Well, traditionally, people with an interest in polar sciences get connected with an existing active group. But unfortunately, at least here at the University of Illinois, polar biology or polar sciences do not have a huge presence. I guess at places like Ohio State with huge polar groups, opportunities to be connected with a lab are probably more plentiful. So, the biggest challenge is to seek out investigators who do work that you’re interested in. I think some sort of polar-focused REU (Research Experience for Undergraduates) program would be an effective way to engage students early on.

This is not yet an option for undergraduates, but I think it would be useful if OPP organized polar REU programs. Scientists from various disciplines could come together and host a summer research opportunity involving different aspects of polar sciences. That could bring in more interested young people, and then they can go on to conduct work in labs that do polar sciences later on in their own career. In terms of existing programs, I think OPP has done a pretty good job in holding office hours and reinstituting the NSF OPP Postdoctoral Research Fellowship Program for polar sciences.

It's important for interested students to join labs that will help them learn the ropes. New people might have ideas, but they don't know how to navigate the logistics, how to go out and cut ice holes, for example. It really is a bit of an apprenticeship, being connected to a group and learning those skills. Then you can then write your own proposal. My current postdoc, Julia York, never went to the Antarctic with me. She did her PhD in another lab and her advisor never went to the Antarctic either. But he wrote this grant to look at membrane channels that are responsible for sensing many different kinds of stimuli, including temperature. The project included work on Antarctic fish, but did not include a field season. What Julia did in order to study notothenioid fish was connect with the people at the British Antarctic Survey. They have one species in captivity, so she went there, collected samples, and did molecular work to understand and to characterize those membrane channel isoforms. So, you can start doing Antarctic science in a lab that doesn’t necessarily conduct Antarctic fieldwork.

So, early scientists: look broadly, go to the SCAR meetings, meet people, see what opportunities are available, and start from there.

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Thank you for the advice, and for your involvement in the Antarctic research community!