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Raising Notothenioidei

Scientists successfully develop icefish embryos for evolutionary studies

The cold-loving fish that inhabit the Southern Ocean are Antarctica’s version of Darwinian finches — an endemic group of different species that evolved special adaptations to fill empty ecological niches.

The eureka moment for Charles Darwin involved the morphology, or shape, of the birds’ beaks, which he found varied around the Galapagos Islands in response to available food sources. In the case of the Notothenioidei, the evolutionary twists and turns that occurred as the Antarctic region cooled more than 30 million years ago resulted in the selection of a number of unusual mutations that allowed various fish species to fill abandoned habitats in the ocean.

Now, researchers are delving into molecular genetics to understand how millions of years of evolutionary adaptations will fare against changes in the Antarctic climate that are predicted to take place on the scale of centuries.

“It’s a very exciting time in terms of evolutionary genetics,” said Bill Detrich, a professor of biochemistry and marine biology at Northeastern University in Boston and principal investigator for an ongoing National Science Foundation-funded project focusing on the function of Antarctic fish proteins in a cold climate and their thermal tolerances.

A veteran polar researcher, Detrich is collaborating with John Postlethwait, a professor in the Institute of Neuroscience at the University of Oregon, and Jeffrey Grim, a National Science Foundation Post Doctoral Research Fellow at Northeastern, on a series of related studies that are looking at different aspects of notothenioid embryo development in one of the fastest warming regions of the planet.

All three biologists and their research teams were at Palmer Station this year to collect specimens and conduct experiments that they hope will help them understand problems as diverse as human bone disease and anemia. Learning more about the molecular underpinnings of notothenioid fish will also tell whether they can stand the heat in the future.

The 120-plus species of Notothenioidei, most of which inhabit waters from about minus 2 degrees Celsius to 4 degrees Celsius, evolved from a common ancestor without a swim bladder, an organ other fish possess that helps them attain neutral buoyancy in the water column. However, millions of years of evolution eventually offered a way for some notothenioids to get off the seafloor. One strategy involves the development of lipids, or fats, that are less dense than water. Another way is to grow a lightweight, brittle skeleton.

“You can accumulate things that are less dense than water — or you can get rid of things that are more dense than water. These animals diminished the densest tissue in their bodies — bone,” explained Postlethwait, whose work is also supported by the National Institute of Aging, which is part of the National Institutes of Health.

What’s an evolutionary advantage for one organism is a human bone density condition called osteopenia, which is believed to be a precursor to the more serious disease known as osteoporosis.

“The problem that we want to solve is to figure out what are the [icefish] genes that have changed over time — what mutations have occurred — that distinguish these animals that have osteopenia from the strong-boned animals that stayed on the bottom,” Postlethwait said. “We need to study what happens over developmental time, as the skeleton initially develops in these animals.”

That’s proven to be the most challenging aspect of the project.

The researchers chose two related but very different Antarctic fish as the principal subjects of their study. In one corner is the Antarctic yellowbelly rock cod (Notothenia coriiceps), which has a standard skeleton and idles away its time on the seafloor. Its opposite is the blackfin icefish (Chaenocephalus aceratus) with its reduced skeletal structure that allows it to enjoy a more pelagic existence.

The former is a red-blooded animal while the latter belongs to icefish family, whose 16 species have no hemoglobin in their blood, a disadvantage just about anywhere else but in the oxygen-rich waters of the Southern Ocean. But it’s the reproductive behavior of the white-blooded icefish that proved to be a challenge to the scientists, who need embryos for their experiments.

In 2008, they successfully mated N. coriiceps, which is a broadcaster spawner, meaning the female releases her eggs and the male his sperm in the water column, where fertilization takes place. In contrast, females of C. aceratus lay a nest of eggs, which the male fertilizes and guards.

Attempts to develop embryos from the white-blooded icefish failed in 2008 and again in 2010. This was a make-or-break season, according to Detrich.

“This year it was really critical for us to do that,” he said. “This year we were very successful. We got not one but two different [white-blooded] icefish species.” In addition to the blackfin icefish, the team was also able to perform in vitro fertilization on Champsocephalus gunnari, known as the mackerel icefish.

“The breeding of the icefish that we accomplished this season is the first time that [white-blooded] icefish have been grown,” Detrich said. “That was a significant accomplishment.”

In all three field seasons, the researchers collected tissues samples from icefish caught while trawling aboard the research vessel Laurence M. Gould. Postlethwait’s lab has been able to isolate messenger RNA, which carries information needed to direct cellular activity. This regulation of activity is called gene expression. His team also instituted a program of genome sequencing for both target species.

They then developed software to analyze and compare the genetic expression between the two species.

“That’s pretty exciting,” Postlethwait said. “We started basically at ground zero and we had to develop the biology and genomic infrastructure and bioinformatics from scratch. It’s pretty exciting that we’re at this stage. … We’re going to be able to make the comparison and learn what genes are expressed differently.”

Two researchers wintered over at Palmer Station this year to continue to monitor the development of fish embryos, according to Grim.

“We’re hopeful with their efforts that we can get them to more advanced developmental stages,” he said.

Grim’s project, while separate from the collaboration between Detrich and Postlethwait, also focuses on gene expression associated with bone and cartilage production, but his research involves studying reactive oxygen species (ROS) in embryonic development.

ROS are chemically reactive molecules that form as a natural byproduct in the normal metabolism of oxygen. They play an important role in cell signaling, as part of the cellular communication process that governs cell activity. Environmental stress can cause ROS levels to spike, leading to cellular damage.

Grim is interested in how ROS production may vary between red- and white-blooded Antarctic fish, as well as how their antioxidant defenses to oxidative stress varies. Grim said there might also be a correlation between ROS activity and skeletal growth.

One of the key questions in all three projects: Will warming in the Antarctic Peninsula affect embryo development? The Southern Ocean has already warmed an average 1 degree Celsius in the last half-century.

“All of the developmental rates and all of the biological processes in these animals are going to be dictated by their environmental temperatures,” Grim said.

Adult notothenioid fish have proven to be more tolerant to spikes in thermal temperature than researchers originally believed, Detrich noted. The same seems to hold true for the embryos so far, at least in experiments that turned up the water temperature by as much as 4 degrees Celsius.

“They’re more tolerant than one might have thought,” said Detrich, whose own research also involves protein folding in Antarctic fish in addition to the thermal tolerance experiments.

Proteins are behind many biological processes. As enzymes, they drive biochemical reactions that make biology work. They are the main constituent of muscles, hair, skin and blood vessels. As antibodies, they recognize intruders and prompt the immune system to get rid of the unwanted invaders.

In order to carry out its specific function, each protein must take on a unique three-dimensional shape, in a process called folding. Detrich is particularly interested in how a complex called CCT chaperonin assists other proteins in the folding process, especially their role in the folding of tubulins that form microtubules. Microtubules are one of the components of the cytoskeleton, a structure that maintains cell shape and cell motility.

Previous work has found that Antarctic fish CCT works very efficiently at the temperature at which the animal evolved, but suffers as it increases.

“That’s very important information,” Detrich said. “It tells us that at some point, as temperature increases, we might expect to see a deficit in protein folding for Antarctic fish. That’s an interesting result that may prove harmful to notothenioid embryos and adults.”

Changes in ocean temperature could have other effects, Grim noted, such as altering the fish growth cycle.

“If the embryos even survive an increase in developmental rate, are they going to hatch out at a time when there’s not food available and they’re going to die once they hatch out?” he asked.

Postlethwait has a different reason to be concerned about climate change affecting the Southern Ocean. His research into the Antarctic fish-human bone disease relationship may eventually lead to therapies to prevent or treat osteopenia.

“I can see a trail that leads there, as we identify genes and gene pathways that have been altered in the light-boned icefish by evolution,” he explained. “If we find in a human being those same genes and gene pathways that have been altered over developmental time and differences between families that are resistant to or susceptible to osteoporosis, then the identification of those genes might suggest targets for substances that might up-regulate those genes to maintain bone mineralization as we get older.”

So a threat to the Antarctic fish from climate change and other human pressures is also a loss to science, Postlethwait noted.

“These animals are unique planetary resources to learn about things like osteoporosis. … As the water starts to warm, all of these animals are going to undergo stresses that are going to make a lot of them go extinct,” he said. “Those animals are unique, and once they’re gone, they’re gone forever. If we’re going to extract secrets from them, we need to do it before they’re gone.”

NSF-funded research in this story: Bill Detrich, Northeastern University, Award No. 0944517; Jeffrey Grim, Northeastern University, Award No. 1019305. NIH-funded research: John Postlethwait, University of Oregon, Project No. 5R01AG031922-04.

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Curator: Peter Rejcek, Antarctic Support Contract | NSF Official: Winifred Reuning, Division of Polar Programs