Uncovering a comic book
critter's biochemistry
By Sarah Osborne

OTTAWA — "An amazing miracle! Create instant life."

Preserved in "instant-life crystals," sea monkeys were the mainstay of most comic book's back cover during the 1960s. Flesh-coloured underwater critters, stick-legged and round-bellied, lounged around the aquarium floor, their pink plastic castle in the background.

Children who bought sea monkey kits were told they had "stepped across the threshold of one of the strange worlds of tomorrow's science... TODAY!"

Tomorrow's "strange world" of sea monkeys – small crustaceans called brine shrimp Artemia, and not three-horned leisure enthusiasts – are actually the product of millions of years of evolution. "Tomorrow's science" has yet to unlock these tiny, feather-finned animals' secret of self-preservation.

The sea monkey's ability to create "instant life crystals" is not just an interesting quirk of nature. Biochemical and genetic research into the brine shrimp's method of self-preservation and the proteins involved may help preserve other animals in the future. This research will not be done, however, if apathy and funding concerns take precedent.

Our knowledge of DNA and genetics is largely due to the sacrifice of millions of fruit flies and C. elegens worms, while mice, rats and rabbits and dogs have helped unlock the mysteries of cancer and diabetes. Yet brine shrimp are still viewed as that quirky little character from the back of comic books. Artemia are nourishing little creatures, used by the bucket in aquaculture, but they are hardly typical laboratory subjects.

Monkey sea, monkey do

Thomas MacRae, a researcher at Dalhousie University in Halifax, has studied brine shrimp Artemia at the cellular level for nearly 30 years. MacRae says he knows brine shrimp will never be the loyal laboratory subject, they still hold secrets worth revealing.

"I don't think shrimp will ever replace Drosophila (fruit flies), mice, rats, or C. elegens, because those systems are now so well established for a lot of studies. " says MacRae.

Brine shrimp are native to some of the harshest ecosystems in the world: inland salt lakes and salt evaporation pools. They thrive in salt concentrations other marine animals could not endure; tolerate low oxygen levels; and survive weather extremes.

Conditions can even become too harsh even for these small crayfish-like creatures, which measure no more than 1 centimetre long. If their lake is too salty, oxygen levels too low, or their food source, algae, too scarce, brine shrimp will slowly die off. When this happens, mother Artemia switch over to a different type of pregnancy. Instead of delivering baby shrimp into an environment that will kill them, mothers protect future shrimp by pausing the gestation process when they reach about 4,000 cells. Baby shrimp develop not as larvae, but as cysts. Imagine if a mammal could choose to lay a dehydrated egg halfway through pregnancy instead of giving birth to a baby.

The cysts are hard, round little capsules that are nearly indestructible. Once they are expelled from the mother Artemia, they may sink to the bottom of the lake, or wash up on shore. The cysts are in (what MacRae describes as) suspended animation – or diapause, as biologists say.

Ken Storey, a Carleton University biochemist, researches animals' different methods of diapause. His particular field of research is squirrel hibernation. While Storey reflects the science community's apathy for brine shrimp ("No one cares about brine shrimp," he says), he reluctantly acknowledges brine shrimp's skill at creating cysts, instead of larvae.

"Brine shrimp are nature's champion's in that they can encyst, but they are not unique to that," Storey says. He points to bacteria 250 million years old encased in salt, which have been revived by biologists in the lab, and insects in Africa which enter diapause every year. Nevertheless, both MacRae and Storey say brine shrimp are perhaps the best animal in the world at creating these cysts.

Dehydrated and containing only two to five per cent water, the cysts can survive temperatures of -273 degrees Celsius or Absolute Zero. This is in contrast to the human body, which is about 72 per cent water. If it loses more than 10 per cent of its water, cells start to die. Hydrated, but without any metabolic activity, cysts can survive temperatures of –18 degrees Celsius to 40 degrees Celsius. Kept safe from oxygen, such as in anaerobic mud at the bottom of a lake, or vacuum-packed, they can survive for years.

Shock to the system

Thomas MacRae studies brine shrimp at the cellular and molecular level.

MacRae conducts his research on the cellular mechanics of the brine shrimp's diapause. Two aspects are particularly interesting: how the cyst can endure such severe dehydration yet rehydrate unscathed, and how the mother brine shrimp actually switches from producing larvae, to producing cysts.

MacRae says his research lab stumbled, almost by accident, over a small heat shock protein that appears to help the cyst endure dehydration and rehydration.

Heat shock proteins, discovered in fruit flies in the late 1970s, are also found in every animal cell including those of humans, except for red blood cells, which have no nucleus. These proteins are abundant in the heart muscle, and prevent cataracts in the eye lens.

Perhaps most importantly, they also act as 'molecular chaperones,' and are especially critical in protecting the Artemia cyst from becoming damaged.

Cells constantly produce proteins to keep themselves in working order. In order to fulfill their task, proteins, which are complex chemical structures, must be folded correctly. When a cell is stressed by heat, dehydration, or lack of oxygen, the proteins start to unfold. The small heat shock proteins, such as p26 in Artemia, prevent these everyday proteins from unfolding irreversibly, and helps refold partially unfolded proteins.

Imagine a slinky toy that has been stretched so much it is started to get kinks and lose its spring. A heat shock protein would be able to see the damage start, move in to prevent the slinky from uncoiling more, and even fix any damage. This is the chaperoning aspect of small heat shock proteins, which keep Artemia's proteins safe, even when it is dehydrated in cyst form.

"It's the term that we all use, but its probably better to call these things molecular chaperones, because heat shock proteins can be induced without heat, and some are produced just normally. Heat shock protein was just a name that stuck and it came about because of how the proteins were discovered," says MacRae

MacRae helped scientists at the University of California Davis, who had the idea of using small heat shock proteins – in this case the sugar trehalose – to freeze-dry and rehydrate viable blood platelets. The average life of platelet cells is five days at no less than 20 degrees Celsius. By inserting trehalose into the platelet cells, they can be dehydrated and rehydrated successfully. Platelets dried with trehalose have a shelf life of up to three months.

Treating cells with small heat shock proteins is one thing, but trying to get mammalian cells to express Artemia's proteins such as p26 is another challenge, one that MacRae says his lab is tackling. If successful, the knowledge could be used to freeze-dry more complex cells in a way that preserves the cell's nucleus for cataloguing or future study.

Mixed signals

While p26 protects brine shrimp cysts while they are dehydrated, MacRae says the actual mechanism of diapause is still only partly understood. Scientists do not know how much of the diapause the cysts take on themselves, and how much is caused by the mother.

"She's responding to some signal from the environment, and producing a hormone which has to effect these embryos. It could be that these decisions are made very early, maybe even before the eggs are fully developed and ready for fertilization," says MacRae.

That is not the only mystery of the brine shrimp's diapause. The cysts have to dehydrate in order to be 'reactivated' from their non-metabolic state, for instance, but nobody knows exactly why. There is speculation it has to do, yet again, with small heat shock proteins.

MacRae says understanding the mechanics of diapause may have an impact on understanding aging, but that aquaculture companies are interested as well. Artemia are already bred for higher yields and longer storage, but they can be farmed even more efficiently once their diapause is fully understood.

Storey says the main impediment to brine shrimp research is that Artemia are not useful enough to entice researchers to bother sequencing the genome, which can cost millions of dollars.

"Brine shrimp are not going to help us make freeze-dried soldiers," he says. "Brine shrimp have a very complicated genome that nobody cares about."

Yet having a complete genome can make researching an organism's biochemistry markedly easier. The technology to sequence genomes is still so expensive and new, that only organisms of special interest to researchers are sequenced – such as C. elegens, mice and rats, the chicken, the potato, and about 14 different types of fruit fly. Storey says when scientists eventually get around to sequencing a crustacean, it will probably be a more marketable one, such as the lobster.

MacRae says he and some other North American colleagues have planned a trip to Beijing in April, where they want to convince Chinese scientists to take on the job of sequencing the brine shrimp genome.

Human connection?

The lack of interest in Artemia biochemistry is also reflected in the difficulties MacRae faces trying to get funding. Although MacRae's work is funded by the Canadian Institute of Health Research, the Nova Scotia Health Research Foundation, and the Heart and Stroke Foundation, it is hard sometimes to show how his research applies directly to human health.

"We're interested in the biology of the organism because its an interesting organism, it's an unusual organism to look at, but we're also trying to apply it to a medical situation or aquaculture... And of course there are many reasons to do that. One, you feel more useful, but two, it's much easier to get money," says MacRae.

"We're studying brine shrimp, which is a semi-obscure organism, and people will ask me, 'well why would you want to be studying that when you could be studying cancer?' My answer is generally, 'well I am studying cancer, because I'm studying cellular biology and chemical biology.' "

Storey says that as long as conducting research into genetics remains expensive, organizations are going to want to see a direct human application to the research they fund.

"I'm not saying it's right, I'm just saying that's the way it is. And right now, the funding gods are smiling on [this] way of seeing things," says Storey.

Ultimately, the proteins MacRae studies are present in humans, and often perform similar functions, acting as molecular chaperones, and preparing the cell membrane to grow with the cell. MacRae says this makes brine shrimp ideal to serve as a "system model," a sort of square one, in the particular functions it excels at, which can then illuminate how the human equivalent works. This is also his approach when requesting funding.

"It's a bit self-serving, but at the same time we are encouraged that whatever our obsessions are, to try and apply them to something useful where everybody else gains, which I think is fair," says MacRae.

"When you work with brine shrimp, it's not perceived so directly as being medical, but if I'm studying a protein which is found in your eye, and I can decide or determine things about my protein, then you can then extrapolate it to your system."

In short, by studying how a protein like p26 works in brine shrimp, scientists have helped discover how p26 may prevent cataracts in the human eye.

MacRae says his lab's work on brine shrimp is fundamental biochemistry and molecular biology, and it adds to the basic literature in those areas. He says studying the basic biology of an organism that is on the sidelines of genetic research can still be useful.

MacRae says science shouldn't always be approached for its direct human impact. "If you want to count the bristles on the legs of an insect, which at first sight doesn't seem to have much use, I'm happy for you to do that, because you don't know what's going to come out of it down the road. And that's more or less what's happening with what we're doing now," he says.