Ocean ultra-ecology

Researchers track the life-cycles of viruses - including human disease organisms - in the sea.
by Eric Mankin
Jed A. Furhman and graduate student Rachel T. Noble collect seawater samples from Santa Monica Pier.

Photo by Irene Fertik
Fishermen have an old saying: "There are bigger things in the sea than ever come out of it." Biological sciences professor Jed A. Fuhrman has found that there are smaller ones, too.

These days, Fuhrman is focusing on a matter of particular local interest - potentially disease-causing organisms in the waters off Southern California beaches.

Fuhrman, a pioneer in the ecology of ocean-borne viruses, and graduate student Rachel T. Noble are conducting a study that will provide the best information yet on controversial questions about the safety of swimming in Santa Monica Bay - the first results of which Noble reported in January.

Ten years ago, little was known about viruses in the oceans. If scientists thought about them at all, it was to dismiss the idea that they were important. But in the late 1980s, while studying marine micro-organisms, Fuhrman grew intrigued by two scientific riddles.

The first was the ocean's persistent failure to achieve ecological balance. Seawater contains a very high concentration of bacteria per liter - a number that remains surprisingly constant. "Yet bacteria reproduce so rapidly that the ocean should very quickly become a solid mass - nothing but bacteria," Fuhrman said. Why had this not happened? Fuhrman speculated that "something was keeping the bacteria in check." The obvious candidate for that "something" was a creature called protista - a one-celled organism that preys on bacteria.

But no matter how carefully Fuhrman counted his bacteria and protista and charted their growth and feeding rates, a discrepancy remained. Protista just weren't numerous or voracious enough to account for the number of bacteria that were disappearing. "Sometimes the gap was bigger," Fuhrman said, "and sometimes it was smaller, but it was always there."

The other riddle was a visual one. Fuhrman studied the bacteria using a special high-resolution technique called epi-fluorescent microscopy, in which bacteria and other living material appear as luminous dots on a dark stage. Looking closely, he noticed that the whole microscopic field swam in a background that was "a haze of fine dots."

Could this haze be clouds of virus particles? And, since viruses were well known to infect and kill bacteria, could viral infection be the explanation for the bacteria gap?

Conventional wisdom of the time argued against the theory. In the open ocean, microorganisms float like isolated planets, surrounded by vast stretches of empty space. Viruses are highly specific, designed to attack only certain types of bacteria. Since viruses are also fragile - little more than tiny, fragile containers holding a coil of DNA or RNA - it seemed likely that they would be killed by sunlight or other decay processes before they could chance upon a suitable host.

The theory soon got a good experimental test. Fuhrman - then at the State University of New York at Stony Brook - teamed up with graduate student Lita Proctor, an expert in the laboratory techniques needed to study viruses. Fuhrman gave Proctor a simple-sounding problem: find out what percentage of the bacteria in a given sample of seawater was infected with a virus.

In a complex, six-step process, Fuhrman and Proctor accomplished this. The task involved taking 40-liter samples of seawater, concentrating down the portion that contained bacterial assemblages, filtering this to separate out the bacteria, fixing these bacteria in a block of plastic, making thin slices of this plasticized sample and then examining the slices under the electron microscope. The goal was to search for bacteria in the only phase of viral infection in which the infection is visible - the moment when an infected bacterium breaks open.

Proctor and Fuhrman found in their samples that, at any given time, about 2 percent of the bacteria were in this phase - a figure that translated into an overall infection rate of about 50 percent. It was enough to explain the bacteria gap.

The resulting Jan. 1990 paper in Nature, coupled with another by a group of Norwegian scientists, established for the first time that viruses are an important part of the ocean ecosystem.

Since then, Fuhrman, now working with Noble at USC, has refined his technique - using isotopic measurements done in an 80-liter bacteria/virus aquarium on the roof of the Alan Hancock Foundation Building. With this equipment, the scientists could follow the fate of labeled DNA, selectively find out how many bacteria were being devoured by protista and calculate how many were succumbing to viral infection.

Using the virus aquarium, the researchers were even able to produce an estimate of the turnover time of viruses in coastal waters. They found that after 18 hours, about one third of an initial batch remained.

This date confirms the earlier work. Although some scholars remain skeptical, Fuhrman said the idea that viruses play a key role in the upper-ocean environment is gaining wider and wider acceptance.

The research has far-reaching consequences in understanding the ocean's ecological bookkeeping. The bacteria population that is destroyed by viral infection forms a separate loop that doesn't go into the main food chain - "which means you'd get fewer larger animals than otherwise," he said, "fewer big fish."

Dead or alive, viruses and bacteria float - unlike large organisms and their waste products, which sink into the deep ocean and become unavailable for recycling. Thus, a highly active viral realm also means that the upper ocean has a larger-than-expected capacity to consume and recycle carbon - a factor in the greenhouse equation.

The virology expertise that Fuhrman and Noble have acquired is also being turned to a more immediate concern. It is known that human viruses can spread by water, and for years there has been speculation that swimmers, surfers and others exposed to the Santa Monica Bay may be at risk of infection by human viruses washed into the sea from storm drains and other sources.

But because of the difficulty in studying viruses, little is known about how much, if any, human virus gets into the ocean, and how long it can survive.

Working with funding from the Santa Monica Bay Restoration Program and the U.S. government Sea Grant Program, Fuhrman, Noble and graduate student John Griffith are engaged in intensive sampling of area waters in an attempt to answer such questions.

They are using a relatively new approach to testing for viruses - one based on looking for the organisms neither through an electron microscope nor through the traditional method of infecting mammalian cells grown in tissue culture.

Instead, Noble takes water samples and filters them to remove larger microorganisms such as bacteria and algae, concentrating the whole sample down to a tiny amount - a single 1/10 milliliter droplet. She then extracts all the hereditary material in what's left and uses molecular biological techniques to test for the presence of the RNA characteristic of the enteroviruses she is looking for. A positive result indicates the presence of virus quickly, cheaply and specifically.

In the large-scale study, which will analyze samples from storm drains as well as samples taken offshore, the scientists are testing for a wide range of human viruses, including ones that cause hepatitis. For the preliminary assays, Noble tested for the presence of the polio-virus strain used in Sabin vaccinations. Though it is harmless, it is extremely widespread - most humans carry it. Its presence in ocean water thus serves as an indicator that other human enteric viruses are likely to be present as well.

Noble reported her results from assays of 20 samples at a February meeting of the Association of Limnology and Oceanography in San Diego.

She found virus in 11 of 20 samples analyzed. A clear (and not unexpected) pattern emerged. The samples taken after rainstorms - when storm drains are full and flowing into the ocean - were much more likely to contain virus. Nine of 11 samples taken after storms were positive for virus; only two of nine samples taken during dry periods were.

Public health authorities traditionally count non-disease-causing bacteria found in human intestines ("coliform" bacteria) as an indicator that human waste is finding its way into the ocean. But at least in the limited series of tests that the USC researchers reported in January, "there was no obvious correlation between the presence of these enteric viruses and coliform levels," Noble said,

Fuhrman and Noble emphasize that much more work remains to be done to answer questions about the dangers posed by human pathogenic viruses in the "urban ocean" off Southern California. "We expect to learn much more as we analyze the large number of samples we've taken," Fuhrman said. He hopes a full report from a current research project will be ready later this year - before the summer swimming season.