CAN A FUNGUS HAVE SEX? RESEARCHERS SAY YES\Reproduction enables Valley Fever spores to create genetic variations.

Byline: Carol Kaesuk Yoon The New York Times

How can you tell if a fungus is having sex? Until recently, not very easily.

Now a team of researchers, borrowing techniques from diverse fields to study the fungus that each year gives thousands of people Valley Fever, say they have found some straightforward ways to answer the question.

They have discovered that the fungus, thought for more than a century to be chastely asexual, is in fact having sex - and quite a bit of it.

Knowing how a species reproduces can alter everything from how a researcher would go about developing a vaccine against it to what sorts of methods can be used to answer even the most basic questions about its biology.

Despite the importance of knowing how both animal and plant pathogens reproduce, many harmful fungi and protozoans, like the Valley Fever fungus, have simply been assumed to be asexual because scientists have never observed them reproducing sexually.

Scientists say the techniques used to figure out indirectly the cryptic sex of Coccidioides immitis, the Valley Fever fungus, could be used to determine which of the many poorly known microbial species assumed to be asexual really are. The researchers reported their results in the Jan. 23 issue of The Proceedings of the National Academy of Sciences.

"This is a really exciting paper," said Dr. James B. Anderson, a fungal specialist at the University of Toronto. "This is the most insightful study on the problem to date."

Dr. Daniel Dykhuizen, a population biologist at the State University of New York at Stony Brook, said that for the great majority of biologists, who work with large, easily observed organisms like birds or fish, the question of whether there is a sexual stage is not really an important issue. "But, as we start to work more on the rest," he said, "the other 99 percent of species - protozoa, fungi and the whole bacterial kingdom - then these become very interesting questions."

The fungus that causes Valley Fever is well-known in the arid Southwest, where it is estimated to infect 50,000 to 100,000 people a year.

People become infected by inhaling spores of the fungus, which can be found living in the soil or blowing in the air. Once in the lungs, the fungus tries to grow and spread throughout the body. Most people, however, suffer minor or no symptoms as their immune systems wall off the fungus in the lungs.

But Dr. John Galgiani, an infectious disease specialist at the Veterans Affairs Medical Center in Tucson, Ariz., said that perhaps a third of those infected suffer Valley Fever along with its fatigue, chest pain and cough for weeks or months, and 50 to 100 people die of the disease each year.

To pin down the reproductive habits of this fungus, five researchers from the University of California at Berkeley and from Roche Molecular Systems at nearby Alameda took advantage of the fact that when organisms have sex, there is evidence, beyond offspring, left behind.

When organisms reproduce sexually, one individual mixes its genes with the genes of another to produce a new, genetically unique offspring. If organisms are instead reproducing asexually - that is, by cloning - they simply reproduce more and more identical copies of themselves, with no mixing of genes. By looking for evidence of mixing, researchers were able to test indirectly whether the fungi were having sex.

In a laboratory designed to contain dangerous pathogens, researchers at Roche Molecular Systems grew cultures of the highly infectious fungus taken from 25 patients.

Then at the University of California, Dr. Austin Burt used the powerful techniques of molecular biologists, and his team added a few innovative twists. Using techniques that allowed them to examine DNA sequences very rapidly, they examined 14 different gene regions in each of the 25 fungal cultures.

If the fungus reproduced clonally, then researchers expected to see only a few of the many possible combinations of the 14 genes, with a few genetic clones showing up again and again in the many different patients, unperturbed by the gene-mixing effects of sex. But what researchers found instead was that each fungus cultured from a different person showed a unique mix of genes. That is, the genes showed up in 25 different combinations, a result researchers say could only be explained by sexual reproduction.

Because the researchers were looking at indirect evidence, they sought to analyze their data by a variety of methods to ensure that they were interpreting the patterns correctly. The researchers even made family trees of the cultures of fungi, borrowing methods from biologists who build evolutionary trees of things like species of snakes or moths. Everything the researchers looked at, even the patterns of the fungal family trees, indicated that the fungi were having sex.

"They used three or four different analyses, and they all pointed to the same thing," said Dr. Michael Milgroom, a plant pathologist at Cornell University. "It was elegantly done."

Dr. John Taylor, a fungal expert at the University of California at Berkeley in whose laboratory much of the work was done, said: "We've always assumed that fungi we couldn't observe (having) sex in the lab . . . were asexual, but you may have always worried a little that you just missed sexual reproduction and that's always been a possibility. This kind of thing may turn out to be quite common."

Researchers say that while they are confident the Valley Fever fungus is reproducing sexually, they have yet to discover exactly how this particular species is doing it.

Fungi can reproduce by normal sexual reproduction in which cells from two different individuals fuse and their nuclei, which contain the chromosomes, fuse as well. The fused cell then goes through an organized process of dividing up the chromosomes from the two parental fungi to produce cells with the original number of chromosomes.

Fungi can also reproduce by another method known as parasexuality, in which two cells fuse but their nuclei may not fuse for some time. When they do fuse, there is a haphazard discarding of chromosomes until the original number is reached.

The bad news is that Coccidioides immitis, able to reproduce sexually and create new arrays of genetic types with each bout of reproduction, is probably evolving more quickly than researchers had suspected, making it a wilier and more dangerous opponent. Taylor explained that during epidemics, like the one from 1991 to 1994 in Bakersfield, when the infection rate was 10 times the norm, researchers typically assumed that the culprit was one particularly virulent strain with asexual pathogens. But once researchers know the fungi can have sex, the picture becomes infinitely more complex and difficult.

"We didn't find a single case where two different people had the same fungus," Taylor said. "If you want to try to develop a vaccine, that's a problem."

But Burt, a population geneticist, now working at Imperial College on the outskirts of London, explained that there is good news, too. Knowing that these fungi can indeed have sex, researchers can begin to try in earnest to induce sexual reproduction in the laboratory. If researchers succeed, they can cross strains as they like, allowing faster, more sophisticated genetic analyses.

Fungal diseases like Valley Fever are most dangerous to those with compromised immune systems, like AIDS patients, whose bodies cannot fight off the fungus. Researchers say that with the numbers of such people on the rise, previously arcane pursuits like studying the sex lives of fungi have begun to get more attention.

Galgiani said: "The tools that have been available for a while for studying bacteria and viruses are just now being applied to fungi, and we're starting to see a lot of things happening. There is a huge revolution going on."