When Dr. Karen Lips trekked deep into the Panamanian rain forest last Christmas to continue her field study of tree frogs, she found a horrifying scene: dead frogs were everywhere.
"I'd go in the morning and see them sitting on the ground along the stream," said Dr. Lips, assistant professor of biology at St. Lawrence University in Canton, N.Y. "They looked perfectly alive, as if they were asleep." But their color was faded, and when Dr. Lips picked up the dead animals, they felt hard and leathery, as if they had been turned into stone by a wicked witch.
Dr. Lips picked up 50 dead frogs representing several species, froze 30 of them and put the others in formalin, a preservative, and sent them to a veterinary pathologist in Maryland for examination. What he found may offer a vital clue to a profound global mystery.
Frogs and toads have been disappearing worldwide for the last 15 years, most often from habitat destruction or from agricultural pollutants. But why they have been dying in near pristine environments in Central America and other protected highlands had remained unknown. The Panamanian frogs, it turns out, had been attacked by a protozoan that may also be killing frogs elsewhere.
Despite widespread frog deaths, this is the first time that a field biologist has come across a large number of frogs in the process of dying, said Dr. David Wake, an evolutionary biologist at the University of California in Berkeley and authority on amphibian extinctions. In the tropics, dead animals are immediately consumed by ants, bees and other creatures, and leave no trace on that spot, he said.
But now the detectives had finally found a corpse. Dr. Earl Green, the pathologist who examined the bodies, said in a telephone interview: "They appeared normal internally and externally. I could find no evidence of widespread viral, bacterial or fungal infections. But it looks like a protozoan of some sort had infiltrated their skin."
Green, who recently left the Maryland Department of Agriculture, said the identity of the protozoan remains unknown, although it resembles a pathogen that kills oysters in Chesapeake Bay. Because amphibians breathe and drink water through their skin, the skin damage caused the frogs to suffocate and dehydrate, he said. How long the process takes is not known, but since entire populations can disappear in months it is not a lingering disease.
Dr. Lips and others believe that the lethal protozoan is sweeping across Central America in a "death wave" moving through the mountains, from one range to the next. Biologists conjecture that the disease first broke out in the Monteverde Cloud Forest Reserve in northwestern Costa Rica in the late 1980s. It has since moved south and east into Panama and may have traveled north into Nicaragua. In July, a tourist found dead and dying frogs on an island in the middle of Lake Nicaragua, but none of the animals were preserved for autopsy. In a finding that only serves to deepen the mystery, Australian biologists last year reported that they believe a similar "death wave" killed amphibians in the lush mountain forests of Queensland in the 1980s and 1990s. They suspected a virus. But when Dr. Green recently shared his slides of Panamanian frog skins with an Australian pathologist, she sent him e-mail saying what he had found was exactly what she was seeing in dying Australian frogs.
Could the same disease be killing frogs and toads in Central America and Australia? If so, where did it come from?
With financing from the Brookfield Zoo in Chicago, Green and his counterparts from several countries plan to meet at Loyola University in Chicago next month to examine slides of frog tissue under a powerful electron microscope. They will look at the most recent specimens as well as older tissues preserved in museum or private collections with the hope of finding common parasites.
Field biologists on all continents first started noticing amphibian declines about 15 years ago, Wake said. They would go back to their sites and find that once abundant animals were gone or greatly diminished in number. The animals evidently died in their burrows or were carried off by carrion eaters.
But the amphibian decline that most galvanized international attention occurred in Costa Rica's Monteverde Reserve, the home of the spectacular golden toad. In 1987, the last year the population was at a normal level, biologists saw hundreds of thousands of animals gleaming like jewels in the dark green forest, Wake said. Two years later, they only found five animals. Since then, not one has been seen. In a paper to be published in December in the journal Conservation Biology, scientists report that the golden toad is almost certainly extinct; its disappearance is not due to some natural fluctuation of amphibian life cycles. Twenty other species of frogs and toads are also missing from the region.
In 1991, scientists formed the Declining Amphibian Population Taskforce to look for underlying causes. Virtually every explanation ran into spirited controversy. One explanation was that the earth had become so polluted that fragile creatures like frogs and toads whose skin is permeable to air and water were dying like canaries in coal mines, said Roderick Mast, vice president of Conservation International, a Washington nonprofit organization dedicated to preserving biodiversity. Perhaps, he said, humans were next.
Others argued that frogs and toads undergo natural population fluctuations. An 18-year study of amphibians in the Southeastern United States has found no dramatic disappearances.
Most scientists agreed that habitat destruction or degradation was the leading cause of amphibian declines in most parts of the world. While Dr. Lips's discovery provides one more explanation -- an infectious disease -- for these extinctions, it raises more questions. Why does disease strike animals in pristine highlands but not, as far as any one knows, in lowlands? Where did the disease come from? Why are so many animals suddenly dying from it?
Once the pathogen or disease causing agent is identified, researchers should have better answers. In the meantime, Green has observed several of its traits. The infection causes a mild swelling or thickening of the skin, especially in the toes and in a part of the pelvis called the "drink patch" where animals take in water, he said. "The organism seems to have stages of development," Green said. "It begins small, about two to three microns in diameter, and then it divides and creates more organisms, expanding to fill an area about 12 to 15 microns across. Then it forms a discharge tube or pore, through which 20 more tiny spores exit into the water" where they might infect other frogs and toads.
"Based on what I can see, this organism has some resemblance to perkinsus, a protozoan that is the biggest cause of death and destruction of oysters in Chesapeake Bay," Green said. "I'm not saying it is the same organism. They just look similar."
If the organism is endemic to streams in Central America, amphibians may be dying from it now because their immune systems have been weakened by pesticides or herbicides carried up from coastal farms in rain clouds, said Dr. Cynthia Carey, a biologist at the University of Colorado in Boulder. Some kind of sublethal stress may make frogs and toads more vulnerable to infection, she said.
To test this idea, Dr. Carey has been exposing amphibians to a wide variety of stresses in the laboratory to see if she can make the animals sick. So far, nothing has worked, she said. Acid-rainlike conditions seem to make their immune systems even stronger.
The rapidity, lethality and wavelike spread of the infection suggests it was introduced, Green said. But by whom?
New fish species like tilapia, stocked in streams or lakes, might bring a disease, as might humans. An ecotourist visiting the Monteverde preserve could have carried the infection on his shoes or it could even have been a scientist -- a kind of typhoid Mary among ecologists -- doing field work in one part of the world, hopping onto a plane, tramping into the Monteverde preserve and releasing an alien organism that then infected the gold toads.
It would be very much like Europeans carrying measles into the new world, killing millions of Indians who had no immunity to the disease, he said.
Whatever the cause, the results are gruesome. As a doctoral student at the University of Miami, Dr. Lips worked in Las Tablas, a protected region about six hours south and east of Monteverde. "In 1990, it was a beautiful site in the cloud forest, wet and rainy, with lots of frogs and clean headwaters," she said. "Only one family lived nearby." In 1993, there were fewer frogs "but I thought, well, maybe I bothered them," she said. The next year was dry and again there were fewer frogs but Dr. Lips was not alarmed. "When I next went down in the summer of 1996 and everything had disappeared, I knew something was going on. Of 30 species I had found there, 3 to 5 were completely missing and the others had declined by 90 percent," she said. "It was weird and depressing, especially at night when the streams were silent."
That summer, Dr. Lips moved her field research farther east and south to a site called La Fortuna, in Panama. She had visited the region in earlier years and knew frogs were still abundant there. She returned to La Fortuna last Christmas, which is when she found all the dead and dying frogs and collected them to be examined.
"I went back again this summer," Dr. Lips said. In her study area, which included nine streams, she found only six living amphibians. The rest had disappeared.