July 9, 2002 Deformities in Pennsylvania wood frogs are linked to the combination of their infection by parasites and a weakening of their immune system caused by exposure to pesticides, according to a study by Penn State researchers to be published in the 9 July issue of the Proceedings of the National Academy of Sciences.
The research includes the first experimental studies of amphibian deformities conducted in ponds where the animals live. The discoveries, which show the effect of environmental stress on disease outbreaks, may help to explain how disease affects the distribution, growth, development, and survival of frogs, which have been disappearing during recent years at alarming rates worldwide.
"It is not uncommon now for 20 to 30 percent of the frogs at many locations to have limb deformities," says Joseph Kiesecker, an assistant professor of biology at Penn State and the leader of the research team. Since the early 1990s, when school children and amateur naturalists first began finding frogs with deformed legs in U. S. wetlands, scientists have been trying to determine the reason for the problem's escalating occurrence. These deformities in frogs have a chilling resemblance to the deformities in human caused decades ago by the drug Thalidomide. "Both the general public and scientists suspect that whatever is causing these problems in frogs may also cause harm to humans," Kiesecker says.
A decade of scientific research has resulted in two prominent hypotheses about the causes of frog deformities: one is that they are caused by human-induced contamination of the frogs' environment with chemicals like pesticides, and the other is that they are caused by a common naturally-occurring parasite, the trematode worm. Scientists had found evidence to support each hypothesis, but the research results were not conclusive enough to resolve the controversy. "More rigorously designed experiments were required to determine which factor is more important and how these two factors work together," Kiesecker explains.
Kiesecker's breakthrough was in designing a study that combined field experiments at the ponds where the frogs live with experiments in the laboratory. "The kind of field experiments that we did in this study have never been done before," Kiesecker says. The Kiesecker team collected tadpoles from ponds in Centre County, Pennsylvania, then used some of those tadpoles in a series of laboratory experiments and some in a series of field experiments conducted in six ponds within the same region. Half of the ponds receive runoff from agricultural fields and contain detectable levels of pesticides, and half are free of pesticides and agricultural runoff.
The Kiesecker team designed the experiments to test four key hypotheses regarding the relationship between pesticides, trematode parasites, and limb deformities in frogs. The first hypothesis was that limb deformities occur in frogs that are infected with the trematode parasite. Trematode parasites inhabit a series of host species during their life cycle, including pond snails. When they leave the snail, in the form of trematode larvae called cercariae, they swim around in the pond in search of a tadpole, which is the next host they need to invade in order to survive. The researchers placed groups of their tadpoles in the six ponds within two kinds of enclosures located side-by-side--one with a fine screen that prevented the trematode larvae from entering the enclosure, and the other with a larger-mesh screen that allowed the trematode larvae to infect the tadpoles.
"These same trematode larvae cause "swimmer's itch," which is a common problem among people who swim in lakes in this part of the country," Kiesecker says, explaining that the swimmer's immune system eventually kicks the larvae out, leaving just an annoying rash. "A more serious problem for people occurs in tropical climates, where trematodes cause an infection known as Schistosomiasis that kills millions of people every year."
The only tadpoles that developed limb deformities in the first experiment were from cages that were exposed to the trematode larvae, while tadpoles in cages that protected them from the larvae did not get any limb deformities. "We learned from the first field experiment that tadpoles have to be exposed to trematode infection for limb deformities to develop," Kiesecker explains.
The second hypothesis the team tested is that limb deformities in trematode-infected tadpoles are affected by pesticides. When they analyzed the rates of limb deformities among their research animals, they found much higher rates of deformities in trematode-infected tadpoles at the three ponds that receive agricultural runoff and contain pesticides than in the ponds that do not.
The team then moved into the lab to test their third hypothesis, which is that pesticide exposure--not some other factor--influenced the increased rates of deformities developed by the trematode-infected tadpoles in the field study. These laboratory experiments involved three groups of tadpoles that the researchers exposed to three different pesticides, plus one group that they did not expose to pesticides. The pesticides were Atrazine--the most commonly used pesticide in North America, Malathion--a common household pesticide that also is used to control insect pests in agricultural fields, and Esfenvalerate--a synthetic pyrethroid pesticide. "Synthetic pyrethroids have become increasingly popular during the last couple of years because they are not very toxic to birds and mammals; however, they are highly toxic to many other kinds of organisms," Kiesecker says.
The researchers also took a blood sample from each tadpole, and then exposed the four groups of tadpoles to trematode larvae under conditions that assured the tadpoles would be invaded by the parasites. Trematode infections can cause limb deformities if the larvae are able to evade the defenses of the tadpole's immune system long enough to transform themselves into hard cysts. If the location of the cyst is on cells that are supposed to develop into legs, the cyst will cause growth disruptions that lead to missing limbs, split limbs, or multiple limbs.
The researchers counted the number of cysts that developed in each infected tadpole and found a higher number of cysts in the animals that were exposed to pesticides. "From this experiment, we learned that a trematode-infected tadpole that is exposed to pesticides is more likely to develop limb-deforming cysts than is an infected tadpole that is not exposed to pesticides," Kiesecker says.
Kiesecker and his team designed their experiments to learn how pesticide exposure affects the immune response of the animals and their ability to fight off trematode parasites. They studied the blood of all the trematode-infected tadpoles--both those that were and were not exposed to pesticides during the laboratory experiments--to determine the prevalence of a type of white blood cell that fights parasites like trematode larvae. Kiesecker's team then compared this measure of immune-system strength with the number of trematode cysts that had formed in each animal. "The tadpoles that we exposed to pesticides had fewer of this particular kind of white blood cell compared to the tadpoles that we did not expose to pesticides, suggesting that pesticides make these animals more susceptible to parasitic infections," Kiesecker reports. "The kicker is that the concentrations that caused deformities were incredibly low for Esfenvalerate and Atrazine--low enough for humans to drink, based on Environmental Protection Agency standards," Kiesecker says.
In summary, Kiesecker says, "The field experiments showed that only the tadpoles that were infected with trematodes developed limb deformities and that these deformities occurred with more frequency in the groups of tadpoles that also were exposed to pesticides." The researchers had found a correlation with pesticides, but they needed to do the laboratory experiments in order to establish with certainty that pesticides were the cause of the different rates of deformities. "The lab experiments show that pesticides can weaken the immune response of the tadpoles, which can result in more infections, making these tadpoles more likely to develop limb deformities," Kiesecker says.
"We can learn a lot from experiments with amphibians because they are particularly sensitive to environmental changes that appear to be associated with the recent emergence of new diseases and resurgence of old diseases that infect humans," Kiesecker comments. "Frogs may be a sentinel species that is warning us about the interplay between human-caused environmental change and disease susceptibility. Hopefully, people will listen."
This research was supported by an Ecology of Infectious Diseases Grant from the National Institutes of Health and the National Science Foundation.
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