ExplainingFrogDeformities An eight-year investigation into the cause of a shocking increase in deformed amphibians has sorted out the roles of three prime suspects

ONE HOT SUMMER DAY in 1995 eight blamed the increase on the amphibians’ greater exposure to ul- middle school children planning a simple study of wetland traviolet radiation, on chemical contamination of water or on ecology began collecting leopard frogs from a small pond near a parasite epidemic. Not surprisingly, every time another re- Henderson, Minn. To their astonishment, one captured frog port appeared, media outlets touted the new view, thus pro- after another had five or more hind legs, some twisted in viding a misleading picture of the situation. It turns out that all macabre contortions. Of the 22 animals they caught that day, these factors probably operate to varying extents, each caus- half were severely deformed. A follow-up search by pollution- ing particular disfigurements, and that all three may at times control officials added to the gruesome inventory. Occasional act in concert. Moreover, all stem in part from human activi- frogs in the pond had no hind limbs at all or had mere nubbins ties such as habitat alteration. where legs should be; others had one or two legs sprouting Deformities undoubtedly impair amphibian survival and from the stomach. A few lacked an eye. most likely contribute to the dramatic declines in populations The story seized national media attention and raised many that have been recognized as a global concern since 1989 [see questions—among them, was this an isolated occurrence or “The Puzzle of Declining Amphibian Populations,” by Andrew one facet of a widespread trend? And what caused the defor- R. Blaustein and David B. Wake; Scientific American, April mities? As researchers elsewhere in the country began investi- 1995]. Both trends are disturbing in their own right and are also gating their local amphibian populations, it became clear that a warning for the planet [see box on page 63]. Amphibians this bizarre collection of ailments was not confined to Min- have long been regarded as important indicators of the earth’s nesota. Since 1995, malformations have been reported in more health because their unshelled eggs and permeable skin make than 60 species, including salamanders and toads, in 46 states. them extremely sensitive to perturbations in the environment. In some local populations 80 percent of the animals are af- Chances are good that factors affecting these animals harshly flicted. International reports show that this phenomenon ex- today are also beginning to take a toll on other species. tends beyond the U.S. Surprising numbers of deformed am- phibians have been found in Asia, Europe and Australia as An Early Suspect well. Worldwide, extra legs and missing legs are most common. ONE PUTATIVE CAUSE of the deformities, excess exposure The aberrations cannot be discounted as being a normal to ultraviolet radiation, came under suspicion almost as soon part of amphibian life. Research dating back to the early 1900s as the malformations were discovered, because it had already indicates that a few individuals in every population have de- been implicated in declines of amphibian populations and be- fects resulting naturally from genetic mutation, injury or de- cause laboratory work had shown it to be capable of disrupt- velopmental problems. In healthy populations, however, usu- ing amphibian development. This form of radiation—which ally no more than 5 percent of animals have missing limbs or can damage immune systems and cause genetic mutations, digits; extreme deformities, such as extra hind legs, are even among other effects—has been reaching the earth in record less common. Moreover, fresh reviews of historical records by doses since chlorofluorocarbons and other human-made Minden Pictures one of us (Johnson) and new field studies indicate that defor- mities have become more prevalent in recent times. ALARMING LEGS: Ecologist Andrew R. Blaustein eyes malformed Pacific

Over the past eight years, dozens of investigators have tree frogs collected from farm ponds in northwestern Oregon. FRANS LANTING

BY ANDREW R. BLAUSTEIN AND PIETER T. J. JOHNSON

60 SCIENTIFIC AMERICAN FEBRUARY 2003 COPYRIGHT 2003 SCIENTIFIC AMERICAN, INC. COPYRIGHT 2003 SCIENTIFIC AMERICAN, INC. Chances are good that the factors affecting amphibians are also taking a toll on other species.

chemicals began thinning the protective can avoid the continuous exposure to ra- much can lead to deformities in embryos. layer of ozone in the stratosphere, a prob- diation studied in the EPA experiments. Indeed, numerous miscarriages and birth lem first measured in the 1970s. Between Juvenile and adult amphibians alike can defects in humans have resulted from 1994 and 1998 one of us (Blaustein) and move in and out of sunlight, often live pregnant women’s use of acne medicines his colleagues demonstrated that expo- in muddy water, or may be nocturnal. that contain a retinoic acid derivative. sure to ultraviolet rays can kill amphibian Some biologists suspected methoprene embryos and larvae, cause serious eye Pollution’s Part might have a similar effect on frogs. In a se- damage in adult frogs, and induce various AS SOME RESEARCHERS were exam- ries of experiments in the late 1990s, the types of bodily deformities in frogs and ining the link between ultraviolet radia- EPA in Minnesota did show that high salamanders. tion and deformities, others were pursu- amounts of retinoic acid could trigger Whether exposure to ultraviolet radi- ing the influence of water pollution, such poor formation of hind limbs in frogs, ation could disrupt leg development re- as pesticide runoff. They focused on pol- but comparable tests with methoprene mained uncertain until the late 1990s, lution because so many of the early re- caused no malformations at all. Separate when Gary Ankley and his co-workers at ports of amphibian ailments came from field measurements also indicated that the Environmental Protection Agency in areas where large amounts of insecticides the pesticide could not be the sole cause. Minnesota carried out the most focused and fertilizers are applied every year. By Methoprene breaks down quickly in the experimental research on this question to the mid-1990s numerous laboratory environment, and investigators found lit- date. When the investigators shielded de- studies had shown that myriad contami- tle evidence that it persists where defor- veloping frogs from ultraviolet rays, the nants can kill amphibians, but it was un- mities are abundant. The same is true for animals grew normal limbs, whereas tad- clear whether they could induce extra or 61 other agricultural chemicals and their poles exposed to full doses of natural lev- incomplete limb formation. breakdown products that have been els of ultraviolet radiation developed with A major challenge for toxicologists is measured in locations that harbor mal- parts of their legs missing or without dig- isolating a single chemical or even a group formed animals throughout the western its. These deformities resembled some of of chemicals as a likely candidate. Mil- U.S. Pesticides are not off the hook, how- those found in wild frogs from several lions of tons of hundreds of different pol- ever. Hundreds remain untested, and sites around the country. lutants are applied annually in regions some evidence implies that certain pesti- The EPA team was quick to point out, where deformed amphibians have been cides can cause bodily damage (albeit not however, that ultraviolet radiation does found. Yet one chemical rose immediate- the formation of extra limbs). not explain all types of leg deformities ly to the top of the list: methoprene. First At the moment, then, laboratory re- seen in nature. Most notably, it does not approved for commercial use in 1975, search tentatively suggests that water lead to the growth of extra legs, one of the methoprene was promoted as a safer re- pollutants and ultraviolet radiation are deformities reported most frequently placement for the banned pesticide DDT. capable of causing disfigurement. But a since 1995. Many laboratory and field ex- Initial concern over methoprene came more potent threat appears to have a periments, several performed by Blaustein from its chemical similarity to compounds much broader impact in nature. and his colleagues, have come to the same called retinoids. These substances, espe- conclusion. Other biologists have also cially retinoic acid, play an integral role in Prolific Parasites pointed out that many wild amphibians vertebrate development; too little or too THE EARLIEST HINTS of this threat— apparently the cause of the widespread hind-leg anomalies—turned up long be- Overview/Amphibian Ailments fore the disturbing findings in Minneso- ■ Since the mid-1990s striking deformities have turned up in more than 60 ta garnered nationwide attention. In the species of frogs, toads and salamanders in 46 states and on four continents. mid-1980s, Stephen B. Ruth, then at Mon- The number of disfigured animals in some populations averages around 25 terey Peninsula College, was exploring percent—significantly higher than in previous decades. ponds in northern California when he ■ Contradictory reports have blamed the deformities on increasing exposure to found hundreds of Pacific tree frogs and ultraviolet radiation, contaminated water or a parasite epidemic. long-toed salamanders with missing legs, ■ New evidence indicates that the parasite epidemic accounts for one of the extra legs and other deformities. He sound- most prevalent deformities—extra hind legs—and strongly suggests that ed no alarm, however, because he assumed human activities such as habitat alteration are exacerbating the problem. he was seeing an isolated oddity. In 1986 Ruth asked Stanley K. Ses-

62 SCIENTIFIC AMERICAN FEBRUARY 2003 COPYRIGHT 2003 SCIENTIFIC AMERICAN, INC. Disfigured and Dwindling Do the deformities explain recent declines in amphibian populations?

FROGS, TOADS AND SALAMANDERS have been including deserts, forests and grasslands, climbing up the long list of creatures in from sea level to high mountains. But as danger of disappearing from the earth diverse as their niches are, few are shielded entirely ever since the first reports of completely from a nearly equal variety of dwindling populations were made 20 years insults that humans inflict on them. Some ago. An obvious question for biologists is to important amphibian habitats have been what degree physical deformities are totally destroyed or are polluted to an contributing to overall population declines. intolerable degree. In other cases, people Most malformed amphibians eventually have introduced foreign animals that either vanish from a population because they can devoured or pushed out the native neither escape their predators nor hunt for amphibians. food efficiently. Events known to increase Some of the most widespread alterations the number of animals that mature into may lead to both population declines and disabled adults—such as the parasite deformities. Many studies have shown, for epidemic that is currently afflicting dozens instance, that excess ultraviolet radiation— of sites across North America—could cause resulting from human-induced ozone loss in CRACKED EARTH signals the temporary — a whole population to crash, particularly if demise of a frog habitat in the U.S. West. the upper atmosphere can inhibit limb the incidence of deformities continues to formation in amphibian juveniles or even kill increase. Although such crashes may be occurring at some sites, embryos inside their vulnerable, unshelled eggs. In the future, numerous amphibian populations have declined severely in the global warming is expected to dry out certain suitable aquatic absence of any deformities, leaving researchers to conclude habitats while elsewhere encouraging the emergence of that deformities are far from the sole basis for the declines. infections that produce abnormal development. When it comes Environmental hazards seem to be a more significant cause. to problems as pressing as these, tackling declines will most Amphibian species inhabit a wide variety of ecosystems, likely help alleviate the deformities as well. —A.R.B. and P.T.J.J.

sions, now at Hartwick College in New deformities in Ruth’s specimens: African trematode, now known to be Ribeiroia York State, to inspect his bizarre am- clawed frogs and axolotls are not known ondatrae. phibians. Sessions agreed and quickly re- to have limb deformities in nature. The re- Thinking that they may well have un- alized that they were all infected with a search that would eventually connect the covered a direct correlation between a parasitic trematode, known commonly dots between trematodes and extra or parasite epidemic and amphibian defor- as a flatworm or a fluke. The California missing legs in frogs was conducted after mities in the wild, Johnson and his team trematodes—whose specific identity was the stir of 1995. At that time, Johnson immediately collected deformed frogs unknown at the time—did not appear to pored over the scientific literature for from the same ponds and dissected them. kill their hosts outright, but Sessions sus- clues to the cause and came across the dis- In every case, they found cysts of the par- pected that their presence in a tadpole coveries made by Ruth and Sessions. asite densely clustered just below the skin mechanically disturbed natural develop- Johnson and his colleagues then conduct- around the base of the hind legs. To test ment wherever the parasites formed cysts ed broader surveys of California wetlands the idea that the trematodes were trig- in the body, most frequently near the between 1996 and 1998 and discovered gering the growth of the extra limbs, the hind legs. To test this hypothesis, he sim- that ponds where tree frogs had abnormal researchers then exposed Pacific tree frog ulated trematode cysts by implanting limbs also had an abundance of the tadpoles to R. ondatrae parasites in the small glass beads in developing limb buds aquatic snail Planorbella tenuis, one in a lab. As expected, the infected tree frogs of African clawed frogs and a salaman- series of hosts colonized by Sessions’s developed deformities identical to those der known as an axolotl. These two spe- cies, which serve as the “white rats” of ANDREW R. BLAUSTEIN and PIETER T. J. JOHNSON began exploring the potential causes of am- amphibian biology because they are easy phibian deformities as a team in 1998. Blaustein, who earned a Ph.D. in 1978 from the Uni- to breed in captivity, developed extra legs versity of California, Santa Barbara, is professor in the zoology department at Oregon State and other abnormalities—much as if they University. A behavioral and population ecologist by training, he has spent the past several Minden Pictures were parasitized. years investigating the dynamics of worldwide declines in amphibian populations, specifi- As intriguing as those experimental cally addressing the effects of ultraviolet radiation, pollutants, pathogens and nonnative THE AUTHORS results were, though, they could not prove species. Johnson, a doctoral candidate at the Center for Limnology at the University of Wis-

FRANS LANTING that trematodes were responsible for the consin– Madison, studies human influences on emerging diseases in aquatic environments.

www.sciam.com SCIENTIFIC AMERICAN 63 COPYRIGHT 2003 SCIENTIFIC AMERICAN, INC. HOW PARASITES CAN CRIPPLE FROGS EXCESS ULTRAVIOLET RADIATION

FERTILIZER RUNOFF LIVESTOCK PESTICIDE RUNOFF MANURE

EGGS

Parasite larvae 1infect snail New larvae hatch, to 6start the cycle again

Adult parasites 5reproduce in bird

INFECTED SNAIL DEFORMED LEG ALGAL BLOOM CYST

Larvae enter 2tadpole Cyst forms in tadpole, 3disrupting development Cyst waits dormant 4for frog to be eaten

LIFE CYCLE of the trematode Ribeiroia ondatrae enables the the bird, which releases trematode eggs into the water with its parasite to induce deformities—including extra hind legs—in feces (5). When larvae hatch (6), they begin the cycle again. generation after generation of frogs. In its first larval form the Human activities can exacerbate this process, especially where trematode infects snails (1). After transforming into a second livestock manure or fertilizers enter a pond and trigger algal free-swimming form inside a snail, the parasite embeds itself blooms that nourish, and thus increase, snail populations. near a tadpole’s future hind leg (2). There it forms a cyst that Excess ultraviolet radiation and pesticide runoff—which might disrupts normal limb development and can cause the tadpole to cause other types of deformities when acting alone—may sprout extra legs as it grows into a frog (3). The disabled frog facilitate the cycle by weakening a tadpole’s immune system then becomes easy prey for the parasite’s final host, often a and making the animal more vulnerable to parasitic infection. heron or egret (4). The parasite matures and reproduces inside —A.R.B. and P.T.J.J. found in nature, including extra limbs of Sessions, Geffrey Stopper of Yale Uni- malformations and Ribeiroia infection. and missing limbs. Higher levels of in- versity and their colleagues showed that In contrast, a field study that we pub- fection led directly to more malforma- Ribeiroia can cause limb deformities in lished last year turned up 25 such habitats tions, whereas uninfected frogs devel- wood frogs and leopard frogs as well. in the western U.S. alone. Among those oped normally. Other evidence indicates that Ribei- sites, six species displayed deformities, This study turned out to be a key roia is almost always found where de- and the proportion of affected individu- breakthrough in solving the mystery of formed amphibians are present, whereas als in each population ranged from 5 to deformed amphibians. Subsequent ex- chemical pollutants are found much less 90 percent. Over the past two years, oth- periments, including one we conducted in frequently. What is more, the parasitic in- er investigators have identified Ribeiroia- 2001 on western toads, provided evidence fection seems to have skyrocketed in re- triggered deformities in Wisconsin, Illi- of Ribeiroia’s major role in disfiguring cent years, possibly reaching epidemic lev- nois, Pennsylvania, New York and Min- amphibians other than Pacific tree frogs. els. An exhaustive literature search we nesota, including the pond where the Two studies reported last summer by conducted early in 2001 identified only eight schoolchildren made headlines. Al- Joseph M. Kiesecker of Pennsylvania seven records prior to 1990 of amphibian though heightened surveillance could ac-

State University and by a team made up populations that exhibited both significant count for some of this increase in report- DAVID FIERSTEIN

64 SCIENTIFIC AMERICAN FEBRUARY 2003 COPYRIGHT 2003 SCIENTIFIC AMERICAN, INC. Parasitic infection seems to have skyrocketed in recent years, possibly reaching epidemic levels.

ing, the vast majority of deformed frogs have been found by people, often children, who were looking for frogs for reasons unrelated to monitoring abnormalities. Not Working Alone SCIENTISTS NOW UNDERSTAND how the life cycle of Ribeiroia helps to perpet- uate the development of deformities in generation after generation of amphibians that are unlucky enough to share a habi- tat with infected snails [see illustration on opposite page]. After the parasite leaves its snail host and enters a tadpole, it embeds itself near the tadpole’s hind leg. Infected tadpoles then sprout extra legs or fail to develop both limbs. In either case, the DEFORMITIES DIFFER in their most likely causes. Trematode parasites can trigger the growth of extra young amphibian becomes unable to move hind legs (left). They can also lead to poorly formed or missing hind legs, although excess ultraviolet radiation, chemical pollutants or injury from predators may also be to blame. Ultraviolet radiation properly and thus becomes easy prey for probably accounts more often for abnormalities of the eyes (right) and skin. the parasite’s final host, often a heron or egret. The parasite matures inside the We recently showed a direct relation abetted by additional factors. At times, bird and becomes reproductively active. between human habitat alteration and water pollutants or excess ultraviolet ra- Through the bird’s feces, trematode eggs sites where Ribeiroia parasites are espe- diation may act alone to cause specific enter the water. When the larvae hatch, cially abundant. Indeed, our survey of the problems, such as disfigured bodies and they find a snail and begin the cycle again. western U.S., reported in 2002, revealed eye or skin abnormalities. At other times, If a spreading epidemic of Ribeiroia that 44 of the 59 wetlands in which am- pollutants or radiation may set the stage accounts for much, or even most, of the phibians were infected by Ribeiroia were for infection by weakening an amphib- increase in frog deformities seen in recent reservoirs, farm ponds or other artificial ian’s immune system and thus leaving the years, what accounts for the epidemic? bodies of water. Fertilizer runoff and cat- animal more vulnerable to a parasitic in- Current environmental trends suggest tle manure near these habitats often en- vasion. In yet another scenario, an in- that human alteration of habitats is at courage overwhelming blooms of algae, crease in amphibian predators, such as fault. In human as well as wildlife popu- which means more food for the snails that fish, leeches or turtles, may create more lations, infectious diseases emerge or be- host Ribeiroia parasites. Larger popula- deformities by biting off tadpole limbs. come more prevalent as features of the tions of snails infected with Ribeiroia lead Clearly, amphibians are subjected to a landscape change in ways that favor the directly to more deformed frogs. Wading cocktail of agents that stress individual an- proliferation of disease-causing organ- birds, the other necessary parasite hosts, imals and then, perhaps, entire popula- isms. Reforestation of the northeastern are usually found in abundance at such tions. The challenge to scientists becomes U.S., for example, has led to the emer- human-made locales. teasing apart these agents to understand gence of Lyme disease by encouraging the Although parasitism by trematodes is their interactions. Humans and other an- proliferation of white-tailed deer, which the likeliest explanation for most out- imals may be affected by the same envi- transport ticks that harbor the Lyme bac- breaks of amphibian deformities, it is cer- ronmental insults harming amphibians. terium. On the other side of the Atlantic, tainly not the only cause and may often be We should heed their warning. the damming of African rivers has led to the spread of human blood flukes that de- MORE TO EXPLORE pend on snails as a host and cause human The Effect of Trematode Infection on Amphibian Limb Development and Survivorship. Pieter T. J. Johnson, Kevin B. Lunde, Euan G. Ritchie and Alan E. Launer in Science, schistosomiasis. During the past several Vol. 284, pages 802–804; April 30, 1999. Stockpix.com decades, alteration of habitats has also en- A Plague of Frogs. William Souder. Hyperion Press, 2000. couraged the expansion of such diseases Ecosystems, Evolution and UV Radiation. Edited by Charles S. Cockell and Andrew R. Blaustein. as hantavirus, Ebola, West Nile virus, Springer-Verlag, 2001.

STEVEN HOLT dengue fever and AIDS. Visit the North American Reporting Center for Amphibian Deformities at www.npwrc.usgs.gov/narcam/

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