Background | Trematode Parasite Infection | An Emerging Disease | Current Research | Malformation Gallery
Our group uses parasite-induced malformations in amphibians as a model system to investigate how environmental change affects the levels of disease pathology. Currently we seek to understand how different forms of environmental change, including pollution, climate change, and biodiversity loss, affect parasite abundance and the frequency of malformations. An additional yet no less important priority is to identify the consequences of such pathology for amphibian population persistence.
Beginning in the mid-1990s, numerous reports of malformed amphibians generated widespread concern among scientists, health officials, and state and federal agencies. In large part, these malformations involved limb deformities in recently metamorphosed frogs. Extra limbs, partially and completely missing limbs, and a variety of other limb malformations (e.g., skin webbings, bony triangles) comprised many of the observed deformities (see Malformation Gallery). Although initial reports mentioned eye abnormalities, internal irregularities, and tumors, these problems were likely over-emphasized and are not discussed here. In the United States, concentrations of hotspots occurred in the western US, the Midwest, and the Northeast (including parts of southern Canada). This issue is still very much alive, and new malformations sites continue to be discovered each year.
Concern over possible threats to human health and ongoing declines in amphibian populations has prompted intensive research by a variety of academic and government organizations. William Souder's book, A Plague of Frogs (Hyperion Press, 2000) presents a detailed history of the early investigation into this issue and some of the ensuing controversies. Broadly, research has focused on the following potential causes: chemical contamination (including retinoids and endocrine disruptors), UV-B radiation, sublethal predation, and parasite infection. For a recent review, see Johnson et al. (in press). I focus here on trematode parasite infection, which has been implicated as an important cause of malformations in amphibians across North America. Readers are advised to consult the primary literature for a more in-depth review of the current state of knowledge.
Trematode Parasite Infection
I first became involved in the issue of amphibian deformities while an undergraduate at Stanford University. Local landowners in Santa Clara County had been observing large numbers of malformed Pacific treefrogs (Pseudacris regilla) around several ponds. Over the next several years, I observed severe malformations in Pacific treefrogs, American bullfrogs (Rana catesbeiana), western toads (Bufo boreas), and California newts (Taricha torosa) (see Johnson et al. 2001a in Publications). However, malformations occurred only at ponds that supported a particular group of pond snails - the planorbid or rams horn snails. As it turned out, these snails are the exclusive first intermediate hosts of a digenetic trematode called Ribeiroia ondatrae.
Digenetic trematodes are parasitic flatworms with complex life cycles, typically involving two or more hosts. Species in the genus Ribeiroia utilize three hosts: planorbid snails, larval amphibians or fishes, and birds or mammals. Planorbid snails function as the first intermediate hosts, in which the parasite undergoes asexual reproduction to produce free-swimming cercariae. Cercariae encyst around the developing limbs of larval amphibians, wherein they form a resting stage (metacercariae) that can interfere with proper limb development. For the parasite to complete its life cycle, an infected amphibian must be consumed by a suitable definitive host. Definitive hosts for Ribeiroia are typically birds, such as herons, raptors, or waterfowl within which the adult parasite reproduces sexually and releases eggs in the host feces. Eggs hatch in water, releasing free-swimming miracidia that infect planorbid snails, continuing the cycle. Malformations, which are suspected to increase the susceptibility of infected frogs to predation, may actually enhance transmission of Ribeiroia between its second intermediate (amphibians) and definitive hosts (birds). A variety of other parasites with complex life cycles are known to alter either the appearance or behavior of their second intermediate hosts, effectively rendering them more vulnerable to predatory definitive hosts.
The idea that trematode parasites could cause malformations in amphibians was first suggested in 1990 by Dr. Stanley Sessions. Dissection of malformed amphibians from our ponds in Santa Clara revealed an abundance of Ribeiroia metacercariae. To test the hypothesis that Ribeiroia could induce the observed malformations, I teamed up with colleagues Kevin Lunde and Euan Ritchie to conduct an experiment. We exposed laboratory-raised tadpoles of Pseudacris regilla to different numbers of Ribeiroia cercariae (0, 12, 24, 48) and allowed them to develop to metamorphosis. Tadpoles exposed to no parasites (control treatment) exhibited high survivorship and no malformations. In contrast, tadpoles exposed to Ribeiroia frequently died before reaching metamorphosis. Moreover, of those that did complete metamorphosis, the majority were severely deformed. The malformations were strikingly similar to those we observed in the field, including supernumerary limbs (up to 6 extra), partially and completely missing limbs, skin webbings, bony triangles, and many others (see Johnson et al. 1999). Subsequently, we completed similar experiments with western toads (Bufo boreas). Similarly, we found that Ribeiroia caused high levels of malformation and mortality in this species, but that the relative abundance of malformation types and sensitivity to infection could differ by species. For example, while extra limbs were the most common malformation observed in Pacific treefrogs (in field and laboratory studies), skin webbings were the most prevalent malformation in western toads and American toads (see Johnson et al. 2001b, Johnson and Hartson 2009). This may help explain why, even at a single site, different amphibian species tend to exhibit different malformation "signatures" (that is, the timing, frequency, and composition of malformations).
Along with many collaborators, Kevin Lunde and I expanded our field research across the western USA. In 1999, we surveyed more than 12,000 amphibians representing 11 species of frogs, toads, and salamanders for malformations. The geographic area encompassed parts of California, Oregon, Washington, Idaho and Montana. At each site visited, we compared the level of malformations in each amphibian species to (1) the concentrations of 61 different pesticides and (2) the intensity of Ribeiroia infection. In brief, we found that Ribeiroia infection was a strong predictor of the presence and frequency of malformations across sites and species (see Johnson et al. 2002). Again, malformations included extra, missing and otherwise misshapen limbs. Pesticide contamination, at least in this study, was not associated with amphibian malformations. It is important to note, however, that parasite infection generally does not explain sites with a high frequency of only missing-legged frogs, as described at some wetlands in other parts of the country (e.g., Vermont).
An Emerging Disease?
One of the most difficult questions to address is whether malformed amphibians have become more common in recent years. Without question, the number of abnormal amphibians reported has skyrocketed since 1995, with over 40 states and at least as many amphibian species included in those reports. However, many of those reports involve only a single animal. In addition, there has been a dramatic increase in surveillance - people have actively searched for malformed amphibians in response to increased media attention. Teasing apart these issues to determine whether the apparent increase is, in fact, a real phenomenon is an ongoing problem that is unlikely to reach definitive resolution.
Nevertheless, we can utilize several pieces of circumstantial evidence to at least evaluate the issue further. Based on the historical scientific literature, it is clear that malformed amphibians are not new; scattered reports of isolated frogs or toads with extra, missing, or abnormal limbs date back over 200 years. Less common, however, are documented occurrences involving high frequencies of malformations in a single population ("mass malformations"), as described in many recent reports. In the USA, only about a dozen historical accounts of mass malformations are known to have occurred prior between 1940 and 1990, relative to more than 50 mass malformation sites associated with Ribeiroia reported since 1990. Increases in surveillance are unlikely to account for all of this increase. We reviewed available information for nine historical accounts from California, Colorado, Idaho, Mississippi, Montana, Ohio, and Texas reported between 1946 and 1988. Between 1999 and 2001, we resurveyed each site, inspected amphibians for malformations, and tested water samples for pesticides. In addition, we re-described original museum specimens and examined them for Ribeiroia. Historical malformations in amphibians from six of the eight sites were associated with Ribeiroia infection, dating back as far as 1946. Malformations recorded historically were consistent with the known effects of Ribeiroia infection, including extra limbs, cutaneous fusion, and bony triangles. Of the six sites that still supported amphibians upon resurvey, three continued to support severe limb malformations at frequencies of 7 to 50% in one or more species. Although no pesticides were detected, amphibians from each of these sites were infected with Ribeiroia metacercariae. Taken together, these results suggest that Ribeiroia infection has historically been an important cause of mass malformations in amphibians (see Johnson et al. 2003).
Current Research: Ecosystem Drivers and Consequences of Parasite Infection
Despite the compelling evidence linking Ribeiroia infection and amphibian malformations, critical questions remains unaddressed. For example, why has the frequency and geographic range of malformed amphibians apparently increased? If parasite infection explains some or even many of the observed malformations, what secondary factor is responsible for increases in parasite abundance or host susceptibility? Previous studies have reported strong associations between malformation hotspots and heavily altered aquatic systems, yet few have investigated mechanisms behind this connection.
Our group uses parasite-induced malformations in amphibian as a model system to investigate how environmental change can affect the levels of disease pathology. Currently we seek to understand how different forms of environmental change, including pollution (especially nutrient runoff), climate change, and biodiversity loss, affect parasite abundance and the frequency of malformations.
Aquatic eutrophication is one of the most widespread and severe problems facing freshwater ecosystems worldwide. Eutrophication is generally caused by excess nutrient (phosphorus and nitrogen) runoff in aquatic environments, which can be associated with atmospheric deposition, agricultural fertilizers, cattle grazing, and other non-point sources (e.g., domestic sewage, urbanization, etc.). Eutrophication can influence trematode life cycles by promoting periphyton, the primary food source of freshwater snails. Through a combination of field surveys and experimental research, we have shown that nutrient runoff can enhance Ribeiroia abundance by increasing snail host density and by increasing the number of infectious parasites produced by each snail (see Johnson and Chase 2004, Johnson et al. 2007). As these snails often limit the distribution and abundance of Ribeiroia, changes in their density will have important consequences on the frequency and severity of malformations in amphibians.
Aquatic communities generally and amphibians in particular stand at the forefront of the current biodiversity crisis, with large numbers of declining and extinct species. Recent attention in disease ecology has focused on how changes in community structure can affect pathogen transmission and disease. The dilution effect hypothesis suggests that biodiversity loss can lead to increases in human and wildlife disease levels. We have found that both amphibians and snails vary considerably in their susceptibility to Ribeiroia infection. As a result, changes in aquatic community structure can strongly influence the levels of parasite transmission. For example, tadpoles of American toads (a sensitive species) raised alongside gray treefrogs (an insensitive species) exhibited fewer malformations and higher survivorship than toads raised alone or with another toad tadpole (see Johnson et al. 2008). In similar work with the human pathogen Schistosoma mansoni, we found that a higher diversity of snail species, which act as first intermediate hosts, led to a reduction in successful infections and a lower production of cercariae, the stage infectious to humans (Johnson et al. 2009).
Current and forecasted increases in temperature will likely have profound effects on host-parasite interactions (Rohr et al. 2011). Because of their small size and high metabolic efficiency, parasites are expected to respond more strongly to increases in temperature than are their hosts. We predict that increases in temperature will enhance and concentrate flatworm infections in early spring when larval amphibians are most vulnerable, leading to an increase in malformations and mortality (see Paull and Johnson 2011; Paull et al. 2012). To investigate the effects of climate change on disease, we are using two novel and complementary approaches. First, a combination of lab experiments and outdoor mesocosms will be used to determine how increases in temperature control amphibian development, parasite production, and their interactions. Second, using latitude and elevation as independent surrogates for shifts in climate and precipitation, we are conducting intensive sampling along latitudinal and elevational gradients to indirectly forecast the effects of shifting climate on disease.
Deformities and Amphibian Conservation
A final yet pressing priority in the study of amphibian malformations is to identify the long-term consequences of Ribeiroia infection and malformations for amphibian population persistence. Malformed frogs do not survive to sexual maturity, likely succumbing to predators and/or starvation, and Ribeiroia infection itself is highly lethal to amphibian larvae. Considering that malformations can affect >50% of the emerging frogs in a population, the net effects of infection and malformations on mortality may be substantial. Although anecdotal reports have linked malformations and amphibian declines in select populations, there have been no attempts to quantify the effects of Ribeiroia relative to other sources of mortality, particularly under field conditions. This is a key issue toward evaluating the conservation significance of amphibian malformations. For more information, see Goodman and Johnson (2011a, 2011b).