Johnson, P, K. Lunde, R. Haight, J. Bowerman, and A. Blaustein The Effect of Trematode Infection on Amphibian Limb Development and Survivorship Johnson, P, K. Lunde, R. Haight, J. Bowerman, and A. Blaustein 30 April, 1999. Science, 284: 802-803

Background Over the past decade there has been increasing reports of deformed amphibians throughout North America North American Reporting Center for Amphibian Malformations (NARCAM) has received over 2100 reports from 1032 sites encompassing 82 species of amphibians. Of these, there are 944 reports with verifiable cases of malformations involving 52 species in 46 states and 4 provinces.

A map of North America showing the counties from which reports have been submitted

Examples of Frog Deformities Polydactyly - the complete duplication of one or more digits. Polymelia - supernumery (extra) limb segments are present.

Common species of frogs reporting malformations

Possible Causes of Frog Deformities Ultraviolet radiation: Continuous exposure of developing frog embryos to UV-B results in bilaterally reduced or missing limb segments (Ankley et al., 1998) Chemical agents: Certain limb malformations can be induced in the laboratory frogs by retinoids (Gardener and Hoppe, 1999) Parasites: Fungal infections suffocate frogs by clogging pores in their skin Positive correlation between ponds with high frequencies of limb malformations and infections with trematodes (Sessions et al., 1999)

The Bottom Line Despite these and other suggested hypotheses, proximate causes of frog limb malformations have not been identified.

Observations by Johnson et al. Surveyed 35 ponds in Santa Clara, CA between 1996-98 to determine the prevalence of deformities among amphibian populations in the field 4 of 13 ponds supporting Pacific treefrogs, Hyla regilla, revealed severely abnormal frogs Intensive monitoring programs (N=8818) at 2 of the ponds revealed 15-45% of frogs with extra limbs (polymely) and other hind limb deformities

Possible Cause(s) Water tests failed to detect unusual concentrations of pesticides, PCBs, or heavy metals from the ponds Eggs (N= 200) collected from the ponds hatched and developed normally in the lab Community analysis of the 35 ponds revealed that the 4 ponds with abnormal frogs were the only ponds to support Hyla regilla and an aquatic snail, Planorbella tenuis, that serves as the 1st intermediate host for the parasitic trematode, Ribeiroia Dissections of frogs from these 4 ponds revealed larval stages (metacercariae) of this parasite in these treefrogs

Ribeiroia Life Cycle

Question Is Ribeiroia infection responsible for the limb abnormalities observed among Hyla regilla?

Experimental Design Collected egg masses of H. regilla from a field site with no known records of abnormal frogs After hatching, tadpoles were held individually in 1-L containers randomly assigned to 1 of 4 treatments: 0 (control; N=35), 16 (light; N=45), 32 (intermediate; N=45), 48 (heavy; N=45) Ribeiroia cercaria A fifth group (N=10) was exposed to 80 cercariae of Alaria mustelae A sixth group (N=10) was exposed to both species: 80 A. mustelae and 32 Ribeiroia

H. regilla survivorship and abnormality frequency with Ribeiroia infections Exposure to Ribeiroia resulted in abnormal limb development in 85% of frogs (N=71) surviving to metamorphosis The frequency of abnormalities was high in all Ribeiroia treatments and showed a positive relationship with parasite density Tadpole survivorship declined with increasing parasite load In the control group 88% of tadpoles survived and all survivors showed normal development Survivorship Abnormality

H. regilla survivorship and abnormality frequency across parasite treatments Alaria did penetrate tadpoles but did not increase mortality or cause limb development, even at when at very high densities The combination of infections with Ribeiroia and Alaria has effects that were similar to those of Ribeiroia alone Survivorship Abnormality

Conclusions The frequency and composition of abnormalities observed in Pacific treefrogs appears to be the result of parasitic infections by trematodes of the genus Ribeiroia Trematode infections appear to represent a substantial source of mortality among amphibians

Future Directions Examine the relationship between parasite infection and the susceptibility to predation among treefrogs, especially as it relates to the transmission success of trematode parasites Examine the degree to which trematode infections are responsible for abnormalities in other populations of frogs Determine the condition(s) leading to a sudden increase in limb abnormalities among frogs parasitized by trematodes Address the mechanism by which Ribeiroia interferes with limb development