1. 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, Science, 284:

2. 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.

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

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

5. Common species of frogs reporting malformations

6. 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)

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

8. Observations by Johnson et al.
Surveyed 35 ponds in Santa Clara, CA between 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

9. 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

10. Ribeiroia Life Cycle

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

12. 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

13. 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

14. 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

15. 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

16. 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