Using an ecosystem-level manipulation to understand host-parasite interactions and how they vary with study venue

We investigated interactions between the virulent trematode Ribeiroia ondatrae, which has been linked to amphibian malformations across the United States, and its amphibian host (Pseudacris regilla) using a hierarchical approach involving multi-year regional field surveys, replicated pond enclosures, and an unreplicated ecosystem manipulation of parasite infection. Results of this multi-tiered approach provide strong evidence of the causal linkage between infection and malformations while offering additional insights about the influence of experimental venue on effect size. Among years and across 18 wetlands in northern California, Ribeiroia infection was a strong predictor of malformation frequency, which ranged from 0 to 77% at all sites (n = 6,511). Correspondingly, the addition of >500 Ribeiroia infected snails to an experimentally divided wetland using a three-year Before After Control Impact (BACI) design caused sharp increases in Ribeiroia infection and severe malformations in P. regilla during the manipulation year (but not pre- or post-manipulation). These results were complemented by the findings from a replicated (n = 16) enclosure/exclosure study conducted on both sides of the divided wetland (Hog Lake), which showed increased infection and malformations only among larvae within cages that allowed parasite entry and only on the manipulated side. No differences in mortality were observed among animals as a function of cage type. A comparison of the slope between observed infection and malformations as a function of venue (previous laboratory work and the two spatial scales of this study (cages and whole-pond)) indicated that small-scale experiments exhibit stronger effects relative to results from larger spatial extents. Multi-year sampling also indicated that malformed frogs were unlikely to return as breeding adults, highlighting the potential for population-level impacts associated with high Ribeiroia infections. Taken together, these results provide support for the causal relationship between Ribeiroia infection and amphibian malformations under realistic conditions while simultaneously emphasizing the influence of study venue on the strength of this relationship.