Treatment Inoculate Preparation
We cultured two strains of Bd (JEL 423 isolated in Panamá fromHylomantis lemur and SRS 810 isolated in the USA from Rana catesbeiana ) on 1% tryptone agar plates. Each amphibian species tested was exposed to the strain that was isolated closest to their native range (Table 1). Immediately before use in the experiments, the Bd+ plates were flooded with ASW to suspend the infectious zoospores. The ASW from all Bd+ plates was homogenized, zoospore concentration was quantified using a hemocytometer, and the stock was diluted with ASW to 1.5 x 105 zoospores/mL (Bd+ stock). Bd-free 1% tryptone agar plates were flooded using the same technique to create a control (Bd- stock).
We used the same methodology to prepare the different treatment inoculates for all the amphibian species. To create Dead zoospores with metabolites , the Bd+ stock was flash frozen with liquid nitrogen. To create Dead zoospores alone , the Dead zoospores with metabolites stock was filtered through a 1.2 µm filter (GE Whatman Laboratory Products, Springhill, PA) to separate the zoospores from the liquid containing the metabolites. We washed the zoospores off the filter with an equivalent amount of ASW. To create Metabolites alone , we used the filtrate separated from the Dead zoospores with metabolites stock (see Dead zoospores alone ). For all treatments we used a hemocytometer to verify that we had what we expected, either live zoospores, dead zoospores, or no zoospores. For the dead Bd and metabolite treatments, we verified there was no live fungus by plating 1 mL of the treatment inoculate on three 1% tryptone agar plates and monitored for growth for two weeks (there was no Bd growth).