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).