Experimental design and animal husbandry
Metamorphic toads (Bufonidae) collected from Arizona, California,
Louisiana, Ohio, and Tennessee, USA, (see Table S1 for specific
collection locations) were shipped overnight to Tampa, Florida. Although
the experiment was designed to cross six host populations with six Bd
strains (a 6x6 design), we could not collect enough Quebec (QC) hosts
and the experiment was reduced to a 5x6 design. Breeding times varied
across collection locations and so we used a randomized block design in
which identical protocols were employed to minimize variation (see
Supplemental Methods and Table S1 for details). To eliminate any
existing Bd infections from field-collected animals, we subjected all
animals to a 10-day 30° C Bd clearance treatment (Supplemental
Methods) (Chatfield & Richards-Zawacki 2011; McMahon et al.2014). Toads were then maintained individually in containers at
18.5° C for a 7-10 day acclimation period (Raffel et al.2013). We fed toads ad libitum with vitamin- and mineral-dusted
crickets and provided a fresh container and bedding twice per week
(Supplemental Methods).
We crossed 5 closely related North American toad species
(HostAZ, HostCA, HostLA,
HostOH, HostTN) with 6 Bd strains
(BdAZ, BdCA, BdLA,
BdOH, BdTN, BdQC; Table
S2) and one sham control (artificial spring water; ASW) for a total of
35 experimental treatment combinations. Strains were pulled from
cryopreservation from the Bd isolate cryopreservation library of Dr.
Joyce Longcore. The number of replicates per treatment varied between 6
and 18 because of variation in the availability of toads across the
populations (Table S1). An experimental unit was an individual toad.
Each toad population was split into seven treatments (6 Bd strains and
one ASW sham). Mortality was monitored daily. All toads were weighed
before the Bd exposure, and again at death or at 70 d post-exposure. At
the end of the experiment, all remaining toads were swabbed for Bd load,
euthanized with phosphate-buffered benzocaine, and stored at
-20° C.