Disease severity metrics
Upon returning to each of the three sites, we recorded the survival
status of each individual. To calculate the on-host pathogen growth rate
of Pseudogymnoascus destructans , we collected swab samples from
each individual bat in both December 2018 and March 2019 using a
standardized swabbing protocol described in 60. Swab
samples were preserved in RNAlater until extraction. We used real-time
polymerase chain reaction (qPCR) targeting the multicopy intergenic
spacer region of the rRNA gene complex to test swab samples for the
presence of P. destructans DNA122. We measured
the quantity of P. destructans DNA in picograms from each swab
sample based on the cycle threshold (Ct) value with a
Ct cutoff of 40 cycles and a previously described
standard quantification curve described in 24.
In both December 2018 and March 2019, we weighed each individual bat
using an electronic scale. We subtracted the late hibernation weight of
each individual from their early hibernation weight to calculate the
amount of weight lost over the course of hibernation in grams.
Additionally, in March 2019, we collected data on the severity of tissue
invasion by transilluminating both wings of each individual using a
9-watt 368nm fluorescent light (BRC-100, Way Too Cool LLC, from
www.fluorescents.com). Infected tissue fluoresces orange when exposed to
ultraviolet light and has been verified by
histology87. During transillumination, we photographed
each wing using a Canon (Tokyo, Japan) EOS Rebel T6 (AF 15-55 mm lens,
F-stop 22, ISO 1600, shutter speed 2.5 seconds, no filter). We used
Adobe (San Jose, USA) Photoshop Elements Editor 15 to quantify the
proportion of the plagiopatagium of each wing that displayed orange
fluorescence indicative of infection with P. destructans . We
first calculated the total number of pixels encompassed by the
plagiopatagium, excluding bone area or area obstructed by fur. Then, we
calculated the total number of orange pixels within the plagiopatagium.
For each bat, pixel values from both wings were summed and proportions
derived from these values. These image analyses were done by an
individual blind to the origin of each bat and the hypotheses and
predictions of the study.