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.