We studied the arrival and establishment of P. destructans at 24 hibernacula (caves and mines where bats spend the winter) in Virginia, Wisconsin, Illinois and Michigan over 7 years (electronic supplementary material, tables S1–S3) [37 (link)]. We visited sites twice per winter and collected data on infection status and body mass of bats. At each site, we sampled individual bats (electronic supplementary material, table S1, mean = 9.2, range: 1–50) stratified across site sections. Because sites used in this study were primarily small mines where it was possible to observe all bats present, in many instances, all individuals in the population were sampled. For each bat, we collected a standardized epidermal swab sample [18 (link)], attached a unique aluminium band and measured body mass using a digital scale (GDealer, accuracy ± 0.03 g). Because common condition indices are no more effective than body mass for estimating fat stores [38 (link)], we did not include information on bat forearm size in order to reduce handling disturbance. At every visit, we recorded and resampled any previously banded bats present. We stored swabs in RNAlater, and samples were kept at 0°C while in the field, and then at −20°C until processing. We tested samples for P. destructans DNA using real-time PCR and quantified fungal loads [21 (link),39 (link)]. Animal handling protocols were approved by Virginia Tech IACUC (no. 17-180, no. 20-150).
We investigated the effect of bat early hibernation (November) body mass on the probability an individual was recaptured overwinter (e.g. within-winter) using a generalized linear mixed model with a binomial distribution and a probit link, with site as a random effect, and body mass and disease phase (epidemic = 1–3 years since pathogen arrival, or established = 4–7 years since pathogen arrival) as interacting fixed effects (electronic supplementary material, table S1, total N individuals = 775). Phases were established based on previous results demonstrating that populations approach stability by year 4 following WNS arrival [6 (link),32 (link)] For analyses of individual survival and body mass, results were similar whether we used categorical disease phase or years since WNS as a continuous variable (electronic supplementary material, appendix) and grouping by phase maximized the number of bats in the epidemic years when mortality was high and the number of recaptured bats was low. For bats that were recaptured overwinter, we examined the effect of early winter body mass and infection on the amount of mass lost overwinter during both the epidemic and established phase using a linear mixed model with site as a random effect and the change in body mass as the response variable and fixed effects of early winter mass interacting with early winter fungal loads with additional additive effect of disease phase (electronic supplementary material, table S2, total N = 158). Finally, we explored changes in mass over time since the invasion of P. destructans on an individual and population level to examine both plasticity and phenotypic change. For bats that were recaptured in multiple years, we used a linear mixed model with mass as a response variable, years since WNS as a fixed effect and bat band ID as a random effect to explore plasticity in whether individual bat mass changed over time (N = 91 observations, 42 unique bands, 1–3 recapture events per bat, electronic supplementary material, appendix 4.0.3). At a population level, bat declines in sites with the best invasion mass data limited our ability to explore changes in mass, so we restricted our analyses to N = 5 sites (electronic supplementary material, table S3) that were measured during invasion and had sufficient bats to estimate during established periods using log10 mass as our response variable (logged to normalize) and years since WNS interacting with season with site as a random effect. All analyses were conducted in R v.4.1.2 using lme4 [40 (link)].
We investigated the effect of bat early hibernation (November) body mass on the probability an individual was recaptured overwinter (e.g. within-winter) using a generalized linear mixed model with a binomial distribution and a probit link, with site as a random effect, and body mass and disease phase (epidemic = 1–3 years since pathogen arrival, or established = 4–7 years since pathogen arrival) as interacting fixed effects (electronic supplementary material, table S1, total N individuals = 775). Phases were established based on previous results demonstrating that populations approach stability by year 4 following WNS arrival [6 (link),32 (link)] For analyses of individual survival and body mass, results were similar whether we used categorical disease phase or years since WNS as a continuous variable (electronic supplementary material, appendix) and grouping by phase maximized the number of bats in the epidemic years when mortality was high and the number of recaptured bats was low. For bats that were recaptured overwinter, we examined the effect of early winter body mass and infection on the amount of mass lost overwinter during both the epidemic and established phase using a linear mixed model with site as a random effect and the change in body mass as the response variable and fixed effects of early winter mass interacting with early winter fungal loads with additional additive effect of disease phase (electronic supplementary material, table S2, total N = 158). Finally, we explored changes in mass over time since the invasion of P. destructans on an individual and population level to examine both plasticity and phenotypic change. For bats that were recaptured in multiple years, we used a linear mixed model with mass as a response variable, years since WNS as a fixed effect and bat band ID as a random effect to explore plasticity in whether individual bat mass changed over time (N = 91 observations, 42 unique bands, 1–3 recapture events per bat, electronic supplementary material, appendix 4.0.3). At a population level, bat declines in sites with the best invasion mass data limited our ability to explore changes in mass, so we restricted our analyses to N = 5 sites (electronic supplementary material, table S3) that were measured during invasion and had sufficient bats to estimate during established periods using log10 mass as our response variable (logged to normalize) and years since WNS interacting with season with site as a random effect. All analyses were conducted in R v.4.1.2 using lme4 [40 (link)].
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