DOI: 10.5061/dryad.sxksn036k
title: Temperature shifts associated with bat arousals during hibernation inhibit the growth of Pseudogymnoascus destructans

description: Temperature is a critically important factor in many infectious disease
 systems because it can regulate responses in both the host and the
 pathogen. White-nose syndrome (WNS) in bats is a severe infectious disease
 caused by the temperature-sensitive fungus, Pseudogymnoascus destructans
 (Pd). One feature of WNS is an increase in the frequency of arousal bouts
 (i.e., when bat body temperatures are elevated) in Pd-infected bats during
 hibernation. While several studies have proposed that increased frequency
 of arousals may play a role in the pathophysiology of WNS, it is unknown
 if the temperature fluctuations might mediate Pd growth. We hypothesized
 that exposure to a high frequency of elevated temperatures would reduce Pd
 growth due to thermal constraints on the pathogen. We simulated the
 thermal conditions for arousal bouts of uninfected and infected bats
 during hibernation (fluctuating from 8 - 25 °C at two different rates) and
 quantified Pd growth in vitro. We found that increased exposure to high
 temperatures significantly reduced Pd growth. Because temperature is one
 of the most critical abiotic factors mediating host-pathogen interactions,
 resolving how Pd responds to fluctuating temperatures will provide
 insights for understanding WNS in bats and other fungal diseases.
description: Culturing and experimental set
 up We obtained a <em>Pd </em>culture from
 American Type Culture Collection number (<em>Geomyces
 destructans</em> ATCC), which was originally isolated from the wing
 of a little brown bat (<em>Myotis lucifugus</em>) in Williams
 Hotel Mine, Ulster County, New York, USA, in 2008 [7,8]. We revived the
 isolate by slowly warming the culture to room temperature (~21-22 °C) in a
 water bath (per ATCC manufacturer’s instructions). We transferred
 the <em>Pd</em> to two Sabourand’s Dextrose agar (SDA) plates
 and incubated it at 15 °C under 24 h darkness until we initiated the
 temperature experiment.   Temperature experimental set
 up To harvest conidia for our experiment, we grew the
 cultures for 6-8 weeks on SDA agar plates or to when mycelia covered ~75%
 of the plates. We collected conidia by flooding the plates with 5 mL 0.5%
 1x Phosphate Buffered Saline with Tween 20 (PBST) and allowed it to sit
 for 5 min. We then drew off 5 mL from the agar plate, counted conidia
 concentrations using a haemocytometer (Hausser Scientific, Horsham, PA,
 USA). We diluted the solution to a concentration of 2 x
 10<sup>4</sup> conidia per 1 mL. We then inoculated 50 μL of
 the solution containing conidia into 30 wells in <em>N</em> =
 8 Falcon 96-well, non-treated, flat-bottom microplates (Fisher Scientific,
 Waltham MA, USA). We also added 100 μL of Sabourand’s Dextrose Solution
 media (for which we used the same methods as for SDA but we omitted agar)
 to each well for all plates. To provide a negative control, we transferred
 the PBST solution containing<em> Pd</em> conidia to a 50 ml
 conical and submerged it in 100 °C water for 10 minutes. We then added 50
 μL of PBST solution that contained heat
 killed <em>Pd </em>conidia and 100 μL of media to 30 negative
 control wells. We placed parafilm around the plates to maintain a tight
 seal on the lid and then incubated the plates in one of four temperature
 conditions and 80% humidity within incubators (Isotemp refrigerated
 incubators, Thermo Fisher, Waltham MA, USA) for 36
 d.   The temperature treatments included: (1) constant 8
 °C, a constant temperature within the thermal breath
 for <em>Pd</em> growth, (2) constant 25 °C, comparable to the
 skin temperature of hibernating bats during arousals [18], (3) fluctuating
 between 8- 25 °C every 16 days, simulating
 the <em>T<sub>b</sub></em> for “low frequency
 arousal” (uninfected, control) bats, and (4) fluctuating between 8- 25 °C
 every 7 days, simulating
 the <em>T<sub>b</sub></em> for “high frequency
 arousal” (i.e., infected bats with advanced WNS). For the fluctuating
 temperature treatments, the experimental plates were exposed to higher
 temperature conditions for 1 hour, which is the approximate duration of
 arousal for multiple bat species. Over the course of the experiment, we
 inspected the wells for contamination using a light microscope, seated the
 plates in a microplate reader (BioTek ELx800 spectrophotometer using the
 540 nm filter), and recorded optical density (OD) every 3 days up to 36
 days.     Statistical analysis We assessed the difference
 in <em>Pd </em>growth over time among different temperature
 treatments using nonlinear mixed effects models with the ‘nlme’ package
 v3.1.131.1 in R v3.4.3 (R Core Team 2017). We selected a three-parameter
 logistic function because logistic growth curves are classically used to
 model the exponential and stationary phases of microbial growth
 <em>in vitro</em>. A nonlinear modeling approach allows for
 meaningful parameter estimates of culture stationary phase (i.e.,
 asymptote), the time at which cultures are in exponential growth phase
 halfway to stationary phase (i.e., inflection point), and a scale
 parameter that determines the steepness of the growth curved. We corrected
 for initial conidia inoculation and media color by subtracting OD values
 of heat-killed controls from OD values of wells
 containing <em>Pd</em>. Due to an error in pipetting, we
 omitted two wells in one of the plates from our analyses. We then built
 models using the adjusted mean OD per plate per day (N = 8 plates per
 temperature treatment).   We compared model fits with analysis of variance
 (ANOVA) and likelihood ratio tests for nested models, and we selected
 models using AIC and principles of parsimony following Pinheiro and Bates
 (2000). <em>Pd </em>incubated at a constant temperature of 25
 °C did not follow a logistic growth pattern (i.e., there was
 no<em> Pd</em> growth at this temperature) and we therefore
 excluded this temperature treatment from the final analysis. Our final
 model included fixed effects of temperature treatment on all three
 logistic growth parameters and the random effect of plate on the
 inflection point and scale parameters. We added an identity variance
 structure and an autoregressive moving average correlation structure to
 the model to address heteroscedasticity. We used an F-test to determine
 the significance of the temperature treatment fixed
 effect.
description: R v3.4.3 (R Core Team 2017)