DOI: 10.25349/d9jg8c
title: Putative resistance and tolerance mechanisms have little impact on disease progression for an emerging salamander pathogen

description: 1. Resistance and tolerance are unique host defense strategies that can
 limit the impacts of a pathogen on a host. However, for most
 wildlife-pathogen systems there are still fundamental uncertainties
 regarding 1) how changes in resistance and tolerance can affect disease
 outcomes and 2) the mechanisms underlying resistance and tolerance in host
 populations. 2. Here, we first compared observed patterns of resistance
 and tolerance and their effects on disease outcomes among salamander
 species that are susceptible to infection and mortality from the emerging
 fungal pathogen Batrachochytrium salamandrivorans (Bsal). We then tested
 whether two putative mechanisms that contribute to host resistance and
 tolerance, skin sloughing and skin lesion reduction, predicted reduced
 Bsal growth rate or increased host survival during infection,
 respectively. 3. We performed multi-dose Bsal challenge experiments on
 four species of Salamandridae found throughout North America. We combined
 the laboratory experiments with dynamic models and sensitivity analysis to
 examine how changes in load-dependent resistance and tolerance functions
 affected Bsal-induced mortality risk. Finally, we used our disease model
 to test whether skin sloughing and lesion reduction predicted variability
 in infection outcomes not described by Bsal infection intensity. 4. We
 found that resistance and tolerance differed significantly among
 salamander species, with the most susceptible species being both less
 resistance and less tolerant of Bsal infection. Our dynamic model showed
 that the relative influence of resistance versus tolerance on host
 survival was species-dependent -- increasing resistance was only more
 influential than increasing tolerance for the least tolerant species where
 changes in pathogen load had a threshold-like effect on host survival.
 Testing two candidate mechanisms of resistance and tolerance, skin
 sloughing and lesion reduction, respectively, we found limited support
 that either of these processes were strong mechanisms of host defense. 5.
 Our study contributes to a broader understanding of resistance and
 tolerance in host-pathogen systems by showing that differences in host
 tolerance can significantly affect whether changes in resistance or
 tolerance have larger effects on disease outcomes, highlighting the need
 for species and even population-specific management approaches that target
 host defense strategies.
description: We performed multi-dose <em>Bsal</em> challenge
 experiments on four Salamandridae species found throughout North America.
 The species tested included <em>Notophthalmus
 viridescens</em> (n=147 individuals), <em>N.
 meridionalis</em> (n=40), <em>N. perstriatus</em> (n=50)
 and <em>Taricha granulosa</em> (n=47). <em>N.
 viridescens</em> were collected from six geographically separated
 populations: three in Tennessee (subsequently labeled as Middle, Ijams,
 and Roan) and one each in Pennsylvania, Vermont and Michigan. <em>T.
 granulosa</em> were captured by the California Fish and Wildlife
 Department. <em>N. meridionalis</em> and <em>N.
 perstriatus</em> were captive bred at the Fort Worth and
 Jacksonville Zoos, respectively, and transferred to the University of
 Tennessee with authorization by U.S. Fish and Wildlife Service.
 Individuals were acclimated for between 2-4 weeks before the experiment
 began.  All animals were housed in environmental
 chambers set at 15 C and 12 hour light and dark cycles. For each challenge
 experiment, we randomly assigned animals to one of four exposure doses
 ranging from 5x10<sup>3</sup> - 5x10<sup>6</sup> 
 <em>Bsal</em> zoospores/mL or to a control group where we
 inoculated animals with autoclaved dechlorinated water (n=5-10 individuals
 per treatment per species). We exposed an animal to
 <em>Bsal</em> by placing it in a 237 mL container with 9 mL of
 autoclaved dechlorinated water and 1mL of the desired exposure dose. We
 mock inoculated control animals by placing them in the same size container
 with 10 mL dechlorinated water. All animals were exposed for 24 hrs.
  Following exposure, each animal was housed individually during the
 experiment inside 710 cm<sup>3</sup> enclosures, containing a
 moist paper towel and a 7.6 cm PVC cover object.  We replaced each
 animal’s container and water every three days. We swabbed each animal
 every six days beginning four days post exposure until the end of each
 experiment (40-70 days, depending on the species).  We chose six days as
 this time step allowed us to capture <em>Bsal</em> growth
 dynamics on individual hosts while minimizing the amount of time we
 handled the animals.  During each swabbing period, we examined the animals
 for signs of skin lesions and skin sloughing. Skin lesions were counted
 grossly along the body during each swabbing period. We counted lesions as
 visible skin erosions and ulcers, occasionally with the aid of a
 magnifying lamp. We also recorded presence or absence of skin sloughing
 during each check by visually examining the animal and the animal’s
 container for signs of skin sloughing.  We extracted
 genomic DNA from skin swabs using a Qiagen DNeasy Blood and Tissue kits
 (Qiagen, Hilden, Germany). We then performed
 <em>Bsal</em> quantitative polymerase chain reaction (qPCR)
 using methods similar to Blooi et al. 2013. All qPCR reactions were
 performed on an Applied Biosystems Quantstudio 6 Flex qPCR instrument
 (Thermo Fisher Scientific, USA). Using qPCR we determined the infection
 status of each animal and estimated the <em>Bsal</em> zoospore
 copies/microL recovered from each swab. Each qPCR reaction was run in
 duplicate and samples were considered positive if both reactions amplified
 prior to 50 cycles.
description: See README.txt file for data description.