DOI: 10.5061/dryad.9s4mw6mjb
title: Challenging a host-pathogen paradigm: Susceptibility to chytridiomycosis is decoupled from genetic erosion

description: The putatively positive association between host genetic diversity and the
 ability to defend against pathogens has long attracted the attention of
 evolutionary biologists. Chytridiomycosis, a disease caused by the chytrid
 fungus Batrachochytrium dendrobatidis (Bd), has emerged in recent decades
 as a cause of dramatic declines and extinctions across the amphibian
 clade. Bd susceptibility can vary widely across populations of the same
 species, but the relationship between standing genetic diversity and
 susceptibility has remained notably underexplored so far. Here, we focus
 on a putatively Bd-naive system of two mainland and two island populations
 of the common toad (Bufo bufo) at the edge of the species’ range, and use
 controlled infection experiments and dd-RAD sequencing of &gt;10,000
 SNPs across 95 individuals to characterise the role of host population
 identity, genetic variation and individual body mass in mediating host
 response to the pathogen. We found strong genetic differentiation between
 populations and marked variation in their susceptibility to Bd. This
 variation was not, however, governed by isolation-mediated genetic
 erosion, and individual heterozygosity was even found to be negatively
 correlated with survival. Individual survival during infection experiments
 was strongly positively related to body mass, which itself was unrelated
 to population of origin or heterozygosity. Our findings underscore the
 general importance of context-dependency when assessing the role of host
 genetic variation for the ability of defence against pathogens.
description: <strong>Experimental
 infections</strong> Experimental procedures took place
 in a temperature controlled room at 18°C with a 12:12 h light:dark cycle.
 Ninety days after the start of metamorphosis, toadlets were weighed to the
 nearest 0.001 g, and transferred to individual housing in 0.7 L
 polypropylene boxes (Really Useful Products LTD, UK) lined with a moist
 paper towel and containing a cover object. Following a two-week period of
 acclimatisation, 202 toadlets were randomly allocated into one of two
 experimental treatments: (I) exposure to an active Bd culture, or (II) a
 control group exposed to culture media alone. Treatments broadly followed
 Garner et al. (2009, 2011). Each toadlet was placed into an individual
 Petri dish filled with 30 ml of aged tap water and 450 μl of culture media
 for four hours. In order to ensure a level of exposure sufficient to cause
 mortality, nine such treatments were carried out across 21 days. All
 individuals in the exposed group received the same dosage of the Bd
 isolate UK CORN'12 3.1, part of the hypervirulent global panzootic
 lineage BdGPL (Farrer et al., 2011). Dosages were calculated by counting
 live spores using a haemocytometer and varied between 40,500 and 382,500
 zoospores depending on session. The experiment proceeded for 50 days after
 the first exposure (i.e., a 21-day period of inoculations followed by a
 further 29 days of daily monitoring of mortality). If a toadlet reached a
 humane end point (i.e., was unresponsive or was unable to support its own
 weight), it was euthanised by licenced personnel in accordance with the
 Animal (Scientific Procedures) Act 1986 using the non-schedule 1 method of
 immersion in buffered tricaine methanesulfonate (MS222) followed by
 fixation in 70% ethanol. All animals surviving to the end of the
 experiment were euthanised and fixed as described
 above.<br><br><strong>SNP
 genotyping</strong> DNA for genotyping was extracted
 from hind leg muscle of toadlets using a Qiagen DNEasy extraction kit
 following the manufacturer's protocol (Qiagen, UK). DNA concentration
 was assessed by fluorometry using a Qubit 3.0 (Thermo Fisher Scientific,
 MA, USA), and standardised to 20 ng/uL. The set of 101 exposed individuals
 (excluding the controls) was reduced to 95 by randomly removing six
 individuals from the best-represented population (MAK). The resulting
 panel of samples constituted 15 individuals from CRO, 31 from MAK, 27 from
 MAT, and 22 from SKB. Preparation of dd-RAD libraries and next generation
 DNA sequencing were performed by Floragenex (OR, USA) following the
 protocol of Truong et al. (2012). In brief, DNA was double digested with a
 combination of rare and frequent cutting endonucleases (PstI and MseI,
 respectively), followed by ligation with adaptors with individual indices.
 1x100bp single end sequencing was performed on the resulting PCR-generated
 library using an Illumina HiSeq 4000 (CA, USA).  Once obtained from Floragenex, a de
 novo catalogue of SNP loci was constructed using STACKS 2.0 (Catchen et
 al., 2013), given that the B. bufo genome (Streicher et al. 2021) was not
 yet available at the time of the analyses. The raw sequences were filtered
 and demultiplexed using the process_radtags pipeline. Reads with an
 uncalled base were discarded, as were reads containing a 15 bp window in
 which the average quality dropped below a phred score of 10 (i.e., a 90%
 probability of being correct). Barcodes and RAD-tags containing one
 mismatch to an expected sequence were retained. To identify an optimal set
 of parameter values in STACKS, we followed procedures described in Paris
 et al. (2017) and Rochette & Catchen (2017). Based on these
 considerations, a value of 4 was chosen for both the M and n parameters,
 which control the number of mismatches permitted between two alleles of an
 individual heterozygote locus, and two alleles in a locus across a
 population, respectively.