10.5061/DRYAD.9S4MW6MJB
Smith, Donal
0000-0002-6093-3406
Institute of Zoology
O'Brien, David
Highland Amphibian and Reptile Project
Hall, Jeanette
Highland Amphibian and Reptile Project
Sergeant, Chris
Institute of Zoology
Brookes, Lola
Institute of Zoology
Harrison, Xavier
Institute of Zoology
Garner, Trenton
Institute of Zoology
Jehle, Robert
University of Salford
Challenging a host-pathogen paradigm: Susceptibility to chytridiomycosis
is decoupled from genetic erosion
Dryad
dataset
2022
FOS: Biological sciences
amphibian populations
Bufo bufo
Batrachochytrium dendrobatidis
SNPs
dd-RADseq
Natural Environment Research Council
https://ror.org/02b5d8509
NE/N009967/1
Natural Environment Research Council
https://ror.org/02b5d8509
NE/S000992/1
Research England
https://ror.org/02wxr8x18
University of Salford
https://ror.org/01tmqtf75
Pathway to Excellence studentship
2022-03-08T00:00:00Z
2022-03-08T00:00:00Z
en
10716548 bytes
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CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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 >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.
Experimental infections 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.SNP genotyping 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.