DOI: 10.5061/dryad.x0k6djhhx
title: Population genetics as a tool to elucidate pathogen reservoirs: Lessons from Pseudogymnoascus destructans, the causative agent of White-Nose disease in bats

description: Emerging infectious diseases pose a major threat to human, animal, and
 plant health. The risk of species-extinctions increases when pathogens can
 survive in the absence of the host. Environmental reservoirs can
 facilitate this. However, identifying such reservoirs and modes of
 infection is often highly challenging. In this study, we investigated the
 presence and nature of an environmental reservoir for the ascomycete
 fungus Pseudogymnoascus destructans, the causative agent of White-Nose
 disease. Using 18 microsatellite markers, we determined the genotypic
 differentiation between 1,497 P. destructans isolates collected from nine
 closely situated underground sites where bats hibernate (i.e.,
 hibernacula) in Northeastern Germany. This approach was unique in that it
 ensured that every isolate and resulting multi-locus genotype was not only
 present, but also viable and therefore theoretically capable of infecting
 a bat. The distinct distribution of multi-locus genotypes across
 hibernacula demonstrates that each hibernaculum has an essentially unique
 fungal population. This would be expected if bats become infected in their
 hibernaculum (i.e., the site they spend winter in to hibernate) rather
 than in other sites visited before they start hibernating. In one
 hibernaculum where both the walls and the hibernating bats were sampled at
 regular intervals over five consecutive winter seasons (1,062 isolates),
 higher genotypic richness was found on walls compared to bats and
 multi-locus genotypes showed a stable frequency over multiple winters.
 This clearly implicates hibernacula walls as the main environmental
 reservoir of the pathogen, from which bats become re-infected annually
 during hibernation.
description: To genotype isolates of <i>P. destructans</i>, 18
 microsatellite markers (Drees et al., 2017b) were used in 4 PCR
 multiplexes as described in Dool et al. (2020). Genotyping was carried out
 using an ABI 3130 Genetic Analyser (Applied Biosystems). GeneMapper®
 Software v.5 (Applied Biosystems) was used for fragment
 analysis. From Dool et al. (2020): "Eighteen
 microsatellite loci and the three mating type markers (apn2_Short,
 sla2_Short with two forward primers and sla2_Long) were optimised into
 four multiplex reactions (Table 2). Reactions were carried out in 6 μL
 volumes containing 1 μL of DNA extract, 1 × Type-it Microsatellite PCR
 Master Mix (QIAGEN) and primer concentrations as reported in Table 2.
 Amplification conditions were: 5 min at 95 °C; 30 cycles of 95 °C for 30
 s, 58 °C for 90 s, 72 °C for 30sec; 30 min at 60 °C. PCR products were run
 on an ABI PRISM 3130XL Genetic Analyser (Applied Biosystems) and sized
 with the internal lane standard 600-LIZ using GeneMapper v.5.0 (Applied
 Biosystems)". Dool SE, Altewischer A, Fischer NM,
 et al. (2020) Mating type determination within a microsatellite multiplex
 for the fungal pathogen <em>Pseudogymnoascus destructans</em>,
 the causative agent of white-nose disease in bats. Conservation Genetics
 Resources 12, 45-48. Drees KP, Parise KL, Rivas SM, et
 al. (2017) Characterization of microsatellites in
 <em>Pseudogymnoascus destructans </em>for White-nose Syndrome
 genetic analysis. Journal of Wildlife Diseases 53, 869-874.
description: Information for the file 'Data_Reservoir_v1.csv'
 containing all the data used in the manuscript.
 culture: name of the culture country: country
 where the sample was collected province: province where
 the sample was collected site: city where the sample
 was collected date: date when the sample was
 collected labNo: name to identify all cultures from the
 same sample (e.g. 'Gd00298': three cultures [Gd_00298-aa,
 Gd_00298-ab, Gd_00298-ac] with the 'Gd00298' identifier have
 been made from sample '20150302_B_ELD_21')
 sample: sample (swab) name species: bat
 species from which the sample was collected (if the sample was collected
 from a bat) substrate: substrate from which the sample
 was collected, here 'bat' or 'wall'
 mtype: mating type inferred from the 'Sla2_Long' and/or
 'Sla2_Short' markers sampling_event: combined
 information on year of sampling, season, and substrate (used in Table 3
 for example) winter: winter season (used in Figure 4
 for example) Pd1- Pd22: microsatellite allele
 ('NA': missing value) Sla2_Long: mating-type
 marker (519: MAT1-1; 547: MAT1-2; 'NA': missing value)
 Sla2_Short: mating-type marker (240: MAT1-1; 208: MAT1-2;
 'NA': missing value)
 ####################################################################### The file 'Rscript_V8.R' containins the R script used to analyse the data contained in the file 'Data_Reservoir_v1.csv'. The script is annotated.