Unlocking the story in the swab: A new genotyping assay for the amphibian chytrid fungus Batrachochytrium dendrobatidis

Abstract

One of the most devastating emerging pathogens of wildlife is the chytrid fungus, Batrachochytrium dendrobatidis (Bd), which affects hundreds of amphibian species around the world. Genomic data from pure Bd cultures have advanced our understanding of Bd phylogenetics, genomic architecture and mechanisms of virulence. However, pure cultures are laborious to obtain and whole-genome sequencing is comparatively expensive, so relatively few isolates have been genetically characterized. Thus, we still know little about the genetic diversity of Bd in natural systems. The most common noninvasive method of sampling Bd from natural populations is to swab amphibian skin. Hundreds of thousands of swabs have been collected from amphibians around the world, but Bd DNA collected via swabs is often low in quality and/or quantity. In this study, we developed a custom Bd genotyping assay using the Fluidigm Access Array platform to amplify 192 carefully selected regions of the Bd genome. We obtained robust sequence data for pure Bd cultures and field-collected skin swabs. This new assay has the power to accurately discriminate among the major Bd clades, recovering the basic tree topology previously revealed using whole-genome data. Additionally, we established a critical value for initial Bd load for swab samples (150 Bd genomic equivalents) above which our assay performs well. By leveraging advances in microfluidic multiplex PCR technology and the globally distributed resource of amphibian swab samples, noninvasive skin swabs can now be used to address critical spatial and temporal questions about Bd and its effects on declining amphibian populations.

Hybrids of amphibian chytrid show high virulence in native hosts

“…Nevertheless, their coexistence with Bd -GPL suggests that they could be adept competitors in Brazilian landscapes. Applying newly developed, non-invasive techniques that can discriminate among Bd genotypes on skin swabs would help to paint a more complete picture of the spatial distribution of Bd lineages in Brazil as well as coinfection dynamics 82 . The same techniques could also be used with contemporary and retrospective sampling of preserved specimens to increase our understanding of which genotypes and lineages are associated with amphibian mortality in Brazil, track the spread of Bd -GPL through time, and determine whether the geographic distributions of hybrid genotypes and Bd -Brazil have changed over time 82 .…”

“…Applying newly developed, non-invasive techniques that can discriminate among Bd genotypes on skin swabs would help to paint a more complete picture of the spatial distribution of Bd lineages in Brazil as well as coinfection dynamics 82 . The same techniques could also be used with contemporary and retrospective sampling of preserved specimens to increase our understanding of which genotypes and lineages are associated with amphibian mortality in Brazil, track the spread of Bd -GPL through time, and determine whether the geographic distributions of hybrid genotypes and Bd -Brazil have changed over time 82 . Another useful avenue for future study is to determine how coexistence and hybridization of Bd genotypes in Brazil could influence host population recoveries through adaptive responses, which have been documented in other regions in the decades following mass declines 83 , 84 .…”

Amphibian skin fungal communities vary across host species and do not correlate with infection by a pathogenic fungus

“…UCLUST), which has a low resolution to resolve finescale variation in amplicon data (Callahan et al, 2017), and can overestimate the number of OTUs in a dataset relative to other algorithms (Callahan et al, 2017;Caruso et al, 2019). In addition, other studies have found that using multiple molecular markers is necessary to accurately identify Bd genotypes (Byrne et al, 2017;O'Hanlon et al, 2018). We think this is worthy of further exploration, as it might influence the design and conclusions of studies using these methods.…”

Opening the file drawer: Unexpected insights from a chytrid infection experiment

“…Initially we could not explain either of these results until we reexamined two key factors in our experiment. First, using a new genotyping assay we developed [ 36 ], we determined that the R . catesbeiana control frogs were infected with a distinct Bd genotype that had not previously been present in our lab.…”

“…catesbeiana , 6 Bd-exposed R . muscosa ) collected during weeks 2, 4, and 8 of the experiment using a custom genotyping assay (see [ 36 ]). Briefly, this assay uses microfluidic multiplex PCR to amplify select regions of the Bd genome using DNA collected from swab samples.…”