Metagenomics analysis of cutaneous microbiome of New Zealand’s endemic frogs (Genus Leiopelma) associated with Batrachochytrium dendrobatidis

Abstract

Hundreds of frog species worldwide have been declining due to a deadly fungal infection known as chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). It has been acknowledged as a major threat to frogs causing mass die-offs and even species extinctions. Interestingly, in New Zealand, species in one of the most distinctive genera of frogs, Leiopelma, may not be at high risk of chytridiomycosis as, in the laboratory at least, they have demonstrated an ability to clear Bd infection. To date, no studies have been conducted as to how these endemic New Zealand frogs are able to counteract the deadly fungus and we do not have information on the current prevalence of Bd in New Zealand. This project explored one of the factors that may contribute to Bd resilience: the role of skin microorganisms and whether they contribute to Bd defence. Leiopelma frogs were swabbed, and the PrepMan Sample Preparation Reagent Kit used to extract DNA. This DNA was screened for the presence of Bd and to describe the skin microbiome through next-generation sequencing. I found that Bd still persists in several wild and captive populations of L. hamiltoni and L. archeyi, but the prevalence was low (6.8%) across six sampling locations in New Zealand. A comprehensive analysis of the cutaneous microbiome in terrestrial Leiopelma species using next-generation sequencing showed that Bd has little influence on the microbial diversity of Bd-positive frogs. Differentially abundant taxa that belong to order Bacillales (bacteria), phylum Ascomycota, class Microbotromycetes, and species Aspergillus flavus (fungi) were found to be associated with the presence of Bd. In addition, Leiopelma skin was rich with putatively inhibitory bacteria based on the antifungal isolates database (Woodhams et al., 2015). This database curates a collection of skin bacteria isolated from different species of frog that were found to inhibit Bd in vitro. Among these putatively inhibitory bacteria, the following cultured bacteria from Leiopelma skin did indeed exhibit actual inhibition of Bd growth in vitro after performing Bd inhibition assays: Chryseobacterium, Flavobacterium, Sphingobacterium, Janthinobacterium lividum, Pseudomonas, Stenotrophomonas, and family Yersinniaceae. Three of these bacterial taxa, Janthinobacterium, Pseudomonas, and Chryseobacterium, demonstrated strong antifungal activity against Bd by clearing the growth of Bd from the entire inhibition assay plate. Taken together, these findings suggest that L. hamiltoni and L. archeyi may be benefiting from protection from its skin microbiome in eliminating Bd growth and preventing chytridiomycosis in wild and captive populations. This study adds to the growing number that support the idea that anti-Bd microbes play an important role in the resistance of Leiopelma species to chytrid infection in populations that coexist with Bd.