Abstract
Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.
Highlights
A major consequence of globalisation has been the increase of invasive species owing to trade in live animals and plants
Batrachochytrium dendrobatidis (Bd), was originally thought to have caused the ongoing panzootic[7], we know that amphibian chytridiomycosis is caused by a much broader swathe of phylogenetic diversity than was previously thought[8,9]
Superimposed upon this background of trade-associated lineages of Bd has come the recent discovery of a new species of pathogenic chytrid, within the Rhizophydiales, B. salamandrivorans[13]
Summary
A major consequence of globalisation has been the increase of invasive species owing to trade in live animals and plants. Next-generation sequencing and phylogenomic analyses have shown that Bd sensu stricto is composed of deep genetic lineages which are emerging through international trade in amphibians[10,11,12] Superimposed upon this background of trade-associated lineages of Bd has come the recent discovery of a new species of pathogenic chytrid, within the Rhizophydiales, B. salamandrivorans[13]. ‘forewarned is forearmed’ and we wished to determine whether the protocol was able to isolate other species of chytrid that are members of the amphibian skin microbiota, and that may present a biosecurity risk This need to more broadly characterise global chytrid biodiversity was met by using resources from RACE to train researchers worldwide in chytrid isolation techniques to provide opportunities to characterise novel chytrids as they were discovered. We describe this method as a refinement to the main isolation protocol
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