Extracting the amphibian chytrid fungus from formalin-fixed specimens

Abstract

Batrachochytrium dendrobatidis is a chytrid fungal pathogen driving many amphibian species extinct. For some species, the only place they can be found is in natural history museum collections. Harnessing molecular tools to uncover this pathogen's date of arrival and movement throughout a region and through time will prove useful to broader B. dendrobatidis research. However, it has remained difficult to access B. dendrobatidis DNA from within preserved host tissues, especially specimens that were originally fixed in formalin. Herein, I describe two methods to extract and detect, via qPCR, B. dendrobatidis DNA from herpetological natural history collections. Both extraction methods use a DNA-binding matrix, either a magnetic bead resin or a silica membrane, that retains and separates the extracted DNA from potentially qPCR-inhibiting contaminates. Nine positive control specimens enabled initial method optimizations and coincidently limited empirical comparisons between each extraction method. A museum study involving 164 formalin-fixed amphibians from Connecticut, either having been originally fixed in the 1960s or 2000s, were swabbed and samples extracted by one of the two presented methods. Each method successfully extracted B. dendrobatidis DNA; of the 164 specimens tested, 37 were found B. dendrobatidis-positive, including six specimens (representing three caudate species) from 1968. In addition, results show that an infected individual stored in a jar of multiple specimens does not always contaminate the other specimens with B. dendrobatidis. These results suggest that qPCR methods are well suited to assess B. dendrobatidis presence but to not assign zoospore loads in preserved specimens. © 2012 The Author. Methods in Ecology and Evolution © 2012 British Ecological Society.