Abstract
Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80–90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis.
Highlights
Natural history collections are becoming increasingly important for ecological and conservation research [1, 2], facilitating studies as diverse as those documenting the effects of environmental contaminants [3] and morphological responses to anthropogenic climate change [4]
None of the optimizations conducted on PM-extracted swabs increased Batrachochytrium dendrobatidis (Bd) detection success or zoospore equivalents (ZE) values
The greatest increase in Bd detection resulted from using the Macherey-Nagel DNA FFPE (MN) spin column-based DNA extraction kit
Summary
Natural history collections are becoming increasingly important for ecological and conservation research [1, 2], facilitating studies as diverse as those documenting the effects of environmental contaminants [3] and morphological responses to anthropogenic climate change [4]. The “spreading pathogen” hypothesis [15] posits that Bd is a novel pathogen that has recently dispersed around the world. This hypothesis has been the focus of much recent research (as reviewed in [16]) and has been supported by both genetic [17,18,19,20] and spatiotemporal data [21, 22]. Museum specimen collections have an increasingly important function in enabling ecologists to address specific questions about the potential role of Bd in historical amphibian populations [2]. Patterns of Bd emergence and spread have been deduced from work with archived amphibian specimens [7, 23,24,25,26,27,28,29,30], using both histological and polymerase chain reaction (PCR) techniques
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