Introduction:
Chytridiomycosis is a skin disease caused by the invasive amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd; Longcore et al., 1999). Bd is affecting amphibian populations worldwide and is linked to mass extirpation and extinction of over 200 amphibians (Berger et al., 1998; Stuart et al., 2004; Skerratt et al., 2007). In the California Sierra Nevada, historical records show that Bd has been present since the 1970s (Fellers et al., 2001). Bd has been linked to the precipitous declines of two endemic and endangered species of yellow-legged frogs, Sierra Nevada yellow-legged frog (Rana sierrae ) and southern mountain yellow-legged frog (R. muscosa ), collectively referred to as the mountain yellow-legged frog complex (Rachowicz et al., 2005; Vredenburg et al., 2009) and referred hereafter as MYLF.
Prior to the arrival of Bd, MYLF populations were already diminished due to the introduction of non-native trout, which prey on tadpoles and adults (Knapp & Mathews, 2000). Nonnative trout also fragment and isolate MYLF populations by occupying and barring dispersal corridors as well as adjacent water bodies (Bradford & Graber, 1993). Bd has caused declines and localized extirpations of many persisting populations in the few remaining fish-free habitats in the Sierra Nevada (Vredenburg et al., 2010). Given these two prominent causes for decline, MYLF species are listed as endangered by the IUCN (2008; IUCN), the state of California (2003; California Department of Fish and Wildlife), and U.S. Fish and Wildlife Service (2014; U.S Fish and Wildlife Service)
There are few remaining Bd-naïve MYLF populations on protected public lands (e.g., National Parks). The remaining populations are likely susceptible to mass die-off events once Bd is introduced (Rachowicz et al., 2005; Vredenburg et al., 2010; Knapp et al., 2016). Bd positive MYLF populations persisting with the disease are thought to have an adaptive immunity response (Knapp et al., 2016), but both population types (naïve and persisting) require monitoring for adaptive management (e.g., translocation and augmentation), and treatment options such as intervention with antifungal agents at the onset of mass die-off events (Harris et al., 2009).
Established techniques for Bd detection include swabbing keratinized skin on frogs and mouthparts of tadpoles, followed by analyzing the swabs for Bd zoospores using quantitative polymerase chain reaction (qPCR) techniques (Boyle et al., 2004). More recently, Bd has been detected using environmental DNA (eDNA) techniques (Kirshtein et al., 2007; Walker et al., 2007; Hyman & Collins, 2012; Chestnut et al., 2014). eDNA is a non-invasive alternative survey tool that is not dependent on finding and handling a host organism and can potentially increase detection of aquatic pathogens when few individuals are infected. Species detection using eDNA methods is accomplished by collection and identification of trace DNA particles that are extracted from water samples (Taberlet et al., 2012). A recent study in the Sierra Nevada detected Bd in water samples prior to a MYLF chytridiomycosis die-off event (Kamoroff & Goldberg, 2017).
The National Park Service (NPS) is actively monitoring and managing populations of endangered MYLF in Yosemite National Park (YNP). The NPS and their partners are currently collecting swabs from a known Bd- naïve population to determine if an outbreak is imminent as well as collecting samples from persisting Bd- positive populations to determine Bd- load and epizootic disease dynamics in park populations. Early and accurate detection as well as reliable quantification of Bd is a critical component in managing overall MYLF recovery, a task compounded by the difficulty of reaching occupied sites as MYLF populations inhabit high elevation (>1830 m) alpine lakes often in remote wilderness or wilderness-like settings.
In this study, we analyzed the effectiveness of using an in-situ DNA extraction method combined with a handheld mobile thermocycler for real-time qPCR analysis in the field (Biomeme Inc. Philadelphia, PA). The NPS currently uses lab-based DNA and eDNA extraction/analysis methods for surveillance of Bd across YNP (Yosemite unpublished data). However, the lab-based approaches require hiking samples ≥10 miles out of the field, followed by additional transport time to a temporary storage facility, and further delay during shipping and lab processing which can result in a minimum turnaround time of weeks to months. Our goal was to circumvent this process by rapidly detecting and quantifying Bd DNA using extracted samples collected and analyzed directly in the field. The field-based platform for DNA extraction and mobile real-time qPCR analysis yield results in less than 60 minutes and does not require hiking samples out of the field for lab analysis. We compared the results of the field-based DNA extraction and analysis approach with lab-based extraction and analysis using two sampling strategies, frog skin swabs and eDNA filtered water samples.