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.