Discussion:
We detected Bd DNA in all positive frog skin swabs collected across
multiple sampling locations and amphibian species using rapid
in-the-field methods. Mobile handheld real-time PCR thermocyclers are
promising tools for rapidly detecting Bd in susceptible amphibian
populations. Previously, to detect Bd in individuals, amphibian skin
swabs had to be stored to prevent DNA degradation, transferred out of
field, and processed in a lab. The difficulty of collecting and
processing swabs in a lab is compounded by species that inhabit remote
wilderness or otherwise difficult to access locations. The rapid,
in-situ method we applied yielded accurate results for all amphibians
swabbed in less than 60 minutes and did not require the transfer of
samples out of the field for lab analysis. Rapid detection of Bd is
critical to predict and/or minimize epizootic outbreaks (e.g., mass
die-off events) and initiating intervention / treatment options such as
salvaging animals for captive rearing efforts or on site anti-fungal
treatments (Harris et al., 2009). The ability to detect Bd presence in a
population in less than 60 min will significantly aid to the recovery of
MYLF and other Bd susceptible amphibians where high loads are expected
if Bd is present.
We also detected Bd DNA in eDNA samples at the same number of sites
using the rapid in-the-field method compared to traditional lab methods.
We did not detect Bd DNA for Site 3 using field analysis and we did not
detect Bd DNA for Site 2 using lab analysis (Table 1); swabs from both
sites tested positive as swabs samples have a higher probability of
detecting Bd DNA than eDNA samples (Fig. 4). The false negative result
in both the lab and field methods suggest that a more sensitive eDNA
surveillance strategy should be used (i.e. increase number and quantity
of water samples collected and/or increase number of technical
replicates), as generally recommended for eDNA surveys (Goldberg et al.,
2016).
We detected Bd DNA in more technical replicates for the samples
processed in the lab compared to samples processed in the field,
suggesting that our field-based methods may not be as sensitive.
Approximately 1.5 technical replicates would have to be analyzed using
the field protocol in order to have the same mean detection probability
of 1 technical replicate using traditional lab protocol. Our findings
are consistent with Sepulveda et al. (2018) who found lower detection of
northern pike in eDNA samples and subsamples processed with field
protocol compared to traditional lab techniques. As a result, the field
extraction approach failed to detect DNA in areas collected with low
densities of northern pike (Sepulveda et al., 2018). Additionally, we
used a liberal positive criterion where a sample was considered positive
if ≥ 1 technical replicate detected Bd DNA. A more conservative approach
where a sample is considered positive if ≥2 or 3 technical replicates
detects the target DNA, as is typically applied to eDNA analysis, would
likely increase false negative rates of the field-based protocol. eDNA
is typically used as a surveillance tool for rare or elusive species
(Rees et al., 2014) or for early detection/ monitoring for invasive
species (Jerde et al., 2011; Hunter et al., 2015; Kamoroff et al.,
2019); being able to detect trace amount of DNA in a water sample is
critical for successful use of eDNA techniques. Prior to the use of
rapid field techniques, further assessment should be made to ensure eDNA
samples (or other low-quality DNA samples) can be detected at low
quantities, and to assess false positive rate at the technical replicate
level.
All quantities of DNA detected using rapid in-the-field techniques were
below the standard curve, further evidence in the field methods lack
sensitivity. Binary detection (i.e. presence/absence) of Bd DNA is an
important metric for understanding disease dynamics and host risk.
However, DNA quantification of both eDNA and swab samples is critical to
the ecological interpretation of the results. Vredenburg et al. (2010)
found Bd prevalence increased rapidly and infection intensity increased
exponentially with declines of MYLF evident after average infection
intensity of ~10,000 zoospores swab-1.
Determining when Bd levels and infection intensities rapid/ exponential
grown before lethal threshold levels is critical for management to
implement conservation strategies. Such determination can only be
accomplished through accurate quantification of Bd load on skin swabs
and potentially eDNA samples.
Bd detection for conservation and management projects needs to be
reliable as well as able to meet budget and time constraints. Typical
costs for lab extraction and analysis of swabs and eDNA samples are
~$10-35 and ~$50-150 respectively
depending on type of lab, extraction method, and number of samples
processed. Typical qPCR machines used in lab analysis have a 96 well
capacity and can multiplex up to five targets per well resulting in a
high-volume throughput per run. The M1 sample prep kit for field-based
DNA and eDNA extraction cost was $15 per sample and the custom
Go-Strips™ were $10 per well (Biomeme inc, Philadelphia PA.). Total
cost of analysis using the Biomeme field methods is $45 for both eDNA
and DNA extractions run in triplicate wells. The two3™ mobile real-time
PCR machine has a three well capacity and can multiplex two targets per
well (the target species and an internal positive control). The limited
wells inherent to a small handheld qPCR machine will take much longer to
run a large number of samples compared to a lab-based machine. As a
result, for projects requiring high numbers of samples, lab-based
extraction and analysis may be more cost and time efficient.