2 Key aspects and challenges in eDNA monitoring of
amphibians
2.1 Sample collection and DNA
capture
Amphibians exhibit a bifurcated life history, comprising both aquatic
and terrestrial phases. In the former, their eDNA is disseminated into
the aquatic milieu via exudates from integumentary secretions, excreta,
and ova deposition. In the latter, eDNA is presumed to derive from
imprints generated during their terrestrial locomotion, manifesting as
soil or sedimentary remnants. Consequently, a comprehensive scrutiny of
amphibian eDNA mandates the simultaneous procurement of aquatic and
terrestrial substrates. The sources of collecting amphibian
environmental DNA samples are mainly water, soil, and other natural
substrates. Some studies have demonstrated that detecting terrestrial
vertebrates from soil samples is challenging and that eDNA testing is
more complex under these circumstances. For example, Walker et al.
cultured salamanders in a mesocosm ecosystem and found that only 1% of
salamander DNA from soil samples could be successfully amplified (Walker
et al., 2017). The success of applying the eDNA method for detection
depends not only on the presence and concentration of eDNA in the
substrate sample but also on capture potency, DNA extraction success,
sample interference (such as inhibition), and assay sensitivity
(Ficetola et al., 2015; MacKenzie et al., 2002; Schultz & Lance, 2015).
Filtration is the standard approach for the capture of DNA from aquatic
environments (Goldberg et al., 2016); generally, volumes between 15 mL
and 10 L of water are sampled to trap eDNA in a filter. In contrast to
fish, amphibians may exhibit comparatively modest eDNA exudation. Some
judicious augmentation of filtrate volume may therefore be considered to
enhance the sensitivity of detection. Common filtration materials
include nitrate cellulose (Caren S Goldberg et al., 2011), glass fiber
(Jerde et al., 2011), polycarbonate (Takahara et al., 2012), nylon
(Philip Francis Thomsen et al., 2012) and polyether sulfone (Renshaw et
al., 2015). Chen et al. evaluated the ability of 12 different types of
adsorption materials and filters to capture eDNA in both laboratory and
field experiments using quantitative PCR (qPCR) analysis. They found
that the ability of a glass fiber filter (GF) to capture eDNA was
superior to other materials, and that the amount of eDNA captured
increased linearly over 72 hours (Chen et al., 2022).
It has been demonstrated that the period of detectability of eDNA in
water samples is limited to between 1 day and 8 weeks (Dejean et al.,
2011; Pilliod et al., 2014; Philip Francis Thomsen et al., 2012; P. F.
Thomsen, J. Kielgast, L. L. Iversen, C. Wiuf, et al., 2012), and
therefore false negative results may be obtained if there is a delay in
the collection of water samples relative to the time of species
presence. In addition, temperature alterations caused by seasonal
changes will also affect the content of eDNA in samples. For example,
Wei et al. studied the variation over time of eDNA in sediments over
periods of one year, one month and one tidal period, and found that eDNA
content was higher in the warm season and lower in the cold season (Wei
et al., 2019). Similarly, the concentration and distribution of eDNA can
be influenced by spatial factors such as spatial dissipation of eDNA and
the flow of the water column. In still water, the eDNA of species
decreases significantly within a radius of 5–10 m from the eDNA point
source (Brys et al., 2021), while in flowing waters, eDNA may move with
the current to new locations hundreds of meters away (Pilliod et al.,
2013), leading to false negatives at the original point as well as false
positives at the new location.
Therefore, biases in results due to temporal and spatial differences
need to be taken into account when designing eDNA experiments, and these
factors also need to be considered when conclusions are drawn about the
range of species present. For example, conclusions drawn from water
samples taken along one side of a river are not directly representative
of the whole river.
2.2 DNA extraction
During amphibian DNA extraction, pollution should be avoided and the
facilities and equipment employed should be disinfected. Standard
autoclave methods are not sufficient to destroy nucleic acid
molecules(Unnithan et al., 2014). Among common stain removal methods,
sodium hypochlorite solvent is the most effective for removing DNA and
PCR products (Champlot et al., 2010; Prince & Andrus, 1992). The choice
of eDNA extraction method after filtration can greatly affect the
detection of eDNA. Deiner et al. compared the effectivity of six
different combinations of sample collection and DNA extraction methods
in biodiversity monitoring in water ecosystems. The results showed that
different environmental DNA collection and extraction methods had a
significant effect on DNA yield and the number of sequences obtained by
high-throughput sequencing techniques (Deiner et al., 2015).
Common DNA extraction methods include liquid phase separation and
commercial DNA extraction kits. Trials have shown that liquid phase
separation methods can in some cases outperform kits in terms of
extraction efficiency. Turner et al. showed that a
cetyltrimethylammonium bromide (CTAB) method using polycarbonate orbital
filters collected more eDNA than a PowerWater DNA isolation kit using GF
membranes (1.5 µm pore size, 934-AH grade) (Turner et al., 2014). It has
also been shown that the phenol-chloroform-isoamyl alcohol extraction
(PCI) method using glass and nitrocellulose membranes extracted more DNA
than the DNeasy kit (Deiner et al., 2015; Renshaw et al., 2015).
However, liquid phase separation methods are difficult to apply widely
due to the utilization of toxic substances such as phenol and chloroform
(Tsuji et al., 2019).
2.3 Primer selection and PCR
amplification
Species-specific detection is carried out the basis of PCR techniques
that use specific primers to amplify and detect short segments of DNA
(typically 80–200 bp) of the target species (Bohmann et al., 2014).
eDNA was first applied to large organisms using specific primers that
amplified only one species during PCR (Jerde et al., 2011; P. F.
Thomsen, J. Kielgast, L. L. Iversen, C. Wiuf, et al., 2012). Advances in
sequencing technology have made it possible to assess the community
composition using universal primers that identify multiple species in a
sample; universal primers are required to amplify all species in a
target taxonomic unit with a minimal bias to avoid preferential
amplification of some species with mismatched primer regions (Ficetola
et al., 2010). The mitochondrial genes 12S and Cytochrome
b and the mitochondrion’s D-loop region are commonly used in amphibian
studies, and the 12S region is often used for metabarcoding eDNA
analysis (Table 1) (Li et al., 2021; Wynne E Moss et al., 2022; Sasso et
al., 2017). For example, Valentini et al. designed a pair of primers
specific for amphibians and scleractinians (batra_F,
5’-ACACCGCCCGTCACCCT-3,’ batra_R, 5’-GTAYACTTACCATGTTACGACTT-3’) and a
human DNA blocking primer (batra_blk, 5’-
TCACCCTCCTCAAGTATACTTCA-AAGGCA-SPC3I-3’); this primer pair can amplify
up to 99% of amphibian species (Valentini et al., 2016). In a previous
study, we employed GF filtration membranes in conjunction with the
“batra” primers to investigate the amphibian diversity within China’s
Chao Lake(Unpublished data). This approach not only facilitated the
successful amplification of all species identified through traditional
sampling methods but also unveiled two species that had previously
eluded detection in historical records and conventional surveys. These
outcomes substantiate the efficacy of the ’batra’ primers for detecting
Chinese amphibians.
Because it is impossible to pinpoint organisms directly based on eDNA
molecules exuded into water, nor to track the movement of specific eDNA
molecules in water, the environmental samples collected often contain
mixed DNA from many different organisms. When amplifying environmental
samples, the specific primers chosen often play an important role in the
successful amplification of the target species. Unsuitable primers have
the potential to generate false positives, which can lead to
overestimates of the presence or abundance of endangered species, or to
false negatives, which can lead to insufficient monitoring of invasive
species. In his study, Kelly pointed out that using a constant number of
PCR cycles, different primers showed different biodiversity structures
for the same simulated community (Kelly et al., 2019), which directly
led to different analysis results. Wilcox tested the factors affecting
the specificity and sensitivity of eDNA detection, and found that
specificity was most affected by the mismatch of base pairs in the
primers, which was much more prevalent than in the probe (Wilcox et al.,
2013). Hence, it is evident that the meticulous selection of appropriate
primers is of paramount importance.
\begin{equation}
Cq=(\text{Cq}_{\text{samples}}-\text{Cq}_{\text{positive\ control}})\nonumber \\
\end{equation}It has been demonstrated that diluting the sample alleviates the
inhibition, but likewise dilutes the target DNA. Especially when the
target DNA is present at very low concentrations, this may lead to
failure of the assay (Goldberg et al., 2013; Anna M McKee et al., 2015;
Strand et al., 2011; Tsai & Olson, 1992). McKee et al. examined this
using a PCR inhibitor removal kit and found some alleviation of
inhibition but also potential loss of target DNA (Anna M McKee et al.,
2015).
2.4 Sequencing and data
analysis
The PCR results are recorded as negative or positive. A negative result
indicates that eDNA of the target species is not present in the
environmental sample, while a positive result indicates its presence.
This result is initially determined by GEP after primer amplification
and then further verified by sequence comparison. In qPCR, a
fluorescence threshold is set using the positive control as a benchmark;
the sample is labelled positive when the amplification exceeds the
fluorescence threshold, and negative when it does not (Venkatesan &
Bashir, 2011). Metabarcoding uses universal PCR primers to amplify DNA
in the environment. A large amount of data containing taxonomic
information on genes can be obtained after high-throughput sequencing,
which requires sequence comparison and species annotation (including
noise removal and assignment of operational taxonomic units),
clustering, and assignment of species annotation.
A complete and high quality reference database is the basis for the
accuracy of amphibian surveys using eDNA technology (Alexander et al.,
2020; West et al., 2022). This can be constructed by consulting
established publicly available databases (e.g. NCBI:
https://www.ncbi.nlm.nih.gov/) or by building custom versions. For
example, Valentini et al. built a reference database containing 53
species of amphibians when using eDNA method to investigate amphibian
diversity (Valentini et al., 2016). The accuracy of self-built databases
is likely to be higher than that of public databases due to the
significant geographic variation in amphibian fauna and the lack of
assurance of the quality of DNA barcoding in publicly available
databases.