Introduction
Effective monitoring of species distribution and abundance is the first
step in the conservation of biodiversity and ecosystems (Margules &
Pressey, 2000), as well as the proper management of fishery resources
(Jackson et al., 2001). However, traditional methods that rely on
capturing and morphological identification of species require
substantial effort and cost, resulting in insufficient and biased
monitoring and damage to individuals and their habitats (Thomsen &
Willerslev, 2015). To overcome these limitations, analysis of
environmental DNA (eDNA), which is defined as the total pool of DNA
isolated from environmental samples (Pawlowski et al., 2020), has been
developed (Ficetola et al., 2008; Minamoto et al., 2012; Deiner et al.,
2017a). Macro-organisms such as fish are reported to produce eDNA from
mucus, scale, feces, and gametes (Barnes & Turner, 2016). The PCR-based
detection of eDNA in water samples enables non-invasive and
cost-effective surveillance of species distribution and composition in
aquatic ecosystems (Takahara et al., 2013; Yamamoto et al., 2017; Lawson
Handley et al., 2019); thus, eDNA analysis is a promising tool for
biological conservation and fishery resource management.
To achieve high accuracy and reliability of eDNA detection and
quantification, eDNA must be preserved as soon as possible after water
sampling because of its rapid degradation. There are a variety of
preservation strategies for aqueous eDNA (Table 1), which primarily
depend on whether water filtration is performed in the field (on-site)
or in the laboratory (in-lab). On-site filtration allows the immediate
storage of filter samples via the addition of a buffer (Renshaw
et al., 2015; Spens et al., 2017) or desiccation (Thomas et al., 2019),
whereas in-lab filtration is generally feasible to maximize the number
of sampling sites per survey, as water filtration in the field is not
required. In case of in-lab processing, to suppress eDNA degradation
during transportation to the laboratory, water samples have been chilled
and frozen (Takahara et al., 2015; Jo et al., 2020a), precipitated using
organic solvents (Doi et al., 2017; Ladell et al., 2019), and directly
added with Longmire’s buffer after collection (Williams et al., 2016).
Recently, benzalkonium chloride (BAC) has been used as an inexpensive
and simple preservative for macrobial eDNA in water samples (Yamanaka et
al., 2017). BAC is a cationic surfactant that inhibits bacterial
function by adsorbing onto their cell surfaces (Ziani et al., 2011). The
preservation strategy does not necessarily require elaborate work (e.g.,
the use of a pipette) and an equipment to chill the sample (e.g., cooler
box and refrigerator). Yamanaka et al. (2017) reported that the addition
of BAC at a final concentration of only 0.01% preserved 92% of
bluegill sunfish (Lepomis macrochirus ) eDNA in water samples
after 8 hours at ambient temperature (~ 25 °C) compared
to only 14% in untreated water samples. Moreover, BAC addition allowed
the retention of 50% of target eDNA in water samples after 10 days
compared to non-detection in untreated water. To the best of our
knowledge, BAC treatment is among the most suitable eDNA preservation
strategies to maximize both the number of sampling sites and sampling
volume (hundreds to thousands of milliliters). It allows intensive
monitoring of species distribution and abundance via eDNA
analysis over a short period of time.
Nevertheless, the performance of BAC in eDNA preservation has not
necessarily been evaluated fully because most eDNA studies using BAC
targeted short fragments (up to 200 bp) of mitochondrial DNA (mtDNA) in
freshwater ecosystems (e.g., Sakata et al., 2017; Yamanaka et al., 2017;
Hayami et al., 2020). Therefore, the present study investigated the
performance of BAC in preserving eDNA in water samples from three
aspects: (i) genetic region, (ii) DNA fragment size (i.e., the length of
PCR amplicon), and (iii) marine ecosystems. First, given the possibility
and prospect of using nuclear DNA (nuDNA) and longer DNA fragments in
eDNA analyses for population-level inferences, such as population status
and genetic diversity (Deiner et al., 2017b; Sigsgaard et al., 2020), it
is important to verify whether BAC can be effective in preserving them
from degradation. Moreover, BAC has only been applied to brackish water
in a single experiment by Takahara et al. (2020); however, no study has
examined its performance in eDNA preservation targeting seawater
samples. Some water chemistry parameters, such as pH, salinity, and
ionic content, are generally higher in marine systems than in freshwater
systems (Okabe & Shimazu, 2007; Collins et al., 2018), which may affect
the performance of BAC in eDNA preservation. Using Japanese jack
mackerel (Trachurus japonicus ), an economically important marine
fish in East Asia, including Japan, we examined the preservative
performance of BAC targeting different fragment sizes of nuDNA and mtDNA
in seawater samples. Furthermore, Yamanaka et al. (2017) anticipated
that BAC should enable the preservation of community information
inferred by eDNA metabarcoding; however, this has not been verified yet.
Thus, we performed eDNA metabarcoding using MiFish primers (Miya et al.,
2015) and examined whether BAC could be effective in preserving genetic
information of fish communities.