Discussion
Although BAC is an effective tool for suppressing eDNA degradation in
water samples, its preservative performance has only been confirmed by
species-specific detection targeting shorter fragments of mitochondrial
genes. In the present study, targeting different fragment sizes of mtDNA
and nuDNA, we demonstrated that BAC suppressed the degradation of
various types of eDNA in seawater samples and increased eDNA yields.
Moreover, BAC addition suppressed the time-series changes in species
richness inferred by eDNA metabarcoding. Taking previous findings of BAC
performance in freshwater and brackish environments into account
(Yamanaka et al., 2017; Takahara et al., 2020), our findings indicated a
high versatility of BAC in preserving aqueous eDNA regardless of genetic
regions, DNA fragment sizes, and environmental conditions.
The tank and field experiments showed that BAC addition increased the
yield of Japanese jack mackerel eDNA at time 0 and suppressed the
degradation of eDNA. Similar tendencies were reported by Takahara et al.
(2020); even at the start of water collection, target eDNA
concentrations were higher in the treatment with BAC addition,
regardless of species. These results imply that both the suppression of
eDNA degradation and the increase in initial eDNA concentrations could
substantially contribute to the preservation of eDNA in water samplesvia BAC. Adding a surfactant such as BAC to water samples might
agglutinate a variety of suspended particles, including eDNA, which may
allow eDNA to be captured by a filter more frequently. The apparent
particle size distribution of eDNA in water samples might shift in the
larger size fraction by adding BAC. On the other hand, depending on
water quality, it is also possible for BAC to agglutinate PCR inhibitory
substances such as humic, fulvic, and tannic acids. Sales et al. (2019)
reported that the number of fish species detected by eDNA metabarcoding
(MOTUs) was slightly lower in samples stored at ambient temperature with
BAC addition than in those stored in a cooler box with ice. Such
tropical freshwater ecosystems are typically characterized by turbidity
due to high sediment loads and algae, and the result might thus have
included the effect of PCR inhibition by BAC.
We observed biphasic degradation of the target eDNA in the tank
experiment. Some previous studies estimating eDNA decay rates reported
similar processes of eDNA degradation and implied that a part of eDNA
degraded rapidly, and subsequently, the rest degraded slowly (Eichmiller
et al., 2016; Bylemans et al., 2018; Shogren et al., 2018). In
particular, Bylemans et al. (2018) reported that the initial rapid
degradation of eDNA might be caused by intra-cellular nuclease
activities and/or microbial digestion, and slower degradation might
reflect other degradation factors such as hydrolytic and oxidative
decomposition of DNA molecules. Considering that BAC inactivates
bacterial functions by adsorbing to their cell surfaces (Ziani et al.,
2011), this hypothesis is consistent with our findings that BAC
substantially suppressed the initial rapid degradation of eDNA but had
little effect on subsequent slower degradation in the tank experiment.
Moreover, Jo et al. (2019) reported that the inflow of degraded eDNA
from larger (e.g., intra-cellular DNA) to smaller size fractions (e.g.,
extra-cellular DNA) could prolong the apparent persistence of
smaller-sized eDNA compared to larger-sized ones. Altogether, BAC mainly
preserves intra-cellular eDNA, such as cell and tissue fragments, by
weakening microbial activities in water.
Conversely, in field experiments, we observed the monophasic degradation
of eDNA. This could simply be explained by a lower concentration of
target eDNA, a shorter experimental period, and fewer sampling time
points relative to those in the tank experiment. Alternatively, an
aerobic environment of seawater samples, where sampling tanks were
continuously aerated, might have inflated the decay rates and influenced
the degradation processes of aqueous eDNA in the tank experiment (Weltz
et al., 2017). In any case, the finding that eDNA in seawater samples
collected from the field scarcely degraded throughout the day by BAC
addition would indicate a high suitability of BAC for preserving eDNA in
marine ecosystems. Unfortunately, longer fragments of eDNA were rarely
detected in field samples, which could be improved by collecting water
samples in the warmer season because Japanese jack mackerels are
abundant in Maizuru Bay from July to August (Masuda, 2008). Jo et al.
(2017) actually detected 719 bp fragments of its mitochondrial eDNA
collected in the summer season here.
In addition to the species-specific analyses using quantitative
real-time PCR described above, we revealed that the richness of fish
communities inferred by eDNA metabarcoding did not vary among sampling
time points by BAC addition, although species richness decreased with
time without BAC addition. Surprisingly, the number of species detected
from seawater samples was not different throughout the day by BAC
addition. Smaller eDNA decay rates by BAC addition would allow the
detection of more fish species with low eDNA concentrations in seawater
samples. Although PERMANOVA tests showed the differences in community
compositions between BAC treatments but not time points, considering the
nMDS plot, it is likely that compositions between BAC treatments were
relatively similar just after seawater sampling (i.e., time 0), followed
by larger differences in compositions between BAC treatments over time
(i.e., time 6 and 24). Our study is the first to show that BAC is
effective in preserving qualitative eDNA information, such as species
richness, as well as quantitative information such as copy number. These
findings would partly support the reasonability of using BAC to preserve
community information inferred by eDNA metabarcoding from water samples,
including previous studies (e.g., Hayami et al., 2020; Sakata et al.,
2020b).