Abundance estimation depending on eDNA particle size and size
fraction
The aquarium experiment using zebrafish revealed that regardless of DNA
marker length, eDNA particles collected by a 10 µm pore size filter
could not explain the fish abundance more accurately than those managed
by other smaller pore size filters. Notably, we observed higherR2 values between the shortest eDNA fragments
(132 bp) and fish abundance by increasing the filter pore sizes.
However, the value radically decreased when using the 10 µm pore size
filter, suggesting that the relationship between eDNA concentration and
species abundance can be worsened when such “huge” eDNA particles and
extremely larger size fractions are targeted. As expected in the
Introduction, massive eDNA particles, such as tissue clumps, reflect the
species abundance less precisely because of their potential
heterogeneous dispersion and distribution in water (Turner et al., 2014;
Furlan et al., 2016; Jo et al., 2020b). Moreover, mainly in natural
environments, eDNA particle sizes can be increased by adsorbing
suspended organic matter, making DNA molecules physiochemically stable
and able to survive longer in water (Levy-Booth et al., 2007; Torti et
al., 2015). Therefore, detecting such eDNA is likely to reflect past
biological information and obscures current species distribution and
abundance (Barnes & Turner, 2016; Jo et al., 2021).
On the contrary, this study indicates that eDNA particles at the 3–10
µm size fractions could be important for better performance of abundance
estimation. First, the zebrafish eDNA particles collected by a 3 µm pore
size filter reflected the fish abundance significantly better than those
managed by a 10 µm pore size filter, corresponding to the result from
the experiment targeting the upside-cumulative eDNA from T.
japonicus . Second, for the downside-cumulative eDNA from Japanese jack
mackerel, the R2 values between the target eDNA
concentration and fish biomass were significantly lower at the 0.4–3 µm
size fraction (i.e., excluding the >3 µm size fraction)
than at the >0.4 µm size fraction, whereas little
difference in the R2 values between the
>0.4 and 0.4–10 µm size fractions existed (i.e., even
excluding the >10 µm size fraction). Third, in the
zebrafish experiment, the slopes of the linear regressions (the
sensitivity of the eDNA concentration in response to changes in species
abundance) were significantly higher for the eDNA particles collected by
a 3 µm pore size filter than those managed by a 10 µm pore size filter.
Summarily, these results suggest that an “appropriately” larger eDNA
particle is suitable for accurate and sensitive estimation of the
species abundance via eDNA.
The 3–10 µm size fraction is the fraction in which macrobial eDNA in
water is most abundant (Turner et al., 2014; Jo et al., 2019; Zhao et
al., 2021). It is possible that the size fraction mainly includes a
single cell and intact nuclei and mitochondria of known sizes.
Therefore, eDNA sampling focused on the specific size fraction where
target eDNA is abundant may lessen false-negative eDNA detection and
variance of eDNA quantification, consequently estimating species
abundance more accurately and sensitively. Alternatively, different
physiological origins and physiochemical structures of eDNA might result
in its different persistence and spatial dispersion, hence influencing
the relationship between eDNA concentration and species abundance.
Additionally, the >3 µm pore size filter generally reduces
filter clogging, increasing the filtration volume of the water samples
considerably compared with smaller pore size filters (e.g., Kumar et
al., 2021). Moreover, Takasaki et al. (2021) recently compared the
pre-filtration performance on eDNA detection using various pore size
filters (10–840 µm pore sizes). The result showed that 10 µm
pre-filtration effectively reduced PCR inhibitors (e.g., humic
substances) in river water samples without decreasing the detectability
of target eDNA. From the above, it would be promising to collect and
analyze target eDNA focusing on the 3–10 µm size fraction to better
estimate species abundance.
Conversely, the performance of abundance estimation was poor for eDNA
particles at the <3 µm size fraction. According to the
experiment targeting size-fractionated eDNA from T. japonicus ,
the selective collection of eDNA particles at the 0.8–3 µm size
fraction tremendously worsened its relationship with fish biomass and
sensitivity in response to changes in fish biomass (i.e., theR2 value and slope). This trend could partly
contribute to the result that the slopes of linear regression tended to
be lower for the Japanese jack mackerel’s eDNA particles at the
>0.8 µm size fraction than that at the >10 µm
size fraction. Furthermore, variations of the abundance estimation
accuracy via eDNA across filter pore sizes in some previous studies are
likely attributed to eDNA particles at the 0.8–3 µm size fraction.
Takahara et al. (2012) assessed the relationships between common carp
(Cyprinus carpio ) eDNA concentration and fish biomass using
different filtration methods, reporting a betterR 2 value for the 3 µm pore size filter (i.e.,
>3 µm size fraction; R2 = 0.93)
than the 0.8 µm pore size filter following a 12-µm pre-filtering (i.e.,
0.8–12 µm size fraction; R2 = 0.85).
Eichmiller et al. (2016) also found that the R2values between common carp eDNA concentration and fish biomass were
higher for 5 µm pore size filters (R2 = 0.86)
than for 1 µm pore size filters (R2 = 0.71).
Moreover, with the frequent non-detection (zero copies) of T.
japonicus eDNA at the 0.4–0.8 µm size fraction, theR2 values and slopes in the linear regressions
at the 0.4–0.8 µm size fraction were biased and overestimated by a
zero-inflation of the target eDNA concentration. Sequel to this, it
agrees to a lower proportion of fish eDNA at the <0.8 µm size
fraction (20% at most; Turner et al., 2014).