Results
In the tank experiment, regardless of BAC treatment, we observed
biphasic exponential degradation of all types of Japanese jack mackerel
eDNA (Fig. 2; Table 3). All types of eDNA concentrations were higher in
the treatment with BAC addition than in those without BAC at time 0,
which lasted throughout the sampling period. The decay rates at the
initial phase (\(k_{1}\)) were substantially lower in the treatment with
BAC addition (31.0 to 53.0% relative to the treatment without BAC),
while those at the following slower phase (\(k_{2}\)) were not
significantly different between BAC treatments (Fig. 3). In contrast, in
field sampling, we observed monophasic exponential degradation of
shorter fragments of eDNA (Fig. 4). Linear models showed a significant
interaction between sampling time points and BAC treatments for nuS,
indicating that eDNA decay rates were significantly lower in the
treatment with BAC than in those without BAC (P < 0.05;
Table S3). Although we did not confirm a significant interaction for
mtS, target eDNA was detected for 24 hours from the seawater samples
with BAC addition, whereas it was hardly detected in the samples without
BAC addition (Fig. 4). We did not evaluate the effect of BAC addition on
the degradation of longer eDNA fragments (mtL and nuL) because of their
poor detection relative to that of shorter eDNA fragments. The overall
PCR efficiencies and R2 values of the standard curves
are shown in Table S4. A few filtration negative controls in the tank
experiment showed PCR amplification, whose concentrations were less than
one copy per PCR and much less than those of the sampling tank at the
corresponding time points. No amplification was observed in any of the
PCR-negative controls throughout the study.
Moreover, the number of fish species detected by eDNA metabarcoding was
higher in the treatment with BAC over time (Fig. 5a). In total, 65
marine and brackish fish were detected in 18 of 1-L seawater samples,
wherein 58 and 45 species were detected in the samples with and without
BAC addition, respectively; 36, 40, and 38 species were detected in
samples with BAC, whereas 36, 28, and 27 species were detected in
samples without BAC at time 0, 6, and 24, respectively, when sampling
triplicates were pooled (Table 4; the number of detected species per
sample is shown in Table S5). Exact McNemer tests showed significant
differences in the number of fish species between BAC treatments at time
6 and 24 (both P < 0.05), while no statistical
difference was observed at time 0 (P = 1.00). In addition,
PERMANOVA tests showed a significant difference in community composition
between BAC treatments (P < 0.05) but not between time
points (P = 0.79) (Fig. 5b). We additionally confirmed that the
variances of the compositions were not statistically different among
treatments (PERMDISP; both P > 0.1). After
preprocessing the iSeq raw reads and removing potential contaminations,
none of the eDNA reads were detected from all filtration and PCR
negative controls (Table S6). All the rarefaction curves, generated byrarecurve function in the package ‘vegan’, showed that the number
of species detected from each sample was saturated and the library
sample was satisfactorily sequenced (Fig. S1).