eDNA extraction and quantitative real-time PCR (qPCR)
We extracted total eDNA on the filter using a DNeasy Blood and Tissue Kit (Qiagen, Germany), according to the method described in Minamoto et al. (2019). The zebrafish eDNA concentration in the water sample was estimated by quantifying the copy number of mitochondrial genes using the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, U.S.). We used the primer/probe sets to amplify the different lengths of mitochondrial genes, including cytochrome b, tRNA-Glu, and ND6 regions of target species (132, 430, 715, and 1021 bp) developed by Hirohara et al. (2021). Each 15 μL of TaqMan reaction mixture contained a 2 μL DNA template, a final 900 nM concentration of each forward and reverse primer, and 125 nM of TaqMan probe in 1 × TaqPath qPCR Master Mix, CG (Thermo Fisher Scientific). Simultaneously, 2 μL of pure water samples were analyzed as the PCR negative controls. Target eDNA concentrations were quantified based on a dilution series of standards containing 3 × 101 to 3 × 104 copies of synthesized artificial DNA fragments from zebrafish mitochondrial genes (Hirohara et al., 2021). All qPCRs were performed in triplicate, including eDNA samples, standards, and negative controls. Thermal conditions of the qPCRs were 2 min at 50°C, 10 min at 95°C, 55 cycles of 15 s at 95°C, and 1 min (132 bp) or 1.5 min (other fragment lengths) at 60°C (Hirohara et al., 2021). We calculated the eDNA concentrations by averaging the triplicates, and each PCR negative replicate (indicating non-amplification) was denoted as containing zero copies (Ellison et al., 2006).