Microfluidic digital PCR 
Quantification of eDNA was also performed by microfluidic digital PCR (dPCR) using the BioMark Real-time System and 12.765 Digital Array (Fluidigm Corporation, South San Francisco, CA, United States) as previously described (Hoshino and Inagaki, 2012). For each sample, 6 µL of PCR mixture was prepared as follows: 3.0 µL of 2 × Probe qPCR mix (Takara Bio, Shiga, Japan), 0.6 µL of 20 × binding dye sample loading reagent (Fluidigm Corporation), 900 nM of forward/reverse primers, 125 nM of TaqMan probe, 0.015 µL of ROX solution, and 1.0 µL of sample DNA. We used three sets of primers and probes for the quantification of H. neglectus , O. latipes , and M. anguillicaudatus  (Table 1). PCR was initiated at 98°C for 2 min, followed by 50 cycles of 98°C for 10 s and 60°C for 1 min. The amplification curves obtained from individual reaction chambers of the microfluidic chip were analysed using Fluidigm Digital PCR analysis software (Fluidigm Corporation) to obtain copy numbers.