PCR Screening of Egg Mass Collars Environmental DNA: Primers were designed to amplify NRPS-A from the eDNA (For: 5' -GACAGCTATTAGCTATGAGCGGTTA GTACAGCAAG -3'; Rev: 5' -GTAACACAGTGTATCACCGCGGCTTCAAGAA TG -3'). DNA was amplified with NEBNext High-Fidelity 2X PCR Master Mix (New England BioLabs, Inc.; Ipswitch, MA) in a BioRad C1000 Touch Thermal Cycler (denaturing at 95 °C, 30 sec, annealing at 55 °C, 30 sec, extension -1 min at 72 °C, repeat 45×). DNA was visualized with SYBR Safe DNA Gel Stain (ThermoFisher). Using this PCR method, the presence of the pseudochelin A NRPS-A gene in the eDNA described above was detected in three of ten samples. To confirm that the PCR product represented the targeted NRPS-A, the band from one sample was gel purified, reamplified, and sequenced through the Illumina platform. The resulting paired-end sequences were imported into QIIME2 (v.2020.6) for demultiplexing, denoising and removal of chimera sequences. Quality filters "--p-trim-left-f", "--p-trimleft-r", "--p-max-ee-f", "--p-max-ee-r" and "--pchimera-method" were set to 10, 10, 1.0, 1.0 and "consensus", respectively. Any sequences < 300 bp and containing a stop codon in all three reading frames were manually excluded. This yielded three unique sequences, all from P. piscicida, including a match to NRPS-A (Table ).