Detection of Seagrass Pathogens Using Environmental DNA (eDNA), North Pacific, 2016-present

Water samples were collected in 500 mL volumes and filtered through 0.22 micron filters (GTTP 04700, Millipore), which were then stored in 5 mL of Longmire Buffer (LMB; Longmire et al 1988) held in 15mL tubes. Sediment samples were collected in 1 mL volumes and stored in 15 mL tubes containing 5 mL of LMB. Approximately eight centimeters of plant tissue (leaf) were collected and stored in 15 mL tubes containing 5 mL of LMB. Cloacal samples, which at times included seeds, were collected opportunistically from sport hunters that shot birds in the Cold Bay, AK area during fall 2016 (Black Brant, Northern Pintail) and fall 2018 (Black Brant, Cackling Goose, Emperor Goose, Northern Pintail, America Wigeon, Eurasian Wigeon, Green-winged Teal, Mallard, and Greater Scaup) using sterile foam tipped applicators and stored in 15 mL tubes containing 5 mL of LMB. Five replicates of each sample type were collected at each location unless otherwise noted in Menning et al 2020 or Menning et al 2021.

Stored environmental samples were vortexed and eDNA extracted using a 400 µL subsample of the LMB-preserved sample using a Qiagen DNeasy Blood and Tissue kit (Qiagen), following manufacturers suggested protocols, with the exception that volumes were doubled. All available Labyrinthula sp., Halophytophthora sp., and Phytophthora sp. sequences were downloaded from NCBI GenBank on 11 October 2018 into locus specific FASTA files (5.8S and 18S), were aligned, and conserved regions identified for primer design. Once candidate primers were identified, they were checked against all locus-specific FASTA sequences on NCBI. The resultant FASTA files were screened to verify that a single species would be identified for each unique sequence and that no potential sequences had more than one unique generic/specific epithet descriptor. Additional methodological details for PCR amplification (including primer sequences), library preparation, and sequencing parameters are documented in Menning et al 2020.