Sea cucumbers were collected at 3 locations (near Juneau, Alaska, near Sitka, Alaska, and Nanoose, British Columbia) to assess the presence and load of PcaFV and other aquatic invertebrate flaviviruses.

Twenty individual Apostichopus californicus specimens were collected by SCUBA divers during routine fisheries surveys by Alaska Department of Fish and Game from near southeastern Alaska (Juneau) in June/July 2021. Specimens were immediately placed into a refrigerated hold on the ship in zip-lock bags before transport to the lab in Juneau later in the day. There, specimens were frozen at -20°C and couriered overnight to Cornell University (Ithaca, NY, USA) where they were frozen at -80°C on arrival.

Thirty-two specimens of Apostichopus californicus were collected by SCUBA divers in Sitka Sound (57.1273 N, 135.4042 W), near Sitka, Alaska on 10 November 2021 and transported together in plastic tubs to the lab at the University of Alaska Southeast (Japonski Island, Sitka). Specimens were immediately sampled for tube feet by pulling them off and placing into RNALater, before freezing at -20^oC for transport to the lab at the University of Alaska Southeast.

Five specimens of Apostichopus californicus were collected from Nanoose, British Columbia, Canada in September 2021 and transported on wet ice to the lab at Cornell University for further analysis.

RT-LAMP was performed in duplicate in 25 µL reactions containing 1X WarmStart Colorimetric RT-LAMP Master Mix (New England Biolabs), 0.5 µL of fluorescent dye, 2.5 µL primers designed using the New England BioLabs primer tool around a portion of the PcaFV NS5 amplicon (GenBank accession MT949664), and 5 µL of extracted RNA. The primer master mix comprised 16 μL of the 100uM PcaFV_NS5_FIP and PcaFV-NS5_BIP primers, 2 μL of 100uM PcaFV_NS5_F3 and PcaFV_NS5_B3 primers, 4 μL of the 100uM PcaFV_NS5_LoopF and PcaFV_NS5_Loop B primers, as well as 56 μL of nuclease-free water (diH₂O). Reactions were performed in optically clear quantitative PCR tubes (Applied Biosystems). Negative controls comprised 5 μL of diH₂O, while positive controls used 5 μL of PcaFV synthetic gene fragment. The tube strips were incubated in a water bath at 65°C for 30 min. After incubation, tubes were removed from the bath and scored based on color change. Positive detections were scored when the reaction appeared yellow and negative detections were scored when the reaction appeared red. Positive controls were consistently yellow, and negative controls were consistently red. When positive controls had not turned yellow, reactions were further incubated for 15 min in the bath, and then reassessed. Possible detections were scored when the reactions turned orange.

Quantitative reverse transcriptase PCR (qRT-PCR) using primers PcaFV_NS5_F3 (5’- CCA GCC ATG GAT GAG TAA TG-3’) and PcaFV_NS5_R3 (5’- GCT GAA CTG CTC CTG AAA CC-3’), and probe PcaFV_NS5_Pr3 (5’-[FAM]CAC GAA TGT ACG GCA ACG GAC G[TAMRA]-3’) was used to determine PcaFV load within individual tissue RNA extracts. qRT-PCR reactions were performed in duplicate for each specimen and compared against duplicate reactions of oligonucleotide standards spanning the amplicon region (from 10² to 10⁷ copies µl^-1) and using 4 negative controls (nuclease free H₂O only). PCR reactions (20 µL) contained 1 X Luna Universal Probe One-Step Reaction Mix (New England Biolabs), 1X Luna WarmStart RT Enzyme Mix (New England Biolabs), 8 µmol each of primers and probes, and 1 µL of template RNA. Reactions were thermal cycled in an ABI StepOne qPCR machine. Reactions were initially held at 55°C for 10 min, followed by an initial denaturation step at 95°C for 1 min. Reactions were then subject to 45 cycles of denature at 95°C and anneal at 56°C, with fluorescence data collected after the annealing step. Linearity of standards was checked during downstream analysis of qPCR data.