eDNA field sampling and sample analysis

Field samples were taken at pre-determined intervals from the Grassland Water District’s Mosquito Ditch, which receives flows via a screw gate immediately downstream of a verified T. gigas population in the Volta Wildlife Area’s Field 10 (Merced County, California; Figs ​Figs22 and ​and3).3). This site was selected due to the paucity of adjacent wetlands from which T. gigas might immigrate and its relative isolation from other occupied locales in the Grasslands Ecological Area ([28–30]; Fig 2). On August 11, 2016, single filter samples were taken within Mosquito Ditch starting downstream at 1000m and working upstream towards Field 10 on the Volta Wildlife Area at site 0m.

Location of the field site within California, USA (top left), location of the study area relative to the Volta Wildlife Area (top right), and the sampling sites (bottom). The black dots represent the sampling site, and the red dots represent historical T. gigas locality records.

Location of the field validation sites on the Mosquito Ditch and their position relative to Field 10 on the Volta Wildlife Area and to one another.

Field sampling and laboratory protocols followed procedures described in [23], with the two exceptions being that samples were taken aboard a kayak and Millipore Sterivex™-GP 0.45μm sterile filter unit (EMD Millipore) were used in place of Millipore Sterivex™-GP 0.22μm. We found that using Millipore Sterivex™-GP 0.45μm in place of the Millipore Sterivex™-GP 0.22μm increases the volume of water that can be filtered. Water samples were collected from either the bank margin, or where this was infeasible due to dense vegetation or steep topography, by kayak at the channel center. All samples taken from the kayak were taken from the upstream side only. For each sampling event, water was filtered directly from the water body at an approximate depth of 6–10 inches below the surface using sterile Saint Gobain XL-60 silicon tubing (Tygon®; internal diameter 6.3mm), and a portable Masterflex1 L/S Easy-Load II peristaltic pump (Cole-Parmer®) powered by a cordless hand drill. Water samples were filtered through a single Millipore Sterivex™-GP 0.45μm sterile filter unit filters clogged. No water was transported or stored during sampling nor was any water transported between sampling sites; instead all filtration occurred directly on the boat at each site. Sample filtrate was captured and measured in graduated flasks to verify the volume of each sample. Filtered water was then poured over the side of the boat after completion of sampling at each site. To ensure that field equipment was free of contamination, DNA field control samples were taken for each sampling day. Each field control consisted of Sterivex™ filtered ultra-pure water transported from the lab and processed in the same fashion as the field samples. The field controls were processed for the presence of T. gigas DNA in parallel with all other samples. To eliminate cross contamination between sites due to equipment or the investigator, sterile gloves and all sampling materials were pre-packaged in the laboratory and discarded after one use. Tubing and gloves were immediately disposed of after each use into a sealed trash bag on board. All filters were likewise considered single use. After filtration, filters were capped at each end, labelled with location ID, placed into a sterile secondary container, sealed, and immediately placed on ice. All filters were kept on ice in a cooler for the duration of the sampling day, after which they were transferred to a -20°C laboratory freezer. The filters were stored within individually sealed secondary containers at -20°C until DNA extraction.

DNA from all samples and controls was extracted using PowerWater Sterivex™ DNA Isolation Kit (Mo Bio Laboratories, Inc.) following the manufacturer’s recommended guidelines. A DNA extraction negative control was processed in parallel to ensure sample integrity throughout extraction procedure. The DNA extraction control consisted of Sterivex™ filtered ultrapure water only. DNA extraction controls were processed using the same equipment used to extract DNA from all samples. Each sample and all controls were analyzed in triplicate, with each qPCR technical replicate consisting of a 10 μl reaction volume, for the presence of the T. gigas DNA using the qPCR T. gigas primer and probe designed as part of this study. Each 10 μl qPCR reaction was composed of 2x Applied Biosystems TaqMan Universal PCR Master Mix, No AmpErase UNG (Thermo Fisher ABI), optimal primer probe concentrations (900 nM initial primer concentrations, 3 μM initial probe concentration for both ND4 and CytB and 4 μl DNA template. Thermocycling was performed using a Bio-Rad CFX 96 Real Time System (Bio-Rad Laboratories, Inc.) with the following profile: 10 min at 95° C, 40 cycles of 15 sec denaturation at 95° C and 1 min extension at 60° C. Six negative template control (NTC) reactions were run on the plate with the control sample templates consisting of 4 μl of ultrapure water replacing DNA template within reaction volume. Three positive control reactions consisting of 20 ng μL^-1 T. gigas genomic DNA template were also tested in parallel to ensure consistent PCR performance. All PCR master mixes were made inside a UV PCR enclosed workstation. DNA template was added to the master mix outside of the UV PCR workstation on a dedicated PCR set up workbench. All PCR reactions were conducted on instruments located outside of the main lab in a separate portion of the building. Results of the qPCR reactions were analyzed using BioRad CFX manager v3.1 (Bio-Rad Laboratories, Inc.).