Unveiling invasive insect threats to plant biodiversity: Leveraging eDNA metabarcoding and saturated salt trap solutions for biosurveillance

were carefully documented. As a negative control, one sample of a saturated salt solution pre- pared in the laboratory underwent filtration as well. The filters were then placed in sterile Petri dishes and stored at -80˚C until further processing for eDNA extraction. eDNA extraction Following the described steps, we performed eDNA extraction on the total number of filter membranes resulting from filtration, including negative controls. The DNeasy Blood and Tis- sue Kit from Qiagen was used with modifications to the manufacturer’s protocol [24]. Each fil- ter was thawed and divided into quarters, then further cut into strips using sterile razor blades. Small strips from each quarter were placed in individual 2 mL microcentrifuge tubes along with approximately 250 mg of glass beads (0.75–1 mm diameter). Buffer ATL (380 μL) was added to each tube, and the tubes were subjected to disruption in a Qiagen TissueLyser II for 1 minute at 30 Hz. After spinning down the samples, Proteinase K (20 μL) was added to each tube, followed by vortexing and overnight incubation at 56˚C at 700 rpm on the orbital shaker. The tubes were vortexed again, spun down, and Buffer AL (400 μL) was added. After vortexing and incubating at 56˚C for 10 minutes, ethanol (96–100%) (400 μL) was added and vortexed for another 30 seconds. All samples, including any residues, were transferred to DNeasy Mini spin columns and centrifuged at 11,000 g for one minute. The columns were then washed twice using AW1 and AW2 buffers, following the manufacturer’s protocol. After each wash,

samples were centrifuged at 11,000 g for one minute after the first wash, and then centrifuged at 17,000 g for 5 minutes after the second wash. Flow-through and collection tubes were dis- carded, and the DNeasy Mini spin columns were placed into clean 1.5 mL LoBind microcen- trifuge tubes (Eppendorf). Buffer AE (100 μL) pre-warmed at 70˚C was added onto the DNeasy membranes, incubated at room temperature for 15 minutes, and finally centrifuged for elution at 11,000 g for five minutes. For the 2019 and 2022 CFIA collections, eDNA extracts from each quarter of the same trap were pooled into a single tube. In contrast, for the 2020 CFIA collection, extracts from each quarter were kept separate. In the latter case, 20 μL of eDNA extract from each quarter belong- ing to the same trap was transferred into a single fifth tube, allowing the evaluation of whether a filter fragment represents the species diversity of the whole filter. Consequently, we kept and analyzed both pooled and unpooled eDNA for each filter of the 2020 collection. If this approach enables the effective detection of invasive alien species (IAS), using only a filter frag- ment in each sample might be enough moving forward, decreasing sample processing costs and time, while the remaining filter material can be stored as a backup for further analysis or validation as needed. The DNA concentrations of the eDNA extracts were determined using a Qubit 4.0 fluorome- ter with the Qubit dsDNA High Sensitivity kit (Thermo Fisher Scientific), using 3 uL of the DNA

extracts and measuring ng/uL of DNA three times (ten minutes apart) for each sample. Qubit quantifications provide a fluorescent-based estimation of DNA concentration while comparing the intensity of fluorescent dye bound to target dsDNA, in this case, to dye binding standards (DNA probes) of known concentration. As there is a dynamic in the dye intercalation and, conse- quently, variation over time in the physical interaction between the dye and the target dsDNA, estimations of DNA concentrations were taken three times and ten minutes apart. Values from each estimation were averaged, and standard deviations were calculated to increase DNA concen- tration estimation accuracy and evaluate dispersion from the mean values. A 5 μL subsample from each extract was also visually assessed for eDNA presence and quality using 1% agarose gel electrophoresis. All eDNA extracts were stored at either 4˚C for immediate use or -20˚C for short-term storage before library preparation for High-Throughput Sequencing (HTS). PLOS ONE Unveiling invasive insect threats to plant biodiversity PLOS ONE | https://doi.org/10.1371/journal.pone.0290036 August 11, 2023 5 / 24