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

species list states that Phytophthora species are regulated except for a listed subset. Therefore, the additional regulated but not listed species were added (see S1 Table). Collection locations Lindgren funnel traps were deployed at four undisclosed locations in southern Ontario, Can- ada, in line with the CFIA’s 2019 regulatory survey program aimed at detecting insects intro- duced to Canada through high-risk pathways. The selected locations were Oshawa, Chatham- Kent, Brantford, and Brampton. For each location, six traps were strategically placed at six dif- ferent sites near tree species known to be hosts of the target invasive alien species (IAS). This process was repeated twice, resulting in a total of 48 field samples collected in 2019. In the sub- sequent year, Lindgren funnel traps were set up in two additional southern Ontario locations: Greater Toronto Area and Hamilton. Similarly, six traps were placed at two sites on two differ- ent dates, yielding 24 samples. Furthermore, three more locations (Peterborough, Windsor, and Fort Erie) were sampled twice in 2022, resulting in 30 additional samples. To enhance the analysis, datasets from traps collected in 2017 and 2018 were also included [4, 23]. S1 Fig. con- tains an interactive map displaying the distribution of all the collected sites from 2017 to 2022. Please refer to the supporting material for further details. Collection protocol Lindgren funnel traps were set up with Ultra High Release (UHR) Ethanol Lures from Synergy Semiochemical Corp, including fuscomol and fuscomol acetate as baits. To preserve trapped

insect specimens and samples for eDNA analysis, the collection cups were filled with a satu- rated saltwater solution (2 kg of NaCl per 5 L of water). These traps were deployed at sampling sites following the CFIA’s Survey Protocol: Invasive Alien Species Forestry Trapping (2019) during the summer of 2019 and 2020. Specimens caught in the traps were removed from the solution and sent to the CFIA’s entomology laboratory. The saturated salt trap solutions were transferred to new Whirl-Pak bags and shipped to the Hanner Laboratory at the University of Guelph, Ontario, Canada. They were temporarily stored at -80˚C until further processing in the laboratory. Unfortunately, the collection fluid from two traps (trap 2 and trap 5) placed on August 26th, 2019, in Brampton could not be successfully collected. Additionally, the sample collection bag for trap number 3, collected on August 16th, 2020, in Hamilton, was empty as the salt trap solu- tion had completely evaporated at the sampling site. Insects collected during the 2019–2020 collections were not retained, while those from the 2022 collection were sent to the Hanner lab for morphological validation. Filtration The samples were taken out from the -80˚C freezer and transferred to decontaminated bea- kers, allowing them to thaw at room temperature overnight. All filtration equipment was steril- ized using a 50% bleach solution or ELIMINase from Decon Labs, Inc., followed by thoroughly rinsing with deionized water. Before filtration, the samples were inverted to resus-

pend any settled particles within the bag. Filtration of salt solutions was carried out using Nitrocellulose Mixed Ester membrane filters (47 mm diameter, 1 μm pore size) from Sterli- tech. The filtration setup consisted of a three-piece manifold connected to a GAST vacuum pump from GAST Manufactured, Inc, utilizing magnetic filtration cups from Pall. The filter cups were covered with Kimwipes throughout the procedure to prevent DNA contamination. Some samples required multiple membranes due to the presence of a high amount of debris, leading to filter clogging. The filtration time for each sample and the number of filters used PLOS ONE Unveiling invasive insect threats to plant biodiversity PLOS ONE | https://doi.org/10.1371/journal.pone.0290036 August 11, 2023 4 / 24