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

collection (Fig 7). On the contrary, eDNA from all four IAS detected in 2022 were recovered from samples taken at the canopy level, and only three IAS were identified during the ground- level sampling effort that year (Fig 7). Discussion Due to their non-destructive and cost-effective nature, environmental DNA-based approaches, such as eDNA metabarcoding, have become the standard molecular method for multispecies identification. However, the routine implementation of eDNA methods in a regulatory context requires thorough validation to be broadly accepted by regulators [17]. In the present study, we pursue eDNA validation for IAS detection on CFIA-sampled salt trap collection fluids using a molecular multi-species identification approach and an experimental, morphological, and bioinformatic validation [17]. Effectiveness of eDNA metabarcoding for IAS detection eDNA from up to five invasive alien species regulated by Canada (Agrilus planipennis, Daktu- losphaira vitifoliae, Lymantria dispar, Popillia japonica, and Trichoferus campestris) was consis- tently found across five years (2017–2022) in high-risk areas sampled by the CFIA. The emerald ash borer (EAB), an invasive beetle from Asia, has caused extensive damage since its introduction to North America in 2002 [31]. The larvae of this wood-boring beetle feed on ash trees, forming tunnels under the bark and ultimately killing the trees within a few years [32]. This has led to a significant loss of ash trees, decreased biodiversity, and increased

vulnerability to other invasive plants [33–35]. The economic losses from EAB are in the bil- lions of dollars [36], making its regulation crucial [37]. In contrast, grape phylloxera, a native Fig 3. Total number of reads (nb_reads) after bioinformatic analyses and number of MOTUs (nb_motus) per collection year and sample type. NA: Represent the data generated from the samples while PCR, sequencing, and extraction indicate different control types. https://doi.org/10.1371/journal.pone.0290036.g003 Fig 4. eDNA invasive alien species detection per collection year. Barplots indicate the proportion of the IAS found per sample. A: 2017 collection; B: 2018 collection; C: 2019 collection; D: 2020 collection; and E: 2022 collection. https://doi.org/10.1371/journal.pone.0290036.g004 PLOS ONE Unveiling invasive insect threats to plant biodiversity PLOS ONE | https://doi.org/10.1371/journal.pone.0290036 August 11, 2023 13 / 24