Unveiling invasive insect threats to plant biodiversity: Leveraging eDNA metabarcoding and saturated salt trap...

The negative global impacts of invasive alien species (IAS) on biodiversity are second only to habitat loss. eDNA metabarcoding allows for a faster and more comprehensive evaluation of community species composition, with a higher taxonomic resolution and less taxonomic expertise required than traditional morphological-based biosurveillance. These advantages have positioned eDNA metabarcoding as the standard method for molecular-based detection of invasive alien species, where fast and accurate detectability allows prompt responses to mitigate their adverse effects. Here, eDNA metabarcoding is used for biosurveillance of invasive alien species regulated by Canada in high-risk areas with four main objectives: i) validate the effectiveness of eDNA metabarcoding of salt trap solutions as a molecular technique for IAS detection, ii) compare detection from DNA extracts obtained from filter quarters versus whole filters, iii) benchmark two different bioinformatic pipelines (MetaWorks and mBRAVE), and iv) compare canopy and ground level trapping. eDNA from up to five IAS (Agrilus planipennis, Daktulosphaira vitifoliae, Lymantria dispar, Popillia japonica, and Trichoferus campestris) were successfully detected across years from 2017 to 2022 in southern Ontario, Canada, with successful morphological validation for all except Lymantria dispar and Trichoferus campestris. Analysis of filter quarters in contrast to whole filters was demonstrated to be insufficient for effective IAS detection in each sample. All IAS were detected in only one filter quarter, suggesting a patchy eDNA distribution on the filter. The MetaWorks and mBRAVE bioinformatics pipelines proved effective in identifying IAS, with MetaWorks yielding a higher success rate when comparing molecular and morphological identifications. Ground-level and canopy-level sampling showed differential IAS recovery rates based on the molecular detection, which also varied per collection year, with all found IAS detected at the canopy level in 2022 while only one (Lymantria dispar) in 2020. The present study ratifies the efficacy and importance of eDNA-based detection in a regulatory context and the utility of adding eDNA metabarcoding of saturated salt trap solutions, a critical tool for IAS detection.