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

Conclusions eDNA metabarcoding of salt trap collection fluids is a valid molecular-based tool for rapid IAS detection, allowing morphological confirmation when needed. Complete filter processing is recommended for higher IAS detection probability instead of filter fragment analysis due to potentially patched eDNA distribution on the filters. MetaWorks and mBRAVE bioinformat- ics pipelines effectively allowed IAS identification, but MetaWorks resulted in a higher success rate. In the context of CFIA molecular biosurveillance, ground-level and canopy-level sam- pling should be considered for a more comprehensive sample regardless of specific IAS, as the effectiveness of trap height continues to be species-dependent and match the behaviour of the particular IAS found. Supporting information S1 Fig. Interactive map displaying the distribution of all the collected sites in southern Ontario from 2017 to 2022. (HTML) S1 Table. Up-to-date species list of terrestrial regulated species by Canada. (CSV) S2 Table. a-f. MetaWorks output tables, including Exact Sequence Variants (ESVs), ESV sizes, taxonomic assignments, and bootstrap support values for each taxonomic rank per data- set analyzed in the present study (2017–2019, 2020 quarters and full, and 2022). (ZIP) Acknowledgments We deeply thank Jenny Gleason for kindly sharing her R scripts with us. We thank Jaeju Yu for providing all the additional information needed to run metabaR for the 2017 dataset. We also thank Robert G. Young for technical assistance during the fluid filtration of the 2019 col-

lections at the Hanner Laboratory. YMG was supported by Mitacs through the Mitacs Elevate Program. Author Contributions Conceptualization: Yoamel Milia´n-Garcı´a, Robert H. Hanner. Data curation: Yoamel Milia´n-Garcı´a, Cassandre Pyne. Formal analysis: Yoamel Milia´n-Garcı´a, Cassandre Pyne. Funding acquisition: Yoamel Milia´n-Garcı´a, Robert H. Hanner. Investigation: Yoamel Milia´n-Garcı´a, Kate Lindsay, Adriana Romero. Methodology: Yoamel Milia´n-Garcı´a. Project administration: Yoamel Milia´n-Garcı´a, Robert H. Hanner. Resources: Robert H. Hanner. Software: Yoamel Milia´n-Garcı´a, Cassandre Pyne. Supervision: Yoamel Milia´n-Garcı´a, Robert H. Hanner. Validation: Yoamel Milia´n-Garcı´a. Visualization: Yoamel Milia´n-Garcı´a, Cassandre Pyne. PLOS ONE Unveiling invasive insect threats to plant biodiversity PLOS ONE | https://doi.org/10.1371/journal.pone.0290036 August 11, 2023 20 / 24