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

of eDNA shed into collection fluid is primarily linked to the number of specimens trapped (e.g., biomass) and the time the specimens remained in the fluid. Additional environmental conditions such as temperature and UV exposure can also influence the amount of eDNA shed by certain IAS, and its subsequent degradation. The rate between eDNA shedding and degradation directly affects the molecular detectability of a species; low rates of shedding/high rates of degradation can lead to specimens being detected morphologically but lacking eDNA identification, as seen in one case here (Trap PB_29_8_22_4). Conversely, there may be cases where a species is detected molecularly but not morphologically. This can be caused by a num- ber of reasons including: an organism escaping a trap after shedding significant amounts of DNA, an organism’s feces, eggs or other eDNA sources falling into the trap without the organ- ism itself, or a soft-bodied organism decays while in the trap fluid. To increase IAS detectabil- ity and to have comprehensive sampling, the present study deployed up to six traps per collection site. However, no level of replication or sampling effort can ensure 100% agreement between morphological and molecular identification in the above-mentioned scenarios. These disagreements should be considered carefully on a sample-by-sample basis, as many factors are at play, including those mentioned above and others, such as sampling bias and taxonomic expertise, which may influence results.

To further validate the eDNA-metabarcoding approach of salt trap solutions for effectively detecting IAS, morphological vouchers can be compared to molecular-based identifications (comparative/ecological validation) in rigorously prepared mock communities resembling complex environmental samples. Choosing eDNA-based detection and/or traditional mor- phological observations for IAS detection should be carefully considered in light of each approach’s known pros and cons. Molecular identifications offer a faster and more sensitive method than traditional approaches, which is critical in the regulatory context. Faster detec- tion allows for detectability before species abundance reaches a point where their damaging effects are difficult to manage. Normally, when IAS are detected via traditional observations, their populations have already grown too large, which makes effective management more Fig 7. Invasive alien species detection at the ground level versus canopy level (lower panel) in the 2020 and 2022 collections. https://doi.org/10.1371/journal.pone.0290036.g007 PLOS ONE Unveiling invasive insect threats to plant biodiversity PLOS ONE | https://doi.org/10.1371/journal.pone.0290036 August 11, 2023 18 / 24

difficult and costly. In the 2022 collection, there were 25 individual detections with 11 of those rat- ified by both molecular and morphological approaches; one detection was morphological only and the remaining 13 were molecular only. The case of morphological detection only was repre- sented by a single specimen, which might have been trapped towards the last moment of collec- tion and may have not shed enough genetic material into the trap fluid before it was removed for morphological identification. Of the 13 detections that were molecular detection only, six of them represented cases where the species was validated in the same collection site but from another trap in that same site. If matching traditional morphological identification to molecular detection is considered the gold standard for validating eDNA results, the current success rate combining both scenarios is 68% (17 out of 25). However, as discussed above, there are well-known scenarios where molecular and morphological detections would not allow for 100% agreement, emphasiz- ing the need to carefully evaluate molecular tools in regular biomonitoring and biosurveillance programs on a sample-by-sample basis. At the bioinformatic level, MetaWorks [19] resulted in more coincidences between molecular and morphological identifications than mBRAVE [20], although the number of IAS detected when using both bioinformatic pipelines was comparable. Altogether, it demonstrates the impact of the data analysis strategy on the final output and the

vitifoliae) is commonly found near ground level since they emerge from the soil. In Ontario, grape phylloxera is suggested to prefer a trap height of 1.3 m in contrast to 0.6 m [64], while another study found no significant difference between traps at 1.5, 2.1, and 2.7 m [65]. Spongy moth (Lymantria dispar) males are strongly attracted to vertical objects and commonly fly up and down tree trunks, where females would most likely be encountered [66]. Various studies note greater success for ground-level traps generally below 4 m [67–72]. Collins and Potts (1932) found that traps placed at 21.3 m height did not capture any males [73] while Richerson et al. (1976a) showed that males primarily search for females at the height of the highest phero- mone source [74], which tends to be below 4 meters [75]. In summary, our study showed greater success for ground-level sampling at 2020 and at the canopy-level in the 2022 collec- tion, which partially corresponds to specific insect behaviour in each case. However, other studies of similar taxa show mixed results, therefore we suggest sampling at both the ground and canopy in future studies for better species representation until more data is gathered. PLOS ONE Unveiling invasive insect threats to plant biodiversity PLOS ONE | https://doi.org/10.1371/journal.pone.0290036 August 11, 2023 19 / 24

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

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