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

It is important to consider that library preparation and High-Throughput Sequencing of a given sample is a sampling process itself [2]. Consequently, sequencing results mainly reflect the diversity in the processed sample under the experimental conditions rather than the site diversity. To get a more accurate representation of site diversity, the sampling effort must be comprehensive enough, there should be no primer biases or taxonomic blind spots, and all prospective species occupying the study area should be properly documented in the reference DNA databases. Failure to meet the mentioned conditions can hinder the capacity for effective eDNA IAS identification in addition to the sampling biases stated above. Experimental, bioinformatic and morphological/ecological validation The five IAS referred to above and detected across years (2017–2022) were successfully con- firmed by two independent bioinformatic pipelines (MetaWokrs and mBRAVE), increasing the accuracy of eDNA-based detections and reducing the probability of false identifications. In addition, all IAS detected in 2022, except Lymantria dispar, were successfully identified mor- phologically among the specimens sampled in that collection year, confirming species presence in the sampling area. However, not every eDNA detection at a given sample was validated with morphological confirmation. Several factors including insect biomass, time in trap fluid, and environmental conditions, can affect the sensitivity of eDNA detection methods. The amount

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