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

North American pest, exclusively feeds on grapevines and has historically caused significant vineyard losses [38, 39]. However, grafting European grapevines onto resistant rootstocks has helped manage this pest [39]. Despite being minor in Ontario, grape phylloxera remains eco- nomically significant worldwide [38, 39], emphasizing the need for early detection and popula- tion regulation. The spongy moth, introduced to Ontario in 1969 [40], defoliates various hardwood and softwood trees during outbreaks, reducing tree health and making them more susceptible to other threats. This moth is one of the most economically damaging insect pests [11], with outbreaks occurring every 7 to 10 years [40]. Spongy moths can also cause acute contact dermatitis when humans come in contact with larvae, which has implications for human health [41]. On the other hand, the Japanese beetle, native to Japan and introduced to North America in 1916 [42], then in Canada in 1939 [43], is highly destructive to turfgrass, landscape plants, and pastures. Its larvae damage grassroots, while adults heavily feed on leaves, impacting over 300 plant species in 79 families [44] and having economic impacts on over 100 of those species [42]. The Japanese beetle causes significant economic losses and requires substantial control efforts [45]. Finally, Trichoferus campestris, a velvet longhorned beetle native to Asia, was detected in Canada in 2002 [46]. T. campestris is native to Asia and feeds on healthy or mildly stressed adults in over 40 genera of broadleaf and conifer trees in its

native range; however, it prefers apple and mulberry trees [47, 48]. While it was initially Table 2. Summary of IAS detections from three sites in the 2022 collection. A = Adult, L = Larval. Specimen size is presented as an average of all lengths when multiple specimens were present. Trap Organism(s) eDNA detection Morphological detection Number of specimens Size of the specimen(s) Lifestage FE_5_8_22_4 Agrilus planipennis YES YES 1 12.2 mm A PB_10_8–22_1 Popillia japonica YES YES 3 10.8 mm A Daktulosphaira vitifoliae YES YES 2 <1 mm L PB_10_8_22_2 Popillia japonica YES YES 6 10.4 mm A Daktulosphaira vitifoliae YES YES 2 <1 mm L PB_10_8_22_4 Popillia japonica YES NO 0 n/a n/a Daktulosphaira vitifoliae YES YES 47 <1 mm L PB_10_8_22_6 Daktulosphaira vitifoliae YES YES 3 <1 mm L PB_29_8_22_1 Popillia japonica YES YES 1 11.5 mm A Daktulosphaira vitifoliae YES YES 2 < 1mm L PB_29_8_22_3 Popillia japonica YES NO 0 n/a n/a Daktulosphaira vitifoliae YES NO 0 n/a n/a PB_29_8_22_4 Daktulosphaira vitifoliae YES YES 2 <1 mm A, L Popillia japonica NO YES 1 11.2 mm A WR_21_7_22_B1 Daktulosphaira vitifoliae YES NO 0 n/a n/a WR_21_7_22_B2 Agrilus planipennis YES NO 0 n/a n/a WR_21_7_22_B3 Agrilus planipennis YES NO 0 n/a n/a Daktulosphaira vitifoliae YES NO 0 n/a n/a WR_21_7_22_G1 Agrilus planipennis YES NO 0 n/a n/a Daktulosphaira vitifoliae YES NO 0 n/a n/a Lymantria dispar YES NO 0 n/a n/a WR_21_7_22_G2 Lymantria dispar YES NO 0 n/a n/a

Daktulosphaira vitifoliae YES NO 0 n/a n/a WR_21_7_22_G3 Lymantria dispar YES NO 0 n/a n/a Agrilus planipennis YES YES 1 12.2 mm A Daktulosphaira vitifoliae YES NO 0 n/a n/a https://doi.org/10.1371/journal.pone.0290036.t002 PLOS ONE Unveiling invasive insect threats to plant biodiversity PLOS ONE | https://doi.org/10.1371/journal.pone.0290036 August 11, 2023 14 / 24