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

Introduction The negative effects of invasive alien species (IAS) are among the top human-mediated drivers of biodiversity loss [1]. Environmental DNA (eDNA) metabarcoding has become the method of choice for molecular-based biosurveillance [2, 3]. It allows for cost-effective, multi-species identification in diverse communities by sampling the DNA shed by the organisms into their environment [4]. Compared to conventional morphology-based identification methods, eDNA metabarcoding can reduce the time spent on species identification, sample sorting and processing, and increase the target taxa’s scope by utilizing High-Throughput Sequencing (HTS) technologies coupled with DNA reference databases. The existence of DNA reference databases (e.g., The Barcode of Life Data Systems [BOLD] [5]), which links genetic and mor- phological information, reduces the necessity of taxonomic expertise by assigning known iden- tities to unclassified organisms based on molecular data. eDNA metabarcoding has the capability to detect rare or elusive species from complex envi- ronmental samples, including species that may be considered pests, vectors of diseases, or spe- cies of conservation concern [6]. Additionally, eDNA metabarcoding causes less ecosystem disturbance than traditional biosurveillance methods and can potentially maintain the mor- phological integrity of detected organisms [7, 8]. The non-destructive capacity of eDNA sam- pling is critical in a regulatory context; it allows for specimens to be preserved for secondary

morphological confirmation, which is crucial for the validation of eDNA-methods through comparison to morphology-based identification biosurveillance. Additionally, taxonomic assessment of immature stages of most arthropods is challenging; characters for identification are often dependent on adult morphology and reproductive structures [9]. DNA-based molec- ular identification tools such as eDNA metabarcoding provide a valuable resource for rapidly and accurately identifying morphologically indistinct IAS [10], as eDNA-based classification is independent of morphological integrity and remains invariable regardless of organism’s life stage. Biological invasions are responsible for tremendous global impacts, including substantial biodiversity and economic losses, and management expenditures [11]. Invasive insects cause long-term environmental effects, including reduced ecosystem stability and loss of native spe- cies [12]. Invasive alien species (IAS) can destroy about 400,000 ha of forest annually in Can- ada [13] and are considered second only to wildfires regarding adverse effects on forests. Previous research has shown that invasions of insects alone cost a minimum of US$70.0 billion per year globally [11]. In Canada, the government is committed to strengthening the regulatory framework to control and prevent IAS. The Canadian Food Inspection Agency (CFIA) is primarily responsi- ble for the biosurveillance of terrestrial IAS [14]. Canada has at least 272 federally terrestrial regulated species, including all the potentially invasive species, where the vast majority are

insects [N = 83] (https://inspection.canada.ca/plant-health/invasive-species/regulated-pests/ eng/1363317115207/1363317187811). Two (Agrilus planipennis and Lymantria dispar) out of those 83 insects are among the top IAS that cost the province of Ontario the most money [15]. Within this framework, IAS DNA-based detection, and the need to integrate molecular-based technology into regulatory policy, are continuously being explored to establish cost-effective protocols for the molecular biosurveillance [4, 16]. The main limitation for faster and more routine integration of DNA-based surveillance into current IAS biosurveillance toolkits has been linked to the lack of a thorough technological validation [17]. Four main validation cate- gories have been recognized to support the efficacy of eDNA-based methods: experimental val- idation, comparative morphological/ecological validation, bioinformatic validation, and statistical validation [17]. In the present case study, we aim to address three of these categories. PLOS ONE Unveiling invasive insect threats to plant biodiversity PLOS ONE | https://doi.org/10.1371/journal.pone.0290036 August 11, 2023 2 / 24 Funding: UoG-BIO acknowledges the financial support of the Canadian Food Inspection Agency (CFIA). [FAP#2122-002 ] The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.