Optimization and validation of a cost-effective protocol for biosurveillance of invasive alien species

Environmental DNA (eDNA) metabarcoding has revolutionized biodiversity monitoring and invasive pest biosurveillance programs. The introduction of insect pests considered invasive alien species (IAS) into a non-native range poses a threat to native plant health. The early detection of IAS can allow for prompt actions by regulating authorities, thereby mitigating their impacts. In the present study, we optimized and validated a fast and cost-effective eDNA metabarcoding protocol for biosurveillance of IAS and characterization of insect and microorganism diversity. Forty-eight traps were placed, following the CFIA's annual forest insect trapping survey, at four locations in southern Ontario that are high risk for forest IAS. We collected insects and eDNA samples using Lindgren funnel traps that contained a saturated salt (NaCl) solution in the collection jar. Using cytochrome c oxidase I (COI) as a molecular marker, a modified Illumina protocol effectively identified 2,535 Barcode Index Numbers (BINs). BINs were distributed among 57 Orders and 304 Families, with the vast majority being arthropods. Two IAS (Agrilus planipennis and Lymantria dispar) are regulated by the Canadian Food Inspection Agency (CFIA) as plant health pests, are known to occur in the study area, and were identified through eDNA in collected traps. Similarly, using 16S ribosomal RNA and nuclear ribosomal internal transcribed spacer (ITS), five bacterial and three fungal genera, which contain species of regulatory concern across several Canadian jurisdictions, were recovered from all sampling locations. Our study results reaffirm the effectiveness and importance of integrating eDNA metabarcoding as part of identification protocols in biosurveillance programs.