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

Abstract

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.

Validation of an Effective Protocol for Culicoides Latreille (Diptera: Ceratopogonidae) Detection Using eDNA Metabarcoding

“…All molecular analyses were performed in the Hanner laboratory at the University of Guelph, Ontario, Canada and using the protocols described in Milián-García et al, 2020 [16]. A modified CTAB buffer [24,25] (2% w/v cetyltrimethyl ammonium bromide, 2% w/v polyvinylpyrrolidone, 1.4M NaCl, 100 mM Tris-HCL, 20 nM EDTA) was used for tissue and cellular lysis due to recurrent success retrieving quality eDNA yield in the literature [26][27][28][29].…”

“…A high-throughput sequencing library for the COI gene was prepared in two PCRs [16,30]. First, a~407 base-pair segment of the mitochondrial genome (mtDNA) was amplified as a single fragment for all samples with the addition of Illumina adaptors.…”

“…First, a~407 base-pair segment of the mitochondrial genome (mtDNA) was amplified as a single fragment for all samples with the addition of Illumina adaptors. The first PCR was conducted in a thermal cycler (Eppendorf Mastercycler) using the forward primers mLepF1_MiSeq/RonMWASPdeg_MiSeq and the reverse primers LepR1_MiSeq/HCO2198_MiSeq [16]. [16,30].…”

“…Glycol-based collection fluids are expensive and recognized as poisonous, limiting their safe use in public spaces. Alcohol-based fluids for transporting specimens are also disadvantageous due to their flammability, and therefore require special handling and transportation restrictions [16,17].…”

“…Using a salt solution as a substitute collection fluid allows for short-term specimen preservation during field placement and transportation to facilities for long-term storage processing. The use of inexpensive saturated salt (NaCl) water solutions has already proven useful for preserving arthropod morphological structures and as a reservoir of eDNA [16,17]. Using a saturated salt solution can eliminate the need for alcohol-based substances as a field collection fluid and removes the requirements of hazardous materials permits for shipping.…”