Biosurveillance for invasive insect pest species using an environmental DNA metabarcoding approach and a high salt trap collection fluid

Abstract

With the increase in global trade and warming patterns, the movement, introduction, and establishment of non‐native insect species has increased. A rapid and effective early detection biosurveillance program to identify species of concern is needed to reduce future impacts and costs associated with introduced non‐native species. One of the challenges facing insect surveillance trapping methods is the sheer volume of individual specimens in the collections. Although molecular identification methods are improving, they currently have limitations (e.g., destructive processing of specimens) and a protocol addressing these limitations can support regulatory applications that need morphological evidence to corroborate molecular data. The novel protocol presented here uses a metabarcoding approach to amplify environmental DNA from a saturated salt solution trap fluid, which retains trap specimens for downstream morphological identifications. The use of a saturated salt solution to preserve specimens in traps addresses issues with the high evaporation rate of ethanol in traps, and public safety concerns with other fluid preservation options with unattended traps in public settings. Using a metabarcoding approach, a 407‐nucleotide segment of the cytochrome c oxidase subunit 1 (COI) animal barcode region was successfully amplified from Lindgren funnel trap collection fluids. These traps were placed in forested areas to survey for wood‐boring beetles of regulatory concern. Our results displayed successful amplification of target taxa, including the molecular identification of the Japanese Beetle Popillia japonica, a species regulated in Canada. A second species, Anisandrus maiche, recently introduced to North America, was identified in every trap. The genus Lymantria, which contains numerous species of concern to North American woodlands, was also detected. Also, there were six other species identified of interest due to their potential impacts on native and crop flora and fauna. Our results show how this protocol can be used as an efficient method for the surveillance of insects using a trap with a saturated salt solution and eDNA metabarcoding to detect species of regulatory concern.

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

“…A saturated salt (NaCl) solution provides many advantages when applied in a metabarcoding biosurveillance approach for flighted insects such as species of Culicoides. One such benefit is that a salt solution protocol has fewer transportation regulations, is non-toxic, is non-flammable, is easier to store, and is less costly than protocols using an alcoholbased collection solution [16,17]. Here, we demonstrate that Culicoides specimens remained morphologically intact in the salt trap solution overnight with no visible morphological interruptions or faults and remained useful for morphological identification methods (Figures 2 and 3).…”

“…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.…”

Computational Evaluation of DNA Metabarcoding for Universal Diagnostics of Invasive Insect Pests

“…High sensitivity across a broad taxonomic scope is a defining feature of DNA metabarcoding assays, facilitating their use as a universal diagnostic assay to rapidly screen mixed samples for a range of target pests or pathogens. Despite several studies applying metabarcoding to certain pest taxa (Batovska et al, 2018(Batovska et al, , 2020Bowser et al, 2019;Young et al, 2021), the diagnostic performance of the required mini-barcodes has not until now been systematically evaluated across the broader diversity of invasive insect species. Using a curated reference database covering 110,676 insect species, including 2,625 species registered on global invasive species lists, we here demonstrate that mini-barcodes can achieve comparable resolution to the full-length COI barcode region already widely accepted within insect diagnostic protocols.…”

Molecular Acquisition, Cleaning and Evaluation in R (MACER) - A tool to assemble molecular marker datasets from BOLD and GenBank

“…The use of molecular sequence data is an essential component of many analyses across various fields of scientific study, including virology ( Radford et al 2012 ), gene expression studies ( Song et al 2021 ), evolutionary biology ( Hudson 2008 ), taxonomy and species identifications ( Hubert and Hanner 2015 ) and biodiversity surveys ( Young et al 2021 ). One of the strengths of molecular approaches is in the comparability of the data, even when obtained by different labs and researchers.…”