Developing a non-destructive metabarcoding protocol for detection of pest insects in bulk trap catches

Abstract

Metabarcoding has the potential to revolutionise insect surveillance by providing high-throughput and cost-effective species identification of all specimens within mixed trap catches. Nevertheless, incorporation of metabarcoding into insect diagnostic laboratories will first require the development and evaluation of protocols that adhere to the specialised regulatory requirements of invasive species surveillance. In this study, we develop a multi-locus non-destructive metabarcoding protocol that allows sensitive detection of agricultural pests, and subsequent confirmation using traditional diagnostic techniques. We validate this protocol for the detection of tomato potato psyllid (Bactericera cockerelli) and Russian wheat aphid (Diuraphis noxia) within mock communities and field survey traps. We find that metabarcoding can reliably detect target insects within mixed community samples, including specimens that morphological identification did not initially detect, but sensitivity appears inversely related to community size and is impacted by primer biases, target loci, and sample indexing strategy. While our multi-locus approach allowed independent validation of target detection, lack of reference sequences for 18S and 12S restricted its usefulness for estimating diversity in field samples. The non-destructive DNA extraction proved invaluable for resolving inconsistencies between morphological and metabarcoding identification results, and post-extraction specimens were suitable for both morphological re-examination and DNA re-extraction for confirmatory barcoding.

Disentangling bias for non-destructive insect metabarcoding

“…The results presented here show that non-destructive metabarcoding analysis can be successfully applied to bulk samples of agriculturally relevant insects to obtain a species identification, in agreement with recent studies ( Carew, Coleman & Hoffmann, 2018 ; Nielsen et al, 2019 ; Batovska et al, 2021 ). This suggests that non-destructive metabarcoding has potential applications not only for biodiversity assessments but also for diagnostics and biosecurity purposes, to determine presence/absence of pests in bulk traps.…”

“…Here, using non-destructive metabarcoding approaches, we successfully recorded all insect species present in pools composed of 100–101 individuals, including those species that were represented by just a single individual insect. At the same time, the use of these non-destructive DNA extraction methods allowed morphological voucher specimens of the insects to be preserved, as previously demonstrated ( Martoni, Valenzuela & Blacket, 2019 ; Batovska et al, 2021 ). This is of paramount importance for many regulatory applications of metabarcoding, where retaining voucher specimens of potential pests or indicator taxa can be required for legal reasons, or to simply provide a morphological specimen preserved in an entomological collection for future taxonomic investigation ( i.e ., Martins et al, 2019 ; Martoni, Valenzuela & Blacket, 2019 ; Martoni, Valenzuela & Blacket, 2021 ).…”

“…Recently, non-destructive DNA extraction has emerged as an alternative to homogenisation-based methods in order to retain voucher specimens for morphological confirmation of metabarcoding detections ( Carew, Coleman & Hoffmann, 2018 ; Martins et al, 2019 ; Batovska et al, 2021 ). This is of particular importance in the context of agricultural biosecurity and other regulatory applications of metabarcoding, allowing DNA sequences to be linked to an insect sample, which can be preserved in entomological collections for future records ( Martoni, Valenzuela & Blacket, 2021 ).…”

DNA Metabarcoding Enables High-Throughput Detection of Spotted Wing Drosophila (Drosophila suzukii) Within Unsorted Trap Catches

“…DNA metabarcoding is a broad-scope molecular diagnostic approach that couples DNA barcoding with high-throughput sequencing (HTS) to simultaneously identify all amplified DNA sequences from complex mixed communities (Taberlet et al, 2012). The resulting data can be compared to both lists of regulated species and baseline knowledge of endemic biodiversity to screen not just for target pests, but also other unanticipated taxa that are not being actively searched for Hardulak et al (2020), Batovska et al (2021). The ability for metabarcoding to be conducted on mixed trap samples without any prior sorting (Nielsen et al, 2019) is particularly appealing for efficiently handling the large number of specimens likely to be produced by an intensive surveillance program.…”

“…Robust metabarcoding protocols thus require both technical replication and use of a detection threshold to resolve true positives from any low-abundance contaminant sequences (Zinger et al, 2019). The required number and type of replicates, and most appropriate manner for deriving this detection threshold remains unclear, however, for protocols which employ non-destructive DNA extractions (i.e., methods which retain morphologically intact specimens) (Carew et al, 2018;Nielsen et al, 2019;Batovska et al, 2021). These recently developed non-destructive protocols allow the high-throughput metabarcoding detections to be confirmed using morphological examination and voucher specimens to be retained according to regulatory requirements (Martins et al, 2019;Batovska et al, 2021), yet come at the expense of reduced DNA concentrations compared to more common destructive tissue-homogenization based protocols (Martoni et al, 2021).…”