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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.

SUBMITTER: Batovska J

PROVIDER: S-EPMC8041782 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Insect identification and preservation of voucher specimens is integral to pest diagnostic and surveillance activities; yet bulk-trapped insects are a diagnostic challenge due to high catch numbers and the susceptibility of samples to environmental damage. Many insect trap catches rely on examination of morphological characters for species identifications, which is a time consuming and highly skilled task, hence there is a need for more efficient molecular approaches. Many bulk DNA extraction methods require destructive sampling of specimens, resulting in damaged, or fully destroyed, voucher specimens. We developed an inexpensive, rapid, bulk DNA isolation method that preserves specimens as pinned vouchers to a standard that allows for post-extraction morphological examination and inclusion in insect reference collections. Our protocol was validated using a group of insects that are time-consuming to identify when trapped in large numbers-the dacine fruit flies (Diptera: Tephritidae: Dacinae). In developing our method, we evaluated existing protocols against the following criteria: effect on morphology; suitability for large trap catches; cost; ease of handling; and application to downstream molecular diagnostic analyses such as real-time PCR and metabarcoding. We found that the optimum method for rapid isolation of DNA extraction was immersing flies in a NaOH:TE buffer at 75°C for 10 minutes, without the need for proteinase K or detergents. This HotSOAK method produced sufficient high-quality DNA whilst preserving morphological characters suitable for species-level identification with up to 20,000 flies in a sample. The lysates performed well in down-stream analyses such as loop-mediated isothermal amplification (LAMP) and real-time PCR applications, while for metabarcoding PCR the lysate required an additional column purification step. Development of this method is a key step required for upscaling our capacity to accurately detect insects captured in bulk traps, whether for biodiversity, biosecurity, or pest management objectives.

Project description: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.

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

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