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

Abstract

The spotted wing drosophila (Drosophila suzukii, Matsumara) is a rapidly spreading global pest of soft and stone fruit production. Due to the similarity of many of its life stages to other cosmopolitan drosophilids, surveillance for this pest is currently bottlenecked by the laborious sorting and morphological identification of large mixed trap catches. DNA metabarcoding presents an alternative high-throughput sequencing (HTS) approach for multi-species identification, which may lend itself ideally to rapid and scalable diagnostics of D. suzukii within unsorted trap samples. In this study, we compared the qualitative (identification accuracy) and quantitative (bias toward each species) performance of four metabarcoding primer pairs on D. suzukii and its close relatives. We then determined the sensitivity of a non-destructive metabarcoding assay (i.e., which retains intact specimens) by spiking whole specimens of target species into mock communities of increasing specimen number, as well as 29 field-sampled communities from a cherry and a stone fruit orchard. Metabarcoding successfully detected D. suzukii and its close relatives Drosophila subpulchrella and Drosophila biarmipes in the spiked communities with an accuracy of 96, 100, and 100% respectively, and identified a further 57 non-target arthropods collected as bycatch by D. suzukii surveillance methods in a field scenario. While the non-destructive DNA extraction retained intact voucher specimens, dropouts of single species and entire technical replicates suggests that these protocols behave more similarly to environmental DNA than homogenized tissue metabarcoding and may require increased technical replication to reliably detect low-abundance taxa. Adoption of high-throughput metabarcoding assays for screening bulk trap samples could enable a substantial increase in the geographic scale and intensity of D. suzukii surveillance, and thus likelihood of detecting a new introduction. Trap designs and surveillance protocols will, however, need to be optimized to adequately preserve specimen DNA for molecular identification.

Contrasting results of multiple species delimitation approaches cause uncertainty in synecological studies

“…With the ongoing employment of integrative approaches using multiple species delimitation methods, it became clear that neither of these always perfectly matches the morphospecies entities (e.g., Ahrens et al, 2013Ahrens et al, , 2016Dalstein et al 2019;Eberle et al, 2019;Lukic et al, 2021) nor do they rarely ever match among each other (Ranasinghe et al, 2022a). Although metabarcoding approaches use mainly distance based clustering algorithms with predefined thresholds for species circumscription (Callahan et al, 2017;Piper et al, 2019), several different pipelines or "cluster parameter values" are in use that may reveal alternative outcome (e.g., Potter et al, 2017;Alberdi et al, 2018;Creedy et al 2019;Bailt et al, 2020) The threshold values used for sequence filtering and the number of reads for the identified MOTUs have an effect on the assessment accuracy of data (Edgar & Flyvbjerg, 2015;Potter et al, 2017;Meyer et al, 2021;Piper et al, 2019Piper et al, , 2022. The choice of the clustering threshold at 3% pairwise distance, as applied by the majority of studies (Elbrecht et al, 2017;Yu et al, 2012;Alberdi et al, 2018), or at 2% for similar taxa (Beentjes et al, 2019;Rossini et al, 2016;Smith et al, 2005) can have a significant impact on taxonomic inferences.…”

Using DNA metabarcoding to assess insect diversity in citrus orchards

“…DNA metabarcoding offers simultaneous identification of multiple species in bulk samples ( Yu et al, 2012 ). Prior studies have used metabarcoding to examine insect biodiversity, pest prevalence ( Piper et al, 2022 ), and to evaluate predator-prey relationships ( Yang et al, 2022 ). For example, ( Huang et al, 2022 ) used metabarcoding to reveal the composition of Diptera communities in a subtropical system, while ( Kirse et al, 2021 ) used this method to analyze seasonal shifts in arthropod diversity in Malaise trap catches.…”

Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar

“…Given the increased utility of genetic data to support species identification, there has been a shift from time-consuming morphological identification to bulk processing of trap catches for downstream genetic characterisation. Downstream approaches, such as metabarcoding identification and real-time quantitative PCR, have become increasingly popular for such bulk insect identification [ 4 , 5 ].…”

“…Taking these considerations into account, our aims were to develop a non-destructive extraction protocol that: (1) had minimal impact on morphology, especially colour, so that voucher specimens can be validated and/or preserved; (2) was cost effective and simple by minimising the use of, or eliminating, reagents such as proteinase K or detergents and is potentially suitable for infield use; (3) uses non-toxic reagents (4); produces high DNA quality and yield; (5) has the capacity to be scaled up for bulk samples; (6) produces extracts that are suitable for use in downstream molecular diagnostic applications (i.e., loop-mediated isothermal amplification [LAMP], real-time PCR and metabarcoding); and finally, (7) was applicable to our chosen study group, the dacine fruit flies, as a means to evaluate the potential for bulk molecular screening of fruit fly trap samples to support Australian biosecurity surveillance and diagnostics operations. We anticipate that successful bulk DNA extraction whilst preserving tephritid fruit fly morphology will translate to other insect groups where diagnostics characters, especially colour, must be preserved.…”