Trap-based surveillance strategies are widely used for monitoring of invasive insect species, aiming to detect newly arrived exotic taxa as well as track the population levels of established or endemic pests. Where these surveillance traps have low specificity and capture non-target endemic species in excess of the target pests, the need for extensive specimen sorting and identification creates a major diagnostic bottleneck. While the recent development of standardized molecular diagnostics has partly alleviated this requirement, the single specimen per reaction nature of these methods does not readily scale to the sheer number of insects trapped in surveillance programmes. Consequently, target lists are often restricted to a few high-priority pests, allowing unanticipated species to avoid detection and potentially establish populations.DNA metabarcoding has recently emerged as a method for conducting simultaneous, multi-species identification of complex mixed communities and may lend itself ideally to rapid diagnostics of bulk insect trap samples. Moreover, the high-throughput nature of recent sequencing platforms could enable the multiplexing of hundreds of diverse trap samples on a single flow cell, thereby providing the means to dramatically scale up insect surveillance in terms of both the quantity of traps that can be processed concurrently and number of pest species that can be targeted. In this review of the metabarcoding literature, we explore how DNA metabarcoding could be tailored to the detection of invasive insects in a surveillance context and highlight the unique technical and regulatory challenges that must be considered when implementing high-throughput sequencing technologies into sensitive diagnostic applications.

DNA barcoding is a modern species identification technique that can be used to distinguish morphologically similar species, and is particularly useful when using small amounts of starting material from partial specimens or from immature stages. In order to use DNA barcoding in a surveillance program, a database containing mosquito barcode sequences is required. This study obtained Cytochrome Oxidase I (COI) sequences for 113 morphologically identified specimens, representing 29 species, six tribes and 12 genera; 17 of these species have not been previously barcoded. Three of the 29 species ─ Culex palpalis, Macleaya macmillani, and an unknown species originally identified as Tripteroides atripes ─ were initially misidentified as they are difficult to separate morphologically, highlighting the utility of DNA barcoding. While most species grouped separately (reciprocally monophyletic), the Cx. pipiens subgroup could not be genetically separated using COI. The average conspecific and congeneric p‐distance was 0.8% and 7.6%, respectively. In our study, we also demonstrate the utility of DNA barcoding in distinguishing exotics from endemic mosquitoes by identifying a single intercepted Stegomyia aegypti egg at an international airport. The use of DNA barcoding dramatically reduced the identification time required compared with rearing specimens through to adults, thereby demonstrating the value of this technique in biosecurity surveillance. The DNA barcodes produced by this study have been uploaded to the ‘Mosquitoes of Australia–Victoria’ project on the Barcode of Life Database (BOLD), which will serve as a resource for the Victorian Arbovirus Disease Control Program and other national and international mosquito surveillance programs.

Effective vector and arbovirus surveillance requires timely and accurate screening techniques that can be easily upscaled. Next‐generation sequencing (NGS) is a high‐throughput technology that has the potential to modernize vector surveillance. When combined withDNAbarcoding, it is termed ‘metabarcoding.’ The aim of our study was to establish a metabarcoding protocol to characterize pools of mosquitoes and screen them for virus. Pools contained 100 morphologically identified individuals, including one Ross River virus (RRV) infected mosquito, with three species present at different proportions: 1, 5, 94%. Nucleic acid extracted from both crude homogenate and supernatant was used to amplify a 269‐bp section of the mitochondrialcytochrome c oxidase subunitI (COI) locus. Additionally, a 67‐bp region of theRRVE2 gene was amplified from synthesizedcDNAto screen forRRV. Amplicon sequencing was performed using an Illumina MiSeq, and bioinformatic analysis was performed using aDNAbarcode database of Victorian mosquitoes. Metabarcoding successfully detected all mosquito species andRRVin every positive sample tested. The limits of species detection were also examined by screening a pool of 1000 individuals, successfully identifying the species andRRVfrom a single mosquito. The primers used for amplification, number ofPCRcycles and total number of individuals present all have effects on the quantification of species in mixed bulk samples. Based on the results, a number of recommendations for future metabarcoding studies are presented. Overall, metabarcoding shows great promise for providing a new alternative approach to screening large insect surveillance trap catches.

With its small size and low cost, the hand-held MinION sequencer is a powerful tool for in-field surveillance. Using a metagenomic approach, it allows non-targeted detection of viruses in a sample within a few hours. This study aimed to determine the ability of the MinION to metagenomically detect and characterise a virus from an infected mosquito. RNA was extracted from an Aedes notoscriptus mosquito infected with Ross River virus (RRV), converted into cDNA and sequenced on the MinION. Bioinformatic analysis of the MinION reads led to detection of full-length RRV, with reads of up to 2.5kb contributing to the assembly. The cDNA was also sequenced on the MiSeq sequencer, and both platforms recovered the RRV genome with >98% accuracy. This proof of concept study demonstrates the metagenomic detection of an arbovirus, using the MinION, directly from a mosquito with minimal sample purification.

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.

Internal Transcribed Spacer 2 (ITS2) is a popular DNA barcoding marker; however, in some animal species it is hypervariable and therefore difficult to sequence with traditional methods. With next-generation sequencing (NGS) it is possible to sequence all gene variants despite the presence of single nucleotide polymorphisms (SNPs), insertions/deletions (indels), homopolymeric regions, and microsatellites. Our aim was to compare the performance of Sanger sequencing and NGS amplicon sequencing in characterizing ITS2 in 26 mosquito species represented by 88 samples. The suitability of ITS2 as a DNA barcoding marker for mosquitoes, and its allelic diversity in individuals and species, was also assessed. Compared to Sanger sequencing, NGS was able to characterize the ITS2 region to a greater extent, with resolution within and between individuals and species that was previously not possible. A total of 382 unique sequences (alleles) were generated from the 88 mosquito specimens, demonstrating the diversity present that has been overlooked by traditional sequencing methods. Multiple indels and microsatellites were present in the ITS2 alleles, which were often specific to species or genera, causing variation in sequence length. As a barcoding marker, ITS2 was able to separate all of the species, apart from members of the Culex pipiens complex, providing the same resolution as the commonly used Cytochrome Oxidase I (COI). The ability to cost-effectively sequence hypervariable markers makes NGS an invaluable tool with many applications in the DNA barcoding field, and provides insights into the limitations of previous studies and techniques.