Discussion

The eDNA method developed here was shown to be highly robust under field conditions, reliably detecting and diagnosing L. sativae DNA from empty leaf mines under tropical conditions. This novel approach significantly increases the surveillance opportunity for this invasive pest and is likely to be highly transferable to other globally important agromyzid flies. Preservation methods of collected leaf samples were found to be important with 100% ethanol being the most reliable collection procedure (but FTA cards also feasible). There was no decline in diagnostic success measured up to 28 days after the emergence of larvae from a mine. Other forms of arthropod eDNA, such as that of the tiger mosquito (Aedes albopictus) , have been shown to persist in the environment for similar lengths of time (Schneider et al. 2016).
Preliminary field applications have already highlighted the flexibility of the eDNA method through identification of new local hosts, includingS. jamaicensis , C. annuum , P. edulis and O. basilicum , and the first occurrence record of L. sativae on Zuna island. These results widen the range of hosts that will be considered during routine surveillance aimed at containing this pest. While the current preference for these host plants appears low, the occurrence patterns of these plants could nevertheless make them important vectors of spread. Passiflora edulis , C. annuum and O. basilicum are common garden plants, with O. basilicum posing a particular risk given it is a popular herb that is routinely transported by tourists and locals as living plants. Stachytarphetajamaicensis is a very common and widely distributed weed found particularly in disturbed areas throughout northern Queensland and other parts of Australia (Atlas of Living Australia 2019). This new knowledge has already been used by the Northern Australian Quarantine Strategy in their regular surveillance programs for exotic pests (B. Waterhouse and S. Cowan, pers. comm.).
The eDNA method developed herein is likely to become an important tool for distinguishing agromyzids that remain a biosecurity threat to Australia’s agricultural industries. It presents a new opportunity to reduce the spread of exotic leafminers, which, historically, have been difficult to detect and contain overseas due to poor host records and overlap with native leafminers (Powell 1981). For example, not only is the leaf mining damage created by L. sativaeindistinguishable from other high-risk exotic agromyzids, includingL. trifolii and L. huidobrensis , it can also be easily confused with the closely related L. brassicae , already common in Australia, which not only creates indistinguishable damage on many of the same plants as L. sativae , but is also indistinguishable as an adult by any means other than dissection or molecular analysis (Shiao 2004; International Plant Protection Convention 2016).
The utility of eDNA in biosecurity programs will extend beyond leafmining species. For example, eDNA approaches using samples collected from water bodies have greater sensitivity compared with traditional surveillance techniques for invasive mosquito larvae (Aedes albopictus , A. japonicus japonicus and A. koreicus ) (Schneider et al. 2016) and the invasive American bullfrog (Rana catesbeiana ) (Dejean et al. 2012). Yet, there remain many unexplored opportunities to apply eDNA to enhance detection of exotic species, particularly in the terrestrial realm. Recently, eDNA approaches have been developed to detect the highly invasive brown marmorated stinkbug (Halyomorpha halys ) in orchards from water reservoirs used to wash fruit as well as bat faecal material; both methods achieved a higher sensitivity than traditional trapping methods such as light and pheromone traps (Maslo et al. 2017; Valentin et al. 2018). Opportunities for early detection of other exotic invertebrate pests also exist and remain to be explored. For instance, the giant African snail (Lissachatina fulica ) leaves a trail of slime that is likely to include their DNA and developing an efficient eDNA sampling protocol could be a more sensitive method of detection at borders (e.g. sea ports). Beyond early detection of exotic species, scalable and high-throughput eDNA techniques should become increasingly valuable for routine monitoring programs. Such technologies will increase the efficiency and sensitivity of both delimiting responses and proof of area freedom assessments, where the large-scale sampling necessary for statistical reliability can be associated with large time and labour costs (Gambley et al. 2009; Abdalla et al. 2012).
Although promising, eDNA will not always result in reliable diagnostics. Derocles et al. (2015) used a mini-barcoding approach to target degraded DNA inside empty leaf mines in a number of plant hosts, but found only 6% of the mines collected from the field (and therefore of an unknown age) allowed for species identification, and only 33% of the mines with a known age (where the emergence of the leafminer was observed and recorded) allowed for identification. This is far lower than the detectability we found for L. sativae . In part, this may be attributed to the use of general primers (designed for a range of lepidopteran and dipteran species) and PCR conditions not optimised to highly degraded DNA (Derocles et al. 2015). Within our study, a highly specific qPCR assay for L. sativae was applied that could detectL. sativae DNA quantities as low as 0.1 pg. The use of specific primers, qPCR detection, as well as improved DNA extraction methods is likely to have accounted for the higher detection rates compared with previous studies.
In many cases eDNA approaches may not replace traditional surveillance techniques, but rather complement them for improved detection (Schneider et al. 2016). In situations where false positives could lead to considerable economic losses (e.g. trade restrictions being imposed), morphological support should ideally complement molecular tests. Further, the success of an eDNA approach will be dependent on the target species lifecycle and biology. To determine the potential for a successful eDNA approach, one must consider what traces a target species might leave behind in its environment, and where those traces are most likely to be concentrated. For example, species with highly localised activity, such as pollinating insects (Thomsen and Sigsgaard 2019), may be more conducive to such an approach than transient species. However, it may be possible to improve detection rates for transient species via ‘eDNA traps’ that take advantage of the target species’ behaviour or industry practices that concentrate eDNA. Burns et al. (in review ) employed a novel eDNA trap to detect the threatened Baw Baw frog (Philoria frosti ) and its’ key threatening pathogen chytridiomycosis. The application of eDNA in terrestrial environments is challenging, but innovative approaches can result in achieving methods being developed that are more sensitive and efficient than traditional approaches.
As the interconnectedness of global economies continues to remove spatial barriers between regions, biosecurity technologies must keep pace to mitigate the enormous pressures from alien species placed on biological systems. Novel eDNA methods based on modern DNA technologies will undoubtedly play a vital role in modern biosecurity efforts to protect both natural and agricultural ecosystems.