1. Understanding how invasive species respond to novel environments is limited by a lack of sensitivity and throughput in conventional biomonitoring methods. Arthropods in particular are often difficult to monitor due to their small size, rapid lifecycles, and/or visual similarities with co-occurring species. This is true for the agromyzid leafminer fly, Liriomyza sativae, a global pest of vegetable and nursery industries that has recently established in Australia.

2. A robust method based on environmental DNA (eDNA) was developed exploiting traces of DNA left inside ‘empty’ leaf mines, which are straightforward to collect and persist longer in the environment than the fly. This extends the window of possible diagnosis to at least 28 days after a leaf mine becomes empty. The test allowed for visually indistinguishable leafmining damage caused by L. sativae to be genetically differentiated from that of other flies.

 3. Field application resulted in the identification of new local plant hosts for L. sativae, including widely distributed weeds and common garden crops, which has important implications for the pest’s ability to spread. Moreover, the test confirmed the presence of a previously unknown population of L. sativae on an island in the Torres Strait.  

4. The developed eDNA method is likely to become an important tool for L. sativae and other leafmining species of biosecurity significance, which, historically, have been difficult to detect, diagnose and monitor. More generally, eDNA is emerging as a highly sensitive and labour-efficient surveillance tool for difficult to survey species to improve outcomes for agricultural industries, global health, and the environment.

Field sites

Field work was conducted in the Torres Strait and the Northern Peninsula Area of Cape York Peninsula, Queensland (Australia) between 2017 and 2019. Trial work took place across three sites on Thursday Island, including the Frog Gully community garden (referred to as “FGG”), a roadside near Thursday Island Hospital (“TIH”) and Green Hill (“GHF”), and one site on the Australian mainland in the town of Injinoo (“INJ”) where L. sativae has never been recorded. Active populations of L. sativae were present at the FGG and TIH sites, while activity was uncommon at the GHF site. The INJ site falls outside the current known range of L. sativae on the mainland. In addition to the trial work, the methods were tested on samples that had been collected as part of regular surveillance activities throughout the Torres Strait, including on Zuna Island, Horn Island and other regions on Thursday Island.

Leaf mine preservation

After collection of plant material, leaf mines were photographed and preserved into either 100% ethanol or onto a Whatman® FTA card, both confirmed as suitable preservation techniques via a pilot study (see Supplementary Figure S2). For mines stored into ethanol, extra leaf material was cut away from the mine, and the mines were placed into a 2 mL Axygen® tube with enough ethanol to submerge the mine (~ 0.75 – 1 mL). Leaf mines were typically up to 2.5 mm wide, and between 20 to 100 mm in length. For mines preserved onto FTA cards, leaves were rubbed, mine side down for about 30 seconds, onto the surface of the card. FTA cards were stored at 4 °C and ethanol samples at -20 °C until analysis.

Experimental groups

‘Positive control’ samples refer to mines that were preserved with the larvae still present within the leaf. ‘Zero day’ samples refer to mines that were collected before larvae had naturally emerged, but the larva was then carefully removed by excision, before the mine was preserved. ‘Unmined’ samples refer to leaves that, after having been isolated in a mesh bag while still on living plants for at least four days, showed no signs of mines, and were therefore taken as absent of larvae. However, while these leaves had no visible signs of mining, L. sativae may still have been present in the area and may have had opportunity to deposit DNA on the leaf in the form of saliva and/or eggs that failed to hatch.

Experiment 1 – Testing of unmined leaves in the field

Trials were undertaken in the Torres Strait and Cape York Peninsula of Australia to investigate the potential for false positives from unmined leaves by the L. sativae eDNA method using siratro weed (Macroptilium atropurpureum), a favoured host of L. sativae in the Torres Strait (Blacket et al. 2015). In 2018, ten random leaves of M. atropurpureum that showed no visible signs of leaf mining were selected at FGG. Each leaf was individually enclosed in a small mesh bag for the duration of the trial. The mesh bags were designed to prevent adult flies accessing the leaf surface, and thus prevent egg laying. After 11 days, each leaf was removed from their respective bag, visibly inspected for the presence of leaf mining, and placed into sealed plastic bags. In 2019, additional field trials were undertaken at three locations. A similar approach to the one described above was used except, at each site, 15 random M. atropurpureum leaves were selected. At two of the three locations, FGG and GHF, leaves were collected four days after mesh bags were first installed, and immediately placed into sealed plastic bags. At the third site, INJ, a revisit was not possible after four days. However, the likelihood of L. sativae presence at this site was extremely low. Thus, random leaves of M. atropurpureum that showed no visible signs of leaf mining were selected and immediately placed into sealed plastic bags. Plant samples from the 2018 and 2019 trials were transported back to the laboratory and stored at -20 °C prior to molecular testing.

Experiment 2 - eDNA persistence trial

To determine the appropriate timeframe to examine the persistence of leafminer eDNA in the field, a pilot trial was conducted on a related and widespread species, L. brassicae (Riley), from which it was found that eDNA remained in leaf mines for at least 28 days under laboratory conditions (see Supplementary Figure S3). A field trial was then conducted at FGG on Thursday Island between July - August 2018 involving L. sativae. Seventy-three active leaf mines were identified on M. atropurpureum. These mines were randomly allocated to experimental groups ranging from 0 to 28 days. Within 24 h, as a result of the rapid lifecycle of L. sativae in this tropical location, some of the larvae in selected leaf mines had already exited the mine. For those that did not emerge within 24 h, the larvae were carefully excised manually, using a thin pair of tweezers, ensuring that the emergence hole created was no larger than for natural emergences. In this way, all larvae emerged, either naturally or artificially, on the same day. A photograph was taken of each mine at this point, for later reference.

Each leaf was then enclosed in a small mesh bag to ensure no further egg lay, and thus no additional leaf mining. Between 9 and 14 leaves were collected on days 0, 1, 3, 7, 14 and 28, and these were placed into separate sealed plastic bags and stored at -20 °C prior to molecular testing.

In the first four days of the trial, all leaves were monitored closely to ensure only one leaf mine developed per leaf. If additional mines were observed forming due to eggs already present in the leaf before the addition of the mesh bag hatching, we immediately excised these additional larvae to prevent further development of the unwanted mines and replaced the mesh bags. To ensure the correct mine was ultimately collected, the photograph taken on day 0 of the desired mine was referenced upon collection. Any leaves for which the original mine was intersected by the formation of a new mine were discarded from the experiment.

A temperature logger (iButton® Maxim Integrated) was placed inside a mesh bag and positioned in the shade among M. atropurpureum leaves. The logger recorded temperature and humidity every 10 minutes for the duration of the trial.

Experiment 3 - eDNA detection under field conditions

The field-based detection of the L. sativae eDNA method was explored on Thursday Island. Field-based detection here refers to the proportion of leaves, all of which are known to have at some point been exposed to L. sativae DNA as a result of leaf mining, but for which the age, concentration, and level of degradation of the DNA is unknown, which yield positive detections via the eDNA test. Thus, the goal of this experiment was not to determine the actual threshold concentration of DNA which could be detected by the eDNA test (this was determined in the laboratory, see below), but rather to determine a realistic field measure of detectability, as the parameters of DNA age, concentration and degradation will almost always be unknown from field collected leaf mine samples. In May 2019, 288 mined leaves of M. atropurpureum were randomly selected from FGG and TIH (144 leaves at each location). Ninety-two leaf mines from each site were excised and placed into 2 mL Axygen® tubes with 100% ethanol. The remaining 52 leaves from each site were preserved onto FTA cards, following the methods described above. Prior to preservation, each mine was scored by its appearance as either fresh, medium or old (since mine age was unknown). To reduce subjectivity in the characterization of mines as fresh, medium or old was relatively subjective, rough guidelines were created based on the appearance of mine colour and distinctiveness of frass trails, and reference mines were selected to provide photographic examples of each category (see Table 1 for specific scoring criteria). Moreover, all mines across all trials and years were scored by the same researcher, who had spent considerable time observing the formation and aging of leaf mines in the field and in the lab. Of course, this appearance score is still not a direct indicator of mine age, as a variety of other factors could possibly influence the appearance of the mine, such as the health of the plant or microclimate conditions of each particular leaf. In additional to the appearance score, each mine was checked under the microscope for any remains of a larva (see Table 1) and the length of each leaf mine was estimated, and categorised as short (< 20 mm), medium (20-50mm) or long (> 50mm).

Unmined leaf samples were the same as those used during the 2019 trials in Experiment 1.

Experiment 4 - Field applications to delimit geographic range and host range

To explore the utility of the L. sativae eDNA method to host plants beyond M. atropurpureum, we applied the test to a range of host plants, selected from field collections between 2018-2019 in Torres Strait where L. sativae is known to be present. In July 2018, leaf mines that looked similar in appearance to L. sativae mining were collected from chilli (Capsicum sp.), passionfruit (Passiflora edulis) and basil (Ocimum basilicum) from FGG. A single leaf mine found on snakeweed (Stachytarpheta jamaicensis) was collected from Horn Island. In May 2019, five mines each from snakebean (Vigna unguiculata ssp. sesquipedalis), tomato (Solanum lycopersicum), wild passionfruit (Passiflora foetida), and yellow alder weed (Turnera ulmifolia) were collected from FGG and stored in 2 mL Axygen® tubes with 100% ethanol. Additionally, ten leaf mines from S. jamaicensis (from a single patch of leaf mines discovered on Thursday Island), were collected and stored in 100% ethanol. These samples were transported back to the laboratory and stored at -20 °C prior to molecular testing.

To further test the application of the eDNA methodology during delimiting survey activities, leaf mines were collected in July 2018 from seven leaf mines found in M. atropurpureum on Zuna Island, Queensland, a sparsely habited island where L. sativae had not been recorded previously and leaf mining activity was known to be very low. The leafmining damage discovered in M. atropurpureum appeared to be old, and none of the seven mines contained any active larvae that could be reared or preserved for identification. In June 2019, an empty leaf mine was also collected in Cairns, Queensland, from an eggplant (Solanum melongena), a known host of L. sativae, but also a host of other leafminer species present in Australia.

In all instances, the empty leaf mines were closely inspected under the microscope prior to preservation, and some samples were found to contain visible remains of dead fly larvae. Sections of the mine that contained these remains were preserved, and analysed, separately from the rest of the empty mines to improve amplification of DNA.