DOI: 10.5061/dryad.v9s4mw6wb
title: A molecular method for biomonitoring of an exotic plant-pest: leafmining for environmental DNA

description: 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.
description: <strong>Field sites</strong> 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. <strong>Leaf mine
 preservation</strong> 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.
 <strong>Experimental groups</strong>
 ‘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. <strong>Experiment 1 –
 Testing of unmined leaves in the field</strong>
 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.
 <strong>Experiment 2 - eDNA persistence
 trial</strong> 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.
 <strong>Experiment 3 - eDNA detection under field
 conditions</strong> 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 (&lt; 20 mm), medium (20-50mm) or
 long (&gt; 50mm). Unmined leaf samples were the
 same as those used during the 2019 trials in Experiment 1.
 <strong>Experiment 4 - Field applications to delimit
 geographic range and host range</strong> 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.