Methods
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 lay. 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. A similar approach to the one described above was used
except 15 random M. atropurpureum leaves were selected, and the
trial repeated at three locations. These were FGG, GHF and INJ. At FGG
and GHF, leaves were collected four days after the mesh bags were first
installed, and immediately placed into sealed plastic bags. At the INJ
site, a revisit was not possible after four days. The likelihoodof L. sativae presence at this site was extremely low, so the
unmined leaves were immediately collected and 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. atropurpureumleaves. The logger recorded temperature and humidity every 10 minutes
for the duration of the trial.
Experiment 3 - eDNA sensitivity under field
conditions
The field-based sensitivity of the L. sativae eDNA method was
explored on Thursday Island. Field-based sensitivity 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 sensitivity, 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) and checked under the microscope for any remains of a larva
(see Table 1 for specific scoring criteria). The length of each leaf
mine was also 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 (Stachytarphetajamaicensis ) 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 fromS. 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. atropurpureumappeared 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.
DNA extraction
Total genomic DNA was extracted using a modified Chelex extraction
protocol (Walsh et al. 1991). Individual leaf mines or 5
mm2 sections of FTA cards were placed into 1.5 ml
tubes along with a 3 mm glass bead (Retsch GmbH, Haan, Germany), 5 µL of
proteinase K and 200 µL of 5% Chelex solution. Each tube was then
shaken in a Mixer Mill (MM300, Retsch GmbH, Haan, Germany) at 30
oscillations /s for 1 min. Samples were subsequently digested at 55 °C
for 60 min, followed by a final incubation at 95 °C for 15 min with
periodic vortexing. Extractions were stored at -20 °C until required.
Prior to real time polymerase chain reaction (qPCR) amplification,
extractions were spun at 10,000 g for 2 min. Aliquots from the bottom
half of the supernatant immediately above the Chelex resin was used for
qPCR amplification.
Molecular assays
Species-specific PrimeTime qPCR assays (Integrated DNA Technologies)
were used to target a 109 base pair (bp) fragment of the mitochondrial
CO1 gene of L. sativae with sequences as described in Sooda et
al. (2017) with the addition of two degenerate bases in the probe to
accommodate sequence variants found within the Torres Strait Islands:
NCBI accession KR476580 Haplotype S.28 (Blacket et al. 2015) and
KR476573 Haplotype S.7 (Blacket et al. 2015). Forward primer SAT-F
ACCCCCTGCTTTAACTCTTTT, reverse primer SAT-R AGCACCACCATGTGCAATAA and
reporter probe SAT-P CAGTATAGTAGAAAATGGRGCTGGRA with a 6-FAM/ZEN/IBFQ
modification.
We also developed a species specific PrimeTime qPCR assay to target a 63
bp fragment of the mitochondrial CO1 gene of L. brassicae for a
pilot trial (see Supplementary Figure S2): NCBI accession KR476570
(Blacket et al. 2015). Forward primer GCCGGAACAGGATGAACAGTTTAT,
reverse primer AGATGCCCCACCGTGAG, and reporter probe CCCCTCTCTTCTATTATTG
with a 6-FAM/ZEN/IBFQ modification. Primer specificity was checked using
a BLAST (Basic Local Alignment Search Tool) search against the National
Institutes of Health NCBI (National Center for Biotechnology
Information) nucleotide database
(https://blast.ncbi.nlm.nih.gov/Blast.cgi), with no close matches found
outside of L. brassicae.
The qPCR assays were tested for specificity against a panel of target
and off-target Liriomyza genomic DNA diluted to 10 picograms.
This included L. sativae , L. brassicae , L. trifoliiand L. huidobrensis , as well as DNA from four other
species, L. yasumatsui , L. katoi , L. chenopodii andL. chinensis . Amplification was only evident in the respective
target species, confirming species specificity of the assays developed
for both L. sativae and L. brassicae .
PrimeTime qPCR assays were conducted using a Roche LightCycler 480
system (Roche Diagnostics Australia, Castle Hill, Australia) in a
384-well format. DNA extraction and qPCR assays were performed in
separate isolated rooms. Reaction volumes were 10 μL, containing 5 μL of
KAPA probe force master mix (KAPA biosystems), 0.5 μL PrimeTime qPCR
assay (final primer and reporter probe concentration of 500 nmol/L and
250 nmol/L, respectively), 2.5 μL ddH2O, and 2 μL of DNA. Each reaction
was prepared in triplicate. Included in each 384-well assay plate were
control reactions containing genomic DNA of L. sativae that was
serially diluted by a factor of 10 (10 ng to 0.01 pg) and a negative
control with no DNA template. Quantitative PCR amplification conditions
were 3 min at 98 °C, followed by 50 cycles of 10 seconds at 95 °C and 20
seconds at 60 °C. The absolute quantification module of the LightCycler
480 software package was used to calculate the assay efficiency and
total amount of DNA in unknown samples based on the genomic DNA
standard. The efficiency of all qPCRs was always >90%. All
extractions and qPCR analyses were undertaken in a room that is
dedicated to low-quantity DNA sources, with qPCR setup undertaken in a
laminar flow-hood. Positive controls and standards were added
immediately prior to placing samples in a Roche LightCycler 480.
Negative controls were also included at all stages (DNA extraction,
qPCR) so that laboratory contamination could be identified if present.
Assay efficiency and
sensitivity
A tenfold dilution series ranging from 100 to 0.001 picogram of tissue
derived gDNA, measured with a Qubit 2.0 fluorometer (Invitrogen,
Carlsbad, CA, USA) was prepared in elution buffer AE (Qiagen). Ten
replicates of each dilution were run and the reported Cq (cycle
quantification) values were used to determine amplification efficiency,
the coefficient of determination (R 2) value as
well as the limit of detection (LOD) and limit of quantification (LOQ)
following the protocol and curve fitting method described in Klymuset al. (2019).
Data analysis
To estimate the required sampling protocol to achieve a predetermined
level of diagnostic confidence under different conditions a hierarchical
probabilistic model was used (Lugg et al. 2018). This model captured the
nested effect of eDNA presence inside a leaf, and subsequent
detectability through technical replicates of the molecular assay.
Positive diagnosis of leafminer DNA was modelled as a nested Bernoulli
variable: eDNA presence in the leaf mine (\(Y_{i,j}=1\)) for each site\(i\) and leafmine \(j\) depends on the probability \(\theta_{i,j}\)and; DNA presence in the extraction (\(X_{i,j,k}\)) for each extraction\(k\) depends on the parameter \(p\) and whether there was extractable
eDNA in the mine (\(Y_{i,j}\)). Thus,
\begin{equation}
X_{i,j,k}\ \sim\ Bern\left(\text{p\ }Y_{i,j}\right)\nonumber \\
\end{equation}\begin{equation}
Y_{i,j}\ \sim\ {Bern(\theta}_{i,j})\nonumber \\
\end{equation}The probability of viable eDNA in a leaf mine is assumed to covary with
site and leaf conditions which are modelled through a logit link
function:
\begin{equation}
\text{logit}\left(\theta_{i,j}\right)=a+B\mathbf{x}_{\mathbf{i,j}}\nonumber \\
\end{equation}The model was fitted to observations using JAGS . Coefficients with 95%
credible intervals were reported model parameters, including the binary
covariates for preservation method (FTA versus ethanol), visible
presence of larval remains (present versus absent), mine age (fresh
versus not fresh), and mine length (long versus short/medium).
The probability of leafminer diagnosis d can be estimated using
the posterior distribution of model parameters as:
\begin{equation}
d\ =\ 1\ -\ \left(\left(1\ -\ \ \theta_{i,j}\right)\left(1\ -\ \left(1\ -\ p\right)^{N_{r}}\ \right)\right)^{N_{l}}\text{\ \ \ }\nonumber \\
\end{equation}where Nr is the number of technical replicates
and Nl is the number of mined leaves tested.