Methods
Field sites
Field work was conducted in the Torres Strait and the Northern Peninsula
Area of Cape York Peninsula, Qld 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 the top of Green Hill (“GHF”), and one
site on the Australian mainland in the town of Injinoo (“INJ”) whereL. sativae has never been recorded. Active populations ofL. 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 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 Supplemental
Figure S1). 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 forming, 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 eggs that failed
to hatch or saliva.
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 specificity of 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 preventing 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
likelihood of 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 Supplemental Figure S2). 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.
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, we immediately excised these additional larvae
and replaced the mesh bags.
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 sensitivity of the L. sativae eDNA method was explored on
Thursday Island in May 2019 where 288 mined leaves of M.
atropurpureum were randomly selected from FGG and TIH. 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 (because mine age was not known) and checked under the
microscope for any remains of a fly larva (see Table 1). 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
of 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 ), a wild
passionfruit (Passiflora foetida ), and an unknown weedy passion
flower (Passiflora sp.) 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 in 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,
Qld, a sparsely habited island where L. sativae had not been
recorded. The leafmining damage discovered appeared to be old, and none
of the 7 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, Qld, 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 mine 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 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 Supplemental Figure S1): 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 (NIH) 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. This included L.
sativae , L. brassicae , L. trifolii and L.
huidobrensis , as well as DNA from four other species, L.
yasumatsui , L. katoi , L. chenopodii and L.
chinensis . Amplification was only evident in the respective target
species, confirming species specificity of the primer sets 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. 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.
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.