Molecular Survey for the Invasive Leafminer Pest Liriomyza
huidobrensis (Diptera: Agromyzidae) in California Uncovers Only the
Native Pest Liriomyza langei
Scheffer, S. J., Lewis, M. L., Gaimari, S. D., & Reitz, S. R. (2014). Molecular
Survey for the Invasive Leafminer Pest Liriomyza huidobrensis (Diptera:
Agromyzidae) in California Uncovers Only the Native Pest Liriomyza langei.
Journal of Economic Entomology, 107(5), 1959-1964. doi:10.1603/EC13279
10.1603/EC13279
Entomological Society of America
Version of Record
http://cdss.library.oregonstate.edu/sa-termsofuse
MOLECULAR ENTOMOLOGY
Molecular Survey for the Invasive Leafminer Pest Liriomyza
huidobrensis (Diptera: Agromyzidae) in California Uncovers Only the
Native Pest Liriomyza langei
SONJA J. SCHEFFER,1,2 MATTHEW L. LEWIS,1 STEPHEN D. GAIMARI,3
AND
STUART R. REITZ4
J. Econ. Entomol. 107(5): 1959Ð1964 (2014); DOI: http://dx.doi.org/10.1603/EC13279
ABSTRACT Liriomyza huidobrensis (Blanchard) is a highly destructive invasive leafminer pest
currently causing extensive damage to vegetable and horticultural crops around the world. Liriomyza
langei Frick is a leafminer pest native to California that cannot currently be morphologically distinguished from L. huidobrensis. We used a DNA-barcoding approach, a published PCR-RFLP method,
and a new multiplex PCR method to analyze 664 ßies matching the morphological description of
huidobrensisÐlangei. We found no evidence for the presence of L. huidobrensis in our extensive samples
from California. In addition to the new molecular method, this work is important because it provides
deÞnitive data that the California “pea leafminer” is currently, and has probably always been, L. langei.
These data will also be important in the event that the highly invasive L. huidobrensis ever becomes
established.
KEY WORDS molecular diagnostics, multiplex PCR, invasive species, crop pest
The polyphagous leafmining ßy Liriomyza huidobrensis
(Blanchard) has become a notorious pest of a wide
variety of vegetable and ßower crops that number in the
hundreds (Spencer 1973, Reitz and Trumble 2002).
Originally described from Argentina (Blanchard
1926), since the 1980s, L. huidobrensis has become
invasive and has spread to Europe, the Middle East,
Asia, and North America (Canada; van der Linden
1990; Weintraub and Horowitz 1995; Head et al. 2000;
Scheffer 2000; Scheffer et al. 2001, 2006; He et al.
2002). Newly established populations are often devastating, and it is currently considered one of the
worldÕs most destructive agromyzid pests.
In 1951, Frick described a leafminer from peas in
California as Liriomyza langei Frick (Frick 1951, Parrella
1982). Upon further examination, this species appeared
to be morphologically indistinguishable from L. huidobrensis and was synonymized with L. huidobrensis by
Spencer (1973). Scheffer (2000) found that South
American and Californian L. huidobrensis s.l. differed
substantially in mitochondrial cytochrome oxidase sequence data. These molecular differences were subThe use of trade, Þrm, or corporation names in this publication is
for the information and convenience of the reader. Such use does not
constitute an ofÞcial endorsement or approval by the United States
Department of Agriculture or the Agricultural Research Service of
any product or service to the exclusion of others that may be suitable.
1 Systematic Entomology Laboratory, USDAÐARS, Bldg. 005, Rm.,
137, 10300 Baltimore Ave., Beltsville, MD 20705.
2 Corresponding author, e-mail: sonja.scheffer@ars.usda.gov.
3 Plant Pest Diagnostics Branch, California Department of Food and
Agriculture, 3294 Meadowview Rd., Sacramento, CA 95832.
4 Malher County Extension, Department of Crop and Soil Science,
Oregon State University, 710 SW 5th Ave., Ontario, OR 97914.
sequently corroborated by differences in two independent nuclear genes, and the name L. langei Frick
was revived for the California (plus Hawaii) populations (Scheffer and Lewis 2001). Invasive populations
around the world sampled to date have invariably carried
mitochondrial haplotypes belonging to the South American L. huidobrensis (e.g., those from Canada, China,
Indonesia, Italy, Israel, Japan, Korea, the Philippines,
South Africa, Sri Lanka, and Taiwan; Scheffer 2000;
Scheffer et al. 2001, 2006; He et al. 2002; Takano et al. 2005
as reported in Takano et al. 2008). Most recently, Takano
et al. (2008) found clear evidence of partial reproductive
isolation between two colonies of ßies molecularly identiÞed as L. langei and L. huidobrensis. Currently, there is
no evidence of these two species occurring in sympatry
in any part of the world.
California is one of the primary vegetable-growing
regions within the United States. Although L. langei
has been known in California as “the pea leafminer”
(at times as L. huidobrensis) and reported as a
polyphagous pest since at least the 1930s (Chaney
1995), it was not until the 1990s that its pest status in
California changed from minor secondary pest to an
economically important crop pest (Chaney 1995, Reitz and Trumble 2002). It has been suggested that this
change has come about due to the presence of a
genetically distinct and more pestiferous population in
the central coastal valleys that is spreading southward
(Morgan et al. 2000, Reitz and Trumble 2002). Given
the invasive nature of South American L. huidobrensis
and the fact that currently it cannot be distinguished
morphologically from L. langei, the question of
whether invasive L. huidobrensis is present but un-
1960
JOURNAL OF ECONOMIC ENTOMOLOGY
documented in California has never been adequately
addressed. The purpose of this study was to conduct
a molecular survey to determine whether L. huidobrensis is present but masquerading as L. langei in vegetable-growing regions of California. To accomplish
this, we used molecular methods to screen extensive
samples of adult ßies collected from several regions
and crops known to harbor L. langei. We paid particular attention to the populations in the central coastal
valleys, where the change in pest status of presumed
L. langei was Þrst observed (Chaney 1995, Reitz and
Trumble 2002).
Materials and Methods
Members of the California Department of Food and
Agriculture collected leafmining ßies by sweep-netting various crop hosts during the period 3Ð24 October
2006 in 11 counties of California: Fresno, Imperial,
Monterey, Riverside, San Benito, San Diego, San Luis
Obispo, San Mateo, Santa Barbara, Santa Cruz, and
Ventura. Specimens were preserved in ethanol and
stored at ⫺80⬚C. Because multiple leafminer species
were present, samples were sorted by S.J.S. to include
only ßies matching the external morphological description of L. langei and L. huidobrensis.
Two molecular methods were used to screen ßies
for evidence of L. huidobrensis. First, total genomic
nucleic acids were extracted from the abdomens of
individual male ßies, and mitochondrial cytochrome
oxidase I was ampliÞed and sequenced. In the second
method, female abdomens were pooled, and the resulting pooled genomic nucleic acids were screened
using two PCR-based screening procedures. Males
and females were treated differently to save genitalia
of individual male specimens for morphological analysis while at the same time processing a large number
of specimens while minimizing the time and expense
of sequencing all specimens in the study.
Males and DNA Sequencing. Male specimens were
dissected and the abdomens removed and subjected to
the DNeasy tissue extraction procedure (Qiagen, Valencia, CA) without grinding. The essentially intact
abdomens were left to soak in the lysis buffer with
proteinase K overnight and were retrieved as vouchers following the extraction procedure. Primers TYJ-1461 (forward, Winkler et al. 2009) and TD-N-3862
(reverse, Simon et al. 1994) were used to amplify the
entire mitochondrial COI and COII region. We sequenced only the COI region of ⬇1,441 bp, including the
DNA “barcode,” as previous work has shown that L.
huidobrensis and L. langei are easily distinguished using even shorter COI sequences (Scheffer 2000, Scheffer et al. 2006). Sequencing was accomplished using an
ABI 3130XL (Life Technologies). The resulting sequences were assembled and the consensus sequences
aligned using Sequencher (Gene Codes, Ann Arbor,
MI). These sequences were compared with previously
published sequences for L. huidobrensis and L. langei
using Neighbor-Joining in Paup* (Swofford 2001).
The closely related Liriomyza bryoniae (Kaltenbach)
was used as the outgroup taxon for rooting the phe-
Vol. 107, no. 5
nogram. DNA sequences have been deposited in
GenBank with accession numbers KJ778702ÐKJ778751.
A subset of voucher specimens for L. langei from this
study have been deposited in the National Museum of
Natural History in Washington, DC.
Females and PCR Screening of Pooled Samples.
To screen a large number of individuals, abdomens
from female specimens were pooled in groups of up to
Þve abdomens before DNA extraction. A maximum of
four pooled samples (20 ßies) were screened from any
particular Þeld collection (location by date by crop).
The head and thorax of each ßy was retained as
voucher material to be investigated further if anomalous results, evidence of L. huidobrensis, or both,
were discovered during the screening. Female abdomens within each pooled sample were ground together for extraction using a micropestle and subjected to the DNeasy tissue extraction procedure.
Two independent PCR-based screening procedures
were used to test pooled female extractions for the
presence of L. huidobrensis DNA. The Þrst method is
a published PCR-RFLP procedure that uses a single
pair of primers to amplify a 1,031-bp fragment of COI
and COII in both L. huidobrensis and L. langei (Scheffer et al. 2001). Restriction digests of the ampliÞcation
fragment using EcoRV and SpeI in separate reactions
result in diagnostic bands of differing lengths for L.
huidobrensis and L. langei that can be visualized on an
agarose gel (for full details see Scheffer et al. 2001).
We tested positive controls (several independent
DNA extractions of a single L. huidobrensis pooled
with four L. langei) to ensure that any L. huidobrensis
present would be detected even at the lowest frequency possible using this procedure. Although initial
results using the positive controls were acceptable
(data not shown), during the course of the actual
screening, we detected unacceptable variation in the
strength of the positive controls.
For this reason, we developed a second screening
procedure using a CO site-speciÞc primer PCR test to
determine whether L. huidobrensis DNA was present
in the pooled samples. This PCR test uses three primers speciÞcally designed to be used together to amplify
either a 356-bp piece of COI of L. langei or a 1,798-bp
piece of COI and COII of L. huidobrensis, depending
on which speciesÕ DNA is present in a genomic template. The forward primer LirHL-F-1 (5⬘-TATAG
TYGAAAACGGAGC-3⬘) was designed to perfectly
match a region of COI of both L. langei and L. huidobrensis. The reverse primer Lang-R-2 (5⬘-ATAAAT
GYTGGTATAGG-3⬘) was designed to perfectly match
L. langei COI sequence and also to be different from
L. huidobrensis sequence. During PCR, the use of this
primer in combination with the forward primer,
LirHL-F-1, selectively ampliÞes a 356-bp piece of only
L. langei COI even in the presence of L. huidobrensis
DNA. A second reverse primer, Huid-R-3 (5⬘-CCTG
GCGTAGCATCAACC-3⬘), was designed similarly to
selectively amplify a 1,798-bp piece of L. huidobrensis
COI and COII. When all three primers are used in a
single PCR reaction containing L. langei, L. huidobrensis, or both, species-speciÞc DNA ampliÞcation
October 2014
SCHEFFER ET AL.: MOLECULAR SURVEY FOR INVASIVE PEA LEAFMINER
1961
A B C D E F G H
L. huidobrensisbands
L. langeibands
Fig. 1. PCR banding patterns resulting from taxon-speciÞc priming reactions using eight different templates of various
species compositions. Lanes A and F: Þve L. langei pooled; Lanes BÐE: four L. langei and one L. huidobrensis, pooled; Lanes
G and H: single L. huidobrensis specimens. DNA ladder in outside lanes.
takes place. Because of differences in the length of the
species-speciÞc ampliÞcation products, visualization
on agarose gels results in size-speciÞc bands that are
diagnostic for each species (Fig. 1, L. langei: Lanes A
and F; L. huidobrensis: Lanes G and H). If both species
are present in the same samples, both diagnostic bands
will be present; we used multiple positive controls
consisting of one L. huidobrensis to four L. langei to
ensure sensitivity of the test to dilute L. huidobrensis
DNA (Fig. 1, Lanes BÐE).
Results
More than 1,000 agromyzid ßies were received from
the California Department of Food and Agriculture.
Field crops and weeds from which the leafminer samples
were swept included beans, broccoli, Brussels sprouts,
Table 1.
cabbage, caulißower, celery, cheeseweed, lettuce, mustard, peas, and spinach. Agromyzid samples from several
of the sampled counties, Fresno, Imperial, Riverside, San
Diego, and Ventura, contained no ßies matching the
external description of L. langei or L. huidobrensis, and
are not considered further in this study.
We screened a total of 48 male specimens and 616
female specimens from the counties of Monterey, San
Benito, San Luis Obispo, San Mateo, Santa Barbara,
and Santa Cruz (Table 1). All DNA sequences from
male specimens clustered with 100% bootstrap support with those previously obtained from L. langei
(Fig. 2). Within the L. langei clade, there was no
obvious clustering of sequences either by county of
origin or by cropÐweed association. Uncorrected pairwise distances between the L. huidobrensis and L.
langei clades ranged from 4.8 to 5.0%. Distances within
California county and crops from which screened flies were sweep-netted during October 2006
County
Crop
Monterey
San Benito
San Luis Obispo
San Mateo
Santa Barbara
Santa Cruz
Total
Broccoli, caulißower, cheeseweed, lettuce
Broccoli, cabbage, celery, lettuce, spinach
Broccoli, cabbage, caulißower, celery, lettuce, spinach
Beans, Brussels sprouts, mustard, peas
Peas
Broccoli, lettuce, spinach
No. of females
screened
No. of males
screened
141
52
129
35
76
183
616
10
11
0
8
8
11
48
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JOURNAL OF ECONOMIC ENTOMOLOGY
Vol. 107, no. 5
Fig. 2. Neighbor-Joining phenogram of L. langei COI sequences from specimens collected in California as well as
previously published COI sequences from L. huidobrensis collected in South and Central America and elsewhere. Bootstrap
values for the L. langei and L. huidobrensis clusters indicated on the branches. Within the L. langei cluster, previously published
sequences are shown in bold. All of the sequences from L. huidobrensis and from L. bryoniae have been previously published;
GenBank accession numbers are indicated on the Þgure for each published sequence.
L. langei ranged from 0 to 1.4% and within L. huidobrensis from 0 to 0.7%, although this latter estimate
does not reßect variation across the native range of L.
huidobrensis (S.J.S. and M.L.L., unpublished data).
Limiting the analysis to only the DNA barcode region
of COI yielded essentially the same results as with the
full-length dataset: the two species formed monophyletic clusters with 100% bootstrap support for each
species. All California specimens clustered with L.
langei (phylogram not shown).
Using the taxon-speciÞc priming approach for
pooled female specimens, the resulting PCR bands in
almost all cases indicated only the presence of L.
langei. Anomalous bands were obtained in a few cases,
and for these samples the voucher heads and thoraxes
were extracted and sequenced. In no case was L.
huidobrensis DNA recovered, although in a couple of
cases the DNA indicated the presence of an additional,
unidentiÞed species (this is not surprising, as several
nonpest agromyzid species in California are externally
quite similar to L. langei or L. huidobrensis).
Discussion
Our molecular survey of 664 male and female ßies
matching the external description of L. huidobrensis
October 2014
SCHEFFER ET AL.: MOLECULAR SURVEY FOR INVASIVE PEA LEAFMINER
and L. langei found no evidence for the presence of L.
huidobrensis in California. The counties from which
leafminer samples were obtained were those known to
or likely to contain “pea leafminers.” That only L.
langei DNA was found in these counties (with the
exception of a few highly divergent sequences from
unidentiÞed species) indicates that L. huidobrensis
was not present in these regions in detectable numbers during October 2006. Given the multivoltine life
cycle of L. huidobrensis, it remains possible that this
species could be present and undetected during yet
unsampled temporal periods. However, our results are
consistent with those of Scheffer (2000; S.J.S. and
M.L.L., unpublished data) that have found only L.
langei sequence data in samples from other times and
locations within California. To date, there is no evidence that L. huidobrensis populations have ever established and spread within California.
Our samples of L. langei originated in six different
counties and were collected from 11 different crops.
No evidence for geographic structure or clustering by
crop was seen, consistent with the general view of L.
langei as a polyphagous pest with at least moderate
dispersal abilities.
The new multiplex PCR screening method using
pooled samples is an ideal approach to screening large
samples of morphologically similar but distinct species
(or populations) that can be diagnosed using sequence data. By aligning sequences from identiÞed
specimens from both groups, divergent sites can be
found from which group (taxon)-speciÞc primers can
be designed. The primer in one direction should be
appropriate for both groups, while primers speciÞc to
one or the other of the two groups should be designed
such that PCR ampliÞcations for the two groups will
be of different sizes, each diagnostic for one or the
other group. One caveat concerning this method is
that false-positive bands may be obtained if nontarget
specimens are inadvertently analyzed.
Pooling specimens before DNA extraction allows
greater efÞciency in terms of both money and time,
due to a reduction in the numbers of both genomic
extractions and PCR reactions; by pooling Þve female
specimens per extraction, we cut costs and bench time
by ⬇80%, thereby allowing us to screen more individuals. In theory, this pooled approach could be used
to screen very large numbers of samples at one time.
However, in the case of agricultural pests, especially
when screening for rare or newly invasive species,
there is a need to be able to corroborate any positive
bands resulting from the test, particularly because of
the possibility of the inclusion of nontarget species. In
our case, we retained the head and thorax of each
female such that if a positive L. huidobrensis band was
obtained, we could go back to the heads and thoraxes
for that pooled sample and sequence for corroboration. Obviously, the more specimens have been
pooled, the more voucher material has to be individually sequenced for conÞrmation of positive bands, so
there is a trade-off between the number of specimens
pooled in a given extraction and the ability to conÞrm
important results.
1963
In summary, we used mitochondrial COI sequences
and two PCR-based screening methods in an extensive, but temporally limited, investigation of whether
the invasive L. huidobrensis is present in California.
Samples originated in a number of different counties
and were associated with a variety of crop species.
From our collections made in 2006, no evidence for
the presence of L. huidobrensis was found. This information and the genetic variation we have documented
provide important baseline data in the event of the
introduction and possible establishment of the highly
invasive L. huidobrensis.
Acknowledgments
We thank Joe Cavey (U.S. Department of AgricultureÐ
Animal Plant Health Inspection Service) for facilitating this
project. Matthew BufÞngton, Allen Norrbom, and Scott Armstrong provided useful comments on the manuscript. Specimens used in this study were collected by D. Alnawi, C.
Barrera, L. Bronson, C. Cannon, J. Dessert, J. Doyle, A.
Gilbert, E. Gomez, L. Irons, S. Khowsudtsi, V. Lopez, E.
Moscoso, Q. Nguyen, B. Oliver, Y. Redler, S. Schmidt, B.
Sidmore, J. Sohal, B. Taylor, J. Willems, and T. Wilson.
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