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 1,2 1 3 4 SONJA J. SCHEFFER, MATTHEW L. LEWIS, STEPHEN D. GAIMARI, AND STUART R. REITZ 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 distin- guished 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 sequently corroborated by differences in two inde- (Blanchard) has become a notorious pest of a wide pendent nuclear genes, and the name L. langei Frick variety of vegetable and ßower crops that number in the was revived for the California (plus Hawaii) popula- hundreds (Spencer 1973, Reitz and Trumble 2002). tions (Scheffer and Lewis 2001). Invasive populations Originally described from Argentina (Blanchard around the world sampled to date have invariably carried 1926), since the 1980s, L. huidobrensis has become mitochondrial haplotypes belonging to the South Amer- invasive and has spread to Europe, the Middle East, ican L. huidobrensis (e.g., those from Canada, China, Asia, and North America (Canada; van der Linden Indonesia, Italy, Israel, Japan, Korea, the Philippines, 1990; Weintraub and Horowitz 1995; Head et al. 2000; South Africa, Sri Lanka, and Taiwan; Scheffer 2000; Scheffer 2000; Scheffer et al. 2001, 2006; He et al. Scheffer et al. 2001, 2006; He et al. 2002; Takano et al. 2005 2002). Newly established populations are often dev- as reported in Takano et al. 2008). Most recently, Takano astating, and it is currently considered one of the et al. (2008) found clear evidence of partial reproductive worldÕs most destructive agromyzid pests. isolation between two colonies of ßies molecularly iden- In 1951, Frick described a leafminer from peas in tiÞed as L. langei and L. huidobrensis. Currently, there is California as Liriomyza langei Frick (Frick 1951, Parrella no evidence of these two species occurring in sympatry 1982). Upon further examination, this species appeared in any part of the world. to be morphologically indistinguishable from L. huido- California is one of the primary vegetable-growing brensis and was synonymized with L. huidobrensis by regions within the United States. Although L. langei Spencer (1973). Scheffer (2000) found that South has been known in California as “the pea leafminer” American and Californian L. huidobrensis s.l. differed (at times as L. huidobrensis) and reported as a substantially in mitochondrial cytochrome oxidase se- polyphagous pest since at least the 1930s (Chaney quence data. These molecular differences were sub- 1995), it was not until the 1990s that its pest status in California changed from minor secondary pest to an The use of trade, Þrm, or corporation names in this publication is economically important crop pest (Chaney 1995, Re- for the information and convenience of the reader. Such use does not itz and Trumble 2002). It has been suggested that this constitute an ofÞcial endorsement or approval by the United States change has come about due to the presence of a Department of Agriculture or the Agricultural Research Service of genetically distinct and more pestiferous population in any product or service to the exclusion of others that may be suitable. 1 Systematic Entomology Laboratory, USDAÐARS, Bldg. 005, Rm., the central coastal valleys that is spreading southward 137, 10300 Baltimore Ave., Beltsville, MD 20705. (Morgan et al. 2000, Reitz and Trumble 2002). Given 2 Corresponding author, e-mail: [email protected]. the invasive nature of South American L. huidobrensis 3 Plant Pest Diagnostics Branch, California Department of Food and and the fact that currently it cannot be distinguished Agriculture, 3294 Meadowview Rd., Sacramento, CA 95832. 4 Malher County Extension, Department of Crop and Soil Science, morphologically from L. langei, the question of Oregon State University, 710 SW 5th Ave., Ontario, OR 97914. whether invasive L. huidobrensis is present but un- 1960 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 5 documented in California has never been adequately nogram. DNA sequences have been deposited in addressed. The purpose of this study was to conduct GenBank with accession numbers KJ778702ÐKJ778751. a molecular survey to determine whether L. huidob- A subset of voucher specimens for L. langei from this rensis is present but masquerading as L. langei in veg- study have been deposited in the National Museum of etable-growing regions of California. To accomplish Natural History in Washington, DC. this, we used molecular methods to screen extensive Females and PCR Screening of Pooled Samples. samples of adult ßies collected from several regions To screen a large number of individuals, abdomens and crops known to harbor L. langei. We paid partic- from female specimens were pooled in groups of up to ular attention to the populations in the central coastal Þve abdomens before DNA extraction. A maximum of valleys, where the change in pest status of presumed four pooled samples (20 ßies) were screened from any L. langei was Þrst observed (Chaney 1995, Reitz and particular Þeld collection (location by date by crop). Trumble 2002). The head and thorax of each ßy was retained as voucher material to be investigated further if anom- alous results, evidence of L. huidobrensis, or both, Materials and Methods were discovered during the screening. Female abdo- Members of the California Department of Food and mens within each pooled sample were ground to- Agriculture collected leafmining ßies by sweep-net- gether for extraction using a micropestle and sub- ting various crop hosts during the period 3Ð24 October jected to the DNeasy tissue extraction procedure. 2006 in 11 counties of California: Fresno, Imperial, Two independent PCR-based screening procedures Monterey, Riverside, San Benito, San Diego, San Luis were used to test pooled female extractions for the Obispo, San Mateo, Santa Barbara, Santa Cruz, and presence of L. huidobrensis DNA. The Þrst method is Ventura. Specimens were preserved in ethanol and a published PCR-RFLP procedure that uses a single stored at Ϫ80ЊC. Because multiple leafminer species pair of primers to amplify a 1,031-bp fragment of COI were present, samples were sorted by S.J.S. to include and COII in both L. huidobrensis and L. langei (Schef- only ßies matching the external morphological de- fer et al. 2001). Restriction digests of the ampliÞcation scription of L. langei and L. huidobrensis. fragment using EcoRV and SpeI in separate reactions Two molecular methods were used to screen ßies result in diagnostic bands of differing lengths for L. for evidence of L. huidobrensis. First, total genomic huidobrensis and L. langei that can be visualized on an nucleic acids were extracted from the abdomens of agarose gel (for full details see Scheffer et al. 2001). individual male ßies, and mitochondrial cytochrome We tested positive controls (several independent oxidase I was ampliÞed and sequenced. In the second DNA extractions of a single L. huidobrensis pooled method, female abdomens were pooled, and the re- with four L. langei) to ensure that any L. huidobrensis sulting pooled genomic nucleic acids were screened present would be detected even at the lowest fre- using two PCR-based screening procedures. Males quency possible using this procedure. Although initial and females were treated differently to save genitalia results using the positive controls were acceptable of individual male specimens for morphological anal- (data not shown), during the course of the actual ysis while at the same time processing a large number screening, we detected unacceptable variation in the of specimens while minimizing the time and expense strength of the positive controls. of sequencing all specimens in the study. For this reason, we developed a second screening Males and DNA Sequencing. Male specimens were procedure using a CO site-speciÞc primer PCR test to dissected and the abdomens removed and subjected to determine whether L. huidobrensis DNA was present the DNeasy tissue extraction procedure (Qiagen, Va- in the pooled samples. This PCR test uses three prim- lencia, CA) without grinding. The essentially intact ers speciÞcally designed to be used together to amplify abdomens were left to soak in the lysis buffer with either a 356-bp piece of COI of L. langei or a 1,798-bp proteinase K overnight and were retrieved as vouch- piece of COI and COII of L. huidobrensis, depending ers following the extraction procedure. Primers TY- on which speciesÕ DNA is present in a genomic tem- J-1461 (forward, Winkler et al. 2009) and TD-N-3862 plate.