COI gene geographic variation of Gypsy moth (Lepidoptera: Lymantriidae) and a TaqMan PCR diagnostic assay

Abstract

Gypsy moth, an important forest/urban pest worldwide, is separated into the European and Asian subspecies, and has important quarantine significance. Diagnostic technique that can accurately and quickly distinguish subspecies is lacking. This study aimed to evaluate genetic difference between the subspecies, and subsequently to develop a reliable and high throughput molecular based diagnostic tool for distinguishing the subspecies. COI genes of 25 gypsy moth samples from China, Russia, Mongolia, Japan and the United States were sequenced. DNASTAR analysis revealed that gypsy moth COI gene was 1531bp long. The UPGM phylogenetic tree constructed based on the COI gene indicated that European subspecies (U.S. population) and Asian subspecies were distinctively divided into two branches. Japanese populations had a far distantly relationship with other Asian populations forming a separate branch. There was a single base substitution (base transformation only) at 14 consistent locations between Asian and American populations, but 13 of them coded the same amino acid. A MGB proper and TaqMan assay was designed base on the base substitution at 406th bp that coded a different amino acid. This allele typing assay took only 4 hours and could accurately distinguish gypsy moth subspecies of Europe and Asia. The study enriches the knowledge basis of genetic differentiation of gypsy moth subspecies. And more importantly the TaqMan assay is the first report of such diagnostic tool that could deliver rapid and accurate results and suitable for routine quarantine inspections to distinguish Asian and European gypsy moth subspecies. This study was supported by the Ministry of Science and Technology of the People’s Republic of China (Science and technology supporting project: 2012BAK11B03; International cooperation project: 2009DFA31950) and Jiangsu Entry and Exit Inspection and Quarantine Bureau (2014KJ45).

A Multi-Species TaqMan PCR Assay for the Identification of Asian Gypsy Moths (Lymantria spp.) and Other Invasive Lymantriines of Biosecurity Concern to North America

“…Other approaches that rely on the amplification and sequencing of mitochondrial markers [e.g., 6 ] or on the analysis of several microsatellite markers (e.g., [ 13 ]) have the potential of achieving levels of accuracy, resolution and scope similar to those of the present set of assays, but will require longer processing time. Conversely, the two AGM TaqMan assays developed earlier by other groups [ 14 , 24 ], although rapid, are limited in scope and subspecies resolution. With respect to cost, the suite of assays proposed here is relatively inexpensive to run (provided the necessary thermocycling equipment is available) and comparable in price to the sequencing of PCR products.…”

“…The first two species are conifer defoliators while the last three are broadleaf defoliators [ 1 ]. For an assessment of the phylogenetic relationships among these species/subspecies, the reader is referred to earlier studies by other groups [ 6 , 8 – 14 ].…”

A needle in a haystack: a multigene TaqMan assay for the detection of Asian gypsy moths in bulk pheromone trap samples

“…Molecular markers and diagnostic tools targeting the gypsy moth have become important to help prevent invasions via early intervention (Arimoto and Iwaizumi 2014;Armstrong and Ball 2005;Armstrong et al 2003;Ball and Armstrong 2006;Bogdanowicz et al 1993Bogdanowicz et al , 1997Bogdanowicz et al , 2000Boykin et al 2012;Chen et al 2013Chen et al , 2015deWaard et al 2010;Garner and Slavicek 1996;Harrison and ODell 1989;Islam et al 2015;Kang et al 2015Kang et al , 2017Keena et al 2008;Lacković et al 2015;Pfeifer et al 1995;Qian et al 2014;Stewart et al 2016;Wu et al 2015). Given that egg masses and larvae of gypsy moth subspecies are impossible to distinguish from one another and from those of other closely related species, we recently developed a suite of TaqMan assays that enables the rapid and accurate identification of 10 Lymantria species and subspecies from intercepted egg masses and older life stages (Stewart et al 2016).…”

Genetic structure and demographic history ofLymantria dispar(Linnaeus, 1758) (Lepidoptera: Erebidae) in its area of origin and adjacent areas

“…Due to the quarantine and danger this invasive species represents, studies of the differences among local populations are actively conducted. In previous decades, population genetic analyses of L. dispar were performed using various methods, such as allozyme detection, amplified fragment length polymorphism, restriction fragment length polymorphism, sequence‐based analysis, and microsatellites (Bogdanowicz et al., , ; deWaard et al., ; George, ; Kang, Lee, & Lee, ; Keena, Cȏté, Grinberg, & Wallner, ; Koshio, Tomishima, Shimizu, Kim, & Takenaka, ; Qian et al., ; Wu et al., ). Area of origin studies, in particular, using microsatellite loci were mainly conducted by North American researchers.…”