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.

Audrey Nisole,Cameron Duff,Brittany Day,Michel Cusson,Catherine Béliveau,Roger C. Lévesque ,Amélie Potvin,Troy Kimoto,Luca Freschi,Josyanne Lamarche,Dave Holden,Darío I. Ojeda ,Sandra Cervantes,Richard Hamelin ,Arnaud Capron,Abdelmadjid Djoumad,Donald J. Stewart ,Hesther Yueh,Reza Zahiri

doi:10.1371/journal.pone.0160878

Abstract

Preventing the introduction and establishment of forest invasive alien species (FIAS) such as the Asian gypsy moth (AGM) is a high-priority goal for countries with extensive forest resources such as Canada. The name AGM designates a group of closely related Lymantria species (Lepidoptera: Erebidae: Lymantriinae) comprising two L. dispar subspecies (L. dispar asiatica, L. dispar japonica) and three closely related Lymantria species (L. umbrosa, L. albescens, L. postalba), all considered potential FIAS in North America. Ships entering Canadian ports are inspected for the presence of suspicious gypsy moth eggs, but those of AGM are impossible to distinguish from eggs of innocuous Lymantria species. To assist regulatory agencies in their identification of these insects, we designed a suite of TaqMan® assays that provide significant improvements over existing molecular assays targeting AGM. The assays presented here can identify all three L. dispar subspecies (including the European gypsy moth, L. dispar dispar), the three other Lymantria species comprising the AGM complex, plus five additional Lymantria species that pose a threat to forests in North America. The suite of assays is built as a “molecular key” (analogous to a taxonomic key) and involves several parallel singleplex and multiplex qPCR reactions. Each reaction uses a combination of primers and probes designed to separate taxa through discriminatory annealing. The success of these assays is based on the presence of single nucleotide polymorphisms (SNPs) in the 5’ region of mitochondrial cytochrome c oxidase I (COI) or in its longer, 3’ region, as well as on the presence of an indel in the “FS1” nuclear marker, generating North American and Asian alleles, used here to assess Asian introgression into L. dispar dispar. These assays have the advantage of providing rapid and accurate identification of ten Lymantria species and subspecies considered potential FIAS.

Highlights

For countries dominated by forested land such as Canada, expanding global trade has increased the risks of introduction and establishment of forest invasive alien species (FIAS)
What is referred to as Asian gypsy moth (AGM) is a group of closely related Lymantria (Lepidoptera: Erebidae: Lymantriinae) moths comprising two L. dispar subspecies (L. dispar asiatica Vnukovskij and L. dispar japonica (Motschulsky)) and three other Lymantria species (L. umbrosa (Butler), L. albescens Hori and Umeno, and L. postalba Inoue [1,2,3]
Their host ranges differ from those of L. dispar asiatica and L. dispar japonica but they are considered a threat to some parts of North America; for this reason, they are regulated in Canada [2,7]

Summary

Introduction

For countries dominated by forested land such as Canada, expanding global trade has increased the risks of introduction and establishment of forest invasive alien species (FIAS). This situation has called for heightened vigilance on the part of plant protection authorities and a strengthening of measures taken to prevent the accidental introduction of unwanted alien pests. The other three species making up the AGM group (L. umbrosa, L. postalba, L. albescens) form a clade with L. dispar [6] and were once considered L. dispar subspecies [1] Their host ranges differ from those of L. dispar asiatica and L. dispar japonica but they are considered a threat to some parts of North America; for this reason, they are regulated in Canada [2,7]. For an assessment of the phylogenetic relationships among these species/subspecies, the reader is referred to earlier studies by other groups [6,8,9,10,11,12,13,14]

Methods

Results

Conclusion