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

Don Stewart,Reza Zahiri,Richard C. Hamelin,Michel Cusson,Abdelmadjid Djoumad,Roger C. Levesque,Audrey Nisole,Josyanne Lamarche

doi:10.1007/s10530-019-01943-9

Abstract

The Asian gypsy moth (AGM) is considered a very serious invasive threat in North America. For this reason, it is subjected to a bio-surveillance program that includes an extensive network of pheromone traps. For regulatory purposes, the term “AGM” designates a group of Asian Lymantria species and subspecies, comprising two L. dispar subspecies (L. d. asiatica and L. d. japonica), and three closely related species (L. umbrosa, L. albescens and L. postalba). These moths are attracted to the same pheromone as the European gypsy moth (EGM), L. dispar dispar, which is already established in North America and typically makes up the bulk of moths caught in gypsy moth pheromone traps. These different Lymantria taxa are difficult to distinguish from one another using morphological characters alone. Here, we designed a TaqMan triplex assay capable of detecting AGM in bulk pheromone trap samples. The assay targets SNPs found in three different mitochondrial genes. Using a DNA dilution series, we show that the assay can detect AGM taxa at AGM:EGM dilution ratios ≥ 1:1000. The assay was validated using batch DNA extractions of moth legs tested at a 1:100 AGM:EGM leg ratio, a proportion that is around the operational limit for a single pheromone trap. The assay provided correct identification for all AGM taxa tested. An experiment examining the integrity of DNA extracted from gypsy moths left in pheromone traps under field conditions for up to 4 months indicated that DNA quality remains sufficient, during that period, for the present assay to remain accurate.

Highlights

The gypsy moth, Lymantria dispar (Linnaeus) (Lepidoptera: Erebidae: Lymantriinae), is considered a major threat to North America’s forest resources and economy
Comparison of this target sequence with equivalent sequences gleaned from public databases indicated that inter-individual sequence homogeneity of the cytb gene in L. dispar asiatica and L. dispar japonica was sufficient to yield discriminatory amplification; discrimination was here provided by a forward Amplification Refractory Mutation System’’ (ARMS) primer and an LNA probe (Fig. 1, alignment provided as an example; see Fig. 3 under the ‘‘Bulk Triplex B’’ tab of Online Resource 1)
Networks of pheromone traps deployed for the detection of Asian gypsy moth (AGM) can produce situations where an AGM individual, if caught, is greatly outnumbered by local European gypsy moth (EGM)

Summary

Introduction

The gypsy moth, Lymantria dispar (Linnaeus) (Lepidoptera: Erebidae: Lymantriinae), is considered a major threat to North America’s forest resources and economy This species comprises three recognized subspecies, including the European gypsy moth (EGM), L. dispar dispar (Linnaeus), which is found over most of continental Europe and north-western Africa (Pogue and Schaefer 2007). Along with three closely related Lymantria species (L. umbrosa Butler, L. albescens Hori and Umeno, and L. postalba Inoue), they form a complex informally known as Asian gypsy moth (AGM) As a group, these insects are considered a much more serious threat than the European subspecies, in large part because AGM females, unlike those of L. dispar dispar, are flightcapable, increasing the probability of a rapid invasion following an unchecked accidental introduction. Such introductions have occurred in the past, but eradication operations appear to have been successful (APHIS 2016; Nealis 2002, 2009)

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Results

Conclusion