A Needle in a Haystack: a Multigene TaqMan Assay for the Detection of Asian Gypsy Moths in Bulk Pheromone Trap Samples

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.