A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World

Abstract

The Old World bollworm, Helicoverpa armigera (Hübner), and the corn earworm, H. zea (Boddie), are two of the most important agricultural pests in the world. Diagnosing these two species is difficult—adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP) analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion) to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2) amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae.

Immigrant Tortricidae: Holarctic versus Introduced Species in North America

“…According to Lovett et al [ 5 ], nonnative insect species have accumulated in United States forests at a rate of approximately 2.5 per year over the last 150 years, with the gypsy moth ( Lymantria dispar (Linnaeus, 1758), Erebidae) being among the most notorious. Examples of important lepidopteran pests that have been introduced recently to new regions around the globe include the fall armyworm, Spodoptera frugiperda (J. E. Smith) (Noctuidae), a native of the New World that has spread to much of Africa and Asia [ 6 , 7 ] and was recently discovered in Australia [ 8 ]; the Old World bollworm, Helicoverpa armigera (Hübner) (Noctuidae), which was first reported in the New World in Brazil and has spread to much of South America and the Caribbean [ 9 , 10 , 11 ]; the light brown apple moth, Epiphyas postvittana (Walker) (Tortricidae), a native of Australia that was documented from California in 2006 and has now spread throughout much of the state [ 12 ]; and the European grapevine moth, Lobesia botrana (Denis and Schiffermüller) ([Tortricidae]), a native of Europe that was inadvertently introduced to the wine-growing regions of Argentina, Chile, and California [ 13 ]. While these contemporary or recent arrivals to new regions are well documented, for many other species, there is considerable ambiguity regarding their origin and/or native distributions.…”

A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples

“…armigera prevalent throughout much of the Old World, including Europe, Asia, Africa, and Australasia [1, 2]. Helicoverpa armigera was first discovered in the New World in Brazil in 2012/2013, and it has since spread to much of South America and the Caribbean, including Puerto Rico [5, 6, 7, 8]. Although not yet present in the continental U.S., three individuals were captured in Florida in 2015 suggesting that establishment is imminent [9, 10].…”

DNA Barcoding the Heliothinae (Lepidoptera: Noctuidae) of Australia and Utility of DNA Barcodes for Pest Identification in Helicoverpa and Relatives

“…DNA barcoding could provide an efficient way to identify heliothine species but it remains to be tested and implemented in a comprehensive manner. Previous studies developing DNA sequence data for Helicoverpa species have focussed on local needs, distinguishing usually just two species although sometimes with up to two other species included as outgroups [ 9 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. On the other hand, Cho et al [ 5 ] sequenced the DNA barcode region of the COI gene for some 70 heliothine species, including about 10 pests, however they sequenced only a single individual for most species as their aim was a phylogenetic analysis of species, not species delimitation and diagnostics.…”