Rapid Identification ofHelicoverpa armigeraandHelicoverpa zea(Lepidoptera: Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1

Abstract

Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.

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

“…armigera sequences were detected, and the results were similar to those in Fig 1F. Peak height for the 18S control is similar to that observed by Perera et al [23] for batch extracted legs of both species, and is directly related to amplicon length and nucleotide composition. The relatively small peak for ITS1 in H .…”

Invasion origin, rapid population expansion, and the lack of genetic structure of cotton bollworm (Helicoverpa armigera) in the Americas

“…Three hundred thirty‐one H. armigera and 61 H. zea individuals, previously identified by the mtDNA COI marker, were genotyped to the Internal Transcribed Spacer 1 (ITS1) region of the nuclear ribosomal DNA (rDNA) using a Helicoverpa spp. forward primer (3373Ha_Hz_ITS1‐F) and two species‐specific reverse primers: for the H. armigera (3374Ha_ITS1‐R) and for H. zea (3377Hz_ITS1‐R) (Perera et al, ; Appendix ). The conditions for the PCR were as follows: 1.0 U of TaqDNA polymerase (Fermentas International Inc., Canada), 56 µM of dNTPs, 2.5 mM of MgCl2, 0.3 mg/ml of BSA, 10× TaqBuffer, 0.16 µM of each primer, and 25 ng of total DNA in a final volume of 25 μl.…”

“…This method produces PCR distinct product sizes between H. armigera (147 bp) and H. zea (334 bp), allowing the identification of putative hybrids when checked in agarose gel electrophoresis (Perera et al, ). H. armigera , H. zea, and one laboratory hybrid, previously confirmed by sequencing, were used as controls for PCR standardization and genotyping of field individuals.…”

Mitochondrial DNA genomes of five majorHelicoverpapest species from the Old and New Worlds (Lepidoptera: Noctuidae)

“…Sequencing libraries were constructed as reported in Walsh (). DNA extraction from H. assulta assulta () was as reported in Perera et al (), and the DNA library was constructed as described in Perera et al (). DNA library sequencing was performed at the Australian National University Biomolecular Resource Facility (Canberra, Australia) and the USDA‐ARS Genomics and Bioinformatics Research Unit, (Stoneville, MS, USA).…”

“…The second (KT626655) was from a sample collected in Thailand in 1986 that was identified and preserved as a pinned reference speci- Walsh (2016). DNA extraction from H. assulta assulta (KT626655) was as reported in Perera et al (2015), and the DNA library was constructed as described in Perera et al (2016). Initial identification of adult H. gelotopoeon specimens from H. zea/H.…”