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Research Project: Innovative Strategies for Insect Resistance Management in Bt Cotton Location:
                                Southern Insect Management Research Title:   Author  item
                                            Perera, Omaththageitem
                                            Allen, Clintitem
                                            JAIN, DEVENDRA - Maharana Pratap University Of Agriculture And Technologyitem
                                            PURCELL, MATTHEW - Commonwealth Scientific And Industrial Research Organisation (CSIRO)item
                                            Little, Nathanitem
                                            Luttrell, Randall
  Submitted to: Journal of Insect Science
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 10/10/2015
                                    Publication  Date: 10/30/2015
                                    Publication URL: https://handle.nal.usda.gov/10113/4577034Citation: Perera, O.P., Allen, K.C., Jain, D., Purcell, M., Little, N., Luttrell, R.G. 2015. Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1. Journal of Insect Science. 15(1):155. doi:1093/jisesa/iev137.Interpretive Summary: Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Invasive old world bollworm, Helicoverpa armigera, has established in some countries in South America and was recently captured in Puerto Rico and Florida. Invasive old world bollworm is morphologically similar to the native bollworm, Helicoverpa zea, and species identification depends on labor intensive and time consuming techniques such as examination of male genitalia or mitochondrial DNA restriction fragment analysis. Conserved nucleotide sequence differences in a segment of the ribosomal RNA genes of two bollworm species were used to develop a method that could be scaled up too screen more than 1,000 insects per day at a low cost.
                                Technical Abstract: Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in some countries of South America has increased the risk of this species invading North America. Differentiation of H. armigera from the native Helicoverpa zea (Boddie) is difficult, since larvae and adults are highly morphologically similar. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA (mtDNA) 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. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt (HRM) analysis to differentiate the two Helicoverpa species.  DNA from at least two insects could be pooled to increase the number of samples processed simultaneously. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. The HRM 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.


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