Molecular Identification of the Light Brown Apple Moth (Lepidoptera: Tortricidae) in California Using a Polymerase Chain Reaction Assay of the Internal Transcribed Spacer 2 Locus

Abstract

A molecular protocol using a hemi-nested polymerase chain reaction (PCR) of the internal transcribed spacer region 2 (ITS2) is reported for the diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in California. This protocol distinguishes the light brown apple moth from other moths in California based on size differences of PCR amplicons that are visualized on agarose gels. The molecular diagnostic tool generated no false negatives based on analysis of 337 light brown apple moths collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 424 moths representing other tortricid species generated correct identification for >95% of the samples and only two false positives. Of the 761 moths tested only fourteen produced no PCR amplicons and five generated inconclusive data.

Research Article ITS2 as a molecular marker for the identification of Diatraea saccharalis and D. flavipennella and possible infection with Cotesia spp.

“…However, Chilo suppressalis and C. hyrax, which are closely related to Diatraea, showed 57.2 and 55.2% GC; the higher values observed until now (Margam et al, 2011;Wan et al, 2011a). When we observe the GC content from other families, we can see a significant increase compared to Crambidae, as in Tortricidae (61%) and Noctuidae (58.6%), but this increase happens in both basal and advanced groups (Barr et al, 2009;Wan et al, 2011b). The GC content to Lepidoptera is considered comparatively high, and this can affect the energy of the secondary structure (Banerjee et al, 2007).…”

“…The sequence size homogenization among individuals, in the case of D. saccharalis and D. flavipennella by breeding, could be due to endogamy that is present in breeding. There was also no report about significant ITS 2 size differences in some lepidopteran species: Chilo sp, Cnaphalocrocis medinalis, Ostrinia sp, Spodoptera spp, Polyommatus spp, and species from the families Geometridae, Lasiocampidae, Pieridae, Tortricidae, and Zygaenidae (Niehuis et al, 2006;Barr et al, 2009;Wiemers et al, 2009;Dincă et al, 2011;Margam et al, 2011;Wan et al, 2011a;Kvie et al, 2012). In other groups as Diptera, ten species belonging to genus Anopheles, from different geographic regions, did not also show intraspecific differences in nucleotide composition and sequence length.…”

“…The ITS 2 region size is specific to each species in the family Crambidae, and this also happens in other families as Tortricidae, Noctuidae, Geometridae, and Lycaenidae (Barr et al, 2009;Wiemers et al, 2009;Wan et al, 2011a;Kvie et al, 2012).…”

“…The amplified ITS 2 fragments visualized on electrophoresis agarose gel showed distinct sizes in each Diatraea species included in this study. This rDNA region has been used to identify many lepidopteran species and other insect orders without the need to be sequenced, but only comparing the amplicon sizes on agarose gel (Barr et al, 2009;Wan et al, 2011a;Sayed et al, 2013;Zomuanpuii et al, 2013). In the sugarcane field, in many cases, we only find pieces of the Diatraea larvae due to cuts in the sugarcane or eggs, but these situations are difficult to identify.…”

“…This nucleotide sequence has been analyzed in many families of Lepidoptera, and in all taxonomic levels, it was observed a variation in length and nucleotide composition of sequences. Therefore, ITS 2 is an efficient barcode for lepidopteran insects and molecular phylogeny of Cotesia species (Kankare and Shaw, 2004;Barr et al, 2009;Wiemers et al, 2009;Wan et al, 2011a).…”