Development of DNA Melt Curve Analysis for the Identification of Lepidopteran Pests in Almonds and Pistachios

SIMPLE SUMMARY: Almonds and pistachios are fed upon by a diverse assemblage of lepidopteran insects, several of which are economically important pests. Unfortunately, identification of these pests can be difficult, as specimens are frequently damaged during collection, occur in traps with non-target species, and are morphologically similar up to their third instar. Here, we present a quantitative PCR based melt curve analysis for simple, rapid, and accurate identification of six lepidopteran pests of almonds and pistachios: navel orangeworm, peach twig borer, oriental fruit moth, obliquebanded leafroller, raisin moth, and Indian meal moth. We demonstrate that the dissociation or the “melt” temperature(s) of a 658 bp section of cytochrome c oxidase subunit 1 provides unambiguous species level identification of these six species and is reproducible in field specimens collected following conventional orchard practices. The melt curve’s simplicity allows it to be performed in any basic molecular biology laboratory with a quantitative PCR. ABSTRACT: Almonds and pistachios are fed upon by a diverse assemblage of lepidopteran insects, several of which are economically important pests. Unfortunately, identification of these pests can be difficult, as specimens are frequently damaged during collection, occur in traps with non-target species, and are morphologically similar up to their third instar. Here, we present a quantitative PCR based melt curve analysis for simple, rapid, and accurate identification of six lepidopteran pests of almonds and pistachios: navel orangeworm (Amyelois transitella), peach twig borer (Anarsia lineatella), oriental fruit moth (Grapholita molesta), obliquebanded leafroller (Choristoneura rosaceana), raisin moth (Cadra figulilella), and Indian meal moth (Plodia interpunctella). In this approach, the dissociation (melt) temperature(s) of a 658 bp section of cytochrome c oxidase subunit 1 was determined using quantitative PCR (qPCR). Within these six species, the distribution and the number of melt peak temperatures provide an unambiguous species level identification that is reproducible when unsheared DNA can be extracted. The test is robust across a variety of sampling approaches including insects removed from sticky card traps, museum specimens, and samples that were left in the field for up to 7 days. The melt curve’s simplicity allows it to be performed in any basic molecular biology laboratory with a quantitative PCR.