Heliothine eggs are commonly found on agricultural commodities and ornamental plants transported through domestic and international commerce. Tobacco budworm [Chloridea (Heliothis) virescens (F.)], Chloridea subflexa (Guenée), and the corn earworm [Helicoverpa zea (Boddie)] are indigenous pests of the American continent. Interceptions of the Old World bollworm [Helicoverpa armigera (Hübner)] at various ports of entry into the United States have been detected due to the invasion of this pest in South America, adding to the complexity and importance of decision making at plant inspection stations. The ability to distinguish the eggs of C. virescens from C. subflexa and Helicoverpa species is a critical component for conducting risk assessments by quarantine authorities, taxonomists, and crop consultants. We developed a simple, rapid (∼60 min), inexpensive ($0.06 per sample), and accurate (100% reliability) technique to distinguish C. virescens eggs from the possibility of being H. armigera based on the presence, number, and/or size of aeropyle holes on the primary ribs of eggs, near the micropylar rosette. In this location, aeropyles were easily visible at 40× magnification in 213 fresh and ethanol-preserved C. virescens eggs once treated with Hoyer's solution. A small number of C. subflexa had one or two aeropyles on a few of the primary ribs, whereas no aeropyles were found on 411 H. zea and 269 H. armigera eggs analyzed using optical, electron, and confocal microscopy. We conclude that in most cases multiple aeropyle holes positively identify H. virescens eggs from H. zea or H. armigera, and C. subflexa.This technique potentially will reduce the number of specimens that need to be sent for molecular identification thereby saving program time and resources.
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