MOLECULAR ENTOMOLOGY Molecular Diagnostic for Boll Weevil (Coleoptera: Curculionidae) Based on Amplification of Three Species-Specific Microsatellites

The boll weevil,Anthonomus grandis grandisBoheman (Coleoptera: Curculionidae), is a serious pest of cultivated cotton,Gossypium hirsutum L., in the Americas, and reinfestation of zones from which they have been eradicated is of perpetual concern. Extensive arrays of pheromone traps monitor for reintroductions, but occasionally the traps collect nontarget weevils that can be misidentiÞed by scouts. For example, the congeneric pepper weevil, Anthonomus eugeniiCano, and other superÞcially similarweevils are attracted to components of the boll weevil lure or trap color. Although morphologically distinguishable by trained personnel, the potential for misidentiÞcation is compounded when captured weevils are dismembered or partially consumed by ants or ground beetles that sometimes feedon them in the traps. BecausemisidentiÞcation canhaveexpensive consequences, a molecular diagnostic tool would be of great value to eradication managers. We demonstrate that a cocktail of three primer pairs in a single polymerase chain reaction (PCR) amplify species-speciÞc microsatellites that unambiguously distinguish the boll weevil from three other weevil species tested, including pepper weevil; cranberry weevil, Anthonomus eugenii musculus Say; and pecan weevil, Curculio caryaeHorn.However, it does not distinguish the bollweevil from the subspeciÞc “thurberia” weevil. A universal internal transcribed spacer primer pair included in the cocktail cross-ampliÞes DNA from all species, serving as a positive control. Furthermore, the diagnostic primers ampliÞed the target microsatellites from various boll weevil adult body parts, indicating that the PCR technology using the primer cocktail is sensitive enough to positively identify a boll weevil even when the body is partly degraded.