Kim Kyung Seok, Szendrei Zsofia, Rodriguez-Saona Cesar, Mulder Phillip G, Sappington Thomas W
USDA-ARS Corn Insects and Crop Genetics Research Unit, Genetics Laboratory, Iowa State University, Ames, IA 50011, USA.
J Econ Entomol. 2009 Apr;102(2):759-66. doi: 10.1603/029.102.0237.
The boll weevil, Anthonomus grandis grandis Boheman (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 misidentified by scouts. For example, the congeneric pepper weevil, Anthonomus eugenii Cano, and other superficially similar weevils are attracted to components of the boll weevil lure or trap color. Although morphologically distinguishable by trained personnel, the potential for misidentification is compounded when captured weevils are dismembered or partially consumed by ants or ground beetles that sometimes feed on them in the traps. Because misidentification can have expensive 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-specific 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 caryae Horn. However, it does not distinguish the boll weevil from the subspecific "thurberia" weevil. A universal internal transcribed spacer primer pair included in the cocktail cross-amplifies DNA from all species, serving as a positive control. Furthermore, the diagnostic primers amplified 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.
棉铃象甲（Anthonomus grandis grandis Boheman，鞘翅目：象甲科）是美洲种植棉花（陆地棉，Gossypium hirsutum L.）的一种严重害虫，其曾经被根除的区域再次出现虫害一直备受关注。大量的性诱捕器用于监测棉铃象甲的重新引入，但诱捕器偶尔会捕获非目标象甲，这些象甲可能会被巡查人员误认。例如，同属的胡椒象甲（Anthonomus eugenii Cano）和其他外观相似的象甲会被棉铃象甲诱捕剂的成分或诱捕器颜色所吸引。尽管经过训练的人员可以通过形态学进行区分，但当捕获的象甲被蚂蚁或有时在诱捕器中取食它们的地甲虫肢解或部分吃掉时，误认的可能性就会增加。由于误认可能会带来昂贵的后果，一种分子诊断工具对根除管理人员来说将具有巨大价值。我们证明，在单一聚合酶链反应（PCR）中使用的三种引物对的混合物可扩增物种特异性微卫星，能明确区分棉铃象甲与另外三种测试象甲物种，包括胡椒象甲；蔓越橘象甲（Anthonomus eugenii musculus Say）；以及山核桃象甲（Curculio caryae Horn）。然而，它无法区分棉铃象甲与亚种“thurberia”象甲。混合物中包含的通用内部转录间隔区引物对可交叉扩增所有物种的DNA，作为阳性对照。此外，诊断引物能从棉铃象甲成虫的各个身体部位扩增出目标微卫星，这表明使用引物混合物的PCR技术足够灵敏，即使身体部分降解也能阳性鉴定棉铃象甲。
