ECOLOGY AND POPULATION BIOLOGY Detection and Differentiation of Parasitoids (Hymenoptera: Aphidiidae and Aphelinidae) of the Brown Citrus Aphid (Homoptera: Aphididae): Species-Specific Polymerase Chain Reaction Amplification of 18S rDNA

The brown citrus aphid, Toxoptera citricida (Kirkaldy), is an important pest of Florida citriculture because it causes feeding damage to citrus and vectors citrus tristeza virus. Parasitoids recovered from brown citrus aphids in Florida include Lysiphlebus testaceipes (Cresson), Lipolexis scutellaris Mackauer, and Aphelinus gossypii Timberlake. Monitoring the levels of parasitism caused byeach species is difÞcult because theparasitoidsmust be rearedoutordissected fromtheaphidhosts. A simple and quick molecular approach was developed to detect and distinguish these parasitoids developing within the host aphid. Total genomicDNAwas extracted from the brown citrus aphid and eachof the threeparasitoids and the18S rRNAgeneof each specieswas ampliÞedbypolymerase chain reaction (PCR). The PCR products were sequenced to obtain complete gene sequences for each species. Thevariable regionsV2of the geneswereused todesign species-speciÞcprimers for detecting and differentiating the three parasitoids. The species-speciÞc PCR ampliÞcations discriminated the parasitoid DNAs from each other and from the host DNA. Detection of L. testaceipesDNAwithin the host aphid was possible in 8% of samples during the Þrst 2 h after parasitoid oviposition; in 66% of samples after 24 h; in 94% of samples after 48 h; and in 100% of samples after 72 h. The PCR approach described in this study provides earlier and more precise detection of parasitism and determination of species than rearing or dissection methods.