Polymerase chain reaction (PCR) technique was used to develop a specific molecular marker for detection of immature stages of the egg parasitoid Anaphes iole Girault developing within its host, Lygus lineolaris (Palisot de Beauvois). Ribosomal DNA sequences for the internal transcribed spacer 2 (ITS2) were cloned and sequenced from adult A. iole and L. lineolaris. PCR amplification of genomic DNA using primers designed on the basis of these rDNA sequences, followed by agarose gel electrophoresis, successfully detected eggs and larvae of the parasitoid within L. lineolaris eggs. This molecular method is highly specific and sensitive. A 661-bp wasp DNA fragment was consistently amplified from pure DNA extracted from a single wasp, from a DNA mixture prepared from a wasp and an L. lineolaris egg, and from a DNA mixture prepared from a wasp, an L. lineolaris egg, and alfalfa tissue. The presence of plant tissue did not hinder detection of A. iole within host eggs. With this PCR technique, as low as 5 × 10−6 wasp DNA equivalent (1.2 pg DNA) could be easily detected. The PCR technique provided more rapid detection of parasitism rates than observed by rearing parasitized host eggs in artificial oviposition packets. PCR technology shows promise for early and sensitive detection and identification of single and multiple species of egg parasitoids in agricultural and natural systems.
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Vol. 95 • No. 3