Species-specific molecular markers were developed to identify and distinguish between two parasitoids, Lipolexis oregmae (=scutellaris) and Lysiphlebus testaceipes. High-fidelity PCR primers were developed based on DNA sequences from the internal transcribed spacer region between the 5.8S and 18S nuclear rRNA. The L. oregmae-specific primer produced a 270 bp band while those of L. testaceipes produced a 520 bp band. 100% of eggs of both parasitoids within the aphid hosts were detected 18 h after oviposition. A sensitivity analysis indicated that 100% of the time, a single aphid containing a parasitoid egg could be detected even if combined with DNA from up to 36 unparasitized aphids. A single first-instar parasitoid could be detected by High-fidelity PCR when the parasitized aphid was combined with up to 500 unparasitized aphids. Both primer sets detected immature parasitoids within Aphis spiraecola, Aphis gossypii, Aphis craccivora, Myzus persicae, Toxoptera citricida and Toxoptera aurantii in the laboratory. Lipoleixs oregmae was detected from T. citricida, A. spiraecola and A. gossypii in a survey of aphids in citrus groves, vegetables and weeds at or near release sites during 2002 and 2003. Lipolexis oregmae also was confirmed in seven of the eight counties surveyed in Florida, indicating L. oregmae is widely distributed and able to overwinter in these aphids. The High-fidelity PCR is an efficient method to determine establishment of L. oregmae in this classical biological control program.
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