Submitted to: Insect Molecular Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/9/2004
Publication Date: 7/1/2004
Citation: Zhu, Y., Riddick, E.W., Williams Iii, L.H., Schotzko, D.J., Logarzo, G.A., Jackson, C.G. 2004. Potential of detection and identification of nymphal parasitoids (Hymenoptera: Braconidae) of Lygus bugs (Heteroptera: Miridae) by using polymerase chain reaction and ITS2 sequence analysis techniques. Insect Molecular Biology. 97(4):743-752. Interpretive Summary: Polymerase chain reaction (PCR) technique achieved a specific detection of parasitic wasp DNA at extremely low level, and it is a useful tool for early evaluation of a biological control program on the tarnished plant bug. This technique may allow us to detect trace DNA of parasitic wasps which died within dead, dying, or surviving hosts, and reflect a more actual oviposition rate by the wasps. As a tarnished bug population might be parasitized by multiple species of parasitic wasps, we developed a molecular technique and several sets of PCR primers for detecting and specifically separating each of four parasitic wasps. The tarnished plant bug nymphs collected from a field in Stoneville, Mississippi, were also analyzed using PCR amplification and DNA sequence comparison. By using this PCR technique, we were able to detect wasp DNA at as low as 0.2 pg DNA level. Each wasp species could be specifically separated from others by amplification of a unique band size or pattern. By using this technique, we were able to determine whether field-collected plant bugs were parasitized. A hard-to-rear-out wasp within plant bugs from Stoneville was determined to be Peristenus pallipes or a closely related species.
Technical Abstract: Ribosomal ITS2 DNA fragments were sequenced from two Peristenus species, two Leiophron species, and two Lygus species. Specific primers were designed from ITS2 DNA sequences. PCR amplification with these primers separated each species from the others. Using this molecular approach, we were able to determine if Lygus hesperus Knight and Lygus lineolaris (Palisot de Beauvois) were parasitized by Peristenus and Leiophron parasitoids. The PCR technique was very sensitive and could detect Peristenus stygicus Loan DNA at a concentration of 0.01 pg/ l or 7.5X10-7 wasp DNA equivalent. Detection of P. stygicus eggs confirmed that early detection of parasitioids was possible. Nearly all L. hesperus nymphs were parasitized by a single P. stygicus after one hr of contact between the parasitoid and putative hosts. Nymphs of L. lineolaris were collected from an uncultivated field in Mississippi; approximately 17% of the nymphs were parasitized (i. e., contained wasp DNA). ITS2 sequence comparison and phylogenetic analysis indicated that L. lineolaris nymphs had been parasitized by a wasp closely related to Peristenus pallipes (Curtis) or P. nr. howardi Shaw. This study demonstrates the effectiveness of a molecular technique for detecting parasitoids developing inside their hosts.