Assessment of parasitism of house fly and stable fly pupae (Diptera: Muscidae) by pteromalid (Hymenoptera: Pteromalidae) parasitoids using polymerase chain reaction (PCR)

I extracted, cloned and sequenced DNA from the house fly, the stable fly, and four parasitoid species in the genus Muscidifurax to develop a method based on the polymerase chain reaction (PCR) to better define the role of pteromalid (Hymenoptera: Pteromalidae) parasitism of house fly and stable fly pupae (Diptera: Muscidae). Based on sequence data, two parasitoid-specific pruners were designed to anneal to the 5' end of the 5.8S rRNA gene in the parasitoid species. Use of the parasitoid specific-primer, 1R, with the universal 18S rRNA primer 1975F allowed amplification from the 3' end of the 18S, the intervening ITS 1, and the 5' end of the 5.8S rRNA gene region. The second parasitoid-specific primer, -3R, was reserved to use with another universal primer (internal to 1975F) for nested PCR. Both 1R and -3R, when paired with a universal primer, amplified the target region in the four species of Muscidifurax (Muscidifurax raptor Girault and Sanders, Muscidifurax zaraptor Kogan and Legner, Muscidifurax raptorellus Kogan and Legner, and Muscidifurax uniraptor Kogan and Legner), and in Spalangia nigroaenea Curtis, Spalangia cameroni Perkins, Spalangia endius Walker, Urolepis rufipes (Ashmead), Nasonia vitripennis (Walker), and Trichomalopsis sarcophagae Gahan.; PCR allowed detection of parasitoid DNA at dilutions as low as 1:10,000 (parasitoid DNA:host DNA) and detection of parasitism within 24 hours after females of S. endius oviposited into house fly puparia. For house fly puparia exposed to a laboratory colony of S. endius, estimates of parasitism, based on PCR assays of puparia that were frozen after 24 hours to suspend parasitoid development, did not differ significantly from estimates based on emergence of adult S. endius. Digestion of the PCR products by restriction enzymes produced restriction fragment length polymorphisms (RFLP) that allowed identification of individual parasitoid species.; Field samples confirmed the usefulness of PCR for detection and identification of parasitism and indicated that parasitoids may play a role in the production of "duds" (puparia from which no adult insect emerges). On two dates in 1997, over 65 percent of the puparia collected and held for emergence were eventually categorized as duds. Estimates of parasitism based on PCR (49.2% and 52.6%) were significantly greater than estimates based on emergence of adult parasitoids (30.6% and 32.2%) for those two dates.; Three puparia collected in 1997 produced double bands prior to digestion of the PCR product by a restriction enzyme. The sizes of the bands corresponded to the sizes produced by amplification of the DNA of Muscidifurax and Spalangia species, possibly indicating hyperparasitism. Comparison of species composition in paired samples indicated higher levels of parasitism by Spalangia spp. in PCR samples (presumably resulting from earlier attach by Spalangia species) and higher levels of parasitism by Muscidifurax spp. in puparia from which adults were allowed to emerge presumably resulting from their ability to win the contest competition. (Abstract shortened by UMI.)...