The internal transcribed spacer (ITS) regions of the ribosomal DNA of house flies, Musca domestica L., the stable flies, Stomoxys calcitrans (L.), and four parasitoid species in the genus Muscidifurax (Hymenoptera: Pteromalidae) were characterized to develop a method based on the polymerase chain reaction (PCR) to better define the role of pteromalid parasitism of pupae of the house fly and stable fly. Two parasitoid-specific primers were designed to anneal to the 5' end of the 5.8S rRNA gene in the parasitoid species. When paired with a universal primer at the 3' end of the 18S rRNA, the primers amplified the target ITS1 region in 10 pteromalid species. PCR allowed detection of parasitoid DNA within 24 h after females of Spalangia endius Walker oviposited into house fly puparia. PCR failed to amplify parasitoid DNA or detect parasitism in puparia that were exposed to parasitoid oviposition, allowed to develop 7 d, then killed by freezing and held at 20-24 degrees C for 4 d to allow DNA degradation. Digestion of the PCR products with restriction enzymes produced restriction fragment length polymorphisms that allowed identification of individual parasitoid species. Significantly greater levels of parasitism (P < 0.05) were detected by PCR for two of the five field collection dates in 1997. On the dates when PCR detected higher levels of parasitism than estimates provided by emergence of adult insects from samples taken at Feedlot M in 1997, more than 65% of all puparia in the emergence samples failed to produce an adult insect. Three puparia collected in 1997 produced double PCR bands that corresponded to PCR band sizes of Muscidifurax spp. and Spalangia sp., possibly indicating multiple parasitism or hyperparasitism.

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Weekly releases of Spalangia nigroaenea Curtis and Muscidifurax zaraptor Kogan & Legner from May through August of 1991-1993 at small, owner-operated cattle feedlots in Illinois provided weekly emergence of 100-300 parasitoids of each species per feedlot animal. In assessments based on fly and parasitoid emergence from > 47,000 stable fly and house fly puparia collected during the 3-yr period, total stable fly mortality was greater in lots where releases were made (60.7%) than in paired, untreated control lots (51.7%) (P = 0.04; paired t-test); parasitism of stable fly pupae by S. nigroaenea averaged 11.6% where releases were made and 6.4% in paired control lots (P = 0.0016). In lots where releases were made, total mortality of house fly pupae was greater (68.7 versus 56.1%; P = 0.0001); unexplained mortality was greater (55.5 versus 46.1%; P = 0.0018); and parasitism by Muscidifurax spp. was greater (2.4 versus 1.4%; P = 0.07) than in paired control lots. Parasitism, unexplained mortality, and total mortality of both fly species varied significantly from 1991 to 1992 in lots that received the same treatment each year, presumably due primarily to weather. Over the 3-yr period, releasing these species, particularly S. nigroaenea, significantly reduced production of stable fly and house fly adults in cattle feedlots. The potential value of such reductions is likely to vary as a result of feedlot conditions and weather.

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