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Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the nuclear ribosomal ITS1 region was used to differentiate Muscidifurax (Hymenoptera: Pteromalidae) species which are parasitoids of filth fly pupae. Three restriction enzymes, Dpn 11, Mse I, and Taq I, produced restriction patterns which were diagnostic for the four species analyzed, M. raptor, M. raptorellus, M. uniraptor, and M. zaraptor. Seven other restriction enzymes were able to differentiate one or more of the species and can be used alone, or in combination with other enzymes, to verify identifications. No intraspecific variation was observed among the populations examined. The utility of the PCR-RFLP technique compared with other moIecuIar and biochemical diagnostic procedures is discussed.