Mutation scanning analysis of microsatellite variability in the second internal transcribed spacer (precursor ribosomal RNA) for three species of Metastrongylus (Strongylida : Metastrongyloidea)

This study investigated sequence variability in the second internal transcr ibed spacer of ribosomal DNA for 3 species of Metastrongylus (porcine lungw orms). The ITS-2 region was amplified by PCR from individuals of M. elongat us, M. pudendotectus and AI. salmi, and them subjected directly to single-s trand conformation polymorphism analysis (SSCP), which allowed the direct d isplay of sequence variation within and among individuals representing each species dagger. There were marked differences in SSCP profiles among speci es, making this approach useful for species identification. For individual species, representative bands were excised from electrophoretic gels, reamp lified by PCR and subjected to direct sequencing. For all 3 taxa, variabili ty in the ITS-2 was related chiefly to the presence of microsatellites. Eig ht different microsatellites were identified, namely (A)n, (TG)n, (TCG)n, ( TA)n, (TATG)n. (G)n, (TACA)n and (T)n. Considerable Variability in microsat ellite repeat number (ranging from 1 to 23) was found among individual nema todes of a species and between species. The microsatellites n ere located t o specific stem or loop regions in the predicted ITS-2 precursor rRNA secon dary structure. The results mag; suggest that slipped-strand mispairing in microsatellite regions contributes to sequence variability in the ITS-2 of Metastrongylus species under structural constraint as a consequence of micr osatellite location in the precursor rRNA. Similar studies of the ITS-2 for a wide range of parasitic nematodes may lead to a better understanding of concerted evolution in these organisms.