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

Abstract

This study investigated sequence variability in the second internal transcribed spacer of ribosomal DNA for 3 species of Metastrongylus (porcine lungworms). The ITS-2 region was amplified by PCR from individuals of M. elongatus, M. pudendotectus and M. salmi, and then subjected directly to single-strand conformation polymorphism analysis (SSCP), which allowed the direct display of sequence variation within and among individuals representing each speciesNucleotide sequences data reported in this paper are available in the EMBL, GenBank^TM accession numbers: AJ305373 to AJ305404.. There were marked differences in SSCP profiles among species, making this approach useful for species identification. For individual species, representative bands were excised from electrophoretic gels, reamplified by PCR and subjected to direct sequencing. For all 3 taxa, variability in the ITS-2 was related chiefly to the presence of microsatellites. Eight 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 microsatellite repeat number (ranging from 1 to 23) was found among individual nematodes of a species and between species. The microsatellites were located to specific stem or loop regions in the predicted ITS-2 precursor rRNA secondary structure. The results may 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 microsatellite 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.