Discrimination among pest species of Bactrocera (Diptera: Tephritidae) based on PCR-RFLP of the mitochondrial DNA

Abstract

A PCR-RFLP based method of species identification was considered for 18 pest species of Bactrocera fruit flies mainly distributed in the Asia-Pacific region. A 1.6 kb portion of the mitochondrial DNA containing portions of 16S and 12S ribosomal RNA genes was determined for eight species for which the mtDNA sequences have not been studied. Comparisons of restriction patterns expected from the sequences obtained in this and previous studies revealed that all 18 species can be discriminated by PCR-RFLP using a small number of restriction enzymes. Based on the highly conserved sequences detected among the 18 species, four sets of PCR primers were designed to amplify shorter, diagnostically informative sections within the mtDNA fragment. Given the simplicity of banding patterns and the ease of amplification of DNA fragments, such sections were considered suitable for PCR-RFLP analysis. Based on the results, a scheme for the Bactrocera pest species identification was proposed. The PCR-RFLP analysis using 83 individuals revealed that the scheme correctly identified most of the 18 species except for two closely related sympatric species, Bactrocera carambolae and B. papayae; the majority of individuals of the former species showed the same banding patterns as the latter species. In addition, molecular phylogenetic analyses were performed based on the nucleotide sequences in order to examine the positioning of each species among many other species of fruit flies.