DNA barcoding and real-time PCR detection ofBactrocera xanthodes(Tephritidae: Diptera) complex

Abstract

AbstractImmature fruit fly stages of the family Tephritidae are commonly intercepted on breadfruit from Pacific countries at the New Zealand border but are unable to be identified to the species level using morphological characters. Subsequent molecular identification showed that they belong toBactrocera xanthodes, which is part of a species complex that includesBactrocera paraxanthodes, Bactrocera neoxanthodesand an undescribed species. To establish a more reliable molecular identification system forB. xanthodes, a reference database of DNA barcode sequences for the 5’-fragment of COI gene region was constructed forB. xanthodesfrom Fiji, Samoa and Tonga. To better understand the species complex,B. neoxanthodesfrom Vanuatu andB. paraxanthodesfrom New Caledonia were also barcoded. Using the results of this analysis, real-time TaqMan polymerase chain reaction (PCR) assays for the detection ofB. xanthodescomplex and for the three individual species of the complex were developed and validated. The assay showed high specificity for the target species, with no cross-reaction observed for closely related organisms. Each of the real-time PCR assays is sensitive, detecting the target sequences at concentrations as low as ten copies µl−1and can be used as either singleplex or multiplex formats. This real-time PCR assay forB. xanthodeshas been successfully applied at the borders in New Zealand, leading to the rapid identification of intercepted Tephritidae eggs and larvae. The developed assays will be useful biosecurity tools for rapid detection of species in theB. xanthodescomplex worldwide.