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

Abstract

Immature 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 to Bactrocera xanthodes, which is part of a species complex that includes Bactrocera paraxanthodes, Bactrocera neoxanthodes and an undescribed species. To establish a more reliable molecular identification system for B. xanthodes, a reference database of DNA barcode sequences for the 5'-fragment of COI gene region was constructed for B. xanthodes from Fiji, Samoa and Tonga. To better understand the species complex, B. neoxanthodes from Vanuatu and B. paraxanthodes from New Caledonia were also barcoded. Using the results of this analysis, real-time TaqMan polymerase chain reaction (PCR) assays for the detection of B. xanthodes complex 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-1 and can be used as either singleplex or multiplex formats. This real-time PCR assay for B. xanthodes has 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 the B. xanthodes complex worldwide.

Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application

“…Real‐time PCR assay for B. tryoni complex and other fruit fly species has been developed and routinely used for diagnostics in New Zealand (Dhami et al, ; Li, Waite, Gunawardana, et al, ), but no real‐time PCR assay for Z. cucumis and B. jarvisi has been reported. Prior to the development of the real‐time PCR assays, if the suspected Tephritidae interceptions were tested negative to B. tryoni complex (Dhami et al, ), the sample needed to be barcoded for the identification to species.…”

“…The amplicons were electrophoresed on 1.2% agarose (in 1× TAE buffer) gel stained with SYBR ® safe (Life Technologies™), and visualized using a Gel Doc Software system (Bio‐Rad, Hercules, CA, USA). Amplified products were sequenced bidirectionally using the amplification primers, and DNA sequences were analysed as previously described (Li et al, ; Li, Waite, Fan, et al, ; Li, Waite, Gunawardana, et al, ; Sooda, Gunawardana, Li, & Kumarasinghe, ). The DNA sequences were submitted to BOLD database under the project of “Barcode of Bactrocera Specimens” (BBS): BBS050‐18 to BBS055‐18 for Z. cucumis and BBS056‐18 to BBS060‐18 for B. jarvisi .…”

“…Plasmid DNA was extracted using the Wizard ® Plus SV Miniprep (Promega, Madison, WI, USA). The plasmid DNA was quantified using a µDrop plate in MultiSkan GO DNA quantification system (Thermo Scientific, USA) and normalized to a concentration of 10 7 copies/µl (Li et al, ; Li, Waite, Fan, et al, ; Li, Waite, Gunawardana, et al, ). A dilution series of the plasmid from 10 7 to 1 copies/µl was created in TE buffer containing 1 mM EDTA (Invitrogen).…”

“…(MacKenzie, Vincent, Burgher‐MacLellan, & Gaul, ), Bactrocera latifrons , Bactrocera philippinensis (current Bactrocera dorsalis ) and Bactrocera occipitalis (MacKenzie et al, ; Yu, Chen, Zhang, & Yin, ; Yu, Zhang, Chen, Zhang, & Yin, ). Several real‐time PCR assays developed for fruit flies are used in the routine diagnostics at the New Zealand borders, including B. tryoni complex , B. dorsalis complex , Dirioxa pornia, Ceratitis capitata (Dhami, Gunawardana, Voice, & Kumarasinghe, ) and Bactrocera xanthodes complex ( B. xanthodes, B. neoxanthodes and B. paraxanthodes ) (Li, Waite, Gunawardana, et al, ) and those real‐time PCR assays could provide rapid identification in 4 hr. However, no real‐time PCR protocols for Z. cucumis and B. jarvisi have been developed yet.…”

“…The application of the molecular techniques has greatly assisted the identification of immature stages of interceptions (Armstrong & Ball, 2005;Armstrong, Cameron, & Frampton, 1997). DNA barcoding of the COI gene is the most commonly used technique in the identification of fruit flies (Armstrong & Ball, 2005;Blacket, Semeraro, & Malipatil, 2012 (Li, Waite, Gunawardana, et al, 2018) and those real-time PCR assays could provide rapid identification in 4 hr. However, no real-time PCR protocols for Z. cucumis and B. jarvisi have been developed yet.…”