Determination of eight economically important fruit fly pests (Diptera: Tephritidae) using PCR-RFLP analyses

عنوان دوره: دومین کنگره بین المللی حشره شناسی ایران

Not only does the Iranian southeastern provinces provide salient ports for importing many agricultural and horticultural crops, but also serving as footholds for many exotic insect pests finally will make their way to other parts of the country. Regarding this issue, accurate identification of pests at species level is considered the first step toward implementing successful strategies against their dispersal. Tephritids have been always a major global threat to the domestic fruit farming industries. Determining tephritid identity, merely morphologically, has still been remained a difficult task requiring high level facilities in terms of expertise and equipment. To effectively solve this issue, various alternative molecular methods have been developed which among them, PCR-RFLP is still a common method being utilized by scientists for tephritid identification (Onah et al. (2015), Ukey et al. (2017)) across the world. In this study, PCR-RFLP was used for quick identification of the eight tephritids; Bactrocera zonata (Saunders), Bactrocera latifrons (Hendel), Bactrocera dorsalis (Hendel), Bactrocera oleae (Rossi), Bactrocera cucurbitae (Coquillett), Dacus ciliates Loew, Ceratitis capitata (Wiedemann) and Carpomya vesuviana Costa. All of them coexist in Sistan and Baluchestan province, southeastern Iran; all are of economic concern and considered the main pests of tropical and subtropical crops (mango, guava, sapodilla, Ziziphus, papaya...) in this area. Total DNA was extracted from thorax and legs of each adult specimen of the species through Chelex method. The COI sequences of the eight species was first examined to recognize the sites of various restriction enzymes by means of CLC Sequence Viewer7 that resulted in selection of three restriction enzymes; MboII, RsaI, and Alu1. PCR-RFLP analysis of COI gene using primers UEA7and UEA10 and the restriction enzymes was performed. An approximate 650 bp of COI gene in the tephritid species was successfully amplified. After electrophoresis of PCR products in a 1.5% agarose gel, the target corresponding bands were excised with a sterile razor blade and purified using QIAQuick Gel Extraction Kit. The purified PCR products (10 µl) were then digested with the restriction enzymes according to the manufacturers’ instructions.The banding profiles for the eight fruit fly speciesin the electrophoresis gel were analyzed. Distinct banding patterns for the examined fruit flies which clearly separated them suggesting the reliability of the molecular markers as effective tools foridentifying the insect pests. The outcome of this research will efficiently help accelerate the identification process of agricultural pests leading to the enhancement of the quarantine standards at borders.

species identification؛ true fruit flies؛ molecular diagnosis