GENETICS Molecular Identification of Ceratitis capitata (Diptera: Tephritidae) Using DNA Sequences of the COI Barcode Region 1,2 3 4 3 N. B. BARR, M. S. ISLAM, M. DE MEYER, AND B. A. MCPHERON Ann. Entomol. Soc. Am. 105(2): 339Ð350 (2012); DOI: http://dx.doi.org/10.1603/AN11100 ABSTRACT The utility of the cytochrome oxidase I gene barcode region for diagnosis of the Downloaded from https://academic.oup.com/aesa/article/105/2/339/120778 by guest on 27 September 2021 Mediterranean fruit ßy, Ceratitis capitata (Weidemann), is evaluated using African fruit ßy collections. The method fails to discern C. capitata from its close relative Ceratitis caetrata Munro, based on genetic distances, parsimony networks, or nucleotide diagnostic characters observed in the DNA barcode sequences. When treated as a single taxon, it is possible to discern the C. capitata ϩ C. caetrata lineage from other Ceratitis species. Levels of intraspeciÞc diversity vary within the genus Ceratitis and multiple copies of the mitochondrial gene are reported for Ceratitis cosyra (Walker). The DNA barcoding method based on genetic distance is compared with a molecular identiÞcation method using restriction fragment length polymorphism. The DNA barcode and restriction fragment-length poly- morphism methods provide similar identiÞcation results, but the DNA sequence information is more suitable for quantitative analysis of the information. KEY WORDS DNA barcode, restriction fragment-length polymorphism, diagnostic, Tephritidae The fruit ßy genus Ceratitis MacLeay (Diptera: Te- Although numerous studies using molecular meth- phritidae) comprises 89 described species that are ods to identify C. capitata have been published, most native to Africa (De Meyer 2000a, De Meyer and include insufÞcient sampling of species to demon- Copeland 2005, Barr and McPheron 2006). The genus strate taxonomic speciÞcity in the techniques. For includes several highly polyphagous species that are of example, Douglas and Haymer (2001) and Kakouli- economic importance (White and Elson-Harris 1992, Duarte et al. (2001) each reported a technique to Yuval and Hendrichs 2000, Copeland et al. 2006) and discern C. capitata from C. rosa Karsch, and Armstrong recognized as invasive or potentially invasive (De and Ball (2005) a technique to discern C. capitata from Meyer et al. 2008). Of these species, Ceratitis capitata C. rosa Karsch and C. cosyra (Walker), but did not test (Wiedemann), commonly called the Mediterranean additional Ceratitis species. Some studies only com- fruit ßy, is regarded as the most serious international pare C. capitata to fruit ßies from other genera (Arm- pest because of its broad range of hosts, nearly world- strong et al. 1997, Huang et al. 2009). Despite the wide distribution, and impact on trade (White and limited taxon sampling, these tools can be of value in Elson-Harris 1992, Copeland et al. 2002, Vera et al. fruit ßy identiÞcation when nonmolecular informa- 2002, De Meyer et al. 2008, Barr 2009). tion can be integrated in the diagnosis process. Accurate identiÞcation of C. capitata is important To date, the most taxonomically comprehensive for implementing proper quarantine and pest man- molecular tool published for Ceratitis species identi- agement practices. Consequently, morphological Þcation is a Polymerase Chain ReactionÐRestriction tools have been developed to diagnose C. capitata and Fragment Length Polymorphism (polymerase chain other Ceratitis species using adult characters (De reaction [PCR]-restriction fragment-length polymor- Meyer 1996, 1998, 2000b; De Meyer and Freidberg phism [RFLP]) method by Barr et al. (2006). This tool 2005a). Unfortunately, at ports of entry, intercepted is based on relatively good, although not complete, larvae cannot be reliably identiÞed to the species-level sampling of the genus and includes collections from its by using morphology. White and Elson-Harris (1992) ancestral home range in Africa (De Meyer et al. 2004). developed keys for third-instar larvae, but these keys These African collections provide a better estimate of include only 11 species and are based on small sample genetic variation within C. capitata than do collections sizes for most of the species. derived from introduced populations (Barr 2009). In addition, the data set includes samples of Ceratitis 1 Center for Plant Health Science and Technology, Mission Labo- caetrata Munro and Ceratitis pinax Munro, two close ratory, USDA-APHIS, Moore Air Base, Edinburg, TX 78541. relatives of C. capitata that are useful for evaluating the 2 Corresponding author, e-mail: [email protected]. speciÞcity of the diagnostic tool (De Meyer 2005, Barr 3 Department of Entomology, Pennsylvania State University, Uni- and McPheron 2006, Barr and Wiegmann 2009). versity Park, PA 16802. 4 Royal Museum for Central Africa, Leuvensesteenweg 13, 3080 The PCR-RFLP method, developed using the 12S Tervuren, Belgium. rRNA, 16S rRNA, and NADH-dehydrogenase subunit 340 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 105, no. 2 6 mitochondrial DNA loci, was not able to distinguish part of a study of Kenyan fauna (De Meyer et al. 2002, C. capitata from C. caetrata but it could distinguish C. Copeland et al. 2006). With the exception of four C. pinax. However, differences in the internal transcribed capitata specimens (codes 620Ð623, Kenyan samples spacer region 1 (ITS-1) of C. capitata and C. caetrata 1382 by R. Copeland), DNA isolates were reported were able to discriminate the two species based on previously in the Barr et al. (2006) and Barr and DNA sequences and PCR amplicon length (Barr et al. McPheron (2006) studies. In addition to Ceratitis, spe- 2006). cies representing the genera Trirhithrum Bezzi, Cap- Many of the DNA samples used in the PCR-RFLP parimyia Bezzi, Carpophthoromyia Austen, and No- study also were used to estimate the phylogeny of the tomma Bezzi are included for comparison to the genus (Barr and McPheron 2006, Barr and Wiegmann restriction fragment-length polymorphism study. De 2009). Although DNA sequences were generated to Meyer and Freidberg (2005b) have shown that the estimate phylogenies and locate restriction enzyme Capparimyia melanspis Bezzi collection included in Downloaded from https://academic.oup.com/aesa/article/105/2/339/120778 by guest on 27 September 2021 recognition sites in the restriction fragment-length Barr et al. (2006) study includes two species: C. mela- polymorphism diagnostic tool, the ability to use the naspis and Capparimyia aenigma De Meyer and DNA sequences as diagnostic information has yet to Freidberg. These samples are treated as a mixture of be evaluated. Recent studies have demonstrated that species and reported as Capparimyia sp. in the current a DNA sequencing approach to identiÞcation of C. study. capitata geographic populations was superior to a Over 600 samples were available for analysis, but PCR-RFLP approach when using the same mitochon- only a subset of 249 samples was selected for DNA drial gene region (Lanzavecchia et al. 2008, Barr barcoding to minimize research costs. For each spe- 2009). cies included in the Barr et al. (2006) study, at least Þve There is growing interest in the use of DNA bar- samples were selected for barcode analysis (Table 1). codes to diagnose pest arthropod species (Armstrong Specimens were selected based on collection location 2010, Floyd et al. 2010). The technology is being in- and restriction fragment-length polymorphism geno- vestigated as a tool for identiÞcation of species that types to maximize geographic and genetic variation have economic, ecological, and health impacts (Ball within each species. In addition, several species rep- and Armstrong 2006, Scheffer et al. 2006, Yancy et al. resented by single specimens in the Barr and 2008, Lowenstein et al. 2009, deWaard et al. 2010, McPheron (2006) phylogenetic study also were in- Naro-Maciel et al. 2010). The Þrst published applica- cluded to increase taxonomic coverage in the data tion of DNA barcoding for tephritid fruit ßy identiÞ- base. Every specimen is numerically coded based on cation focused on the genus Bactrocera Macquart position in the database (Supp. Table S1). (Armstrong and Ball 2005). Currently, DNA barcod- All material was identiÞed to species based on adult ing of tephritid fruit ßies is an objective of interna- morphology (Supp. Table S1). Pinned vouchers (of tional barcode campaigns such as the Quarantine Bar- coding of Life (QBOL, www.qbol.org/UK/) and the entire adult body) representative of each Kenyan col- Tephritid Barcode Initiative (TBI, http://www. lection series are maintained at the Royal Museum for barcoding.si.edu/major_projects.html). Although the Central Africa (KMMA, Tervuren, Belgium), the number of public DNA barcode records is increasing Frost Entomological Museum at the Pennsylvania for Ceratitis species (e.g., 317 specimens with barcode State University (PSUC, University Park, PA), or both. records representing 45 taxa were reported on the For most DNA barcoded samples (Table 1), a tissue Barcode of Life Database, BOLD, on 3 May 2011), no voucher is maintained in ethanol at the APHIS- publications have evaluated the utility of these se- CPHST lab in Edinburg, TX. The tissue vouchers in- quences. clude wings and abdomens from each specimen (the We sequence the proposed barcode region (Hebert head, thoraces, and legs were used in the DNA isola- et al. 2003) of the cytochrome oxidase subunit 1 gene tion process). A description of DNA isolation proce- (COI) using the Ceratitis specimens included