Species Determination of Malaysian Bactrocera Pests Using PCR-RFLP Analyses (Diptera: Tephritidae) Tock H Chua,A∗ Yi Vern Chonga and Saw Hoon Limb
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Research Article Received: 15 January 2009 Revised: 21 August 2009 Accepted: 5 October 2009 Published online in Wiley Interscience: (www.interscience.wiley.com) DOI 10.1002/ps.1886 Species determination of Malaysian Bactrocera pests using PCR-RFLP analyses (Diptera: Tephritidae) Tock H Chua,a∗ Yi Vern Chonga and Saw Hoon Limb Abstract BACKGROUND: Identification of Bactrocera carambolae Drew and Hancock, B. papayae Drew and Hancock, B. tau Walker, B. latifrons Hendel, B. cucurbitae Coquillett, B. umbrosa Fabricius and B. caudata Fabricius would pose a problem if only a body part or an immature stage were available. Analysis of polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) of cytochrome oxidase I (COI) gene using primers COIR, COIF, UEA7 and UEA10 and restriction enzymes (MseI, RsaI and Alu1) was carried out. The banding profiles in the electrophoresis gel were analysed. RESULTS: The COI gene in six Bactrocera spp. was successfully amplified by COIR and COIF, as well as UEA7 and UEA10, while B. caudata was amplified successfully only by UEA primers. Using COI amplified PCR products and restriction enzymes, distinct banding profiles for B. tau, B. latifrons, B. cucurbitae and B. umbrosa were observed, but not for B. carambolae and B. papayae. However, using UEA7, UEA10 and RsaI, B. caudata could be identified, while B. carambolae and B. papayae might possibly be separated from one another. It was also shown that adult body parts or immature life stages of B. carambolae, B. papayae, B. latifrons and B. cucurbitae produced the same banding profiles as the adults. CONCLUSION: PCR-RFLP analyses are able to identify positively five Bactrocera species, while B. papayae and B. carambolae might possibly be separated from one another, even if immature life stages or adult body parts are used. c 2009 Society of Chemical Industry Keywords: Bactrocera spp.; PCR-RFLP; species determination; COI 1INTRODUCTION polymorphism (PCR-RFLP) failed to separate between B. papayae The Tephritid fruit flies are almost ubiquitous and found in and B. carambolae.8 In recent studies, researchers have studied all regions of the world.1 Some of these fruit flies possess a speciesidentificationbasedmostlyon16SandITSsequencesusing great potential to cause damage to agricultural and horticultural PCR-RFLP.9–11 Similarly, the mitochondrial control region has been production.1–3 The medfly, Ceratitis capitata (Wiedemann), the used as a marker to detect genetic variations in B. dorsalis.12 Molec- olive fruit fly, Bactrocera oleae (Rossi), and the melon fly, Bactrocera ular diagnostics has also been employed by insect researchers to cucurbitae Coquillett, are some of the well-known examples. infer phylogenetic relationships and for identification to species Bactrocera, a genus found mostly in tropical Asia, Australia and level.13–16 the South Pacific regions, is known to be a major tropical fruit pest In this study, cytochrome oxidase (COI) gene was chosen as a causing heavy losses in fruit and vegetable cultivation. In Malaysia, marker, as it has not been widely used for PCR-RFLP analyses. A there are possibly at least a hundred Bactrocera species, of which 1.3 kb variable region of the COI gene from seven Bactrocera only about half have been recorded.4 Of these, the melon fly, pest species found in Malaysia and neighbouring countries B. cucurbitae, the papaya fruit fly, B. papayae Drew and Hancock, was sequenced. These belong to two subgenera: Bactrocera the carambola fruit fly, B. carambolae Drew and Hancock, the and Zeugodacus. They were Bactrocera (Bactrocera) papayae, nangka fruit fly, B. umbrosa Fabricius, and the Malaysian fruit fly, B. (B.) carambolae, B. (B.) umbrosa, B. (B.) latifrons, Bactrocera B. latifrons Hendel, are major agricultural pests. (Zeugodacus) cucurbitae, B. (Z.) tau Walker and B. (Z) caudata In quarantine work, it is quite common to encounter immature life stages or body parts of suspected pests that require species determination. The current method of identification, based ∗ Correspondence to: Tock H Chua, School of Science, University of Monash, 5 on morphological characters of adult insects, may encounter Sunway Campus, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor difficulty in separating sibling species such as B. papayae and Darul Ehsan, Malaysia. E-mail: [email protected] B. carambolae or identifying immature stages. In these cases, molecular determination would be an advantage. a School of Science, University of Monash, Sunway Campus, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia Mitochondrial DNA is used as a common marker for identi- 6–8 fication of species in fruit flies. However, a previous study b Consultant, Transforming Science Sdn Bhd, 23, Jalan Putra Mahkota 7/3A, using polymerase chain reaction–restriction fragment length Putra Heights, 47650 Subang Jaya, Selangor Darul Ehsan, Malaysia Pest Manag Sci (2009) www.soci.org c 2009 Society of Chemical Industry www.soci.org TH Chua, YV Chong, SH Lim Table 1. List of specimens of Bactrocera spp. collected in 2007–2008 and used in the study: their collection sites, preservation methods and number of individuals. The number of individuals sequenced is indicated in brackets Subgenus Species Collection site Preservation method Number of individuals ◦ Bactrocera B. (B.) papayae Kerinchi −20 C freezer 19 (3) Pantai Hillpark 96% alcohol Kota Kinabalu 96% alcohol ◦ Seremban −20 C freezer ◦ B. (B.) carambolae Kerinchi −20 C freezer 16 (2) Pantai Hillpark 96% alcohol ◦ B. (B.) umbrosa Damansara Jaya −20 C freezer 4 (2) Pantai Hillpark 96% alcohol ◦ B. (B.) latifrons Petaling Jaya −20 C freezer 10 (2) ◦ Lukut −20 C freezer Zeugodacus B. (Z.) caudata Lukut 96% alcohol 2 (2) ◦ B. (Z.) cucurbitae Kerinchi −20 C freezer 8 (2) ◦ Lukut −20 C freezer ◦ B. (Z.) tau Tanah Rata −20 C freezer 6 (2) Kerichi 96% alcohol Gombak 96% alcohol Table 2. Oligonucleotides used for DNA amplification and sequencing of the Bactrocera spp Primer Sequence Reference Set 1 COIF 5-TACAATTTATCGCCTAAACTTCAGCC-3 Han and Ro16 COIR 5-CATTTCAAGTTGTGTAAGCATC-3 Set 2 UEA7 5-TACAGTTGGAATAGACGTTGATAC-3 Lunt et al.;17 Jamnongluk et al.15 UEA10 5-TCCAATGCACTAATCTGCCATATTA-3 ◦ Fabricius. The restriction site variation across the COI gene for extension at 72 C for 15 min. The PCR conditions for the second the seven species was analysed through PCR-RFLP. Digestion of set of primers (UEA7 and UEA10)17 were as follows:18 an initial ◦ ◦ ◦ the PCR products with restriction enzymes produced different denaturation at 94 C for 3 min, 35 cycles at 94 Cfor1min,50 C ◦ ◦ banding profiles in the gel that could be used for identification of for 1 min, extension at 72 C for 1 min and final extension at 72 C the Bactrocera species. for 30 min. PCR products were electrophoresed on a 1% SeaKem LE agarose gel. The DNA fragment sizes were estimated by comparing 2 MATERIALS AND METHODS with 1 kb, 50 bp and 25 bp commercial markers (Promega, USA). 2.1 Fruit fly collection and handling The band corresponding to the target PCR product was excised Adult specimens of the seven Bactrocera species were collected and purified using Qiagen QIAquick Gel Extraction Kit. All samples from different parts of Malaysia using chemical lures (methyl were then sent to First BASE Laboratories Sdn. Bhd. (Malaysia) for eugenol and cuelure) (Table 1), while the immature stages were sequencing. collected from host fruits. The specimens were either stored at To test whether this molecular method can be used to identify ◦ −20 C in a freezer or preserved in 96% alcohol prior to analysis. A a species when only a small body part or an immature stage is total of 65 individuals were used in this analysis. available, which may be the case in quarantine work, DNA was also extracted from different body parts of B. papayae (head, thorax and legs) and from immature stages (egg, larvae and pupae) of 2.2 DNA extraction, amplification and sequencing B. carambolae,B.papayae,B.latifrons andB.cucurbitae forPCR-RFLP Total DNA was extracted using DNeasy Tissue Kit (Qiagen Inc., analyses. USA), following the manufacturer’s protocol for animal tissue with slight modifications to increase DNA yield. Either a whole body or one leg was used for each extraction. 2.3 Restriction analysis Two sets of primers were used for polymerase chain reaction The sequences of seven Bactrocera spp. amplified using both (PCR)amplificationofcytochromeoxidaseI(COI)markers(Table 2). primer sets were aligned using ClustalW19 andexaminedfortheir PCR amplifications for the first set of primers (COIR and COIF) were recognition sites of 50 restriction enzymes using the BioEdit performed following the conditions as described by Han and program.20 Three restriction enzymes were then chosen for ◦ ◦ Ro:16 an initial denaturation at 95 C for 3 min, 40 cycles at 93 C further work, and their recognition sites are depicted in Fig. 1. ◦ ◦ for 1 min, 55 C for 1 min, extension at 72 Cfor2minandfinal The restriction fragment lengths of PCR-amplified section were www.interscience.wiley.com/journal/ps c 2009 Society of Chemical Industry Pest Manag Sci (2009) Determining Bactrocera spp. using PCR-RFLP www.soci.org Figure 1. Recognition sites of chosen restriction enzymes predicted for seven Bactrocera spp. from COI fragments amplified using the two primer sets. The primers and restriction enzymes were respectively for: (A) COIR, COIF, AluI; (B) COIR, COIF, MseI; (C) UEA7, UEA1, RsaI. then predicted (Table 3). Polymerase chain reaction products volume of 20 µL containing 1 U restriction enzyme, MseI, AluI (NEW obtained from each species were directly used for the PCR- ENGLAND Biolabs) or RsaI (Promega Corporation) for 2 h (as per RFLP analyses, without further purification. For B. papayae and manufacturer’s protocol) or overnight (for confirmation purposes) ◦ B. carambolae, however, the PCR-RFLP analyses were also carried at 37 C. For further confirmation of the banding patterns of out with purified polymerase chain reaction products.