Reliable molecular identification of nine tropical whitefly species Tatiana M. Ovalle1, Soroush Parsa1,2, Maria P. Hernandez 1 & Luis A. Becerra Lopez-Lavalle1,2 1Centro Internacional de Agricultura Tropical (CIAT), Km 17, Recta Cali-Palmira, Cali, Colombia 2CGIAR Research Program for Root Tubers and Bananas, Lima, Peru Keywords Abstract COI, RFLP-PCR, Tropical whiteflies, Molecular identification. The identification of whitefly species in adult stage is problematic. Morphologi- cal differentiation of pupae is one of the better methods for determining identity Correspondence of species, but it may vary depending on the host plant on which they develop Luis A. Becerra Lopez-Lavalle, Centro which can lead to misidentifications and erroneous naming of new species. Poly- Internacional de Agricultura Tropical (CIAT) merase chain reaction (PCR) fragment amplified from the mitochondrial cyto- Km 17, Recta Cali-Palmira, Cali, Colombia. chrome oxidase I (COI) gene is often used for mitochondrial haplotype Tel: +57 2445 0000; Fax: +57 2445 0073; E-mail: [email protected] identification that can be associated with specific species. Our objective was to compare morphometric traits against DNA barcode sequences to develop and Funding Information implement a diagnostic molecular kit based on a RFLP-PCR method using the This should state that the CGIAR Reseach COI gene for the rapid identification of whiteflies. This study will allow for the Program for Root Tubers and Bananas rapid diagnosis of the diverse community of whiteflies attacking plants of eco- provided the resources to do this work. nomic interest in Colombia. It also provides access to the COI sequence that can be used to develop predator conservation techniques by establishing which Received: 4 April 2014; Revised: 20 June predators have a trophic linkage with the focal whitefly pest species. 2014; Accepted: 2 July 2014 Ecology and Evolution 2014; 4(19): 3778– 3787 doi: 10.1002/ece3.1204 Introduction 2000). Whitefly pupae, however, can exhibit significant phe- notypic plasticity in response to differences in leaf architec- Whiteflies (Hemiptera: Aleyrodidae) are globally considered ture and to environmental or physical factors (Guershon as one of the most important pests in agriculture (Bellotti and and Gerling 2001), potentially confounding research out- Arias 2001; Morales and Anderson 2001). They can attack a comes. On the other hand, adult characters have been used wide range of crop hosts often reducing their yields by more successfully in the subfamily Aleurodicinae, but much work than 50% due to the combined effects of their feeding from is still needed before they can be more broadly used in phloem and their vectoring of plant viruses (Byrne and whitefly systematics (Ghahari et al. 2009). Modern molecu- Bellows 1991). Most species of whiteflies are oligophagous or lar techniques can assist whitefly systematics research also polyphagous; however, a few species are monophagous (feed facilitating species identifications (Oliveira et al. 2000; Cal- on only one host plant; Resh and Carde 2009). The most vert et al. 2001, 2005; Shatters et al. 2009). notorious whitefly species, Bemisia tabaci, can feed from over Mitochondrial DNA (mtDNA) has been extensively used 500 different plant hosts worldwide (Greathead 1986). in phylogenetic studies of animals because it evolves much Today, 1556 valid species are known to compose the Aley- more rapidly than nuclear DNA, favoring the accumulation rodidae family, the only included family in the Aleyrodidae of nucleotide differences (i.e., polymorphisms) between subgroup of Hemiptera (Forero 2008). The Aleyrodidae is closely related species (Brown et al. 1979; Lunt et al. 1996). further subdivided into three subfamilies: Aleurodicinae, The accumulation of these polymorphisms is primarily Aleyrodinae, and Udamoselinae (Forero 2008). The system- caused by the loss or gain of restriction sites without a atics of these subfamilies is based almost entirely on mor- detectable change in genome size (Hebert et al. 2003a). phological characters of the forth-instar nymph (i.e., pupae), The nature of the sequence changes within the mito- the most conspicuous developmental stage (Martin et al. chondrial cytochrome oxidase I (COI) gene make it an 3778 ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. T. M. Ovalle et al. Whitefly Molecular Identification ideal candidate to be used as a DNA barcode system Observations were carried out from January of 2011 to (Hebert et al. 2003b; Kress and Erickson 2008). Generally, August of 2012 on CIAT’s fields and laboratories located sequencing of the polymerase chain reaction (PCR) frag- in Palmira, Valle del Cauca, Colombia. Pupae were col- ment amplified from the COI gene is used for mitochon- lected from infested leaves of seven plant species and drial haplotype identification that can be then associated brought to the laboratory. Nine whitefly species were with specific species (Shatters et al. 2009). This approach identified and 10–20 nymphs of each species (Table 1). can be particularly useful when rapid whitefly detection The pupae samples were then placed vials and allowed and identification is required for regulatory purposes to develop to adults. Once emerged, 10–20 adults per spe- especially when specimens are physically damaged. To be cies were introduced in 1.5-mL microcentrifuge tube and most valuable, the test needs to be highly accurate while placed in liquid nitrogen for DNA isolation. maintaining high rates of throughput. A basic approach to develop such type test has been to DNA isolation and PCR amplification employ universal PCR primers to amplify the COI gene and to subject the products to restriction fragment length Total genomic DNA was obtained from nine whiteflies polymorphism (RFPL) analysis (Vidigal et al. 2002; species using a modified cetyltrimethylammonium bro- Caldeira et al. 2003; Thyssen et al. 2005). In addition, a mide method (CTAB; Doyle and Doyle 1990) by adding large number of species-specific COI sequences (nearly 2.5 M potassium acetate (pH 5.5) to precipitate protein. 150,000 species) have now been submitted to public The DNA barcode region was amplified from all whitefly sequence databases such as GenBank or The Barcode of species using 10 lM of universal DNA primers LCO1490 Life Data System (BOLD) for use in taxonomic and (50-GGTCAACAAATCATAAAGATATTGG-30) and HCO phylogenetic studies of insects (Kwong et al. 2012; 2198 (50-TAAACTTCAGGGTGACCAAAAAATCA-30)for Ptaszynska et al. 2012; Smith et al. 2012). the amplification of mitochondrial cytochrome c oxidase Our objective was to compare morphometric traits subunit I – COI (Folmer et al. 1994). PCR was conducted â against DNA barcode sequences to develop and imple- in a Mastercycler pro (Eppendorf, Hamburg, Germany) ment a diagnostic molecular kit based on a RFLP-PCR for five cycles (40 sec at 94°C, 40 sec at 45°C, and 60 sec method using the COI gene for the rapid identification of at 72°C) followed by 35 cycles (40 sec at 94°C, 40 sec at whiteflies. This study will allow for the rapid diagnosis 51°C, and 60 sec at 72°C) with a final extension period of of the diverse community of whiteflies attacking plants of 72°C for 10 min. All PCRs were performed in a 12 lL â economic interest in Colombia. It will also provide access reaction volume using 0.25 U of Platinum Taq (Invitro- â to the COI sequence that can be used to development gen Carlsbad, CA), 19 PCR Buffer (buffer composition), predator conservation techniques. For instance, Lundgren 2.5 mM of each dNTP (Promega Corp. Madison, WI), et al. (2013) were able to establish which predators have a 2.5 mM MgCl2, and 20 ng of DNA. trophic linkage with Aleurotrachelus socialis in Colombia. Sequencing of the COI region Materials and Methods Amplified COI region from each white fly species was â cloned into the PGEM -T Easy vector (Promega Corp.). Morphological study Plasmid DNA from Escherichia coli used for sequence Over 40 years, CIAT’s entomology research group has col- analysis was purified using a SV minipreps preparation lected or received hundreds of whitefly voucher specimens (Promega Corp.). DNA inserts in this vector were from tropical and subtropical regions of the world, where sequenced in both directions using the Big DyeTM Termi- beans, cassava, rice, tropical forages and fruits crops are nator Cycle Sequencing kit with an Applied Biosystems 377 grown by small holder farmers. These specimens deposited (Foster City, CA) DNA fragment analyzer by the Cornell in the Arthropod Reference Collection at CIAT (CIAT- University Life Sciences Core Laboratories Center (Ithaca, ARC) headquarters in Cali, Colombia constitute an invalu- NY), using vector primers SP6 and T7. The resulting DNA â able resource for the identification and characterization of sequences were edited and analyzed using Sequencher 4.5 current, as well as potentially new tropical crop pest. Ini- (Genes Codes Corp. Ann Arbor, MI). tially, pupae of whitefly were collected from multiple crops in the area of Palmira – Valle del Cauca (Table 1). Pupae at DNA sequence analysis the 4th instar nymph stage were morphologically examined under a light microscope and identified using the keys of DNA Sequences were edited to the whitefly species-spe- Martin et al. (2000), Martin (2004), Hodges and Evans cific COI sequence by eliminating vector and universal (2005), Martin (2005), and Dooley (Dooley 2006). primer sequence and analyzed for identity with sequences ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3779 Whitefly Molecular Identification T. M. Ovalle et al. Table 1. List of whitefly species found associated with seven plant species in Palmira/Valle del Cauca (Colombia) based on morphological and anatomical features of the puparia.