Molecular Entomology Single-Target and Multiplex Discrimination of Whiteflies (Hemiptera: Aleyrodidae) Bemisia tabaci and Trialeurodes vaporariorum With Modified Priming Oligonucleotide Thermodynamics Sharon A. Andreason, 1,2 Mohammad Arif, 3 Judith K. Brown, 4 Francisco Ochoa-Corona, 1 Jacqueline Fletcher, 1 and Astri Wayadande 1,5 1 Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078 (sharona@ucr.edu; ochoaco@okstate.edu; jacqueline.fletcher@okstate.edu; a.wayadande@okstate.edu), 2 Current Address: Department of Entomology, University of California, Riverside, CA 92521, 3 Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, HI 96822 (arif@hawaii.edu), 4 School of Plant Sciences, University of Arizona, Tucson, AZ 85721 (JBrown@ag.arizona.edu), and 5 Corresponding author, e-mail: a.wayadande@okstate.edu Subject Editor: John Trumble Received 16 December 2016; Editorial decision 6 April 2017 Abstract The whitefly species Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood) are worldwide ag- ricultural pests and virus vectors. Bemisia tabaci, in particular, is often transported internationally via trade routes leading to potential introductions of exotic whiteflies or plant viruses. Quick identification of agricultur- ally important whiteflies can facilitate interventions that prevent these cross-border introductions. Polymerase chain reaction (PCR) primers were designed to amplify the mitochondrial cytochrome oxidase I gene (mtCOI) sequence of members of the B. tabaci complex, MEAM1, MED, and NW, and T. vaporariorum. Primers incorpo- rated an A/T-rich overhang sequence at the 5 0 terminus (5 0 flap) to test for increased primer sensitivity and assay efficiency. Single-target and multiplex endpoint PCR assays with the eight primer sets were performed using genomic DNA template extracted from individual adult whiteflies. Resultant PCR amplicons obtained for B. tabaci MEAM1, MED, and NW, and T. vaporariorum primers with the 5 0 flap were 559-, 717-, 353-, and 258-bp, respectively, and without the 5 0 flap were 550-, 712-, 329-, and 252-bp in length, respectively. In single-target and multiplex reactions, specific amplification was achieved using both the unmodified and 5 0 flap-modified pri- mers. Sequencing and phylogenetic analysis confirmed primer-target amplification specificity. Using these pri- mer sets in single-target or multiplex PCR allows for quick discrimination and specific identification of B. tabaci complex members and T. vaporariorum, and the addition of 5 0 A/T-rich overhang sequences increases the sensi- tivity and amplification of some primer sets. Key words: Aleyrodidae, whitefly cryptic species, mitochondrial cytochrome oxidase I gene, multiplex polymerase chain reaction, 5 0 A/T-rich flap The whitefly (Hemiptera: Aleyrodidae) species Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood) are agri- cultural pests and vectors of plant viruses infecting food, fiber, and ornamental crops globally. The greenhouse whitefly, T. vaporario- rum, is a pest of >850 plant species and the insect vector of certain viruses within the Crinivirus (Closteroviridae) and Torradovirus (Secoviridae) genera (Duffus 1965, Wintermantel 2004, Amari et al. 2008, Centre for Agriculture and Bioscience International [CABI] 2015). The sweetpotato whitefly, B. tabaci, is a highly polyphagous pest and vector of a number of economically important begomovi- ruses (Geminiviridae), as well as some criniviruses, torradoviruses, carlaviruses (Betaflexiviridae), and ipomoviruses (Potyviridae) (Costa 1976, Jones 2003, Navas-Castillo et al. 2011). Both species commonly occur in greenhouse or controlled environment facilities where ornamental or vegetable plants are grown (Jones 2003, Cicero and Brown 2016, Wintermantel 2016). The high volume of plants shipped from one geographic location to another has resulted in the introduction of various B. tabaci haplotypes and T. vaporariorum into new regions. Furthermore, exotic plant viruses harbored by B. tabaci or T. vaporariorum have been introduced to nonendemic loca- tions by these same routes (Brown and Poulos 1990, Brown and Bird 1992, Brown et al. 1995, Gill and Brown 2010). Identification and V C The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com 1821 Journal of Economic Entomology, 110(4), 2017, 1821–1830 doi: 10.1093/jee/tox125 Advance Access Publication Date: 11 May 2017 Research article Downloaded from https://academic.oup.com/jee/article/110/4/1821/3817180 by guest on 24 April 2023