Biol Invasions DOI 10.1007/s10530-017-1546-6 ORIGINAL PAPER Barcode index numbers expedite quarantine inspections and aid the interception of nonindigenous mealybugs (Pseudococcidae) Jing-Mei Ren . Muhammad Ashfaq . Xu-Nan Hu . Jun Ma . Fan Liang . Paul D. N. Hebert . Li Lin . Jean François Germain . Muhammad Z. Ahmed Received: 2 February 2017 / Accepted: 18 August 2017 © Springer International Publishing AG 2017 Abstract Quarantine interception of invasive and (836) of these specimens were assigned to a named nonindigenous insect pests at ports of entry is often species, but others were only identified to a genus or impeded by the lack of robust identification methods. family. Their sequence analysis revealed substantial Because of their inconspicuous morphology and wax- COI diversity with maximum divergences reaching covered bodies, mealybugs present a particular chal- 27%. While the identified specimens included repre- lenge. The present study employs DNA barcoding sentatives of 62 species, the Barcode Index Number (658 base pairs near the 5′-terminus of the cyto- (BIN) system assigned the 914 sequences to 120 chrome c oxidase I gene) as a tool for their BINs, nearly doubling the putative species count and discrimination because of its proven utility in revealing cases of potential cryptic species and discriminating closely related species. The current misidentifications. With a single exception, study considers DNA barcodes from 914 mealybugs intraspecific divergence values for named species (Pseudococcidae) collected in 31 countries. Most were less than their nearest-neighbor distances, but 13 species showed BIN splits and two species were merged in a BIN. High genetic diversity and presence Electronic supplementary material The online version of this article (doi:10.1007/s10530-017-1546-6) of cryptic species in the known mealybugs, revealed contains supplementary material, which is available to in this study, underscore the limitations of morphol- authorized users. ogy and potential utility of BINs for the rapid recognition of nonindigenous insect pests. Jing-Mei Ren and Muhammad Ashfaq have contributed equally to this work. M. Ashfaq · P. D. N. Hebert J.-M. Ren · X.-N. Hu (&) · J. Ma (&) · F. Liang · L. Lin Centre for Biodiversity Genomics, Biodiversity Institute Plant Quarantine Laboratory, Guangdong Inspection and of Ontario, University of Guelph, Guelph, ON, Canada Quarantine Technology Center, Guangzhou 510623, People’s Republic of China J. F. Germain e-mail: [email protected] ANSES, Laboratoire de la Sante´ des Ve´ge´taux, Unite´ J. Ma d’entomologie et Plantes Invasives, Campus International e-mail: [email protected] de Baillarguet, CS 30016, 34988 Montferrier-sur-Lez Cedex, France J.-M. Ren Department of Entomology, South China Agricultural M. Z. Ahmed University, Guangzhou 510642, People’s Republic of Florida Department of Agriculture and Consumer China Services, Division of Plant Industry, 1911 SW 34th Street, Gainesville, FL 32608, USA 123 J.-M. Ren et al. Keywords Invasive species · Biosecurity · morphology and genetic data of well recognized Plant pests · Cryptic species · DNA barcoding mealybug species (Malausa et al. 2011). DNA sequences provide a reliable option to resolve complex taxa but the technique is limited by non-availability of Introduction reference data. For example, China has documented 154 species of mealybugs, some representing alien With increasing global trade, the cross-border move- invasive species (Wang et al. 2016), but most of them ment of nonindigenous and invasive insect pests is on lack genetic data. The use of a morphology-based the rise (Westphal et al. 2008; Koch et al. 2014; identification approach for the quarantine inspection of http://www.eea.europa.eu/). Dwindling expertise in mealybugs, particularly their immature stages, is conventional identification techniques and the lack of difficult and time-consuming because it often requires robust methods for species recognition have created slide mounting and rearing (Gullan 2000; Johnson and new challenges for the quarantine interception of Giliomee 2013). Moreover, the perishable nature of invading insects (Bacon et al. 2012; Wan and Yang fruits and vegetables demands the capacity to rapidly 2016). With their inconspicuous morphology, mealy- and reliably recognize nonindigenous pests at quaran- bugs (Hemiptera: Pseudococcidae) are a particular tine inspection stations. challenge for identification (Qin et al. 2010). Many of Because DNA-based methods overcome the imita- the nearly 2000 described species of Pseudococcidae tions associated with morphology-based are important plant pests (Abd-Rabou et al. 2012; identifications, they are gaining increasing adoption Garcı´a Morales et al. 2016) and several species have in resolving mealybug species (Saccaggi et al. 2008; shown their capacity to adapt to new hosts (Chellap- Ashfaq et al. 2010; Daane et al. 2011). A range of DNA pan et al. 2013). Over the past decade, there have markers, such as elongation factor 1α (EF-1α), 18S been damaging invasions by mealybugs (Ahmed rRNA, 28S rRNA, internal transcribed spacer (ITS), et al. 2015a; Mendel et al. 2016) which have resulted and cytochrome c oxidase I (COI), have been used to in substantial economic losses for agriculture and discriminate mealybug species (Gullan et al. 2010; horticulture (Zeddies et al. 2001; Hodgson et al. Pieterse et al. 2010; Ashfaq et al. 2011; Abd-Rabou 2008). For example, the solenopsis mealybug et al. 2012; Beltra` et al. 2012; Beuning et al. 2014), but (Phenacoccus solenopsis) has invaded South Asia the lack of marker standardization has limited the (Hodgson et al. 2008; Ashfaq et al. 2010), China utility of sequence data. The implementation of a (Ahmed et al. 2015b) and Egypt (Ibrahim et al. 2015) DNA-based approach for species identification at a where it has become a major pest on cotton. global scale requires the generation of a sequence Likewise, invasions of the pine mealybug (Oracella library based upon a standard gene region to enable acuta) in China, the papaya mealybug (Paracoccus identifications. The adoption of COI-5′ as the standard marginatus) in West Africa, and the hibiscus mealy- barcode region (Hebert et al. 2003) for species bug (Maconellicoccus hirsutus) in the Caribbean have identification in the animal kingdom has led to the been documented (Sagarra and Peterkin 1999; Goer- assembly of more than four million barcode records, gen et al. 2011; You et al. 2013). including 1500 for the Pseudococcidae (accessed 16 The reduction of introductions of exotic species July 2017), in the Barcode of Life Data System linked to international trade is one of the main (BOLD) (www.boldsystems.org) (Ratnasingham and functions of plant quarantine programs and mealybugs Hebert 2007). Those sequences meeting designated represent an important group of concern. The taxo- criteria ([507 bp, no stop codons, \1% ambiguous nomic situation in Pseudococcidae is complex as some bases) have each been assigned to a Barcode Index genera, such as Antonina, Chorizococcus, Dysmicoc- Number (BIN) (Ratnasingham and Hebert 2013) that is cus, Pseudococcus, and Spilococcus are currently an effective species proxy (Telfer et al. 2015; Ashfaq recognized as artificial assemblages of species created et al. 2017). BINs have been frequently used to resolve merely for ease in identification than for phylogenetic cryptic species complexes and to analyze their distri- relationships (McKenzie 1967; Gavrilov-Zimin 2016). butions (Ashfaq et al. 2014; Mutanen et al. 2015; This is also evidenced by incongruence between Ashfaq and Hebert 2016). 123 Barcode index numbers expedite quarantine inspections China imports fresh produce from more than 34 from 24 nations were identified and sequenced at countries, and international travelers often carry IQTC (Table S1; Fig. 1). These specimens were undeclared plant material, creating an ongoing chal- preserved in 95–100% ethanol and stored at −20 °C lenge for quarantine inspections. Each year more than until DNA extraction. Morphological identifications 30 mealybug species, some regulated and nonindige- were based upon taxonomic keys in Tang (1992) and nous, are intercepted and identified at the Plant Williams (2004). Voucher specimens are held in the Quarantine Laboratory at the Guangdong Inspection Insect Collection of Plant Quarantine Laboratory at and Quarantine Technology Center (IQTC). The IQTC. current study aimed to establish a platform to support DNA extraction, polymerase chain reaction (PCR), barcode-based quarantine inspections for all inter- and sequencing: cepted mealybug species by testing the efficacy of DNA was extracted from individual specimens BINs as a tool for the prompt detection of newly using DNeasy Blood & Tissue Kit (Qiagen, Shang- encountered species. This is the first study on hai, China) following the manufacturer’s protocol. mealybugs which examines the congruence between DNA elution was performed with 50–80 μlofAE morphological species and BINs, and then maps their buffer. PCR amplification of COI-5′ (DNA barcode) distribution by integrating barcode data for Chinese (Hebert et al. 2003) was performed with primers mealybugs with those from other countries. PcoF1 (5′-CCTTCAACTAATCATAAAAATATYA G-3′)/LepR1 (5′-TAAACTTCTGGATGTCCAAA AAATCA-3′) (Park et al. 2011) or C1-1554F (5′- Materials and methods CAGGAATAATAGGAACATCAATAAG-3′)/C1-23 42R (5′-ATCAATGTCTAATCCGATAGTAAATA- Mealybugs were obtained from collections within 3′) (Deng et al. 2012) using 2 μl of DNA template. China, quarantine interceptions