New primers for DNA barcoding of digeneans and cestodes (Platyhelminthes) NIELS VAN STEENKISTE,* SEAN A. LOCKE, † 1 MAGALIE CASTELIN,* DAVID J. MARCOGLIESE † and CATHRYN L. ABBOTT* *Aquatic Animal Health Section, Fisheries and Oceans Canada, Pacific Biological Station, 3190 Hammond Bay Road, Nanaimo, BC, Canada V9T 6N7, †Aquatic Biodiversity Section, Watershed Hydrology and Ecology Research Division, Water Science and Technology Directorate, Science and Technology Branch, Environment Canada, St. Lawrence Centre, 105 McGill, 7th Floor, Montreal, QC, Canada H2Y 2E7 Abstract Digeneans and cestodes are species-rich taxa and can seriously impact human health, fisheries, aqua- and agriculture, and wildlife conservation and management. DNA barcoding using the COI Folmer region could be applied for spe- cies detection and identification, but both ‘universal’ and taxon-specific COI primers fail to amplify in many flat- worm taxa. We found that high levels of nucleotide variation at priming sites made it unrealistic to design primers targeting all flatworms. We developed new degenerate primers that enabled acquisition of the COI barcode region from 100% of specimens tested (n = 46), representing 23 families of digeneans and 6 orders of cestodes. This high success rate represents an improvement over existing methods. Primers and methods provided here are critical pieces towards redressing the current paucity of COI barcodes for these taxa in public databases. Keywords: Cestoda, COI, Digenea, DNA barcoding, Platyhelminthes, Primers Received 18 February 2014; revision received 18 November 2014; accepted 21 November 2014 Introduction Digenea (flukes) and Cestoda (tapeworms) are among the most species-rich groups of parasitic metazoans. Although involved in major disease in humans and wild- life, the identity of pathogenic species is often poorly characterized (F€ urst et al. 2012; Th etiot-Laurent et al. 2013). Traditional morphology-based detection and iden- tification is often hampered by the small size and inac- cessibility within hosts in these organisms. A lack of distinctive morphological features in larval stages and even adults in some groups (e.g. see Kol a rov a 2007 and references therein) further confound identification to species level. DNA barcoding is a widely used tool for specimen identification to species level, but despite early success with ‘universal’ Folmer primers (Folmer et al. 1994) in a diverse range of animal taxa, including 14 flatworms (six digeneans and eight cestodes; Hebert et al. 2003), it was soon recognized that primer modification would be needed for reliable amplification of the COI barcode in many taxa (Hajibabaei et al. 2005). Primer development efforts in COI barcoding thus far have only involved a limited number of flatworm taxa and low representation of nucleotide variation. Moszczynska et al. (2009) developed degenerate prim- ers targeting the Folmer region for digeneans and ces- todes that were reasonably successful within a limited number of groups therein. These included the Strigeida (particularly Clinostomidae, Diplostomidae and Strigei- dae; Locke et al. 2010; Caffara et al. 2011; Locke et al. 2011) and isolated taxa within the Echinostomida (Psilos- tomidae; Bergmame et al. 2011) and Plagiorchiida (Hete- rophyidae and Paragonimidae; see Ferguson et al. 2012; L opez-Caballero et al. 2013). However, the success rate was only 5% in cestodes (1/20 specimens) and 40% in digeneans (231/572 specimens; see Table S1 (Supporting information) in Moszczynska et al. 2009). Moreover, important lineages of medical, veterinary or zoonotic importance, either were not tested (e.g. Hemiuridae, Bucephalidae, Proteocephalidea, Caryophyllidea) or failed to amplify (e.g. Taenia, Diphyllobothrium, Fasciolidae, Schistosomatidae). Correspondence: Niels Van Steenkiste, Fax: +1 250 756 7053; E-mail: niels_van_steenkiste@hotmail.com 1 Present address: Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON, Canada N1G 2W1 Reproduced with the permission of the Minister of Fisheries and Oceans Canada. © 2014 Her Majesty the Queen in Right of Canada Molecular Ecology Resources © 2014 John Wiley & Sons Ltd Molecular Ecology Resources (2015) 15, 945–952 doi: 10.1111/1755-0998.12358