Biocontrol Science and Technology, August 2005; 15(5): 481Á/495 A single-step multiplex PCR assay for the detection of European Peristenus spp., parasitoids of Lygus spp. TARA D. GARIEPY1Á3, ULRICH KUHLMANN2, TIM HAYE2, CEDRIC GILLOTT3, & MARTIN ERLANDSON1 1Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, SK, Canada, 2CABI Bioscience Switzerland Centre, Dele´mont, Switzerland, and 3Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada (Received 8 August 2004; returned 14 October 2004; accepted 13 November 2004) Abstract Lygus Hahn (Hemiptera: Miridae) are serious pests of agricultural and greenhouse crops throughout North America. In Europe, bivoltine Peristenus Fo¨rster (Hymenoptera: Braconidae) species have a significant impact on Lygus populations. Release and establishment of European P. digoneutis Loan in Lygus lineolaris (Palisot de Beauvois) populations in northeastern USA has renewed interest in the intended liberation of European parasitoids for Lygus control in Canada. Accurate identification of natural enemies is the cornerstone of biological control but conventional methods for identifying Peristenus species and estimating parasitism rates rely on tedious and time-consuming dissection and rearing methods. The present study describes species-specific PCR primers for three species of Peristenus, and the use of a multiplex PCR assay to detect P.digoneutis and P.stygicus Loan eggs and larvae from Lygus rugulipennis Poppius nymphs. Results indicate that the primers amplify uniquely sized, species-specific PCR products for the three species and are capable of detecting single eggs in parasitized nymphs within 3 days post-parasitism. Using a multiplex PCR assay, the primers maintain specificity and sensitivity, and allow detection of each of the three species in a single reaction. Although molecular diagnostics have previously been used in the identification of parasitoids and estimation of parasitism rates, this is the first time a single-step multiplex PCR protocol has been described. Keywords: Peristenus digoneutis, P. stygicus, P. pallipes, Lygus, biological control, molecular diagnostics, multiplex PCR Introduction Species of Lygus Hahn (Hemiptera: Miridae) are serious pests of crops grown in agricultural and greenhouse environments throughout North America. Feeding damage by Lygus nymphs and adults can cause flower bud and fruit abortion, side- shoot abscission, leaf tissue perforation and deformation or death of meristem tissue (Strong 1970; Broadbent et al. 2002). In North America, Lygus have historically been recorded as destructive pests (Craig & Loan 1984; Young 1986; Coulson 1987), and recent literature demonstrates their continued pest status on a variety of crops, Correspondence: M. Erlandson, Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, SK, Canada S7N 0X2. Tel: 1 306 956 7276. Fax: 1 306 956 7247. E-mail: erlandsonm@ agr.gc.ca ISSN 0958-3157 print/ISSN 1360-0478 online # 2005 Taylor & Francis Group Ltd DOI: 10.1080/09583150500086771 482 T. D. Gariepy et al. including wheat (Wise et al. 2000), oilseed flax (Wise & Lamb 2000), cotton (Layton 2000), canola (Boyd & Lentz 1999; Braun et al. 2001; Carcamo et al. 2002), alfalfa (Braun et al. 2001; May et al. 2003;) and strawberry (Rhainds & English-Loeb 2003). In North America, native Lygus parasitoids in the genus Peristenus Fo¨rster (Hymenoptera: Braconidae) are primarily univoltine, attacking only the first Lygus generation. As Lygus species are generally multivoltine, subsequent generations are not attacked by native parasitoids (Kuhlmann et al. 1998; Coutinot & Hoelmer 1999). Several bivoltine and multivoltine Peristenus species have a significant impact on populations of Lygus rugulipennis Poppius in Europe (Loan & Bilewicz-Pawinska 1973; Bilewicz-Pawinska 1976; Kuhlmann et al. 1998). Thus, the introduction of bivoltine European Peristenus species as classical biological control agents into Canada may fill an unoccupied niche and suppress native Lygus populations throughout the season. Attempts have been made since the 1960s to introduce and establish populations of P. digoneutis Loan and P. stygicus Loan in the USA (Coulson 1987). Peristenus digoneutis was released in the 1980s in New Jersey and has become established in Lygus lineolaris (Palisot de Beauvois) populations in alfalfa and strawberry fields in northeastern USA (Day 1999; Day et al. 1990, 2000; Tilmon & Hoffmann 2003). The success of this program has renewed interest in the intended liberation of European parasitoids for Lygus control in Canada. Accurate identification of potential natural enemies is the cornerstone of biological control, and methods that can definitively identify potential biological control agents are essential, especially when morphological variation among species is slight. Members of the genus Peristenus can be difficult to distinguish because species differences are small (Loan & Bilewicz-Pawinska 1973; Bilewicz-Pawinska & Pankanin 1974). As such, PCR-based methods for distinguishing between species would be advantageous for identifying adult Peristenus species and for detecting immature stages of Peristenus in parasitized Lygus nymphs. Recently, molecular techniques have been investigated for their utility in identifica- tion of some species of Peristenus (Tilmon et al. 2000; Erlandson et al. 2003; Ashfaq et al. 2004; Zhu et al. 2004). These molecular marker systems include PCR-based methods that potentially preclude the need for tedious and time-consuming dissection and rearing procedures. This would be advantageous as dissection provides no information as to which parasitoid species is present (Carignan et al. 1995), and rearing methods often require several months because of diapause prior to adult emergence and identification (Loan & Bilewicz-Pawinska 1973). Tilmon et al. (2000) developed molecular markers based on the cytochrome oxidase 1 (CO1) gene for P. pallipes Curtis, P. digoneutis, and P. conradi Marsh in order to assess parasitism rates in L. lineolaris in the field. Similarly, Erlandson et al. (2003) developed molecular markers based on sequences from the internal transcribed spacer (ITS) regions for P. digoneutis, P. stygicus, and P. pallipes. However, the molecular markers developed in both these studies amplify fragment sizes that are identical for all Peristenus species investigated, and therefore require the additional step of restriction enzyme digestion or additional PCRs in order to distinguish between species. Recently, Zhu et al. (2004) screened a collection of single, short oligonucleotide primers and selected several which produced PCR product patterns that distinguish between P. pallipes, Single-step multiplex PCR for detection of European Peristenus spp. 483 P. pseudopallipes and P. howardi without the need for additional digestion with restriction enzymes. Multiplex PCR involves the use of multiple species-specific primer pairs in a single reaction to amplify unique fragment sizes for different DNA targets, allowing samples to be screened for several organisms simultaneously (Bej et al. 1991; Henson & French 1993). Although this type of system has yet to be implemented in host- parasitoid studies to identify multiple parasitoid species, multiplex PCR is routinely used in the diagnosis of medical and veterinary diseases (Rossiter & Caskey 1993; Zarlena & Higgins 2001; Favia et al. 2003) as well as in the detection of plant pathogens (Hamelin et al. 1996; Cullen et al. 2000; Taylor et al. 2001; Bertolini et al. 2003; Gariepy et al. 2003; Lopez et al. 2003). The purpose of this study was to design PCR primers for P. digoneutis, P. stygicus, and P. pallipes that produce unique fragment sizes, and that could be applied in multiplex to improve their diagnostic utility. Two of these species, P. digoneutis and P. stygicus, have been released as biological control agents for Lygus spp. in the USA (Coulson 1987; Day 1999), and are currently being considered for release in Canada. Peristenus pallipes, however, is an holarctic species occuring in Europe and North America (Loan 1965, 1974; Loan & Bilewicz-Pawinska 1973; Hormchan 1977) where it is known to parasitize several mirid species (Craig 1963; Loan 1965; Bilewicz- Pawinka 1977, 1982). A multiplex PCR protocol for these species would be particularly useful in North America and Europe for detecting immature stages of Peristenus in parasitized Lygus nymphs in order to determine Peristenus-host associations, which are particularly important for non-target risk assessment studies. Materials and methods Acquisition of voucher specimens Cocoons of the Lygus parasitoids P. digoneutis and P. stygicus were reared from L. rugulipennis nymphs collected from geographically dispersed red clover (Trifolium pratense L.) and camomile (Matricaria recutita L.) fields in Schleswig-Holstein, northern Germany. Peristenus pallipes cocoons were obtained from Liocoris tripustulatus Fabricius (Hemiptera: Miridae) and Closterotomus norvegicus Gmelin (Hemiptera: Miridae) collected from stinging nettles (Urtica dioica L.) in the same region. Emerged adults were put in 95% ethanol and identified based on morphological characteristics by H. Goulet (Systematic Entomology Unit, Agriculture and Agri-Food Canada, Ottawa, Canada). Twenty identified specimens for each of the Peristenus species investigated were randomly selected from collections made in different habitats and locations in northern Germany. These specimens were used as voucher samples for molecular