A Single-Step Multiplex PCR Assay for the Detection of European Peristenus Spp., Parasitoids of Lygus Spp

Biocontrol Science and Technology, August 2005; 15(5): 481/495

A single-step multiplex PCR assay for the detection of European Peristenus spp., parasitoids of Lygus spp.

TARA D. GARIEPY13, 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 identification 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 analysis, and were kept in 95% ethanol.

DNA extraction Peristenus adults preserved in 95% ethanol were rinsed in sterile, distilled water, and placed individually in 1.5-mL centrifuge tubes. Individual Peristenus adults were homogenized using disposable pestles (Kimble Kontes, Vineland, NJ, USA) in 250 mL of Lifton buffer [0.2 M sucrose, 50 mM ethylene-diaminetetraacetic acid, 100 mM

Tris /HCl (pH 7.5), and 0.5% sodium dodecyl sulfate], and incubated for 2 h at room temperature. Proteinase K (1.25 mLof20mg/mL) was added, and samples were 484 T. D. Gariepy et al. incubated for 4 h at 658C. Samples were put on ice for 30 min following the addition of 35 mL of 5 M potassium acetate, and centrifuged for 20 min at 20 000/g.

The supernatant was extracted once with 200 mL of phenol/chloroform/isoamyl alcohol (25:24:1), and once with 200 mL chloroform/isoamyl alcohol (24:1). The aqueous phase was precipitated with two volumes of cold 95% ethanol by overnight incubation at /208C. Precipitated DNA was pelleted by centrifugation (30 min at 20 000/g), washed with cold 70% ethanol, and air-dried for 15 min. DNA samples were resuspended in 50 mLTris/EDTA (pH 7) with 0.5 mL of 10 mg/mL RNase A.

DNA sequencing and primer design The oligonucleotide primers inDNA44 (5?-TCCTCCGCTTATTGATATGC-3?) and inDNA45 (5?-GGAAGTAAAAGTCGTAACAAGG-3?) (Nucleic Acid / Protein Service, University of British Columbia, Vancouver, BC, Canada) were used in PCR to amplify conserved rRNA gene sequences and ITS regions of nuclear DNA from voucher specimens. PCR conditions were as follows: 25-mL reactions contained

2.5 mLof10/ reaction buffer, 2.5 mM MgCl2, 0.25 mM dNTPs, 0.5 mM of each primer, 1 U of Ta q DNA polymerase (Invitrogen, Burlington, ON, Canada), and approximately 20 ng of DNA template. PCR reactions were carried out in a Strategene Robocycler thermocycler (LaJolla, CA, USA) with an initial denaturation at 948C for 120 s, followed by 30 cycles at 948C for 60 s, 558C for 60 s, and 728C for 90 s. A final extension period at 728C for 5 min followed the 30 cycles. PCR products were electrophoresed on 1% agarose gels containing 0.5 mg/mL ethidium bromide at

75 V for 45/60 min and products were visualized by UV transillumination. PCR † products were directly cloned into pGem / T Easy vectors (Promega, Madison, WI, USA) using the manufacturer’s protocol. Cloned PCR products were sequenced at the DNA Services Laboratory, Plant Biotechnology Institute (NRCC, Saskatoon, SK, Canada) using universal primers to sequence from both ends of the cloned insert. DNA sequence data were assembled and aligned using SeqMan (DNAStar Inc., Madison, WI, USA) and Vector NTI (InfoMax, Fredrick, MD, USA). Areas of sequence variation determined by visual inspection of the sequence data were used to design primers specific for P. digoneutis, P. stygicus and P. pallipes.

Specificity of Peristenus species-specific primers The specificity of the primer pairs designed for P. digoneutis, P. stygicus and P. pallipes was evaluated by amplifying 20 voucher specimens of each species using each of the primer pairs. Adult Lygus DNA was used as a negative control to ensure that the primer sets did not amplify Lygus sequences. Amplification of DNA was performed in a Stratagene Robocycler thermocycler using the same reagents as above, except the primer pairs used were Per R1 (a presumed universal Peristenus primer) in combination with either dig F1096 for P. digoneutis, sty F1230 for P. stygicus, or pal F517 for P. pallipes. Primer concentrations were as described above. Amplification conditions were: initial denaturation at 948C for 120 s, followed by 35 cycles of 948C for 45 s, 548C for 45 s, and 728C for 60 s. A final extension period of 5 min at 728C followed. Single-step multiplex PCR for detection of European Peristenus spp. 485

Multiplex PCR assay and detection sensitivity In order to enhance the diagnostic utility of Peristenus spp. primers, a multiplex PCR protocol was tested. For this approach, the reagents mentioned above were used with the following modifications: 0.2 mM dig F1096, 0.2 mM sty F1230, 0.2 mM pal F517 and 0.4 mM Per R1 were combined in the reaction mixture. Amplification conditions were as described above for specificity testing of the Peristenus species- specific primers. DNA from adult P. digoneutis, P. stygicus, and P. pallipes voucher specimens was serially diluted 10-fold, from 20 to 0.02 ng, and used in a multiplex PCR assay to determine the sensitivity of the primers.

Detection of immature stages of Peristenus in parasitized Lygus nymphs To determine the ability of the molecular markers to detect immature stages of the biological control agents, P. digoneutis and P. stygicus inside their host, L. rugulipennis nymphs were parasitized in the laboratory. Between 20 and 25 experienced, mated P. digoneutis and P. stygicus females (5/10 days old) were provided with second or third instar Lygus nymphs in a 5-cm diameter Petri dish. Each wasp was transferred into a Petri dish containing five nymphs, and attacks were observed. When an attack occurred, the nymph was immediately removed to prevent superparasitism. In total, 100/125 nymphs attacked by P. digoneutis and

100/125 nymphs attacked by P. stygicus were obtained. Attacked nymphs were either placed immediately in 95% ethanol (0 h) or reared for fixed periods at approximately 229/28C, 16L:8D photoperiod, and 50% RH in rearing cylinders with sprouting potatoes, lettuce, and green beans. Rearing periods were chosen to cover the documented range of developmental stages of P. digoneutis and P. stygicus within Lygus spp. nymphs (Drea et al. 1973; Carignan et al. 1995). Rearing periods included: 3 days (egg), 6 days (first instar), 9 days (second instar), and 12 days (third instar). After the specified rearing period, attacked nymphs were placed in vials containing 95% ethanol. DNA was extracted from the attacked nymphs (20 nymphs per rearing period for each Peristenus species), and amplified using the multiplex PCR assay.

Application of the multiplex PCR assay to field-collected Lygus nymphs Lygus rugulipennis nymphs were collected from sweep-net samples from three different sites [two camomile, Matricaria recutita L. (GPS coordinates: N 54824.757? E 010809.919?;N54825.484? E 010801.349?) and one red clover, Trifolium pratense L. (GPS coordinates: N 54805.191? E 010804.353?)] in Schleswig-Holstein, Germany. From each site, 300 late-instar Lygus nymphs were collected: 100 nymphs from each site were reared, 100 from each site were dissected, and 100 from each site were used for molecular analysis using the multiplex PCR assay. DNA extraction and amplification using the multiplex assay was as described above, and PCR products from individual nymphs were electrophoresed at 65 V for 60 min on 1% agarose gels containing 0.5 mL/mL ethidium bromide. Parasitism and/or species composition based on rearing, dissection and molecular analysis were compared. 486 T. D. Gariepy et al.

Results DNA sequencing and primer design DNA sequences were obtained for cloned PCR products generated using inDNA44 and inDNA45 from at least four individuals (one to three clones per individual) for each of the Peristenus species investigated. Sequences were obtained for the ITS1 regions of P. digoneutis (GenBank accession no. AY605234) and P. stygicus (GenBank accession no. AY605233). ITS1 and ITS2 sequences were obtained for P. pallipes (GenBank accession no. AY608602). Sequences were aligned to analyse sequence variation among species. Several areas of variation were identified, allowing the design of species-specific primers for each species (Table I). Primer sets consisted of a species-specific reverse primer (dig F1096, sty F1230, or pal F517) used in combination with a genus-specific forward primer (Per R1) common to all three species investigated. The combination of Per R1 with dig F1096 for P. digoneutis, with sty F1230 for P. stygicus, and with pal F517 for P. pallipes produced PCR products of 515, 330, and 1060 bp, respectively (Figure 1).

Specificity of Peristenus species-specific primers Each primer set selectively amplified the DNA of the species for which it was designed, and yielded the expected product sizes when using the PCR conditions described above. Species-specific amplification was consistent for all voucher specimens tested (20 specimens per species), and a representative for each species is shown (Figure 2). In addition to the 20 voucher specimens of P.pallipes from European mirid populations, the P. pallipes primers (Per R1 and palF517) also amplified P. pallipes (25 individuals) obtained from three widely dispersed geographic regions in western Canada. All samples yielded the expected 1060-bp PCR product for P. pallipes (Erlandson, Ashfaq and Gariepy, data not shown), indicating that the primers designed for European P. pallipes were also specific for representative North American populations. None of the primer sets amplified Lygus DNA.

Multiplex PCR assay and detection sensitivity Combining the three species-specific reverse primers (dig F1096, sty F1230, pal F517) and the genus-specific forward primer (Per R1) in a multiplex PCR protocol distinguished each of the three species, and amplified the appropriate species-specific fragment; 515 bp for P. digoneutis, 330 bp for P. stygicus, and 1060 bp for P. pallipes (Figure 3). These results were consistent with those obtained using the species- specific primer sets individually (Figure 2). When used in multiplex, the lower limit of

Table I. Peristenus species-speciﬁc primers, their respective sequences, and fragment sizes when used in combination with a genus-speciﬁc reverse primer (Per R1).

Species Primer Primer sequence Fragment size

Peristenus spp. Per R1 5?-ACAAGGTTTCCGTAGGTG-3?/ P. digoneutis dig F1096 5?-GAACATAAAAACTTCTTCTACGC-3? 515 P. stygicus sty F1230 5?-CAGGTAGAGATACATGGCTGT-3? 330 P. pallipes pal F517 5?-TAAACTTTGGCCAGATAAATG-3? 1060 Single-step multiplex PCR for detection of European Peristenus spp. 487

Figure 1. Diagrammatic representation of the ITS region of P. digoneutis, P. stygicus and P. pallipes, showing location of species-speciﬁc primers. detection appears to be 0.2 ng for P. digoneutis primers, and 0.02 ng for P. stygicus and P. pallipes primers (Figure 4).

Detection of immature stages of Peristenus in parasitized Lygus nymphs Using the multiplex PCR protocol, primers successfully detected Peristenus eggs and larvae in L. rugulipennis nymphs 3, 6, 9, and 12 days post-parasitism. For these periods, detection of both parasitoid species was consistent among Lygus nymphs (20 attacked nymphs per rearing period for P. digoneutis and P. stygicus), with 90/100% of observed attacks resulting in confirmed parasitism; representa- tives for each rearing period are presented (Figure 5). Detection of Peristenus within nymphs placed in 95% ethanol immediately after an attack (0 h) was inconsistent. Faint bands of the expected size were occasionally present, but required 4/5 times more DNA template than was required for samples from the other post-parasitism periods. The primer sets did not amplify Lygus DNA.

Figure 2. Diagnostic PCR using species-speciﬁc primers for P.digoneutis (dig F1096 and Per R1), P.stygicus (sty F1230 and Per R1), and P.pallipes (pal F517 and Per R1). D, S, and P designate P.digoneutis, P.stygicus, and P. pallipes, respectively. M, 100-bp marker (Invitrogen); NEG, negative control (no DNA). 488 T. D. Gariepy et al.

Figure 3. Diagnostic multiplex PCR of P.digoneutis, P.stygicus and P. pallipes and Lygus adult DNA (NEG, negative control) using a combination of dig F1096, sty F1230, pal F517, and Per R1 in each reaction. M, 100-bp marker (Invitrogen).

Application of the multiplex PCR assay to field-collected Lygus nymphs Results from rearing, dissection, and molecular analysis of Lygus nymphs collected from three sites are summarized in Table II. For each site, the percent parasitism based on molecular analysis, rearing, and dissection were not significantly different 2 2 2 (Chi-square test, site 1: x /0.36, P B/0.05; site 2: x /1.5, P B/0.05; site 3: x /1.02,

P B/0.05). Multiplex PCR results indicated the occurrence of multiparasitism in some Lygus nymphs, as demonstrated by the presence of P. digoneutis and P. stygicus specific PCR products in single nymphs (Figure 6). When the incidence of multiple parasitism detected by PCR analysis was taken into account, the relative proportions of P. digoneutis and P. stygicus in the overall species composition were similar when determined by rearing and multiplex PCR.

Discussion The PCR primers developed for P.digoneutis, P.stygicus,andP.pallipes are specific and sensitive when used individually or in multiplex with DNA extracted from Peristenus adults or from parasitized Lygus nymphs. When used individually or in multiplex, the primers yield unique fragment sizes for each species, allowing the identification of

Figure 4. Multiplex PCR using 20, 2, 0.2, and 0.02 ng of DNA template from P. digoneutis, P. stygicus, and P. pallipes adults. M, 100-bp marker (Invitrogen). Single-step multiplex PCR for detection of European Peristenus spp. 489

Figure 5. Multiplex PCR of Lygus nymphs parasitized by P.digoneutis and P.stygicus and killed 0 h and 3, 6, 9, and 12 days after attack. NEG 1 and NEG 2 are negative controls containing no DNA and Lygus DNA, respectively. M, 100-bp marker (Invitrogen).

Peristenus eggs (3 days post-parasitism), larvae, and adults based on the size of the PCR fragment generated. The inability of molecular markers to detect newly laid parasitoid eggs in their host is not uncommon. Primers developed by Amornsak et al. (1998) for detection of Trichogramma australicum Girault in Helicoverpa armigera Hubner and H. punctigera Wallengren were unable to detect the parasitoid immediately after oviposition; PCR products were only visible 12 h after parasitism. Within the first 24 h after oviposition, Peristenus eggs swell and further embryonic development occurs (Carignan et al.

1995). Presumably within 12/24 h post-parasitism, our multiplex PCR system would be capable of detecting eggs, but this remains to be tested. The fact that electrophoresis of PCR product from the 3-day post-parasitism stage produced very strong bands indicates that this is certainly not the lower limit of detection. Amplification of serial dilutions of adult Peristenus DNA suggests that the lower limit of detection is between 0.2 and 0.02 ng for P.digoneutis and approximately 0.02 ng for P. stygicus. However, this represents the ability of the primers to detect pure Peristenus DNA, and may not reflect the minimum quantity detectable in samples containing parasitoid and host DNA. In order to determine the lower-limit of parasitoid detection within Lygus nymphs, it would be prudent to expand these experiments to include 12, 24, and 48 h post-parasitism periods. In addition to being used previously to distinguish between Peristenus species (Tilmon et al. 2000; Erlandson et al. 2003; Zhu et al. 2004), molecular diagnostic methods have been used to separate morphologically similar parasitoid species using PCR or PCR followed by restriction enzyme digestion. For example, these techniques have been used by Silva et al. (1999) and Stouthamer et al. (1999) to distinguish between species of Trichogramma Westwood (Hymenoptera: Trichogrammatidae), by Zhu and Greenstone (1999) and Zhu et al. (2000) to detect different species and strains of Aphelinus Dalman (Hymenoptera: Aphelinidae) in Diuraphis noxia Mordvilko (Homoptera: Aphididae), and by Ratcliffe et al. (2002) to detect eight pteromalid (Hymenoptera: Pteromalidae) species parasitizing Musca domestica L. and Stomoxys calcitrans L. However, none of these studies use techniques that allow detection and identification of more than one species in a single reaction. 490 .D aip tal. et Gariepy D. T.

Table II. Total percent parasitism and parasitoid species composition based on molecular analysis, rearing, and dissection of ﬁeld-collected Lygus rugulipennis nymphs from northern Germany. From each site, 100 nymphs were tested per method (i.e. 100 for molecular analysis, 100 for rearing, and 100 for dissection).

Site 1 Site 2 Site 3

Molecular Rearing Dissections Molecular Rearing Dissections Molecular Rearing Dissection

Total% parasitism: 68 68 62 70 70 58 60 67 56 Species composition:

P. digoneutis (%) 53 66 / 83 100 / 50 58 / P. stygicus (%) 29 34 / 60/ 47 42 / Multiparasitism (%) 18 / / 11 / / 3 / /

/, information not available using this technique. Single-step multiplex PCR for detection of European Peristenus spp. 491

Figure 6. Application of the multiplex PCR assay to ﬁeld collected Lygus rugulipennis nymphs. PCR products speciﬁc to P. digoneutis (515 bp) and P. stygicus (330 bp) are indicated by D and S, respectively. Lanes with no bands indicate nymphs that are not parasitized (NP), and lanes showing both 515- and 330-bp products simultaneously indicate multiparasitism (DS). M, 100-bp marker (Invitrogen).

Lopez et al. (2003) list multiplex PCR methods among the innovative tools currently used in pathogen diagnosis based on the sensitivity of the technique and the ability to simultaneously detect different DNA targets in a single reaction. In insect parasitology, molecular diagnostic tools incorporating multiplex PCR have been used to identify different mosquito species and strains that vector malaria (Kengne et al. 2001; Phuc et al. 2003), to detect animal viruses and plant pathogens in insect vectors (Wilson & Chase 1993; Rodrigues et al. 2003), to identify microsporidian infection in ants (Valles et al. 2002) and bacterial infection in insect cadavers (Bourque et al. 1993). However, the implementation of multiplex assays to detect and identify insect parasitoid species within their hosts has not previously been reported. The application of the multiplex PCR protocol to field-collected Lygus samples demonstrates the applicability and potential of this method for screening field- collected samples for the presence of Peristenus spp. Percent parasitism by rearing, dissection and molecular analysis were not significantly different; however, molecular methods provide additional information pertaining to multiple parasitism. Such information is not evident by rearing or dissection, but could become important to assess inter-specific competition between indigenous and exotic biological control agents in target and non-target hosts. Furthermore, the use of a multiplex PCR assay for Peristenus species improves upon the conventional and molecular techniques currently used to identify Peristenus spp. and quantify parasitism levels. It allows rapid and accurate identification of multiple Peristenus species and eliminates mortality issues encountered during the rearing process. Furthermore, unlike dissections, the multiplex assay allows the determination of species composition, and unlike rearing, it does so without the time-delay due to the lengthy diapause period of the parasitoid prior to adult emergence. This is a novel approach that could be valuable in insect biological control programs, particularly for pre-release and post-release studies on potential biological control agents. Pre-release studies, including the determination of ecological and physiological host ranges, are strongly recommended by van Lenteren et al. (2003) prior to importation and release of exotic parasitoids for inundative biological control programs. Furthermore, recommendations by Louda et al. (2003) suggest that pre-release field research generates better ecological data for biological control agents in order to reduce the risk of non-target effects. Insect molecular systematics has supplemented and improved the value of morphological and ecological data (Caterino et al. 2000), 492 T. D. Gariepy et al. and in fact, molecular data may complement and enhance ecological and physiological studies on parasitoid host range. The single-step multiplex PCR assay described here would be useful in conducting pre-release studies in the area of origin; nymphs can be screened to determine

Peristenus/ host associations as well as the distribution of Peristenus species. In addition, following introduction, molecular tools could facilitate post-release monitoring studies. In fact, recent post-introduction studies in New York State using molecular markers for the COI gene have been used to map the distribution of P. digoneutis in Lygus populations in alfalfa and strawberry fields (Tilmon et al. 2000; Tilmon & Hoffmann 2003). However, as previously mentioned, the markers used in these studies produce identical fragment sizes for each species and require additional processing to obtain species-specific identifications. The use of the single-step multiplex PCR protocol described here could facilitate similar post-introduction studies in the future. Furthermore, it would allow samples to be screened for the introduced Peristenus species and indigenous North American P. pallipes simultaneously to assess establishment, distribution, potential displacement of native P. pallipes, and realized host range in the area of introduction.

Acknowledgements This work was supported by the Biocontrol Network (Canada), CABI Bioscience, and Agriculture and Agri-Food Canada. We thank Dr H. Goulet (AAFC, ECORC, Ottawa, ON, Canada) for voucher specimen identiﬁcation, and Dr P. Mason (AAFC, ECORC, Ottawa, ON, Canada) for useful discussions and advice.

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