This article was downloaded by: [USDA National Agricultural Library] On: 1 October 2008 Access details: Access Details: [subscription number 790740294] Publisher Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Biocontrol Science and Technology Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713409232 PCR-based, rapid diagnosis of parasitism of Lygus spp. (Hemiptera: Miridae) by Peristenus relictus (Hymenoptera: Braconidae) M. C. Bon a; K. Hoelmer a; D. Coutinot a; N. Ramualde a a USDA-ARS European Biological Control Laboratory, Campus International de Baillarguet, Montferrier sur Lez, France First Published:2008 To cite this Article Bon, M. C., Hoelmer, K., Coutinot, D. and Ramualde, N.(2008)'PCR-based, rapid diagnosis of parasitism of Lygus spp. (Hemiptera: Miridae) by Peristenus relictus (Hymenoptera: Braconidae)',Biocontrol Science and Technology,18:5,505 — 516 To link to this Article: DOI: 10.1080/09583150802005149 URL: http://dx.doi.org/10.1080/09583150802005149 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Biocontrol Science and Technology, Vol. 18, No. 5, 2008, 505Á516 PCR-based, rapid diagnosis of parasitism of Lygus spp. (Hemiptera: Miridae) by Peristenus relictus (Hymenoptera: Braconidae) M.C. Bon, K. Hoelmer*, D. Coutinot and N. Ramualde USDA-ARS European Biological Control Laboratory, Campus International de Baillarguet, Montferrier sur Lez, France (Received 7 December 2007; returned 16 January 2008; accepted 20 February 2008) Several species of Lygus (Heteroptera: Miridae: Mirini) are serious crop pests in North America where their parasitism rate by native nymphal parasitoids is generally lower than in Europe. Peristenus relictus (Ruthe) (formerly P. stygicus Loan) (Hymenoptera: Braconidae: Euphorinae) is the predominant nymphal parasitoid of several Lygus spp. in the warm Mediterranean region and has been a candidate for introduction against Lygus hesperus Knight and L. lineolaris (Palisot de Beauvois) in the southern US. We report a rapid, sensitive, and specific PCR-based assay for diagnosis of P. relictus immature stages within Lygus nymphs that entails three steps: DNA extraction, PCR of the partial mitochondrial COI gene, and agarose gel electrophoresis. The PCR-based methodology is species-specific because the target DNA of other sympatric, congeneric species was not amplified with use of the primers developed for P. relictus diagnosis. The sensitivity of the PCR method, assessed through spike tests, was established by the detection of a ratio of 1:10,000 P. relictus DNA to Lygus DNA. Molecular diagnosis of parasitism of field collected nymphs is achievable in one day, eliminating the need to rear nymphs to obtain adult parasitoids for morphological identification. Keywords: Peristenus relictus; P. stygicus; Peristenus spp.; Lygus spp.; PCR; parasitoid; natural enemy; cytochrome oxidase I Introduction Lygus (Heteroptera: Miridae: Mirini) is a genus of 50 species of plant bugs distributed throughout North America, Europe and Asia (Schwartz and Foottit 1998). Several species, including Lygus hesperus Knight and Lygus lineolaris (Palisot de Beauvois), are serious pests of cotton, Gossypium hirsutum L., seed crops, and various fruit and vegetable crops in North America (Nordlund 2000). Lygus species are attacked by numerous predators, parasitoids, and pathogens in North America and Europe (Hedlund 1987; Day 1999; Downloaded By: [USDA National Agricultural Library] At: 17:19 1 October 2008 Ruberson and Williams 2000). Because parasitism by native species in North America is generally low, especially after the first generation of Lygus, more effective nymphal parasitoids in the genus Peristenus (Hymenoptera: Braconidae: Euphorinae) that attack exotic Lygus species have been imported into North America on several occasions (Ruberson and Williams 2000). The European species Peristenus digoneutis Loan was released in the eastern US during the 1980s and became established on L. lineolaris, resulting in reductions in L. lineolaris populations in the northeastern US on alfalfa (Medicago sativa L.), a preferred host plant (Day, Hedlund, Saunders, and Coutinot 1990; Day 1996). The successful establishment of P. digoneutis has resulted in continued efforts to establish Peristenus species in other regions of North America where indigenous nymphal *Corresponding author. Email: [email protected] ISSN 0958-3157 print/ISSN 1360-0478 online # 2008 Taylor & Francis DOI: 10.1080/09583150802005149 http://www.informaworld.com 506 M.C. Bon et al. parasitoids do not significantly impact Lygus populations, such as in California (Pickett et al. 2007). One of several species of nymphal parasitoids that attack Lygus species in Europe, Peristenus relictus (Ruthe) [formerly P. stygicus Loan (Varis and van Acherberg 2001)] has been regarded as a promising agent for reducing populations of US Lygus species, especially in southern regions (Drea, Dureseau, and Rivet 1973) because of its predominance in warm southern parts of Europe (Coutinot and Hoelmer 1999; Coutinot, Hoelmer, and Pickett 2005), and it has been released recently on both coasts of the US in California and in New Jersey (Pickett et al. 2007; Hoelmer et al. 2008). Because natural populations of Lygus species and their parasitoids tend to be patchy in southern Europe, and because several different Peristenus species and hyperparasitoids occur together at many locations, it has been necessary for foreign collectors to devote considerable time and effort to field collections in order to obtain sufficient numbers of the desired species of parasitoids for shipment to cooperators in the US (Coutinot and Hoelmer 1999; Coutinot et al. 2005). Given that plant protection regulations governing importation of foreign agents are becoming increasingly stringent, methods for quickly verifying the identity of live biological control agents for prompt shipment to cooperators are not only very useful, they are increasingly important to comply with international and regional regulatory standards for exportation, importation, and release of biological control agents (OECD 2004; FAO 2005). Detailed information that characterises these agents is often a requisite for compliance (Coutinot 2006). Monitoring the presence and identity of Lygus parasitoids in host populations traditionally relied on dissections of nymphs or rearing adult parasitoids from parasitised nymphs. Dissections provide good estimates of actual parasitism levels, but do not allow the identification of parasitoid species because parasitoid eggs and larvae lack distinguishing morphological features (Day 1994). Rearing adults cannot be completed until the following spring following an overwintering diapause, and is often subject to high levels of mortality. Both procedures are time consuming and labour intensive. This has hindered the understanding of patterns of parasitism in the field, the evolution of parasitoid populations (such as newly established biological control agents), and the interaction of native and exotic Peristenus species with overlapping geographic and host ranges. Such studies and mass field collections have benefited from the development of DNA-based techniques for detecting and identifying species of parasitoids in field samples. The sensitivity and specificity of the polymerase chain reaction (PCR) Downloaded By: [USDA National Agricultural Library] At: 17:19 1 October 2008 techniques for detection of trace quantities of target DNA in heterogeneous samples is ideal for identifying parasitoids present as immature stages within their hosts. This method was first applied to parasitoids of Lygus by Tilmon, Danforth, Day, and Hoffmann (2000) to detect and distinguish among the introduced P. digoneutis and several indigenous Peristenus species attacking L. lineolaris in the northeastern US and their host, L. lineolaris. These authors selected the mitochondrial gene cytochrome oxidase I (COI) for their two-step assay because of its demonstrated interspecific variability but moderate intraspecific variability in many animal phyla, including Hymenoptera (Simon et al. 1994; Dowton and Austin 2001). Mowry and Barbour (2004) extended the use of this assay to include additional North American Peristenus species. Erlandson et al. (2003) used primers designed from the nuclear rRNA gene ITS2 (internal transcribed spacer) to distinguish among P. digoneutis, P. relictus and the indigenous P. pallipes (Curtis) present within three Lygus species occurring on Canadian prairies. Parasitism levels detected by