Development of species-specific PCR assays for detection and identification of parasitoids associated with Lygus plant bugs
1Agriculutre and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, SK, Canada
2Dept. Biology, University of Saskatchewan, Saskatoon, SK, Canada and
3CABI Europe-Switzerland, Delémont, Switzerland
Correspondence: erlandsonm@agr.gc.ca
Lygus plant bugs (Hemiptera: Miridae) are hosts to an array of primary parasitoids which in turn serve as hosts for hyperparasitoids. Members of the genus Peristenus (Hymenoptera: Braconidae) are key primary parasitoids in Lygus spp. in North America and Europe. In Europe Peristenus digoneutis and P. relitcus have a significant impact on Lygus rugulipennis and have been released in North America in a classical biological control approach against mirid pest species in various crop habitats. The traditional approaches to estimating parasitism in Lygus spp. populations include dissection and rearing of field collected nymphs. Although the dissection approach yields relatively quick results, collection of species composition data for the parasitoids is virtually impossible due to the lack of diagnostic morphological characters for Peristenus spp. larval stages. Rearing is a time consuming and expensive procedure taking up to 6–9 months to break pupal diapause and for adult eclosion and identification to species based on morphological characters. The rearing approach also suffers from the loss of information due to host and parasitoid mortality during the initial rearing period and overwintering pupal parasitoid mortality. Additionally, the recently described species in the P. pallipes complex are difficult for the non-expert to identify even based on adult characters. Thus we have undertaken the development of sensitive and species-specific DNA markers for the detection and identification of key Peristenus species associated with Lygus pests. A one-step multiplex PCR system based on nuclear rRNA - ITS sequences has been developed and used to detect and distinguish P. digoneutis, P. relictus and members of the P. pallipes complex (Gariepy et al., 2005, Biocontrol Science and Technology 15: 481–495). The multiplex PCR assay was validated in a comprehensive study of parasitism in L. rugulipennis populations. Subsequently the multiplex PCR assay has been used in various applications including: ecological host-range testing for P. digoneutis and P. relictus in their area of origin; detecting the establishment P. digoneutis and P. relictus in areas of recent releases; and tracking the dispersal of these species from areas of original release. For example, the multiplex PCR system was used to identify Peristenus parasitoid species associated with mirid nymphs collected in southern Ontario, including a confirmation of P. digoneutis establishment. A number of parasitoid larvae recovered from these mirid nymphs could not be identified using the Peristenus multiplex PCR assay; however, the use of universal PCR primers for nuclear rRNA - ITS2 regions and sequencing of the PCR products generated identified these as Leiophron uniformis another braconid parasitoid. Further sequence analysis of nuclear rRNA - ITS DNA sequences of geographic populations of the P. pallipes complex and other North American Peristenus species indicates that the P. pallipes complex PCR primer set from the multiplex PCR assay will detect most North American species attacking Lygus spp. Finally, to more completely address the potential for species-specific PCR primers to assist in the investigation of host-parasitoid associations, PCR primer sets have been developed for the associated Mesochorus spp. hyperparasitoids. Sequence analysis of Mesochorus spp. from European mirid populations from different geographic locations and habitats suggest a complex of strains or subspecies of Mesochorus attack specific primary parasitoids associated with host mirid species that utilize similar habitats.
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