A single-step multiplex PCR assay for the detection of EuropeanPeristenusspp., parasitoids ofLygusspp.

Abstract

Lygus Hahn (Hemiptera: Miridae) are serious pests of agricultural and greenhouse crops throughout North America. In Europe, bivoltine Peristenus Fö 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.

A molecular key to the common species of Trichogramma of the Mediterranean region

“…In such a multiplex PCR, for each species a specific primer is designed, so that when combined in a single reaction with a complementary universal primer, all the species can be identified by the size of the resulting PCR product alone (Gariepy et al 2005;Davies et al 2006). Development of such multiplex primers is very feasible but the choice of primers depends on the species that are present.…”

Does host plant influence parasitism and parasitoid species composition inLygus rugulipennis? A molecular approach

“…Lygus rugulipennis DNA was extracted from individual L. rugulipennis nymphs and amplified using a multiplex PCR assay for P. digoneutis, P. relictus and P. pallipes (Gariepy et al, 2005). As parasitized nymphs provide a positive PCR result and unparasitized nymphs provide a negative result, amplification of DNA from individual nymphs was used to calculate the parasitism level at each site.…”

“…Molecular methods to screen host insects for the presence of multiple parasitoid species can be used to estimate parasitism level, parasitoid species composition and incidence of multiparasitism in mirid populations (Gariepy et al, 2005;Greenstone, 2006;Gariepy et al, 2007). Thus, molecular methods could be used to screen L. rugulipennis nymphs collected from different host plants to investigate potential host plant-parasitoid associations.…”

“…Molecular methods would facilitate such studies because they provide rapid and accurate detection of parasitoids within host insects and are not plagued by the host and parasitoid mortality encountered in rearing. A single-step multiplex PCR assay developed for the simultaneous detection of P. digoneutis, P. relictus and P. pallipes (see Gariepy et al, 2005) was used to screen DNA extracted from L. rugulipennis nymphs collected in red clover (Trifolium pratense L.; Fabaceae), alfalfa (Medicago sativa L.; Fabaceae) and chamomile (Matricaria recutita L.; Asteraceae) in two European ecoregions (see DMEER, 2000). The parasitism levels and parasitoid species composition in the host populations from these different plant species were compared to investigate plant species-host-parasitoid interactions.…”

Taxon-specific multiplex-PCR for quick, easy, and accurate identification of encyrtid and aphelinid parasitoid species attacking soft scale insects in California citrus groves

“…This technique involves the use of one PCR primer located in a region shared by several taxa (a universal primer) alongside several opposing primers, each of which lies in a region that is specific to only one of the taxa under consideration, and which when it amplifies, produces a PCR product of a size that is unique to that taxon. As a result, taxa can be identified directly following gel electrophoresis of the PCR product (e.g., Cornel et al 1996;Fritz et al 2004;Gariepy et al 2005;Rugman-Jones et al 2009b).…”