A molecular diagnostic tool for the preliminary assessment of host-parasitoid associations in biological control programmes for a new invasive pest.

Evaluation of host-parasitoid associations can be tenuous using conventional methods. Molecular techniques are well placed to identify trophic links and resolve host-parasitoid associations. Establishment of the highly invasive brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), outside Asia has prompted interest in the use of egg parasitoids (Hymenoptera: Scelionidae) as biological control agents. However, little is known regarding their host ranges. To address this, a DNA barcoding approach was taken wherein general PCR primers for Scelionidae and Pentatomidae were developed to amplify and sequence >500-bp products within the DNA barcoding region of the cytochrome oxidase I (COI) gene that would permit the identification of key players in this association. Amplification of DNA from Pentatomidae and Scelionidae was consistent across a broad range of taxa within these families, and permitted the detection of Scelionidae eggs within H. halys 1 h following oviposition. In laboratory assays, amplification and sequencing of DNA from empty, parasitized eggs was successful for both host (100% success) and parasitoid (50% success). When applied to field-collected, empty egg masses, the primers permitted host identification in 50-100% of the eggs analysed, and yielded species-level identifications. Parasitoid identification success ranged from 33 to 67% among field-collected eggs, with genus-level identification for most specimens. The inability to obtain species-level identities for these individuals is due to the lack of coverage of this taxonomic group in public DNA sequence databases; this situation is likely to improve as more species are sequenced and recorded in these databases. These primers were able to detect and identify both pentatomid host and scelionid parasitoid in a hyperparasitized egg mass, thereby clarifying trophic links otherwise unresolved by conventional methodology.

Investigation of the population structure of the tick vector of Lyme disease Ixodes scapularis (Acari: Ixodidae) in Canada using mitochondrial cytochrome C oxidase subunit I gene sequences.

Genotyping of Ixodes scapularis Say (Acari: Ixodidae) ticks could enhance understanding of the occurrence and genotypes of I. scapularis-borne pathogens. We investigated the utility of mitochondrial (mt) Cytochrome C Oxidase subunit I gene (cox1) sequences as a tool for understanding the population structure of I. scapularis collected in Canada, where we also investigated the geographic occurrence of different cox1 haplotypes. Sequences obtained from 414 ticks were one of 55 unique haplotypes, most of which grouped into one of six clades. Demographic analysis suggested that cox1 sequences have haplotype and nucleotide diversity comparable to other mt genes. All haplotypes were connected in a single minimum spanning network tree. Despite low fixation index values there were significant differences in the frequency of occurrence of haplotypes of different clades among four geographic regions: 1) Alberta to western Ontario, 2) eastern Ontario, 3) Quebec, and 4) Atlantic Provinces; suggesting that cox1 sequences could reveal population structure differences between I. scapularis in geographically separated populations of northeastern and midwestern North America. Spatial clusters of ticks of the same haplotype identified in regions of southern Quebec and southern Ontario where I. scapularis is invading were consistent with population bottlenecks associated with founder events. These findings suggest that cox1 sequences are useful for the study of I. scapularis population structure, are of sufficient diversity that spatial analyses of haplotypes can be used to identify where I. scapularis is emerging in southern Canada, and may be useful for exploring differences between northeastern and midwestern populations of I. scapularis.

Identifying the last supper: utility of the DNA barcode library for bloodmeal identification in ticks.

Ticks are among the most important vectors of disease in the Northern Hemisphere, and a better understanding of their feeding behaviour and life cycle is critical to the management and control of tick-borne zoonoses. DNA-based tools for the identification of residual bloodmeals in hematophagous arthropods have proven useful in the investigation of patterns of host use in nature. Using a blind test approach, we challenged the utility of the DNA barcode library for the identification of vertebrate bloodmeals in engorged, field-collected Ixodes scapularis. Universal vertebrate primers for the COI barcode region successfully amplified DNA from the host bloodmeal and only rarely amplified tick DNA. Of the 61 field-collected ticks, conclusive genus- and species-level identification was possible for 72% of the specimens. In all but two cases, barcode-based identification of the bloodmeal was consistent with the morphological identification of the vertebrate host the ticks were collected from. Possible explanations for mismatches or ambiguities are presented. This study validates the utility of the DNA barcode library as a valuable and reliable resource for the identification of unknown bloodmeals in arthropod vectors of disease. Future directions aimed at the refinement of these techniques to gain additional information and to improve the amplification success of digested vertebrate DNA in tick bloodmeals are discussed.

Does host plant influence parasitism and parasitoid species composition in Lygus rugulipennis? A molecular approach.

Lygus Hahn plant bugs (Hemiptera: Miridae) are serious pests of a wide variety of economically important crops in North America. European Peristenus digoneutis Loan and P. relictus Ruthe (Hymenoptera: Braconidae) are being considered for release in Canada as part of a classical biological control program for Lygus. The attractiveness of different host plants to European Peristenus has not been addressed, but may be an important consideration prior to parasitoid release. Lygus rugulipennis Poppius nymphs were collected in the Northern Temperate Atlantic (NTA) ecoregion on red clover (Trifolium pratense L.; Fabaceae) and chamomile (Matricaria recutita L.; Asteraceae), and in the Western European Broadleaf Forest (WEBF) ecoregion on red clover and alfalfa (Medicago sativa L.; Fabaceae). Parasitism levels and parasitoid species were determined using a multiplex PCR assay for P. digoneutis, P. relictus, and P. pallipes Curtis. Mean parasitism levels in L. rugulipennis were 45-49% in the NTA ecoregion and 25-32% in the WEBF ecoregion. However, in neither ecoregion were parasitism levels and parasitoid species compositions significantly different in nymphs from different host plant species. Furthermore, multiparasitism was low despite the fact that P. digoneutis and P. relictus share the same host species.