Confirmation of species when morphological characteristics are indistinguishable between native and introduced congeners

Laricobius nigrinus Fender was introduced into the eastern United States as a biological control agent against the exotic hemlock woolly adelgid (HWA) in 2003. Evaluation of L. nigrinus effectiveness involves sampling release areas for the presence of immatures on HWA infested hemlock branches. A native congener, L. rubidus LeConte, can be co-located within the samples; though its primary prey is pine bark adelgid. Larvae of the two Laricobius species are morphologically indistinguishable below the genus level and there is sufficient overlap of key morphological characters to compromise the reliability of adult identification. Molecular diagnostics based on a 700 base pair portion of the COI gene has distinguished field collected larvae specimen. Effectiveness of two PCR applications were verified based on 20 beetles of each L. nigrinus and L. rubidus, collected from geographically separated populations and morphologically identified. The first application, PCR-RFLP, utilized primers developed for Lepidoptera and suitable for Coleoptera, two restriction enzymes, and electrophoresis. Targeting the same gene, the second application utilized a custom-made TaqMan SNP genotyping assay, and real-time PCR. The multiplexing assay contains two species-specific fluorescing probes, each displayed distinct clusters on an allelic discrimination plot. Sequences from the PCR products of the 40 specimens were compared with sequence data collected as part of ongoing Laricobius phylogenic research. Depending on time, financial, or equipment constraints one of the two described protocols will meet the needs of cooperators involved with evaluation of the biological control agent, L. nigrinus.