Leland M. Humble

Towards a global barcode library for Lymantria (Lepidoptera: Lymantriinae) tussock moths of biosecurity concern

Background Detecting and controlling the movements of invasive species, such as insect pests, relies upon rapid and accurate species identification in order to initiate containment procedures by the appropriate authorities. Many species in the tussock moth genus Lymantria are significant forestry pests, including the gypsy moth Lymantria dispar L., and consequently have been a focus for the development of molecular diagnostic tools to assist in identifying species and source populations. In this study we expand the taxonomic and geographic coverage of the DNA barcode reference library, and further test the utility of this diagnostic method, both for species/subspecies assignment and for determination of geographic provenance of populations. Methodology/Principal Findings Cytochrome oxidase I (COI) barcodes were obtained from 518 individuals and 36 species of Lymantria, including sequences assembled and generated from previous studies, vouchered material in public collections, and intercepted specimens obtained from surveillance programs in Canada. A maximum likelihood tree was constructed, revealing high bootstrap support for 90% of species clusters. Bayesian species assignment was also tested, and resulted in correct assignment to species and subspecies in all instances. The performance of barcoding was also compared against the commonly employed NB restriction digest system (also based on COI); while the latter is informative for discriminating gypsy moth subspecies, COI barcode sequences provide greater resolution and generality by encompassing a greater number of haplotypes across all Lymantria species, none shared between species. Conclusions/Significance This study demonstrates the efficacy of DNA barcodes for diagnosing species of Lymantria and reinforces the view that the approach is an under-utilized resource with substantial potential for biosecurity and surveillance. Biomonitoring agencies currently employing the NB restriction digest system would gather more information by transitioning to the use of DNA barcoding, a change which could be made relatively seamlessly as the same gene region underlies both protocols.

Application of a real-time PCR method for the detection of pine wood nematode, Bursaphelenchus xylophilus, in wood samples from lodgepole pine

A real-time PCR polymerase chain reaction (real-time PCR) method was developed to detect and differentiate Bursaphelenchus xylophilus (pine wood nematode, PWN) from other wood-inhabiting nematode species. A primer set and a specific Taqman® fluorescent probe were designed to amplify target B. xylophilus heat shock protein 70 sequences. After optimization, this real-time PCR assay was shown to be highly specific and sensitive, detecting at least 0.005 ng of B. xylophilus genomic DNA, as well as DNA extracted from single nematodes. The practical application of this real-time PCR diagnostic method for the detection of B. xylophilus from actual wood samples of lodgepole pine (Pinus contorta, Dougl. var. latifolia) trees containing a heterogeneous population of nematodes, rather than pure cultures or individual nematodes, is demonstrated. This method works well in the presence of potential inhibitors associated with wood after Baermann extraction and thus eliminates the need to produce pure nematode samples through further culturing and/or isolation of nematodes with a high-power microscope.

A Comprehensive DNA Barcode Library for the Looper Moths (Lepidoptera: Geometridae) of British Columbia, Canada

Background The construction of comprehensive reference libraries is essential to foster the development of DNA barcoding as a tool for monitoring biodiversity and detecting invasive species. The looper moths of British Columbia (BC), Canada present a challenging case for species discrimination via DNA barcoding due to their considerable diversity and limited taxonomic maturity. Methodology/Principal Findings By analyzing specimens held in national and regional natural history collections, we assemble barcode records from representatives of 400 species from BC and surrounding provinces, territories and states. Sequence variation in the barcode region unambiguously discriminates over 93% of these 400 geometrid species. However, a final estimate of resolution success awaits detailed taxonomic analysis of 48 species where patterns of barcode variation suggest cases of cryptic species, unrecognized synonymy as well as young species. Conclusions/Significance A catalog of these taxa meriting further taxonomic investigation is presented as well as the supplemental information needed to facilitate these investigations.

In the dark in a large urban park: DNA barcodes illuminate cryptic and introduced moth species

To facilitate future assessments of diversity following disturbance events, we conducted a first level inventory of nocturnal Lepidoptera in Stanley Park, Vancouver, Canada. To aid the considerable task, we employed high-throughput DNA barcoding for the rough sorting of all material and for tentative species identifications, where possible. We report the preliminary species list of 190, the detection of four new exotic species (Argyresthia pruniella, Dichelia histrionana, Paraswammerdamia lutarea, and Prays fraxinella), and the potential discovery of two cryptic species. We describe the magnitude of assistance that barcoding presents for faunal inventories, from reducing specialist time to facilitating the detection of native and exotic species at low density.

An effective PCR-based diagnostic method for the detection of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) in wood samples from lodgepole pine

A molecular diagnostic method has been designed for the detection and identification of Bursaphelenchus xylophilus. Heat shock protein 70 gene sequences from B. xylophilus and the closely related B. mucronatus were compared and used to design primers Bx701F and Bx701R which amplify a 171 base pair fragment from B. xylophilus by PCR. As a control, primers Bm701F and Bm701R were designed which specifically amplify a 168 base pair fragment from B. mucronatus. After optimisation, B. xylophilus primers were shown to be highly sensitive and could easily detect 23 target copies, or less than one nematode. Species-specific detection of B. xylophilus was carried out directly from concentrated Baermann funnel extracts using wood samples from lodgepole pine (Pinus contorta var. latifolia) trees from British Columbia, Canada, containing an unknown nematode population, thus bypassing the need for culturing or recovering the nematode before analysis.

Ophiostomatoid fungi associated with the northern spruce engraver, Ips perturbatus, in western Canada

A number of ophiostomatoid fungi were isolated from the spruce-infesting bark beetle, Ips perturbatus and its galleries collected from felled spruce trees and logs in northern BC and the Yukon Territory. Isolates were identified to species using morphological characteristics, nuclear ribosomal DNA and partial β-tubulin gene sequences. Thirteen morphological and phylogenetic species were identified among the isolates. Leptographium fruticetum, Leptographium abietinum, Ophiostoma bicolor, Ophiostoma manitobense, O. piceaperdum, and eight undescribed species of the genus Ophiostoma and the anamorph genera Leptographium, Hyalorhinocladiella, Ambrosiella and Graphium. A number of these species, i.e. L. fruticetum, Hyalorhinocladiella sp. 2, O. bicolor and O. manitobense, were isolated repeatedly from I. perturbatus, while others, i.e. Graphium sp. 1 and O. piceaperdum, seemed to be␣sporadic associates. Among all the isolates, L. fruticetum had the highest relative dominance in this survey. A high frequency of occurrence of this species with the beetle may indicate a specific relationship between the two partners.

First North American records for Heterarthrus vagans (Hymenoptera: Tenthredinidae), a Palaearctic leafmining sawfly of alder

Abstract The discovery in southwestern British Columbia of widespread established populations of the Palaearctic leafminer Heterarthrus vagans (Fallén) feeding on native red alder, Alnus rubra Bong. (Betulaceae), is reported. A preliminary survey suggests that it is currently confined to the Fraser Valley west of Hope, its distribution extending north along Howe Sound almost to Squamish. It was not found in the greater Victoria area. No other regions of the province have been surveyed. This introduced sawfly completes at least two generations a year. Diagnostic characters to aid the recognition of adults and feeding larvae of H. vagans and a modified key to adult Heterarthrinae on Betulaceae are provided.

Application of Conventional PCR and Real-Time PCR Diagnostic Methods for Detection of the PineWood Nematode, Bursaphelenchus xylophilus, in Wood Samples from Lodgepole Pine

Molecular diagnostic methods have been designed for the detection and identification of the pinewood nematode, Bursaphelenchus xylophilus. Heat shock protein 70 (Hsp 70) gene sequences from B. xylophilus and the closely related B. mucronatus, were compared and used to design primers Bx701F and Bx701R which amplify a 171 base pair fragment from B. xylophilus by polymerase chain reaction (PCR). As a control, primers Bm701F and Bm701R were designed which specifically amplify a 168 base pair fragment from B. mucronatus. After optimization, B. xylophilus primers were shown to be highly sensitive and could easily detect 23 target copies, or less than 1 nematode. In addition, a real-time PCR method was developed to detect and differentiate B. xylophilus from other wood-inhabiting nematode species. A primer set and a specific Taqman® fluorescent probe were designed to amplify target B. xylophilus Hsp 70 sequences. After optimization, this real-time PCR assay was shown to be highly specific and sensitive, detecting at least 5 pg of B. xylophilus genomic DNA, as well as DNA extracted from individual nematodes. The species-specific detection of B. xylophilus was carried out directly from concentrated Baermann funnel extracts using wood samples from lodgepole pine (Pinus contorta, Dougl. var. latifolia) trees.