A TaqMan Assay for the Detection and Monitoring of Potentially Invasive Lasiocampids, With Particular Attention to the Siberian Silk Moth, Dendrolimus sibiricus (Lepidoptera: Lasiocampidae).

The Siberian silk moth, Dendrolimus sibiricus Tschetverikov, is a very serious pest of conifers in Russia and is an emerging threat in North America where an accidental introduction could have devastating impacts on native forest resources. Other Dendrolimus Germar species and related Eurasian lasiocampids in the genus Malacosoma (Hubner) could also present a risk to North America's forests. Foreign vessels entering Canadian and U.S. ports are regularly inspected for Lymantria dispar (Linnaeus) and for the presence of other potentially invasive insects, including suspicious lasiocampid eggs. However, eggs are difficult to identify based on morphological features alone. Here, we report on the development of two TaqMan (Roche Molecular Systems, Inc., Rotkreuz, Switzerland) assays designed to assist regulatory agencies in their identification of these insects. Developed using the barcode region of the cytochrome c oxidase I (COI) gene and run in triplex format, the first assay can detect Dendrolimus and Malacosoma DNA, and can distinguish North American from Eurasian Malacosoma species. The second assay is based on markers identified within the internal transcribed spacer 2 (ITS2) region and was designed to specifically identify D. sibiricus, while discriminating closely related Dendrolimus taxa. In addition to providing direct species identification in the context of its use in North America, the D. sibiricus assay should prove useful for monitoring the spread of this pest in Eurasia, where its range overlaps with those of the morphologically identical D. superans (Butler) and similar D. pini (Linnaeus). The assays described here can be performed either in the lab on a benchtop instrument, or on-site using a portable machine.

New Canadian and Provincial Records of Coleoptera Resulting from Annual Canadian Food Inspection Agency Surveillance for Detection of Non-Native, Potentially Invasive Forest Insects.

The arrival and establishment of adventive, invasive forest insects are a threat to the health, diversity, and productivity of forests in Canada and the world at large, and their early detection is essential for successful eradication and management. For that reason, the Canadian Food Inspection Agency (CFIA) conducts annual surveys at high risk sites such as international ports and freight terminals, industrial zones, and disposal sites for solid wood packaging material using two methods: (1) semiochemical-baited traps deployed in a total of about 63-80 sites per year in British Columbia (BC), Ontario (ON), Quebec (QC), New Brunswick (NB), Nova Scotia (NS), and Newfoundland and Labrador (NL); and (2) rearing of insects from bolts collected from stressed trees and incubated in modified shipping containers in four cities (Vancouver, Toronto, Montreal, and Halifax). We report 31 new Canadian provincial records of Coleoptera from surveys conducted in 2011-2021, including 13 new records for Canada and 9 species adventive to North America (indicated by †). Nine of the new Canadian records were native North American species previously detected only south of the border. All but three species belong to the Curculionidae family and most of these were in the subfamily Scolytinae. The records include: Xenomelanophila miranda (LeConte) (Canada, BC) (Buprestidae: Buprestinae); Neoclytus mucronatus mucronatus (Fabricius) (BC) (Cerambycidae: Cerambycinae); Amphicerus cornutus (Pallas) (Canada, BC) (Bostrichidae: Bostrichinae); Mecinus janthinus (Germar)† (ON) (Curculionidae: Curculioninae); Aulacobaris lepidii (Germar)† (Canada, ON); Buchananius striatus (LeConte) (ON) (Curculionidae: Baridinae); Cylindrocopturus binotatus LeConte (Canada, ON) (Curculionidae: Conoderinae); Himatium errans LeConte (ON); Phloeophagus canadensis Van Dyke (ON); Rhyncolus spretus Casey (Canada, BC); Stenomimus pallidus (Boheman) (Canada, ON); Tomolips quercicola (Boheman) (Canada, ON) (Curculionidae: Cossoninae); Strophosoma melanogrammum (Forster)† (NB) (Curculionidae: Entiminae); Conotrachelus aratus (Germar) (ON) (Curculionidae: Molytinae); Anisandrus maiche Stark† (Canada, ON, QC); Cnesinus strigicollis LeConte (Canada, ON); Cyclorhipidion pelliculosum (Eichhoff)† (Canada, ON, QC); Hylesinus fasciatus LeConte (QC); Hylesinus pruinosus Eichhoff (QC); Hypothenemus interstitialis (Hopkins) (Canada, ON); Lymantor alaskanus Wood (BC); Pityogenes bidentatus (Herbst)† (Canada, ON); Scolytus mali (Bechstein)† (BC); Scolytus schevyrewi Semenov† (QC); Trypodendron scabricollis (LeConte) (Canada, ON); Trypophloeus populi Hopkins (QC); Xylechinus americanus Blackman (NFLB); and Xylosandrus crassiusculus (Motschulsky)† (BC, QC) (Curculionidae: Scolytinae). We also provide additional data confirming the presence of the adventive Hylastes opacus Erichson† in NS. Rearing of insects from bolts accounted for two new records (H. pruinosus, R. spretus) and trapping accounted for the remainder. These surveys not only assist our efforts to manage forest insects by documenting new species introductions and apparent range expansions but also increase our knowledge of biodiversity.

High-Throughput Sequencing to Investigate Phytopathogenic Fungal Propagules Caught in Baited Insect Traps.

Studying the means of dispersal of plant pathogens is crucial to better understand the dynamic interactions involved in plant infections. On one hand, entomologists rely mostly on both traditional molecular methods and morphological characteristics, to identify pests. On the other hand, high-throughput sequencing (HTS) is becoming the go-to avenue for scientists studying phytopathogens. These organisms sometimes infect plants, together with insects. Considering the growing number of exotic insect introductions in Canada, forest pest-management efforts would benefit from the development of a high-throughput strategy to investigate the phytopathogenic fungal and oomycete species interacting with wood-boring insects. We recycled formerly discarded preservative fluids from the Canadian Food Inspection Agency annual survey using insect traps and analysed more than one hundred samples originating from across Canada. Using the Ion Torrent Personal Genome Machine (PGM) HTS technology and fusion primers, we performed metabarcoding to screen unwanted fungi and oomycetes species, including Phytophthora spp. Community profiling was conducted on the four different wood-boring, insect-attracting semiochemicals; although the preservative (contained ethanol) also attracted other insects. Phytopathogenic fungi (e.g., Leptographium spp. and Meria laricis in the pine sawyer semiochemical) and oomycetes (mainly Peronospora spp. and Pythium aff. hypogynum in the General Longhorn semiochemical), solely associated with one of the four types of semiochemicals, were detected. This project demonstrated that the insect traps' semiochemical microbiome represents a new and powerful matrix for screening phytopathogens. Compared to traditional diagnostic techniques, the fluids allowed for a faster and higher throughput assessment of the biodiversity contained within. Additionally, minimal modifications to this approach would allow it to be used in other phytopathology fields.

Screening for Exotic Forest Pathogens to Increase Survey Capacity Using Metagenomics.

Anthropogenic activities have a major impact on the global environment. Canada's natural resources are threatened by the spread of fungal pathogens, which is facilitated by agricultural practices and international trade. Fungi are introduced to new environments and sometimes become established, in which case they can cause disease outbreaks resulting in extensive forest decline. Here, we describe how a nationwide sample collection strategy coupled to next-generation sequencing (NGS) (i.e., metagenomics) can achieve fast and comprehensive screening for exotic invasive species. This methodology can help provide guidance to phytopathology stakeholders such as regulatory agencies. Several regulated invasive species were monitored by processing field samples collected over 3 years (2013 to 2015) near high-risk areas across Canada. Fifteen sequencing runs were required on the Ion Torrent platform to process 398 samples that yielded 45 million reads. High-throughput screening of fungal and oomycete operational taxonomic units using customized fungi-specific ribosomal internal transcribed spacer 1 barcoded primers was performed. Likewise, Phytophthora-specific barcoded primers were used to amplify the adenosine triphosphate synthase subunit 9-nicotinamide adenine dinucleotide dehydrogenase subunit 9 spacer. Several Phytophthora spp. were detected by NGS and confirmed by species-specific quantitative polymerase chain reaction (qPCR) assays. The target species Heterobasidion annosum sensu stricto could be detected only through metagenomics. We demonstrated that screening target species using a variety of sampling techniques and NGS-the results of which were validated by qPCR-has the potential to increase survey capacity and detection sensitivity, reduce hands-on time and costs, and assist regulatory agencies to identify ports of entry. Considering that early detection and prevention are the keys in mitigating invasive species damage, our method represents a substantial asset in plant pathology management.

A Multi-Species TaqMan PCR Assay for the Identification of Asian Gypsy Moths (Lymantria spp.) and Other Invasive Lymantriines of Biosecurity Concern to North America.

Preventing the introduction and establishment of forest invasive alien species (FIAS) such as the Asian gypsy moth (AGM) is a high-priority goal for countries with extensive forest resources such as Canada. The name AGM designates a group of closely related Lymantria species (Lepidoptera: Erebidae: Lymantriinae) comprising two L. dispar subspecies (L. dispar asiatica, L. dispar japonica) and three closely related Lymantria species (L. umbrosa, L. albescens, L. postalba), all considered potential FIAS in North America. Ships entering Canadian ports are inspected for the presence of suspicious gypsy moth eggs, but those of AGM are impossible to distinguish from eggs of innocuous Lymantria species. To assist regulatory agencies in their identification of these insects, we designed a suite of TaqMan® assays that provide significant improvements over existing molecular assays targeting AGM. The assays presented here can identify all three L. dispar subspecies (including the European gypsy moth, L. dispar dispar), the three other Lymantria species comprising the AGM complex, plus five additional Lymantria species that pose a threat to forests in North America. The suite of assays is built as a "molecular key" (analogous to a taxonomic key) and involves several parallel singleplex and multiplex qPCR reactions. Each reaction uses a combination of primers and probes designed to separate taxa through discriminatory annealing. The success of these assays is based on the presence of single nucleotide polymorphisms (SNPs) in the 5' region of mitochondrial cytochrome c oxidase I (COI) or in its longer, 3' region, as well as on the presence of an indel in the "FS1" nuclear marker, generating North American and Asian alleles, used here to assess Asian introgression into L. dispar dispar. These assays have the advantage of providing rapid and accurate identification of ten Lymantria species and subspecies considered potential FIAS.