Identification of Spruce Budworm Natural Enemies Using a qPCR-Based Molecular Sorting Approach
| Autor: | Michel Cusson, Véronique Martel, Audrey Nisole, Abdelmadjid Djoumad, Simon Trudeau, Stefaniya Kamenova, George Kyei-Poku, Rob C. Johns, M. Alex Smith, Eldon S. Eveleigh, Don Stewart, Marianne Nadeau, Paule Huron |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
Tortricidae biology fungi TaqMan® molecular identification Forestry Single-nucleotide polymorphism Computational biology lcsh:QK900-989 natural enemies biology.organism_classification 010603 evolutionary biology 01 natural sciences Choristoneura fumiferana Lepidoptera genitalia 010602 entomology qPCR TaqMan lcsh:Plant ecology Multiplex spruce budworm Gene Spruce budworm |
| Zdroj: | Forests, Vol 11, Iss 621, p 621 (2020) Forests Volume 11 Issue 6 |
| ISSN: | 1999-4907 |
| Popis: | Annual monitoring of mortality agents in the course of a spruce budworm (Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)) population cycle is essential to understanding the factors governing the rise and collapse of outbreaks. To date, assessments of causes of budworm mortality have relied on laboratory rearing of field-collected larvae, followed by visual identification of emerging parasitoids and/or microscopic analysis of pathogens in larval carcasses. Although this approach has provided vital information on the abundance and identity of mortality agents, the procedure is labor-intensive and has limits in terms of accuracy. To overcome these shortcomings, we developed a molecular identification tool that makes use of real-time quantitative PCR (qPCR) and TaqMan® technologies. The tool relies on taxon-specific molecular variants (single nucleotide polymorphism [SNP] markers) found in mitochondrial (COI) and nuclear (28S rDNA) genes, for parasitoids, and in the nuclear SSU rDNA gene for microsporidian pathogens these are then used as molecular signatures targeted by qPCR primers and TaqMan probes. Thus, the design of several sets of primers and probes deployed in multiplex format enables the identification of natural enemies via a molecular sorting process, bypassing barcode sequencing. Crude budworm DNA extracts are processed through a first module that detects dipteran and hymenopteran parasitoids, and microsporidian infections. Positive samples are then processed for species determination using three additional modules, enabling the identification of 20 common natural enemies of the spruce budworm. The tool has been fully validated using DNA samples from all comprised taxa, and both its sensitivity and accuracy compared favorably with the rearing-based method in an analysis of field-collected budworms. Using this tool, sample processing can be completed within two days, does not require larval rearing, provides accurate species identification, and can be conducted by technical staff without extensive molecular biology or insect taxonomy training. |
| Databáze: | OpenAIRE |
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