Abstract
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.
Highlights
Effective management of eruptive forest defoliators rests on a thorough characterization of the factors responsible for the rise and decline of population outbreaks
We developed a suite of quantitative real-time PCR TaqMan® assays analogous to those we designed earlier for the identification of gypsy moths and their lymantriine relatives [17,18]
To develop a molecular identification tool that builds on an existing DNA library, but does not require the technical expertise necessary to complete DNA sequencing, we capitalized on the quantitative PCR (qPCR) TaqMan® technology, used in multiplex format
Summary
Effective management of eruptive forest defoliators rests on a thorough characterization of the factors responsible for the rise and decline of population outbreaks. This type of knowledge is relevant to the fight against the spruce budworm (SBW), Choristoneura fumiferana, Canada’s most iconic and devastating lepidopteran defoliator. This pest ranges from coast to coast across the North American boreal forest, its epidemics have historically been most severe in the provinces of Quebec, New Brunswick, and Ontario (i.e., its eastern range), with a recurrence of outbreaks every ~35–45 years. The success of EIS depends at least partially on whether suppressed areas are reinvaded by immigrant moths and whether control tactics can be deployed in ways that avoid impacting the natural enemies that keep populations low [1]
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