BackgroundThe mountain pine beetle (Dendroctonus ponderosae) is a significant coniferous forest pest in western North America. It relies on aggregation pheromones to colonize hosts. Its three major pheromone components, trans-verbenol, exo-brevicomin, and frontalin, are thought to arise via different metabolic pathways, but the enzymes involved have not been identified or characterized. We produced ESTs from male and female midguts and associated fat bodies and used custom oligonucleotide microarrays to study gene expression patterns and thereby made preliminary identification of pheromone-biosynthetic genes.ResultsClones from two un-normalized cDNA libraries were directionally sequenced from the 5' end to yield 11,775 ESTs following sequence cleansing. The average read length was 550 nt. The ESTs clustered into 1,201 contigs and 2,833 singlets (4,034 tentative unique genes). The ESTs are broadly distributed among GO functional groups, suggesting they reflect a broad spectrum of the transcriptome. Among the most represented genes are representatives of sugar-digesting enzymes and members of an apparently Scolytid-specific gene family of unknown function. Custom NimbleGen 4-plex arrays representing the 4,034 tentative unique genes were queried with RNA from eleven different biological states representing larvae, pupae, and midguts and associated fat bodies of unfed or fed adults. Quantitative (Real-Time) RT-PCR (qRT-PCR) experiments confirmed that the microarray data accurately reflect expression levels in the different samples. Candidate genes encoding enzymes involved in terminal steps of biosynthetic pathways for exo-brevicomin and frontalin were tentatively identified.ConclusionsThese EST and microarray data are the first publicly-available functional genomics resources for this devastating forestry pest.
The mountain pine beetle (Dendroctonus ponderosae Hopkins) is the most destructive pest of western North American pine forests. Adult males produce frontalin, an eight-carbon antiaggregation pheromone, via the mevalonate pathway, as part of several pheromones that initiate and modulate the mass attack of host trees. Frontalin acts as a pheromone, attractant, or kairomone in most Dendroctonus species, other insects, and even elephants. 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to originate from 10-carbon geranyl diphosphate (GPP), 15-carbon farnesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate (GGPP) via a dioxygenase-or cytochrome P450-mediated carboncarbon bond cleavage. To investigate the role of isoprenyl diphosphate synthases in pheromone biosynthesis, we characterized a bifunctional GPP/FPP synthase and a GGPP synthase in the mountain pine beetle. The ratio of GPP to FPP produced by the GPP/FPP synthase was highly dependent on the ratio of the substrates isopentenyl diphosphate and dimethylallyl diphosphate used in the assay. Transcript levels in various tissues and life stages suggested that GGPP rather than GPP or FPP is used as a precursor to frontalin. Reduction of transcript levels by RNA interference of the isoprenyl diphosphate synthases identified GGPP synthase as having the largest effect on frontalin production, suggesting that frontalin is derived from a 20-carbon isoprenoid precursor rather than from the 10-or 15-carbon precursors. (Fig. 1), is an antiaggregation pheromone known to be synthesized de novo via the mevalonate pathway in male midgut tissue (3, 4). Frontalin acts as a pheromone, attractant, or kairomone in most other Dendroctonus species, other insects (5), and even elephants (6, 7) and has been studied in bark beetles for more than 40 y (8). Its biosynthetic precursor, 6-methylhept-6-en-2-one (6MHO), found in both insects and elephants (6, 9), is hypothesized to originate from cleavage of geranyl diphosphate (GPP), farnesyl diphosphate (FPP), or geranylgeranyl diphosphate (GGPP) by a dioxygenase or cytochrome P450 (10). These isoprenyl diphosphates are biosynthesized from the five-carbon precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by the isoprenyl diphosphate synthases (IDSs) GPP synthase (GPPS), FPP synthase (FPPS), and GGPP synthase (GGPPS). However, it is not known which of these isoprenyl diphosphates is used as a precursor in frontalin biosynthesis.A few IDSs have been characterized in Coleoptera, including an FPPS from the cotton boll weevil (Anthonomus grandis) (11), a GPPS/FPPS from the horseradish leaf beetle (Phaedon cochleariae) (12), and a unique GPPS from the pine engraver beetle (Ips pini) that also produces myrcene (13,14). To assess their importance in pheromone biosynthesis, we identified and functionally characterized two IDSs in MPB. Based on three different lines of evidence, gene-expression patterns of the IDSs, enzyme activity of recombinant proteins, and the frontalin content of dsRNA-trea...
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