Geranyl diphosphate synthase (GPPS) catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to form geranyl diphosphate. Geranyl diphosphate is the precursor of monoterpenes, a large family of natural occurring C 10 compounds predominately found in plants. Similar to plants but unique to animals, some bark beetle genera (Coleoptera: Scolytidae) produce monoterpenes that function in intraspecific chemical communication as aggregation and dispersion pheromones. The release of monoterpene aggregation pheromone mediates host colonization and mating. It has been debated whether these monoterpene pheromone components are derived de novo through the mevalonate pathway or result from simple modifications of dietary precursors. The data reported here provide conclusive evidence for de novo biosynthesis of monoterpene pheromone components from bark beetles. We describe GPPS in the midgut tissue of pheromone-producing male Ips pini. GPPS expression levels are regulated by juvenile hormone III, similar to other mevalonate pathway genes involved in pheromone biosynthesis. In addition, GPPS transcript is almost exclusively expressed in the anterior midgut of male I. pini, the site of aggregation pheromone biosynthesis. The recombinant enzyme was functionally expressed and produced geranyl diphosphate as its major product. The threedimensional model structure of GPPS shows that the insect enzyme has the sequence structural motifs common to E-isoprenyl diphosphate synthases.isoprenyl diphosphate synthase ͉ monoterpene biosynthesis ͉ ipsdienol

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.

Juvenile hormone III (JH III) stimulates biosynthesis of the monoterpenoid aggregation pheromone component, ipsdienol, in the anterior midgut of the male pine engraver beetle, Ips pini (Say). To understand better the hormonal regulation of pheromone biosynthesis in this forest pest, and identify JH III-responsive genes, microarrays were prepared and hybridized to cDNA from midguts of JH III-treated beetles. Expression patterns were confirmed by quantitative real-time RT-PCR. JH III co-ordinately regulated mevalonate pathway genes and many other genes implicated in pheromone biosynthesis. Sex differences in basal levels of mevalonate pathway genes were consistent with their role in male-specific pheromone biosynthesis. This is the first microarray-based study of the developmental and hormonal regulation of insect pheromone biosynthesis.

Bark beetles use aggregation pheromones to coordinate host colonization and mating. These monoterpenoid chemical signals are produced de novo in midgut cells via the mevalonate pathway, and pheromone production is induced when an adult beetle feeds on phloem of a host tree. In Ips pini, juvenile hormone (JH) III influences key regulatory enzymes along the mevalonate pathway that leads to pheromone production. In fact, topically applied JH III is sufficient to stimulate pheromone production in unfed males. In this study, we explore the influence of feeding and JH III treatment on pheromone production in male Ips confusus, the pinyon Ips. We also characterize the influence of feeding and JH III treatment on transcript levels and activity of three key enzymes involved in pheromone biosynthesis: 3-hydroxy-3-methylglutaryl-CoA (HMG) synthase (HMGS), HMG-CoA reductase (HMGR) and geranyl diphosphate synthase (GPPS). We also extend the current understanding of the regulation of pheromone biosynthesis in I. pini, by measuring the influence of feeding and JHIII treatment on enzymatic activity of HMGS and GPPS. Feeding on host phloem alone strongly induces pheromone production in male I. confusus, while JH III treatment has no effect. However, feeding and JH III both significantly up-regulate mRNA levels of key mevalonate pathway genes. Feeding up-regulates these genes to a maximum at 32 h, whereas with JH III-treatment, they are up-regulated at 4, 8, and 16 h, but return near to non-treatment levels at 32 h. Feeding, but not JH III treatment, also increases the activity of all three enzymes in I. confusus, while both feeding or treatment with JH III increase HMGS and GPPS activity in I. pini. Our data suggest that pheromone production in Ips is not uniformly controlled by JH III and feeding may stimulate the release of some other regulatory factor, perhaps a brain hormone, required for pheromone production.

Myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation is likely one of the final reactions involved in the production of the Ips spp. (Coleoptera: Scolytidae) aggregation pheromone components, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol). To gain insight into the evolution of pheromone production, we isolated a full-length cDNA from the pinyon ips, Ips confusus (LeConte), that encodes a pheromone-biosynthetic cytochrome P450, I. confusus CYP9T1 (IcCYP9T1). The recovered cDNA is 1.70 kb, and the open reading frame encodes a 532 amino acid protein. IcCYP9T1 is 94% identical to the pine engraver, Ips pini (Say), CYP9T2 ortholog that hydroxylates myrcene. Quantitative real-time PCR experiments showed that IcCYP9T1, as does CYP9T2, has an expression pattern similar to other pheromone-biosynthetic genes in I. pini. Basal expression levels were higher in males than females, and expression was significantly induced in male, but not in female, anterior midguts by feeding on host phloem. Microsomes, prepared from Sf9 cells co-expressing baculoviral-mediated recombinant IcCYP9T1 and house fly (Musca domestica) NADPH-cytochrome P450 reductase, converted myrcene to ~85%-(R)-(-)-ipsdienol. These results are consistent with IcCYP9T1 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway. Since the I. confusus pheromone blend contains >90%-(S)-(+)-ipsdienol, these results confirm further that Ips spp. myrcene hydroxylases do not control the final ipsdienol enantiomeric blend. Other enzymes are required following myrcene hydroxylation to achieve the critical quantity and enantiomeric composition of pheromonal ipsenol and ipsdienol used by different Ips spp.

We isolated a full-length cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) from the pine engraver beetle, Ips pini (Say), and examined its genomic structure. The intron-less gene has a predicted 460 amino acid cytosolic protein product with 73% identity to HMG-S from Dendroctonus jeffreyi, and high identity (58-64%) with other insect HMG-Ss. Topically applied juvenile hormone (JH) III induced HMG-S mRNA levels up to 6.5-fold in both sexes, mostly in the anterior midgut, though there were differences between males and females in the timing, sensitivity to JH III dose and tissue distribution of HMG-S mRNA. These data further validate the coordinate regulation of mevalonate pathway genes for de novo isoprenoid pheromone production in bark beetles.

Bark beetles use monoterpenoid aggregation pheromones to coordinate host colonization and mating. These chemical signals are produced de novo in midgut cells via the mevalonate pathway, and pheromone production may be regulated by a negative feedback system mediated through the antennae. In this study, we explored the effect of antennectomy on pheromone production and transcript levels of key mevalonate pathway genes in juvenile hormone III-treated male pine engraver beetles, Ips pini (Say). Antennectomized males produced significantly greater amounts of pheromone than podectomized males and those with intact antennae. Likewise, mRNA levels of three mevalonate pathway genes important in pheromone biosynthesis were measured by quantitative real-time PCR and found to be induced to a greater extent with antennectomy, suggesting a transcriptional regulation of pheromone production.

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