RNA-Seq used to identify ipsdienone reductase (IDONER): A novel monoterpene carbon-carbon double bond reductase central to Ips confusus pheromone production.

The pinyon ips beetle, Ips confusus (LeConte) is a highly destructive pest in pine forests in western North America. When colonizing a new host tree, I. confusus beetles coordinate a mass attack to overcome the tree's defenses using aggregation pheromones. Ips confusus, as with other Ips spp. beetles, biosynthesize ipsdienol and ipsenol in a specific enantiomeric blend and ratio as aggregation pheromones. While several of the initial steps in the pheromone biosynthetic pathway have been well defined, the final steps were unknown. We used comparative RNA-Seq analysis between fed and unfed male I. confusus midgut tissue to identify candidate genes involved in pheromone biosynthesis. The 12,995 potentially unique transcripts showed a clear separation based on feeding state. Differential expression analysis identified gene groups that were tightly connected. This analysis identified all known pheromone biosynthetic genes and suggested a novel monoterpene double bond reductase, ipsdienone reductase (IDONER), with pheromone biosynthetic gene expression patterns. IDONER cDNA was cloned, expressed, and functionally characterized. The coding DNA sequence has an ORF of 1101 nt with a predicted translation product of 336 amino acids. The enzyme has a molecular weight of 36.7 kDa with conserved motifs of the medium chain dehydrogenases/reductase (MDR) superfamily in the leukotriene B4 dehydrogenases/reductases (LTB4R) family. Tagged recombinant protein was expressed and purified. Enzyme assays and GC/MS analysis showed IDONER catalyzed the reduction of ipsdienone to form ipsenone. This study shows that IDONER is a monoterpene double bond reductase involved in I. confusus pheromone biosynthesis.

Cytochromes P450: terpene detoxification and pheromone production in bark beetles.

Bark beetles (family: Curculionidae; subfamily: Scolytinae) in the Dendroctonus and Ips genera are the most destructive forest pests in the Northern hemisphere. They use cytochromes P450 (P450s) to detoxify tree-produced terpenes to produce pheromones, in de novo pheromone production and to oxidize odorants on antennae. Many Dendroctonus spp. use trans-verbenol as an aggregation pheromone, and it is formed from host-tree produced α-pinene hydroxylated by CYP6DE1 during larval stages, stored as verbenyl ester of fatty acids, and then released when the female begins feeding on a new host tree. Ips spp. hydroxylate de novo produced myrcene to form ipsdienol. Subsequent steps form the appropriate enantiomeric composition of ipsdienol and convert ipsdienol to ipsenol. In this article we review recent progress in elucidating the functions of P450s in Ips and Dendroctonus species and in doing so provide insights into the role of these enzymes in host phytochemical detoxification and pheromone production.

Mountain pine beetle (Dendroctonus ponderosae) CYP4Gs convert long and short chain alcohols and aldehydes to hydrocarbons.

Hydrocarbon biosynthesis in insects involves the elongation of fatty acyl-CoAs to very-long chain fatty acyl-CoAs that are then reduced and converted to hydrocarbon, with the last step involving the oxidative decarbonylation of an aldehyde to hydrocarbon and carbon dioxide. Cytochromes P450 in the 4G family decarbonylate aldehydes to hydrocarbon. All insect acyl-CoA reductases studied to date reduce fatty acyl-CoAs to alcohols. The results of the work reported herein demonstrate that CYP4G55 and CYP4G56 from the mountain pine beetle, Dendroctonus ponderosae, expressed as fusion proteins with house fly cytochrome P450 reductase (CPR), convert both long chain aldehydes and long chain alcohols to hydrocarbons. CYP4G55 and CYP4G56 appear to prefer primary alcohols to aldehydes as substrates. These data strongly suggest that hydrocarbon biosynthesis in insects occurs by the two-step reduction of very long chain fatty acyl-CoAs to alcohols, which are then oxidized to aldehydes and then oxidatively decarbonylated to hydrocarbon by CYP4G enzymes. In addition, both CYP4G55 and CYP4G56 fusion proteins convert C10 alcohols and aldehydes to hydrocarbons, including the conversion of (Z)-7-decenal, a putative intermediate in the exo-brevicomin pheromone biosynthetic pathway, to (Z)-3-nonene. These data demonstrate that the highly conserved CYP4G enzymes accept a broad range of carbon chain lengths, including C10 and C18, and have evolved to function in cuticular hydrocarbon biosynthesis and pheromone production.

De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug.

Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (1S,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication.

Pheromone biosynthesis in bark beetles.

Pine bark beetles rely on aggregation pheromones to coordinate mass attacks and thus reproduce in host trees. The structural similarity between many pheromone components and those of defensive tree resin led to early suggestions that pheromone components are metabolic derivatives of ingested precursors. This model has given way to our current understanding that most pheromone components are synthesized de novo. Their synthesis involves enzymes that modify products from endogenous metabolic pathways; some of these enzymes have been identified and characterized. Pheromone production is regulated in a complex way involving multiple signals, including JH III. This brief review summarizes progress in our understanding of this highly specialized metabolic process.

Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling.

Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes.

Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of (14)C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An gambiae.

High substrate specificity of ipsdienol dehydrogenase (IDOLDH), a short-chain dehydrogenase from Ips pini bark beetles.

Ips spp. bark beetles use ipsdienol, ipsenol, ipsdienone and ipsenone as aggregation pheromone components and pheromone precursors. For Ips pini, the short-chain oxidoreductase ipsdienol dehydrogenase (IDOLDH) converts (-)-ipsdienol to ipsdienone, and thus likely plays a role in determining pheromone composition. In order to further understand the role of IDOLDH in pheromone biosynthesis, we compared IDOLDH to its nearest functionally characterized ortholog with a solved structure: human L-3-hydroxyacyl-CoA dehydrogenase type II/ amyloid-ß binding alcohol dehydrogenase (hHADH II/ABAD), and conducted functional assays of recombinant IDOLDH to determine substrate and product ranges and structural characteristics. Although IDOLDH and hHADH II/ABAD had only 35% sequence identity, their predicted tertiary structures had high identity. We found IDOLDH is a functional homo-tetramer. In addition to oxidizing (-)-ipsdienol, IDOLDH readily converted racemic ipsenol to ipsenone, and stereo-specifically reduced both ketones to their corresponding (-)-alcohols. The (+)-enantiomers were never observed as products. Assays with various substrate analogs showed IDOLDH had high substrate specificity for (-)-ipsdienol, ipsenol, ipsenone and ipsdienone, supporting that IDOLDH functions as a pheromone-biosynthetic enzyme. These results suggest that different IDOLDH orthologs and or activity levels contribute to differences in Ips spp. pheromone composition.

exo-Brevicomin biosynthetic pathway enzymes from the Mountain Pine Beetle, Dendroctonus ponderosae.

exoBrevicomin (exo-7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octane) is an important semiochemical for a number of beetle species, including the highly destructive Mountain Pine Beetle (Dendroctonus ponderosae). It is also found in other insects and the African elephant. Despite its significance, very little is known about its biosynthesis. A recent microarray analysis implicated a small cluster of three D. ponderosae genes in exo-brevicomin biosynthesis, two of which had identifiable open reading frames (Aw et al., 2010; BMC Genomics 11:215). Here we report further expression profiling of two genes in that cluster and functional analysis of their recombinantly-produced enzymes. One encodes a short-chain dehydrogenase that used NAD(P)(+) as a co-factor to catalyze the oxidation of (Z)-6-nonen-2-ol to (Z)-6-nonen-2-one. We therefore named the enzyme (Z)-6-nonen-2-ol dehydrogenase (ZnoDH). The other encodes the cytochrome P450, CYP6CR1, which epoxidized (Z)-6-nonen-2-one to 6,7-epoxynonan-2-one with very high specificity and substrate selectivity. Both the substrates and products of the two enzymes are intermediates in the exo-brevicomin biosynthetic pathway. Thus, ZnoDH and CYP6CR1 are enzymes that apparently catalyze the antepenultimate and penultimate steps in the exo-brevicomin biosynthetic pathway, respectively.

exo-Brevicomin biosynthesis in the fat body of the mountain pine beetle, Dendroctonus ponderosae.

exo-Brevicomin (exo-7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octane) is an important semiochemical for a number of beetle species, including the highly destructive mountain pine beetle, Dendroctonus ponderosae. It also has been found in other insects and even in the African elephant. Despite its significance, little is known about its biosynthesis. In order to fill this gap and to identify new molecular targets for potential pest management methods, we performed gas chromatography-mass spectrometry analyses of cell cultures and in vitro assays of various D. ponderosae tissues with exo-brevicomin intermediates, analogs, and inhibitors. We confirmed that exo-brevicomin was synthesized by "unfed" males after emerging from the brood tree. Furthermore, in contrast to the paradigm established for biosynthesis of monoterpenoid pheromone components in bark beetles, exo-brevicomin was produced in the fat body, and not in the anterior midgut. The first committed step involves decarboxylation or decarbonylation of ω-3-decenoic acid, which is derived from a longer-chain precursor via ß-oxidation, to (Z)-6-nonen-2-ol. This secondary alcohol is converted to the known precursor, (Z)-6-nonen-2-one, and further epoxidized by a cytochrome P450 to 6,7-epoxynonan-2-one. The keto-epoxide is stable at physiological pH, suggesting that its final cyclization to form exo-brevicomin is enzyme-catalyzed. exo-Brevicomin production is unusual in that tissue not derived from ectoderm apparently is involved.

Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae, and the role of isoprenyl diphosphate synthases.

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 carbon-carbon 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.

Functional characterization of myrcene hydroxylases from two geographically distinct Ips pini populations.

Ips pini bark beetles use myrcene hydroxylases to produce the aggregation pheromone component, ipsdienol, from myrcene. The enantiomeric ratio of pheromonal ipsdienol is an important prezygotic mating isolation mechanism of I. pini and differs among geographically distinct populations. We explored the substrate and product ranges of myrcene hydroxylases (CYP9T2 and CYP9T3) from reproductively-isolated western and eastern I. pini. The two cytochromes P450 share 94% amino acid identity. CYP9T2 mRNA levels were not induced in adults exposed to myrcene-saturated atmosphere. Functional assays of recombinant enzymes showed both hydroxylated myrcene, (+)- and (-)-α-pinene, 3-carene, and R-(+)-limonene, but not α-phellandrene, (-)-ß-pinene, γ-terpinene, or terpinolene, with evidence that CYP9T2 strongly preferred myrcene over other substrates. They differed in the enantiomeric ratios of ipsdienol produced from myrcene, and in the products resulting from different α-pinene enantiomers. These data provide new information regarding bark beetle pheromone evolution and factors affecting cytochrome P450 structure-function relationships.

An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis.

Insects use hydrocarbons as cuticular waterproofing agents and as contact pheromones. Although their biosynthesis from fatty acyl precursors is well established, the last step of hydrocarbon biosynthesis from long-chain fatty aldehydes has remained mysterious. We show here that insects use a P450 enzyme of the CYP4G family to oxidatively produce hydrocarbons from aldehydes. Oenocyte-directed RNAi knock-down of Drosophila CYP4G1 or NADPH-cytochrome P450 reductase results in flies deficient in cuticular hydrocarbons, highly susceptible to desiccation, and with reduced viability upon adult emergence. The heterologously expressed enzyme converts C(18)-trideuterated octadecanal to C(17)-trideuterated heptadecane, showing that the insect enzyme is an oxidative decarbonylase that catalyzes the cleavage of long-chain aldehydes to hydrocarbons with the release of carbon dioxide. This process is unlike cyanobacteria that use a nonheme diiron decarbonylase to make alkanes from aldehydes with the release of formate. The unique and highly conserved insect CYP4G enzymes are a key evolutionary innovation that allowed their colonization of land.

Ipsdienol dehydrogenase (IDOLDH): a novel oxidoreductase important for Ips pini pheromone production.

Ipsdienone (2-methyl-6-methylene-2,7-octadien-4-one) is an important intermediate in the biosynthesis of pheromonal ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol) in male pine engraver beetles, Ips pini (Say). A novel ipsdienol dehydrogenase (IDOLDH) with a pheromone-biosynthetic gene expression pattern was cloned, expressed, functionally characterized, and its cellular localization analyzed. The cDNA has a 762nt ORF encoding a 253 amino acid predicted translation product of 28kDa and pI 5.8. The protein has conserved motifs of the Cp2 subfamily of "classical" short-chain dehydrogenases. Transcript levels were highest in pheromone producing tissue: the anterior midgut of fed males. The protein was detected only in male midguts and localized in the cytosolic fraction of midgut cells. Recombinant IDOLDH was produced in Sf9 cells using a baculovirus expression system. Enzyme assays of protein preparations showed IDOLDH used both NAD⁺ and NADP⁺ as coenzymes with specific activities in the nanomole range. Enzyme assays and GC/MS analysis showed that IDOLDH catalyzed the oxidation of racemic ipsdienol and (4R)-(-)-ipsdienol to form ipsdienone, while (4S)-(+)-ipsdienol was not a substrate. These data strongly implicate IDOLDH as an enzyme involved in terminal pheromone-biosynthetic steps, likely functioning to "tune" ipsdienol enantiomeric ratios.

Pheromone production in bark beetles.

The first aggregation pheromone components from bark beetles were identified in 1966 as a mixture of ipsdienol, ipsenol and verbenol. Since then, a number of additional components have been identified as both aggregation and anti-aggregation pheromones, with many of them being monoterpenoids or derived from monoterpenoids. The structural similarity between the major pheromone components of bark beetles and the monoterpenes found in the host trees, along with the association of monoterpenoid production with plant tissue, led to the paradigm that most if not all bark beetle pheromone components were derived from host tree precursors, often with a simple hydroxylation producing the pheromone. In the 1990 s there was a paradigm shift as evidence for de novo biosynthesis of pheromone components began to accumulate, and it is now recognized that most bark beetle monoterpenoid aggregation pheromone components are biosynthesized de novo. The bark beetle aggregation pheromones are released from the frass, which is consistent with the isoprenoid aggregation pheromones, including ipsdienol, ipsenol and frontalin, being produced in midgut tissue. It appears that exo-brevocomin is produced de novo in fat body tissue, and that verbenol, verbenone and verbenene are produced from dietary α-pinene in fat body tissue. Combined biochemical, molecular and functional genomics studies in Ips pini yielded the discovery and characterization of the enzymes that convert mevalonate pathway intermediates to pheromone components, including a novel bifunctional geranyl diphosphate synthase/myrcene synthase, a cytochrome P450 that hydroxylates myrcene to ipsdienol, and an oxidoreductase that interconverts ipsdienol and ipsdienone to achieve the appropriate stereochemistry of ipsdienol for pheromonal activity. Furthermore, the regulation of these genes and their corresponding enzymes proved complex and diverse in different species. Mevalonate pathway genes in pheromone producing male I. pini have much higher basal levels than in females, and feeding induces their expression. In I. duplicatus and I. pini, juvenile hormone III (JH III) induces pheromone production in the absence of feeding, whereas in I. paraconfusus and I. confusus, topically applied JH III does not induce pheromone production. In all four species, feeding induces pheromone production. While many of the details of pheromone production, including the site of synthesis, pathways and knowledge of the enzymes involved are known for Ips, less is known about pheromone production in Dendroctonus. Functional genomics studies are under way in D. ponderosae, which should rapidly increase our understanding of pheromone production in this genus. This chapter presents a historical development of what is known about pheromone production in bark beetles, emphasizes the genomic and post-genomic work in I. pini and points out areas where research is needed to obtain a more complete understanding of pheromone production.

Functional genomics of mountain pine beetle (Dendroctonus ponderosae) midguts and fat bodies.

BACKGROUND: The 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. RESULTS: Clones 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. CONCLUSIONS: These EST and microarray data are the first publicly-available functional genomics resources for this devastating forestry pest.

Two regulatory mechanisms of monoterpenoid pheromone production in Ips spp. of bark beetles.

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.

Isolation and characterization of farnesyl diphosphate synthase from the cotton boll weevil, Anthonomus grandis.

Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full-length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three-dimensional structure of coleopteran FPPS was determined and compared to the X-ray crystal structure of avian FPPS. The alpha-helical fold is conserved in AgFPPS and the size of the active site cavity is consistent with the enzyme being a FPPS.

Unique animal prenyltransferase with monoterpene synthase activity.

Monoterpenes are structurally diverse natural compounds that play an essential role in the chemical ecology of a wide array of organisms. A key enzyme in monoterpene biosynthesis is geranyl diphosphate synthase (GPPS). GPPS is an isoprenyl diphosphate synthase that catalyzes a single electrophilic condensation reaction between dimethylallyl diphosphate (C(5)) and isopentenyl diphosphate (C(5)) to produce geranyl diphosphate (GDP; C(10)). GDP is the universal precursor to all monoterpenes. Subsequently, monoterpene synthases are responsible for the transformation of GDP to a variety of acyclic, monocyclic, and bicyclic monoterpene products. In pheromone-producing male Ips pini bark beetles (Coleoptera: Scolytidae), the acyclic monoterpene myrcene is required for the production of the major aggregation pheromone component, ipsdienol. Here, we report monoterpene synthase activity associated with GPPS of I. pini. Enzyme assays were performed on recombinant GPPS to determine the presence of monoterpene synthase activity, and the reaction products were analyzed by coupled gas chromatography-mass spectrometry. The functionally expressed recombinant enzyme produced both GDP and myrcene, making GPPS of I. pini a bifunctional enzyme. This unique insect isoprenyl diphosphate synthase possesses the functional plasticity that is characteristic of terpene biosynthetic enzymes of plants, contributing toward the current understanding of product specificity of the isoprenoid pathway.

Myrcene hydroxylases do not determine enantiomeric composition of pheromonal ipsdienol in Ips spp.

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.