Neofunctionalization in an ancestral insect desaturase lineage led to rare Δ6 pheromone signals in the Chinese tussah silkworm

The Chinese tussah silkworm, Antheraea pernyi (Lepidoptera: Saturniidae) produces a rare dienoic sex pheromone composed of (E,Z)-6,11-hexadecadienal, (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-4,9-tetradecadienyl acetate, and for which the biosynthetic routes are yet unresolved. By means of gland composition analyses and in vivo labeling we evidenced that pheromone biosynthesis towards the immediate dienoic gland precursor, the (E,Z)-6,11-hexadecadienoic acid, involves desaturation steps with Δ⁶ and Δ¹¹ regioselectivity. cDNA cloning of pheromone gland desaturases and heterologous expression in yeast demonstrated that the 6,11-dienoic pheromone is generated from two biosynthetic routes implicating a Δ⁶ and Δ¹¹ desaturase duo albeit with an inverted reaction order. The two desaturases first catalyze the formation of the (E)-6-hexadecenoic acid or (Z)-11-hexadecenoic acid, key mono-unsaturated biosynthetic intermediates. Subsequently, each enzyme is able to produce the (E,Z)-6,11-hexadecadienoic acid by accommodating its non-respective mono-unsaturated product. Besides elucidating an unusually flexible pheromone biosynthetic pathway, our data provide the first identification of a biosynthetic Δ⁶ desaturase involved in insect mate communication. The occurrence of this novel Δ⁶ desaturase function is consistent with an evolutionary scenario involving neo-functionalization of an ancestral desaturase belonging to a gene lineage different from the Δ¹¹ desaturases commonly involved in moth pheromone biosynthesis.     

Sex pheromone biosynthesis in the tortricid moth Planotortrix excessana (Walker) involves chain-shortening of palmitoleate and oleate

Biosynthesis of the sex pheromone components, (Z)-5-tetradecenyl acetate (Z5-14:OAc) and (Z)-7-tetradecenyl acetate (Z7-14:OAc), was investigated in the New Zealand tortricid moth Planotortrix excessana (Walker) by fatty acid methyl ester (FAME) analysis of base-methanolyzed extracts of lipids in the sex pheromone gland and through application of various labelled fatty acids. Analysis of the base-methanolyzed gland extracts revealed common FAMEs, including methyl oleate and methyl palmitoleate, as well as the FAMEs of the putative precursors, methyl (Z)-5-tetradecenoate and methyl (Z)-7-tetradecenoate. Application of labelled, saturated fatty acids, myristic, palmitic, and stearic did not result in any significant incorporation of label into either of the unsaturated pheromone components, although label was incorporated into tetradecyl acetate (14:OAc). In contrast, application of labelled oleic acid resulted in incorporation of label into Z5-14:OAc but not into Z7-14:OAc or into 14:OAc, whereas application of labelled palmitoleic acid resulted in incorporation of label into Z7-14:OAc but not into Z5-14:OAc or 14:OAc. These data support a route for biosynthesis of Z5-14:OAc and Z7-14:OAc in this species by limited β-oxidation of the common fatty acyl moieties, respectively, oleate (involving two cycles of 2-carbon chain-shortening) and palmitoleate (involving only one cycle of 2-carbon chain-shortening), and apparently involving no desaturase (other than the common Δ9) specific to sex pheromone biosynthesis. Interestingly, P. excessana females biosynthesize the same component (Z5-14:OAc) from an entirely different route from that of the related species Ctenopseustis obliquana (which biosynthesizes Z5-14:OAc by Δ5-desaturation of myristate). Additionally, the pheromone biosynthesis activating neuropeptide (PBAN) stimulates pheromone biosynthesis in this species. Arch. Insect Biochem. Physiol. 37:158–167, 1998. © 1998 Wiley-Liss, Inc.     

The synergistic attractiveness effect of plant volatiles to sex pheromones in a moth

The effects of plant-derived chemicals (volatiles) on the attraction of the Spodoptera litura moth to sex pheromones were evaluated using an electroantennogram (EAG). Neuronal responses of male moths to sex pheromone mixtures (SPs) (a 9:1 mixture of synthetic (9Z,11E)-9,11-tetraddecadienyl acetate (Z9E11-14:OAc) and (9Z,12E)-9,12-tetradecadienyl acetate (Z9E12-14:OAc)) and to SPs mixtures with eight plant volatiles (benzaldehyde, (E)-β-caryophyllene, phenylacetaldehyde, 2,6-nonadienal, benzyl alcohol, racemic linalool, longifolene, and (E)-β-ocimene) were also measured. Then, wind tunnels and field trapping bioassays were conducted to determine the influence of plant volatiles on S. litura moth behavioral responses to SPs. The results indicated that benzaldehyde, phenylacetaldehyde, and benzyl alcohol significantly enhanced, and longifolene, (E)-β-caryophyllene, and (E)-β-ocimene had no significant effect on the attractions to SPs, whereas racemic linalool significantly decreased the attraction of male S. litura moths to SPs throughout the olfactory pathway. 2,6-Nonadienal significantly enhanced olfactory responses, but had no significant effect on output behavior. These findings provide foundations in utilization of plant volatiles and sex pheromones to manage the pest and other agricultural pests.     

Sex pheromone biosynthesis in the Asian corn borer Ostrinia furnacalis (II): Biosynthesis of (E)- and (Z)-12-tetradecenyl acetate involves Δ14 desaturation

Sex pheromone biosynthesis in the Asian corn borer Ostrinia furnacalis was studied by topical application of deuterium labelled fatty acids to the pheromone gland. The incorporation of the labelled acids into pheromone components and precursors was determined by gas chromatography with flame ionization detection and mass spectrometry in the selected ion monitoring mode. The labelling experiments suggest that the pheromone components (E)- and (Z)-12-tetradecenyl acetates are biosynthesized from palmitic acid by δ14 desaturation, followed by chain shortening (β-oxidation), reduction, and acetylation. This is the first confirmation of a Δ14 desaturase in an eukaryotic system.     

Acid-catalyzed transformation of 13(S)-hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids

Acid treatment of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid in tetrahydrofuran-water solvent afforded mainly (11R,12R,13S)-(Z)-12,13-epoxy-11-hydroxy-9-octadecenoic acid, diastereomeric (Z)-11,12,13-trihydroxy-9-octadecenoic acids and four isomers of (E)-9,12,13(9,10,13)-trihydroxy-10(11)-octadecenoic acid. Other minor products were oxooctadecadienoic, (E)-9(13)-hydroxy-13(9)-oxo-10(11)-octadecenoic and (E)-12-oxo-10-dodecenoic acids. A heterolytic mechanism for acid catalysis was indicated, even though most of the products characterized also have been observed as a result of homolytic decomposition of the hydroperoxide via an oxy radical. Most of the products found in this study have been observed as metabolites of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadenoic acid in biological systems, and analogous compounds have been reported as metabolites of (12S)-(5Z,8Z,10E, 14Z)-12-hydroperoxy-5,8,10,14-hydroperoxy-5,8,10,14-eicosatetraenoic acid in either blood platelets or lung tissue.     

Key biosynthetic gene subfamily recruited for pheromone production prior to the extensive radiation of Lepidoptera

Background  

Moths have evolved highly successful mating systems, relying on species-specific mixtures of sex pheromone components for long-distance mate communication. Acyl-CoA desaturases are key enzymes in the biosynthesis of these compounds and to a large extent they account for the great diversity of pheromone structures in Lepidoptera. A novel desaturase gene subfamily that displays Δ11 catalytic activities has been highlighted to account for most of the unique pheromone signatures of the taxonomically advanced ditrysian species. To assess the mechanisms driving pheromone evolution, information is needed about the signalling machinery of primitive moths. The currant shoot borer, Lampronia capitella, is the sole reported primitive non-ditrysian moth known to use unsaturated fatty-acid derivatives as sex-pheromone. By combining biochemical and molecular approaches we elucidated the biosynthesis paths of its main pheromone component, the (Z,Z)-9,11-tetradecadien-1-ol and bring new insights into the time point of the recruitment of the key Δ11-desaturase gene subfamily in moth pheromone biosynthesis.     

(10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic Acid Methyl Ester as an Anti-inflammatory Compound from Ehretia dicksonii

The methanol extract of Ehretia dicksonii provided (10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid methyl ester (1) which was isolated as an anti-inflammatory compound. Compound 1 suppressed 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 μg (the inhibitory effect (IE) was 43%). Linolenic acid methyl ester did not inhibit this inflammation at the same dose. However, the related compounds of 1, (9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (5) and (9Z,11E)- 13-oxo-9,11-octadecadienoic acid (6), showed potent activity (IE_{500 μg} of 63% and 79%, respectively). Compounds 1, 4 ((9Z,12Z,14E)-16-hydroxy-9,12,14-octadecatrienoic acid), 5 and 6 also showed inhibitory activity toward soybean lipoxygenase at a concentration of 10 μg/ml.     

Female sex pheromone of a lichen moth Eilema japonica (Arctiidae, Lithosiinae): Components and control of production

Seven candidates for components of the female sex pheromone of Eilema japonica (Arctiidae, Lithosiinae) were detected in an extract of pheromone glands with a gas chromatograph-electroantennographic detector. The compounds were identified as (Z,Z)-6,9-icosadiene (D20), (Z,Z)-6,9-henicosadiene (D21), (Z,Z,Z)-3,6,9-henicosatriene (T21), (Z,Z)-6,9-docosadiene (D22), (Z,Z,Z)-3,6,9-docosatriene (T22), (Z,Z)-6,9-tricosadiene (D23), and (Z,Z,Z)-3,6,9-tricosatriene (T23). Assays using synthetic lures in a wind tunnel showed that D21 (proportion, 0.39), T21 (0.08), D22 (0.27), and T22 (0.26) are important for evoking full behavioral responses from the males. Titers of the pheromone components did not show clear temporal fluctuations. Moreover, decapitation of the female moth had no effect on the titers of pheromone components in the pheromone gland, suggesting that cephalic endocrine factors such as pheromone biosynthesis activating neuropeptide (PBAN) are not involved in the control of pheromone biosynthesis in this species.     

Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids

Allylic hydroxylated derivatives of the C18 unsaturated fatty acids were prepared from linoleic acid (LA) and conjugated linoleic acids (CLAs). The reaction of LA methyl ester with selenium dioxide (SeO₂) gave mono-hydroxylated derivatives, 13-hydroxy-9Z,11E-octadecadienoic acid, 13-hydroxy-9E,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid and 9-hydroxy-10E,12E-octadecadienoic acid methyl esters. In contrast, the reaction of CLA methyl ester with SeO₂ gave di-hydroxylated derivatives as novel products including, erythro-12,13-dihydroxy-10E-octadecenoic acid, erythro-11,12-dihydroxy-9E-octadecenoic acid, erythro-10,11-dihydroxy-12E-octadecenoic acid and erythro-9,10-dihydroxy-11E-octadecenoic acid methyl esters. These products were purified by normal-phase short column vacuum chromatography followed by high-performance liquid chromatography (HPLC). Their chemical structures were characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). The allylic hydroxylated derivatives of LA and CLA exhibited moderate in vitro cytotoxicity against a panel of human cancer cell lines including chronic myelogenous leukemia K562, myeloma RPMI8226, hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cells (IC₅₀ 10-75 μM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD₅₀ values in the range of 2.30-13.8 μM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 μg/bee.     

Sex pheromone biosynthetic pathway in Spodoptera littoralis and its activation by a neurohormone

Deuterium-labeled fatty acids have been used to elucidate the sex pheromone biosynthetic pathway in Spodoptera littoralis. Label from palmitic acid was incorporated during the scotophase into all the pheromone acetates and their corresponding fatty acyl intermediates. (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone blend, is synthesized from palmitic acid via tetradecanoic acid, which, by the action of a specific (E)-11 desaturase and subsequently a (Z)-9 desaturase, is converted into (Z,E)-9,11-tetradecadienoate. By further reduction and acetylation, this compound leads to the dienne acetate. Deuterated precursors applied to the pheromone gland during the photophase were also incorporated into the pheromone. The percentage of labeled (Z,E)-9,11-tetradecadienyl acetate relative to natural compound was significantly higher during the light period. Label incorporation from different intermediates into the pheromone was stimulated by injection of brain-subesophageal ganglion extract during the photophase. The influence of the pheromone biosynthesis-activating neuropeptide on the biosynthetic pathway is discussed.     

Identification and behavioral evaluation of sex pheromone components of the Chinese pine caterpillar moth, Dendrolimus tabulaeformis

Background

The Chinese pine caterpillar moth, Dendrolimus tabulaeformis Tsai and Liu (Lepidoptera: Lasiocampidae) is the most important defoliator of coniferous trees in northern China. Outbreaks occur over enormous areas and often lead to the death of forests during 2–3 successive years of defoliation. The sex pheromone of D. tabulaeformis was investigated to define its chemistry and behavioral activity.

Methodology/Principal Findings

Sex pheromone was collected from calling female D. tabulaeformis by headspace solid phase microextraction (SPME) and by solvent extraction of pheromone glands. Extracts were analyzed by coupled gas chromatography/mass spectrometry (GC-MS) and coupled GC-electroantennographic detection (GC-EAD), using antennae from male moths. Five components from the extracts elicited antennal responses. These compounds were identified by a combination of retention indices, electron impact mass spectral matches, and derivatization as (Z)-5-dodecenyl acetate (Z5-12:OAc), (Z)-5-dodecenyl alcohol (Z5-12:OH), (5Z,7E)-5,7-dodecadien-1-yl acetate (Z5,E7-12:OAc), (5Z,7E)-5,7-dodecadien-1-yl propionate (Z5,E7-12:OPr), and (5Z,7E)-5,7-dodecadien-1-ol (Z5,E7-12:OH). Behavioral assays showed that male D. tabulaeformis strongly discriminated against incomplete and aberrant blend ratios. The correct ratio of Z5,E7-12:OAc, Z5,E7-12:OH, and Z5,E7-12:OPr was essential for optimal upwind flight and source contact. The two monoenes, Z5-12:OAc and Z5-12:OH, alone or binary mixtures, had no effect on behavioral responses when added to the optimal three-component blend.

Conclusions/Significance

The fact that deviations from the optimal ratio of 100∶100∶4.5 of Z5,E7-12:OAc, Z5,EZ7-12:OH, and Z5,E7-12:OPr resulted in marked decreases in male responses suggests that biosynthesis of the pheromone components is precisely controlled. The optimal blend of the sex pheromone components of D. tabulaeformis worked out in this study should find immediate use in monitoring this pest in Chinese forests.     

Reidentification of pheromone composition of Sparganothis sulfureana (Clemens) and evidence of geographic variation in male responses from two US states

GC-EAD analyses of pheromone gland extracts of calling female Sparganothis sulfureana revealed at least 6 compounds that consistently elicited antennal responses from male antennae. In addition to the major pheromone compound, (E)-11-tetradecenyl acetate (E11–14:OAc), which was previously reported, the other compounds were found to be (E)-9-dodecenyl acetate (E9–12:OAc), (Z)-9-dodecenyl acetate (Z9–12:OAc), (Z)-9-tetradecenyl acetate (Z9–14:OAc), (Z)-11-tetradecenyl acetate (Z11–14:OAc), and (E)-11-tetradecenol (E11–14:OH). Tetradecyl acetate, hexadecyl acetate and hexadecenyl acetates were also present in the extracts, but elicited no EAG response from male antennae. Wind tunnel tests demonstrated that males from New Jersey responded equally well to a blend containing five pheromone components in relative to the pheromone glands of calling females. Different male-response profiles from field-trapping tests conducted in the states of Wisconsin and New Jersey were observed, respectively. Significantly higher numbers of male S. sulfureana were caught in New Jersey in traps baited with the binary blend of E11–14:OAc (30 μg) with 1% of Z11–14:OAc, but males from Wisconsin responded equally well to traps containing blends of E11–14:OAc with 0–10% of Z11–14:OAc. The addition of more than 10% of Z11–14:OAc to the primary pheromone compound reduced male captures significantly in both states. Male catches were doubled by adding E9–12:OAc and E11–14:OH to the most attractive binary blend in both states. The trapping test with caged live virgin female moths showed that males in Wisconsin preferred females from the local population than those from New Jersey. The differences in male responses observed may indicate the existence of pheromone polymorphism in this species.     

Mimicking Insect Communication: Release and Detection of Pheromone,Biosynthesized by an Alcohol Acetyl Transferase Immobilized in a Microreactor

Infochemical production, release and detection of (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone of the moth Spodoptera littoralis, is achieved in a novel microfluidic system designed to mimic the final step of the pheromone biosynthesis by immobilized recombinant alcohol acetyl transferase. The microfluidic system is part of an “artificial gland”, i.e., a chemoemitter that comprises a microreactor connected to a microevaporator and is able to produce and release a pre-defined amount of the major component of the pheromone from the corresponding (Z,E)-9,11-tetradecadienol. Performance of the entire chemoemitter has been assessed in electrophysiological and behavioral experiments. Electroantennographic depolarizations of the pheromone produced by the chemoemitter were ca. 40% relative to that evoked by the synthetic pheromone. In a wind tunnel, the pheromone released from the evaporator elicited on males a similar attraction behavior as 3 virgin females in most of the parameters considered.     

Novel prostaglandin D2-derived activators of peroxisome proliferator-activated receptor-γ are formed in macrophage cell cultures

Incubation of RAW 264.7 murine macrophages with 9,15-dihydroxy-11-oxo-, (5Z,9α,13E,15(S))-Prosta-5,13-dien-1-oic acid [prostaglandin D₂ (PGD₂)] induced formation of considerable peroxisome proliferator-activated receptor-γ (PPARγ) activity [Nature 391 (1998) 79]. Because PGD₂ itself is a poor PPARγ ligand, we incubated RAW 264.7 macrophage cultures with prostaglandin D₂ for 24 h and studied the ability of the metabolites formed to activate PPARγ. PGD₂ products were extracted and fractionated by reverse phase high-performance liquid chromatography. Chemical identification was achieved by UV spectroscopy, gas–liquid chromatography/mass spectrometry and chemical syntheses of reference compounds. PGD₂ was converted to eight products, six of which were identified. Ligand-induced interaction of PPARγ with steroid receptor coactivator-1 was determined by glutathione-S-transferase pull-down assays and PPARγ activation was investigated by transient transfection of RAW 264.7 macrophages. In addition to the previously known ligand 11-oxo-(5Z,9,12E,14Z)-Prosta-5,9,12,14-tetraen-1-oic acid (15-deoxy-Δ^12,14-PGJ₂), a novel PPARγ ligand and activator viz. 9-hydroxy-11-oxo-, (5Z,9α,12E,14Z)-Prosta-5,12,14-trien-1-oic acid (15-deoxy-Δ^12,14-PGD₂) was identified. The biological significance of these results is currently under investigation.     

Moth sex pheromone receptors and deceitful parapheromones

The insect''s olfactory system is so selective that male moths, for example, can discriminate female-produced sex pheromones from compounds with minimal structural modifications. Yet, there is an exception for this “lock-and-key” tight selectivity. Formate analogs can be used as replacement for less chemically stable, long-chain aldehyde pheromones, because male moths respond physiologically and behaviorally to these parapheromones. However, it remained hitherto unknown how formate analogs interact with aldehyde-sensitive odorant receptors (ORs). Neuronal responses to semiochemicals were investigated with single sensillum recordings. Odorant receptors (ORs) were cloned using degenerate primers, and tested with the Xenopus oocyte expression system. Quality, relative quantity, and purity of samples were evaluated by gas chromatography and gas chromatography-mass spectrometry. We identified olfactory receptor neurons (ORNs) housed in trichoid sensilla on the antennae of male navel orangeworm that responded equally to the main constituent of the sex pheromone, (11Z,13Z)-hexadecadienal (Z11Z13-16Ald), and its formate analog, (9Z,11Z)-tetradecen-1-yl formate (Z9Z11-14OFor). We cloned an odorant receptor co-receptor (Orco) and aldehyde-sensitive ORs from the navel orangeworm, one of which (AtraOR1) was expressed specifically in male antennae. AtraOR1•AtraOrco-expressing oocytes responded mainly to Z11Z13-16Ald, with moderate sensitivity to another component of the sex pheromone, (11Z,13Z)-hexadecadien-1-ol. Surprisingly, this receptor was more sensitive to the related formate than to the natural sex pheromone. A pheromone receptor from Heliothis virescens, HR13 ( = HvirOR13) showed a similar profile, with stronger responses elicited by a formate analog than to the natural sex pheromone, (11Z)-hexadecenal thus suggesting this might be a common feature of moth pheromone receptors.     

Discovery of a disused desaturase gene from the pheromone gland of the moth Ascotis selenaria, which secretes an epoxyalkenyl sex pheromone

Female Ascotis selenaria (Geometridae) moths use 3,4-epoxy-(Z,Z)-6,9-nonadecadiene, which is synthesized from linolenic acid, as the main component of their sex pheromone. While the use of dietary linolenic or linoleic fatty acid derivatives as sex pheromone components has been observed in moth species belonging to a few families including Geometridae, the majority of moths use derivatives of a common saturated fatty acid, palmitic acid, as their sex pheromone components. We attempted to gain insight into the differentiation of pheromone biosynthetic pathways in geometrids by analyzing the desaturase genes expressed in the pheromone gland of A. selenaria. We demonstrated that a Δ11-desaturase-like gene (Asdesat1) was specifically expressed in the pheromone gland of A. selenaria in spite of the absence of a desaturation step in the pheromone biosynthetic pathway in this species. Further analysis revealed that the presumed transmembrane domains were degenerated in Asdesat1. Phylogenetic analysis demonstrated that Asdesat1 anciently diverged from the lineage of Δ11-desaturases, which are currently widely used in the biosynthesis of sex pheromones by moths. These results suggest that an ancestral Δ11-desaturase became dysfunctional in A. selenaria after a shift in pheromone biosynthetic pathways.     

Toward the Identification of the Sex Pheromone of Diatraea indigenella Dyar & Heinrich (Lepidoptera: Crambidae): Calling Behavior and Chemical Structure of a Major Component

Behavioral patterns and pheromone titer in females of the steam borer Diatraea indigenella Dyar & Heinrich were assessed for 7 days during scotophase under a 13:11 L:D photoperiod. Calling was observed from the first scotophase for most of the females. The highest percentage of calling females was recorded 6 h after the onset of scotophase. Calling bouts and length of calling were affected by age, decreasing dramatically after the sixth scotophase. By coupling chemical and electrophysiological techniques (GC–EAD, GC–MS) and olfactometer bioassays (Z,E)-9,11-hexadecadienal was identified as the main sex pheromonal component in the extracts of female glands. The concentration varied from 2.53 to 13.7 ng gland^?1 with a peak at the 6th hour of scotophase. In addition, two unidentified secondary active components were detected at very low concentrations. Behavioral assays showed that 86% of the male moths were more attracted to the gland extracts than to hexane (control), and 77% of the male moths were more attracted to the gland extract than to the synthetic major component (Z,E)-9,11-hexadecadienal. However, 68% of males preferred the synthetic major component over hexane. The identification of the major sex pheromonal component represents an important step toward the complete elucidation of the composition of the sex pheromone of D. indigenella in order to develop monitoring tools.