Cloning and functional characterization of a fatty acid transport protein (FATP) from the pheromone gland of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone

Sex pheromones of moths are largely classified into two types based on the presence (Type I) or absence (Type II) of a terminal functional group. While Type-I sex pheromones are synthesized from common fatty acids in the pheromone gland (PG), Type-II sex pheromones are derived from hydrocarbons produced presumably in the oenocytes and transported to the PG via the hemolymph. Recently, a fatty acid transport protein (BmFATP) was identified from the PG of the silkworm Bombyx mori, which produces a Type-I sex pheromone (bombykol). BmFATP was shown to facilitate the uptake of extracellular fatty acids into PG cells for the synthesis of bombykol. To elucidate the presence and function of FATP in the PG of moths that produce Type-II sex pheromones, we explored fatp homologues expressed in the PG of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone (Type II). A fatp homologue cloned from E. japonica (Ejfatp) was predominantly expressed in the PG, and its expression is upregulated shortly after eclosion. Functional expression of EjFATP in Escherichia coli enhanced the uptake of long chain fatty acids (C₁₈ and C₂₀), but not pheromone precursor hydrocarbons. To the best of our knowledge, this is the first report of the cloning and functional characterization of a FATP in the PG of a moth producing a Type-II sex pheromone. Although EjFATP is not likely to be involved in the uptake of pheromone precursors in E. japonica, the expression pattern of Ejfatp suggests a role for EjFATP in the PG not directly linked to pheromone biosynthesis.     

Molecular mechanisms underlying sex pheromone production in the silkmoth, Bombyx mori: characterization of the molecular components involved in bombykol biosynthesis

Many species of female moths produce sex pheromones to attract conspecific males. To date, sex pheromones from more than 570 moth species have been chemically identified. Most moth species utilize Type I pheromones that consist of straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester and usually with several double bonds. In contrast, some moth species use unsaturated hydrocarbons or hydrocarbon epoxides, classified as Type II lepidopteran pheromones, as sex pheromones. Studies over the past three decades have demonstrated that female moths usually produce sex pheromones as multi-component blends where the ratio of the individual components is precisely controlled, thus making it possible to generate species-specific pheromone blends. As for the biosynthesis of Type I pheromones, it is well established that they are de novo synthesized in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways. However, as many of the molecular components within the PG cells (i.e., enzymes, proteins, and small regulatory molecules) have not been functionally characterized, the molecular mechanisms underlying sex pheromone production in PG cells remain poorly understood. To address this, we have recently characterized some of the molecules involved in the biosynthesis of the sex pheromone bombykol in the silkmoth, Bombyx mori. Characterization of these, and other, key molecules will facilitate our understanding of the precise mechanisms underlying lepidopteran sex pheromone production.     

Incorporation of fatty acids into cuticular hydrocarbons of male and female Drosophila melanogaster

The biosynthesis of cuticular hydrocarbons was investigated in male and female Drosophila melanogaster (Canton-S strain), especially in those with a pheromonal role i.e. male 7-tricosene and female 7, 11- heptacosadiene. The incorporation of radioactivity was followed after topical application of (14)C-labelled myristic, palmitic and stearic acid and (3)H-labelled cis-vaccenic acid on one to ten day old flies. The incorporation levels into unsaturated hydrocarbons are similar in both sexes, depending markedly on the chain length of the saturated precursor, with a maximum level from myristic acid. Cis-vaccenic acid leads only to unsaturated compounds. With this precursor, there is an enhanced incorporation into female monoenes and dienes, maximum in two to three day old females. The total fatty acid composition shows the highest abundance of fatty acids with 16 carbon atoms and the presence of a major position for double bond, Delta9. Moreover, cis-vaccenic acid and 17-tetracosenoic acid are identified by GC-MS analysis. These data support an elongation-decarboxylation mechanism for the biosynthesis of D. melanogaster cuticular hydrocarbons. Its early steps for male monoenes and female monoenes and dienes might involve a Delta9 desaturase transforming palmitic acid into palmitoleic acid which would then be elongated into vaccenic acid, an important common precursor for all pheromones.     

Involvement of desat1 gene in the control of Drosophila melanogaster pheromone biosynthesis

Cuticular pheromones in Drosophila melanogaster are unsaturated hydrocarbons with at least one double bond in position 7: 7-tricosene and 7-pentacosene in males and 7,11-heptacosadiene and 7,11-nonacosadiene in females. We have previously shown that a desaturase gene, desat1, located in chromosome region 87 C could be involved in this process: the Desat1 enzyme preferentially leads to the synthesis of palmitoleic acid, a precursor of 7 fatty acids and 7-unsaturated hydrocarbons. Therefore, we have searched for P–elements in the 87 region and mapped them. One was found inserted into the first intron of the desat1 gene. Flies heterozygous for this insertion showed a large decrease in the level of 7-unsaturated hydrocarbons, comparable to that observed in flies heterozygous for a deficiency overlapping desat1. Less than 1% of flies homozygous for this insertion were viable. They were characterized by dramatic pheromone decreases. After excision of the transposon, the pheromone phenotype was reversed in 69% of the lines and the other excision lines had more or less decreased amounts of 7-unsaturated hydrocarbons. All these results implicate desat1 in the synthesis of Drosophila pheromones.     

Production of moth sex pheromones for pest control by yeast fermentation

The use of insect sex pheromones is an alternative technology for pest control in agriculture and forestry, which, in contrast to insecticides, does not have adverse effects on human health or environment and is efficient also against insecticide-resistant insect populations. Due to the high cost of chemically synthesized pheromones, mating disruption applications are currently primarily targeting higher value crops, such as fruits. Here we demonstrate a biotechnological method for the production of (Z)-hexadec-11-en-1-ol and (Z)-tetradec-9-en-1-ol, using engineered yeast cell factories. These unsaturated fatty alcohols are pheromone components or the immediate precursors of pheromone components of several economically important moth pests. Biosynthetic pathways towards several pheromones or their precursors were reconstructed in the oleaginous yeast Yarrowia lipolytica, which was further metabolically engineered for improved pheromone biosynthesis by decreasing fatty alcohol degradation and downregulating storage lipid accumulation. The sex pheromone of the cotton bollworm Helicoverpa armigera was produced by oxidation of fermented fatty alcohols into corresponding aldehydes. The resulting yeast-derived pheromone was just as efficient and specific for trapping of H. armigera male moths in cotton fields in Greece as a conventionally produced synthetic pheromone mixture. We further demonstrated the production of (Z)-tetradec-9-en-1-yl acetate, the main pheromone component of the fall armyworm Spodoptera frugiperda. Taken together our work describes a biotech platform for the production of commercially relevant titres of moth pheromones for pest control via yeast fermentation.     

Stereochemical studies on algal pheromone biosynthesis. A model study with the flowering plant Senecio isatideus (Asteraceae)

Unsaturated C8 and C11 hydrocarbons act as chemical signals (chemotaxis) during sexual reproduction of many marine brown algae. One of these compounds, namely (+)-(6S)-6-(1Z-butenyl)cyclohepta-1,4-diene (= ectocarpene) is also formed as a major hydrocarbon by the flowering plant Senecio isatideus (Asteraceae). On administration of enantiospecifically labelled (8R)- or (8S)-[7,8-2H2]trideca-3,6,9-trienoic acid instead of the natural precursor dodeca-3,6,9-trienoic acid to this plant, the artificial C12 analogue of ectocarpene is formed. Mass spectroscopic analysis of the metabolites revealed this process to be enantiospecific for the C(8)-HRe atom of the precursor acid. The stereochemical course of the overall reaction is in agreement with a precise, U-shaped embedding of the trienoic acid into the active center of the enzyme(s) and Re-attack onto a hydrogen at the C(8) methylene group of this precursor. The reactive intermediate cyclizes by pi-orbital interaction between C(4) and C(6) of the acid and is accompanied by decarboxylation. The first product is a thermally unstable (1S,2R)-cis-1-(1E,3Z-hexadienyl)-2-vinylcyclopropane, which immediately rearranges to (+)-(6S)-ectocarpene (homo-Cope rearrangement). The present work provides first experimental evidence for a homo-Cope rearrangement as a naturally occurring electrocyclic reaction.     

Pheromone biosynthetic pathways in the moths Heliothis subflexa and Heliothis virescens

Sex pheromones of many moth species have relatively simple structures consisting of a hydrocarbon chain with a functional group and one to several double bonds. These sex pheromones are derived from fatty acids through specific biosynthetic pathways. We investigated the incorporation of deuterium-labeled tetradecanoic, hexadecanoic, and octadecanoic acid precursors into pheromone components of Heliothis subflexa and Heliothis virescens. The two species utilize (Z)11-hexadecenal as the major pheromone component, which is produced by Delta11 desaturation of hexadecanoic acid. H. subflexa also produced (Z)11-hexadecanol and (Z)-11-hexadecenyl acetate via Delta11 desaturation. In H. subflexa, octadecanoic acid was used to biosynthesize the minor pheromone components (Z)9-hexadecenal, (Z)9-hexadecenol, and (Z)9-hexadecenyl acetate. These minor components are produced by Delta11 desaturation of octadecanoic acid followed by one round of chain-shortening. In contrast, H. virescens used hexadecanoic acid as a substrate to form (Z)11-hexadecenal and (Z)11-hexadecenol and hexadecenal. H. virescens also produced (Z)9-tetradecenal by Delta11 desaturation of the hexadecanoic acid followed by one round of chain-shortening and reduction. Tetradecanoic acid was not utilized as a precursor to form Z9-14:Ald in H. virescens. This labeling pattern indicates that the Delta11 desaturase is the only active desaturase present in the pheromone gland cells of both species.     

Selectivity and neuroendocrine regulation of the precursor uptake by pheromone glands from hemolymph in geometrid female moths, which secrete epoxyalkenyl sex pheromones

Macrolepidopteran female moths in families such as Geometridae produce epoxyalkenyl sex pheromones, which are biosynthesized via epoxidation of polyunsaturated hydrocarbons in their pheromone glands. The precursors, however, are expected to be produced outside of the pheromone glands, probably in oenocytes or in the fat body, and transported to the glands via hemolymph. Based on these facts, the selectivity of the epoxidation substrates and of the precursor uptake by pheromone glands was examined with two geometrid species, Hemerophila artilineata and Ascotis selenaria cretacea, using binary mixtures of deuterated precursors and their analogs, which were topically applied to the pheromone glands or injected into the abdomen. GC-MS measurements of pheromone extracts showed equal epoxidation of two polyenes, indicating a low selectivity for both processes, while the epoxidation proceeded at only one double bond specific to each species. This result makes it possible to conclude that the formation of species-specific epoxyalkenyl pheromones results from the rigid formation of polyunsaturated precursors and their epoxidation at a fixed position. Next, the neuroendocrine regulation of these processes was studied with in vivo and in vitro experiments using decapitated females. The epoxy pheromones disappeared completely within 36 h of decapitation, and epoxidation of the injected precursors was not detected in the decapitated females, which restarted the reaction by treatment with a pheromone biosynthesis-activating neuropeptide (PBAN). The precursors topically applied to glands of the decapitated females, however, were converted into epoxy pheromones without PBAN, indicating that this neuropeptide hormone accelerated the precursor uptake by pheromone glands but not the epoxidation already underway in the glands.     

Qualitative and quantitative determination of pheromone secretion in female gametes of Ectocarpus siliculosus (Phaeophyceae)

Pheromone secretion in living female gametes of Ectocarpus siliculosus was examined by closed-loop extraction and gas chromatography. The pheromone (ectocarpene) recovery efficiency was calibrated with synthetic ectocarpene and was found to depend strongly on the volume of the closed-loop system. Maximum extraction efficiency of ectocarpene (17.4%) was obtained with a small-volume system. In addition to the main compound ectocarpene, the pheromone bouquet of E. siliculosus contained minor amounts of hormosirene, multifidene and dictyotene, which until now are known only as sperm-attractants in other brown algae. The pheromone bouquet was identical in 4 clones of different geographic origins. Settled female gametes of Ectocarpus siliculosus continued to secrete pheromone and to attract male gametes for up to 7 h after their release with an ectocarpene secretion rate of 1 x 10(5) molecules s-1 cell-1.     

Expression profiling reveals differential gene induction underlying specific and non-specific memory for pheromones in mice

Memory for the mating male’s pheromones in female mice is thought to require synaptic changes in the accessory olfactory bulb (AOB). Induction of this memory depends on release of glutamate in response to pheromonal exposure coincident with release of norepinephrine (NE) in the AOB following mating. A similar memory for pheromones can also be induced artificially by local infusion of the GABA_A receptor antagonist bicuculline into the AOB. The natural memory formed by exposure to pheromones during mating is specific to the pheromones sensed by the female during mating. In contrast, the artificial memory induced by bicuculline is non-specific and results in the female mice recognizing all pheromones as if they were from the mating male. Although protein synthesis has been shown to be essential for development of pheromone memory, the gene expression cascades critical for memory formation are not known. We investigated changes in gene expression in the AOB using oligonucleotide microarrays during mating-induced pheromone memory (MIPM) as well as bicuculline-induced pheromone memory (BIPM). We found the set of genes induced during MIPM and BIPM are largely non-overlapping and Ingenuity Pathway Analysis revealed that the signaling pathways in MIPM and BIPM also differ. The products of genes induced during MIPM are associated with synaptic function, indicating the possibility of modification at specific synapses, while those induced during BIPM appear to possess neuron-wide functions, which would be consistent with global cellular changes. Thus, these results begin to provide a mechanistic explanation for specific and non-specific memories induced by pheromones and bicuculline infusion respectively.     

昆虫神经肽PBAN的细胞内信号转导

昆虫性信息素多数为长链的不饱和醇、醋酸酯、醛或酮类,链长一般为10-20碳,主要在性信息素腺体内由乙酰辅酶A经过脂肪酸合成、碳链缩短、去饱和以及碳酰基的还原修饰等步骤合成的;而性信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)是由昆虫食管下神经节中的部分神经细胞合成和分泌的神经肽,通常由33个氨基酸组成,在C-末端有一个相同的五肽序列,主要调控性信息素的生物合成。有关PBAN的细胞内信号转导是近几年的研究热点,研究显示 PBAN首先与性信息素腺体细胞表面的G蛋白偶联受体结合,随后依据昆虫种类的不同,其细胞内信号转导方式主要有三种:(1)以cAMP信号传导途径进行信号转导;(2)以cAMP和磷脂酰肌醇信号传导途径共同进行信号转导;(3)主要以Ca2 为第二信使进行信号传导。     

Polyunsaturated hydrocarbons in the hemolymph: biosynthetic precursors of epoxy pheromones of geometrid and arctiid moths

Female moths of many species in Geometridae, Arctiidae and some other macrolepidopteran families produce epoxy pheromones, which are probably derived from polyunsaturated hydrocarbons. In order to understand a biosynthetic site, hemolymph from both sexes of two geometrid species, Ascotis selenaria cretacea and Hemerophila artilineata, and one arctiid species, Spilosoma imparilis, was shaken with n-hexane and the solvent extracts were analyzed by GC-MS. Each extract of the female hemolymph sex-specifically included polyunsaturated hydrocarbons corresponding to the pheromonal epoxy components in addition to many saturated hydrocarbons, but no epoxy compounds were detected in it. Based on this analysis, deuterated polyunsaturated hydrocarbons were injected into the abdomens of two geometrid females, and the labeled epoxy components were successfully yielded from the pheromone glands. This result indicated that the polyunsaturated hydrocarbons occurring in the female hemolymph were direct pheromone precursors, which might be produced outside the pheromone gland, probably in oenocytes associated with abdominal epidermal cells or in the fat body, and transported to the pheromone gland via the hemolymph for their epoxydation and emission into the atmosphere.     

Pheromone biosynthetic pathways in the moths Helicoverpa zea and Helicoverpa assulta

Sex pheromones of many Lepidopteran species have relatively simple structures consisting of a hydrocarbon chain with a functional group and usually one to several double bonds. The sex pheromones are usually derived from fatty acids through a specific biosynthetic pathway. We investigated the incorporation of deuterium-labeled palmitic and stearic acid precursors into pheromone components of Helicoverpa zea and Helicoverpa assulta. The major pheromone component for H. zea is (Z)11-hexadecenal (Z11-16:Ald) while H. assulta utilizes (Z)9-hexadecenal (Z9-16:Ald). We found that H. zea uses palmitic acid to form Z11-16:Ald via delta 11 desaturation and reduction, but also requires stearic acid to biosynthesize the minor pheromone components Z9-16:Ald and Z7-16:Ald. The Z9-16:Ald is produced by delta 11 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. The Z7-16:Ald is produced by delta 9 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. H. assulta uses palmitic acid as a substrate to form Z9-16:Ald, Z11-16:Ald and 16:Ald. The amount of labeling indicated that the delta 9 desaturase is the major desaturase present in the pheromone gland cells of H. assulta; whereas, the delta 11 desaturase is the major desaturase in pheromone glands of H. zea. It also appears that H. assulta lacks chain-shortening enzymes since stearic acid did not label any of the 16-carbon aldehydes.     

Flexible origin of hydrocarbon/pheromone precursors in Drosophila melanogaster

In terrestrial insects, cuticular hydrocarbons (CHCs) provide protection from desiccation. Specific CHCs can also act as pheromones, which are important for successful mating. Oenocytes are abdominal cells thought to act as specialized units for CHC biogenesis that consists of long-chain fatty acid (LCFA) synthesis, optional desaturation(s), elongation to very long-chain fatty acids (VLCFAs), and removal of the carboxyl group. By investigating CHC biogenesis in Drosophila melanogaster, we showed that VLCFA synthesis takes place only within the oenocytes. Conversely, several pathways, which may compensate for one another, can feed the oenocyte pool of LCFAs, suggesting that this step is a critical node for regulating CHC synthesis. Importantly, flies deficient in LCFA synthesis sacrificed their triacylglycerol stores while maintaining some CHC production. Moreover, pheromone production was lower in adult flies that emerged from larvae that were fed excess dietary lipids, and their mating success was lower. Further, we showed that pheromone production in the oenocytes depends on lipid metabolism in the fat tissue and that fatty acid transport protein, a bipartite acyl-CoA synthase (ACS)/FA transporter, likely acts through its ACS domain in the oenocyte pathway of CHC biogenesis. Our study highlights the importance of environmental and physiological inputs in regulating LCFA synthesis to eventually control sexual communication in a polyphagous animal.     

CHEMISTRY OF ATTRACTION AND DEFENSE IN MACRO- AND MICROALGAE

Boland, W. & Pohnert, G. Max-Planck-Institut of Chemical Ecology, Carl-Zeiss-Promenade 10, 07745 Jena, Germany A diverse group of brown seaweeds produce bouquets of C₁₁ and C₈ metabolites, some of which act as pheromones that trigger gamete release or attract sperm to eggs following release [1]. The same compounds and, especially, their oxidative degradation products frequently and strongly deter feeding by mezograzers (Ampithoe longimana) which often consume seaweed spores, zygotes, and juveniles [2]. Besides macroalgae also several microalgae (ca. 20 Gomphonema spp., Asterionella formosa; Diatomophyceae) produce C11 and C8 hydrocarbons along with a toxic polar compound from the pool of highly unsaturated fatty acids (eicosanoids) [3]. In biosynthetic studies with cell free extracts of the diatom G. parvulum (9S)-hydroperoxy-eicosatetraenoic (9S-HPETE) acid was shown to be converted by a novel type of a lyase into the defensive com-pound 9-oxo-nona-5Z,7E-dienoic acid along with cyclic and linear C₁₁ hydrocarbons [4]. If the eicosanoid pre-cursors are first con-verted into C-12 hydroperoxides, subsequent lyase activity produces C₈ trienes together with 12-oxo-dodeca-5Z,8Z,10E-dienoic acid (A. formosa). Both unsaturated acids serve as efficient chemi-cal defenses against attacking feeders and microorganisms. The reaction cascade is triggered by damage of the diatom, initiating rapid release of the free fatty acid from phospholipids followed by lipoxygenation and oxidative cleavage into the unsaturated hydrocarbon and the oxo acid [5]. 1. Boland W 1995 Proc. Natl. Acad. Sci. 92:31-43. 2. Hay ME, Piel J, Boland W, Schnitzler I 1998 Chemoecology 8:91-98 3. Pohnert G, Boland W 1997 Tetrahedron 53:13681-13694. 4. Hombeck M, Pohnert G, Boland W 1999 J. Chem. Soc., Chem. Commun., 243-244. 5. Pohnert G 2000 Angew. Chem. 112:4506-4508.     

Nonadecadienone, a new termite trail-following pheromone identified in Glossotermes oculatus (Serritermitidae)

Within the multitude of chemical signals used by termites, the trail marking by means of pheromones is ubiquitous. Chemistry and biology of the trail-following communication have been described in more than 60 species from all families except for the Neotropical Serritermitidae. The chemical ecology of Serritermitidae is of special interest not only as a missing piece of knowledge on the diversity and evolution of isopteran pheromones but also because it may contribute to the debate on the phylogenetic position of this family, which is still unresolved. Therefore, we aimed in this study to identify the trail-following pheromone of the serritermitid Glossotermes oculatus. Based on a combined approach of analytical chemistry, electrophysiology, and behavioral bioassays, we propose (10Z,13Z)-nonadeca-10,13-dien-2-one to be the trail-following pheromone of G. oculatus, secreted by the sternal gland of pseudergates. Thus, we report on a new termite trail-following pheromone of an unexpected chemical structure, a ketone with 19 carbons, contrasting with unsaturated alcohols containing 12 carbons as trail-following pheromones in other advanced termite families. In addition to this unique trail-following pheromone, we also describe the sternal gland in pseudergates as an organ of unusual shape, size, and structure when compared with other isopteran species. These results underline the peculiarity of the family Serritermitidae and prompt our interest in the chemistry of pheromones in the other genus of the family, Serritermes.     

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.     

蛾类昆虫性信息素生物合成的研究进展

综述了各种不同化学结构类型的蛾类雌性信息素生物合成途径。此外还叙述了特定比例的性信息素成分在雌蛾体内产生的机理以及某些蛾类中信息素生物合成酶类与物种进化间的关系。