Fatty acyl pheromone analogue-containing lipids and their roles in sex pheromone biosynthesis in the lightbrown apple moth, Epipyhas postvittana (Walker)

The pheromone gland of the moth Epiphyas postvittana was analysed for lipids containing the fatty acyl pheromone analogue (FAPA) of the component, (E)-11-tetradecenyl acetate. The FAPA was found predominantly in the triglycerides (TGs), and to a lesser extent in the choline phosphatides. The FAPA was found to be exclusively on the sn-1 or sn-3 position (probably the latter) of the TGs. When pheromone gland lipid extracts were eluted through silica solid phase extraction, a significant proportion of the FAPA was not recovered. Changes in titre of this non-recoverable FAPA paralleled changes in pheromone titre in females. In contrast, changes in recoverable FAPA (mostly in the TGs) titre showed a gradual increase with time after eclosion. The properties of this non-recoverable FAPA were consistent with it being the CoA ester of the FAPA. Thus, it appears that the FAPA-CoA ester is the immediate lipid precursor of the pheromone, and that the FAPA-containing TGs are formed by reaction of the FAPA-CoA with 1,2-DGs, as a consequence of the rate-limiting reduction of the FAPA-CoA. Finally, injection of PBAN into females decapitated for 3 days resulted in a decrease in recoverable FAPA and an increase in non-recoverable FAPA, suggesting that PBAN influences the lipolysis of TGs. Overall these data suggest that there are two routes for biosynthesis of the pheromone component E11-14:OAc in E. postvittana: a de novo route, directly via the CoA esters of the various fatty acid intermediates, and a less direct route via the lipolysis of FAPA-containing TGs.     

Evidence for two-step regulation of pheromone biosynthesis by the pheromone biosynthesis-activating neuropeptide in the moth Heliothis virescens

The control of pheromone biosynthesis by the neuropeptide PBAN was investigated in the moth Heliothis virescens. When decapitated females were injected with [2-(14)C] acetate, females co-injected with PBAN produced significantly greater quantities of radiolabeled fatty acids in their pheromone gland than females co-injected with saline. This indicates that PBAN controls an enzyme involved in the synthesis of fatty acids, probably acetyl CoA carboxylase. Decapitated females injected with PBAN showed a rapid increase in native pheromone, and a slower increase in the pheromone precursor, (Z)-11-hexadecenoate. Total native palmitate and stearate (both pheromone intermediates) showed a significant decrease after PBAN injection, before their titers were later restored to initial levels. In contrast, the acyl-CoA thioesters of these two saturated fatty acids increased during the period when their total titers decreased. When a mixture of labeled palmitic and heptadecanoic (an acid that cannot be converted to pheromone) acids was applied to the gland, PBAN-injected females produced greater quantities of labeled pheromone and precursor than did saline-injected ones. The two acids showed similar time-course patterns, with no difference in total titers of each of the respective acids between saline- and PBAN-injected females. When labeled heptadecanoic acid was applied to the gland alone, there was no difference in titers of either total heptadecanoate or of heptadecanoyl-CoA between PBAN- and saline-injected females, suggesting that PBAN does not directly control the storage or liberation of fatty acids in the gland, at least for this fatty acid. Overall, these data indicate that PBAN also controls a later step involved in pheromone biosynthesis, perhaps the reduction of acyl-CoA moieties. The control by PBAN of two enzymes, near the beginning and end of the pheromone biosynthetic process, would seem to allow for more efficient utilization of fatty acids and pheromone than control of only one enzyme.     

Biosynthesis of lepidopteran pheromones

Research on lepidopteran sex pheromone component biosynthetic pathways has revealed general systems that may have significance in understanding the evolution of these moth mating communication signals. Studies with the redbanded leafroller moth, cabbage looper moth, and the domestic silkworm showed that they all possess a unique delta-11 unsaturated acid precursor. Radiolabeled precursor acids were used to show that various combinations of limited beta-oxidation chain-shortening or chain-elongation steps with the desaturase enzyme could produce most of the pheromone components identified for noctuid, pyralid, and tortricid moths. Evolution of the delta-11 desaturase enzyme from the ubiquitous delta-9 desaturase enzyme was suggested by finding primitive species that use the intermediate delta-10 desaturase enzyme. It is suggested that pheromone components of other primitive species are produced by using only the chain-shortening steps on available oleate, linoleate, and linolenate. Pheromone componets of some more advanced species appear to be produced by chain elongation of these available acids, with subsequent reductive decarboxylation to hydrocarbon.     

Functional characterization of a desaturase from the tobacco hornworm moth (Manduca sexta) with bifunctional Z11- and 10,12-desaturase activity

The pheromone blend produced by the tobacco hornworm moth (Manduca sexta) (L.) female is unusually complex and contains two conjugated dienals and trienals together with two monounsaturated alkenals. Here, we describe the identification and construction of two genes encoding MsexKPSE and MsexAPTQ desaturases from a cDNA library prepared from the total RNA of the M. sexta pheromone gland. The MsexKPSE desaturase shares a high degree of similarity with Delta(9)-desaturases from different moth species. The functional expression of MsexAPTQ desaturase in Saccharomyces cerevisiae followed by a detailed GC-MS analysis of fatty acid methyl esters (FAME) and their derivatized products and gas-phase Fourier transform infrared (FTIR) spectroscopy of the extracted FAME confirms that this enzyme is a bifunctional Z-Delta(11)-desaturase. MsexAPTQ desaturase catalyses the production of Z11-hexadecenoate (Z11-16) and Z10E12- and E10E12-hexadecadienoates (Z10E12-16) via 1,4-desaturation of the Z11-16 substrate. The stereochemistry of 1,4-desaturation and formation of isomers is discussed.     

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.     

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.     

cDNA cloning and in situ hybridization of Delta11-desaturase, a key enzyme of pheromone biosynthesis in Ostrinia scapulalis (Lepidoptera: Crambidae)

Female sex pheromones are considered to be produced in a "pheromone gland" located in the terminal abdominal segments (8th-10th, TAS) of a moth; however, in many moth species, the cells that produce pheromones have not actually been specified. We investigated cells in the TAS that synthesize pheromones in the adzuki bean borer Ostrinia scapulalis, by locating pheromones and their precursors, and mRNA for Delta11-desaturase, a key enzyme in pheromone biosynthesis. We demonstrated that the pheromone components, (E)-11- and (Z)-11-tetradecenyl acetates, and their fatty acyl precursors were specifically contained in the dorsal part of the TAS. A cDNA (OscaZ/E11) that encodes a Delta11-desaturase was cloned from the TAS. RT-PCR and in situ hybridization unequivocally showed that OscaZ/E11 is specifically expressed in the modified epidermal cells located at the dorsal end of the 8th-9th intersegmental membrane.     

Phenotypic plasticity in sexual communication signal of a noctuid moth

Variability within sex pheromone signalling systems is generally believed to be low because of strong stabilizing selection; yet the noctuid moth Heliothis subflexa (Hs) shows significant intraspecific variation. One possible explanation is that females may alter their sex pheromone blend depending on prevailing olfactory cues in the habitat, which we termed the 'experience hypothesis'. This could be adaptive if Hs females experiencing the pheromone of another species, Heliothis virescens (Hv), responded to reduce the frequency of heterospecific matings. We exposed Hs females to no pheromone, Hs pheromone or Hv pheromone in the first 3 days of their adult lives. Hs females in the latter treatment produced significantly more of the acetate Z11-16:OAc, which inhibits the attraction of Hv males. To our knowledge, this is the first study showing adaptive phenotypic plasticity in a moth sex pheromone and suggests that behavioural differentiation may precede genetic divergence in the sexual signals of moths.     

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.     

Improved monitoring of female codling moth (Lepidoptera: Tortricidae) with pear ester plus acetic acid in sex pheromone-treated orchards

The performance of clear delta traps baited with 3.0 mg of pear ester, ethyl (E,Z)-2,4-decadienoate, and 5.0 ml of acetic acid in separate lures was compared with orange delta traps baited with a single lure containing 3.0 mg of both pear ester and the sex pheromone, (E,E)-8,10-dodecadien-1-ol (codlemone) for codling moth, Cydia pomonella (L.), in apple, Malus domestica (Borkhausen). Residual analyses and field tests demonstrated that both the pear ester and acetic acid lures were effective for at least 8 wk. The two trap-lure combinations caught a similar number of total moths in an orchard treated with sex pheromone dispensers during short-term trials in 2008. However, the mean catch of female moths was significantly higher and male moths significantly lower in clear traps baited with pear ester and acetic acid versus orange traps baited with pear ester and codlemone. Season-long studies were conducted with these two trap-lure combinations in orchards treated with (n = 6) and without (n = 7) sex pheromone dispensers during 2009. The two trap-lure combinations caught similar numbers of moths in dispenser-treated orchards. In contrast, total catch was significantly higher (>2-fold) in the orange compared with the clear traps in untreated orchards. The clear caught >6-fold more females than the orange trap in both types of orchards. These studies suggest that deploying clear delta traps baited with pear ester and acetic acid can be an effective monitoring tool for female codling moth and an alternative to codlemone-baited traps in sex pheromone-treated orchards.     

Sex pheromone biosynthesis in the processionary moth Thaumetopoea pityocampa by delta-13 desaturation

In vivo treatments of female sex pheromone glands of the processionary moth, Thaumetopoea pityocampa, with mass-labeled fatty acids showed that (Z)-13-hexadecen-11-ynyl acetate, the main sex pheromone component, is biosynthesized from palmitic acid by the combined action of delta-11 and delta-13 desaturases. The involvement of this unusual delta-13 has been proven by application of [16,16,16-2H3] [1,2-13C2]-hexadecanoic acid to the glands with a resultant incorporation of all labeled atoms into the pheromone and each one of the corresponding intermediates. These results seem to exclude alternative biosynthetic pathways, such as chain shortening and elongation combined with delta-11 desaturation. The delta-11 desaturase responsible for the formation of the triple bond in both the 11-hexadecynoyl and (Z)-13-hexadecen-11-ynoyl intermediates is also an unusual enzyme not previously reported in lepidopteran sex pheromone biosynthesis.     

A single sex pheromone receptor determines chemical response specificity of sexual behavior in the silkmoth Bombyx mori

In insects and other animals, intraspecific communication between individuals of the opposite sex is mediated in part by chemical signals called sex pheromones. In most moth species, male moths rely heavily on species-specific sex pheromones emitted by female moths to identify and orient towards an appropriate mating partner among a large number of sympatric insect species. The silkmoth, Bombyx mori, utilizes the simplest possible pheromone system, in which a single pheromone component, (E, Z)-10,12-hexadecadienol (bombykol), is sufficient to elicit full sexual behavior. We have previously shown that the sex pheromone receptor BmOR1 mediates specific detection of bombykol in the antennae of male silkmoths. However, it is unclear whether the sex pheromone receptor is the minimally sufficient determination factor that triggers initiation of orientation behavior towards a potential mate. Using transgenic silkmoths expressing the sex pheromone receptor PxOR1 of the diamondback moth Plutella xylostella in BmOR1-expressing neurons, we show that the selectivity of the sex pheromone receptor determines the chemical response specificity of sexual behavior in the silkmoth. Bombykol receptor neurons expressing PxOR1 responded to its specific ligand, (Z)-11-hexadecenal (Z11-16:Ald), in a dose-dependent manner. Male moths expressing PxOR1 exhibited typical pheromone orientation behavior and copulation attempts in response to Z11-16:Ald and to females of P. xylostella. Transformation of the bombykol receptor neurons had no effect on their projections in the antennal lobe. These results indicate that activation of bombykol receptor neurons alone is sufficient to trigger full sexual behavior. Thus, a single gene defines behavioral selectivity in sex pheromone communication in the silkmoth. Our findings show that a single molecular determinant can not only function as a modulator of behavior but also as an all-or-nothing initiator of a complex species-specific behavioral sequence.     

Effect of a neuroendocrine factor on sex pheromone biosynthesis in the tomato looper,Chrysodeixis chalcites (Lepidoptera: Noctuidae)

The presence of a pheromone biosynthesis activating neurohormone in the head gandlia, and its effect on the sex phermone biosynthetic pathway, were investigated in the tomato looper, Chrysodeixis chalcites (Esper). Comparison of pheromone components and precursor levels in the presence and absence of the factor was performed using untreated, ligated and ligated and injected virgin females. Pheromone glands of treated and untreated moths were extracted and analyzed by capillary gas chromatography for their most abundant pheromone components, (Z)-7-dodecenyl acetate and (Z)-9-tetradecenyl acetate, and the putative biosynthetic precursors hexadecanoate, (Z)-11-hexadecenoate, (Z)-9-tetradecenoate and (Z)-7-dodecenoate. Comparison of the amounts of the pheromone and precursor components in the three groups of females indicated that a neuroendocrine factor is involved in the regulation of the pheromone biosynthesis in C. chalcites. Lack of such a factor resulted in a marked decrease of the sex pheromone components as well as the three unsaturated putative biosynthetic precursors. However, no decrease was observed in the content of palmitoate, suggesting that the Δ11 desaturation step is affected by the neuroendocrine factor. Injection of head ganglia extracts into ligated females resulted in a recovery of unsaturated precursor and phermone content. Both male and female head ganglia were found to contain a sex pheromone biosynthesis regulatory factor. However, the stimulatory pattern of the factor from the two sexes was different, suggesting that the two factors are quantitatively and/or qualitatively distinct.     

Fatty acid and sex pheromone changes and the role of glandular lipids in the Z-strain of the European corn borer, Ostrinia nubilalis (Hübner)

Lipids in the sex pheromone gland of females of the Z-strain of Ostrinia nubilalis were analyzed for fatty acyl pheromone analogs (FAPAs) and other potential biosynthetic intermediates. More than 80% of the FAPAs were found in the triacylglycerols (TGs), with smaller amounts found in the phosphatidyl cholines, ethanolamines, and serines. Analysis of the TGs by lipase revealed that the two FAPAs were distributed fairly evenly among all three stereospecific positions. Comparison of changes in titers of key glandular fatty acids with those of pheromone components, with respect to photoperiodic time and age of females, showed that both FAPA and pheromone titers exhibited a cyclical pattern with peaks in the scotophase and valleys in the photophase. However, whereas pheromone titer tended to peak in the first half of the scotophase, FAPA titer peaked at the end of the scotophase. Significantly, the titer of the FAPA of the minor component, (E)-11-tetradecenyl acetate (3% of pheromone), was always much greater than the titer of the FAPA of the major component, (Z)-11-tetradecenyl acetate (97%), of the pheromone. Titer of myristate, an intermediate in pheromone biosynthesis, was also higher during the scotophase than the photophase. However, myristate titer showed a pronounced dip in the middle of the scotophase. These data suggest two roles for glandular lipids in sex pheromone biosynthesis in O. nubilalis. Firstly, they remove excess FAPA of the minor component so the fatty acid reductase system is not presented with a high ratio of this isomer (which would otherwise result from the reductase's own selectivity), which could cause changes in the final pheromone ratio. Secondly, hydrolysis of the large amounts of stored saturated fatty acids from the TGs may provide substrate for pheromone biosynthesis.     

Properties of the Δ11-desaturase enzyme used in cabbage looper moth sex pheromone biosynthesis

The biosynthesis of a large number of sex pheromone components of various moth species can be explained by invoking a Δ 11-desaturation of common fatty acids. A Δ11-desaturase system from Trichoplusia ni, the cabbage looper moth, is identified and partially purified. Some of its properties are defined and compared with those of the ubiquitous Δ-9 desaturase enzyme. Similarities between the two systems include subcellular location (microsomal), substrate specificity (16- and 18-carbon acids), and lack of sensitivity to carbon monoxide, while differences include cofactor preference (NADH rather than NADPH), sensitivity to cyanide ion, pH optimum (7.4-7.8 vs 6.8-7.2), and location in the organism (in the pheromone gland compared to generally distributed). The effects of insect age were also investigated.     

枣镰翅小卷蛾性信息素通讯系统

采用单个雌蛾性信息素腺体分析技术对枣镰翅小卷蛾Ancylis sativa Liu雌蛾性信息素的组分和组分间的精确比例进行了测定,结果表明,枣镰翅小卷蛾雌蛾的性信息素系统由二个顺反异构体组分组成,即反-9-十二碳烯醋酸酯(E9-12∶Ac)和顺-9-十二碳烯醋酸酯(Z9-12∶Ac)组成,E9-12∶Ac与Z9-12∶Ac的比例为6.5∶3.5;雌蛾产生和释放性信息素具有时辰节律性,在光周期14L∶10D、温度为21℃时,性信息素产生的高峰期为进入黑暗期6.5 h;不同日龄雌蛾产生的性信息素有差异,1日龄最低,2日龄最高,3～5日龄居中,不同日龄雌蛾产生的性信息素组分间的比例无显著差异;对3个世代雌蛾产生的性信息素的量及组分间的比例的研究表明,越冬代含量最高,为(10.1±7.0)ng/头,第2代为(9.5±4.6)ng/头,第1代仅为(1.4±1.0)ng/头,3个世代性信息素组分间的比例无显著差异,在63.1%～64.3%(E)范围内。     

Terminal fatty-acyl-CoA desaturase involved in sex pheromone biosynthesis in the winter moth (Operophtera brumata)

The winter moth (Operophtera brumata L., Lepidoptera: Geometridae) utilizes a single hydrocarbon, 1,Z3,Z6,Z9-nonadecatetraene, as its sex pheromone. We tested the hypothesis that a fatty acid precursor, Z11,Z14,Z17,19-nonadecanoic acid, is biosynthesized from ??-linolenic acid, through chain elongation by one 2-carbon unit, and subsequent methyl-terminus desaturation. Our results show that labeled ??-linolenic acid is indeed incorporated into the pheromone component in vivo. A fatty-acyl-CoA desaturase gene that we found to be expressed in the abdominal epidermal tissue, the presumed site of biosynthesis for type II pheromones, was characterized and expressed heterologously in a yeast system. The transgenic yeast expressing this insect derived gene could convert Z11,Z14,Z17-eicosatrienoic acid into Z11,Z14,Z17,19-eicosatetraenoic acid. These results provide evidence that a terminal desaturation step is involved in the winter moth pheromone biosynthesis, prior to the decarboxylation.