Sex pheromone biosynthesis in the leafroller moth Planotortix excessana by ╬Ф10 desaturation.код для вставкиСкачать
Archives of insect Biochemistry and Physiology 8:l-9 (1988) Sex Pheromone Biosynthesis in the Leafroller Moth Planotortrix excessana by A10 Desaturation S.P. Foster and W.L. Roelofs Department of Entomology, New York State Agricultural Experiment Station, Geneva With the use of deuterium-labeled saturated fatty acids coupled with gas chromatography-mass spectrometric analysis, biosynthesis of the sex pheromone component (a-8-tetradecenyl acetate in the greenheaded leafroller moth Planotortrix excessana was shown t o proceed via A10 desaturation of palmitate. The resultant (Z)-lO-hexadecenoate is two carbon chain-shortened t o the precursor (a-8-tetradecenoate. The minor component (a-10-tetradecenyl acetate i s biosynthesized by A10 desaturation of myristate. This is the first confirmation of A10 desaturation in an eukaryotic system. Key words: deuterium-labelling, 0-8-tetradecenyl acetate, 0-lo-tetradecenyl acetate, Planofortrix excessana (greenheaded leafroller moth), delta-10 desaturation, sex pheromone biosynthesis INTRODUCTION Insect sex pheromones, particularly in the Lepidoptera, have been extensively studied over the last 20 years [l], especially with regard to chemical identification and behavioral responses. In recent years, one area of this field that has received more attention is the biosynthesis of insect sex pheromone chemicals. The similarity of many Lepidopteran sex pheromone chemicals to naturally occurring fatty acids suggests that sex pheromone biosynthesis in the Lepidoptera occurs by a similar fatty-acid synthesis. Acknowledgments: We thank Wanda Hansen and Dr J.R. Clearwater for rearing and shipping the insects to New York. We thank the New Zealand-United States of America Cooperative Science Program for a grant (to SPF) aiding this cooperation. This research was supported by National Science Foundation grant PCM-8406348. Dr. Foster i s a visiting scientist from the Entomology Division, DSIR, Mt. Albert Research Centre, Private Bag, Auckland, New Zealand. Received September 9,1987; accepted March 7,1988. Address reprint requests to Dr. S.P. Foster, Department of Entomology, New York Agricultural Experiment Station, Geneva, N Y 14456. 0 1988 Alan R. Liss, Inc. 2 Foster and Roelofs Early radiolabelling studies on Trichoplusia ni, the cabbage looper , confirmed that the major component (Z)-7-dodecenyl acetate was biosynthesized from acetate. More-recent work has begun to determine the different processes involved in pheromone biosynthesis. A two-carbon chain-shortening  enzyme and a A l l desaturase  have been identified in several moths, and their general use has been invoked to explain the biosynthesis of many known sex pheromone chemicals in moths [5,6]. Although the use of these two classes of enzymes in sex pheromone biosynthesis is probably common in the Lepidoptera, there are also a number of sex pheromone chemicals that are unlikely to be explained by biosynthesis involving A l l desaturation. One such chemical is Z8-14:OAc, which among other examples is produced by the endemic New Zealand leafroller moth Planotortrix excessanat (Walker). Galbreath et al. [q identified the sex pheromone of a Christchurch (midcoastal South Island) population of this species as a mixture of Z8-14:OAc and 14:OAc. Following this, Lofstedt and Roelofs  analyzed the sex pheromone gland of this same population for fatty acyl intermediates and found the likely precursors, Z8-14:Acyl and 14:Acyl, as well as relatively large quantities of the unusual Z10-16:Acyl. They proposed that the sex pheromone component Z8-14:OAc is biosynthesized from Z1016:Acyl, which is itself formed by A10 desaturation of palmitate. We have now studied this system using deuterium-labelled fatty acids coupled with GC-MS* [9,10], to show that all known pheromone components can be biosynthesized from palmitic acid. Moreover, the unsaturated component, Z8-14:OAc, is shown to be biosynthesized by the route proposed by Lofstedt and Roelofs . METHODS AND MATERIALS Insects and Gland Extracts P. excessuna were reared in New Zealand from a colony originally collected from Christchurch. They were fed on a synthetic diet incorporating dried Acrnena srnithii (Poiret) [q.Pupae were sexed in New Zealand, and young female pupae only were shipped to Geneva, New York, by airfreight. Upon arrival, pupae were placed in a 16:8 1ight:dark cycle and were used 2 days after emergence, approximately half an hour before the onset of the scotophase period. Insects that had emerged en route were used immediately upon arrival. Pheromone extracts were made by dissecting the extruded pheromone glands with fine forceps under a binocular microscope and placing the +Tortricidae: Tortricinae *Abbreviations: CI = chemical ionization; DMSO = dimethyl sulfoxide; FAMEs = fatty acid methyl esters; CC = gas chromatograph; CC-MS = gas chromatography-mass spectrometry; D3-14:COOH = [14,14,14-D3]myristic acid; D3-16:COOH = [16,16,16-D3]palmitic acid; 12:OAc = dodecyl acetate; 13:OAc = tridecyl acetate; 14:OAc = tetradecyl acetate; Z8-14:OAc = 8-tetradecenyl acetate; Z10-14:OAc = (a-IO-tetradecenyl acetate; 16:OAc = hexadecyl acetate; 14Acyl = tetradecanoate; Z8-14Acyl = (a-8-tetradecenoate; Z10-14:Acyl = (a-lo-tetradecenoate; Z10-16:Acyl = (a-IO-hexadecenoate. (a- Sex Pheromone Biosynthesis in the Leafroller Moth 3 excised glands in approximately 10-20 pl of distilled Skelly B (petroleum ether fraction). The glands were left to extract for approximately 16 h at ambient temperature before analysis. Extracts for analysis of FAMEs were made by the same method, except that glands were allowed to extract in distilled dichloromethane for 16 h at 3°C. The fatty acyl groups present in the gland extract were converted to the corresponding methyl esters by base methanolysis [ll]. The dichloromethane extract was decanted away from the glands and evaporated to apparent dryness with a gentle stream of nitrogen. The residue was allowed to react with 25 p1 of 0.5 M KOH in methanol for at least 30 min at ambient temperature. The products were acidified by the addition of 25 pl of 1.0 M HC1 (aqueous). The resultant FAMEs were extracted with 50 p1 of Skelly B. The pheromone gland of the female moth was extruded and held in this position by a closed alligator clip. The labelled acids (see below) were applied topically as DMSO solutions (ca. 10 pg per p1) to the extruded gland. Approximately 0.2 pl of DMSO was applied to the gland by a 1.0 pl syringe. The insect was placed in the dark with the gland extruded for about an hour, to allow the DMSO to absorb into the gland, after which the clip was removed and the gland allowed to return to its normal position. The insects were placed in the dark for an additional 3 h until the glands were dissected and extracted. In these experiments a large amount was applied to the gland, and losses of the liquid occurred as a result of absorption into other parts of the insect. As a result, no attempts were made to quantify the fate of the labelled fatty acid. Extracts of 5-7 female pheromone glands were analysed by GCMS. Chemicals Omega labelled D3-16:COOH and D3-14:COOH were purchased from KOR Isotopes, Cambridge, MA, and ICON Services Inc., Summit, NJ, respectively. They were both greater than 98% isotopically pure, as determined by mass spectrometry. Synthetic reference acetates and FAMEs were generally available from this laboratory. FAMEs that were not available were prepared from the corresponding carboxylic acid by acid methanolysis [l2],by reaction with a methano1:benzene:sulfuric acid (30:15:1) solution at 100°C. The carboxylic acids, if not available, were synthesized from the alcohol by reaction with a solution of pyridinium dichromate in dimethylformamide [B] . Analyses Before mass spectral analysis, extracts were analyzed by capillary gas chromatography with a Hewlett-Packard 5880 GC with splitless injector and flame ionization detector. A polar 50 m x 0.25 mm i.d. Silar lOC, fused silica column (Quadrex Corporation, New Haven, CT was used. The carrier gas was nitrogen at a linear flow velocity of l2 cm s-! The GC was programmed 80-140°C at 5°C min-' after an initial delay of 1min, then to 180°C at 2°C min-l. These conditions allowed some resolution of synthetic standards of isotopomers of both acetates and FAMEs. An internal reference standard of 13:OAc was used. 4 Foster and Roelofs To follow the fate of the deuterium label a Hewlett-Packard 5985 Quadrupole mass spectrometer interfaced with a Hewlett-Packard 5840 GC (with splitless injection) was used in the CI mode with isobutane as the reactant gas. For increased sensitivity, the mass spectrometer was used in the selected ion mode. The mass spectrometer was capable of monitoring up to five ion groups (at different time periods), with up to four ions per group. Each ion in a time group was scanned for 250 ps consecutively every second. For the types of chemicals studied here, CI mass spectrometry with isobutane gives the molecular ion plus one mass unit [(M+l)+] as the most intense ion of the spectrum. Therefore, the ion corresponding to the (M+4)+ ion of the unlabeled compound was scanned to observe incorporation of the D3-label. In addition to the two aforementioned ions, the corresponding (M+2)+ and (M+3)' ions were also generally scanned, primarily to observe the diminution of intensity and increase of the corresponding (M+4)+ ion when label was detected. The interfaced GC was equipped with a 30 m x 0.25 mm i.d Sulpelcowax 10 (Supelco Inc., Bellefonte, PA) fused silica column. Helium at 20 cm s-l was used as carrier gas, with a temperature program of 80-200°C at 4°C min-l after an initial delay of 3 min. RESULTS Gas Chromatography-Flame Ionization Detection Analysis of synthetic mixtures of the methyl esters of D3-16:COOH and unlabeled palmitic acid on the Silar 1OC column showed the deuterated isotopomer eluted some 0.2 min earlier than the nondeuterated isotopomer. This difference was insufficient to obtain baseline separation of the two isotopomers, but mixtures of greater than approximately 10% of one component in the other could be detected. Analysis of an extract of P. excessunu pheromone glands treated with D3-16:COOH gave leading edges on peaks, with the retention times of Z8-14:OAc and 14:OAc (relative to internal 13:OAc standard), indicating some incorporation of the label into these two pheromone components. Mass Spectrometric Analyses The results of mass spectrometric analyses of P. excessunu pheromone glands untreated and treated with D3-16:COOH or D3-14:COOH are illustrated in Figures 1and 2. Application of D3-16:COOH to the sex pheromone gland of P. excessunu resulted in an increased intensity of the corresponding (M+4)+ ion of each of the main pheromone components Z8-14:OAc (mlz = 255) and 14:OAc (ml z = 257), relative to those ions monitored in extracts of control (untreated) glands (see Fig. 1).An average incorporation of the D3-label into the two pheromone components, Z8-14:OAc and 14:0Ac, of 14.7% and 12.4%, respectively, was obtained from two runs. Additionally, the corresponding (M+4)+ ion of ZlO-l4:OAc, a minor component of the pheromone (Clearwater and Foster, unreported data) was enhanced relative to its intensity in untreated glands. Sex Pheromone Biosynthesis in the Leafroller Moth CONTROL +D3- 14:COOH X10 ( M f 3 ) (Mf3)- 12 14 28 Z10 16 +D3-16:COOH X10 ( 12 14 Z8 Z10 16 5 M 4 3 ) L XI0 12 14 Z8 210 NR Fig. 1. Relative intensities of the corresponding ( M + l ) + , ( M + 3 ) + , and (M+4)+ ions of compounds found in pheromone gland extracts from female Planotortrix excessana for untreated glands (control), glands treated with 14,14,14-D3-myristic acid (D3-14:COOH), and glands treated with 16,16,16-D3-palmitic acid (D3-16COOH). The control and D3-16:COOH diagrams are the means of two CC-MS separate runs each, whereas the D3-14COOH diagram i s from one CC-MS run. Abbreviations used: 12 = dodecyl acetate; 14 = tetradecyl acetate; 28 = (Z)-tl-tetradecenyl acetate; Z10 = (Z)-lO-tetradecenyl acetate; 16 = hexadecyl acetate; M = molecular ion of a chemical. In contrast, application of D3-14:COOH to the pheromone gland of P. excessana resulted in no significant enhancement of the corresponding (M+4)+ ion of Z8-14:OAc. However, the corresponding (M+4)+ ions of 14:OAc and Z10-14:OAc were enhanced in this treatment, relative to these ions in untreated glands. In addition, the corresponding (M+4)+ ion of l2:OAc was enhanced, but no detectable enhancement of the (M+4)+ ion of 16:OAc was observed. When the resulting FAMEs were analyzed (see Fig. 2) after base methanolysis of glands treated with D3-16:COOH, incorporation of the D3-label into the methyl esters of the putative precursors Z8-14:Acyl and 14:Acyl was observed. Furthermore, the label was incorporated into the methyl esters of laurate and palmitate and, most important, into Z10-16:Acyl. The incorporation of label into the precursors Z8-14:Acyl and 14:Acyl was much lower (1.4% and l.8%, respectively) than into the corresponding pheromone components. DISCUSSION Our results with deuterium-labeled saturated fatty acids show that the sex pheromone components Z8-14:OAc and 14:OAc of P. excessma are both 6 Foster and Roelofs +D3- 16:COOH CONTROL P+3) ,A X10 14 28-14 210-14 210-16 (M+3) d x10 14 28-14 210-14 210-16 Fig. 2. Relative intensities of the corresponding (M + I ) + ,(M+3)+,and (M +4)+ ions of fatty acid methyl esters from base-methanolysed pheromone gland extracts from female Planotortrix excessam for untreated glands (control) and glands treated with 16,16,16-D3-palmiticacid (D3-16:COOH). The control diagram is from one run, whereas t h e D3-16COOH diagram is the mean of two separate runs. Abbreviations used: 14 = methyl myristate; 28-14 = methyl (3-8-tetradecenoate; 210-14 = methyl (3-IO-tetradecenoate; 210-16 = methyl (a-IO-hexadecenoate; M = molecular ion of a chemical. biosynthesized from palmitic acid. In contrast, only 14:OAc can be biosynthesized directly from myristic acid. Another sex pheromone component of P. excessunu, ZlO-l4:OAc, was also biosynthesized from myristic acid, as was the saturated l2:OAc also found in the gland [S]. However, no label from myristic acid was detected in the other saturated acetate found in the gland, 16:0Ac, (Foster, unreported data), suggesting that it cannot be biosynthesized from myristic acid (at least to the detectable level of ca. 0.5% incorporation). The incorporation of label from palmitic acid into the fatty-acyl precursors of these chemicals is consistent with the results described here. The label is incorporated into both putative precursors and additionally into the unsaturated hexadecenoate, Z10-16:Acyl. Our interpretation of these results (Fig. 3) is the same as that of Lofstedt and Roelofs [S] and invokes the use of both desaturation and two-carbon chain-shortening. Our evidence for biosynthesis of ZS-14:OAc from palmitic acid is consistent with the first step being A10 desaturation of palmitate to produce Z1016:Acyl, which is chain-shortened by two carbons to the putative precursor ZS-14:Acyl. This precursor in turn is reduced and acetylated to the pheromone component. The alternative route of AS desaturation to produce ZS-14:Acyl directly from myristate is highly unlikely because of the nonincorporation of label from myristic acid into the pheromone component. Further evidence for a Sex Pheromone Biosynthesis in the Leafroller Moth HMMEWOATE DELTA-10 DESANRAllON / // ----__ 7 II 0 BnA-OXIDAllON ------+ 'WR ETPJDEWOATE 1 \ II 0 o 1 R BEIA-OXIDATION 1 1 1 DELTA-10 OES4NRAllON I1 R R (2)- 10-TEIWDECPIOATE (Z)--&TEIRADECENOATE 1, REOUCTIDN AN0 ACEMATlON 1 (Z)-O-TEIWDECPM ACElATE 0 0 I1 I REDUCTION AND ACEMAllON (Z)-lO-TEIWDECWrL 0 It ACETATE Fig. 3. Proposed pathways for biosynthesis of the sex-pheromone components, (a-8-tetradecenyl acetate (a-10-tetradecenyl acetate, and tetradecyl acetate in the leafroller moth Planotortrix excessana. A10 desaturase in the gland is provided by the presence of small amounts of Z10-14:OAc and its precursor, Z10-14:Acyl, which are probably biosynthesized directly from myristate (see Fig. 3). The chain shortening of Z10-16:Acyl to Z8-14:Acyl and of palmitate to myristate and laurate probably occurs by &oxidation of the carboxyl group, as reported for the chain shortening of palmitate to myristate in Argyvofaeniu citrana sex pheromone biosynthesis . In this chain-shortening process the position of unsaturation shifts two carbons closer to the carboxyl group, leaving the carbon atom in the omega position unchanged. We have no evidence for chain elongation of added palmitic or myristic acids in the gland, although it is possible that it occurs to a small extent, as in A. velutinana , but remains undetected. An interesting observation from these results is that the label was incorporated in relatively greater quantity into the pheromone components than into the fatty acyl precursors. This phenomenon is also apparent in the results from a similar study on A. velutinana . Although free fatty acids have not been found in pheromone glands of moths , this observation implies that they are preferentially converted to a lipid that is more readily converted to the pheromone components than other lipids containing the fatty acyl precursors of the pheromone present in the gland. This would tend to support the work of Bjostad and Roelofs , who failed to obtain incorporation of label from labelled triacylglycerols into the pheromone components 8 Foster and Roelofs of A . velutinana. Although the triacylglycerols contain the greatest amount of the pheromone precursors in this insect, this result led Bjostad and Roelofs  to speculate that the triacylglycerols act as a “dumping ground’’ for the excess acyl moieties not used in pheromone biosynthesis. Our results are obtained from the entire lipid content of the gland; therefore, the isotopomer content of the fatty acyl moieties in all of the lipid classes is averaged. If one lipid class (or chemical) is preferentially converted to the pheromone, we may see incorporation of label in this class to the same extent as observed in the pheromone components. This investigation is the first demonstration of A10 desaturase activity in an insect sex pheromone system and, to our knowledge, in any eukaryotic system [15,16]. This unique A10 desaturase activity may be considerably more widespread in moth sex pheromone biosynthesis. Although many femaleproduced moth pheromones may be explained by the action of a A l l desaturase, a number of species, especially in the Olethreutinae subfamily of the Tortricidae, use even-numbered A8 and A10 unsaturated 12- and 14-carbon compounds . These chemicals may be formed directly by A10 desaturation of a saturated intermediate, or less directly in combination with two-carbon @-oxidationchain shortening. We hope to further characterize this desaturase activity and make comparisons with the A9 and A l l desaturases found in other systems [17,16]. LITERATURE CITED 1. Tamaki Y: Sex pheromones. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, New York, Vol9, pp 145-191 (1985). 2. Jones IF, Berger RS: Incorporation of [l-’4C]acetate into cis-7-dodecen-1-01 acetate, a sex pheromone in the cabbage looper (Trichoplusia ni). Envir Ent 7, 666 (1978). 3. Wolf WA, Roelofs WL: A chain-shortening reaction in orange tortrix moth sex pheromone biosynthesis. Insect Biochem 23, 375 (1983). 4. Bjostad LB, Roelofs WL: Sex pheromone biosynthesis in Trichoplusia ni: Key steps involve delta-11 desaturation and chain shortening. Science 220, 1387 (1983). 5. Roelofs WL, Brown RL: Pheromones and evolutionary relationships of Tortricidae. Annu Rev Ecol Syst 23, 395 (1982). 6. Roelofs WL, Bjostad LB: Biosynthesis of Lepidopteran pheromones. 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Corey EJ, Schmidt G: Useful procedures for the oxidation of alcohols involving pyridinium dichromate in aprotic media. Tetrahedron Lett 5, 399 (1979). Sex Pheromone Biosynthesis in the Leafroller Moth 9 14. Bjostad LB, Wolf WA, Roelofs WL: Total lipid analysis of the sex pheromone gland of the redbanded leafroller moth, Argyrofueniu velufinunu, with reference to pheromone biosynthesis. Insect Biochem 11, 73 (1981). 15. James AT: The specificity of mammalian desaturases. Adv Exp Med Biol83, 51 (1977). 16. Wolf WA, Roelofs WL: Properties of the All-desaturase enzyme used in cabbage looper moth sex pheromone biosynthesis. Arch Insect Biochem Physiol3, 45 (1986). 17. Wang DL, Dillwith JW, Ryan RO, Blomquist GJ, Reitz RC: Characterization of the acylCoA desaturase in the housefly, Muscu dornesficuL. Insect Biochem 12, 545 (1982).