Sex pheromone biosynthesis in the Asian corn borer Ostrinia furnacalis IIBiosynthesis of E- and Z-12-tetradecenyl acetate involves ╬Ф14 desaturation.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 15:57-65 (1 990) Sex Pheromone Biosynthesis in the Asian Corn Borer Osfrinia furnacalis (II): Biosynt h es is of (€)- and (Z)-I2-Tet radece nyI Acetate Involves A14 Desaturation Chenghua Zhao, Christer Lofstedt, and Xuying Wang Institute of Zoology, Academia Sinica, Beijing, China (Z.C., W.X.); Department of Ecology, Lund University, Helgonav. 5, S-223 62 Lund, Sweden (C.L.) Sex pheromone biosynthesis in the Asian corn borer Ostrinia furnacafis 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 i n the selected ion monitoring mode. The labelling experiments suggest that the pheromone components (€)- and (Z)-12-tetradecenyl acetates are biosynthesized from palmitic acid by A14 desaturation, followed by chain shortening (P-oxidation), reduction, and acetylation. This i s the first confirmation of a A14 desaturase in an eukaryotic system. Key words: pyralidae, deuterium labelling, topical application, selected ion monitoring, (E)-l2-tetradecenoate, (Z)-12-tetradecenoate, (f)-1 &hexadecenoate, (Z)-14hexadecenoate, p-oxidation INTRODUCTION Studies of the biosynthesis of lepidopteran sex pheromones have shown that a few different desaturases in various combinations with chain shortening (limited P-oxidation) or chain elongation enzymes can produce most of the pheromone components identified so far [l].A l l desaturation, a key step in the production of A l l , A9, A7, and A5 pheromone components, has been demonstrated in several species [2-61. A A10 desaturase, involved in Acknowledgments: We thank Dr. Liu Xun for valuable suggestionsand Guo Xingyu for insect maintenance. This work was supported by the National Natural Science Foundation of China, the Swedish Natural Science Research Council, and the Swedish Royal Academy of Forestry and Agriculture. Received October 18,1989;accepted May 29,1990. Address reprint requests to Dr. Chenghua Zhao, Institute of Zoology, Academia Sinica, Beijing, China. 0 1990 Wiley-Liss, Inc. 58 Zhaoetal. the biosynthesis of the pheromone components (Z)-8-tetradecenyl acetate and (Z)-lO-tetradecenyl acetate, has been found in Planotorfrix excessana [7,8]. In addition to these two types of desaturases, an unusual A9 desaturase has been found in the codling moth Cydia pornonella .This enzyme interacts with dodecanoate to produce (EhPdodecenoate, which is subsequently converted into the conjugated precursor of the pheromone component ( E ,E M , 10-dodecadienol. The sex pheromone of the Asian corn borer Ostriniu furnucalis (Guenke) (Lepid0ptera:Pyralidae)was identified by Klun et al. [lo] and Cheng et al. [ll] as E and Z12-14:OAc." These unusual pheromone components, which cannot easily be derived from any known desaturation system, are released by females in a 47:53 ratio [ll].In addition, the female moth produces 14:0Ac, but its biological function remains to be elucidated. In a preliminary study , we found a group of likely pheromone precursors, including E12- and Z12-14:Acyl as well as E14- and Z14-16:Acyl, in gland extracts of 0. furnacalis. We thus hypothesized two possible pathways for the pheromone biosynthesis in 0.furnacalis. The most simple would be that E12- and Zl2-14:OAc are produced by A12 desaturation of 14:Acyl, followed by reduction and acetylation. This pathway would be similar to that in the production of E l l - and Z11-14:OAc in the European corn borer 0. nubilalis (Hiibner) by interaction of a A l l desaturase with 14:Acyl . An alternative pathway would be that the pheromone components of 0. furnacalis are formed by A14 desaturation of 16:Acyl, followed by P-oxidation chain shortening, reduction, and final acetylation. A third possible route would start with A10 desaturation of 12:Acyl and continue with chain elongation to produce the E12- and Z12-14:Acyl precursors. We now report a series of labelling experiments, using deuterated fatty acids as precursors and GC-FID and GC-MS for monitoring of incorporation, which test these hypotheses. MATERIALS AND METHODS Chemicals Omega-labelled D3-16:COOH was purchased from ICN Biomedicals, Inc. (Cambridge, MA). Omega-labelled D3-14:COOHand D3-12:COOHwere purchased from Larodan Fine Chemicals, Malmo, Sweden. The deuterium enrichment of these acids was 99% for D3-14COOHand D3-12:COOH, and 98% for D3-16:COOH. Reference acetates were available from the laboratory collections. Reference fatty acid methyl esters were prepared from the corresponding fatty acids with diazomethane in ether . *Abbreviations used: €12-14:OAc = (€)-12-tetradecenylacetate; Zl2-14: OAc = (ZJ-12-tetradecenyl acetate; 14:OAc = tetradecyl acetate; E12-14:Acyl = (E)-l2-tetradecenoate;Zl2-14:Acyl = (ZJ-12-tetradecenoate; E14-16:Acyl = (E)-14-hexadecenoate; Z14-16:A~yl= (ZJ-14-hexadecenoate; 14:Acyl = tetradecanoate; 16:Acyl = hexadecanoate; CC = gas chromatography; FID = flame ionization detection; GC-MS = gas chromatography-mass spectrometry; D3-16:COOH = [16,16,16-D3] palmitic acid; D3-14:COOH = [14,14,14-D31-rnyristic acid; D3-12:COOH = [12,12,12-D3]-lauricacid; DMSO = dimethyl suifoxide; D3-14:Me = [14,14, 14-D3]-methyltetradecanoate; 14:Me = methyl tetradecanoate; 214-16: M e = methyl (Z)-14hexadecenoate; etc. - Pheromone Biosynthesisin Ostrinia furnacalis 59 Insect Source and Application of Labelled Fatty Acids to Insects Asian corn borers were collected from Hengshui county of Hubei pravince, China and reared on an artificial diet . Pupae and adults were kept under a 15:9 h light-dark cycle and 24- to 48-h old females were used for topical application. Approximately 6 h into the scotophase the females were taken out into the light, their pheromone glands were extruded with alligator clips and then 4 pg of the labelled fatty acids in 0.05 pl DMSO were applied topically to each gland. After the DMSO had been absorbed into the glands (ca.30min), the insects were released and returned into the dark. About 1.5 h after the application of the labelled acids, the female glands were excised for extraction using fine forceps. In control experiments, the females were removed from the dark 7-8 h into the scotophase and the glands were excised directly as above. Gland Extracts and Methylation Pheromone extracts were prepared by soaking 15-20 excised glands in approximately 10 p1 distilled hexane for 1-2 h at room temperature. For the analysis of fatty acids, a total lipid extract of 25-60 pheromone glands was prepared by soaking them in 10-20 p1 of 2:l chloroform and methanol for 8-10 h at room temperature. Base methanolysis and acetylation were performed to convert fatty acyl moieties to the corresponding methyl esters and the alcohols to the corresponding acetates, as described by Bjostad et al. . The samples were analyzed immediately by GC or stored in the freezer ( - 20°C) until analysed by GC-MS. GC and GC-MS Analyses GC analyses were carried out on Pye Unicam 204 GC equipped with a unijector (ScientificGlass Engineering Pty. Ltd.) and a flame ionization detector. A 50 m x 0.22 mrn i.d. BP-20 column (crosslinked polyethylene glycol, Scientific Glass Engineering Pty. Ltd.) was used for separation. The oven temperature was programmed from 80”to 190°Cat 2”C/minafter an initial delay of 1 min. Hydrogen carrier gas linear velocity was 50 cmls at: 80°C. The split valve was opened 0.25 min after splitless injection. Under these conditions, omega-labelled (D3)fatty acid methyl esters and acetates are separated from the corresponding unlabelled compounds [5,9]. For example, D3-14:Me eluted 0.17 rnin earlier than 14:Me. Although baseline separation was not obtained, the occurrence of Dpl4Me could be detected if the relative amount of D3-14:Me to 14:Me was larger than 3%. GC-MS with electron impact ionization (70eV) was performed on a Hewlett Packard model 5970B GC-MS system equipped with a 59970B computer system, and interfaced with a Hewlett Packard model 5890 GC. The GC-MS was operated in the selected ion monitoring mode for the detection of incorporation of labelled precursors into pheromone components and intermediates. An acquisition program was designed to monitor diagnostic ions (DI) of native and D3-labelled (DI + 3) methyl esters and acetates. Selected ions were monitored in groups of four and the groups were changed at preset times in course of the separation, based on the retention times of synthetic standards. This allowed selective and sensitive detection of each of the different compounds of interest. The following diagnostic ions were chosen for the detection of 60 Zhaoetal. unlabelled and D3-labelled specimen, respectively: 12:Me mlz 214.20 and 217.20; 14:Me mlz 242.25 and 242.25; monounsaturated 14:Me rnlz 208.20 and 211.20; 14:OAc mlz 196.20 and 199.20; monounsaturated 14:OAc mlz 194.20 and 197.20; 15:Me rnlz 256.25 and 259.25; 16:Me mlz 270.25 and 273.25; monounsaturated 16:Me rnlz 236.25 and 239.25; 18:Me mlz 298.30 and 301.30; monounsaturated 18:Me rnlz 264.25 and 267.25; Z9,Z12-18:Me rnlz 262.25 and 265.25; and Z9,Z12,Z15-18:Me mlz 260.25 and 263.25, RESULTS GC Analyses No incorporation into the pheromone components or into 14:OAc was observed in the extracts of the glands incubated with D3-12:COOH (N = 2 batch extracts, each containing 15-20 female equivalents). When glands were incubated with D3-14:COOH(N = 3), label was incorporated only into 14:0Ac, but not into E12- or Z12-14:OAc. In these experiments, the peak with the predicted retention time of labelled 14:OAc was even larger than the peak corresponding to native 14:OAc. By contrast, the GC traces of extracts of glands treated with D3-16:COOH showed that the label from this precursor was not only incorporated into 14:0Ac, but also into E12- and Z12-14:OAc (Fig. 1).The average incorporation into the pheromone components was 15.7% for E1214:OAc and 13.3%for Z12-14:OAc, respectively (N = 3 ) . GC-MS Analyses The results of the GC analyses were confirmed and extended by GC-MS analyses (Fig. 2) of extracts of untreated pheromone glands and glands inC control a 4 L Fig. I. GC-FID traces from the analyses of gland extracts from untreated female 0. furnacalis and females treated with D3-16:COOH. a = 14:OAc; b = E12-14:OAc; c = Z12-14:OAc. Peaks labelled a', b', and c' have the predicted retention times of the respective omega-labelled (D3)analogues. Pheromone Biosynthesisin Ostrinia furnacalis 61 * iI ~ B 16:Me 1.0E51 I e. 0 ~ 4 DI 6.0E4 4 -'""""II/ -40000 V D1+3 17 18 C 15 16 Time 17 ( m l n . ) Fig. 2. CC-MS analyses of pheromone gland extracts from female 0. furnacalis. A: Treated with D3-l6:COOH. B: Treated with D3-14:COOH.C: Untreated glands (control). The extracted ion current profiles were constructed by adding diagnostic ions of native (DI)and D,-labelled (DI+3) specimen respectively. The DI + 3 abundance was multiplied by a factor 3. Labelled specimen elute approximately 0.05 min earlier than the native ones. 62 Zhaoet al. cubated with D3-12:COOH, D3-14:COOH, and D3-16:COOH, respectively. Treatment of glands with D3-16:COOH resulted in a significantly increased abundance of the DI + 3 ions for each of the following compounds: 14:0Ac, E12- 14:0Ac, Z12-14:OAc, E12-14Me, Z12-14:Me, 16:Me, and E14-16:Me. The glands treated with D3-14:COOH contained a significant DI + 3 peak only for 14:OAc and its corresponding methyl ester (Table 1).Extracts of glands treated with D3-12:COOH were no different from controls with respect to pheromone components and potential precursors (2 replicates only, no statistical evaluation). DISCUSSION Our interpretation of the labelling experiments is that the pheromone components of 0.furnacalis are biosynthesized along a pathway which starts with the production of E and Z14-16:Acyl by A14 desaturation of palmitate. These intermediates are, in turn, chain shortened by P-oxidation to form the irnmediate fatty acid precursors E12- and Z12-14:Acyl, which are finally reduced and acetylated to form the pheromone components (Fig. 3). The fact that there was no significant incorporation of D3-14:COOH into the pheromone components or their precursors, but only into 14:0Ac, rules out the alternative pathway that involves the production of the immediate precursors E12- and 212-14: Acyl by A12 desaturation of myristate . Neither was D3-12:COOH incorporated into the pheromone components, which rules out the second alternative that E12- and 212-14:Acyl may be biosynthesized by A10 desaturation of laurate, followed by carbon chain elongation. The low but statistically insignificant increase in abundance of DI + 3 ions upon treatment with D3-14: COOH may be due to some carbon chain elongation of this precursor to D3-16:COOHtaking place in the gland, followed by A14 desaturation of 16:Acyl and the subsequent reactions outlined above. However, the confirmation of this hypothesis would require additional experiments. The average incorporation of D3-16:COOH into the pheromone components (approximately 15%)is not high, but it is comparable to that which has been observed in A , velutinana  and P. excessma . It is interesting to note that D3-16:COOH was incorporated into the pheromone components E12- and Z12-14:OAc in approximately the same ratio as into the precursors E12- and Z12-14:Acyl, and E14- and Z14-16:Acyl (Fig. 2). This is quite different from what was observed in A . velutinana  and P. excessam . In these species, the label was incorporated in relatively greater quantity into the pheromone components than into the fatty acyl precursors. Foster and Roelofs  speculated that the labelled fatty acids 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. In 0. furnacalis, this does not seem to be the case. The ratio of E12- to Z12-14:OAc (47:53) is substantially different from the ratio of E12- to Z12-14:Acyl as well as the ratio of E14- to Z14-16:Acyl (Fig. 2). The ratio of E12- to 212-14:Acyl is 12:88, and the ratio of E14- to Z14-16:Acyl is 66:34 [12, this study]. A substantial difference in E/Z ratios between the pheromone components and their corresponding precursors has also been observed 0 78 s 27 s 3 4 4 Control D,y14:COOH D3-16:COOH 0 4 ns 29 s 26 s 2 ns 0 E12-14OAc Z12-14:OAc 2 391 s 11ns 14Me 16 s 4 ns 0 2 3 ns 22 s 0 Ins 5s 16:Me 0 Ins 16 s 9 6ns 25 ns E14-16:Me Z1416:Me *Abundanceof labelled specimen relative to abundance of native compound (+ 100by definition). +Statisticalsignificance of increased abundance over control samples tested by one-sided Mann-Whitney U-test at P < 0.05, s = significant, 11s = nonsignificant. All differences between D3-14:COOHand D3-16:COOH treatments were sigruhcantly different for all compounds (twosided M a n n - W h e y U-test). "Each sample containing the pooled extracts of 25-60 females. 14:OAc N" Treatment Compound E12-14Me Z12-14:Me TABLE 1. Relative Abundance of Diagnostic Ions Indicating Deuterium-Labelled Pheromone Components and Precursors in Gland Extracts of Female OsfriniafurnacaZis**' 64 Zhaoet al. 16L O R hcxadccanoate A14-dcsaturation I (a-14-hexadccenoate 1 (E)- 14-hexadecenoate I &oxidation H-oxidation 0 0 OR 14 ( E ) -12-ictradccenoare Q-12-teuadeccnoate reduction and acetylauon 14 (Z)-12-tcrradecenyI acctate 0 (E)-I 2-iciradeccnyl acetate Fig. 3. Proposed pathways for the biosynthesis of the sex pheromone components E12- and Z12-14:OAc in the Asian corn borer 0. furnacalis. in, for instance, A. velutinana , P. gossypiella , and 0. nubilulis . The specific ratio of E12- to Z12-14:OAc might be produced by selective enzymatic reduction of the geometric isomers of the precursors as suggested for A . velutinana . In 0.furnacalis, reduction and acetylation of E12-14:acyl seems to be favored over the conversion of the corresponding Z-isomer. However, the operation of this mechanism also requires that chain shortening of the E14- and Z14-16:acyl moieties is selective, favoring shortening of the Z-isomer, otherwise the insect would produce an E-biased acetate ratio. This is the first report that a A14 desaturase exists in an insect pheromone system or in any other eukaryotic system . Interestingly, our results imply a major biosynthetic difference between the pheromone of 0. furnacalis and that of its close European relative 0. nubilulis, although the pheromone components appear very similar. The pheromone of 0. nubilulis, (€1 and (Zklltetradecenyl acetate, is produced by A l l desaturation of myristate, directly followed by reduction and acetylation, whereas biosynthesis of E12- and Z12-14:OAc involves both a different desaturase and subsequent chain shortening of the fatty acid precursors. Desaturases (and other enzymes) involved in the biosynthesis of pheromones may be useful characters for phylogenetic analysis of moths [MI. However, such an approach requires that the homology of the various desaturases and the polarity of the character change can first be established. The fact that A l l desaturases are commonly distributed within the pyralids and other lepidopteran families, whereas the A14 desaturase in 0.furnacalis is the first case reported, suggests that the latter is the derived state. Pheromone Biosynthesis in Ostrinia furnacalis 65 LITERATURE CITED 1. Roelofs WL, Bjostad LB: Biosynthesis of Lepidopteran pheromones. Bioorg Chem 12, 279 (1984). 2. Bjostad LB, Roelofs WL: Sex pheromone biosynthesis in Trichoplusia ni: key steps involve delta-11 desaturation and chain shortening. Science 220,1387 (1983). 3. Bjostad LB, Roelofs WL: Sex pheromone biosynthesis in the redbanded leafroller moth, studies by mass labeling with stable isotopes and analysis with mass spectrometry. J Chem Ecol 22,431 (1986). 4. Wolf WA, Roelofs WL: A chain shortening reaction in orange tortrix moth sex pheromone biosynthesis. Insect Biochem 13,375 (1983). 5. Lofstedt C, Elmfors A, Sjogren M, Wijk E: Confirmation of sex pheromone biosynthesis from 16-D3palmitic acid in the turnip moth using capillary gas chromatography. Experientia 42, 1059 (1986). 6. Foster SP, Roelofs WL: Pink bollworm sex pheromone biosynthesis from oleic acid. Insect Biochem 18,281 (1988). 7. Foster SP,Roelofs WL: Sex pheromone biosynthesis in the leafroller moth Planotortrix excessana by A10 desaturation. Arch Insect Biochem Physiol8,1(1988). 8. Lofstedt C, Roelofs WL: Sex pheromone precursors in two primitive New Zealand tortricid moth species. Insect Biochem 25,729 (1985). 9. Lofstedt C, Bengtsson M: Sex pheromone biosynthesis of (E,E)-8,10-dodecadienol in codling moth Cydiu pornonella involves E9 desaturation. J Chem Ecol14,903 (1988). 10. N u n JA, Bierl-Leonhardt BA, Schwarz M, Litsinger LA, Barrion AT, Chiang HC, Zhungxie J: Sex pheromone of the Asian corn borer moth. Life Sci 27,1603 (1980). 11. Cheng Z-Q, Xiao J-C, Huang X-T, Chen D-L, Li J-Q, He Y-S, Huang S-R, Lug Q-C, Yang G-M, Yang T-H Sex pheromone components isolated from China corn borer, Ostrinia furnacalis Guenee (Lepid0ptera:Pyralidae) ( E ) and (E)-12-tetradecenylacetates. J Chem Ecol 7,841 (1981). 12. Zhao C, Wang X: Sex pheromone biosynthesis in the Asian comborer Ostriniufurnaculis (1)Sex pheromone biosynthetic precursors. Kexue Tongbao (Sci Bull) 14,1110 (1989). 13. Wolf WA, Roelofs WL: Reinvestigation confirms action of All-desaturases in spruce budworm moth sex pheromone biosynthesis. J Chem Ecol13,1019 (1987). 14. Zhou D, Wang Y, Liu B, Ju Z : Studies on the mass rearing of corn borer. 1. Development of a satisfactory artificial diet for larval growth. Acta Phytophyl Sin 7,113 (1980). 15. Bjostad LB, Linn CE, Du J-W, Roelofs WL: Identification of new sex pheromone components in Trichoplusia ni predicted from biosynthetic precursors. J Chem Ecol 20,1309 (1984). 16. Stanley-Samuelson DW, Jurenka RA, Cripps C, Blomquist GJ, Renobales M: Fatty acids in insects: composition, metabolism and biological significance. Arch Insect Biochem Physiol 9, l(1988). 17. Bjostad LB, Koelofs WL: Biosynthesis of sex pheromone components and glycerolipid precursors from sodium [1-l4C]acetatein redbanded leafroller moth. J Chem Ecol10,681 (1984). 18. Roelofs WL, Brown R L Pheromones and evolutionary relationships of tortricidae. Annu Rev Ecol Syst 23,395 (1982).