Inhibitory effect of 10 11-methyl-enetetradec-10-enoic acid on a Z9-desaturase in the sex pheromone biosynthesis of Spodoptera littoralis.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 26:279-286 (1 994) Inhibitory Effect of 10,11 -Methylenetetradec-10-Enoic Acid on a Z9-Desaturase in the Sex Pheromone Biosynthesis of Spodoptera littoralis Laura Gosalbo, Gemma FabrSs, and Francisco Camps Departament de Quimica Organica Biologica, CID-CSIC, Barcelona, Spain The effect of 1 0 , l l -methylenetetradec-l O-enoic acid on the sex pheromone biosynthetic pathway of Spodoptera littoralis is reported. This new cyclopropene fatty acid inhibited the biosynthesis of the main pheromone component from labeled myristic acid. The study of each Z desaturation step revealed that the Z9-desaturase of E l 1-14:Acid was inhibited, whereas the Z11-desaturase of 16:Acid was not affected. The results presented in this article agree with our hypothesis that the methylenehexadecenoic acids are beta-oxidized in the pheromone gland to the corresponding methylenetetradecenoic acids. (0 1994 WiIey-Liss, Inc. Key words: sex pheromone biosynthesis, desaturase, inhibition, Spodoptera iiftoralis INTRODUCTION In the Egyptian armyworm, Spodoptera liftoralis, the main sex pheromone component, Z9,Ell-l4:OAc*, is synthesized from 16:Acid by chain-shortening followed by sequential Ell-desaturation, Z9-desaturation, reduction, and acetylation (Martinez et al., 1990). Likewise, the Z11-desaturation of this 16:Acid Acknowledgments: We thank Isabel Millan for rearing the insects used in this study, the Ministerio de Educacion y Ciencia for a predoctoral fellowship to L.G., and CICYT for financial support (grant ACF-92-0178). Received June29, 1993; accepted October 8, 1993 Address reprint requests to Gemma Fabrids, Departament de Quimica Organica Biologica, CID-CSIC, Jordi Girona, 18-26, 08034 Barcelona, Spain. *Abbreviations used: DMSO = dimethylsulfoxide; d314:Acid = [14,14,1 4-2H3] myristic acid; dSE1114:Acid = [13,13,14,14,1 4 - 2 H ~(E)-l1 ] -tetradecenoic acid; d2g11-16:Acid = perdeuterated ( Z ) - l l hexadecenoic acid; d3116:Acid = perdeuterated palmitic acid; E l 1-14:Acid = (E)-l1-tetradecenoic acid; FAME =fatty acid methyl ester; FID-GC = flame ionization detector gas chromatography; GC-MS = gas chromatography coupled to mass spectrometry; MHAs = methylenehexadecenoic acids; MTAs = methylenetetradecenoic acids; Z9,Ell-l4:Acid = (Z,E)-9,11 -tetradecadienoic acid; Z9-14:Acid = (Z)-g-tetradecenoic acid; Z11-16:Acid = ( Z ) - l l-hexadecenoic acid; 10-MHA = 10,11 -methylenehexadec-10-enoic acid; 10- MTA = 10,ll -methylenetetradec-10-enoic acid; 12-MHA = 12,13methylenehexadec-12-enoic acid; 14:Acid = myristic acid; 16:Acid = palmitic acid; the same kind of nomenclature has been used for both methyl esters and acetates replacing Arid by Me and OAc, respectively. 0 1994 Wiley-Liss, Inc. Gosalbo et al. 280 Z11-16:Acyl Z9-14:Acyl 3 16:Acyl Y 14Acyl .u. E-ll+ Ell-14Acyl u '-' 29,Ell-l4:Acyl j Fig. 1. Biosynthetic pathway of S. liftoralis sex pheromone. Z-11,Zll -desaturation; E-I 1, E l l-desaturation; Z-9.Z-desaturation; -2C, chain shortening. Open arrows indicate reduction and acetylation. followed by beta-oxidation and further reduction and acetylation gives rise to Z9-14:OAc, a minor pheromone component (Fig. 1). Previously (Arsequell et al., 1989; Gosalbo et al., 1992)we demonstrated that the production of selected components of S. littoralis sex phermone can be inhibited by several C-16 cyclopropene fatty acids with the ring at positions 10, 11, or 12. This effect is brought about by inactivation of two desaturases: Z11-desaturase of 16:Acid and ZPdesaturase of Ell-14:Acid (Gosalbo et al., 1992). On the basis of previously reported structure-activity relationships (Fogerty et al., 1972), the effect of the above MHAs on the latter enzyme was unexpected. As a possible explanation, we suggested that these compounds might be beta-oxidized to the corresponding C-14 cyclopropene acids, which would be the actual inhibitors. Although this beta-oxidation in pheromone glands has not been so far experimentally proven, the ability of selected MTAs to inhibit the Z9 desaturase of Ell-14:Acid would indirectly support this assumption. In this paper we report on the biological activity of one of these compounds, namely ZO-MTA, on the sex pheromone biosynthetic pathway of S. littoralis. MATERIALS AND METHODS Insects Insects were maintained at 25 f 1°C with a 1ight:dark cycle of 16 h:8 h. Larvae were reared on an artificial diet (Poitut et al., 1972).Pupae were sexed and adults kept in separate containers. Adult females were separated daily before the onset of the scotophase. Only virgin females that emerged within 5 h before lights-off were used throughout this study. Chemicals DMSO was obtained from Sigma (St. Louis, MO), and Bom-PBAN was obtained from Peninsula Laboratories (Belmont, CA). d31 16:Acid was purchased from Fluorochem Ltd. (Derbyshire, UK) and d314:Acid from IC Chemikalien (Munich, Germany). dsEll-14:Acid and 10-MTA were prepared in our laboratory as described previously (Martinez et al., 1990; Gosalbo et al., 1993). inhibition of a Z9-Desaturase 281 Treatments Effect of 10-MTA on Z11-desaturation of d3116:Acid. Insects were briefly anesthetized and immobilized 3 0 4 0 min before the onset of their second scotophase and their pheromone glands topically treated with 0.1 pl of DMSO (controls) or 0.1 pl of DMSO containing 0.1 pg of inhibitor. After a 30 rnin incubation, 4 pg of d3116:Acid was topically applied to the glands, and the insects, still immobilized, were placed back in the rearing chamber and the glands excised 2 h later and processed for FAME analysis as indicated below. Effect of 10-MTA on ZPdesturation of ds-Ell-14 Acid. These experiments were performed as described above, using a dose of 1 pg of dsEll-14:Acid and serial dilutions containing 0.001-1 pg of inhibitor. Effect of PBAN on pheromone production. All the experiments that involved PBAN stimulation of pheromone production were performed 5 h before lights-off. Females were anesthetized by brief cooling and injected with 4 p1 of a PBAN solution in Meyers and Miller’s saline (Meyers and Miller, 1969) containing the doses indicated. To study the effect of PBAN on the incorporation of d3 14:Acid into pheromone, the glands were topically treated with 1 pg of d314:Acid in DMSO (0.1 pl), and the insects were released 15 min later and injected with 4 p1 of either saline or saline containing PBAN (12.5 pmols/female). The pheromone glands were excised 2 h after injection, and the amounts of both natural and labeled pheromone were determined as described below. Effect of 10-MTA on incorporation of d314:Acid into pheromone and intermediates. In these experiments, 5 h before the onset of the scotophase, the glands were treated with DMSO (0.1 pl) with or without 10- MTA (0.1 pg) and, after a 30 min incubation, with 1pg of d314:Acid. Insects were freed 15min after the last topical application and injected with 4 p1 of a PBAN solution (12.5 pmol/female). The pheromone glands were excised 2 h later and extracted and analyzed for pheromone and intermediate contents as described below. Tissue Extraction and Analytical Methods The Z11-desaturation of d3116:Acid was monitored by FID-GC using the equipment and conditions previously reported (Arsequell et al., 1989; Gosalbo et al., 1992). Individual pheromone glands were extracted with CHC13:MeOH 2:l (ST, overnight), and the d29Zll-l6:Me/d3116:Me ratios were calculated after usual base methanolysis of the lipidic extracts. The ZPdesaturation of d5El l-14:Acid was determined by GC-MS analysis of methanolyzed extracts with the same equipment and conditions described in a previous paper (Gosalbo et al., 1992). Groups of three glands were extracted and methanolyzed under standard conditions, and the ratios between ions 243 and 245 (molecular ions for dsZ9,Ell-l4:Me and d5El1-l4:MeTrespectively) were determined. For pheromone titer determinations, individual glands were extracted with 40 pl of hexane containing 10 ng of 12:OAc as internal standard (1 h, room temperature), and the samples were analyzed by FTD-GC as reported previously (Martinez and Camps, 1988). The incorporation of d314:Acid into pheromone and intermediates was determined in groups of three pheromone glands, which were first extracted with 282 Cosalbo et al. 100 pl of hexane containing 60 ng of 12:OAc as internal standard (1 h, room temperature). After pheromone extraction, the tissues were transferred and soaked in 200 pl of CHC13:MeOH 2:l (SOC, overnight), and lipids thus extracted were methanolyzed under usual conditions, adding 100 ng of 12:Me to the samples before workup for quantification. The analyses were carried out by GC-MS, using a Fisons gas chromatograph (8000 series) coupled to a Fisons MD800 mass selective detector. The system was equipped with a Hewlett Packard HP-1 capillary column (30 m x 0.20 mm), which was programmed either from 60-300°C at 8"C/min (pheromone analysis) or from 80-220°C at 5"C/min and then to 280°C at 10"C/min (FAME analysis). The selected ions monitored in pheromone analysis were 168 (M.+-60for 12:OAc),252 (M.+for natural Z9,Ell14:OAc), and 255 (M.+for d39,Ell- 14:OAc).In analysis of intermediates, the selected ions were 238 (M.7 for natural Z9,Ell-l4:Me), 241 (M.+for d a 9 , E l l 14:Me),240(M.+fornatural E11-14:Me),243 (M-+for d3Ell-l4:Me), and 245 (M.+ for d314:Me). To quantify the amounts of d39,Ell-14:OAc1 an aliquot of each sample was also analyzed by FID-GC under the conditions previously reported (Martinez and Camps, 1988). The actual amount of d3Z9,Ell-l4:OAc was calculated as [255/(252 + 255)] multiplied by the total amount obtained in the FID-GC analysis. To assess the effect of 10-MTA on the Z9-desaturase of Ell-l4:Acid, the ratios between ions 241 and 243 were determined. Likewise, the effect of 10-MTA on the Ell-desaturase of 14:Acid was estimated from the ratios between ions 243 and 245. Statistics Data were analyzed by the unpaired two-tail t test, after log (x + 1)transformation of the data when variances were unequal. RESULTS The effect of 10-MTAon the different desaturases involved in the biosynthetic pathway of S. Iitforalis sex pheromone was studied using several tracers and experimental conditions. In a first set of experiments, the effect of 10-MTA on Z11-desaturation of 16:Acid was investigated with d3116:Acid as labeled precursor, following the same procedure previously reported (Arsequell et al., 1989; Gosalbo et al., 1992).No significant difference was found in the amounts of d29Z11-16:Acid produced by controls and insects treated with 0.1 pg of 10-MTA, as concluded from the ratios between the corresponding perdeuterated methyl esters in the FID-GC analyses (Fig. 2). In a second group of experiments, the effect of 10-MTA on the ZPdesaturation of Ell-14:Acid was studied using dsEll-14:Acid as tracer. The inhibitory activity of 10-MTA on this step was found to be dose-dependent (Fig. 31, and statistically significant effects were observed with all the doses tested, except for 1 ng. Finally, the effect of 10-MTA on sex pheromone production was studied with d314:Acid as tracer. In order to stimulate its conversion into d3Z9,E11-14:OAc during the photophase, PBAN was also injected into these females. In a first set of experiments, we found that PBAN stimulated an increase in pheromone titer in a dose-dependent manner when injected into whole females at mid-photo- Inhibition of a Z9-Desaturase 283 DMSO 10-MTA Fig. 2. Effect of 10-MTA on Z11 -desaturation of djil6:Acid. Ratios between dmZ11-16:Me and d3116:Me, obtained from the GC traces, are given in the ordinate. Data are expressed as mean k SEM of 8-1 0 individual replicates. The difference between control and treatment is not statistically significant ( P > 0.1). phase (Fig. 4A). After analysis of the dose-response curve, a dose of 12.5 pmols/female was chosen for further treatments. As found for natural pheromone, amounts of d39,Ell-l4:OAc formed from d314:Acid were higher in females injected with 12.5 pmols of PBAN than in controls (Fig. 4B). However, the females that had been treated with 10-MTA produced significantly lower amounts of d$9,Ell-l4:0Ac from d314:Acid than controls after PBAN stimulation, as concluded from both FID-GC and GC-MS analyses (Fig. 5A). The analyses of FAME profiles revealed that the ratios between ions 243 and 245 and those between ions 241 and 243 were significantlylower in treatments than in controls (Fig. 58). Thus, both the Ell-desaturase of l4:Acid and the Z9-desaWase of Ell-14:Acid were apparently inhibited by 10-MTA in these experiments. DISCUSSION Our investigations on the development of desaturase inhibitors (Arsequell et al., 1989; Gosalbo et al., 1992) have led us to a new cyclopropene fatty acid, Fig. 3 . Effect of different doses of 10-MTA on Z9-desaturation of dsEl1- 14:Acid. Determinations were performed as indicated in Materials and Methods. Abscissa: The amount of inhibitor administered. Ordinate: Ratios between the abundances of ions 243 (dsZ9,Ell-l4:Me) and 245 (dsEl114:Me) from the GC-MS analyses. Each point represents mean i SEM of eight determinations with groups of three glands. Asterisks indicate statistical significance: * P < 0.05; **P i0.0005. "': cd C 30 - 20 - bd fb 10 - 01 :a, 0 i , , , 10 20 , , . 30 , 40 . , , XI dose (pmols) B T 1.0 0.5 0.0 Fig. 4. A: Dose-dependence of PBAN-stimulation of sex pheromone production in S. littoralis. Abscissa: The amount of PBAN injected. Ordinate: Amounts of Z9,E11-14:OAc calculated from the GC traces. Data are mean f SEM of six individual females, except for 50 pmols, with which only four replicates were obtained. Statistical significance among means is indicated by different letters beside each bar ( P < 0.05). B: PBAN effect on production of d3Z9,Ell-l4:OAc from d314:Acid. Bars represent mean SE of six groups of three females. Differences between saline and PBAN-injected females are statistically significant at P i 0.02. + H DMSO E3 10-MTA Fig. 5. A: Effect of 10-MTA on production of d3Z9,Ell-l4:OAc from dj14:Acid. Amounts of d3Z9,Ell-l4:0Ac were obtained from both FID-GC and GC-MS analyses. B: Effect of 10-MTA on the Z9-desaturase of E l 1-14:Acid. Ratios between ions 241 and 243 were obtained from the GC-MS analyses. C: Effect of 10-MTA on the E l 1-desaturase of 14:Acid. Ratios between ions 243 and 245 were obtained from the CC-MS analyses. In all figures, data are means SE of nine groups of three females/group. Differences between controls and treatments are statistically significant at P < 0.05 in all cases. The same glands were used for the analysis of both pheromone and intermediates. + Inhibition of a Z9-Desaturase 285 10-MTA, which interferes with the Z9-desaturase of E l 1-14:Acid in the biosynthesis of the sex pheromone of S. littoralis. In previous articles (Arsequell et al., 1989; Gosalbo et al., 1992) we reported the ability of selected MHAs to inhibit both the desaturation of 16:Acid to Z11-16:Acid and that of Ell-14:Acid to Z9,Ell-l4:Acid. Although the former effect was expected, the latter was somewhat surprising, since, except for 10-MHA, these compounds did not have one of the critical structural requirements to inhibit the Z9-desaturase enzyme (Fogerty et al., 1972), namely neither C-9 nor C-10, which are the positions desaturated in E l l - 14:Acid, included in the ring. This requirement would be present in the C-14 cyclopropene fatty acids that would result from beta-oxidation of these MHAs. Thus, we anticipated that the MHAs might get chain-shortened in the pheromone gland to the corresponding MTAs, which would be the actual inhibitors of the Z9-desahrase enzyme. Although final confirmation of this assumption awaits the detection of MTAs in pheromone glands incubated with MHAs, the results presented in this article support this possibility. Thus, 10-MTA, which would result from chain-shortening of 12-MHA, inhibited the ZPdesaturase of E l I-14:Acid in similar extent to that previously described for 12-MHA (Gosalbo et al., 1992) under similar experimental conditions. However, it is noteworthy that the activities exhibited by any of the MHAs assayed or by 10-MTA on the ZPdesaturase of Ell-l4:Acid are lower than those of the MHAs on the Z11-desaturase of 16:Acid (ArsequeI1 et al., 1989; Gosalbo et al., 1992). On the other hand, 10-MTA had no effect on the Z11-desaturation of 16:Acid.This lack of activity, as compared to the effectiveness of the C-16 analog 10-MHA (Gosalbo et al., 1992), suggests that the length of the alkyl substituent at C-12 is relevant for this inhibitory activity. The effect of 10-MTA on pheromone production was studied using d314:Acid as tracer. In previous papers (Arsequell et al., 1989; Gosalbo et al., 1992), treatments with both inhibitor and precursor were performed shortly before the onset of the scotophase, and pheromone gland excision was carried out 2 h into the dark period, when maximum pheromone titers are reached (Martinez and Camps, 1988). However, since we found that insect handling close to the scotophase caused a threefold reduction in pheromone production and that pheromone biosynthesis could be stimulated in the photophase by injection of brain-subesophageal ganglion extracts (Martinez et al., 19901, we decided to carry out these inhibition experiments in the photophase, using synthetic PBAN to stimulate pheromone production. In a first series of experiments, we confirmed that synthetic Born-PBAN was able to cause an increase in pheromone titer in S. littoralis. Likewise, we found that, as previously observed for different labeled precursors and with brain-subesophageal ganglion extracts (Martinez et al., 19901, females treated with dsl4:Acid and further injected with PBAN produced higher amounts of labeled pheromone than saline-injected animals. In these experiments, amounts of natural Z9,Ell- 14:OAc were also higher in PBAN than in saline-injected insects (mean k SE: 23.4 k 3.5 and 6.8 k 2.1, respectively; n = 6; P < 0.002). In the inhibition studies, insects that had been treated with 10-MTA produced significantly less d3Z9,Ell-l4:OAc from d314:Acid than controls after PBAN stimulation. The analysis of intermediates confirmed that inhibition of the Z9-desaturase of Ell-14:Acid was involved in 286 Gosalbo et al. the observed reduction in labeled pheromone production. In these experiments, inhibition of the Ell-desaturase of 14:Acid did apparently occur as well. However, we must not disregard the possibility that inhibition of this E l l-desaturase may not be caused by 10-MTA itself, but by the dsEll-14:Acid that accumulates as a result of inhibition of the Z9-desaturase. In summary, we have prepared a new cyclopropene fatty acid, structurally related to myristic acid, which inhibits the Z9-desaturation of Ell-14:Acid to the corresponding diene system, but it does not affect the Z11-desaturation of 16:Acid. Further studies about the mode of action of these inhibitors on the different desaturases involved in the biosynthetic pathway of S. littoralis sex pheromone are underway in our laboratory. LITERATURE CITED Arsequell G, Fabrias G, Camps F (1989): Inhibition of a delta-11 desaturase in Spodoptera littoralis by 12,13-methylenehexadec-12-enoic acid. Insect Biochem 19:623427. Fogerty AC, Johnson AR, Pearson JA (1972): Ring position in cyclopropene fatty acids and stearic acid desaturation in hen liver. Lipids 7:335-338. 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