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Acetylation of alcohols in the pheromone biosynthesis of the tomato looper, Chrysodeixis chalcites (Lepidoptera: Noctuidae)
Insect Biochem. Vol. 19, No. 5, pp. 523-526, 1989 Printed in Great Britain. All rights reserved
0020-1790/89 $3.00 + 0.00 Copyright © 1989Pergamon Press plc
ACETYLATION OF ALCOHOLS IN THE PHEROMONE BIOSYNTHESIS OF THE TOMATO LOOPER, CHR YSODEIXIS C H A L C I T E S (LEPIDOPTERA: NOCTUIDAE)* EZRA DUNKELBLUM1, HAVA MAMANE2, MIRIAM ALTSTEIN1 and ZEEV GOLDSCHMIDT2 qnstitute of Plant Protection, ARO, The Volcani Center, Bet Dagan 50250 and 2Department of Chemistry, Bar Ilan University, Ramat Gan 52100, Israel
(Received 5 December 1988; revised and accepted 19 April 1989) Abstract--A series of C11-C14 alcohols, varying in the number, position and geometry of double bonds, was applied topically to the sex pheromone glands of the tomato looper, Chrysodeixis chalcites, in order to study the acetylation step in the pheromone biosynthesis of this moth. Each application contained one of the alcohols and (Z)-I 1-tetradecenol, in equimolar amounts, as a metabolic standard for comparison of the relative conversion of the alcohols to acetates in the terminal biosynthetic step. One secondary and one tertiary alcohol were also included in the study. All alcohols were converted to the corresponding acetates at similar relative rates indicating that this step has a very low substrate specificity. One alcohol, (Z)-9-dodecenol was applied to the glands of head ligated females, which produces very small amounts of pheromone, in order to investigate the relation between the total biosynthesis of the pheromone and the acetylation step. The decrease in pheromone biosynthesis due to ligation of the head did not affect the acetylation step.
Key Word Index: Chrysodeixis chalcites, terminal acetylation, sex pheromone biosynthesis, PBAN
INTRODUCTION
acetate (ll-12:Ac) (3.6%), (Z)-8-tridecenylacetate (Z8-13:Ac) (1.4%) and (Z)-9-tetradecenyl acetate (Z9-14:Ac) (15.8%). In addition one alcohol, (Z)-7dodecenol (Z7-12:OH) was detected in very small amounts (1.1%) (Dunkelblum et al., 1987). The total amount of the pheromonal blend at optimal conditions was approx. 500 ng/female (Snir et al., 1986). These findings indicate a very high capability of enzymatic acetylation in the glands of C. chalcites. In the present study we analyzed the substrate specificity of the terminal acetylation step in C. chalcites by applying various alcohols to the glands and determining their relative conversion to the corresponding acetates. In addition we investigated the effectof P B A N on the bioacetylationby applying a representative alcohol, namely, (Z)-9-dodecenol (Z9-12:OH), to ligated females.
The study of the biosynthesis of Lepidoptera sex pheromones is relatively new. Nonetheless, considerable progress has been achieved in recent years, in particular in the formation of specific fatty acids which are precursors of pheromone components (Roelofs and Bjostad, 1984). Very recently, two reports were published describing the terminal acetylation step in the biosynthesis of acetate pheromone components in Hyraecia micacea (Teal and Tumlinson, 1987) and Mamestra brassicae (Bestmann et al., 1987). In both eases the results indicated that the acetyl transferase responsible for the conversion of alcohols into the corresponding acetates had low substrate specificity. • Another important development in the biosynthesis of sex pheromones in Lepidoptera has been the discovery of a neurohormone factor designated as "pheromone biosynthesis-activating neuropeptide" (PBAN), which induces pheromone biosynthesis (Raina and Klun, 1984). The activity of P B A N was demonstrated in two moths, Heliothis zea (Raina and Klun, 1984) and Bombyx mori (Ando et al., 1988). The sex pheromone glands of Chrysodeixis chalcites (previously named Plusia chalcites) contain a series of acetates; six of them were found in gland volatiles and are considered to be pheromone components. These are: dodecyl acetate (12:Ac) (3,0%), (Z)-7-dodecenyl acetate (Z7-12:Ac) (100%), (Z)-9dodecenyl acetate (Z9-12:Ac) 1.3%, l l-dodecenyl
MATERIALS AND
*Contribution from the Agricultural Research Organization ( A R O ) , Bet Dagan, Israel. No. 2547-E, 1988 series.
METHODS
Insects, topical application and gland extracts Insects were reared on an artificial medium (Shorey and Hale, 1965). Pupae were sexed and the females were placed in a separate room with a dark-light regime of 10:14 h at 25 + 2°C. The moths were kept in 30 x 30 x 30 cm screen cages on a 10% sugar-solution in groups of the same age. Topical application of alcohols in dimethyl sulfoxide (DMSO) was performed as described by Bjostad and Roelofs (1983). Females, 3-5 days old, were used for these experiments. Smooth aluminium hair-pins were used to clamp the glands. Test alcohols together with an equimolar amount of (Z)-ll-tetrad~nol (ZII-14:OH) in 0.5#1 of DMSO were applied. The amount of each alcohol was 4 x 10 -9 real which is in the range of 688-848 ng according to the molecular weight. The females were clamped for 1 h
523
524
EZRA DUNKELBLUM et al.
prior to the onset of scotophase, during this time most of the droplets absorbed into the gland. Then the clips were removed and the moths were kept in the dark for an additional 3 h. Individual glands were excised and extracted with hexane for 15 min. To each sample 200 ng of an internal standard, either 10-undecenyl acetate (10-11: Ac) or (E)-8-tridecenyl acetate (E8-13:Ac), was added. All samples were concentrated to a small volume (I0-20~1) by letting them stand in a fume hood and then stored at 4°C until used. Females 10-12 h were ligated I h prior to the onset of scotophase between the head and the thorax as described by Raina and Klun (1984). After 24 h, Z9-12:OH in DMSO was topically applied to the pheromone glands as described for the nonligated females and the gland extracts were prepared as described above. Chemicals All primary alcohols and reference acetates were from our pheromone collection. The secondary alcohol 2-undecanol (ll:2OH) was prepared from the corresponding ketone by LAH reduction in boiling THF for 18 h. Usual workup gave the crude alcohol which was purified by column chromatography on silica with hexane + 10% ethyl acetate, affording 80% yield. The secondary alcohol was converted to the corresponding acetate with acetic anhydride and pyridine. The tertiary alcohol 3-methyl-3-dodecanol (3Me-12:3OH) was prepared by a Grignard reaction of ethylmagnesium iodide and 2-undecanone. Workup with aqueous NH4CI and subsequent purification by column chromatography on silica with hexane+ 5% ethyl acetate gave the desired alcohol in 80% yield. The tertiary acetate was prepared from the alcohol with acetic anhydride, triethylamine and 4-dimethylamino pyridine. All compounds were analyzed by capillary GC and had correct MS and NMR spectra. Alcohols, used in this study, were 95% > pure. Gas chromatography and mass spectrometry All samples were analyzed by capillary gas chromatography (CGC) on either a 30m x 0.25 mm DB5 column or a 30m x 0.25 mm DB225 column, both purchased from J&W, Rancho Cordova, Calif. They were injected in the splitless mode and the compounds were detected by FID. The temperature of the injectors and detectors was 220°C. The purge valve was opened I rain after injection and helium (carrier gas) pressure was maintained at 15 psi. The DB5 column was kept at 60°C for 2 min and then programmed at 10°C/min to 140°C; after 30 rain the temperature w a s raised at the same rate to 160°C. The DB225 column w a s programmed similarly but the temperatures were 60°C for 2 min, then 130°C, and raised to 150°C after 35 rain. Quantification of peaks was performed by either a Spectraphysics 4270 integrator or by a 4290 model equipped with a memory module enabling replay of chromatograms. Combined GC-MS was performed on a Finnigan 5100 machine equipped with a 30 m x 0.25 mm DB5 column in the splitless mode. The column was kept for 2 rain at 60°C
and then programmed at 10-180°C. The purge valve was opened 30 s after injection and helium pressure was maintained at 10 psi. RESULTS A series of pheromonal alcohols including, (E)-7dodecenol (E7-12:OH), (Z)-9-dodccenol (Z912:OH), (E,E)-8,10-dodacadienol (Eg,E10-12:OH), tridecanol (13:OH), (E)-9-tetradecenol (E9-14:OH), one secondary alcohol 2-undecanol ( l I : 2 O H ) and one tertiary alcohol 3-methyl-3-dodecanol (3Me12:3OH), was topically applied to the pheromone glands of C. chalcites. All these alcohols, with the exception of Z9-12:OH, are unrelated to the pheromone components of C. chalcites. Each application also contained an equimolar amount of (Z)-11tetradecenol (Z 11-14:OH) as a metabolic competition standard for the bioacetylation reaction. After the incubation, the gland extracts were analyzed by G C and the amount of the formed acetate from the tested alcohols and from Z I I - 1 4 : O H was determined. The molar amounts of the observed acetates and (Z)- 11tetradecenyl acetate (ZI 1-14:Ac) were calculated for each replicate and the corresponding relative conversion factor K was found by dividing the molar amount of R : A c by Z11-14:Ac. For each alcohol, the mean relative conversion factor K + SD is presented in Table 1. Clamping of the gland resulted in a considerable decrease of the pheromone as compared with glands from nonclamped females. The amount of the major pheromone component Z7-12:Ac declined from an average of 300 ng/female (Snir et al., 1986) to an average of 100 ng/female under the same conditions. All tested alcohols were acetylated by the gland at approximately the same relative rate (Table 1), with E7-12:OH having the largest relative conversion factor K of 1.24 _+ 0.30 and 13:OH having the smallest one of 0.65 + 0.11. Representative G C analyses of nontreated and treated gland extracts are shown in Fig. 1. One sample of the topical application of the tertiary alcohol was analyzed by G C - M S and the presence of 3Me-12:3OH and 3Me-12:3Ac as well as other pheromonal components was confirmed. Tridccanol was tested both under the usual conditions of scotophase and in the photophase. The results (Table 1) indicated that the relative rate of alcohol/acetate conversion K remained the same. In the photophase, the amount of pheromone was low, an average of 4 8 n g as compared with l l 6 n g in scotophase. However, the bioacetylation proceeded
Table 1. Bioacetylation of topically applied alcohols in the pheromone gland of Chrysodeixis chalcites females* Relative Z7-12:Ac Applied (I :1) R:Ac, rig/female± SD ZI 1-14:Ac ng/female± SD conversion ng/female ± SD R:OH + ZI 1-14:OH (10-N tool amount) (10-~ tool amount) factor Kt 127 + 60 11:2OH 18 ± 2 (8.41 ± 0.93) 17 ± 3 (6.69 ± 1.18) 1.12 ± 0.07 103+69 E7-12:OH 24±12 (10,62±5.30) 21±9 (8.27±3.54) 1.24 ±0.30 106+51 Z9-12:OH 33±14 (14.60±6,19) 32+9 (12.60+3.54) 1.17±0.40 120+29 E8, E10-12:OH 14+7 (6.25±3.12) 15±10 (5.90±3.94) 1.14+0.30 116 + 10 13:OH 12 ± 1 (4.96 + 0.41) 19 + 4 (7.48 + 1.57) 0.65 + 0.11 48 _+22 13:OH (light) 29 + 16 (11.98 + 6.61) 38 + 25 (14.96 ± 9.84) 0.56 ± 0.20 101 ± 35 3Me-12:3OH 13 + 2 (5.37 :t: 0.83) 14 ± 4 (5.51 ± 0.39) 0.91 ± 0.22 110+30 E9-14:OH 20+5 (7.87+1.97) 27+4 (10.62±1.57) 0.82+0.10 *Each test was performed on a single moth and was replicated 4--6 times. tThe mean relative conversion factors were obtained by dividing the molar ratios of R:Ac by ZI 1-14:Ac for each replicate and then calculating the mean ± SD value.
Acetylation in the pheromone biosynthesis of C. chalcites
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_o.Lt Fig. l. (A) GC profile of a gland extract from a clamped nontreated female. (B) GC profile of a gland extract from an E8, E10-12:OH treated female. Both performed on the DB 225 column. normally and both 13:Ac and Z I I - 1 4 : A c were formed in the light even in larger amounts than in the dark. One of the alcohols, Z9-12:OH was chosen to test the dependence of the bioacetylation on the presence of PBAN. In previous experiments it was shown that ligation of the head in C. chalcites interrupted the transport of PBAN to the gland and inhibited the formation of the sex pheromone (Altstein et al., 1989). The present results (Table 2) showed that ligation of the head did not stop the bioacetylation. Topical application of Z9-12:OH resulted in a large increase of Z9-12:Ac both in 3-5-day-old females (Table 1) and in 34-36-h-old females as compared to untreated females (Table 2, and Fig. 2). In the ligated females the amount of Z9-12:Ac, formed from the topically applied Z9-12:OH, remained as high as in the nonligated treated moths, despite the fact that the amount of the main pheromone component, Z712:Ac, declined by 50% in the ligated and clamped females as compared to clamped females (Table 2). The basal amounts of Z7-12:Ac in the pheromone glands in the ligation experiment were lower (Table 2) than those in the bioacetylation experiments (Table 1) because the females used in the ligation work were c. 1.5 days old, whereas the other females were 3-5 days old. DISCUSSION
The results of our study indicate that C. chalcites is capable of acetylating primary, secondary and even Table 2. Bioacetylationof topically applied Z9-12:OH in the pheromone gland of ligated Chrysodetxts chalcttes females* Z7-12:Ac,
Z9-12:Ac,
Condition of female ng + SD ng + SD Untreated 180 ± 80 < 3.5 Ligated 61 + 34 < 1.0 Clamped+ Z9-12:OH 49+11 25:1:10 Ligated and clamped+ Z9-12:OH 24 + I I 25 + I I *Each test was performedon a singlemoth and was replicated4-6 times.
525
tertiary aliphatic long-chain alcohols. All alcohols were topically applied to clamped glands. This procedure was introduced by Bjostad and Roelofs in 1983 and is now a standard method. It is used widely for the application of radiolabeled precursors (Bjostad and Roelofs, 1983) and deutero-labeled precursors (Lofsted et al., 1986) in biosynthesis studies of moths. Clamping does not inhibit drastically the biosynthesis as evidenced by the incorporation of labeled precursors, although it might have some effect on the yields of the pheromone components. Most of the decrease in the amounts of the pheromone in clamped C. chalcites females is probably due to a fast release of the gland content. Incorporation of a metabolic internal standard in each test enabled the determination of the relative rates of acetylation. The Z11-14:OH alcohol was chosen as the standard since its corresponding acetate, Z 11-14: Ac, was detected in gland extracts of C. chalcites in trace amounts, c. 0.2% relative to Z7-12:Ac, which is below detection limit in the present one gland extracts (Dunkelblum et al., 1987). It was assumed that this alcohol would be readily accepted by the acetylation enzyme system. Each experiment was run as a competition test with one of the alcohols together with ZI 1-14:OH. This procedure enabled us to obtain a quantitative comparison of the acetylation of various alcohols in the pheromone gland of C. chalcites. All conversion factors K were very similar, in the range of 0.6-1.2 relative to the rate of acetylation of ZI 1-14:OH. The bioacetylation proceeded in the light similarly to that observed by Teal and Tumlinson (1987). The amounts of the formed 13:Ac and Z I I - 1 4 : A c were larger at photophase than at scotophase, but the quantity of pheromone was much lower (Table 1). This may indicate that less natural alcohol precursors were competing with the topically applied alcohols. The yield of the acetates was 1.5--4% comparable to the yield of acetates obtained in H. micacea (Teal and Tumlinson, 1987). Surprisingly, only small amounts of alcohols were recovered in the extracts, and in several cases the applied alcohols were not observed in the GC analysis, although normal yields of acetates were obtained. The reasons for this phenomenon were not investigated. The present experiments demonstrated that in C. chalcites the bioacetylation step has low specificity and can utilize various pheromone-like alcohols. The narrow range of relative conversion factors indicated that different aliphatic alcohols, in the range of C l l - C 1 4 , were converted to their corresponding acetates at approximately the same rate. On the other hand, some differences in the rates of bioacetylation of various alcohols were found in Choristoneura fumiferana (Morse and Meighen, 1987), in particular between the group of C12-C15 and the C10 and C18 alcohols. Unsaturated alcohols were acetylated faster than the corresponding saturated ones. However, in C. fumiferana, the acetylation of alcohols is an intermediate step in the biosynthesis of the aldehydic pheromone components and not the terminal step, as in H. micacea (Teal and Tumlinson, 1987), M. brasicae (Bestmann et al., 1987) and C. chalcites. Different enzymatic systems may be involved in these transformations. In C. fumiferana, both pheromone intermediates, namely alcohols and acetates are
EZRADUNKELBLUMet al.
526
the terminal step in the biosynthesis of the pheromone in C. chalcites is not under hormonal control. The technique of using ligated females for the study of the biosynthetic steps with topically applied compounds may be very helpful in the study of these transformations. More work is needed to confirm that the biosynthesis of the intermediate fatty acids and alcohols, and the terminal acetylation step in C. chalcites are completely separate sequences.
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Acknowledgements--This work was supported by BARD grant No. US 1208-86. The results are a part from the M.Sc thesis of Mrs Hava Mamane.
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REFERENCES
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Fig. 2. (A) GC profile of gland extract from a ligated nontreated female. (B) GC profile of a gland extract from a ligated and Z9-12:OH treated female. Both performed on the DB5 column. stored in the pheromone gland (Silk et al., 1980), whereas in C. chalcites only one alcohol was found in very small amounts. This alcohol, Z7-12:OH, detected in C. chalcites in c. 1% amount relative to Z7-12:Ac (Dunkelblum et al., 1987) was not found in higher quantities in any of the present experiments. The second part of this study was a preliminary investigation of the bioacetylation step in ligated females. It has been shown by Raina and Klun (1984) in H. zea and by Ando et al. (1988) in B. mori that ligation or decapitation of the virgin female caused the interruption of the pheromone biosynthesis. Recently, the same phenomenon was demonstrated in C. chalcites in our laboratory. By analyzing the profiles of the fatty acid precursors, we could demonstrate that the formation of the first monounsaturated fatty acid precursor is affected by ligation (Altstein et al., 1899). In the present experiment, we could show that ligation of the head did not stop the formation of Z9-12:Ac after application of the corresponding alcohol. The amount of the pheromone, as monitored with Z7-12:Ac, declined considerably but the bioacetylation capability remained intact. Normally, the relative amount of Z9-12:Ac, as compared with the major component Z7-12:Ac, is 1-2% which is at most several ng/female. This was found previously in gland extracts and volatiles (Dunkelblum et al., 1987) and in the present study in both nonligated and ligated females. When Z9-12:OH was topically applied to ligated females the amount of formed Z9-12:Ac remained as high as that in treated nonligated moths (Table 2). This amount was much higher than that in untreated ligated females (Fig. 2). These results indicate strongly that PBAN is probably not involved in the bioacetylation and that
Altstein M., Harel M. and Dunkelblum E. (1989) Effect of a neuroendocrine factor on sex pheromone biosynthesisin the tomato looper, Chrysodeixis chalcites (Lepidoptera: Noctuidae). Insect Biochem. In press. Ando T., Arima R., Uchiyama M., Nagasawa H., Inoue T. and Suzuki A. 0988) Pheromone biosynthesis activating neuropeptide hormone in heads of the silkwork moth. Agric. Biol. Chem. 52, 881-883. Bestmann H. J., Herrig M. and Attygale A. B. (1987) Terminal acetylation in pheromone biosynthesis by Mamestra brassicae L. (Lepidoptera: Noctuidae). Experientia 43, 1033-1034. Bjostad L. and Roelofs W. L. (1983) Sex pheromone biosynthesis in Trichoplusia hi: Key steps involve AH desaturation and chain shortening. Science 220, 1387-1389. Dunkelblum E., Snir R., Gothilf S. and Harpaz I. (1987) Identification of sex pheromone components from pheromone gland volatiles of the tomato looper, Plusia chalcites (Esp.). J. chem. Ecol. 13, 991-1003. Lofsted C., Elmfors A., Sjorgen M. and Wijk E. (1986) Confirmation of sex pheromone biosynthesis from (16-D3) palmitic acid in the turnip moth using capillary gas chromatography. Experientia 42, 1059-1061. Morse E. and Meighen E. (1987) Pheromone biosynthesis: Enzymatic studies in Lepidoptera. In Pheromone Biochemistry (Edited by Prestwich G, D. and Blomquist G. I.), pp. 121-158. Academic Press, Orlando, Fla. Raina A. K. and Klun J. A. (t984) Brain factor control of sex pheromone production in the female corn earworm moth. Science 225, 531-533. Roelofs W. L. and Bjostad L. 0984) Biosynthesis of Lepidopteran pheromones. Bioorg. Chem. 12, 279-298. Shorey H. H. and Hale R. H. (1985) Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. J. econ. Ent. 58, 522-524. Silk P. J., Tan S. H., Wiesner C. J., Ross R. J. and Lonergan G. C. (1980) Sex pheromone chemistry of the Eastern Spruce Budworm, Choristoneura fumiferana. Envir. Ent. 9, 640-644.
Snir R., Dunkelblum E., Gothilf S. and Harpaz I. (1986) Sexual behaviour and pheromone titre in the tomato looper, Plusia chalcites (Esp.) (Lepidoptera: Noctuidae). J, Insect Physiol. 32, 735-739. Teal P. E. A. and Tumlinson J. H. 0987) The role of alcohols in pheromone biosynthesis by two noctuid moths that use acetate pheromone components. Archs Insect Biochem. Physiol. 4, 261-269.
0020-1790/89 $3.00 + 0.00 Copyright © 1989Pergamon Press plc
ACETYLATION OF ALCOHOLS IN THE PHEROMONE BIOSYNTHESIS OF THE TOMATO LOOPER, CHR YSODEIXIS C H A L C I T E S (LEPIDOPTERA: NOCTUIDAE)* EZRA DUNKELBLUM1, HAVA MAMANE2, MIRIAM ALTSTEIN1 and ZEEV GOLDSCHMIDT2 qnstitute of Plant Protection, ARO, The Volcani Center, Bet Dagan 50250 and 2Department of Chemistry, Bar Ilan University, Ramat Gan 52100, Israel
(Received 5 December 1988; revised and accepted 19 April 1989) Abstract--A series of C11-C14 alcohols, varying in the number, position and geometry of double bonds, was applied topically to the sex pheromone glands of the tomato looper, Chrysodeixis chalcites, in order to study the acetylation step in the pheromone biosynthesis of this moth. Each application contained one of the alcohols and (Z)-I 1-tetradecenol, in equimolar amounts, as a metabolic standard for comparison of the relative conversion of the alcohols to acetates in the terminal biosynthetic step. One secondary and one tertiary alcohol were also included in the study. All alcohols were converted to the corresponding acetates at similar relative rates indicating that this step has a very low substrate specificity. One alcohol, (Z)-9-dodecenol was applied to the glands of head ligated females, which produces very small amounts of pheromone, in order to investigate the relation between the total biosynthesis of the pheromone and the acetylation step. The decrease in pheromone biosynthesis due to ligation of the head did not affect the acetylation step.
Key Word Index: Chrysodeixis chalcites, terminal acetylation, sex pheromone biosynthesis, PBAN
INTRODUCTION
acetate (ll-12:Ac) (3.6%), (Z)-8-tridecenylacetate (Z8-13:Ac) (1.4%) and (Z)-9-tetradecenyl acetate (Z9-14:Ac) (15.8%). In addition one alcohol, (Z)-7dodecenol (Z7-12:OH) was detected in very small amounts (1.1%) (Dunkelblum et al., 1987). The total amount of the pheromonal blend at optimal conditions was approx. 500 ng/female (Snir et al., 1986). These findings indicate a very high capability of enzymatic acetylation in the glands of C. chalcites. In the present study we analyzed the substrate specificity of the terminal acetylation step in C. chalcites by applying various alcohols to the glands and determining their relative conversion to the corresponding acetates. In addition we investigated the effectof P B A N on the bioacetylationby applying a representative alcohol, namely, (Z)-9-dodecenol (Z9-12:OH), to ligated females.
The study of the biosynthesis of Lepidoptera sex pheromones is relatively new. Nonetheless, considerable progress has been achieved in recent years, in particular in the formation of specific fatty acids which are precursors of pheromone components (Roelofs and Bjostad, 1984). Very recently, two reports were published describing the terminal acetylation step in the biosynthesis of acetate pheromone components in Hyraecia micacea (Teal and Tumlinson, 1987) and Mamestra brassicae (Bestmann et al., 1987). In both eases the results indicated that the acetyl transferase responsible for the conversion of alcohols into the corresponding acetates had low substrate specificity. • Another important development in the biosynthesis of sex pheromones in Lepidoptera has been the discovery of a neurohormone factor designated as "pheromone biosynthesis-activating neuropeptide" (PBAN), which induces pheromone biosynthesis (Raina and Klun, 1984). The activity of P B A N was demonstrated in two moths, Heliothis zea (Raina and Klun, 1984) and Bombyx mori (Ando et al., 1988). The sex pheromone glands of Chrysodeixis chalcites (previously named Plusia chalcites) contain a series of acetates; six of them were found in gland volatiles and are considered to be pheromone components. These are: dodecyl acetate (12:Ac) (3,0%), (Z)-7-dodecenyl acetate (Z7-12:Ac) (100%), (Z)-9dodecenyl acetate (Z9-12:Ac) 1.3%, l l-dodecenyl
MATERIALS AND
*Contribution from the Agricultural Research Organization ( A R O ) , Bet Dagan, Israel. No. 2547-E, 1988 series.
METHODS
Insects, topical application and gland extracts Insects were reared on an artificial medium (Shorey and Hale, 1965). Pupae were sexed and the females were placed in a separate room with a dark-light regime of 10:14 h at 25 + 2°C. The moths were kept in 30 x 30 x 30 cm screen cages on a 10% sugar-solution in groups of the same age. Topical application of alcohols in dimethyl sulfoxide (DMSO) was performed as described by Bjostad and Roelofs (1983). Females, 3-5 days old, were used for these experiments. Smooth aluminium hair-pins were used to clamp the glands. Test alcohols together with an equimolar amount of (Z)-ll-tetrad~nol (ZII-14:OH) in 0.5#1 of DMSO were applied. The amount of each alcohol was 4 x 10 -9 real which is in the range of 688-848 ng according to the molecular weight. The females were clamped for 1 h
523
524
EZRA DUNKELBLUM et al.
prior to the onset of scotophase, during this time most of the droplets absorbed into the gland. Then the clips were removed and the moths were kept in the dark for an additional 3 h. Individual glands were excised and extracted with hexane for 15 min. To each sample 200 ng of an internal standard, either 10-undecenyl acetate (10-11: Ac) or (E)-8-tridecenyl acetate (E8-13:Ac), was added. All samples were concentrated to a small volume (I0-20~1) by letting them stand in a fume hood and then stored at 4°C until used. Females 10-12 h were ligated I h prior to the onset of scotophase between the head and the thorax as described by Raina and Klun (1984). After 24 h, Z9-12:OH in DMSO was topically applied to the pheromone glands as described for the nonligated females and the gland extracts were prepared as described above. Chemicals All primary alcohols and reference acetates were from our pheromone collection. The secondary alcohol 2-undecanol (ll:2OH) was prepared from the corresponding ketone by LAH reduction in boiling THF for 18 h. Usual workup gave the crude alcohol which was purified by column chromatography on silica with hexane + 10% ethyl acetate, affording 80% yield. The secondary alcohol was converted to the corresponding acetate with acetic anhydride and pyridine. The tertiary alcohol 3-methyl-3-dodecanol (3Me-12:3OH) was prepared by a Grignard reaction of ethylmagnesium iodide and 2-undecanone. Workup with aqueous NH4CI and subsequent purification by column chromatography on silica with hexane+ 5% ethyl acetate gave the desired alcohol in 80% yield. The tertiary acetate was prepared from the alcohol with acetic anhydride, triethylamine and 4-dimethylamino pyridine. All compounds were analyzed by capillary GC and had correct MS and NMR spectra. Alcohols, used in this study, were 95% > pure. Gas chromatography and mass spectrometry All samples were analyzed by capillary gas chromatography (CGC) on either a 30m x 0.25 mm DB5 column or a 30m x 0.25 mm DB225 column, both purchased from J&W, Rancho Cordova, Calif. They were injected in the splitless mode and the compounds were detected by FID. The temperature of the injectors and detectors was 220°C. The purge valve was opened I rain after injection and helium (carrier gas) pressure was maintained at 15 psi. The DB5 column was kept at 60°C for 2 min and then programmed at 10°C/min to 140°C; after 30 rain the temperature w a s raised at the same rate to 160°C. The DB225 column w a s programmed similarly but the temperatures were 60°C for 2 min, then 130°C, and raised to 150°C after 35 rain. Quantification of peaks was performed by either a Spectraphysics 4270 integrator or by a 4290 model equipped with a memory module enabling replay of chromatograms. Combined GC-MS was performed on a Finnigan 5100 machine equipped with a 30 m x 0.25 mm DB5 column in the splitless mode. The column was kept for 2 rain at 60°C
and then programmed at 10-180°C. The purge valve was opened 30 s after injection and helium pressure was maintained at 10 psi. RESULTS A series of pheromonal alcohols including, (E)-7dodecenol (E7-12:OH), (Z)-9-dodccenol (Z912:OH), (E,E)-8,10-dodacadienol (Eg,E10-12:OH), tridecanol (13:OH), (E)-9-tetradecenol (E9-14:OH), one secondary alcohol 2-undecanol ( l I : 2 O H ) and one tertiary alcohol 3-methyl-3-dodecanol (3Me12:3OH), was topically applied to the pheromone glands of C. chalcites. All these alcohols, with the exception of Z9-12:OH, are unrelated to the pheromone components of C. chalcites. Each application also contained an equimolar amount of (Z)-11tetradecenol (Z 11-14:OH) as a metabolic competition standard for the bioacetylation reaction. After the incubation, the gland extracts were analyzed by G C and the amount of the formed acetate from the tested alcohols and from Z I I - 1 4 : O H was determined. The molar amounts of the observed acetates and (Z)- 11tetradecenyl acetate (ZI 1-14:Ac) were calculated for each replicate and the corresponding relative conversion factor K was found by dividing the molar amount of R : A c by Z11-14:Ac. For each alcohol, the mean relative conversion factor K + SD is presented in Table 1. Clamping of the gland resulted in a considerable decrease of the pheromone as compared with glands from nonclamped females. The amount of the major pheromone component Z7-12:Ac declined from an average of 300 ng/female (Snir et al., 1986) to an average of 100 ng/female under the same conditions. All tested alcohols were acetylated by the gland at approximately the same relative rate (Table 1), with E7-12:OH having the largest relative conversion factor K of 1.24 _+ 0.30 and 13:OH having the smallest one of 0.65 + 0.11. Representative G C analyses of nontreated and treated gland extracts are shown in Fig. 1. One sample of the topical application of the tertiary alcohol was analyzed by G C - M S and the presence of 3Me-12:3OH and 3Me-12:3Ac as well as other pheromonal components was confirmed. Tridccanol was tested both under the usual conditions of scotophase and in the photophase. The results (Table 1) indicated that the relative rate of alcohol/acetate conversion K remained the same. In the photophase, the amount of pheromone was low, an average of 4 8 n g as compared with l l 6 n g in scotophase. However, the bioacetylation proceeded
Table 1. Bioacetylation of topically applied alcohols in the pheromone gland of Chrysodeixis chalcites females* Relative Z7-12:Ac Applied (I :1) R:Ac, rig/female± SD ZI 1-14:Ac ng/female± SD conversion ng/female ± SD R:OH + ZI 1-14:OH (10-N tool amount) (10-~ tool amount) factor Kt 127 + 60 11:2OH 18 ± 2 (8.41 ± 0.93) 17 ± 3 (6.69 ± 1.18) 1.12 ± 0.07 103+69 E7-12:OH 24±12 (10,62±5.30) 21±9 (8.27±3.54) 1.24 ±0.30 106+51 Z9-12:OH 33±14 (14.60±6,19) 32+9 (12.60+3.54) 1.17±0.40 120+29 E8, E10-12:OH 14+7 (6.25±3.12) 15±10 (5.90±3.94) 1.14+0.30 116 + 10 13:OH 12 ± 1 (4.96 + 0.41) 19 + 4 (7.48 + 1.57) 0.65 + 0.11 48 _+22 13:OH (light) 29 + 16 (11.98 + 6.61) 38 + 25 (14.96 ± 9.84) 0.56 ± 0.20 101 ± 35 3Me-12:3OH 13 + 2 (5.37 :t: 0.83) 14 ± 4 (5.51 ± 0.39) 0.91 ± 0.22 110+30 E9-14:OH 20+5 (7.87+1.97) 27+4 (10.62±1.57) 0.82+0.10 *Each test was performed on a single moth and was replicated 4--6 times. tThe mean relative conversion factors were obtained by dividing the molar ratios of R:Ac by ZI 1-14:Ac for each replicate and then calculating the mean ± SD value.
Acetylation in the pheromone biosynthesis of C. chalcites
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_o.Lt Fig. l. (A) GC profile of a gland extract from a clamped nontreated female. (B) GC profile of a gland extract from an E8, E10-12:OH treated female. Both performed on the DB 225 column. normally and both 13:Ac and Z I I - 1 4 : A c were formed in the light even in larger amounts than in the dark. One of the alcohols, Z9-12:OH was chosen to test the dependence of the bioacetylation on the presence of PBAN. In previous experiments it was shown that ligation of the head in C. chalcites interrupted the transport of PBAN to the gland and inhibited the formation of the sex pheromone (Altstein et al., 1989). The present results (Table 2) showed that ligation of the head did not stop the bioacetylation. Topical application of Z9-12:OH resulted in a large increase of Z9-12:Ac both in 3-5-day-old females (Table 1) and in 34-36-h-old females as compared to untreated females (Table 2, and Fig. 2). In the ligated females the amount of Z9-12:Ac, formed from the topically applied Z9-12:OH, remained as high as in the nonligated treated moths, despite the fact that the amount of the main pheromone component, Z712:Ac, declined by 50% in the ligated and clamped females as compared to clamped females (Table 2). The basal amounts of Z7-12:Ac in the pheromone glands in the ligation experiment were lower (Table 2) than those in the bioacetylation experiments (Table 1) because the females used in the ligation work were c. 1.5 days old, whereas the other females were 3-5 days old. DISCUSSION
The results of our study indicate that C. chalcites is capable of acetylating primary, secondary and even Table 2. Bioacetylationof topically applied Z9-12:OH in the pheromone gland of ligated Chrysodetxts chalcttes females* Z7-12:Ac,
Z9-12:Ac,
Condition of female ng + SD ng + SD Untreated 180 ± 80 < 3.5 Ligated 61 + 34 < 1.0 Clamped+ Z9-12:OH 49+11 25:1:10 Ligated and clamped+ Z9-12:OH 24 + I I 25 + I I *Each test was performedon a singlemoth and was replicated4-6 times.
525
tertiary aliphatic long-chain alcohols. All alcohols were topically applied to clamped glands. This procedure was introduced by Bjostad and Roelofs in 1983 and is now a standard method. It is used widely for the application of radiolabeled precursors (Bjostad and Roelofs, 1983) and deutero-labeled precursors (Lofsted et al., 1986) in biosynthesis studies of moths. Clamping does not inhibit drastically the biosynthesis as evidenced by the incorporation of labeled precursors, although it might have some effect on the yields of the pheromone components. Most of the decrease in the amounts of the pheromone in clamped C. chalcites females is probably due to a fast release of the gland content. Incorporation of a metabolic internal standard in each test enabled the determination of the relative rates of acetylation. The Z11-14:OH alcohol was chosen as the standard since its corresponding acetate, Z 11-14: Ac, was detected in gland extracts of C. chalcites in trace amounts, c. 0.2% relative to Z7-12:Ac, which is below detection limit in the present one gland extracts (Dunkelblum et al., 1987). It was assumed that this alcohol would be readily accepted by the acetylation enzyme system. Each experiment was run as a competition test with one of the alcohols together with ZI 1-14:OH. This procedure enabled us to obtain a quantitative comparison of the acetylation of various alcohols in the pheromone gland of C. chalcites. All conversion factors K were very similar, in the range of 0.6-1.2 relative to the rate of acetylation of ZI 1-14:OH. The bioacetylation proceeded in the light similarly to that observed by Teal and Tumlinson (1987). The amounts of the formed 13:Ac and Z I I - 1 4 : A c were larger at photophase than at scotophase, but the quantity of pheromone was much lower (Table 1). This may indicate that less natural alcohol precursors were competing with the topically applied alcohols. The yield of the acetates was 1.5--4% comparable to the yield of acetates obtained in H. micacea (Teal and Tumlinson, 1987). Surprisingly, only small amounts of alcohols were recovered in the extracts, and in several cases the applied alcohols were not observed in the GC analysis, although normal yields of acetates were obtained. The reasons for this phenomenon were not investigated. The present experiments demonstrated that in C. chalcites the bioacetylation step has low specificity and can utilize various pheromone-like alcohols. The narrow range of relative conversion factors indicated that different aliphatic alcohols, in the range of C l l - C 1 4 , were converted to their corresponding acetates at approximately the same rate. On the other hand, some differences in the rates of bioacetylation of various alcohols were found in Choristoneura fumiferana (Morse and Meighen, 1987), in particular between the group of C12-C15 and the C10 and C18 alcohols. Unsaturated alcohols were acetylated faster than the corresponding saturated ones. However, in C. fumiferana, the acetylation of alcohols is an intermediate step in the biosynthesis of the aldehydic pheromone components and not the terminal step, as in H. micacea (Teal and Tumlinson, 1987), M. brasicae (Bestmann et al., 1987) and C. chalcites. Different enzymatic systems may be involved in these transformations. In C. fumiferana, both pheromone intermediates, namely alcohols and acetates are
EZRADUNKELBLUMet al.
526
the terminal step in the biosynthesis of the pheromone in C. chalcites is not under hormonal control. The technique of using ligated females for the study of the biosynthetic steps with topically applied compounds may be very helpful in the study of these transformations. More work is needed to confirm that the biosynthesis of the intermediate fatty acids and alcohols, and the terminal acetylation step in C. chalcites are completely separate sequences.
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Acknowledgements--This work was supported by BARD grant No. US 1208-86. The results are a part from the M.Sc thesis of Mrs Hava Mamane.
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REFERENCES
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Fig. 2. (A) GC profile of gland extract from a ligated nontreated female. (B) GC profile of a gland extract from a ligated and Z9-12:OH treated female. Both performed on the DB5 column. stored in the pheromone gland (Silk et al., 1980), whereas in C. chalcites only one alcohol was found in very small amounts. This alcohol, Z7-12:OH, detected in C. chalcites in c. 1% amount relative to Z7-12:Ac (Dunkelblum et al., 1987) was not found in higher quantities in any of the present experiments. The second part of this study was a preliminary investigation of the bioacetylation step in ligated females. It has been shown by Raina and Klun (1984) in H. zea and by Ando et al. (1988) in B. mori that ligation or decapitation of the virgin female caused the interruption of the pheromone biosynthesis. Recently, the same phenomenon was demonstrated in C. chalcites in our laboratory. By analyzing the profiles of the fatty acid precursors, we could demonstrate that the formation of the first monounsaturated fatty acid precursor is affected by ligation (Altstein et al., 1899). In the present experiment, we could show that ligation of the head did not stop the formation of Z9-12:Ac after application of the corresponding alcohol. The amount of the pheromone, as monitored with Z7-12:Ac, declined considerably but the bioacetylation capability remained intact. Normally, the relative amount of Z9-12:Ac, as compared with the major component Z7-12:Ac, is 1-2% which is at most several ng/female. This was found previously in gland extracts and volatiles (Dunkelblum et al., 1987) and in the present study in both nonligated and ligated females. When Z9-12:OH was topically applied to ligated females the amount of formed Z9-12:Ac remained as high as that in treated nonligated moths (Table 2). This amount was much higher than that in untreated ligated females (Fig. 2). These results indicate strongly that PBAN is probably not involved in the bioacetylation and that
Altstein M., Harel M. and Dunkelblum E. (1989) Effect of a neuroendocrine factor on sex pheromone biosynthesisin the tomato looper, Chrysodeixis chalcites (Lepidoptera: Noctuidae). Insect Biochem. In press. Ando T., Arima R., Uchiyama M., Nagasawa H., Inoue T. and Suzuki A. 0988) Pheromone biosynthesis activating neuropeptide hormone in heads of the silkwork moth. Agric. Biol. Chem. 52, 881-883. Bestmann H. J., Herrig M. and Attygale A. B. (1987) Terminal acetylation in pheromone biosynthesis by Mamestra brassicae L. (Lepidoptera: Noctuidae). Experientia 43, 1033-1034. Bjostad L. and Roelofs W. L. (1983) Sex pheromone biosynthesis in Trichoplusia hi: Key steps involve AH desaturation and chain shortening. Science 220, 1387-1389. Dunkelblum E., Snir R., Gothilf S. and Harpaz I. (1987) Identification of sex pheromone components from pheromone gland volatiles of the tomato looper, Plusia chalcites (Esp.). J. chem. Ecol. 13, 991-1003. Lofsted C., Elmfors A., Sjorgen M. and Wijk E. (1986) Confirmation of sex pheromone biosynthesis from (16-D3) palmitic acid in the turnip moth using capillary gas chromatography. Experientia 42, 1059-1061. Morse E. and Meighen E. (1987) Pheromone biosynthesis: Enzymatic studies in Lepidoptera. In Pheromone Biochemistry (Edited by Prestwich G, D. and Blomquist G. I.), pp. 121-158. Academic Press, Orlando, Fla. Raina A. K. and Klun J. A. (t984) Brain factor control of sex pheromone production in the female corn earworm moth. Science 225, 531-533. Roelofs W. L. and Bjostad L. 0984) Biosynthesis of Lepidopteran pheromones. Bioorg. Chem. 12, 279-298. Shorey H. H. and Hale R. H. (1985) Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. J. econ. Ent. 58, 522-524. Silk P. J., Tan S. H., Wiesner C. J., Ross R. J. and Lonergan G. C. (1980) Sex pheromone chemistry of the Eastern Spruce Budworm, Choristoneura fumiferana. Envir. Ent. 9, 640-644.
Snir R., Dunkelblum E., Gothilf S. and Harpaz I. (1986) Sexual behaviour and pheromone titre in the tomato looper, Plusia chalcites (Esp.) (Lepidoptera: Noctuidae). J, Insect Physiol. 32, 735-739. Teal P. E. A. and Tumlinson J. H. 0987) The role of alcohols in pheromone biosynthesis by two noctuid moths that use acetate pheromone components. Archs Insect Biochem. Physiol. 4, 261-269.