Regulation of pheromone biosynthesis in the Z strain Э of the European corn borer Ostrinia nubilalis.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 65:29–38 (2007) Regulation of Pheromone Biosynthesis in the “Z Strain” of the European Corn Borer, Ostrinia nubilalis H.S. Eltahlawy, J.S. Buckner, and S.P. Foster* The regulation of pheromone biosynthesis by the neuropeptide PBAN in the Z strain of the European corn borer, Ostrinia nubilalis, was investigated using labeled intermediates. Injection of radiolabeled acetate showed PBAN did not influence the de novo synthesis of saturated fatty acids in the gland. When deuterium-labeled myristic acid was topically applied to the gland, females injected with PBAN produced more labeled pheromone than did control females, indicating that PBAN controls one of the later steps of pheromone biosynthesis. Although more myristic acid was ∆11-desaturated in the gland in the presence of PBAN, this was counterbalanced by less ∆11-desaturation of palmitic acid, indicating that desaturase activity did not change overall. This change in flux of myristic acid through to pheromone was shown to be caused by increased reduction of fatty acid pheromone precursors occurring in the presence of PBAN. Arch. Insect Biochem. Physiol. 65:29–38, 2007. © 2007 Wiley-Liss, Inc. KEYWORDS: pheromone biosynthesis-activating neuropeptide; PBAN; Lepidoptera; Pyralidae; fatty acid reduction INTRODUCTION In most species of moths, the female produces and releases a volatile sex pheromone that attracts conspecific males. To date, the sex pheromones or attractants (i.e., Witzgall et al., 2004) of roughly 1,600 species of moths have been identified, with the chemicals sorting into two major types of compounds: those with structural similarity to common fatty acids and those with a hydrocarbon structure (Witzgall et al., 2004). The fatty acid–like compounds appear to be more common, being used by 42 out of the 50 families of Lepidoptera for which pheromones or attractants have been identified (Witzgall et al., 2004). The fatty acid–like compounds typically have an even number of carbon atoms (8–18) in a straight chain, with one or more double bonds and terminal oxygenated functionality of an alcohol, acetate ester, or aldehyde (Tillman et al., 1999). These compounds are biosynthesized in a specialized gland, located between the 8th and 9th abdominal segments of the female. Their biosynthesis involves de novo synthesis of medium-chain Department of Entomology, North Dakota State University, Fargo, North Dakota Contract grant sponsor: USDA-NRI; Contract grant sponsor: NSF; Contract grant number: EPS-0132289. Abbreviations used: ACCase = acetyl CoA carboxylase; D3-14:COOH = [14,14,14-D3]-tetradecanoic acid; D4-16:COOH = [7,7,8,8-D4]-hexadecanoic acid; D5-Z11-14:COOH = [13,13,14,14,14,14-D5]-(Z)-11-tetradecenoic acid; E11-14-OAc = (E)-11-tetradecenyl acetate; E11-14:Acyl = (E)-11-tetradecenoate; FAME = fatty acid methyl ester; GC-MS = gas chromatography-mass spectrometry; HPTLC = high performance thin layer chromatography; PBAN = pheromone biosynthesis-activating neuropeptide; TGs = triacylglycerols; TLC = thin layer chromatography; Z11-14:OAc = (Z)-11-tetradecenyl acetate; Z1114:Acyl = (Z)-11-tetradecenoate;; Z11-16:Acyl = (Z)-11-hexadecenoate; 14:Acyl = tetradecanoate; 14:Me = methyl tetradecanoate; 15:Me = methyl pentadecanoate; 16:Acyl = hexadecanoate; 16:Me = methyl hexadecanoate. *Correspondence to: Dr. S.P. Foster, Department of Entomology, North Dakota State University, P.O. Box 5346, Fargo, ND 58105-5346. E-mail: Stephen.Foster@ndsu.edu Received 25 July 2006; Accepted 20 January 2007 © 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20175 Published online in Wiley InterScience (www.interscience.wiley.com) 30 Eltahlawy et al. length, saturated fatty acids, usually stearic or palmitic, which are partially metabolized to produce the pheromone components. Specific steps in this metabolism include desaturation, cytosolic β-oxidation (2-carbon chain-shortening), reduction (to an alcohol), and acetylation (to produce acetate esters) or oxidation (to produce aldehydes) (reviewed in Bjostad et al., 1987, Tillman et al., 1999, Jurenka, 2003). In many species of moths, biosynthesis of such pheromone chemicals is under the regulation of PBAN (Raina, 1995, Rafaeli and Jurenka, 2003). PBAN was first isolated and sequenced from Helicoverpa zea (Raina et al., 1989). Since this first identification, other PBANs have been sequenced from a number of other moths (for a review, see Rafaeli and Jurenka, 2003). All PBANs have a high degree of similarity at the C-terminus, in which an amidated terminal FXPRL (X = S, T, G, or V) motif appears to confer activity (Rafaeli and Jurenka, 2003). PBAN is produced in the brainsubesophageal ganglion complex and is released into the hemolymph, probably from the corpora cardiaca (Rafaeli and Jurenka, 2003). It is then transported to the pheromone gland, where it interacts with a G protein–coupled receptor (Choi et al., 2003). PBAN is thought to regulate pheromone biosynthesis in moths by controlling the activity of a single pheromone biosynthetic enzyme. To date, only a handful of studies have investigated which enzyme is controlled by PBAN. Somewhat surprisingly, given the similarity of moth pheromone biosynthetic systems and the consistency of PBAN in regulating pheromone biosynthesis (Rafaeli and Jurenka, 2003), different studies have indicated that PBAN may control different enzymes, according to the species. In several species (Tang et al., 1989; Jacquin et al., 1994; Jurenka et al., 1991a; Zhao et al., 2002), PBAN influences a step in fatty acid synthesis, probably the conversion of acetyl-CoA to malonyl-CoA by the enzyme acetyl CoA carboxylase. In other species, it influences the metabolism of fatty acids including their reduction (Martinez et al., 1990, Fabrias et al., 1995, Ozawa and Matsumoto, 1996) or acetylation following reduc- tion (Mas et al., 2000). One other study (Fang et al., 1996) suggested that PBAN may control the mobilization of pheromone precursor fatty acids from TGs. Together, these data indicate that either PBAN is capable of controlling different biosynthetic enzymes in different species, perhaps by a common mechanism (e.g., phosphorylation/dephosphorylation), or that the effect of PBAN on pheromone biosynthesis for a given species is more complex than thought, and has yet to be elucidated fully. The European corn borer, Ostrinia nubilalis Hübner (Pyralidae), was introduced from Italy into North America in the early 1900s, and has become a major pest of corn in the United States (Becker, 2000). In the 1970s, two distinct pheromone strains of O. nubilalis were identified, a socalled “Z-strain” using a mixture of Z11-14-OAc and E11-14-OAc in a ratio of roughly 97:3, and an “E-strain,” which uses the opposite ratio of isomers (3:97) (Klun, 1975, Cardé et al., 1978). In both strains, the two pheromone components are biosynthesized from myristic acid by the same ∆11-desaturase (Roelofs et al., 2002), which produces a roughly equal amount of the two precursor acids, Z11-14:Acyl and E11-14:Acyl (Wolf and Roelofs, 1987). The distinct pheromonal blends produced by the females of the two strains are determined by differences in the specificity of their respective fatty acid reductase, which, in the Z-strain, shows greater selectivity for Z11-14:Acyl and, in the E strain, for E11-14: Acyl (Zhu et al., 1996). Pheromone biosynthesis in both strains of O. nubilalis is under the regulation of PBAN (Ma and Roelofs, 1995). However, which enzyme(s) is controlled by PBAN has not been investigated in either strain. In this article, we report the results of our study on the effect of PBAN on pheromone biosynthesis in the “Z-strain” of O. nubilalis. MATERIALS AND METHODS Insects “Z-strain” O. nubilalis were obtained as pupae from Mass Rearing Laboratories (Madrid, IA). The two sexes were separated from each other as puArchives of Insect Biochemistry and Physiology May 2007 doi: 10.1002/arch. PBAN Regulation in O. nubilalis pae, and the female pupae maintained at 25 ± 0.5°C under a 16:8 (light: dark) photoperiod. Adults were collected daily, just before the start of the scotophase, and placed in 500-ml plastic containers, along with a 10% sugar solution absorbed on cotton wool, under the same temperature and photoperiodic conditions as the pupae. For the experiments, females, 1–2 days after collection, were decapitated and left for 24 h before use (i.e., when 2–3 days old). This age corresponds to the time in intact females when pheromone production is maximal (Foster, 2004b). Chemicals The sodium salt of [2-14 C] acetic acid (51 mCi/ mmol) and BetaMax (liquid scintillation cocktails) were purchased from MP Biomedicals (Aurora, OH). Deuterium-labeled D3-14:COOH and D416:COOH were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA) and were both greater than 99% isotopically pure. [13,13,14, 14,14,14-D 5]-Z11-14:OAc was a gift from Dr. Christer Lofstedt (Lund University, Sweden). It was converted to the corresponding acid, D5-Z1114:COOH, by hydrolysis with methanolic KOH (O.5 M), followed by oxidation by pyridinium dichromate in dimethyl formamide (Corey and Schmidt, 1979). The acid was purified through a small silica gel column constructed from a Pasteur pipette. GC-MS analysis indicated that it was free of any corresponding acetate or alcohol and was >97% isotopically pure. Synthetic Helicoverpa zea PBAN was purchased from Bachem, Inc. (Torrance, CA), and tripentadecanoin and other chemical standards were from Sigma Chemical Co. (St Louis, MO). De Novo Synthesis of Fatty Acids Radiolabeled 2-14C acetate was dissolved in Manduca saline (4 mM NaCl, 40 mM KCl, 243 mM sucrose, 18 mM MgCl, 3 mM CaCl, 0.1 mM polyvinylpyrollidone, 1.5 mM PIPES, pH 6.5), and 2.5 µl (0.3µci) injected into the abdomen of a decapitated female, along with either saline or 5 pmol Archives of Insect Biochemistry and Physiology May 2007 doi: 10.1002/arch. 31 PBAN in saline. Females were left for 2 h in the dark at their rearing temperature before the pheromone gland was dissected. Ten glands were pooled for either treatment and extracted with chloroform/ methanol (2/1 vol./vol.) for 24 h at –15°C. Following extraction, the gland extract was transferred into a 1 ml Reacti-vial and the solvent removed with a gentle stream of N2. The residue was then base methanolyzed (Litchfield, 1972) by adding 100 µl of 0.5M methanolic KOH and allowing it to react for 3–4 h at room temperature with occasional mixing. FAME were formed by adding 100 µl 1.0 M HCL (aq), and extracted with chloroform. Aliquots (5% of total) of the extracts were transferred into separate 7-ml glass vials, the chloroform evaporated, and 6.5 ml of BetaMax added. Radioactivity in each vial was counted using a Packard model 2300TR liquid scintillation analyzer (Packard Instrument, Co., Meriden, CT) at counting efficiency of 97% for 14C. A further aliquot (5% of total) of the extract was subjected to normal phase TLC, using silica HPTLC plates (EM Science, Gibbstown, NJ) developed with hexane/ethyl ether/ formic acid (80:20:1). Radioactivity on the developed plates was counted using a Bioscan System 200 radio-TLC scanner (Bioscan Inc., Washington, DC). Following counting, spots of samples and various synthetic standards were visualized with iodine vapor. Topical Application of Fatty Acids The pheromone gland of a female was extruded by placing an alligator clip near the posterior end of the abdomen, and a deuterium-labeled acid (in DMSO) applied topically to the gland. Ten minutes after application, the clip was removed and 2.5 µl of either saline (NaCl, 188 mM, KCL, 4.8 mM, CaCl2, 2.6 mM, Hepes, 10.0 mM, glucose, 13.9 mM, pH 6.8) or PBAN (5 pmol in saline) injected into the abdomen of the female. Insects were left at ambient temperature for the assigned time, after which their pheromone gland was dissected and extracted in either n-heptane (for pheromone analyses) or 1:2 dichloromethane:methanol (for glycerolipid analyses) for at least 16 h at –15°C. Either 25 32 Eltahlawy et al. ng (E)-4-tetradecenyl acetate or 200 ng tripentadecanoin was added as an internal standard to the respective solvents. The following experiments were performed: 1. Effect on production of pheromone and pheromone precursor acid. D3-14:COOH (10 µg/µl in DMSO) was applied to the gland and the amount of label in both pheromone components and precursor acids determined 2 h following application 2. Effect on fatty acid chain-shortening. D416:COOH (10 µg/µl in DMSO) was applied to the gland and the amount of label in 14:Acyl determined 2 h following application. 3. Effect on fatty acid desaturation. The products of ∆11-desaturation in the pheromone gland of female O. nublialis are Z11-14:Acyl and E11-14:Acyl. However, since these moieties are readily reduced to pheromone (Zhu et al., 1996), their quantities do not represent the total amount of ∆11-desaturation. The ∆11-desaturase of O. nubilalis is also capable of desaturating 16:Acyl to Z11-16:Acyl, which is not reduced but stored in the gland (Roelofs et al., 2002, Foster, 2004b). Therefore, we tested the effect of PBAN on ∆11desaturation by analyzing for labeled Z1116:Acyl 2 h following application of D416:COOH (10 µg/µl in DMSO). 4. Effect on fatty acid reduction. D5-Z11-14: COOH (5 µg/µl in DMSO) was applied to the gland and the amount of label in Z1114:OAc and Z11-14:Acyl determined at 3 times (0.25, 0.75, 1.5 h). Pooled extracts of 10–12 pheromone glands were analyzed by GC-MS using a Hewlett-Packard 5890 Series II gas chromatograph/5972 mass selective detector. The GC was operated with splitless injection and fitted with a 30 m × 0.25 mm id ZB Wax column (Phenomenex Inc., Torrance, CA). The column oven was programmed from 80– 220°C at 15°C min–1 after an initial delay of 1 min. Helium was the carrier gas at a linear flow velocity of 30 cm sec–1. The MS was used in the selected ion mode, monitoring the following m/ z: 194 (for tetradecenyl acetates; = M+-acetic acid), 197 (D3-labeled tetradecenyl acetates), 198 (D4labeled tetradecenyl acetates), and 199 (D5-labeled tetradecenyl acetates). Pooled glycerolipid extracts of 6–10 dissected pheromone glands were analyzed as FAME, generated by base methanolysis (Litchfield, 1972), as previously described. GC-MS conditions were as for the pheromone analyses except the following m/z were monitored: 242 (M+ of 14:Me), 246 (M+ of D4labeled 14:Me), 240 (M+ of methyl tetradecenoates), 244 (M+ of D4-labeled methyl tetradecenoates), 245 (M+ of D5-labeled methyl tetradecenoates), 270 (M+ of 16:Me ), 274 (M+ of D4-labeled 16:Me), 268 (M+ of methyl hexadecenoates), 272 (M+ of D4-labeled methyl hexadecenoates), and 256 (M+ of 15:Me). Statistical Analyses The experiment testing the de novo synthesis of fatty acids was replicated five times. In all other experiments, at least 6 replicates of each treatment were conducted. PBAN and saline treatments in the various experiments were compared by Wilcoxon Rank Sum tests. Differences between treatments are reported at P < 0.05. RESULTS De Novo Fatty Acid Synthesis There was no significant difference in mean radioactivity of the derivatized extracts between females injected with PBAN (mean = 1,083.4 ± 138 DPM) and those injected with saline (mean = 1,385 ± 106 DPM). Radio-TLC analysis showed two major peaks at Rf = 0.22 and Rf = 0.52, as well as a smaller peak at the origin. In both the PBAN and saline samples, these two peaks accounted for roughly 65% of the total radioactivity (the peak at the origin accounted for roughly 6% of the total). No peak at an Rf value (=0.13) corresponding to that of synthetic (Z)-11-tetradecenol (which would have resulted from hydrolysis of the major pheromone component) was discernible in samples from both the PBAN- and saline-injected females. Archives of Insect Biochemistry and Physiology May 2007 doi: 10.1002/arch. PBAN Regulation in O. nubilalis Pheromone and Fatty Acid Pheromone Precursor Production From Myristic Acid Females injected with PBAN and with D314:COOH topically applied to their pheromone gland had significantly greater titers of both native (Fig. 1a) and labeled (Fig. 1b) Z11-14:OAc than did the corresponding females injected with saline. The titers of native E11-14:OAc were very low in all the samples, and no significant difference in the respective mean titers of this compound between PBAN- and saline-injected females was observed (data not shown). Labeled E11-14:OAc was undetectable in all the samples. FAME analyses of glands treated with D314:COOH showed no significant differences in titers of labeled or native (data not shown) 14:Acyl, Z11-14:Acyl and E11-14:Acyl between PBAN- and saline-injected females (Fig. 2). 33 Fatty Acid Desaturation Saline-injected females (mean = 0.095 ± 0.009 ng/female) had a significantly higher mean titer of labeled Z11-16:Acyl than did PBAN-injected ones (mean = 0.062 ± 0.009 ng/female). Fatty Acid Reduction Maximum titer of labeled Z11-14:OAc was reached at the shortest time (0.25 h) analyzed following application of D5-Z11-14:OAc, for both PBAN- and saline-injected females (Fig. 3a); there was no significant difference between the respective titers of the two types of females at this time. For the PBAN-injected females, labeled Z11-14;OAc titer remained at this higher level until 0.75 h after application, before declining significantly by 1.5 h. In contrast, in the saline-injected females, the Fatty Acid Chain-Shortening No difference in mean titer of labeled 14:Acyl was observed between PBAN- (mean = 0.18 ± 0.04 ng/female) and saline-injected females (mean = 0.21 ± 0.04 ng/female). Fig. 1. Mean titers (and SEM) of (a) labeled Z11-14:OAc and (b) native Z11-14:OAc in the pheromone gland of decapitated Z-strain Ostrinia nubilalis females, topically applied with D3-14:COOH, and injected with either PBAN or saline. Means were derived from 10 replicates, with 10– 12 glands per replicate. Different letters atop bars indicate means that are significantly different (P < 0.05). Archives of Insect Biochemistry and Physiology May 2007 doi: 10.1002/arch. Fig. 2. Mean titers (and SEM) of labeled 14:Acyl, Z1114:Acyl, and E11-14:Acyl in the pheromone gland of decapitated Z-strain Ostrinia nubilalis females, topically applied with D3-14:COOH, and injected with either PBAN or saline. Means were derived from 10 replicates, with 8– 10 glands per replicate. Different letters (with the same subscript) atop bars indicate means that are significantly different (P < 0.05). 34 Eltahlawy et al. Fig. 3. Mean titers (and SEM) of (a) labeled Z11-14:OAc and (b) native Z11-14: OAc in the pheromone gland of decapitated Z-strain Ostrinia nubilalis females, topically applied with D5-Z11-14:COOH, and injected with either PBAN or saline. Means were derived from 10 replicates, with 10–12 glands per replicate. Different letters atop bars indicate means that are significantly different (P < 0.05). titer of labeled Z11-14:OAc titer had significantly declined between 0.25 and 0.75 h. At the latter time, there was a significant difference in the titer of labeled Z11-14:OAc between PBAN- and salineinjected females. Native Z11-14:OAc titers were similar at all three times for saline-injected females. However, for PBAN-injected females native Z1114:OAc titers increased significantly from 0.25 to 0.75 h and remained at this higher level at 1.5 h. At the two longer times, titers of native Z11-14:OAc were significantly greater in PBAN-injected females than in controls. There were no differences in labeled (Fig. 4a) or native (Fig. 4b) Z11-14:Acyl titers, in females over time, or between females injected with PBAN or saline at any given time. DISCUSSION The neuropeptide PBAN regulates the biosynthesis of sex pheromone in O. nubilalis (Ma and Roelofs, 1995). Our data indicate that, in the “Zstrain” of O. nubilalis, PBAN affects biosynthesis during the later steps, specifically following the production of myristic acid in the gland. Unlike several other species (Tang et al., 1989, Jurenka et al., 1991b, Jurenka, 1997, Jacquin et al., 1994), PBAN does not appear to control the biosynthesis of saturated fatty acids directly. In O. nubilalis, there are several steps involved in the biosynthesis of pheromone from myristic acid: ∆11 desaturation, in which myristate is converted to the pheromone precursor acids, Z11Archives of Insect Biochemistry and Physiology May 2007 doi: 10.1002/arch. PBAN Regulation in O. nubilalis 35 Fig. 4. Mean titers (and SEM) of (a) labeled Z11-14:Acyl and (b) native Z11-14:Acyl in the pheromone gland of decapitated Z-strain Ostrinia nubilalis females, topically applied with D5Z11-14:COOH, and injected with either PBAN or saline. Means were derived from 6–8 replicates, with 6–8 glands per replicate. There were no significant differences among means of any of the treatments. 14:Acyl and E11-14:Acyl (Roelofs et al., 2002), fatty acid reduction, in which the pheromone precursor acids are converted to alcohols (Zhu et al., 1996) and acetylation (Jurenka and Roelofs, 1989), in which the pheromone acetate esters are produced. With regard to desaturation, our data show that increased desaturation of myristate occurs in the gland in the presence of PBAN, with more labeled pheromone and similar amounts of the precursor acids produced, compared to the saline control. However, this increased desaturation of myristate apparently comes at the expense of ∆11desaturation of palmitate, with PBAN-injected females showing a concomitant decrease (relative to the saline controls) in labeled Z11-16:Acyl, a fatty acid normally produced, but not reduced, in the Archives of Insect Biochemistry and Physiology May 2007 doi: 10.1002/arch. gland. This suggests that PBAN does not increase the activity of ∆11-desaturation per se, but somehow increases the flux of myristate through ∆11desaturation. Since PBAN does not cause an increase in production of saturated fatty acids, nor does it increase the activity of chain-shortening of palmitate, the increase in flux of myristate through ∆11-desaturation suggests that PBAN affects the activity of a downstream enzyme in the biosynthetic pathway. Alternatively, PBAN could cause increased mobilization of myristate from stores (predominantly triacylglycerols; Foster, 2004b). However, this would probably increase the concentration of palmitate (as palmitoyl-CoA) more, since triacylglycerol lipases in general tend to be stereospecific 36 Eltahlawy et al. rather than substrate-specific (Benlohr and Simpson, 1996), and palmitate is far more abundant in the gland of female O. nubilalis than myristate (Foster, 2004b). Thus, although more myristate may be produced from increased chain-shortening, the increase in palmitate concentration should also yield increased production of Z11-16:Acyl, which was not observed. Of the two downstream biosynthetic processes, fatty acid reduction and acetylation, an increase in activity of the former seems more likely since (Z)-11-tetradecenol titer does not increase in the gland following decapitation (Foster, unpublished data). Further support for the activity of fatty acid reduction being influenced by PBAN was provided by the topical application of labeled precursor acid (D5-Z11-14:COOH). This showed that over the first 0.75 h of the experiment, PBAN-injected females produced more labeled pheromone than did saline-injected ones. At 0.25 h, control females produced similar levels of labeled Z1114:OAc as PBAN-injected ones. However, they produced significantly less native Z11-14:OAc. Thus, total (labeled and native) Z11-14:Acyl reduced was greater in PBAN-injected, than in control, females. Moreover, while labeled Z11-14:Acyl titer was fairly constant over the 1.5 h of the experiment for saline-injected females, it increased from 0.75–1.5 h for PBAN-injected ones; i.e., in the same period that labeled Z11-14:OAc decreased. This suggests that the principal effect of PBAN on fatty acid reduction occurred within the first 0.75 h after injection, allowing increased flux of myristate through ∆11-desaturation and therefore increased production of pheromone. Then, as the effect of PBAN on fatty acid reduction decreased, probably due to degradation of the neuropeptide (Weirich et al., 1995), the level of labeled Z11-14:Acyl increased until the flux of myristate through ∆11-desaturation slowed, possibly due to inhibition by increased glandular acyl-CoA concentrations (Foster, 2004a). Thus, the flux of myristate through ∆11-desaturation appears to be controlled indirectly by fatty acid reduction. Control of fatty acid reduction by PBAN has also been demonstrated in several other species of moths (Martinez et al., 1990, Fabrias et al., 1995, Ozawa and Matsumoto, 1996). Through controlling fatty acid reduction, PBAN is able to control the flux of fatty acids metabolized to pheromone in the gland. However, there is no direct control of de novo fatty acid synthesis. Although saturated fatty acid titers in the pheromone gland of female O. nubilalis show some diel changes, with higher titers in the photophase than in the scotophase, titers remain fairly constant for a given time from day to day (Foster, 2004b). This suggests that fatty acids are not synthesized continuously (otherwise titers would increase over time), but that some modulation of this process occurs. In O. nubilalis, when PBAN release ceases, acyl-CoA concentrations should increase in the gland, since the upstream processes continue producing fatty acids that will not be metabolized directly to pheromone. High acyl-CoA concentrations inhibit the activity of acetyl CoA carboxylase, the enzyme that catalyzes the first committed step in fatty acid synthesis (Salati and Goodridge, 1996). Such an inhibition would, therefore, modulate the synthesis of fatty acids in the gland. Thus, we believe that pheromone biosynthesis in O. nubilalis is modulated by a combination of hormonal factors and product inhibition. Future work will investigate potential product inhibition of fatty acid synthesis. ACKNOWLEDGMENTS Thanks are due to Dr. Wendell Roelofs for the suggestion of analyzing desaturase activity on palmitic acid. LITERATURE CITED Becker H. 2000. Ugly duckling corn repels borers. Agric Res 48:17. Benlohr DA, Simpson MA 1996. Adipose tissue and lipid metabolism. In: Vance DE, Vance J, editors. Biochemistry of lipids, lipoproteins and membranes. Amsterdam: Elsevier. p 257–281. Bjostad LB, Wolf WA, Roelofs WL. 1987. 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