inhibition of hydrocarbon biosynthesis in the housefly by 2-octadecynoate

Archives of Insect Biochemistry and Physiology 3:75-86 (1986)

Inhibition of Hydrocarbon Biosynthesis in the Housefly by 2-Octadecynoate Mertxe de Renobales, Edgar J. Wakayama, Premjit P. Halarnkar, Ronald C. Reitz, J. George Pomonis, and Gary J. Blomquist Department of Biochemist y, University of Nevada, Reno (M.de R., E.J. W., P.P.H., R.C.R., G.J.B.), and Metabolism and Radiation Research Laboratory USDA-ARS Fargo, North Dakota (J.G.P.)

The elongation of [9,10-3H]oleoyl-CoA with malonyl-CoA to form 20, 22, and 24 carbon monounsaturated fatty acids was demonstrated in housefly microsomes by radio-GLC. These elongation reactions, which have been postulated to be involved in hydrocarbon biosynthesis, have not been previously demonstrated in insects. 2-Octadecynoate (18:l A2-) inhibited the in vivo incorporation of [l-14C]acetate into both fatty acids and hydrocarbons in a dose-dependent manner. At doses of 10 p g per female housefly of the alkynoic acid, the incorporation of [l-14C]acetate into hydrocarbon was inhibited 93%, the incorporation of [9,10-3H]oleate into hydrocarbon was inhibited 64%, and the incorporation of [l-14C]acetate into total internal lipid was inhibited 65%. Partially purified FAS was inhibited 50% and 95% at 15 pM and 40 pM, respectively, of the alkynoic acid. These results show that 2- octadecynoate inhibits hydrocarbon biosynthesis in the housefly by inhibiting FAS, and the in vivo data suggest that the elongation of 18:l to longer chain fatty acids is also inhibited.

Key words: hydrocarbon biosynthesis, 2-octadecynoate, housefly, fatty acid synthetase, fatty acid elongation

INTRODUCTION

Hydrocarbons are the major components of the cuticular lipids of many insects [l], including the housefly, Muscu dornesficu L. 121. In addition to the critical function of preventing desiccation, the cuticular hydrocarbons of the female housefly serve as components of the sex pheromone [3 ] . (Z)-PTrico- sene and methylalkanes are part of the sex pheromone [3,4], and production

Acknowledgments: This work was supported in part by National Science Foundation grant DC5-8416558. This i s a contribution from the Nevada Agriculture Experiment Station.

Received February 25, 1985; accepted July 9,1985.

Address reprint requests to Dr. Mertxe de Renobales, Department of Biochemistry, University of Nevada, Reno, NV 89557.

0 1986 Alan R. Liss, Inc.

76 de Renobales et al

of these components is regulated by an ovarian product, 20-hydroxyecdysone [5-7l.

The biosynthesis of hydrocarbons, both in plants [8] and in insects [9], is postulated to occur by the elongation of 16 and 18 carbon fatty acids followed by reductive decarboxylation. The in vivo incorporation of 18 carbon saturated and unsaturated fatty acids into cuticular hydrocarbons has been demonstrated in a number of insect species [l,lO]. In the housefly, oleic acid is efficiently and specifically incorporated into C23-C33 alkenes [HI.

In insects, the decarboxylation of C24 and C26 fatty acids to yield C23 and CZ hydrocarbons has been shown to occur in vivo in a cockroach [12] and in vivo and by a microsomal preparation in a termite [13]. Fatty acids longer than 18 or 20 carbons are not usually observed in insects, and the elongation reactions leading to the putative hydrocarbon precursors have not been previously demonstrated in insect. In this work, the microsomal elongation of oleoyl-CoA to 20:1, 22:1, and 24:l is demonstrated.

2-Alkynoic acids are inhibitors of lipid synthesis. The shorter-chain alkynoic acids are potent, irreversible inhibitors of FAS* [14], and it has been suggested that the longer-chain 2-hexadecynoate inhibits the elongation of 16 carbon fatty acids in rats [15,16]. In this paper we examine the effect of 2- octadecynoate on fatty acid and hydrocarbon biosynthesis in vivo and on partially purified FAS.

MATERIALS AND METHODS Insects

Housefly pupae were supplied by the Biology Section, S.C. Johnson and Son (Racine, WI), and the adults were maintained as previously described [lq.

Radioactive Precursors

Sodium [l-14C]acetate (57 mCilmmol) was purchased from Research Prod- ucts International (Elk Grove, IL). [9, 10-3H]Oleic acid (6 Ciimmol) and [9,10- 3H]palmitic acid (6 Ciimmol) were obtained from New England Nuclear (Boston, MA). The CoA derivatives were prepared as previously described [W.

Synthesis of 2-Octadecynoate A lithium acetylide-ethylene diamine complex (8.7 g, 0.0946 mol) in DMSO

(47.3 ml, 2 M solution) was placed in a 500-ml round-bottom flask under argon. An ice bath was used to maintain a temperature of 19-20°C. 1- Bromopentadecane (25 g, 23.4 ml, 0.086 mol) was added dropwise over 3 h.

'Abbreviations: GLC = gas l iquid chromatography; FAS = fatty acid synthetase; CoASH = free coenzyme A; ATP = adenosine triphosphate; NADPH = reduced nicotinarnide adenine dinucleotide phosphate; DTE = dithioerythritol; DMSO = dirnethylsulfoxide; THF = tetrah- ydrofuran; TLC = thin layer Chromatography; EDTA = ethylenediarninetetraacetic acid.

Hydrocarbon Biosynthesis in the Housefly 77

The mixture was allowed to warm to room temperature and remain there for 1 h. The solution was added to 250 ml of brine, extracted three times with 100 ml of hexane, and dried over Na2S04. The 1-heptadecyne was purified by vacuum distillation (yield 17.9 g, 88%).

1-Heptadecyne (1.0 g, 4.26 mmol) in 5 ml of dry THF was placed in a 25- ml three-necked round-bottom flask equipped with a dropping funnel, stir bar, argon source, and drying tube. Butyl lithium (5.3 ml of 0.087 M in hexane) was added dropwise over 30 min at 0°C. After 1 h at 0°C the mixture was poured over crushed solid C02 in THF and slowly warmed to room temperature. The resulting mixture was extracted with diethyl ether to yield 2-octadecynoic acid, which was purified by silica TLC developed in hexane:ether:formic acid (80:40:2). Yield was 85% by GLC.

In Vivo Studies Four- or five-day-old insects were used in all studies. Insects were immo-

bilized at -20°C and, as soon as movement ceased, they were placed in Petri dishes on ice. 2-Octadecynoate (1-10 pglpl in 0.5 mg Tween-80lml water) was injected through the cervix into the thorax with a 10-pl syringe fitted with a 33-gauge needle. Control insects were injected with the Tween-80 solution. At the times indicated, sodium [l-14C]acetate in water, [l-14C]propionate in water, [G-3H]valine in water, or [9,10-3H]oleate in a 0.5 mglml Tween-80 solution were injected as described above. Insects were placed in small bottles at 27°C. After 1 h, the insects were killed by freezing.

The cuticular hydrocarbons were extracted with hexane, isolated, and separated by column chromatography on BioSil A and BioSil A impregnated with 20% (wlw) silver nitrate as previously described [ll]. The internal lipids were extracted by the method of Bligh and Dyer [19]. Aliquots of each sample were placed in scintillation vials with 10 mlo.4% diphenyloxazole in toluene, and radioactivity was assayed by liquid scintillation counting. Counting efficiency was 86-88% for 14C and 4546% for 3H.

Microsomal Elongation Assay

About 1,000 houseflies were used 4 +_ 0.5 days after emergence to adults. The insects were homogenized with a mortar and pestle at 4°C in 0.1 M potassium phosphate buffer, pH 7.2, containing 25 mM sucrose. The homogenate was centrifuged at 12,0009 for 20 min, and the resulting supernatant was centrifuged at 105,OOOg for 1.5 h. The pellets were resuspended in the above phosphate buffer.

The in vitro chain elongation assay was similar to that of Cassagne and Darriet [20]. The total volume of the incubation mixture was 3 ml and contained 0.1 M potassium phosphate, 25 mM sucrose, 1 mM NADPH, 0.2 mM malonyl-CoA, 0.5 mh4 CoASH, 2 mM ATP, 2 mh4 MgC12, 1 mh4 DTE, 2 mh4 sodium ascorbate, 1 mM KCN, and 40 pM KF at pH 7.2. One to ten milligrams of microsomal protein in phosphate buffer, MgC12, sucrose, NADPH, ATP, CoA, KCN, and KF was added to the main compartment of a Warburg flask. The side arm contained phosphate buffer, MgCI2, sucrose, malonyl-CoA, and [3H]oleoyl-CoA. In the Warburg flask, the manometers


were replaced by a stopcock device for successively removing air by vacuum and flushing with pure nitrogen. The flask was preincubated at 30°C for 15 min. The incubation, initiated by tipping the flask, was performed at 30°C for 1 h.

The reaction was stopped by adding 5 ml of 5% (wh) of KOH in methanol and incubated at 80°C for 60 min. The solution was acidified with HCI, and the lipid was extracted by the method of Bligh and Dyer [19]. BF3-methanol was added to the lipid extract and kept at 60°C for 15 min. The methyl esters were extracted with chloroform and analyzed by radio-GLC using a Packard 894 combustion flow-through proportional counter interfaced with a Hew- lett-Packard 5710A gas chromatograph with thermal conductivity detectors. The fatty acid methyl esters were separated on a 1.8 m x 3 mm i.d. 3% (wi w) Dexsil300 on Supelcoport column, temperature programmed from 180°C to 260°C at 2"Clmin and then held at 260°C for 4 min. Counting efficiency was approximately 5% for tritium. Quantitation was accomplished by triangulation.

Partially Purfied Fatty Acid Synthetase Ten milliliters of insects (mixed sexes) were homogenized in 15 ml buffer

(0.1 M potassium phosphate, 1 mM EDTA, pH 7.2, containing 1 mM DTE). The crude homogenate was centrifuged at 10,OOOg for 20 min to remove mitochondria and cell debris. The supernatant was centrifuged again at 105,OOOg for 90 min and the particle-free supernatant was applied to a Se- pharose CL-6B column (110 x 2.8 cm) previously equilibrated with the above buffer. The column was eluted with the same buffer, and 4 ml fractions were collected. FAS-containing fractions were pooled and concentrated using an Amicon ultrafiltration apparatus with a PM-30 membrane. All steps above were done at 4°C. This FAS preparation will be referred to subsequently as partially purified FAS.

Enzyme Assays FAS activity was assayed spectrophotometrically by monitoring the change

in absorbance at 340 nm [21] in a total volume of 0.3 ml. One unit of enzymic activity is defined as 1 nmol NADPH oxidized per min per mg protein. Octadecanoic acid (1 pglpl), 2-octadecynoic acid (1 pgipl) and 5,8,11,14-eicosatetraynoic acid (10 pglpl) were dissolved in 0.5 mgiml Tween-80 solution. Unless otherwise indicated, aliquots of these solutions were added to the above assay mixture with the substrates. Reactions were started by adding aliquots of partially purified FAS preparations.

Protein concentrations were determined by the method of Bradford [22] with bovine serum albumin as standard. The concentrated dye reagent was purchased from Bio-Rad (Richmond, CA).

RESULTS

Microsomal preparations from the housefly elongated [9, 10-3H]oleoyl-CoA to 20:1, 22:l and perhaps 24:l (Fig. 1A) when incubated anaerobically with

A

w 16:O

B

1;

2'


Fig. 1. Radio-CLC traces from the rnicrosornal elongation of [9,10-3H]oleoyl-CoA (A) and [9,10- 3H]palrnitoyl-CoA (B). The substrates were incubated with rnicrosornal aliquots as described in Materials and Methods. After 1 h, lipids were extracted, rnethylated, and analyzed by radio- CLC as described in Materials and Methods.


malonyl-CoA, ATP, and NADPH. The radioactive products were identified by radio-GLC by comparing their retention times to those of standards. The elongation activity was quite variable among different microsomal preparations, varying from almost a 50% conversion of starting material to elongated fatty acids to less than one-tenth conversion of oleoyl-CoA to longer chain products. Similarly, the chain length of the products varied from almost exclusively 20:l to mixtures of 20:1, 22:1, and 24:l in decreasing amounts. To detect the longer-chain components formed from oleoyl-CoA, it was necessary to inject a large enough sample into the radio-GLC such that the radioactivity peak from 18:l was considerably off scale. In all preparations, 20:l was a major product and was clearly shown by radio-GLC analysis. Lesser amounts of 22:l and 24:l were formed, and the amounts of these were more variable. No fatty acids longer than 24:l were detected in any preparation.

Substitution of aliquots of the 12,OOOg pellet, or the 105,OOOg supernatant for microsomes resulted in no detectable elongated products, indicating that the elongating activity was confined to the microsomes.

Incubation of [9,10-3H]stearoyl-CoA (18:O-CoA) with the microsomal preparation resulted in products that cochromatographed with 20:0, 22:0, and probably 24:O (data not shown). [9,10-3H]Palmitoyl-CoA was efficiently elongated to 18:O by housefly microsomes, but longer-chain products were not observed from 16:O CoA (Fig. 1B). At present, it is not known why palmitoyl- CoA is not elongated beyond an 18 carbon fatty acid. These data were consistent with in vivo observations that [9, 10-3H]oleic acid was efficiently incorporated into hydrocarbons of 23-33 carbons, whereas [9, 10-3H]palmitic acid was not efficiently incorporated into hydrocarbon [ll]. These results suggest that there are two or more separate elongating enzyme systems.

In Vivo Inhibition of Hydrocarbon Biosynthesis Preliminary studies showed that up to 10 &insect of 2-octadecynoate

could be injected into 4 or 5-day-old female insects with less than 10% mortality and that up to 5 &insect could be administered to males. Four- day-old females weigh almost twice as much as males.

The incorporation of [l-14C]acetate into hydrocarbon was inhibited by 2- octadecynoate in both a preincubation time- and dose-dependent manner. When [l-14C]acetate was injected into the insect at the same time as 5 pg of alkynoic acid, there was a 75% reduction in the incorporation of label into hydrocarbon in female insects in a 1 h incubation period (Fig. 2A). Maximum inhibition (over goo/,) of hydrocarbon synthesis occurred when the [l- 14C]acetate was injected 2 h after the alkynoic acid (Fig. 2A). This inhibition declined to 55% when the insects were preincubated for 24 h before injecting them with [l-14C]acetate (Fig. 2A).

2-Octadecynoate also inhibited the incorporation of [l-14C]acetate into total internal lipids, which include predominantly 16 and 18 carbon fatty acids in the form of triacylglycerols, phospholipids, and free fatty acids. This effect was less preincubation time-dependent and ranged from 60% to 80% inhibition when acetate was injected from 0 to 24 h after injecting the inhibitor. Figure 2B shows data from female insects.


A

% OF

CONTROL

B % OF

CONTROL

T IME(HOURS)

Fig. 2. In vivo study of the effect of 2-octadecynoate on the biosynthesis of cuticular hydrocarbons (A) and total internal lipids (B) in female houseflies. Insects were injected with 1 pl of a 2-octadecynoate solution in Tween-80 or a Tween-80 solution (controls), incubated for different time periods, and then injected with 1 pCi of [l-'4C]acetate. After 1 h, insects were killed and lipids extracted and analyzed as described in Materials and Methods.

Using a 2 h period between injection of the alkynoate and [l-14C]acetate and a 1 h incubation period with the radioactive substrate, the effect of dose on the synthesis of hydrocarbon and internal lipid was determined (Table 1). The 2-octadecynoate caused over a 90% inhibition in hydrocarbon synthesis at doses of 10 &insect in females and 5 &insect in males. The only difference in the inhibition of the synthesis of saturated or unsaturated

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hydrocarbons occurred at the 2 pg insect dose in females. In all cases except one, there was greater inhibition of hydrocarbon synthesis than of internal lipids synthesis.

The accumulation of 16 carbon fatty acids has been reported in rats after feeding of 2-alkynoic acids [15]. Therefore, we examined the distribution of label from [l-14C]acetate into the fatty acids of control insects and insects treated with 2-octadecynoate to determine whether or not a similar phenom- enon occurred in the housefly. Although the incorporation of label into the total fatty acid fraction was decreased markedly, there was no difference in the percent distribution of radioactivity among each fatty acid in control or treated insects.

The unsaturated hydrocarbons from control females and females treated with 5 pg per insect of 2-octadecynoate were analyzed by radio-GLC to determine whether or not the alkynoate affected the chain length distribution of the products. The results showed that in control females 71% of the radioactivity incorporated into the alkene fraction was in the Z-9-tricosene (23:l) component, with 29% in the 27:l component. In females treated with 5 pg of 2-octadecynoate per insect, these values shifted slightly to 81% of the radioactivity in 23:l and 19% in 27:l. Only trace amounts of radioactivity were in components longer than 27 carbons. Thus the major effect of the alkynoate appears to be to reduce total hydrocarbon synthesis rather than change the chain length of the products.

The methyl branches of methylalkanes (arising from propionate and valine [11,23]) are inserted during the early stages of hydrocarbon synthesis at the level of FAS [23]. 2-Octadecynoate inhibited incorporation of propionate and valine into methyl branched hydrocarbons in females (Table 2). The incorporation of oleic acid was inhibited to a lesser extent than propionate, valine, or acetate (Tables 1 and 2). This, along with the lesser inhibition of the incorporation of [l-14C]acetate into internal lipids (primarily 16 and 18 carbon fatty acids) than into hydrocarbon, suggests that in addition to inhibiting the elongation reaction 2-octadecynoate can inhibit FAS.

Inhibition of FAS by 2-Octadecynoic Acid Partially purified housefly FAS was severely inhibited by 2-octadecynoic

acid (Fig. 3). Fifty percent and 95% inhibition were achieved with 15 pM and

TABLE 2. Effect of 2-Octadecynoate on Hydrocarbon Synthesis From [l-14C]Propionate, [G-3H]Valine, and [9,10-3H]Oleic Acid in Female Housefliesa

0 pgiinsect 5pglinsect 10 pgiinsect YO YO O h

Substrate injected cpm Inhibition cpm Inhibition cpm Inhibition

[l-'4C]propionate 3,288 f 330b 0 471 f 171 85 354 51 89 [ G-3H]valine 2,190 k 330 0 486 f 36 78 261 f 30 88 (9,10-3H]oleic acid 1,270 k 130 0 600 f 70 53 450 f 90 64

'Four-day-old female insects were injected with appropriate amounts of alkynoic acid in 1 pl of an aqueous Tween-80 solution (0.5 mgiml). After 1 h, insects were injected with 1 pCi of substrate in 0.5 p1 water (propionate, valine) or an aqueous Tween-80 solution (oleic acid). After a 1 h incubation period, the insects were killed and the cuticular hydrocarbons extracted and analyzed as described in Methods. bMean f SD; n = 3 groups of five insects per group.

84 de Renobales et a1

I

20 4 0 60 p M

Fig. 3. Effect of 2-octadecynoic acid on the activity of partially purified FAS from the housefly. Assays were done as described in Materials and Methods. The specific activity of FAS preparations ranged between 200 and 250 units and the enzyme concentration varied between 20 and 40 p g h l in the assay cuvette.

40 pM, respectively. Preincubation of the multifunctional enzyme with the inhibitor resulted in a faster loss of activity. Tween-80 was added with the inhibitor to a maximum concentration of 8.3 pglml. Thus the effect of Tween- 80 on FAS was studied. In the presence of 167 pg Tween 80lm1, FAS retained 80% of its activity, indicating that the inhibition observed with the 2-octadecynoic acid solution was due mainly to this compound rather than to the detergent. This inhibition due to 2-octadecynoate could not be reversed by dialysis in the cold against 500 mlO.1 M potassium phosphate buffer, pH 7.2, containing 1 mM EDTA, 1 mM DTE, and 0.5 mglml Tween-80 (four changes of buffer). The inhibited FAS exhibited no activity, whereas a control, di- alyzed in the same flask, retained 44% of its original activity. These results indicate that 2-octadecynoate is tightly bound (perhaps covalently) to FAS.

FAS lost only 15% of its activity in the presence of 12 pM octadecanoic acid, a closely related saturated fatty acid. 5,8,11,14-Eicosatetraynoic acid, in concentrations up to 1 mM, did not inhibit FAS. These results indicate that the triple bond in the second position is necessary for inhibition.

DISCUSSION

A number of lines of indirect evidence have been presented favoring the occurrence of the elongation-decarboxylation pathway for hydrocarbon bio-


synthesis in insects [9,10], and the direct decarboxylation of 24 and 26 carbon fatty acids has been demonstrated in insects [12,13]. However, in most insects, including the housefly, fatty acids longer than 18 or 20 carbons are not observed, and the in vitro elongation of 18 carbon fatty acids to longer- chain components has not been previously reported in insects. The demon- stration that oleoyl-CoA and stearoyl-CoA are elongated to 20, 22, and 24 carbon fatty acids provides strong evidence that the elongation-decarboxylation pathway in insects is indeed operative. The fact that very-long-chain fatty acids have not been detected in most insects has been suggested to indicate tight coupling between the elongation and decarboxylation reactions [9]. The reductive decarboxylation of tetracosanoic acid to n-tricosane in a termite occurred primarily in the microsomal fraction [13], which is consistent with the microsomal elongation of oleoyl-CoA and stearoyl-CoA.

A comparison of the in vivo incorporation of [9,10-3H]oleate and [9,10- 3H]palmitate of similar specific activities into hydrocarbon showed that the 18 carbon fatty acid was incorporated at a much higher rate [ll]. The data presented here show that oleoyl-CoA and stearoyl-CoA are elongated in vitro by a microsomal preparation to 20, 22, and 24 carbon components, whereas palmitoyl-CoA is elongated only to an 18 carbon fatty acid. These results are consistent with the in vivo data on hydrocarbon biosynthesis. They also suggest the possibility that two different microsomal elongation systems are operative, perhaps one in hydrocarbon-synthesizing tissue and the other in nonhydrocarbon-synthesizing tissues.

The chain elongation reactions leading to fatty acids longer than 18 carbon atoms are analogous to some of the partial reactions on the multifunctional protein FAS [24]. In vertebrates, it was demonstrated that 2-hexadecynoic acid inhibited only the chain elongation reactions but not FAS, resulting in an accumulation of 16 and 18 carbon fatty acids [15,16]. However, it appears that in the housefly both FAS and the subsequent elongation reactions are inhibited by 2-octadecynoate. The biosynthesis of the sex pheromone component Z-9-tricosene in the housefly occurs sequentially in a manner in which the entire molecule is formed de novo rather than by the elongation of preformed fatty acids [23]. Therefore, the inhibition of hydrocarbon biosynthesis by 2-octadecynoate could occur at either the cytoplasmic FAS level or the subsequent microsomal elongation state. The in vivo inhibition of the incorporation of [l-14C]acetate into internal lipids along with the in vitro inhibition of partially purified FAS demonstrated that the 18 carbon alkynoic acid inhibits FAS.

Because of the variability in the results from the microsomal elongation studies, it was not possible to obtain unequivocal data that the alkynoate inhibited these reactions. However, the observation that the CIS alkynoate inhibits hydrocarbon biosynthesis from [l-14C]acetate to a greater extent than it does the biosynthesis of total internal lipids suggests that the elongation reactions are also affected. Additional evidence for this is the observation that the incorporation of [9,10-3H]oleate into hydrocarbon is inhibited by the alkynoate, although not as efficiently as is the incorporation of [l-14C]acetate into hydrocarbon. Furthermore, 2-octadecynoate inhibits the synthesis of Z- 9-tricosene to about the same extent as that of the alkenes with longer chain lengths. These data, taken together, suggest that both the cytoplasmic FAS


and the microsomal elongation reactions involved in hydrocarbon biosynthesis are inhibited by 2-octadecynoate.

LITERATURE CITED

1. Blomquist GJ, Dillwith JW: Cuticular lipids. In: Comprehensive Insect Physiology, Bio- chemistry and Pharmacology. GA Kerkut, LI Gilbert, eds. Pergamon Press, Elmsford,

2. Nelson DR, Dillwith JW, Blomquist GJ: Cuticular hydrocarbons of the housefly Muscu domesticu. Insect Biochem 11, 187 (1981).

3. Carlson DA, Mayer MS, Silhacek DL, James JD, Beroza MM, Bier1 BA: Sex attractant pheromone of the housefly: Isolation, identification and synthesis. Science 174, 76 (1971).

4. Uebel EC, Sonnet PE, Miller RW: Housefly sex pheromone: Enhancement of mating strike activity by combination of (Z)-9-tricosene with branched saturated hydrocarbons. J Econ Entomol5, 905 (1976).

5. Dillwith JW, Adams TS, Blomquist GJ: Correlation of housefly sex pheromone production with ovarian development. J Insect Physiol29, 377 (1983).

6. Adams TS, Dillwith JW, Blomquist GJ: The role of 20-hydroxyecdysone in housefly sex pheromone synthesis. J Insect Physiol30, 287 (1984).

7. Blomquist GJ, Adams TS, Dillwith JW: Induction of sex pheromone production in male houseflies by ovary implants or 20-hydroxyecdysone. J Insect Physiol30, 295 (1984).

8. Kolattukudy PE, Croteau R, Buckner JS: Biochemistry of plant waxes. In: Chemistry and Biochemistry of Natural Waxes. Kolattukudy, PE, ed. Elsevier, New York, p 289 (1976).

9. Blomquist GJ, Jackson LL: Chemistry and biochemistry of insect waxes. Prog Lipid Res 27, 319 (1979).

10. Howard RW, Blomquist GJ: Chemical ecology and biochemistry of insect hydrocarbons. Annu Rev Entomol27, 149 (1982).

11. Dillwith JW, Blomquist GJ, Nelson DR: Biosynthesis of the hydrocarbon components of the sex pheromone of the housefly, Musca dornesticu. Insect Biochem 22, 247 (1981).

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inhibition of hydrocarbon biosynthesis in the housefly by 2-octadecynoate

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