Unusual mechanism of hydrocarbon formation in the housefly: cytochrome P450 converts aldehyde to the sex pheromone component (Z)-9-tricosene and CO2.

An unusual mechanism for hydrocarbon biosynthesis is proposed from work examining the formation of (Z)-9-tricosene (Z9-23:Hy), the major sex pheromone component of the female housefly, Musca domestica. Incubation of (Z)-15-[1-14C]- and (Z)-15-[15,16-3H2]tetracosenoic acid (24:1 fatty acid) with microsomes from houseflies gave equal amounts of [3H]Z9-23:Hy and 14CO2. The formation of CO2 and not CO, as reported for hydrocarbon formation in plants, animals, and microorganisms [Dennis, M. & Kolattukudy, P. E. (1992) Proc. Natl. Acad. Sci. USA 89, 5306-5310], was verified by trapping agents and by radio-GLC analysis. Incubation of (Z)-15-[15,16-3H2]tetracosenoyl-CoA with microsomal preparations in the presence of NADPH and O2 gave almost equal amounts of (Z)-15-3H2]tetrasosenal (24:1 aldehyde) and Z9-23:Hy. Addition of increasing amounts of hydroxylamine (aldehyde trapping agent) caused a decrease in hydrocarbon formation with a concomitant increase in oxime (aldehyde derivative) formation. The 24:1 aldehyde was efficiently converted to (Z)-9-tricosene only in the presence of both NADPH and O2. Bubbling carbon monoxide (20:80 CO/O2) or including an antibody against housefly cytochrome P450 reductase inhibited the formation Z9-23:Hy from 24:1 aldehyde. These data demonstrate an unusual mechanism for hydrocarbon formation in insects in which the acyl-CoA is reduced to the corresponding aldehyde and then carbon-1 is removed as CO2. The requirement for NADPH and O2 and the inhibition by CO and the antibody to cytochrome P450 reductase strongly implicate the participation of a cytochrome P450 in this reaction.

Identification of compounds in an HPLC fraction from female extracts that elicit mating responses in male screwworm flies,Cochliomyia hominivorax.

When hexane extracts of mature screwworm females were chromatographed on a silica gel column, mating stimulant activity was concentrated in a fraction that eluted with hexane-ether (94∶6, v/v). Separation of this fraction with HPLC (acetonitrile-acetone; 60∶40, isocratic) resulted in a chromatogram of some 20 peaks. Only peaks 4-11 elicited mating responses. Peaks 5-10 had most of the activity, with peak 8 producing the highest response. Sixteen compounds were characterized from peak 8 by gas chromatography-mass spectrometry: six unbranched secondary acetates (C31H62O2); seven previously unreported methyl-branched secondary acetates (C32H64O2); one unbranched ketone (C31H62O); and one methyl-branched ketone (C32H64O). The isomeric acetates were not completely resolved from each other by capillary gas chromatography (CGC) on methyl silicone columns. The sixteenth compound was an aldehyde (C30H60O) that was present only in occasional peak 8 preparations. These compounds and several derivatives were characterized by capillary gas chromatography-mass spectrometry (CGC-MS). The position of the acetate group was ascertained by conversion to a keto group or by replacement of the acetate with a methyl group. Pheromone activity was not observed in peaks trapped either from CGC or by recombination of the trapped CGC peaks from HPLC peak 8. This apparent loss of activity from CGC peaks or from TLC cannot currently be explained.

Synthesis of omega 9-tetracosynoic and omega 9-octacosynoic acids as entries into tritiated metabolic precursors of cis-9-tricosene and cis-9-heptacosene in the housefly.

The syntheses of 15-tetracosynoic acid (omega 9-tetracosynoic acid) and 19-octacosynoic acid (omega 9-octacosynoic acid) are described. These alkynoic acids are to be tritiated to the corresponding alkenoic acids, which will be used as metabolic precursors of housefly pheromone components. The final step in each synthesis involved the coupling of 1-decyne to the lithio-salt of the appropriate omega-bromoacid. Homologation of dibromoalkanes was accomplished with triphasic catalytic displacement of bromide by cyanide ion. Oxidation of a bromo-alcohol to a bromoacid was performed in benzene with KMnO4 and 18-crown-6 ether.

Multicomponent attractant for female screwworm flies,Cochliomyia hominivorax, in bovine blood.

An olfactometer bioassay was used to follow attractant for screwworm flies,Cochliomyia hominivorax, in steam distillates of bovine blood under different distillation and storage conditions and after HPLC separation of components in a water-methanol gradient. In addition, fly responsiveness was examined in relation to sex and ovarian stage. Gravid and vitellogenic nullipars were attracted to the blood, although the former predominated four to one. Males did not respond at a dose that attracted 76% of gravid females. Maximum attractiveness occurred when distillate was stored in sealed glass ampoules. An argon atmosphere made storage at ambient temperatures feasible, but offered no advantage during storage at ca. -60°C or during distillation. The HPLC separation produced four fractions that duplicated the attractiveness of the distillate when recombined but showed little activity when presented as two-fraction, and most three-fraction, mixtures. Availability of the HPLC fractions for combination with other samples will facilitate location via bioassay of attractant components in samples obtained from subsequent or alternate isolations that preserve only one or two elements of the multicomponent mixture.

Cuticular hydrocarbons of the screwworm,Cochliomyia hominivorox (Diptera: Calliphoridae) : Isolation, identification, and quantification as a function of age, sex, and irradiation.

A novel series of 2,X-dimethylalkanes were isolated and identified. The nonpolar fraction of the surface lipids secreted by the adult (5-day-old) screwworm,Cochliomyia hominivorax, contains over 130 different hydrocarbons comprising normal alkanes (32% of the total hydrocarbon), branched alkanes (53%), and monoalkenes (11%). Branched alkanes included monomethylalkanes with substitution in all possible positions except for 4-methylalkanes, internally branched dimethylalkanes, and 2,X- and 3,X-dimethylalkanes. At emergence, adults of both sexes of the 009 strain have nearly identical gas Chromatographic profiles, which diverge as the insect ages. Irradiation of pharate pupae does not affect the hydrocarbon produced.

Long chain oxoaldehydes and oxoalcohols from esters as major constituents of the surface lipids of Manduca sexta pupae in diapause.

Lipid constitutents of diapausing pupae of the tobacco hornworm, Manduca sexta (L), were identified by thin layer and gas-liquid chromatography, IR spectroscopy, and gas-liquid chromatography-mass spectrometry. Surface wax was a mixture of long chain polar compounds: oxoalcohol esters, oxoaldehydes, primary alcohol esters, and oxoalcohols, as listed in descending order of abundance. The distribution of the alcohols and aldehydes was C28 (75-85%), C27 (5%), and C26 (10-15%). The C26 compounds were largely 11-oxo isomers, but the C28 compounds consisted of similar amounts of 11- and 12-oxo isomers. The identities of the oxoaldehydes were confirmed by selective and complete NaBH4 reductions to yield oxoalcohols and diols, respectively. Mass spectral interpretations were verified with mass spectra of the oxoaldehyde, oxoalcohol, and diol synthesized from 12-hydroxystearic acid. Reduction of the total lipids with NaBH4 and hydrolysis of the product with ethanolic KOH gave oxoalcohols (85%), primary alcohols (8%), and oxoacids (5%); 30-40% of the oxoalcohols were derived from NaBH4-reduced oxoaldehydes, 5-10% were from free oxoalcohols, and 50% were from esters. Primary alcohols only existed as esters. Large quantities of fatty oxoalcohols relative to fatty oxoacids in the saponified mixture suggested the presence of esters other than those composed of long chain acids and alcohols. Oxoacids appeared to be mainly oxidation products of the oxoaldehydes.

Novel surface lipids of diapausing Manduca sexta pupae. Long chain oxoalcohol esters of acetoacetic, hydroxybutyric, and acetic acids.

Ester components in the surface wax from diapausing tobacco hornworm pupae, Manduca sexta L., were separated by thin layer chromatography and gas-liquid chromatography, and characterized by infrared spectroscopy and gas-liquid chromatography-mass spectrometry. Three groups of esters were identified as natural derivatives of acetic acid, acetoacetic acid, and 3-hydroxybutyric acid. The major ester fraction was identified as a mixture of C26 (10%), C27 (5%), and C28 (85%) oxoalcohol esters of acetoacetic acid. The major homolog consisted of equal amounts of 11-oxooctacosanyl 3-oxobutanoate and 12-oxooctacosanyl 3-oxobutanoate. Lesser amounts of 11- and 12-oxooctacosanyl and n-octacosanyl esters of acetic and 3-hydroxybutyric acids were also identified. The chain length distributions of these C26, C27, and C28 oxoalcohol and n-primary alcohol ester moieties, as well as the isomeric ratios for the 11- and 12-oxoalcohol isomers, were similar to the oxoaldehydes and unesterified oxoalcohols previously identified by Buckner et al (Buckner, J. S., Nelson, D. R., Haak, H., and Pomonis, J. G. (1984) J. Biol. Chem. 259, 8452-8470) as lipid components of the surface wax of M. sexta pupae.

Alkanes from surface lipids of sunflower stem weevil,Cylindrocopturus adspersus (LeConte).

The stem weevil,Cylindrocopturus adspersus (LeConte) (Coleoptera: Curculionidae) yields 3% of its body weight as extractable lipids (40 µg/ weevil). The alkane fraction was composed ofn-alkanes (38%) and branched alkanes (62%). The compounds were characterized by gas chromatography-mass spectrometry (GC-MS). The chromatogram contained several single-component peaks (9 of 25). Only seven dimethylalkanes were isolated (17.8%): 9,19- and 9,21-dimethylheptacosane; 9,19- and 9,21-dimethylnonacosane; 9,21- and 11,21-dimethylhentriacontane; and 11,21-dimethyltritriacontane. Important methylalkanes were: 2-methyltetra- and hexacosanes and 10-methylhexa- and octacosanes. Late-eluting gas chromatography peaks were composed of simple alkane mixtures or a single component.

A 13C NMR study of methyl-branched hydrocarbon biosynthesis in the housefly.

Carbon 13 NMR, radiotracer and mass spectrometry studies were performed to confirm that propionate and methylmalonate are incorporated into long chain branched hydrocarbons as the methyl branch unit, to determine whether the branching methyl group was added initially or toward the end of the elongation process, to determine the precursor of methylmalonate, and to examine the metabolism of propionate in the housefly, Musca domestica. The labeled carbons from [3-13C]propionate and [methyl-13C]methylmalonate were incorporated into the branching methyl carbon of the methylalkanes, and into the even numbered carbons in alkanes and alkenes. The labeled carbons from [2-13C]propionate and [1-13C]propionate labeled the tertiary carbon and the carbon adjacent to the teritary carbon, respectively, in the methylalkanes. Mass spectral analysis of the methylalkanes after enrichment from [1-13C]propionate showed that propionate was incorporated during the initial stages of chain synthesis of the terminally branched alkanes. Sodium [2-13C]propionate labeled the odd numbered carbons of both the alkanes and alkenes. These data suggest that propionate is converted to an acetyl derivative, with carbon 3 of propionate converted to the carboxyl carbon of acetate, and carbon 2 of propionate converted to the methyl carbon of acetate. This pathway of propionate metabolism has hitherto only been reported in plants. The labeled carbons from DL-[3,4,5-13C3]valine were incorporated intact (as determined by 13C--13C coupling) into the branching methyl carbon, tertiary carbon, and carbon adjacent to the tertiary carbon, respectively, demonstrating that valine serves as a precursor to the methylmalonate used in branched alkane biosynthesis.

A 13C-NMR study of the biosynthesis of 3-methylpentacosane in the American cockroach.

13C-NMR spectrometry was used to examine the in vivo incorporation of 13C-labeled precursors into 3-methylpentacosane in the cockroach Periplaneta americana. Natural abundance 13C-NMR of 3-methylpentacosane showed that carbons 1 through 6, 23 through 25 and the branching methyl carbon (C26) each gave distinct signals, with carbons 7 through 22 indistinguishable from each other. The label from dipotassium 2-[methyl-13C]methylmalonate was incorporated primarily into the methyl branch of 3-methylpentacosane, demonstrating the 2-methylmalonate is the precursor to the methyl branch unit. The carboxyl carbon from sodium [1-13C]propionate was incorporated exclusively into the 4-position. This indicates that propionate, as a 2-methylmalonyl derivative, is incorporated as the second unit during chain synthesis rather than toward the end of the elongation process. The labeled carbon from sodium [1-13C]acetate was incorporated into carbons 2, 6 and 24 and the labeled carbon from [2-13C]acetate was incorporated into carbons 1, 5, 23 and 25 of 3-methylpentacosane, respectively. These data are consistent with an elongation-decarboxylation pathway for 3-methylpentacosane biosynthesis.