Proc. Natl. Acad. Sci. USAVol. 89, pp. 6050-6054, July 1992Physiology

Biosynthesis and endocrine control of the production of the Germancockroach sex pheromone 3,11-dimethylnonacosan-2-one

(pheromone biosynthesis/hydrocarbon metabolism/methyl ketone biosynthesis)

JODY CHASE*, KAZUSHIGE TOUHARAt, GLENN D. PRESTWICHt, COBY SCHAL*, AND GARY J. BLOMQUIST*§*Department of Biochemistry, University of Nevada, Reno, NV 89557-0014; tDepartment of Chemistry, State University of New York, Stony Brook, NY11794-3400; and tDepartment of Entomology, Cook College, Rutgers University, New Brunswick, NJ 08903

Communicated by Wendell L. Roelofs, March 30, 1992 (received for review January 16, 1992)

ABSTRACT The biosynthesis and endocrine regulation ofsex pheromone production in the female German cockroach(Blattela germanica) were determined. Radio-TLC and radio-GLC were used to demonstrate the metabolism of 3,11-dimethylnonacosane, a major cuticular lipid component, to thecorresponding alkan-2-ol and methyl ketone. [11,12-3H2J-3,11-Dimethylnonacosan-2-ol was efficiently metabolized to themethyl ketone, and radio-GLC showed that the methyl ketoneproduct from both experiments was coeluted with a methylketone standard. A comparison ofthe metabolism of the labeleddimethylalkane and dimethylalkan-2-ol by age and sex showedthat both males and females from day 1 through day 9 afteradult emergence readily metabolized the alcohol to the corre-sponding methyl ketone, whereas only females of 5-9 dayspostemergence efficiently converted the labeled dimethylal-kane to the corresponding methyl ketone. Application of thejuvenile hormone analog hydroprene induced significant in-creases in the conversion of the labeled hydrocarbon to themethyl ketone in starved adult females as well as in females feda protein-free diet, conditions under which endogenous juve-nile hormone biosynthesis is nearly undetectable. These datashow that the methyl ketone sex pheromone is formed by thehydroxylation and oxidation of the 3,11-dimethylalkane at the2 position, show that the age- and sex-specific step in thisprocess is the conversion of 3,11-dimethylnonacosane to 3,11-dimethylnonacosan-2-ol, and provide evidence that juvenilehormone regulates sex pheromone production in the Germancockroach.

The biosynthesis of the sex pheromones from many insectsinvolves the addition of only one or two ancillary enzymes.For example, the carbon chains of many of the lepidopteransex pheromones arise from a unique A1' desaturase andhighly specific chain-shortening reactions (1). The houseflysex pheromone arises from a change in the chain lengthspecificity of the enzymes that produce cuticular alkenes (2),and many coleopteran pheromones are produced by thespecific hydroxylation of dietary precursors (3). The contactsex pheromone of the female German cockroach (Blattellagermanica) contains three oxygenated derivatives of 3,11-dimethylnonacosane-(3S,11S)-3,11-dimethylnonacosan-2-one, (3S,11S)-29-hydroxy-3,11-dimethylnonacosan-2-one,and 29-oxo-3,11-dimethylnonacosan-2-one (4)-and 3,11-dimethylheptacosan-2-one (5). Each of the pheromone com-pounds elicits the full range of behavioral responses in males(5). The identification of 3,11-dimethylnonacosane as a majorcomponent of the cuticular hydrocarbons (6, 7) led to thesuggestion (8) that the C29 sex pheromone components andthe dimethylalkane have a common biosynthetic origin. Theoxygenated components could be derived from the hydro-carbon or both the dimethylalkanes and methyl ketone could

arise from a common pathway, perhaps one where there wasa failure to reduce the keto group in position 2 during chaininitiation. Data are presented herein demonstrating that themethyl ketone pheromone component is produced by asex-specific hydroxylation of 3,11-dimethylnonacosane tothe corresponding 3,11-dimethylnonacosan-2-ol, which isthen converted to the methyl ketone. Further, evidence isprovided that the hydroxylation reaction is the key sex-specific regulatory step in pheromone biosynthesis in thisinsect and that both males and females can convert thealcohol intermediate to methyl ketone.Three different types of endocrine regulation of sex pher-

omone production have been demonstrated in insects. Insome Lepidoptera, a pheromone biosynthesis-activating neu-ropeptide induces sex pheromone production (9). In thehousefly, ecdysteroids produced by the maturing ovarychange the chain length specificity of the enzymes thatproduce cuticular alkenes, so that (Z)-9-tricosene (musca-lure) is produced (2). In some Blattaria (10, 11), Coleoptera(3), and Lepidoptera (12) juvenile hormone (JH) regulates sexpheromone production. In none of the systems studied todate is it known which enzymatic steps are affected byhormones. In this paper we present evidence that JH inducesthe sex-specific oxidation ofdimethylalkane to methyl ketonevia the alkan-2-ol.

MATERIALS AND METHODSRadiolabeled Substrates. The radiolabeled substrates were

prepared (Fig. 1) as follows. Ethyl 2,10-dimethyl-10-octacosenoate (3). To a solution oftriphenyloctadecanephos-phonium bromide (4.11 g, 6.9 mmol, prepared from 10-octadecanol in two steps) in dry Et2O (60 ml) was addedn-BuLi (1.6 M, 3.75 ml, 6.0 mmol) at room temperature. After20 min of stirring at room temperature, a solution of 2 (0.56g, 2.3 mmol) in dry Et2O was added. After 20 min of stirring,excess ylide was quenched with water. The precipitate wasfiltered off and the filtrate was concentrated in vacuo. Theresidue was chromatographed over SiO2 (40 g). Elution withn-hexane/EtOAc (70:1 to 50:1) gave 0.6 g (54%) of 3: P.l(film) 2925 (s), 2855 (s), 1735 (br s), 1465 (m), 1375 (m), 1180(m) cm-1; 300-MHz 1H NMR (C2HC13) 8 0.88 (t, 3, J = 6.6Hz), 1.13 (d, 3, J = 7.0 Hz) 1.2-1.5 (m, 45), 1.57 (s, 3 transH), 1.66 (s, 3 cis H), 1.9-2.1 (m, 4), 2.40 (q, 1, J = 7.0 Hz),4.12 (q, 2, J = 7.2 Hz), 5.10 (t, 1, J = 7.0 Hz).

2,10-Dimethyl-10-octacosenoic acid (4). A solution of 3(0.55 g, 1.15 mmol) in EtOH (12 ml) and aqueous 50%o KOH(1 ml) was refluxed for 30 min. Two-thirds of the EtOH wasremoved by distillation, and then five drops of concentratedH2SO4 was added and the mixture was extracted with Et2O.The extract was dried with Na2SO4 and concentrated invacuo. The residue was chromatographed over SiO2 (10 g).

Abbreviations: JH, juvenile hormone; JHA, JH analog.§To whom reprint requests should be addressed.

6050

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 89 (1992) 6051

o 1) conc H2SO4, EtOH

2) LDA, CH3IHO

3) CuCl, PdC121 02, DMF-H20

0C18H37Br-PPh3'

EtOn-BuLi

2 ° Et2O

0 0 T2CH3Li, Me3SiCl Pd/C

R - C17H35 >-*- C17H35 - >NH4Cl EtOH

3: R=OEt 54: R=OH

0 T T

C17H35

6a: T=H6b: T=3H

OHNaBH4

MeOH

1) HS(Ci2)2SHBF3-OEt2

2) Raney NiEtOH

T T

C17H35

8a: T=H8b: T='H

FIG. 1. Scheme for the synthesis of [11,12-3H2]-3,11-dimethylnonacosane, [11,12-3H2]-3,11-dimethylnonacosan-2-ol, and [11,12-3H2]-3,11-dimethylnonacosan-2-one. LDA, lithiodiisopropylamine; DMF, N,N-dimethylformamide; C18H37Br-PPh', triphenyloctadecanephosphoniumbromide; T2, 3H2.

Elution with n-hexane/EtOAc (30:1 to 10:1) gave 0.46 g

(89%) of 4: l . . (film) 2925 (s), 2855 (s), 1710 (s), 1465 (m)cm-'; 300-MHz 1H NMR (C2HC13) 8 0.88 (t, 3, J = 6.5 Hz),1.1-1.4 (m, 45), 1.57 (s, 3 trans H), 1.66 (s, 3 cis H), 1.9-2.1(m, 4), 2.3-2.6 (m, 1), 5.1 (t, 1, J = 7.0 Hz).3,11-Dimethyl-JJ-nonacosen-2-one (5). To a solution of 4

(0.45 g, 1.0 mmol) in dry tetrahydrofuran (8 ml) was addedMeLi (1.4 M in diethyl ether, 1.57 ml, 2.2 mmol) at 0°C. Themixture was stirred for 30 min at 0°C, Me3SiCl (0.63 ml, 4.96mmol) was added rapidly, and the temperature was raised toroom temperature. At this stage, the reaction mixture was

poured into saturated aqueous NH4CI (20 ml). The mixturewas extracted with Et2O. The extract was washed with waterand brine, dried with K2CO3, and concentrated in vacuo. Theresidue was chromatographed over SiO2 (20 g). Elution withn-hexane/EtOAc (60:1 to 50:1) gave 0.33 g (74%) of 5:trans/cis ratio, 37:40 by GC analysis; Zma., (film) 2925 (s),2855 (s), 1715 (i), 1465 (i), 1355 (i) cm-1; 300-MHz 1HNMR (C2HCl3) 8 0.87 (t, 3, J = 6.6 Hz), 1.07 (d, 3, J = 6.9Hz), 1.2-1.4 (m, 42), 1.56 (s, 3 trans H), 1.66 (s, 3 cis H),1.9-2.1 (m, 4), 2.13 (s, 3), 2.49 (q, 1, J = 6.9 Hz), 5.10 (t, 1,J = 6.9 Hz); 75-MHz 13C NMR (C2HCl3) 8 14.1, 15.8, 16.1,22.7, 23.4, 27.2, 27.8, 28.0, 29.4, 29.5, 29.7, 29.9, 30.1, 31.7,31.9, 32.9, 39.7, 47.2, 124.6, 125.3, 135.2, 213.0; MS m/z 448MW.3,11-Dimethylnonacosan-2-one (6a). A mixture of 5 (55

mg) and Pd/C (5%) (12 mg) in absolute EtOH (1 ml) wasstirred vigorously under H2 gas for 2 hr. (The completion ofthe reaction was detected by GC.) After this period, thecatalyst was filtered off and the filtrate was concentrated invacuo. The residue was chromatographed over SiO2 (4 g).Elution with n-hexane/EtOAc (90: 1) gave 54 mg (%%) of 6a:vmax (film) 2920 (s), 2850 (s), 1710 (s), 1470 (s), 1375 (m) cm-1;300-MHz 1H NMR (C2HC13) 8 0.82 (t, 3, J = 6.3 Hz), 0.88 (t,3, J = 6.6 Hz), 1.08 (d, 3, J = 6.9 Hz), 1.2-1.4 (mi, 49), 2.13(s, 3), 2.50 (t, q, 1, J = 6.9 Hz).3,11-Dimethylnonacosane (7a). A mixture of 6a (20 mg),

1,2-ethanedithiol (0.03 ml), and BF3-OEt2 (0.03 ml) wasstirred for 17 hr at room temperature. The reaction mixturewas diluted with water and extracted with Et2O. The extractwas washed with water, saturated aqueous NaHCO3, andbrine, dried with Na2SO4, and concentrated in vacuo to givethe desired dithioketal, which was used without purification;vma,, (film) 2920 (s), 2850 (s), 1465 (m) cm-'. The dithioketal

was added to a suspension of Raney nickel catalyst (0.5 g) inEtOH (10 ml). After 5 min of stirring, the catalyst was filteredoff and the filtrate was concentrated in vacuo. The residuewas chromatographed over SiO2 (5 g). Elution with n-hexane(only) gave 12 mg (62%) of 7a: Vmax (film) 2920 (s), 2850 (s),1465 (m), 1375 (w), 720 (w) cm-'; 300-MHz 1H NMR(C2HCl3) 8 0.75-0.9 (m, 12), 1.0-1.5 (m, 52).3,11-Dimethylnonacosan-2-ol (8a). A mixture of 6a (5 mg)

and NaBH4 (3 mg) in dry MeOH (0.5 ml) was stirred for 10min. MeOH was removed by evaporation. The residue waschromatographed over SiO2. Elution with n-hexane/EtOAc(40:1) gave 4 mg (80%) of 8a: 300-MHz 1H NMR (C2HC13) 80.83 (d, 3, J = 6.4 Hz), 0.85-0.90 (m, 3), 1.05-1.2 (m, 6),1,2-1.5 (in, 51), 3.6-3.75 (m, 1).[11,12-3H21-3,11-DimethyInonacosan-2-one (6b). A 20-mg

portion of 5 was reductively tritiated with excess no-carrier-added 3H2 and Pd/C (5%) (4 mg) in absolute EtOH (0.5 ml)to give 570 mCi of 6b, specific activity > 60 Ci/mmol (1 Ci= 37 GBq), by New England Nuclear using the conditionsdescribed above for 6a. All radiolabeled materials werepurified to >99% radiochemical purity as determined byradio-TLC.

[11 ,12-3H2]-3,11-Dimethylnonacosane (7b). A 150-mCiportion of 6b was reduced to the hydrocarbon withHS(CH2)2SH in BF3-OEt2 and Raney Ni in EtOH as de-scribed above for 7a, to give 110 mCi of the labeled hydro-carbon 7b, specific activity > 60 Ci/mmol.

[11 ,12-3HJI1-3,11-Dimethylnonacosan-2-oI (8b). A 100-mCiportion of 6b was reduced to the alcohol with NaBH4 inMeOH as described above for 8a, to give 73 mCi of 8b,specific activity > 60 Ci/mmol.

Insects. German cockroaches were reared in glass jars andfed dog chow (13). Males and females were separated within1 day of adult emergence and females were reared in groups.In Vivo Radiotracer Experiments. Males or females of

specific ages were treated in triplicate groups of three or fiveinsects per group with 0.1-0.5 ,uCi of [11,12-3H2]-3,11-dimethylnonacosane or [11,12-3H2]-3,11-dimethylnona-cosan-2-ol in 0.5 ,ul of hexane by applying the solution to theventral abdomen. The insects were killed at the times indi-cated by freezing at -20°C.Treatment with Juvenile Hormone Analog (JHA). Hydro-

prene [ZRS12 (ethyl (2E,4E)-3,7,11-trimethyl-2,4-dodecadi-enoate; Zoecon, Palo Alto, CA] was applied topically (100,g

Physiology: Chase et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

in 2 1.l of acetone). Control insects were treated with 2 Al ofacetone. The radiolabeled dimethylalkane was injected intothe hemolymph in 0.4 ul of acetone on day 2. On day 6, theinsects were killed, and the cuticular lipids were extractedand analyzed as described below.

Extraction and Separation of Lipids. Cuticular lipids wereextracted with 2 ml of hexane for 10 min followed by twoextractions with 1.5 ml for 1 min each, and the extracts werecombined. Lipids were separated by TLC in hexane/diethylether/formic acid (80:20:2) or hexane/diethyl ether (90:10).For some experiments, specific bands were extracted indiethyl ether or CHC13/MeOH/H2O/HOAc (polar lipidband).

Assay of Radioactivity. Aliquots of each fraction of ex-tracted lipid were assayed for radioactivity by liquid scintil-lation counting on a Beckman LS 1701 at 54% efficiency orBeckman 3801 at 59% efficiency. Alternatively, sampleswere assayed for radioactivity by radio-TLC scanning on aBioscan system 200 imaging scanner. In some experiments,the hydrocarbon and methyl ketone bands were analyzed byradio-GLC on a Hewlett-Packard chromatograph interfacedwith a Radiomatic Flo One/,feta combustion flow-throughproportional counter.

Conversion ofUnlabeled Alcohol to Methyl Ketone by Males.Males of mixed ages 0-8 days after emergence were treatedwith 3,11-dimethylnonacosan-2-ol, incubated for 24 hr, andkilled by freezing. Cuticular lipids were isolated and analyzedby GLC and GC-MS as described earlier (8).

RESULTSTo determine whether 3,11-dimethylnonacosane was a pre-cursor to the methyl ketone, 7-day-old adult virgin femaleswere treated topically with [11,12-3H2]-3,11-dimethylnona-cosane. After 24 hr, the insects were killed and the cuticular

3 oj 10 JL- 0 ~ ~ BMUFHY KEMNE

ALCOHOL

o~~~~~f 200 10 2c0DIT (Cn)

SUBgIRTh PMKIIL0,* ALCOHOL M0

o 0LB X

- 0 ~~~10 20

ALCOHOL,

0 10 20DIANCE (cm)

FIG. 2. Radio-TLC ofthe cuticular extract from 7-day-old femaleGerman cockroaches treated with [11,12-3H2]-3,11-dimethylnona-cosane (A) or [11,12-3H2]-3,11-dimethylnonacosan-2-ol (B). Insectswere treated with radiolabel and lipids were extracted after 24 hr andanalyzed as described in Materials and Methods. (Insets) Radio-TLCtraces of the substrates.

lipids were extracted and analyzed. A representative chro-matogram (Fig. 2A) showed that 19.4% of the recoveredradiolabel was metabolized to material that was present in themethyl ketone fraction, with 10.4% of the recovered radio-activity found in the band that comigrated with 3,11-dimethylnonacosan-2-ol. The rest of the recovered radioac-tivity was associated mainly with unmetabolized hydrocar-bon. Approximately 60% of the applied radiolabel wasrecovered in hexane extracts. When the methyl ketone bandwas analyzed by radio-GLC, all of the radioactivity detectedwas present in a peak that was coeluted with the [11,12-3H2]-3,11-dimethylnonacosan-2-one standard (Fig. 3). Very littleradioactivity from the dimethylalkane was recovered in thepolar lipid fraction. If the major pheromone component isproduced from hydrocarbon through a cytochrome P-450system, it is likely to occur as a two-stage process in whichthe P450 hydroxylation of the hydrocarbon at the 2 positionis followed by oxidation to the methyl ketone. When 5-day-old females were treated topically with [11,12-3H21-3,11-dimethylnonacosan-2-ol, most of the recovered radioactivitywas found in the methyl ketone band (Fig. 2B). Analysis ofthis fraction by radio-GLC showed that all the radioactivitywas found in one peak with retention time identical to that ofthe methyl ketone standard. Only 14% of [11,12-3H21-3,11-dimethylnonacosan-2-ol applied to females remained un-changed in the alcohol fraction, indicating a very efficientconversion of this apolar material.Female German cockroaches produce pheromone at a very

low rate for the first few days immediately following adultemergence (14). In isolated females, the rate of productionincreases after day 5 and reaches a maximum on day 8, afterwhich pheromone production drops dramatically and thefemales ovulate and produce an egg case. There has been noevidence that the males are able to produce any of the

A

z0

09

0

u)

F-w

a

B

wV)z0

LO

0

0

20 30TIME (MIN)

FIG. 3. Radio-GLC of the methyl ketone band from 5-day-oldfemale German cockroaches treated with [11,12-3H2]-3,11-dimethylnonacosane (A) and of [11,12-3H2]-3,11-dimethylnona-cosan-2-one standard (B). Insects were treated with radiolabeledhydrocarbon and incubated for 4 days before lipid extraction.

6052 Physiology: Chase et al.

Proc. Natl. Acad. Sci. USA 89 (1992) 6053

pheromone components, indicating that the biosynthesis of3,11-dimethylnonacosan-2-one is sex-specific. To determinewhich step in the conversion of hydrocarbon to methylketone is rate-limiting, females were treated with either[11,12-3H2]-3,11-dimethylnonacosane or the correspondingradiolabeled alcohol at specific ages, and then, after a 24-hrincubation, the products were analyzed. Similarly, groups ofmale insects of the same ages were treated with [11,12-3H2]-3,11-dimethylnonacosane and, in separate experiments, with[11,12-3H2]-3,11-dimethylnonacosan-2-ol.Females of all ages examined converted hydrocarbon to

material recovered in the methyl ketone fraction, but thisconversion was the highest on days 4-7 and declined by day 9.Male German cockroaches also metabolized small amounts oflabeled dimethylalkane to material that migrated in the methylketone fraction. However, radio-GLC showed that this materialwas not eluted with the methyl ketone standard. The chemicalidentity of this material from males is not known.When males or females were treated topically with [11,12-

3H2]-3,11-dimethylnonacosan-2-ol, the conversion to methylketone occurred at a high rate at all ages (Fig. 4). Thissuggests that the hydroxylation step converting the dimeth-ylalkane to alcohol is both age- and sex-specific in this insectand that the enzyme catalyzing this step regulates contact sexpheromone production.To verify that males can convert 3,11-dimethylnonacosan-

2-ol to the corresponding ketone, males of mixed ages weretreated topically with microgram quantities of unlabeled3,11-dimethylnonacosan-2-ol. Analysis of the products byGLC showed a peak with a retention time identical to that ofthe methyl ketone standard. Analysis of this component byGC-MS yielded a fragmentation pattern characteristic of theC29 methyl ketone, with a parent ion peak ofm/z 451 (data notshown), thus confirming that males can convert the dimethylalcohol to the corresponding ketone.Pheromone production is significantly suppressed in

starved females, as it is in females from which the corporaallata (which produce JH) have been excised (15). Similarly,females fed a protein-free diet accumulate significantly lesscuticular 3,11-dimethylnonacosan-2-one than females fed a25% protein diet (23.7 ng per female vs. 238.9 ng per femaleon day 6). An in vitro assay for JH biosynthesis by thecorpora allata (see ref. 16 for methods) showed that nodetectable JH was released from corpora allata of 6-day-oldstarved females (n = 6), and only 0.05 + 0.03 pmol/hr per pairof corpora allata (n = 6) in females fed a protein-free diet. Therespective release rates for control females were 5.4 + 0.89 (n= 6) for dog chow-fed females and 12.07 ± 0.95 (n = 6) for

1001

80

60 ALCOHOL-TREATED

40

HYDROCARBON-TREATED

20

0 2 4 6 8 10

AGE (DAYS AFTER ADULT EMERGENCE)

FIG. 4. Metabolism of [11,12-3H2]-3,11-dimethylnonacosane and[11,12-3H2]-3,11-dimethylnonacosan-2-ol to methyl ketone band byfemales of various ages. Female insects of appropriate ages were

treated with [11,12-3H2]-3,11-dimethylnonacosane (A) or [11,12-3H2]-3,11-dimethylnonacosan-2-ol (o) and incubated for 24 hr beforeextraction. Radioactivity in the methyl ketone band was assayed byliquid scintillation counting.

2 11)

(11

7- =6aI X

.

5J

li

St St + JHIA 01-; Pr O% Pr ± JHA

FIG. 5. Effect of the JHA hydroprene on the metabolism of[11,12-3H2]-3,11-dimethylnonacosane to methyl ketone pheromonein starved (St) and protein-deprived (0%o Pr) females. The JHA wasapplied topically in acetone and the labeled alkane was injected.Cuticular lipids were extracted on day 6. Radioactivity in the methylketone band was assayed by liquid scintillation counting.

females fed a control 25% protein diet (P < 0.01 for both; t test).Since topical application of 100 gg ofJHA on females fed a dietlacking protein, on starved, or on allatectomized females in-duced 14-, 26-, and 12-fold increases, respectively, in theamount of cuticular pheromone, it was likely that the metabo-lism of the dimethylalkane to the ketone was JH-induced.

Topical treatment of starved or protein-deprived femaleswith 100 pg of hydroprene resulted in significantly higheramounts of injected 3H-labeled dimethylalkane converted tomethyl ketone (Fig. 5). The conversion of dimethylalkane tomethyl ketone was 1.44-fold higher in starved JHA-treatedfemales and 2.76-fold higher in protein-deprived JHA-induced females than in the respective acetone-treated con-trols (P < 0.03 for both; t test). These data clearly indicatethat JH induces 3,11-dimethylnonacosan-2-one sex phero-mone production from 3,11-dimethylnonacosane in B. ger-manica females and, taken with the fact that the oxidation ofthe alkan-2-ol to the ketone occurs at all ages, stronglyindicate that JH induces the 2-hydroxylation of the dimeth-ylalkane.

DISCUSSIONThe results show that 3,11-dimethylnonacosane, a majorcuticular hydrocarbon component of the German cockroach,is a precursor to 3,11-dimethylnonacosan-2-one, the femalesex pheromone. The presence of radioactivity in the fractioncorresponding to 3,11-dimethylnonacosan-2-ol from [11,12-3H2]-3,11-dimethylnonacosane and the efficient conversionof [11,12-3H2]-3,11-dimethylnonacosan-2-ol to the corre-sponding methyl ketone show that the first step involves thehydroxylation of the alkane to 3,11-dimethylnonacosan-2-ol.The secondary alcohol is then efficiently converted to thecorresponding ketone (Fig. 6). The absence of significantamounts of radioactivity in the polar lipid fraction and in theC27 methyl ketone demonstrates that the labeled alkane wasnot p-oxidized with the radiolabel and then incorporatedduring resynthesis. If that were the case, significant amountsof radioactivity should be recovered in the polar lipids and inthe C27 methyl ketone.By using modified cuticular lipids for its sex pheromone,

the female German cockroach is able to produce its sexpheromone with a minimum of enzymes different from thoseof "normal metabolism," thus conserving genetic material.Most insects produce methyl-branched hydrocarbons, with-20 species shown to date to have the 3,x-dimethyl type (17).Thus, the German cockroach fits the proposed model inwhich the addition of one or a few ancillary enzymes resultsin sex pheromone production.The presence of three isomers of the dimethylnonacosane in

the cuticular lipids of the German cockroach with methyl

Physiology: Chase et A

---F--

Proc. Natl. Acad. Sci. USA 89 (1992)

VAL

MLE 0

m;gr '02C" 1SCoAt SUCCINYL-CoA

02 ''0~o- 'o2CESCoAMCHE

METHYLMALONYL-CoA

0

MALONYL-CoA

*CHs CHs

3,11-DIMETHYLNONACOSANE

A AGE- & SEX-SPECIFICtSTEP

CHE CHE

OH3,11-DIMETHYL-2.NONACOSANOL

CH3 CH3

3,11.DIMETHYL-2.NONACOSANONE

FIG. 6. Biosynthesis and regulation of German cockroach sexpheromone.

branches at the 3,11, 3,9, and 3,7 positions (5) compared withthe methyl ketone with only the 3,11-dimethyl branch positionsimplies that a remarkable specificity is required for the positionsofthe methyl branches in the hydroxylation reaction. The chainlength specificity is apparently not as exacting, as a 3,11-dimethylnonacosan-2-one is also part of the pheromone (5, 8),presumably derived from the corresponding 3,11-dimethylhep-tacosane, also a component of the cuticular lipids (8).

In the housefly, a microsomal cytochrome P-450 converts(Z)-9-tricosene to 9,10-epoxytricosane and (Z)-14-tricosen-10-one (18), with the ketone formed presumably via hydrox-ylation followed by oxidation. In several species of beetles,sex pheromone is produced by the hydroxylation of dietaryterpenoids to active material, and the incorporation of 1802suggests that a cytochrome P-450 is involved in this process(D. Vanderwel and A. C. Oehlschlager, personal communi-cation). It is very likely that a P-450 system also converts thehydrocarbon to a secondary alcohol that is then oxidized tothe methyl ketone in the German cockroach.Hydrocarbon synthesis occurs at relatively high rates from

day 0 to 8 in adult female German cockroaches (C.S., unpub-lished data). Methyl ketone pheromone production as mea-sured by the accumulation of pheromone on the surface of theinsect or the incorporation of [1-14C]propionate into phero-mone is the highest from day 6 through 9 (14). The appearanceof relatively large amounts of the C29 dimethylalkane severaldays prior to the appearance of the sex pheromone suggeststhat the availability of the precursor hydrocarbon is not afactor in regulating sex pheromone production in normalfemales. Rather, the data indicate that the hydroxylation of3,11-dimethylnonacosane to the corresponding secondary al-cohol is the control step in sex pheromone production. Bothmales and females of all ages readily metabolize 3,11-dimethylnonacosan-2-ol to the corresponding ketone, whereasonly females of ages 5-9 days efficiently convert the dimeth-ylalkane to the methyl ketone sex pheromone.The in vivo synthesis of pheromone and its accumulation

on the cuticle are correlated with the synthesis of JH by thecorpora allata in vitro and with oocyte development, sug-gesting a regulation of sex pheromone production by JR (14).The increase in the amounts of methyl ketone productionfrom injected methylalkane after treatment with JHA pro-

vides further evidence that JH regulates sex pheromoneproduction in the German cockroach. Somewhat surpris-ingly, JHA did not increase the amount ofmethyl ketone fromlabeled hydrocarbon applied to the surface of the insect (J.C.and G.J.B., unpublished data), suggesting that JHA mayexert its action partially by regulating hydrocarbon transport.Recent studies have led to the following conclusions on the

biosynthesis and regulation of the German cockroach sexpheromone (Fig. 6). The methyl-branched alkane is formedby the insertion of methylmalonyl units derived from succi-nate and the carbon skeletons of isoleucine, valine, andmethionine early in chain elongation (13) by a microsomalfatty acid synthetase (19). The 3,11-dimethylalkane is thenselectively hydroxylated in reproductively mature females,and JH induces this process. The in vivo synthesis of pher-omone and its accumulation on the cuticle are correlated withthe synthesis of JH by the corpora allata in vitro and withoocyte development, suggesting common regulation by JH ofpheromone production and other reproductive events (14).Work is needed to clarify the exact role of JH in regulationof sex pheromone production, to determine the transport ofthe hydrocarbon precursor and methyl ketone in the he-molymph and through the cuticle, to characterize the hy-droxylase that converts the dimethylalkane to the secondaryalcohol, to examine the role of JH in inducing hydroxylaseactivity, and to examine the metabolism of the methyl ketoneto the 29-hydroxy and 29-oxo derivatives.

We thank Marion Page and Lori J. Nelson for GC-MS analyses,Dr. Bachir Latli (Stony Brook) for assistance in radiosynthesis andspectroscopy, and Patrick Smith (Reno) for technical assistance.This work was supported in part by National Science FoundationGrants DCB-8900088 (G.J.B.) and CHE-8809588 (G.D.P.) and by aHerman Frasch Foundation grant (G.D.P.). This is a contribution ofthe Nevada and New Jersey Agriculture Experiment Stations.

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6054 Physiology: Chase et al.