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Biosynthesis of internally branched monomethylalkanes in the cockroach Periplaneta fuliginosa
ARCHIVES
OF
BIOCHEMISTRY
Biosynthesis
GARY Chemistry
Department,
AND
BIOPHYSICS
of Internally Cockroach
173, 546-553 (1976)
Branched Monomethylalkanes Periplaneta fuliginosal
J. BLOMQUIST2 University
GEORGE
AND
of Southern Mississippi, Southern Mississippi 39401 Received August
in the
P. KEARNEY Station,
Box 5347, Hattiesburg,
21, 1975
Sodium [1-14C]acetate, sodium [l-Wlpropionate, sodium [2-Wlpropionate, sodium [3“Clpropionate and sodium [methyE-14Clmethylmalonate were readily incorporated into the cuticular hydrocarbons of nymphal stages of the cockroach Periplaneta fzdiginosa both in vivo and in vitro, whereas no incorporation of [methyl-Wlmethionine was observed. The alkanes of the nymphal stages of this insect are 25+% n-alkanes, 14% 3methylalkanes, and 59+% internally branched monomethylalkanes, principally 13methylpentacosane. Sodium [l-Wlacetate was incorporated into each class of alkane at about its percentage composition. In contrast, labeled sodium propionate and sodium methylmalonate were preferentially incorporated into the branched fractions. Radiogas-liquid chromatography showed that sodium [l-Wpropionate was incorporated almost exclusively into 3-methyltricosane and 13-methylpentacosane, whereas sodium ll-Wacetate was incorporated into each glc peak at about its percentage composition. These data suggest that propionate, incorporated during chain elongation, serves as the branching methyl group donor for both the 3-methyl and the internally branched monomethylalkanes in insects. The location of hydrocarbon synthesis in P. fuliginosa was studied using an in vitro tissue slice system. Excised cuticle slices, with adhering fat body tissue removed, gave good incorporation of labeled substrates into the hydrocarbon fraction. No hydrocarbon synthesis was observed in fat body preparations.
Methyl branched hydrocarbons occur in many biological systems, including plants (l), algae (2), bacteria (31, and insects (4). The biosynthesis of both the normal and branched hydrocarbons has received considerable attention in plants and microorganisms, with convincing evidence on the pathways leading to the major components reported (1, 3). In plants, n-alkanes are formed by an elongation of fatty acids followed by decarboxylation (5, 6) and the 2and 3-methylalkanes originate from the appropriately branched starter chains derived from branched amino acids (1). In the algae, the active methyl group from methionine serves as the branching
methyl group donor for the internally branched monomethylalkanes (7, 8). In contrast to the situation for plants and microorganisms, the biosynthetic pathways for insect cuticular hydrocarbons have not received extensive study. Acetate has been shown to be readily incorporated into the hydrocarbons of live insects (4, 9, lo), excised cuticles (41, and oenocyte-rich fat body preparations (11). Indirect evidence on n-alkane synthesis obtained from studying the formation of secondary alcohols (12-14) suggests that an elongation-decarboxylation pathway is operative, although definitive work has not been done. Recent evidence indicates that the 3-methylalkanes in insects are formed by a pathway different from that reported in other organisms. In insects, propionate is apparently converted to a methylmalonyl derivative and incorpo-
1 This work was supported in part by a Cottrell Grant from Research Corporation and a Sigma Xi Research Grant. 2 Author to whom inquiries should be directed. 546 Copyright 8 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOSYNTHESIS
OF MONOMETHYLALKANES
rated as the penultimate biosynthetic unit (15). This observation suggested the possibility of a similar pathway for the biosynthesis of the internally branched monomethylalkanes in insects, in which additional acetyl units are added after propionate is incorporated, followed by decarboxylation. The experiments reported in this paper were designed to determine if the branching methyl group of the internally branched monomethylalkanes in insects is inserted by addition to a preformed chain by the active methyl group of methionine, as observed in algae (7, 8), or if propionate or a derivative such as methylmalonate is incorporated during chain elongation. No information on the biosynthesis of this type of alkane in insects has been previously reported. The internally branched monomethylalkanes comprise a major fraction of the cutitular hydrocarbons from a number of insects (16-19) and are often present in complex mixtures. Their chain length distribution ranges from 24 to 28 carbons in a number of cockroaches (19, 20), 28 to 36 carbons in two species of Melanoplus (17), and up to 50 carbons in the grasshopper Schistocerca uaga (21). The majority of the internally branched monomethylalkanes in insects have the methyl branch on oddnumbered carbons, and this is usually on carbon number 11, 13, or 15, although isomers with methyl branches on all oddnumbered carbons have been reported (18). In contrast to the complex mixtures of these alkanes observed in many insects, much of the cuticular hydrocarbon of the cockroach Periplaneta fuliginosa is comprised of 13-methylpentacosane (19), making this an excellent experimental animal for studying the biosynthesis of these alkanes. MATERIALS
AND
METHODS
Materials Sodium [l-14Clacetate (55 mCi/mmol), DL-[Z“Clmevalonic acid lactone (10.3 mCi/mmol), sodium [methyZ-~4Clmethylmalonate (10.5 mCi/mmol), sodium [l-L4C]propionate (0.55 mCi/mmol), sodium [2Wlpropionate (27 mCi/mmol) and sodium [3Wlpropionate (0.8 mCi/mmol) were purchased from New England Nuclear. L-[G-3Hlisoleucine (2.7 Ci/
IN INSECTS
547
mmol), L-[G-3Hlvaline (6.8 Ci/mmol), L-[methyl“Clmethionine (50 mCi/mmol) and [U-Wlglucose (180 mCi/mmol) were purchased from ICN. Grace’s insect medium was obtained from the Grand Island Biological Co. All solvents were redistilled from glass prior to use. A starter colony of P. fuliginosa was obtained from Dr. Mary Ross. Colonies of P. fuliginosa were reared in 20-gal metal containers on a diet of ground dry dog food and an agar-water (1:99) gel fed ad lib. Middle instar nymphs (fourth through sixth instars) were used in all studies.
Methods In uiuo studies. Labeled substrates in 2 ~1 of water were injected just beneath the cuticle between the eighth and ninth abdominal segments. The insects were kept at room temperature and sacrificed after 6 h. In some cases larger amounts of labeled substrates and longer incubation times were used where greater amounts of labeled hydrocarbons were needed for radio-gas-liquid chromatography (radio-glc). The cuticular lipids were extracted by immersion of the insects in hexane for 10 min and then taken to dryness under nitrogen. Time studies indicate that this gave essentially complete extraction of cuticular hydrocarbon while not extracting internal lipid. Three to five insects were used in each experimental group. In vitro studies. Middle instar insects were anesthesized with carbon dioxide, placed on ice, and kept at O-4% during subsequent manipulation. The abdomens were excised. Cuticle tissue was isolated by separating the ventral and dorsal halves after an incision on each side of the abdomen, and the fat body was removed by scraping gently with a razor blade. The isolated cuticles were cut into strips, the tissues pooled, and 25 mg of tissue placed in incubation vials with appropriate labeled substrates containing 250 ~1 of Grace’s insect medium, distilled water, or a saline solution containing: NaCl (12 g/ liter), KC1 (0.5 g/liter), CaCl, (1.5 g/liter), MgCl, (0.53 g/liter) and NaHCO, (0.2 g/liter). The vials were thoroughly mixed and incubated for 2 h, unless otherwise indicated, at 32°C in a shaking water bath (100 oscillationslmin). At the end of the incubation period, lipids were extracted from the cuticle tissue with chloroform, and the solvent was removed under nitrogen. Separation of lipid components. The lipid samples were transferred to a Pasteur pipet containing 50 mg of BioSil A in 1 ml of hexane. The hydrocarbons were eluted into a vial with 8 ml of hexane and the polar lipids eluted with 8 ml of chloroform:methanol (2:l). Polar lipids, as used here, include all lipids more polar than hydrocarbons. The hydrocarbon sample was divided into two equal fractions, and all
548
BLOMQUIST
AND
fractions were taken to dryness under nitrogen. One fraction contained one-half of the total hydrocarbons. The branched hydrocarbons were isolated by adding molecular sieve 5 A (22) to the other fraction, followed by shaking for 6 h in 1 ml of 2,2,4- trimethylpentane. The branched hydrocarbons in 2,2,4-trimethylpentane were transferred to another vial, the molecular sieve washed twice with an additional l-ml aliquot of 2,2,4-trimethylpentane and the washes combined and taken to dryness under nitrogen. Gas-liquid chromatography of the total and branched alkanes indicated complete inclusion of n-alkane into the molecular sieve. Determination of radioactivity. Samples were transferred to counting vials in hexane, the solvent removed under nitrogen, and 10 ml of a fluor solution (0.4% PP03 in toluene) added. Samples were counted for 10 min on a Packard Tri-Carb liquid scintillation counter at 88% efficiency. Efficiency was determined with internal standards. All data points are the averages of three to six experiments. Gas-liquid chromatography. The same procedure described above was used to isolate total and branched hydrocarbon fractions from insects not injected with labelled substrates. Glc was performed on a 6-ft x l/8-in. column containing 3% SE-30 on Gas Chrom Q programmed from 150 to 280°C at 8”C/ min. Integration was obtained by disc integration. Radio-glc of the hydrocarbons isolated after incubation with sodium [I-Wlacetate and sodium [l“C]propionate was run under identical conditions. A 9:l stream splitter was used, and each peak was collected in a Pasteur pipet. The sample was washed into a scintillation vial with fluor solution and counted. About 65% of the sample injected into the glc was recovered. RESULTS
Cuticular
Hydrocarbons
The major cuticular hydrocarbons of adult P. fuliginosa are cis-9-tricosene, ntricosane, 11-methyltricosane, 3-methyltricosane, 13-methylpentacosane, and nheptacosane, with considerable differences noted between the male and female insects (19). Table I shows the hydrocarbon composition of the P. fuliginosa nymphs used in this study. n-Alkanes comprise 25+% of the hydrocarbon sample, 3-methylalkanes 14%, and internally branched monomethylalkanes 59+%. Several groups of three nymphs each from the fourth through sixth instars were studied with no appreciable difference observed in the hydrocarbon composition. 3Abbreviation
used: PPO, 2,5-diphenyloxazole.
KEARNEY TABLE I CUTICULAR HYDROCARBONS OF PeripZaneta fuliginosa MIDDLE INSTAR NYMPH@ Hydrocarbonb
cis-9-Tricosene n-Tricosane ll-Methyltricosane 3-Methyltricosane n-Tetracosane 12-Methyltetracosane n-Pentacosane 13-Methylpentacosane n-Hexacosane 13-Methylhexacosane n-Heptacosane n-Octacosane n-Nonacosane n-Tricontane
ComF$tion 0 TraceC 15 1 14 Trace 1 Trace 51 Trace Trace I Trace 3 Trace
a Cuticular lipids were extracted from PeripZaneta fuliginosa nymphs by immersion in hexane for 10 min and the hydrocarbons isolated by column chromatography on BioSil A eluting with 8 ml of hexane. Gas-liquid chromatography of the hydrocarbon fraction was performed on a 6-ft x ‘/e-in. 3% SE-30 column programmed from 150 to 280°C at 8”C/ min. Integration was obtained by disc integration. b Hydrocarbons were identified by comparison with standards, and the branched and unsaturated components were identified as reported by Jackson (19). c Trace equals less than 0.5% but greater than 0.1%.
In Vivo Studies Labeled glucose, acetate, propionate, and methylmalonate injected beneath the cuticle were readily incorporated into the cuticular hydrocarbons of P. fuliginosa (Table II). Acetate and propionate were incorporated most readily into the cuticular hydrocarbons with 0.4-1.9% of the injected label recovered in the hydrocarbon fraction, followed by methylmalonate and glucose, with 0.6 and 0.2% incorporated. Of the labeled glucose and acetate incorporated into hydrocarbon, 63 f 4 and 66 +3% were found in the branched alkanes, compared to 78 2 6,77 f 8, and 90 f 3% for sodium [3J4Cl-, [2J4Cl-, and [lJ4Clpropionate and 81 f 4% for sodium [methyl14Clmethylmalonate. Labeled methionine, isoleucine, valine, mevalonic acid lactone, and sodium palmitate were not readily incorporated into the
BIOSYNTHESIS
OF MONOMETHYLALKANES
cuticular hydrocarbon of P. fuliginosa (Table III). Less than 0.001% of labeled methionine and less than 0.01% of labeled mevaIonic acid lactone were found in the hydrocarbon fraction. Although 97 2 2 and 97 2 3% of labeled isoleucine and valine incorporated into hydrocarbon were incorporated into the branched fraction, only 0.12 and 0.08% of these precursors were recovered in the hydrocarbon fraction. PalTABLE
549
IN INSECTS
mitate labeled at either the l-14C or 16-14C position was incorporated into hydrocarbon at low levels, with 62 & 3 and 61 * 6% of the label incorporated into hydrocarbon found in the branched fraction. The selective incorporation of [l14Clpropionate into the branched alkanes over that of [lJ4C]acetate prompted a closer examination of the labeled hydrocarbons resulting from these precursors. II
In Vivo INCORPORATION OF LABELED GLUCOSE, ACETATE, PROPIONATE, AND METHYLMALONATE INTO THE CUTICULAR HYDROCARBONS OF Periplanetu fuliginosa” Substrate
[II-Wlglucose Sodium llJ4C1acetate Sodium [3-‘Qpropionate Sodium 12-Wlpropionate Sodium [I-Wlpropionate Sodium [meth~l-14Clmethylrnalonate
Percentage of injected labe1 incorporated into hydrocarbon 0.2 1.9 0.4 1.9 1.0 0.6
f + f +k k
0.13 0.6 0.1 0.6 0.4 0.3
Label incorporated Percentage in branched 63 66 78 77 90 81
-e 4 t 3 -+- 6 ? 8 + 3 + 4
into hydrocarbon Percentage in straight chain 37 34 22 23 10 19
5 k t r 2 +
4 3 6 8 3 4
n Appropriate labeled precursors were injected into P. fuliginosa nymphs. After 6 h the insects were sacrificed, extracted by immersion in hexane, the solvent reduced in volume, and hydrocarbon isolated by elution with 8 ml of hexane from BioSil A columns. Isolated hydrocarbon was divided into two equal fractions and taken to dryness. One-half was counted to determine incorporation of labeled substrates into hydrocarbon. Molecular sieve 5 A was added to the other fraction with 1 ml of 2,2,4-trimethylpentane and the sample placed on a shaker for 6 h. The branched hydrocarbons in 2,2,4-trimethylpentane were transferred to another vial, the molecular sieve washed twice with an additional 1 ml of 2,2,4-trimethylpentane, and the washes combined and taken to dryness. Radioactivity was determined as described in Methods. TABLE
III
In Viuo INCORPORATION OF LABELED METHIONINE, MEVALONIC ACID LACTONE, ISOLEUCINE, VALINE, AND PALMITIC ACID INTO THE CUTICULAR HYDROCARBON OF Periplaneta fuliginosa” Substrate
L-[Methyl-Wlmethionine L[G-3H1isoleucine L-[G-3H1valine nn-[2-‘4Clmevalonic acid lactone Sodium [I-%]palmitate Sodium [16-L4C]palmitate
Percentage of label incorporatred into hydrocarbon
Label incorporated
into hydrocarbon
Percentage in branched
Percentage in straight chain
97 + 2 97 k 3 62 + 3 61 + 6
322 3?3 38 + 3 39 f 6
a Appropriate labeled precursors were injected into P. fuliginosa nymphs. After 6 h the insects were sacrificed, extracted by immersion in hexane, the solvent reduced in volume, and hydrocarbon isolated by elution with 8 ml of hexane from BioSil A columns. Isolated hydrocarbon was divided into two equal fractions and taken to dryness. One-half was counted to determine incorporation of labeled substrates into hydrocarbon. Molecular sieve 5 A was added to the other fraction with 1 ml of 2,2,4-trimethylpentane and the sample placed on a shaker for 6 h. The branched hydrocarbons in 2,2,4-trimethylpentane were transferred to another vial, the molecular sieve washed twice with an additional 1 ml of 2,2,4-trimethylpentane, and the washes combined and taken to dryness. Radioactivity was determined as described in Methods.
550
BLOMQUIST
AND
KEARNEY
tate into the hydrocarbon fraction (Table IV), compared to a much lower incorporation in distilled water. Excised cuticle slices synthesized both hydrocarbon and polar lipids, whereas no hydrocarbon synthesis was observed in the fat body tissue (Table IV). However, fat body tissue did synthesize polar lipids. The excised cuticle system showed a linear increase in hydrocarbon synthesis with time up to at least 2 h (Fig. 2), the incubation time used for all further studies. After 2 h, the variance in incorporation among In Vitro Studies different samples increased considerably. An in vitro tissue slice system was de- Incorporation of label into hydrocarbon inveloped in order to confirm the selective creased linearly with increasing tissue incorporation of labeled propionate into weight up to at least 25 mg (Fig. 21, and the branched fractions and to determine this was the tissue weight used for all the location of the hydrocarbon biosyn- subsequent studies. thesis in insects. Cuticle slices in either To verify the selective incorporation of Grace’s insect medium or a saline solution propionate into the methyl branched algave good incorporation of label from ace- kanes of P. fuliginosa observed in the in Figure 1 shows a radio-glc of the hydrocarbon fraction after the metabolism of sodium [l-14Clacetate and sodium [l-14Clpropionate. The results show that acetate is incorporated into each component in about the same proportion as the percentage composition of hydrocarbon. Sodium [lJ4Clpropionate, however, is preferentially incorporated into 3-methyltricosane and 13-methylpentacosane, with little of the injected radioactivity found in the straight chain components.
FIG. 1. Radio-gas-liquid chromatogram of the cuticular hydrocarbons of Periplanetu fuliginosa after injection with sodium [l-Wlacetate (A) and sodium [l-“Clpropionate (B). Glc peaks are identified as n-tricosane (A), 11-methyltricosane (B), 3-methyltricosane (0, n-tetracosane (D), 12-methyltetracosane (E), n-pentacosane (F), 13-methylpentacosane (G), n-hexacosane (H), 13-methylhexacosane (I), n-heptacosane (J), n-octacosane (K), n-nonacosane CL), and ntricontane (M) (19). Sodium [l-Wlacetate and sodium [l-Wlpropionate (1.2 &i per insect) were injected into Pe+aneta fdiginosa nymphs. After 6 h the insects were sacrificed by immersion in hexane and hydrocarbon isolated by column chromatography on BioSil A eluting with hexane. Radio-gas-liquid chromatography was performed on 6-ft x ‘/e-in. 3% SE-30 column programmed from 150 to 280°C using a 9 to 1 stream splitter, and each peak was collected in a Pasteur pipet. Sample was eluted with a scintillation solution and counted as described in Methods.
BIOSYNTHESIS
OF MONOMETHYLALKANES TABLE
In Vitro
INCORPORATION
OF LABELED
ACETATE
551
IN INSECTS
IV INTO HYDROCARBON
Tissue
OF Periplaneta
Radioactivity
(dpm)
Hydrocarbon Cuticle slices in Cuticle slices in Cuticle slices in Fat body tissue Fat body tissue
Hz0 Grace’s insect medium salt solution in H,O in Grace’s medium
450 5,000 7,000 10 14
-t f t k 2
fuliginosa”
Polar lipids
100 500 2,100 4 8
7,000 13,000 18,000 5,800 16,500
+ ? c t +
900 3,200 5,200 650 6,200
a Excised cuticles or fat body tissue were obtained as described in Methods and incubated with 1.2 &i of sodium [l-‘4Clacetate for 2 h at 32°C in 250 ~1 of appropriate solutions. The incubation was stopped by adding 1 ml of chloroform. kid was extracted, seDarated on columns of BioSil A and counted as described in Methods. _
0
I
-hr
2
FIG. 2. In vitro incorporation of sodium [lWlacetate into cuticular hydrocarbon of the cockroach Periplanetn fuliginosa with increased time and tissue weight. Twenty-five milligrams of excised cuticle tissue was incubated with 1.2 &i of sodium [l-Wlacetate in Grace’s medium for appropriate time periods in the time study. Appropriate amounts of pooled cuticle tissue were incubated for 2 h in the weight study. All incubations were carried out at 32°C and stopped by the addition of chloroform. Lipid was extracted with chloroform, separated by column chromatography on BioSil A, and counted as described in Methods.
uivo studies, incubation of sodium [1J4Clacetate, sodium [1J4Cl- and [2J4C]propionate, L-[methyZJ4C]methionine, L-[G-~H]valine, and L-[G-3Hlisoleucine with excised cuticles was carried out. Sodium [2J4Clpropionate was incorporated most readily, with 1.1 -c 0.3% of the label recovered in the hydrocarbon fraction, compared to 0.2 2 0.1 and 0.07 f 0.03% for sodium [lJ4Clacetate and sodium [lJ4Clpropionate (Table V). Labeled methionine, isoleucine, and valine were incorporated into hydrocarbon at less than 0.001%. So-
dium [l-14Clpropionate and sodium [2-14C]propionate were selectively incorporated into the branched hydrocarbon fraction, with 81 + 3 and 65 2 9% of the label incorporated into hydrocarbon found in the branched alkanes. In contrast, only 31 2 5% of sodium [lJ4C]acetate incorporated into hydrocarbon was found in the branched fraction (Table V). Sodium [314Clmethylmalonate were also preferentially incorporated into the branched hydrocarbons, although less than 0.01% of the label was incorporated into the hydrocarbon fraction. DISCUSSION
Results of these experiments suggest that insects utilize a different pathway for the biosynthesis of the internally branched monomethylalkanes than demonstrated in which algae. L-[MethyZ-14C]methionine, serves as the branching methyl group donor for 7- and 8-methyl-heptadecanes in algae (7, 8), was not incorporated into the cuticular hydrocarbons of P. fuliginosa either in uiuo or in vitro. The preferential incorporation of the labeled sodium propionates and sodium methylmalonate over that of labeled glucose and sodium [l14Clacetate into the branched hydrocarbons suggests that 13-methylpentacosane is synthesized by the pathway indicated in Fig. 3. Propionate is apparently converted to a methylmalonyl derivative which is incorporated into the alkyl chain during elongation. Additional acetyl units are then added, followed by decarboxylation. This proposed pathway is consistent with the predominance of the branching methyl
BLOMQUIST
552
AND KEARNEY
TABLE V INCORPORATION OF LABELED ACETATE, PROPIONATE, METHIONINE, ISOLEUCINE, AND VALINE INTO HYDROCARBON OF THE COCKROACH Periplaneta fuliginosa in Vitro”
Substrate
Sodium [l-‘4C1acetate Sodium [I-Wlpropionate Sodium [2-Wlpropionate L-[MethyZ-14C]methionine L-[GJHlisoleucine c[G3H1valine
Percentage incorporated into hydrocarbon
Label incorporated into hydrocarbon Percentage in branched
0.2 + 0.1 0.07 + 0.03 1.1 * 0.03 <0.0001
31 f 5 81 f 3 65 2 9 89 + 10 -
Percentage in straight chain 69 k 19 f 35 2 11 2 -
5 3 9 10
a Appropriate labeled precursors (0.6 @i) were incubated with 25 mg of excised cuticle tissue in 250 ~1 of Grace’s insect medium for 2 h at 32°C. The reaction was stopped by addition of chloroform, lipids extracted, and the hydrocarbons isolated, separated by column chromatography and counted as described in Methods.
CH3 - (CH2)lO
E
EF- 9 - x
FIG. 3. Proposed biosynthetic pathway for l3-methylpentacosane neta fuliginosa.
groups on odd-numbered carbons in insect cuticular lipids, is similar to the proposed pathway for the 3-methylalkanes in Periplaneta americana (15), and is consistent with the biosynthetic pathways proposed for multibranched fatty acids in the uropygial glands of goose (23,24), where methylmalonyl-CoA serves as the precursor to the branching methyl groups. A comparison of the data on the incorporation of the sodium [l-14C]-, [2-14C]-, and [3-14Clpropionate into normal and methyl branched alkanes suggests that propionate can be metabolized to other precursors, and these precursors are then incorporated into hydrocarbon. The number of meta-
in the cockroach Peripla-
bolic pathways for propionate is suffrciently great to preclude any simple resolution of this problem. However, the data suggest that a portion of the propionate incorporated into hydrocarbon is metabolized to an acetyl unit with loss of the carboxyl carbon. This would account for the preferential incorporation of sodium [lJ4Clpropionate and the lesser but nearequal incorporation of sodium [2-14Cland sodium [3J4Clpropionate into the branched alkanes, as the labeled carboxyl carbon would be lost during conversion to an acetyl unit. Similar data were reported for the incorporation of labeled propionate into 3-methylpentacosane in Periplaneta
BIOSYNTHESIS
OF MONOMETHYLALKANES
(15) and for the incorporation of propionate into juvenile hormones in Mun-
americana
duca se&u (26).
The preferential incorporation of sodium [lJ4Clpropionate over that of sodium [l14Clacetate into 3-methyltricosane lends additional support to the proposed pathway for the 3-methylalkanes in insects, in which propionate is converted to a methylmalonyl derivative and incorporated near the end of the elongation process. The incorporation of the branching unit near the end of the elongation process could explain the absence of ante-is0 branched fatty acids in insects (4), whereas anteiso fatty acids commonly occur associated with 3methylalkanes in plants (1). The similarity in the biosynthetic pathways for the 3methyl- and internally branched monomethylalkanes might also explain the occurrence of monomethylalkanes in which the methyl branch occurs on every odd-numbered carbon (18). The similarity in the biosynthetic pathways for the 3-methyl- and internally branched monomethylalkanes in insects suggests the possibility of a similar pathway for the dimethylalkanes. The dimethylalkanes often occur in conjunction with the internally branched monomethylalkanes (16, 17, 21) and sometimes with both the 3-methylalkanes and the internally branched monomethylalkanes (17). The branching methyl groups are commonly separated by three methylene units. One could envision elongation occurring by the sequential incorporation of a methylmalonyl derivative, a malonyl derivative, and then another methylmalonyl derivative. No experimental evidence is available on the biosynthesis of this type of branched alkane. Hydrocarbon synthesis appears to occur very close to the cuticle surface in P. fuliginosa. Nelson (25) reported that in Periplanetu americana the epidermal cells are responsible for the synthesis of hydrocarbons. However, Diehl(11, 26) has recently presented convincing evidence that the oenocytes are the site of hydrocarbon synthesis in the desert locust Schistocercu gregaria. This apparent contradiction could be due to the close association of the oenocytes with the epidermis in the cock-
IN INSECTS
553
roach (27), and both Nelson’s work (25) and the data presented in this paper suggesting that hydrocarbon synthesis occurs near the surface of the insect could be synthesis occurring in oenocytes associated with the epidermis. REFERENCES 1. KOLATTUKUDY, P. E., AND WALTON, T. J. (1973) PFOgF. Ch‘?m. ht.S &lidS 13, 121. 2. GELPI, E., SCHNEIDER, H., MANN, J., AND ORO, J. (1970) Phytochemistry 9, 603. 3. ALBRO, P. W. 1970. Lipids 5, 320. 4. JACKSON, L. L., AND BAKER, G. L. (1970) Lipids 5, 239. 5. KOLATTUKUDY, P. E., CROTEAU, R., AND BROWN, L. (1974) Plant Physiol. 54, 670. 6. KHAN, A. A., AND KOLATTUKUDY, P. E. (1974) Biochem. Biophys. Res. Commun. 61, 1379. 7. FEHLER, S. W. G., AND LIGHT, R. J. (1970) Biochemistry 9, 418. 8. HAN, J., CHAN, H. W. S., AND CALVIN, M. J. (1969) J. Amer. Chem. Sot. 91, 5156. 9. CONRAD, C., AND JACKSON, L. L. (1971) J. Insect Pysiol. 17, 1907. 10. ROBBINS, W. E., KAPLANIS, J. N., LOUOUDES, S. J., AND MONROE, R. E. (1960) Ann. Entomol. sot. Amer. 53, 128. 11. DIEHL, P. A. (1973) Nature (London) 243, 468. 12. BLOMQUIST, G. J., AND JACKSON, L. L. (1973) J. Insect. Physiol. 19, 1639. 13. BLOMQUIST, G. J., AND JACKSON, L. L. (1973) Biochem. Biophys. Res. Commun. 53, 703. 14. BLOMQUIST, G. J., MCCAIN, D. C., AND JACKSON, L. L. (1975) Lipids 10, 303. 15. BLOMQUIST, G. J., MAJOR, M. A., AND LOK, J. B. (1975) Biochem. Biophys. Res. Commun. 64, 43. 16. NELSON, D. R., AND SUKKESTAD, D. R. (1970) Biochemistry 9, 4601. 17. SOLIDAY, C. L., BLOMQUIST, G. J., AND JACKSON, L. L. (1974) J. Lipid Res. 15, 399. 18. JACKSON, L. L., ARMOLD, M. T;, AND REGNIER, F. E. (1974) Insect Biochem. 4, 369. 19. JACKSON, L. L. (1970) Lipids 5, 38. 20. TARTIVITA, K., AND JACKSON, L. L. (1970) Lipids 5, 35. 21. NELSON, D. R., AND SUKKESTAD, D. R. (1974) J. Lipid Res. 16, 12. 22. O’CONNOR, J. G., BURROW, F. H., AND NORRIS, M. S. (1962) Anal. Chem. 34, 82. 23. BUCKNER, J. S., AND KOLATTUKLJDY, P. E. (1975) Biochemistry 14, 1768. 24. BUCKNER, J. S., AND KOLATTUKUDY, P. E. (1975) Biochemistry 14, 1774. 25. NELSON, D. R. (1969) Nature (London) 221, 854. 26. DIEHL, P. A. (1975) J. Insect Physiol. 21, 1237. 27. KRAMER, S., AND WIGGLESWORTH, V. B. (1950) @art. J. Microsc. Sci. 91, 63.
OF
BIOCHEMISTRY
Biosynthesis
GARY Chemistry
Department,
AND
BIOPHYSICS
of Internally Cockroach
173, 546-553 (1976)
Branched Monomethylalkanes Periplaneta fuliginosal
J. BLOMQUIST2 University
GEORGE
AND
of Southern Mississippi, Southern Mississippi 39401 Received August
in the
P. KEARNEY Station,
Box 5347, Hattiesburg,
21, 1975
Sodium [1-14C]acetate, sodium [l-Wlpropionate, sodium [2-Wlpropionate, sodium [3“Clpropionate and sodium [methyE-14Clmethylmalonate were readily incorporated into the cuticular hydrocarbons of nymphal stages of the cockroach Periplaneta fzdiginosa both in vivo and in vitro, whereas no incorporation of [methyl-Wlmethionine was observed. The alkanes of the nymphal stages of this insect are 25+% n-alkanes, 14% 3methylalkanes, and 59+% internally branched monomethylalkanes, principally 13methylpentacosane. Sodium [l-Wlacetate was incorporated into each class of alkane at about its percentage composition. In contrast, labeled sodium propionate and sodium methylmalonate were preferentially incorporated into the branched fractions. Radiogas-liquid chromatography showed that sodium [l-Wpropionate was incorporated almost exclusively into 3-methyltricosane and 13-methylpentacosane, whereas sodium ll-Wacetate was incorporated into each glc peak at about its percentage composition. These data suggest that propionate, incorporated during chain elongation, serves as the branching methyl group donor for both the 3-methyl and the internally branched monomethylalkanes in insects. The location of hydrocarbon synthesis in P. fuliginosa was studied using an in vitro tissue slice system. Excised cuticle slices, with adhering fat body tissue removed, gave good incorporation of labeled substrates into the hydrocarbon fraction. No hydrocarbon synthesis was observed in fat body preparations.
Methyl branched hydrocarbons occur in many biological systems, including plants (l), algae (2), bacteria (31, and insects (4). The biosynthesis of both the normal and branched hydrocarbons has received considerable attention in plants and microorganisms, with convincing evidence on the pathways leading to the major components reported (1, 3). In plants, n-alkanes are formed by an elongation of fatty acids followed by decarboxylation (5, 6) and the 2and 3-methylalkanes originate from the appropriately branched starter chains derived from branched amino acids (1). In the algae, the active methyl group from methionine serves as the branching
methyl group donor for the internally branched monomethylalkanes (7, 8). In contrast to the situation for plants and microorganisms, the biosynthetic pathways for insect cuticular hydrocarbons have not received extensive study. Acetate has been shown to be readily incorporated into the hydrocarbons of live insects (4, 9, lo), excised cuticles (41, and oenocyte-rich fat body preparations (11). Indirect evidence on n-alkane synthesis obtained from studying the formation of secondary alcohols (12-14) suggests that an elongation-decarboxylation pathway is operative, although definitive work has not been done. Recent evidence indicates that the 3-methylalkanes in insects are formed by a pathway different from that reported in other organisms. In insects, propionate is apparently converted to a methylmalonyl derivative and incorpo-
1 This work was supported in part by a Cottrell Grant from Research Corporation and a Sigma Xi Research Grant. 2 Author to whom inquiries should be directed. 546 Copyright 8 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOSYNTHESIS
OF MONOMETHYLALKANES
rated as the penultimate biosynthetic unit (15). This observation suggested the possibility of a similar pathway for the biosynthesis of the internally branched monomethylalkanes in insects, in which additional acetyl units are added after propionate is incorporated, followed by decarboxylation. The experiments reported in this paper were designed to determine if the branching methyl group of the internally branched monomethylalkanes in insects is inserted by addition to a preformed chain by the active methyl group of methionine, as observed in algae (7, 8), or if propionate or a derivative such as methylmalonate is incorporated during chain elongation. No information on the biosynthesis of this type of alkane in insects has been previously reported. The internally branched monomethylalkanes comprise a major fraction of the cutitular hydrocarbons from a number of insects (16-19) and are often present in complex mixtures. Their chain length distribution ranges from 24 to 28 carbons in a number of cockroaches (19, 20), 28 to 36 carbons in two species of Melanoplus (17), and up to 50 carbons in the grasshopper Schistocerca uaga (21). The majority of the internally branched monomethylalkanes in insects have the methyl branch on oddnumbered carbons, and this is usually on carbon number 11, 13, or 15, although isomers with methyl branches on all oddnumbered carbons have been reported (18). In contrast to the complex mixtures of these alkanes observed in many insects, much of the cuticular hydrocarbon of the cockroach Periplaneta fuliginosa is comprised of 13-methylpentacosane (19), making this an excellent experimental animal for studying the biosynthesis of these alkanes. MATERIALS
AND
METHODS
Materials Sodium [l-14Clacetate (55 mCi/mmol), DL-[Z“Clmevalonic acid lactone (10.3 mCi/mmol), sodium [methyZ-~4Clmethylmalonate (10.5 mCi/mmol), sodium [l-L4C]propionate (0.55 mCi/mmol), sodium [2Wlpropionate (27 mCi/mmol) and sodium [3Wlpropionate (0.8 mCi/mmol) were purchased from New England Nuclear. L-[G-3Hlisoleucine (2.7 Ci/
IN INSECTS
547
mmol), L-[G-3Hlvaline (6.8 Ci/mmol), L-[methyl“Clmethionine (50 mCi/mmol) and [U-Wlglucose (180 mCi/mmol) were purchased from ICN. Grace’s insect medium was obtained from the Grand Island Biological Co. All solvents were redistilled from glass prior to use. A starter colony of P. fuliginosa was obtained from Dr. Mary Ross. Colonies of P. fuliginosa were reared in 20-gal metal containers on a diet of ground dry dog food and an agar-water (1:99) gel fed ad lib. Middle instar nymphs (fourth through sixth instars) were used in all studies.
Methods In uiuo studies. Labeled substrates in 2 ~1 of water were injected just beneath the cuticle between the eighth and ninth abdominal segments. The insects were kept at room temperature and sacrificed after 6 h. In some cases larger amounts of labeled substrates and longer incubation times were used where greater amounts of labeled hydrocarbons were needed for radio-gas-liquid chromatography (radio-glc). The cuticular lipids were extracted by immersion of the insects in hexane for 10 min and then taken to dryness under nitrogen. Time studies indicate that this gave essentially complete extraction of cuticular hydrocarbon while not extracting internal lipid. Three to five insects were used in each experimental group. In vitro studies. Middle instar insects were anesthesized with carbon dioxide, placed on ice, and kept at O-4% during subsequent manipulation. The abdomens were excised. Cuticle tissue was isolated by separating the ventral and dorsal halves after an incision on each side of the abdomen, and the fat body was removed by scraping gently with a razor blade. The isolated cuticles were cut into strips, the tissues pooled, and 25 mg of tissue placed in incubation vials with appropriate labeled substrates containing 250 ~1 of Grace’s insect medium, distilled water, or a saline solution containing: NaCl (12 g/ liter), KC1 (0.5 g/liter), CaCl, (1.5 g/liter), MgCl, (0.53 g/liter) and NaHCO, (0.2 g/liter). The vials were thoroughly mixed and incubated for 2 h, unless otherwise indicated, at 32°C in a shaking water bath (100 oscillationslmin). At the end of the incubation period, lipids were extracted from the cuticle tissue with chloroform, and the solvent was removed under nitrogen. Separation of lipid components. The lipid samples were transferred to a Pasteur pipet containing 50 mg of BioSil A in 1 ml of hexane. The hydrocarbons were eluted into a vial with 8 ml of hexane and the polar lipids eluted with 8 ml of chloroform:methanol (2:l). Polar lipids, as used here, include all lipids more polar than hydrocarbons. The hydrocarbon sample was divided into two equal fractions, and all
548
BLOMQUIST
AND
fractions were taken to dryness under nitrogen. One fraction contained one-half of the total hydrocarbons. The branched hydrocarbons were isolated by adding molecular sieve 5 A (22) to the other fraction, followed by shaking for 6 h in 1 ml of 2,2,4- trimethylpentane. The branched hydrocarbons in 2,2,4-trimethylpentane were transferred to another vial, the molecular sieve washed twice with an additional l-ml aliquot of 2,2,4-trimethylpentane and the washes combined and taken to dryness under nitrogen. Gas-liquid chromatography of the total and branched alkanes indicated complete inclusion of n-alkane into the molecular sieve. Determination of radioactivity. Samples were transferred to counting vials in hexane, the solvent removed under nitrogen, and 10 ml of a fluor solution (0.4% PP03 in toluene) added. Samples were counted for 10 min on a Packard Tri-Carb liquid scintillation counter at 88% efficiency. Efficiency was determined with internal standards. All data points are the averages of three to six experiments. Gas-liquid chromatography. The same procedure described above was used to isolate total and branched hydrocarbon fractions from insects not injected with labelled substrates. Glc was performed on a 6-ft x l/8-in. column containing 3% SE-30 on Gas Chrom Q programmed from 150 to 280°C at 8”C/ min. Integration was obtained by disc integration. Radio-glc of the hydrocarbons isolated after incubation with sodium [I-Wlacetate and sodium [l“C]propionate was run under identical conditions. A 9:l stream splitter was used, and each peak was collected in a Pasteur pipet. The sample was washed into a scintillation vial with fluor solution and counted. About 65% of the sample injected into the glc was recovered. RESULTS
Cuticular
Hydrocarbons
The major cuticular hydrocarbons of adult P. fuliginosa are cis-9-tricosene, ntricosane, 11-methyltricosane, 3-methyltricosane, 13-methylpentacosane, and nheptacosane, with considerable differences noted between the male and female insects (19). Table I shows the hydrocarbon composition of the P. fuliginosa nymphs used in this study. n-Alkanes comprise 25+% of the hydrocarbon sample, 3-methylalkanes 14%, and internally branched monomethylalkanes 59+%. Several groups of three nymphs each from the fourth through sixth instars were studied with no appreciable difference observed in the hydrocarbon composition. 3Abbreviation
used: PPO, 2,5-diphenyloxazole.
KEARNEY TABLE I CUTICULAR HYDROCARBONS OF PeripZaneta fuliginosa MIDDLE INSTAR NYMPH@ Hydrocarbonb
cis-9-Tricosene n-Tricosane ll-Methyltricosane 3-Methyltricosane n-Tetracosane 12-Methyltetracosane n-Pentacosane 13-Methylpentacosane n-Hexacosane 13-Methylhexacosane n-Heptacosane n-Octacosane n-Nonacosane n-Tricontane
ComF$tion 0 TraceC 15 1 14 Trace 1 Trace 51 Trace Trace I Trace 3 Trace
a Cuticular lipids were extracted from PeripZaneta fuliginosa nymphs by immersion in hexane for 10 min and the hydrocarbons isolated by column chromatography on BioSil A eluting with 8 ml of hexane. Gas-liquid chromatography of the hydrocarbon fraction was performed on a 6-ft x ‘/e-in. 3% SE-30 column programmed from 150 to 280°C at 8”C/ min. Integration was obtained by disc integration. b Hydrocarbons were identified by comparison with standards, and the branched and unsaturated components were identified as reported by Jackson (19). c Trace equals less than 0.5% but greater than 0.1%.
In Vivo Studies Labeled glucose, acetate, propionate, and methylmalonate injected beneath the cuticle were readily incorporated into the cuticular hydrocarbons of P. fuliginosa (Table II). Acetate and propionate were incorporated most readily into the cuticular hydrocarbons with 0.4-1.9% of the injected label recovered in the hydrocarbon fraction, followed by methylmalonate and glucose, with 0.6 and 0.2% incorporated. Of the labeled glucose and acetate incorporated into hydrocarbon, 63 f 4 and 66 +3% were found in the branched alkanes, compared to 78 2 6,77 f 8, and 90 f 3% for sodium [3J4Cl-, [2J4Cl-, and [lJ4Clpropionate and 81 f 4% for sodium [methyl14Clmethylmalonate. Labeled methionine, isoleucine, valine, mevalonic acid lactone, and sodium palmitate were not readily incorporated into the
BIOSYNTHESIS
OF MONOMETHYLALKANES
cuticular hydrocarbon of P. fuliginosa (Table III). Less than 0.001% of labeled methionine and less than 0.01% of labeled mevaIonic acid lactone were found in the hydrocarbon fraction. Although 97 2 2 and 97 2 3% of labeled isoleucine and valine incorporated into hydrocarbon were incorporated into the branched fraction, only 0.12 and 0.08% of these precursors were recovered in the hydrocarbon fraction. PalTABLE
549
IN INSECTS
mitate labeled at either the l-14C or 16-14C position was incorporated into hydrocarbon at low levels, with 62 & 3 and 61 * 6% of the label incorporated into hydrocarbon found in the branched fraction. The selective incorporation of [l14Clpropionate into the branched alkanes over that of [lJ4C]acetate prompted a closer examination of the labeled hydrocarbons resulting from these precursors. II
In Vivo INCORPORATION OF LABELED GLUCOSE, ACETATE, PROPIONATE, AND METHYLMALONATE INTO THE CUTICULAR HYDROCARBONS OF Periplanetu fuliginosa” Substrate
[II-Wlglucose Sodium llJ4C1acetate Sodium [3-‘Qpropionate Sodium 12-Wlpropionate Sodium [I-Wlpropionate Sodium [meth~l-14Clmethylrnalonate
Percentage of injected labe1 incorporated into hydrocarbon 0.2 1.9 0.4 1.9 1.0 0.6
f + f +k k
0.13 0.6 0.1 0.6 0.4 0.3
Label incorporated Percentage in branched 63 66 78 77 90 81
-e 4 t 3 -+- 6 ? 8 + 3 + 4
into hydrocarbon Percentage in straight chain 37 34 22 23 10 19
5 k t r 2 +
4 3 6 8 3 4
n Appropriate labeled precursors were injected into P. fuliginosa nymphs. After 6 h the insects were sacrificed, extracted by immersion in hexane, the solvent reduced in volume, and hydrocarbon isolated by elution with 8 ml of hexane from BioSil A columns. Isolated hydrocarbon was divided into two equal fractions and taken to dryness. One-half was counted to determine incorporation of labeled substrates into hydrocarbon. Molecular sieve 5 A was added to the other fraction with 1 ml of 2,2,4-trimethylpentane and the sample placed on a shaker for 6 h. The branched hydrocarbons in 2,2,4-trimethylpentane were transferred to another vial, the molecular sieve washed twice with an additional 1 ml of 2,2,4-trimethylpentane, and the washes combined and taken to dryness. Radioactivity was determined as described in Methods. TABLE
III
In Viuo INCORPORATION OF LABELED METHIONINE, MEVALONIC ACID LACTONE, ISOLEUCINE, VALINE, AND PALMITIC ACID INTO THE CUTICULAR HYDROCARBON OF Periplaneta fuliginosa” Substrate
L-[Methyl-Wlmethionine L[G-3H1isoleucine L-[G-3H1valine nn-[2-‘4Clmevalonic acid lactone Sodium [I-%]palmitate Sodium [16-L4C]palmitate
Percentage of label incorporatred into hydrocarbon
Label incorporated
into hydrocarbon
Percentage in branched
Percentage in straight chain
97 + 2 97 k 3 62 + 3 61 + 6
322 3?3 38 + 3 39 f 6
a Appropriate labeled precursors were injected into P. fuliginosa nymphs. After 6 h the insects were sacrificed, extracted by immersion in hexane, the solvent reduced in volume, and hydrocarbon isolated by elution with 8 ml of hexane from BioSil A columns. Isolated hydrocarbon was divided into two equal fractions and taken to dryness. One-half was counted to determine incorporation of labeled substrates into hydrocarbon. Molecular sieve 5 A was added to the other fraction with 1 ml of 2,2,4-trimethylpentane and the sample placed on a shaker for 6 h. The branched hydrocarbons in 2,2,4-trimethylpentane were transferred to another vial, the molecular sieve washed twice with an additional 1 ml of 2,2,4-trimethylpentane, and the washes combined and taken to dryness. Radioactivity was determined as described in Methods.
550
BLOMQUIST
AND
KEARNEY
tate into the hydrocarbon fraction (Table IV), compared to a much lower incorporation in distilled water. Excised cuticle slices synthesized both hydrocarbon and polar lipids, whereas no hydrocarbon synthesis was observed in the fat body tissue (Table IV). However, fat body tissue did synthesize polar lipids. The excised cuticle system showed a linear increase in hydrocarbon synthesis with time up to at least 2 h (Fig. 2), the incubation time used for all further studies. After 2 h, the variance in incorporation among In Vitro Studies different samples increased considerably. An in vitro tissue slice system was de- Incorporation of label into hydrocarbon inveloped in order to confirm the selective creased linearly with increasing tissue incorporation of labeled propionate into weight up to at least 25 mg (Fig. 21, and the branched fractions and to determine this was the tissue weight used for all the location of the hydrocarbon biosyn- subsequent studies. thesis in insects. Cuticle slices in either To verify the selective incorporation of Grace’s insect medium or a saline solution propionate into the methyl branched algave good incorporation of label from ace- kanes of P. fuliginosa observed in the in Figure 1 shows a radio-glc of the hydrocarbon fraction after the metabolism of sodium [l-14Clacetate and sodium [l-14Clpropionate. The results show that acetate is incorporated into each component in about the same proportion as the percentage composition of hydrocarbon. Sodium [lJ4Clpropionate, however, is preferentially incorporated into 3-methyltricosane and 13-methylpentacosane, with little of the injected radioactivity found in the straight chain components.
FIG. 1. Radio-gas-liquid chromatogram of the cuticular hydrocarbons of Periplanetu fuliginosa after injection with sodium [l-Wlacetate (A) and sodium [l-“Clpropionate (B). Glc peaks are identified as n-tricosane (A), 11-methyltricosane (B), 3-methyltricosane (0, n-tetracosane (D), 12-methyltetracosane (E), n-pentacosane (F), 13-methylpentacosane (G), n-hexacosane (H), 13-methylhexacosane (I), n-heptacosane (J), n-octacosane (K), n-nonacosane CL), and ntricontane (M) (19). Sodium [l-Wlacetate and sodium [l-Wlpropionate (1.2 &i per insect) were injected into Pe+aneta fdiginosa nymphs. After 6 h the insects were sacrificed by immersion in hexane and hydrocarbon isolated by column chromatography on BioSil A eluting with hexane. Radio-gas-liquid chromatography was performed on 6-ft x ‘/e-in. 3% SE-30 column programmed from 150 to 280°C using a 9 to 1 stream splitter, and each peak was collected in a Pasteur pipet. Sample was eluted with a scintillation solution and counted as described in Methods.
BIOSYNTHESIS
OF MONOMETHYLALKANES TABLE
In Vitro
INCORPORATION
OF LABELED
ACETATE
551
IN INSECTS
IV INTO HYDROCARBON
Tissue
OF Periplaneta
Radioactivity
(dpm)
Hydrocarbon Cuticle slices in Cuticle slices in Cuticle slices in Fat body tissue Fat body tissue
Hz0 Grace’s insect medium salt solution in H,O in Grace’s medium
450 5,000 7,000 10 14
-t f t k 2
fuliginosa”
Polar lipids
100 500 2,100 4 8
7,000 13,000 18,000 5,800 16,500
+ ? c t +
900 3,200 5,200 650 6,200
a Excised cuticles or fat body tissue were obtained as described in Methods and incubated with 1.2 &i of sodium [l-‘4Clacetate for 2 h at 32°C in 250 ~1 of appropriate solutions. The incubation was stopped by adding 1 ml of chloroform. kid was extracted, seDarated on columns of BioSil A and counted as described in Methods. _
0
I
-hr
2
FIG. 2. In vitro incorporation of sodium [lWlacetate into cuticular hydrocarbon of the cockroach Periplanetn fuliginosa with increased time and tissue weight. Twenty-five milligrams of excised cuticle tissue was incubated with 1.2 &i of sodium [l-Wlacetate in Grace’s medium for appropriate time periods in the time study. Appropriate amounts of pooled cuticle tissue were incubated for 2 h in the weight study. All incubations were carried out at 32°C and stopped by the addition of chloroform. Lipid was extracted with chloroform, separated by column chromatography on BioSil A, and counted as described in Methods.
uivo studies, incubation of sodium [1J4Clacetate, sodium [1J4Cl- and [2J4C]propionate, L-[methyZJ4C]methionine, L-[G-~H]valine, and L-[G-3Hlisoleucine with excised cuticles was carried out. Sodium [2J4Clpropionate was incorporated most readily, with 1.1 -c 0.3% of the label recovered in the hydrocarbon fraction, compared to 0.2 2 0.1 and 0.07 f 0.03% for sodium [lJ4Clacetate and sodium [lJ4Clpropionate (Table V). Labeled methionine, isoleucine, and valine were incorporated into hydrocarbon at less than 0.001%. So-
dium [l-14Clpropionate and sodium [2-14C]propionate were selectively incorporated into the branched hydrocarbon fraction, with 81 + 3 and 65 2 9% of the label incorporated into hydrocarbon found in the branched alkanes. In contrast, only 31 2 5% of sodium [lJ4C]acetate incorporated into hydrocarbon was found in the branched fraction (Table V). Sodium [314Clmethylmalonate were also preferentially incorporated into the branched hydrocarbons, although less than 0.01% of the label was incorporated into the hydrocarbon fraction. DISCUSSION
Results of these experiments suggest that insects utilize a different pathway for the biosynthesis of the internally branched monomethylalkanes than demonstrated in which algae. L-[MethyZ-14C]methionine, serves as the branching methyl group donor for 7- and 8-methyl-heptadecanes in algae (7, 8), was not incorporated into the cuticular hydrocarbons of P. fuliginosa either in uiuo or in vitro. The preferential incorporation of the labeled sodium propionates and sodium methylmalonate over that of labeled glucose and sodium [l14Clacetate into the branched hydrocarbons suggests that 13-methylpentacosane is synthesized by the pathway indicated in Fig. 3. Propionate is apparently converted to a methylmalonyl derivative which is incorporated into the alkyl chain during elongation. Additional acetyl units are then added, followed by decarboxylation. This proposed pathway is consistent with the predominance of the branching methyl
BLOMQUIST
552
AND KEARNEY
TABLE V INCORPORATION OF LABELED ACETATE, PROPIONATE, METHIONINE, ISOLEUCINE, AND VALINE INTO HYDROCARBON OF THE COCKROACH Periplaneta fuliginosa in Vitro”
Substrate
Sodium [l-‘4C1acetate Sodium [I-Wlpropionate Sodium [2-Wlpropionate L-[MethyZ-14C]methionine L-[GJHlisoleucine c[G3H1valine
Percentage incorporated into hydrocarbon
Label incorporated into hydrocarbon Percentage in branched
0.2 + 0.1 0.07 + 0.03 1.1 * 0.03 <0.0001
31 f 5 81 f 3 65 2 9 89 + 10 -
Percentage in straight chain 69 k 19 f 35 2 11 2 -
5 3 9 10
a Appropriate labeled precursors (0.6 @i) were incubated with 25 mg of excised cuticle tissue in 250 ~1 of Grace’s insect medium for 2 h at 32°C. The reaction was stopped by addition of chloroform, lipids extracted, and the hydrocarbons isolated, separated by column chromatography and counted as described in Methods.
CH3 - (CH2)lO
E
EF- 9 - x
FIG. 3. Proposed biosynthetic pathway for l3-methylpentacosane neta fuliginosa.
groups on odd-numbered carbons in insect cuticular lipids, is similar to the proposed pathway for the 3-methylalkanes in Periplaneta americana (15), and is consistent with the biosynthetic pathways proposed for multibranched fatty acids in the uropygial glands of goose (23,24), where methylmalonyl-CoA serves as the precursor to the branching methyl groups. A comparison of the data on the incorporation of the sodium [l-14C]-, [2-14C]-, and [3-14Clpropionate into normal and methyl branched alkanes suggests that propionate can be metabolized to other precursors, and these precursors are then incorporated into hydrocarbon. The number of meta-
in the cockroach Peripla-
bolic pathways for propionate is suffrciently great to preclude any simple resolution of this problem. However, the data suggest that a portion of the propionate incorporated into hydrocarbon is metabolized to an acetyl unit with loss of the carboxyl carbon. This would account for the preferential incorporation of sodium [lJ4Clpropionate and the lesser but nearequal incorporation of sodium [2-14Cland sodium [3J4Clpropionate into the branched alkanes, as the labeled carboxyl carbon would be lost during conversion to an acetyl unit. Similar data were reported for the incorporation of labeled propionate into 3-methylpentacosane in Periplaneta
BIOSYNTHESIS
OF MONOMETHYLALKANES
(15) and for the incorporation of propionate into juvenile hormones in Mun-
americana
duca se&u (26).
The preferential incorporation of sodium [lJ4Clpropionate over that of sodium [l14Clacetate into 3-methyltricosane lends additional support to the proposed pathway for the 3-methylalkanes in insects, in which propionate is converted to a methylmalonyl derivative and incorporated near the end of the elongation process. The incorporation of the branching unit near the end of the elongation process could explain the absence of ante-is0 branched fatty acids in insects (4), whereas anteiso fatty acids commonly occur associated with 3methylalkanes in plants (1). The similarity in the biosynthetic pathways for the 3methyl- and internally branched monomethylalkanes might also explain the occurrence of monomethylalkanes in which the methyl branch occurs on every odd-numbered carbon (18). The similarity in the biosynthetic pathways for the 3-methyl- and internally branched monomethylalkanes in insects suggests the possibility of a similar pathway for the dimethylalkanes. The dimethylalkanes often occur in conjunction with the internally branched monomethylalkanes (16, 17, 21) and sometimes with both the 3-methylalkanes and the internally branched monomethylalkanes (17). The branching methyl groups are commonly separated by three methylene units. One could envision elongation occurring by the sequential incorporation of a methylmalonyl derivative, a malonyl derivative, and then another methylmalonyl derivative. No experimental evidence is available on the biosynthesis of this type of branched alkane. Hydrocarbon synthesis appears to occur very close to the cuticle surface in P. fuliginosa. Nelson (25) reported that in Periplanetu americana the epidermal cells are responsible for the synthesis of hydrocarbons. However, Diehl(11, 26) has recently presented convincing evidence that the oenocytes are the site of hydrocarbon synthesis in the desert locust Schistocercu gregaria. This apparent contradiction could be due to the close association of the oenocytes with the epidermis in the cock-
IN INSECTS
553
roach (27), and both Nelson’s work (25) and the data presented in this paper suggesting that hydrocarbon synthesis occurs near the surface of the insect could be synthesis occurring in oenocytes associated with the epidermis. REFERENCES 1. KOLATTUKUDY, P. E., AND WALTON, T. J. (1973) PFOgF. Ch‘?m. ht.S &lidS 13, 121. 2. GELPI, E., SCHNEIDER, H., MANN, J., AND ORO, J. (1970) Phytochemistry 9, 603. 3. ALBRO, P. W. 1970. Lipids 5, 320. 4. JACKSON, L. L., AND BAKER, G. L. (1970) Lipids 5, 239. 5. KOLATTUKUDY, P. E., CROTEAU, R., AND BROWN, L. (1974) Plant Physiol. 54, 670. 6. KHAN, A. A., AND KOLATTUKUDY, P. E. (1974) Biochem. Biophys. Res. Commun. 61, 1379. 7. FEHLER, S. W. G., AND LIGHT, R. J. (1970) Biochemistry 9, 418. 8. HAN, J., CHAN, H. W. S., AND CALVIN, M. J. (1969) J. Amer. Chem. Sot. 91, 5156. 9. CONRAD, C., AND JACKSON, L. L. (1971) J. Insect Pysiol. 17, 1907. 10. ROBBINS, W. E., KAPLANIS, J. N., LOUOUDES, S. J., AND MONROE, R. E. (1960) Ann. Entomol. sot. Amer. 53, 128. 11. DIEHL, P. A. (1973) Nature (London) 243, 468. 12. BLOMQUIST, G. J., AND JACKSON, L. L. (1973) J. Insect. Physiol. 19, 1639. 13. BLOMQUIST, G. J., AND JACKSON, L. L. (1973) Biochem. Biophys. Res. Commun. 53, 703. 14. BLOMQUIST, G. J., MCCAIN, D. C., AND JACKSON, L. L. (1975) Lipids 10, 303. 15. BLOMQUIST, G. J., MAJOR, M. A., AND LOK, J. B. (1975) Biochem. Biophys. Res. Commun. 64, 43. 16. NELSON, D. R., AND SUKKESTAD, D. R. (1970) Biochemistry 9, 4601. 17. SOLIDAY, C. L., BLOMQUIST, G. J., AND JACKSON, L. L. (1974) J. Lipid Res. 15, 399. 18. JACKSON, L. L., ARMOLD, M. T;, AND REGNIER, F. E. (1974) Insect Biochem. 4, 369. 19. JACKSON, L. L. (1970) Lipids 5, 38. 20. TARTIVITA, K., AND JACKSON, L. L. (1970) Lipids 5, 35. 21. NELSON, D. R., AND SUKKESTAD, D. R. (1974) J. Lipid Res. 16, 12. 22. O’CONNOR, J. G., BURROW, F. H., AND NORRIS, M. S. (1962) Anal. Chem. 34, 82. 23. BUCKNER, J. S., AND KOLATTUKLJDY, P. E. (1975) Biochemistry 14, 1768. 24. BUCKNER, J. S., AND KOLATTUKUDY, P. E. (1975) Biochemistry 14, 1774. 25. NELSON, D. R. (1969) Nature (London) 221, 854. 26. DIEHL, P. A. (1975) J. Insect Physiol. 21, 1237. 27. KRAMER, S., AND WIGGLESWORTH, V. B. (1950) @art. J. Microsc. Sci. 91, 63.