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Synthesis and metabolic conversion of fatty acids by the larval boll weevil
Comp. Biochem. Physiol., 1965, Vol. 16, pp. 289 to 302. Pergamon Press Ltd. Printed in Great Britain
S Y N T H E S I S A N D M E T A B O L I C C O N V E R S I O N OF F A T T Y A C I D S BY T H E LARVAL B O L L WEEVIL* E D W A R D N. L A M B R E M O N T , J " C A R R O L L I. STEIN:I: and A N D R E A F. B E N N E T T ' j Entomology Research Division, Agricultural Research Service, U.S.D.A., and Department of Entomology, Louisiana State University, Baton Rouge, La., U.S.A. (Received 29 M a r c h 1965)
A b s t r a c t - - 1 . The boll weevil (Anthonomus grandis Boheman) synthesized longchain fatty acids under sterile conditions from C1~-1 and CX4-2-acetate placed in its larval diet. Larvae, pupae and newly molted unfed adults had an identical labeling pattern. Oleic acid possessed 60 per cent of the incorporated radioactivity. 2. The weevil also desaturated dietary palmitic and stearic acids to corresponding mono-unsaturated fatty acids, palmitoleic and oleic acid. A significant portion of dietary palmitic acid underwent chain elongation to stearic acid, which subsequently was desaturated. Dietary oleic acid was not hydrogenated to any appreciable extent, because oleic acid was incorporated unchanged in the insects' tissues. 3. The boll weevil was unable to form linoleic acid from acetate and, furthermore, could not convert closely related long-chain fatty acids into linoleic acid. INTRODUCTION RESEARCH in this laboratory has shown that the newly emerged adult boll weevil contained fatty acids that were in general representative of those the larva had consumed and carried through metamorphosis ( L a m b r e m o n t & Blum, 1963). In addition, fed adults contained fatty acids that approximately matched those in their food ( L a m b r e m o n t et al., 1964). However, some fatty acids not detected in the diet were easily detected in both the larval and adult weevil. I t was then shown ( L a m b r e m o n t , 1965) that the adult under both aseptic and nonaseptic conditions could synthesize some long-chain fatty acids, and therefore had the ability to modify its body fat composition to some extent. T h e principal acids synthesized * In cooperation and located with the Department of Entomology and the Nuclear Science Center, Louisiana State University, and the Louisiana Agricultural Experiment Station. A portion of this work was supported by funds granted to the Nuclear Science Center jointly by the National Science Foundation (GE 3043) and the Atomic Energy Commission (AT-40-1-3205), and to the Department of Entomology from the National Institutes Health (AI 05881-TMP). t Entomology Research Division, Agric. Res. Serv., U.S. Department of Agriculture, Department of Entomology, Louisiana State University, Baton Rouge, La., U.S.A. ++Former Research participant, N.S.F. College Teachers Isotope Institute, and late Professor of Biology, Cambellville College, Cambellville, Ky. Deceased 29 March 1965. 289
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EDWARD N . LAMBREMONT, CARROLL I. STEIN AND ANDREA F. BENNETT
from C14-1abeled acetate possessed either sixteen or eighteen carbon atoms, and were either saturated or mono-unsaturated. A small amount of the C18 trienoic acid appeared to be formed, but no synthesis of the C18 dienoic acid (linoleic) took place. Two minor labeled components in the C20 range were also found. Although we had acquired a large amount of information, several questions still needed answering before we could properly interpret the effect of diet on body fatty acid content. Does the boll weevil larva synthesize fatty acids from acetate as does the adult, and to what extent can the larva modify the dietary fatty acids by such metabolic alterations as saturation or desaturation or changes in carbon chain length ? We also wished to know whether the larva, which cannot produce linoleic acid from acetate, could synthesize this important metabolite from closely related fatty acids. This paper presents the answers to some of these questions. MATERIALS AND METHODS Sterile rearing on diets containing labeled materials" Asepsis was practiced throughout all procedures in which microbial contamination could take place. All instruments, gloves and towels and the like were sterilized. Filling of diet vials and transfer of boll weevil eggs into the diet were TABLE
1--STANDARD LARVAL DIETS WITH C14-ISOTOPICALLY LABELED MATERIALS FOR STUDYING CONVERSION OF THE DIETARY FATTY ACIDS
Diet No.
Labeled compound
Amount (/xc)
Added to diet in
1 2 3 4 5
Acetate-l-C 14 Acetate-2-C 14 Tripalmitin-l-C ~4 Stearic-l-C 14 acid Oleic-l-C 14 acid
20 20 5 20 30
Aqueous solution Aqueous solution Ethyl ether solution 95% hot ethanol solution 95% hot ethanol solution
performed in a sterile glove box. Earlier work in our laboratory had shown that boll weevils reared under these conditions remained free of microorganisms as evidenced by several standard checks for bacteria, molds and yeasts (Lambremont, 1965). Boll weevil larval diet was prepared by adding 10 g of an acetone powder of cotton buds and 2 g of soybean protein to the standard diet constituents described by Earle et al. (1959). The isotopically labeled materials, amounts, chemical forms and method of adding the material to each of the five diets used are given in Table 1. Approximately 0.5 ml of diet was pipetted from disposable sterile plastic syringes into heated glass vials in the sterile glove box and the vials were wrapped in aluminum foil. All vials were autoclaved for 20-30 min at 15 lb pressure and 121°C, cooled in a refrigerator and used within 24 hr of preparation. Boll weevil eggs, freshly disected from cotton buds, were surface sterilized in 18% aqueous copper sulfate and a 0.025% mercuric chloride solution in 25O/o sterile ethanol. The eggs were rinsed once in 25% sterile ethanol and three times in sterile water and then implanted singly in the diet vials. The implanting needle
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and each vial were flamed after each transfer and each vial was sealed with aluminum foil. The vials were maintained at 21°C in a closed, aerated canister equipped with a fluorescent lamp programmed to give a 13 hr photophase. The vials were checked daily for development of weevils and for signs of microbial contamination. From a total of 780 vials inoculated with boll weevil eggs, 414 (53 per cent) adults were obtained from vials with no visible evidence of contamination. In about 5 per cent of the vials a larva had begun to develop, but growth had ceased. Contamination appeared in only eight vials (I per cent) in the form of a brownishgreen organism that was tenatively identified as a species of Pseudomonas. These vials were disposed of immediately. In each of the contaminated vials, the point of disease origin could easily be traced to the location where egg was deposited and the chorion broken by the hatching larva. Preliminary research in our laboratory had shown that surface contaminants on boll weevil eggs were almost entirely eliminated by sterilization, and although an occasional egg could be found to possess contaminants, these were easily detected. The balance of the vials (41 per cent) appeared to remain sterile, but no growth of weevils or other living organisms were observed.
Separation and identification o/fatty acids The newly emerged adults were taken from the vials and frozen in groups according to the particular diet on which they were reared. The total lipids from groups of thirty adults from each type of diet were extracted, and fatty acids methylated as previously described (Lambremont & Blum, 1963). We also extracted lipids from samples of fresh diet, as well as from diet in which an insect had fed to determine whether decomposition of the labeled precursors took place or whether metabolic changes had resulted from the insect's feeding and digestive activities. In determining the labeling pattern in the immature stages, last-instar larvae were removed from their vials and starved in sterile gauze containers for 24 hr before extraction of lipids. Those that pupated during this time were extracted separately for estimating the labeling pattern in the pupal stage. The methyl esters of fatty acids obtained from the weevils were analyzed carrier free by gas-liquid chromatography (GLC). However, since very small quantities of total fatty acids were obtained from the diet and frass their methyl esters were introduced into the gas-liquid chromatograph along with 10 tA of nonlabeled carrier methyl esters in n-hexane. The composition of the carrier approximated that of the major fatty adds known to be present in the diet (Lambremont & Blum, 1963).
Conditions used for gas-liquid chromatography and radioassay Radioactive methyl esters were separated on a 18 ft stainless steel column conraining acid-washed Chromosorb-W* initially coated with 25% diethyleneglycol adipate and 2% phosphoric acid. However, since the column was used repeatedly * Mention of a trade name does not necessarily imply its indorsement by the U.S. Department of Agriculture.
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the amount of liquid phase was some indeterminate quantity less than that at the start. Helium flow measured 75 ml/min at the out]et with a pressure drop across the column from 37-5 lb/in2g to atmospheric pressure. The injection port was held at 245°C and the column at 220°C. The esters were detected by a conventional, nondestructive thermal conductivity detector at 285°C and 150 mA. The separated esters were transferred to anthracene cartridges through a heated line which had a dead space of less than 0-5 ml, thus assuring collection of all components matching a detector response with minimum delay. Readings of C14radioactivity were made in a liquid scintillation spectrometer directly on the anthracene cartridges at - 20°C.
Radiochemical purity of C14-labeled precursors Radiochemical purity of the tripalmitin in diet 3 was 98 per cent as stated by the manufacturer. We assumed that sodium acetate samples in diets 1 and 2 were not likely to contain long-chain labeled fatty acids as contaminants since we obtained them in crystalline form and used them as aqueous solutions. T h e manufacturer's assay indicated a radiochemical purity of at least 99 per cent, as demonstrated by one-dimensional paper chromatography. We first purified the C14-stearic acid used in diet 4 by column chromatography on Florisil using the solvent schedule of Carroll (1961). Its radiochemical purity was 95 per cent, as determined by G L C of its methyl ester. The remaining 5 per cent of the C 14 radioactivity corresponded to short-chain esters below palmitate (0.7 per cent), palmitate (0"6 per cent), heptadecanoate (0"4 per cent), oleate (2.2 per cent) and linoleate (0.9 per cent). The detector mass record showed only a trace of linoleate, an amount too small for accurate quantitative analysis. The oleic acid in diet 5 was 93 per cent pure, with the remaining radioactivity distributed to two unidentified short-chain components (0.2 per cent)and to two components whose peaks showedthem to be palmitate and palmitoleate (2"7 per cent). Although we observed no other peaks, 1.0 per cent of the C 14 had a retention time corresponding to heptadecanoate, and 2 per cent eluted within 5 min after oleate and an additional 1-6 per cent within 15 min after the oleate peak. These latter observations indicated that some tailing of C ~4 from the large oleate peak was taking place beyond the limits of detector sensitivity. Dispersion of radioactivity beyond the limits of the observable mass trace would result in misleading identification of the newly synthesized fatty acids. Meinertz & Dole (1962) showed that rechromatography of collected peaks still displayed a similar dispersion pattern both before and after emergence of a single mass peak, thus indicating that even a pure substance emerges over a wider time interval than shown on the mass detector record. For these reasons extreme care must be taken in correlating low intensity radioactivity with unknowns separated by gas-liquid chromatography. In general, losses of about 3 4 per cent are spread over the entire analysis. We feel that positive identifications of synthesized C '4 fatty acids can be made only when a significant increase in C '4 count matches the detector response.
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As a check to determine whether C ~4 radioactivity was being held back by the column which could be "swept" off by a later ester band, we injected 5/~1 of unlabeled esters (fourteen component mixture) immediately after having separated a labeled sample. The unlabeled esters were collected individually in anthracene cartridges and none possessed radioactivity more than a few disintergrations per minute (d/min) above background. This result indicated that little or no significant C 14 contamination of unlabeled esters was taking place in the gas chromatographic system. In our experience, from 5 to 10 d/min of C14/min of effluent will elute from the GLC column, especially from a column that has been used for separating many radioactive samples. RESULTS
Synthesis of fatty acids by the boll weevil from acetate placed in the larval diet Table 2 summarizes the distribution of C 14 radioactivity in the principal fatty acids from newly emerged unfed adults reared from standard larval diet containing C14-acetate (diets 1 and 2, Table 1). Close agreement existed in the labeling pattern of the acids synthesized from either carboxyl- or methyl-labeled acetate by TABLE
2--DISTRIBUTION
O F R A D I O A C T I V I T Y I N F A T T Y ACIDS S Y N T H E S I Z E D B Y T H E LARVAL
BOLL WEEVIL FROM INGESTED C14-1
A N D C 1 4 - 2 - A C E T A T E A N D CARRIED OVER T O T H E N E W L Y
EMERGED~ U N F E D A D U L T
Fatty acid Short-chain below palmitic Palmitic Palmitoleic Heptadecanoic Stearic Oleic Linoleic Linolenic Baseline cartridges past linolenic
Cx4 Radioactivity (%) C14-1-acetate C14-2-acetate 1"7 8"8 11-1 0-8 7"6 60'0 0"9 3"1 6"1
1"7 11"6 10"3 0-8 11"8 58-0 0"6 3.3 1'9
Only major components and groupings are given. the larva, which matched earlier results with adults that we injected with two radioactive forms of acetate (Lambremont, 1965). Oleic acid in each instance contained about 60 per cent of the label and was the predominant acid synthesized, whereas stearic, palmitic and palmitoleic acids were of a secondary order and contained approximately equal C ~4 radioactivity. Linolenic acid possessed a low level of radioactivity, however, the C18 diene, linoleic acid, possessed even less, which was indicative of a lack of any significant C 14 being incorporated. The G L C effluent between oleic and linoleic acid contained as much radioactivity and often more than that found in the linoleic cartridge. This finding suggested that the readings
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EDWARD N . LAMBREMONT, CARROLL I. STEIN AND ANDREA F . BENNETT
from the linoleic fraction were due to carry-over from the highly radioactive oleic component, and not from the inherent radioactivity of linoleic acid. To resolve this question, we repeatedly collected the methyl linoleate peak from a sample of fatty acids synthesized from C14-acetate by aseptic adults. The methyl linoleate was trapped in cartridges of glass wool wetted with toluene. Rechromatography by GLC along with the standard mixture of unlabeled methyl esters showed that the only.significant C 1~ radioactivity/min of G L C effluent matched the methyl oleate peak and not the methyl linoleate peak. This observation indicated that the small amount of C 14 generally associated with linoleate was, in fact, due to carry-over from Cl~-methyl oleate.
Assimilation of dietary fatty acids and their metabolic conversion to other fatty acids Table 3 summarizes the'mass analysis of all methyl esters. In this Table we combined both labeled and unlabeled esters that were obtained from newly emerged unfed adults reared as larvae on the standard autoclaved diet. The results of this analysis are representative of the gross fatty acids contained in all TABLE 3--DETECTOR MASS COMPOSITION ( % ) OF THE PRINCIPAL FATTY ACID METHYL ESTERS OF NEWLY EMERGED ADULT WEEVILS REARED ON AUTOCLAVED LARVAL DIET
Fatty acid Short-chain below palmitic Palmitic Palmitoleic Heptadecanoic Stearic Oleic Linoleic Linolenic
Detector mass composition (%) 39'7 9' 5 7' 5
1"9 4' 8 28'0 6"3 2'6
weevils used in the present study. With regard to total fatty acids, the weevils possessed a large amount of the shorter chain components between C 6 and C15. This finding was not unusual, since we reported earlier (Lambremont & Blum, 1963) that a disproportionately large amount of these acids was in the diet used in this laboratory, and in all life stages of boll weevils reared from this diet. Since the short-chain acids from all three diets with C 14 fatty acids possessed insignificant C 1~ radioactivity (1.9 per cent or less), we may disregard these for the present. Also background cartridges obtained for each analysis before injection of the methyl esters showed that traces of C 14 in the G L C effluent could easily have accounted for nearly all the C 14 in the short-chain components. Figures 1, 2 and 3 summarize the G L C analysis and radioassay of C 14 in the fatty acids of diet, weevils and frass from the C14-tripalmitin, C14-stearic and C14-oleic acid diets respectively (diets 3, 4 and 5 of Table 1). The frass samples consisted of uneaten diet, feces, exhuviae, excreta and all associated debris representing an accumulation of all components left by the larva throughout its developmental period.
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The important points to observe in Figs. 1, 2 and 3 involve the relative interconversions of C ~4 radioactivity in the various acids from the weevils reared on different labeled diets. Figure 1 shows that weevils reared from the diet containing labeled tripalmitin were capable of absorbing and storing a significant portion of this component (34.6 per cent) as unchanged palmitic acid. Further, they could desaturate the dietary palmitic to palmitoleic acid (17.2 per cent) and in addition
enl
80 m TRIPALMITIN 6 0 - -
- 1 - C 14 DIET
DIET
I~
WEEVIL
H
FRASS
H
I--
m I,--
o
40 - -
i-
20
16
16:1
17
18
18:1
18;2
18:3
FATTY ACIDS
FIG. 1. Distribution of C 1~ in the principal fatty acids from diet 3, and weevils and frass from this diet which contained tripalmitin-l-C 14. T h e fatty acid numbers at the lower ordinate represent: 16-palmitie, 16 : 1-palrnitoleic, 17-heptadecanoic, 18-stearic, 18 : 1-oleie, 18 : 2-linoleic, 18 : 3-1inolenic. Baseline cartridges for the weevil body fat collected at points 1, 2, 3 and 4 (black circles) represented 1"9, 1"3, 1 "2 and 7"8 per cent of the total radioactivity, respectively.
add two carbon atoms to the palmitic chain to form stearic acid (12.2 per cent), which in turn could be desaturated to oleic acid (18.3 per cent). We found no significant radioactivity in linoleic acid and very slight C 14 in linolenic. The 0.7 per cent in the linoleic could be accounted for as trailing over from the oleic peak inasmuch as the baseline cartridge preceding linoleic contained 1-3 per cent of the collected C 14.
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EDWARDN . LAMBREMONT, CARROLL ]. STEIN AND ANDREA F. BENNETT
Figure 2 further shows that when the weevils fed on labeled stearic acid, they absorbed a significant amount of this component unchanged (26-8 per cent), and similarly converted large amounts by desaturation to oleic acid (58.1 per cent). This one-directional accumulation of oleic acid is seen even more clearly in Fig. 3, which shows that most of the C 14 in weevils fed labeled oleic acid was absorbed and assimilated as unchanged oleic acid, which possessed 85-3 per cent of the collected C1L Further, essentially little of the dietarv oleic acid was saturated to form stearic. Slightly higher C H readings were obtained from the linoleic fractions with dietary stearic and oleic, but these represented carrv-over.
80
STEARIC - I - C i4
ACiD
DiET
DiET
60 >
O m 40
WEEVfL
M
FRASS
[~
ix
20
16
16:1
17
18
18:1
18:2
18:3
FATTY ACIDS
FIG. 2. Distribution of C 14in the principal fatty acids from diet 4, and weevils and frass from this diet which contained C14-1-stearic acid. See Fig. 1 for identities of fatty acids.
Labeling pattern of larvae and pupae from radioactive diets Most of the analyses in the present report were of the newly emerged unfed adults that had been feeding as larvae on labeled diets. T w o additional experiments were performed as a further check of the ability of the larva itself to synthesize fatty acids, and to carry these through the pupal stage. Larvae and pupae from Cl~-l-acetate diet possessed an identical labeling pattern to that of the newly
SYNTHESIS AND METABOLIC CONVERSION OF FATTY ACIDS BY THE LARVAL BOLL WEEVIL
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emerged unfed adults from the same diet (as in Table 2). T h e same situation held true for larvae and pupae reared in a diet containing C14-1-palmitic acid, in which palmitic, palmitoleic, stearic and oleic acids were the principal radioactive components, and were quite similar to the results of Fig. 1 with C14-1-tripalmitin. These observations demonstrated that the larva truly possessed the capacity to synthesize fatty acids from acetate and to convert dietary fatty acids to closely related ones. The acids, furthermore, were carried essentially unchanged through the pupal stage, and appeared in the newly molted adult.
80
OLEIC-I- cl4 ACID DIET 60
DIET
II
WEEVIL
H
FRASS
40
20
@ 16
16:1
17
18 FATTY
18: I
18:2
18:3
ACIDS
Fxc 3. Distribution of C x4in the principal fatty acids from diet 5, and weevils and frass from this diet which contained C14-1-oleic acid. Baseline cartridges taken for the weevil body fat at points 5, 6 and 7 (black circles) represented 1-9, 0"4 and 1"8 per cent of the total radioactivity, respectively. See Fig. 1 for identities of the fatty acids.
Radioactivity @fatty acids in the diet and frass Analysis of radioactivity in the diets and frass is also included in Figs. 1, 2 and 3. The only principal radioactive components appeared to be those that were placed there intentionally during diet preparation. The small amounts of the other fatty acids were equivalent to the amounts of trace contaminants discovered in our checks of the radiochemical purity of the starting compounds. Also, we found no
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E D W A R D N . L A M B R E M O N T , CARROLL I . S T E I N AND ANDREA F . BENNETT
radioactive fatty acids in the diet or frass when C14-acetate was the starting labeled compound. Those observations are important from two standpoints. First, no decomposition or alteration of the diet resulted from the preparative procedures or of feeding activity by the insect itself. Second, the metabolic alterations did not take place in the insects' alimentary tracts because we could detect no differences between the labeling pattern of the frass fatty acids and that of the fresh diet except that the frass contained much less total radioactivity. This finding indicated that the conversion occurred in the insect itself after digestion and absorption of fatty acid through the midgut. Presumably, the sites of action for these conversions are the cells of the fat body. This point will be considered in a later paper.
Catalytic hydrogenation and bromination of the unsaturated acids of body fat As a confirmatory check on the unsaturated nature of some of the weevil's fatty acids, we hydrogenated a nonradioactive sample (Farquhar et al., 1959). T h e analysis of this sample before and after hydrogenation is given in Table 4. All unsaturated components disappeared after hydrogenation, and a concomitant TABLE
4 - - - R E L A T I V E COMPOSITION OF BODY FAT OF BOLL WEEVIL BEFORE AND CATALYTIC HYDROGENATION~ AS DETERMINED BY GLC OF THE METHYL ESTERS
AFTER
% Fatty acid Myristic Myristoleic Pentadecanoic Palmitic Palmitoleic Palmitolenic (?) Heptadecanoic Stearic Oleic Linoleic Linolenic Nonadecanoic (?) Arachidic
Carbon Double chain length bonds 14 14 15 16 16 16 17 18 18 18 18 19 20
0 1 0 0 1 2 0 0 1 2 3 0 0
Before hydrogenation
After hydrogenation
1"3 0'4 0-3 27' 5 6"0 0'7 1"1 4-2 27"3 19"0 12"5 NS* NS
1"5 R 0"8 34"2 R R 1' 5 61 "4 R R R 0"4 0.2
* Not seen on unhydrogenated sample ; probably obscured by polyunsaturated C~, esters. t Removed by hydrogenation. increase in the saturated analogs of equal chain length resulted. We detected two minor components, tentatively identified as palmitolenic acid (C16:2) and nonadecanoic acid (C19). These were not observed in earlier work ( L a m b r e m o n t & Blum, 1963) and were probably obscured by larger components. Both must be regarded as minor constituents and accounted for only about 1 per cent of the methyl esters.
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Bromination by the method of James (1960) also resulted in disappearance of all components identified by GLC as containing one or more double bonds. DISCUSSION The results with labeled acetate are significantfrom severalstandpoints. First, with this informationwe now know that both the larval and adult boll weevilhave the capacity to synthesize fatty acids from acetate. Also, the chain lengths and types of fatty acids synthesizedby the two life stages correspondquite closely, and both stages appeared to be unable to form linoleic acid. Furthermore, the almost identical labeling pattern obtained with methyl- and carboxyl-labeledacetate supported the generally accepted view that fatty acid synthesis proceeds by the successive condensationof a two-carbon unit. Second, the 3 weeks from the egg stage to adult emergenceof the weevils, as planned in these experiments,provided a vastly longer time for synthesisthan the 2 hr period used in previous experiments to determine fatty acid synthesis in injected adults (Lambremont, 1965). We previouslyspeculated that linoleic acid might be synthesizedso slowlythat no significant CI~ could be incorporatedwithin 2 hr. Our present data eliminated this possibility. That no significant C14 was present in the short-chain acids from the weevils indicated that these componentswere not produced and stored as intermediatesin the synthesis of long-chain acids although they were present in large amounts in the diet as well as in body fat (Table 3). The nonradioactiveshort-chainacidsin the insects, therefore, represented direct incorporation from the diet. One must conclude that substantial amounts of palmitic, palmitoleic, stearic and oleic acids are synthesized de novo from the acetate precursor or through the condensation of acetate with short-chain intermediates,and once condensationof two-carbon units begins, it continuesto completionto produce C1s and Cls fatty acids. Coniglio& Cate (1958) pointed out that in rats, palmitic acid was the principal intermediate of acetate condensationand only a few short-chain acids were synthesized. Chain elongation of short-chain acids occurred to a greater extent in fasted rats. Once palmitic acid was formed it was then convertedto other long-chain acids. Finally, although both the larvae and adults synthesizedthe same kinds of fatty acids from acetate, the relativeamounts differedconsiderably. Oleicacid contained a very large share (60 per cent) of the C14incorporatedby the larva, but only about 25 per cent of that by the adult. Synthesis of palmitic acid by the larva, on the other hand, was only about a third that observed in the adult, and stearic only about half. We do not know all of the reasons for these differences,although the results with labeled fatty acids in diets 3, 4 and 5 offered a possible explanation of the mechanismfor oleic acid build-up in the larva, and permitted us to come to several additional conclusionsas follows. The larva appeared to absorb a large portion of the dietaryfatty acids unchanged but it could a]so readily modify the remainder to a certain degree, depending on the original acid. If the acid had sixteen carbon atoms, it could be dehydrogenated
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E D W A R DN. LAMBREMONT, CARROLL [. STEIN AND ANDREA F. BENNETT
to a mono-unsaturated acid or its chain lengthened by one acetate unit to eighteen carbons and dehydrogenated to oleic acid. Thus, in a manner of speaking, all pathways led toward oleic acid, which appeared to be the endpoint of fatty acid incorporation in the larva. These observations suggested why we found higher amounts of radioactivity (up to 60 per cent) in the oleic acid fraction of fat synthesized by larvae on the C 14 acetate diets 1 and 2. We must also conclude that the boll weevil is unable to form linoleic acid either from acetate or from closely related long-chain fatty acids. The suggestion put forth by Vonk & Zandee (1963) that invertebrates have independent pathways for building up saturated and unsaturated fatty acids was not upheld by our present work. These authors noted several instances of higher labeling of the unsaturated than the saturated analogs and concluded that two routes of biosynthesis existed. Sedee (1961), who worked with larvae of the blowfly Calliphora erythrocephela (Meigen), stated that if, as in higher animals, the saturated and unsaturated fatty acids were interconvertible, they should have attained the same isotope content. Since they did not, he too concluded that separate pathways of biosynthesis were in operation. Yet, the present results argue for the existence of a single pathway, with desaturation to mono-enoic acids being the principal step. Because the reverse saturation reaction did not occur, once oleic acid was formed it could not be changed back into stearic; and this situation is clearly shown by the data in Fig. 3. Mead & Howton (1960), in discussing the biosynthetic pathways of unsaturated fatty acids pointed out that hydrogenation of unsaturated acids to the corresponding saturated analogs did not appear to occur in the animal body to any appreciable extent. This situation held true for the boll weevil. The quantitative relationships of precursor and product, especially those shown in Fig. 2, also strongly suggested direct desaturation. The decrease in labeled stearic acid in the weevil below that in its diet was almost exactly offset by the increase in its C 14 oleic content. A similar relationship held for the three radioactive acids formed from precursor C 14palmitate as shown, in Fig. 1. It is clear from the data in Table 2 and Fig. 2 that considerably more oleic than stearic was deposited in the body fat of the boll weevil feeding on a diet of C 1~ acetate or stearic acid. Recent studies showing a direct conversion of stearate to oleate were reported by Bade (1964) for aseptic roaches, Eurycotisfloridana (Walker). Decarboxylation of the carbon atom in the first position of both precursor and product removed all radioactivity from the C14-stearic acid and the oleic acid formed from it. Our present results with boll weevil larvae suggested that the direct desaturation pathway is in operation on all long-chain fatty acids obtained from the diet and on fatty acids synthesized from acetate. Furthermore, no conversion of oleate to linoleate took place. These features were characteristic of the animal-type biosynthesis mechanism described by Erwin et al. (1964) for some protozoa and for the vertebrates, and supported an earlier observation (Lambremont, 1965) that the weevils, like other metazoans, are not capable of forming linoleic acid. The linoleate component (6.3 per cent) given in Table 3 for the boll weevil fatty acids therefore apparently arose solely from dietary linoleate.
SYNTHESIS AND METABOLIC CONVERSION OF FATTY ACIDS BY THE LARVAL BOLL WEEVIL 301
Unpublished results from research we conducted in our laboratory in cooperation with Dr. Erma Vanderzant further strengthened our belief that the weevil's linoleic acid was derived from the diet. G L C analyses of adults reared on a variety of chemically defined larval media showed that linoleic acid in the body fat only when there was a direct dietary supply, and that no conversion of oleate or metabolic rearrangement of linolenate appeared to be taking place. T h a t we found a small amount of label in the linolenate fraction was not sufficient evidence to substantiate weevil biosynthesis of this polyunsaturated acid. In our experiments with CX4-acetate-injected adult weevils, consistent labeling of linolenate might have been due to the addition of two-carbon C 14 unit to nonradioactive heptadecanoic acid a small amount of which is known to be present in this insect. This process would have yielded C xa nonadecanoic acid, which is also a trace component of the boll weevil (Table 4). Both linolenic acid and nonadecanoic acid have very similar retention times by G L C . It was evident from the small amounts of radioactivity in the linolenic acid that synthesis of linolenate (if it occurs at all) could not have accounted for the amounts (2.6 per cent in Table 3) we found in the weevil's body fat. Most, if not all, of the linolenic acid must, therefore, have come from the diet. T h e lack of synthesis of the Cls polyenoic fatty acids was significant in the light of recent work by Vanderzant & Richardson (1964) on the lipid requirements of the adult boll weevil. These workers reported that adults reared from a fat-deficient larval diet depended heavily upon the adult food for those lipid constituents needed for the synthesis of eggs. Addition of either linoleic or linolenic acids increased the n u m b e r of eggs laid and females lived longer. Oleic acid also partially replaced dietary fat, but was not as effective as the two polyunsaturated acids, and possibly spared traces of these latter acids already in the insect or its diet. T h e observations of these workers in conjunction with ours presented here, support the general view that the polyunsaturated C18 fatty acids are not formed by the tissues of insects. Acknowledgements--We thank L. D. Newsom, H. D. Richardson and Rosamond Killebrew of Louisiana State University for their cooperative support of this project. We also thank Dr. Erma S. Vanderzant of the Entomology Research Division, at College Station, Texas, for sending the adults reared on defined media, and Rhonda Frayer and Opal Colvin, also of this Division, for their technical assistance. REFERENCES BADE M. L. (1964) Biosynthesis of fatty acids in the roach Eurycotis floridana. J. Insect Physiol. 10, 333-341. CARROLLK. K. (1961) Separation of lipid classes by chromatography on Florisil. ft. Lipid Res. 2, 135-141. CONIGLIO J. G. ~ fATE D. L. (1958) The distribution and biosynthesis of palmitic and stearic acids in liver, intestine and carcass of intact normal and fasted rats. ft. Biol. Chem. 232, 361-368. EARLE N . W . , GAINES R. C. & ROUSSEL J. S. (1959) A larval diet for the boll weevil containing an acetone powder of cotton squares. J. Econ. Ent 52, 710-712. ERWIN J., HULANICKAB. & BLOCH K. (1964) Comparative aspects of unsaturated fatty acid synthesis. Comp. Biochem. Physiol. 12, 191-207. 2r
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FARQUHAR J. W., iNSUL W., ROSEN P., STOFFEL W. • AHRENS E. H. (1959) T h e analysis of fatty acid mixtures by gas liquid chromatography. Nutr. Rev. (Suppl.) 17, 1-30. JAMES A. T. (1960) Qualitative and quantitative determination of the fatty acids by gasliquid chromatography. In Methods of Biochemical Analysis (Edited by GLICK D.), Vol. 8, pp. 1-59. LAMBREMONT E. N. (1965) Biosynthesis of fatty acids in aseptically reared insects. Comp. Biochem. Physiol. 14, 419-424. LAMBREMONT E. N. & BLUM M. S. (1963) Fatty-acids of the boll weevil. Ann. Ent. Soc. Amer. 56, 612-616. LAMBREMONT E. N., BLUM M. S. & SCHRADER R. M. (1964) Storage and fatty acid composition of triglycerides during adult diapause of the boll weevil. Ann. Ent. Soc. Amer. 57, 526-532. MEAD J. F. (1960) T h e metabolism of unsaturated fatty acids. In Lipide Metabolism (Edited by BLOCH K.), pp. 41-68. John Wiley, N.Y. MEAD J. F. & HOWTON D. R. (1960) Radioisotope Studies of Fatty Acid Metabolism, pp. 141. Pergamon Press, N.Y. MEINERTZ H. & DOLE V. P. (1962) Radioassay of low activity fractions encountered in gas-liquid chromatography of long-chain fatty acids. J . Lipid Res. 3, 140-144. SEDEE P. D. J. W. (1961) Intermediary metabolism in aseptically reared blowfly larvae, CaUiphora erythrocephala (Meig). II. Biosynthesis of fatty acids and amino acids. Arch. Int. Physiol. Biochem. 69, 295-309. VANDERZANT E. S. & RICHARDSON C. D. (1964) Nutrition of the adult boll weevil: lipid requirements. J . Insect Physiol. 10, 267-272. VONK H. J. & ZANDEE D. I. (1963) Intermediary metabolism of lipids and amino acids in invertebrates. Proc. X V I Int. Congr. Zool., p. 34.
S Y N T H E S I S A N D M E T A B O L I C C O N V E R S I O N OF F A T T Y A C I D S BY T H E LARVAL B O L L WEEVIL* E D W A R D N. L A M B R E M O N T , J " C A R R O L L I. STEIN:I: and A N D R E A F. B E N N E T T ' j Entomology Research Division, Agricultural Research Service, U.S.D.A., and Department of Entomology, Louisiana State University, Baton Rouge, La., U.S.A. (Received 29 M a r c h 1965)
A b s t r a c t - - 1 . The boll weevil (Anthonomus grandis Boheman) synthesized longchain fatty acids under sterile conditions from C1~-1 and CX4-2-acetate placed in its larval diet. Larvae, pupae and newly molted unfed adults had an identical labeling pattern. Oleic acid possessed 60 per cent of the incorporated radioactivity. 2. The weevil also desaturated dietary palmitic and stearic acids to corresponding mono-unsaturated fatty acids, palmitoleic and oleic acid. A significant portion of dietary palmitic acid underwent chain elongation to stearic acid, which subsequently was desaturated. Dietary oleic acid was not hydrogenated to any appreciable extent, because oleic acid was incorporated unchanged in the insects' tissues. 3. The boll weevil was unable to form linoleic acid from acetate and, furthermore, could not convert closely related long-chain fatty acids into linoleic acid. INTRODUCTION RESEARCH in this laboratory has shown that the newly emerged adult boll weevil contained fatty acids that were in general representative of those the larva had consumed and carried through metamorphosis ( L a m b r e m o n t & Blum, 1963). In addition, fed adults contained fatty acids that approximately matched those in their food ( L a m b r e m o n t et al., 1964). However, some fatty acids not detected in the diet were easily detected in both the larval and adult weevil. I t was then shown ( L a m b r e m o n t , 1965) that the adult under both aseptic and nonaseptic conditions could synthesize some long-chain fatty acids, and therefore had the ability to modify its body fat composition to some extent. T h e principal acids synthesized * In cooperation and located with the Department of Entomology and the Nuclear Science Center, Louisiana State University, and the Louisiana Agricultural Experiment Station. A portion of this work was supported by funds granted to the Nuclear Science Center jointly by the National Science Foundation (GE 3043) and the Atomic Energy Commission (AT-40-1-3205), and to the Department of Entomology from the National Institutes Health (AI 05881-TMP). t Entomology Research Division, Agric. Res. Serv., U.S. Department of Agriculture, Department of Entomology, Louisiana State University, Baton Rouge, La., U.S.A. ++Former Research participant, N.S.F. College Teachers Isotope Institute, and late Professor of Biology, Cambellville College, Cambellville, Ky. Deceased 29 March 1965. 289
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EDWARD N . LAMBREMONT, CARROLL I. STEIN AND ANDREA F. BENNETT
from C14-1abeled acetate possessed either sixteen or eighteen carbon atoms, and were either saturated or mono-unsaturated. A small amount of the C18 trienoic acid appeared to be formed, but no synthesis of the C18 dienoic acid (linoleic) took place. Two minor labeled components in the C20 range were also found. Although we had acquired a large amount of information, several questions still needed answering before we could properly interpret the effect of diet on body fatty acid content. Does the boll weevil larva synthesize fatty acids from acetate as does the adult, and to what extent can the larva modify the dietary fatty acids by such metabolic alterations as saturation or desaturation or changes in carbon chain length ? We also wished to know whether the larva, which cannot produce linoleic acid from acetate, could synthesize this important metabolite from closely related fatty acids. This paper presents the answers to some of these questions. MATERIALS AND METHODS Sterile rearing on diets containing labeled materials" Asepsis was practiced throughout all procedures in which microbial contamination could take place. All instruments, gloves and towels and the like were sterilized. Filling of diet vials and transfer of boll weevil eggs into the diet were TABLE
1--STANDARD LARVAL DIETS WITH C14-ISOTOPICALLY LABELED MATERIALS FOR STUDYING CONVERSION OF THE DIETARY FATTY ACIDS
Diet No.
Labeled compound
Amount (/xc)
Added to diet in
1 2 3 4 5
Acetate-l-C 14 Acetate-2-C 14 Tripalmitin-l-C ~4 Stearic-l-C 14 acid Oleic-l-C 14 acid
20 20 5 20 30
Aqueous solution Aqueous solution Ethyl ether solution 95% hot ethanol solution 95% hot ethanol solution
performed in a sterile glove box. Earlier work in our laboratory had shown that boll weevils reared under these conditions remained free of microorganisms as evidenced by several standard checks for bacteria, molds and yeasts (Lambremont, 1965). Boll weevil larval diet was prepared by adding 10 g of an acetone powder of cotton buds and 2 g of soybean protein to the standard diet constituents described by Earle et al. (1959). The isotopically labeled materials, amounts, chemical forms and method of adding the material to each of the five diets used are given in Table 1. Approximately 0.5 ml of diet was pipetted from disposable sterile plastic syringes into heated glass vials in the sterile glove box and the vials were wrapped in aluminum foil. All vials were autoclaved for 20-30 min at 15 lb pressure and 121°C, cooled in a refrigerator and used within 24 hr of preparation. Boll weevil eggs, freshly disected from cotton buds, were surface sterilized in 18% aqueous copper sulfate and a 0.025% mercuric chloride solution in 25O/o sterile ethanol. The eggs were rinsed once in 25% sterile ethanol and three times in sterile water and then implanted singly in the diet vials. The implanting needle
SYNTHESIS AND METABOLIC CONVERSION OF FATTY ACIDS BY THE LARVAL BOLL WEEVIL
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and each vial were flamed after each transfer and each vial was sealed with aluminum foil. The vials were maintained at 21°C in a closed, aerated canister equipped with a fluorescent lamp programmed to give a 13 hr photophase. The vials were checked daily for development of weevils and for signs of microbial contamination. From a total of 780 vials inoculated with boll weevil eggs, 414 (53 per cent) adults were obtained from vials with no visible evidence of contamination. In about 5 per cent of the vials a larva had begun to develop, but growth had ceased. Contamination appeared in only eight vials (I per cent) in the form of a brownishgreen organism that was tenatively identified as a species of Pseudomonas. These vials were disposed of immediately. In each of the contaminated vials, the point of disease origin could easily be traced to the location where egg was deposited and the chorion broken by the hatching larva. Preliminary research in our laboratory had shown that surface contaminants on boll weevil eggs were almost entirely eliminated by sterilization, and although an occasional egg could be found to possess contaminants, these were easily detected. The balance of the vials (41 per cent) appeared to remain sterile, but no growth of weevils or other living organisms were observed.
Separation and identification o/fatty acids The newly emerged adults were taken from the vials and frozen in groups according to the particular diet on which they were reared. The total lipids from groups of thirty adults from each type of diet were extracted, and fatty acids methylated as previously described (Lambremont & Blum, 1963). We also extracted lipids from samples of fresh diet, as well as from diet in which an insect had fed to determine whether decomposition of the labeled precursors took place or whether metabolic changes had resulted from the insect's feeding and digestive activities. In determining the labeling pattern in the immature stages, last-instar larvae were removed from their vials and starved in sterile gauze containers for 24 hr before extraction of lipids. Those that pupated during this time were extracted separately for estimating the labeling pattern in the pupal stage. The methyl esters of fatty acids obtained from the weevils were analyzed carrier free by gas-liquid chromatography (GLC). However, since very small quantities of total fatty acids were obtained from the diet and frass their methyl esters were introduced into the gas-liquid chromatograph along with 10 tA of nonlabeled carrier methyl esters in n-hexane. The composition of the carrier approximated that of the major fatty adds known to be present in the diet (Lambremont & Blum, 1963).
Conditions used for gas-liquid chromatography and radioassay Radioactive methyl esters were separated on a 18 ft stainless steel column conraining acid-washed Chromosorb-W* initially coated with 25% diethyleneglycol adipate and 2% phosphoric acid. However, since the column was used repeatedly * Mention of a trade name does not necessarily imply its indorsement by the U.S. Department of Agriculture.
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EDWARD N. LAMBREMONT, CARROLL I. STEIN AND ANDREA F. BENNETT
the amount of liquid phase was some indeterminate quantity less than that at the start. Helium flow measured 75 ml/min at the out]et with a pressure drop across the column from 37-5 lb/in2g to atmospheric pressure. The injection port was held at 245°C and the column at 220°C. The esters were detected by a conventional, nondestructive thermal conductivity detector at 285°C and 150 mA. The separated esters were transferred to anthracene cartridges through a heated line which had a dead space of less than 0-5 ml, thus assuring collection of all components matching a detector response with minimum delay. Readings of C14radioactivity were made in a liquid scintillation spectrometer directly on the anthracene cartridges at - 20°C.
Radiochemical purity of C14-labeled precursors Radiochemical purity of the tripalmitin in diet 3 was 98 per cent as stated by the manufacturer. We assumed that sodium acetate samples in diets 1 and 2 were not likely to contain long-chain labeled fatty acids as contaminants since we obtained them in crystalline form and used them as aqueous solutions. T h e manufacturer's assay indicated a radiochemical purity of at least 99 per cent, as demonstrated by one-dimensional paper chromatography. We first purified the C14-stearic acid used in diet 4 by column chromatography on Florisil using the solvent schedule of Carroll (1961). Its radiochemical purity was 95 per cent, as determined by G L C of its methyl ester. The remaining 5 per cent of the C 14 radioactivity corresponded to short-chain esters below palmitate (0.7 per cent), palmitate (0"6 per cent), heptadecanoate (0"4 per cent), oleate (2.2 per cent) and linoleate (0.9 per cent). The detector mass record showed only a trace of linoleate, an amount too small for accurate quantitative analysis. The oleic acid in diet 5 was 93 per cent pure, with the remaining radioactivity distributed to two unidentified short-chain components (0.2 per cent)and to two components whose peaks showedthem to be palmitate and palmitoleate (2"7 per cent). Although we observed no other peaks, 1.0 per cent of the C 14 had a retention time corresponding to heptadecanoate, and 2 per cent eluted within 5 min after oleate and an additional 1-6 per cent within 15 min after the oleate peak. These latter observations indicated that some tailing of C ~4 from the large oleate peak was taking place beyond the limits of detector sensitivity. Dispersion of radioactivity beyond the limits of the observable mass trace would result in misleading identification of the newly synthesized fatty acids. Meinertz & Dole (1962) showed that rechromatography of collected peaks still displayed a similar dispersion pattern both before and after emergence of a single mass peak, thus indicating that even a pure substance emerges over a wider time interval than shown on the mass detector record. For these reasons extreme care must be taken in correlating low intensity radioactivity with unknowns separated by gas-liquid chromatography. In general, losses of about 3 4 per cent are spread over the entire analysis. We feel that positive identifications of synthesized C '4 fatty acids can be made only when a significant increase in C '4 count matches the detector response.
S Y N T H E S I S A N D M E T A B O L I C C O N V E R S I O N O F F A T T Y A C I D S B Y T H E LARVAL B O L L W E E V I L
293
As a check to determine whether C ~4 radioactivity was being held back by the column which could be "swept" off by a later ester band, we injected 5/~1 of unlabeled esters (fourteen component mixture) immediately after having separated a labeled sample. The unlabeled esters were collected individually in anthracene cartridges and none possessed radioactivity more than a few disintergrations per minute (d/min) above background. This result indicated that little or no significant C 14 contamination of unlabeled esters was taking place in the gas chromatographic system. In our experience, from 5 to 10 d/min of C14/min of effluent will elute from the GLC column, especially from a column that has been used for separating many radioactive samples. RESULTS
Synthesis of fatty acids by the boll weevil from acetate placed in the larval diet Table 2 summarizes the distribution of C 14 radioactivity in the principal fatty acids from newly emerged unfed adults reared from standard larval diet containing C14-acetate (diets 1 and 2, Table 1). Close agreement existed in the labeling pattern of the acids synthesized from either carboxyl- or methyl-labeled acetate by TABLE
2--DISTRIBUTION
O F R A D I O A C T I V I T Y I N F A T T Y ACIDS S Y N T H E S I Z E D B Y T H E LARVAL
BOLL WEEVIL FROM INGESTED C14-1
A N D C 1 4 - 2 - A C E T A T E A N D CARRIED OVER T O T H E N E W L Y
EMERGED~ U N F E D A D U L T
Fatty acid Short-chain below palmitic Palmitic Palmitoleic Heptadecanoic Stearic Oleic Linoleic Linolenic Baseline cartridges past linolenic
Cx4 Radioactivity (%) C14-1-acetate C14-2-acetate 1"7 8"8 11-1 0-8 7"6 60'0 0"9 3"1 6"1
1"7 11"6 10"3 0-8 11"8 58-0 0"6 3.3 1'9
Only major components and groupings are given. the larva, which matched earlier results with adults that we injected with two radioactive forms of acetate (Lambremont, 1965). Oleic acid in each instance contained about 60 per cent of the label and was the predominant acid synthesized, whereas stearic, palmitic and palmitoleic acids were of a secondary order and contained approximately equal C ~4 radioactivity. Linolenic acid possessed a low level of radioactivity, however, the C18 diene, linoleic acid, possessed even less, which was indicative of a lack of any significant C 14 being incorporated. The G L C effluent between oleic and linoleic acid contained as much radioactivity and often more than that found in the linoleic cartridge. This finding suggested that the readings
294
EDWARD N . LAMBREMONT, CARROLL I. STEIN AND ANDREA F . BENNETT
from the linoleic fraction were due to carry-over from the highly radioactive oleic component, and not from the inherent radioactivity of linoleic acid. To resolve this question, we repeatedly collected the methyl linoleate peak from a sample of fatty acids synthesized from C14-acetate by aseptic adults. The methyl linoleate was trapped in cartridges of glass wool wetted with toluene. Rechromatography by GLC along with the standard mixture of unlabeled methyl esters showed that the only.significant C 1~ radioactivity/min of G L C effluent matched the methyl oleate peak and not the methyl linoleate peak. This observation indicated that the small amount of C 14 generally associated with linoleate was, in fact, due to carry-over from Cl~-methyl oleate.
Assimilation of dietary fatty acids and their metabolic conversion to other fatty acids Table 3 summarizes the'mass analysis of all methyl esters. In this Table we combined both labeled and unlabeled esters that were obtained from newly emerged unfed adults reared as larvae on the standard autoclaved diet. The results of this analysis are representative of the gross fatty acids contained in all TABLE 3--DETECTOR MASS COMPOSITION ( % ) OF THE PRINCIPAL FATTY ACID METHYL ESTERS OF NEWLY EMERGED ADULT WEEVILS REARED ON AUTOCLAVED LARVAL DIET
Fatty acid Short-chain below palmitic Palmitic Palmitoleic Heptadecanoic Stearic Oleic Linoleic Linolenic
Detector mass composition (%) 39'7 9' 5 7' 5
1"9 4' 8 28'0 6"3 2'6
weevils used in the present study. With regard to total fatty acids, the weevils possessed a large amount of the shorter chain components between C 6 and C15. This finding was not unusual, since we reported earlier (Lambremont & Blum, 1963) that a disproportionately large amount of these acids was in the diet used in this laboratory, and in all life stages of boll weevils reared from this diet. Since the short-chain acids from all three diets with C 14 fatty acids possessed insignificant C 1~ radioactivity (1.9 per cent or less), we may disregard these for the present. Also background cartridges obtained for each analysis before injection of the methyl esters showed that traces of C 14 in the G L C effluent could easily have accounted for nearly all the C 14 in the short-chain components. Figures 1, 2 and 3 summarize the G L C analysis and radioassay of C 14 in the fatty acids of diet, weevils and frass from the C14-tripalmitin, C14-stearic and C14-oleic acid diets respectively (diets 3, 4 and 5 of Table 1). The frass samples consisted of uneaten diet, feces, exhuviae, excreta and all associated debris representing an accumulation of all components left by the larva throughout its developmental period.
SYNTHESIS AND M E T A B O L I C CONVERSION OF FATTY ACIDS BY THE LARVAL B O L L W E E V I L
295
The important points to observe in Figs. 1, 2 and 3 involve the relative interconversions of C ~4 radioactivity in the various acids from the weevils reared on different labeled diets. Figure 1 shows that weevils reared from the diet containing labeled tripalmitin were capable of absorbing and storing a significant portion of this component (34.6 per cent) as unchanged palmitic acid. Further, they could desaturate the dietary palmitic to palmitoleic acid (17.2 per cent) and in addition
enl
80 m TRIPALMITIN 6 0 - -
- 1 - C 14 DIET
DIET
I~
WEEVIL
H
FRASS
H
I--
m I,--
o
40 - -
i-
20
16
16:1
17
18
18:1
18;2
18:3
FATTY ACIDS
FIG. 1. Distribution of C 1~ in the principal fatty acids from diet 3, and weevils and frass from this diet which contained tripalmitin-l-C 14. T h e fatty acid numbers at the lower ordinate represent: 16-palmitie, 16 : 1-palrnitoleic, 17-heptadecanoic, 18-stearic, 18 : 1-oleie, 18 : 2-linoleic, 18 : 3-1inolenic. Baseline cartridges for the weevil body fat collected at points 1, 2, 3 and 4 (black circles) represented 1"9, 1"3, 1 "2 and 7"8 per cent of the total radioactivity, respectively.
add two carbon atoms to the palmitic chain to form stearic acid (12.2 per cent), which in turn could be desaturated to oleic acid (18.3 per cent). We found no significant radioactivity in linoleic acid and very slight C 14 in linolenic. The 0.7 per cent in the linoleic could be accounted for as trailing over from the oleic peak inasmuch as the baseline cartridge preceding linoleic contained 1-3 per cent of the collected C 14.
296
EDWARDN . LAMBREMONT, CARROLL ]. STEIN AND ANDREA F. BENNETT
Figure 2 further shows that when the weevils fed on labeled stearic acid, they absorbed a significant amount of this component unchanged (26-8 per cent), and similarly converted large amounts by desaturation to oleic acid (58.1 per cent). This one-directional accumulation of oleic acid is seen even more clearly in Fig. 3, which shows that most of the C 14 in weevils fed labeled oleic acid was absorbed and assimilated as unchanged oleic acid, which possessed 85-3 per cent of the collected C1L Further, essentially little of the dietarv oleic acid was saturated to form stearic. Slightly higher C H readings were obtained from the linoleic fractions with dietary stearic and oleic, but these represented carrv-over.
80
STEARIC - I - C i4
ACiD
DiET
DiET
60 >
O m 40
WEEVfL
M
FRASS
[~
ix
20
16
16:1
17
18
18:1
18:2
18:3
FATTY ACIDS
FIG. 2. Distribution of C 14in the principal fatty acids from diet 4, and weevils and frass from this diet which contained C14-1-stearic acid. See Fig. 1 for identities of fatty acids.
Labeling pattern of larvae and pupae from radioactive diets Most of the analyses in the present report were of the newly emerged unfed adults that had been feeding as larvae on labeled diets. T w o additional experiments were performed as a further check of the ability of the larva itself to synthesize fatty acids, and to carry these through the pupal stage. Larvae and pupae from Cl~-l-acetate diet possessed an identical labeling pattern to that of the newly
SYNTHESIS AND METABOLIC CONVERSION OF FATTY ACIDS BY THE LARVAL BOLL WEEVIL
297
emerged unfed adults from the same diet (as in Table 2). T h e same situation held true for larvae and pupae reared in a diet containing C14-1-palmitic acid, in which palmitic, palmitoleic, stearic and oleic acids were the principal radioactive components, and were quite similar to the results of Fig. 1 with C14-1-tripalmitin. These observations demonstrated that the larva truly possessed the capacity to synthesize fatty acids from acetate and to convert dietary fatty acids to closely related ones. The acids, furthermore, were carried essentially unchanged through the pupal stage, and appeared in the newly molted adult.
80
OLEIC-I- cl4 ACID DIET 60
DIET
II
WEEVIL
H
FRASS
40
20
@ 16
16:1
17
18 FATTY
18: I
18:2
18:3
ACIDS
Fxc 3. Distribution of C x4in the principal fatty acids from diet 5, and weevils and frass from this diet which contained C14-1-oleic acid. Baseline cartridges taken for the weevil body fat at points 5, 6 and 7 (black circles) represented 1-9, 0"4 and 1"8 per cent of the total radioactivity, respectively. See Fig. 1 for identities of the fatty acids.
Radioactivity @fatty acids in the diet and frass Analysis of radioactivity in the diets and frass is also included in Figs. 1, 2 and 3. The only principal radioactive components appeared to be those that were placed there intentionally during diet preparation. The small amounts of the other fatty acids were equivalent to the amounts of trace contaminants discovered in our checks of the radiochemical purity of the starting compounds. Also, we found no
298
E D W A R D N . L A M B R E M O N T , CARROLL I . S T E I N AND ANDREA F . BENNETT
radioactive fatty acids in the diet or frass when C14-acetate was the starting labeled compound. Those observations are important from two standpoints. First, no decomposition or alteration of the diet resulted from the preparative procedures or of feeding activity by the insect itself. Second, the metabolic alterations did not take place in the insects' alimentary tracts because we could detect no differences between the labeling pattern of the frass fatty acids and that of the fresh diet except that the frass contained much less total radioactivity. This finding indicated that the conversion occurred in the insect itself after digestion and absorption of fatty acid through the midgut. Presumably, the sites of action for these conversions are the cells of the fat body. This point will be considered in a later paper.
Catalytic hydrogenation and bromination of the unsaturated acids of body fat As a confirmatory check on the unsaturated nature of some of the weevil's fatty acids, we hydrogenated a nonradioactive sample (Farquhar et al., 1959). T h e analysis of this sample before and after hydrogenation is given in Table 4. All unsaturated components disappeared after hydrogenation, and a concomitant TABLE
4 - - - R E L A T I V E COMPOSITION OF BODY FAT OF BOLL WEEVIL BEFORE AND CATALYTIC HYDROGENATION~ AS DETERMINED BY GLC OF THE METHYL ESTERS
AFTER
% Fatty acid Myristic Myristoleic Pentadecanoic Palmitic Palmitoleic Palmitolenic (?) Heptadecanoic Stearic Oleic Linoleic Linolenic Nonadecanoic (?) Arachidic
Carbon Double chain length bonds 14 14 15 16 16 16 17 18 18 18 18 19 20
0 1 0 0 1 2 0 0 1 2 3 0 0
Before hydrogenation
After hydrogenation
1"3 0'4 0-3 27' 5 6"0 0'7 1"1 4-2 27"3 19"0 12"5 NS* NS
1"5 R 0"8 34"2 R R 1' 5 61 "4 R R R 0"4 0.2
* Not seen on unhydrogenated sample ; probably obscured by polyunsaturated C~, esters. t Removed by hydrogenation. increase in the saturated analogs of equal chain length resulted. We detected two minor components, tentatively identified as palmitolenic acid (C16:2) and nonadecanoic acid (C19). These were not observed in earlier work ( L a m b r e m o n t & Blum, 1963) and were probably obscured by larger components. Both must be regarded as minor constituents and accounted for only about 1 per cent of the methyl esters.
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Bromination by the method of James (1960) also resulted in disappearance of all components identified by GLC as containing one or more double bonds. DISCUSSION The results with labeled acetate are significantfrom severalstandpoints. First, with this informationwe now know that both the larval and adult boll weevilhave the capacity to synthesize fatty acids from acetate. Also, the chain lengths and types of fatty acids synthesizedby the two life stages correspondquite closely, and both stages appeared to be unable to form linoleic acid. Furthermore, the almost identical labeling pattern obtained with methyl- and carboxyl-labeledacetate supported the generally accepted view that fatty acid synthesis proceeds by the successive condensationof a two-carbon unit. Second, the 3 weeks from the egg stage to adult emergenceof the weevils, as planned in these experiments,provided a vastly longer time for synthesisthan the 2 hr period used in previous experiments to determine fatty acid synthesis in injected adults (Lambremont, 1965). We previouslyspeculated that linoleic acid might be synthesizedso slowlythat no significant CI~ could be incorporatedwithin 2 hr. Our present data eliminated this possibility. That no significant C14 was present in the short-chain acids from the weevils indicated that these componentswere not produced and stored as intermediatesin the synthesis of long-chain acids although they were present in large amounts in the diet as well as in body fat (Table 3). The nonradioactiveshort-chainacidsin the insects, therefore, represented direct incorporation from the diet. One must conclude that substantial amounts of palmitic, palmitoleic, stearic and oleic acids are synthesized de novo from the acetate precursor or through the condensation of acetate with short-chain intermediates,and once condensationof two-carbon units begins, it continuesto completionto produce C1s and Cls fatty acids. Coniglio& Cate (1958) pointed out that in rats, palmitic acid was the principal intermediate of acetate condensationand only a few short-chain acids were synthesized. Chain elongation of short-chain acids occurred to a greater extent in fasted rats. Once palmitic acid was formed it was then convertedto other long-chain acids. Finally, although both the larvae and adults synthesizedthe same kinds of fatty acids from acetate, the relativeamounts differedconsiderably. Oleicacid contained a very large share (60 per cent) of the C14incorporatedby the larva, but only about 25 per cent of that by the adult. Synthesis of palmitic acid by the larva, on the other hand, was only about a third that observed in the adult, and stearic only about half. We do not know all of the reasons for these differences,although the results with labeled fatty acids in diets 3, 4 and 5 offered a possible explanation of the mechanismfor oleic acid build-up in the larva, and permitted us to come to several additional conclusionsas follows. The larva appeared to absorb a large portion of the dietaryfatty acids unchanged but it could a]so readily modify the remainder to a certain degree, depending on the original acid. If the acid had sixteen carbon atoms, it could be dehydrogenated
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to a mono-unsaturated acid or its chain lengthened by one acetate unit to eighteen carbons and dehydrogenated to oleic acid. Thus, in a manner of speaking, all pathways led toward oleic acid, which appeared to be the endpoint of fatty acid incorporation in the larva. These observations suggested why we found higher amounts of radioactivity (up to 60 per cent) in the oleic acid fraction of fat synthesized by larvae on the C 14 acetate diets 1 and 2. We must also conclude that the boll weevil is unable to form linoleic acid either from acetate or from closely related long-chain fatty acids. The suggestion put forth by Vonk & Zandee (1963) that invertebrates have independent pathways for building up saturated and unsaturated fatty acids was not upheld by our present work. These authors noted several instances of higher labeling of the unsaturated than the saturated analogs and concluded that two routes of biosynthesis existed. Sedee (1961), who worked with larvae of the blowfly Calliphora erythrocephela (Meigen), stated that if, as in higher animals, the saturated and unsaturated fatty acids were interconvertible, they should have attained the same isotope content. Since they did not, he too concluded that separate pathways of biosynthesis were in operation. Yet, the present results argue for the existence of a single pathway, with desaturation to mono-enoic acids being the principal step. Because the reverse saturation reaction did not occur, once oleic acid was formed it could not be changed back into stearic; and this situation is clearly shown by the data in Fig. 3. Mead & Howton (1960), in discussing the biosynthetic pathways of unsaturated fatty acids pointed out that hydrogenation of unsaturated acids to the corresponding saturated analogs did not appear to occur in the animal body to any appreciable extent. This situation held true for the boll weevil. The quantitative relationships of precursor and product, especially those shown in Fig. 2, also strongly suggested direct desaturation. The decrease in labeled stearic acid in the weevil below that in its diet was almost exactly offset by the increase in its C 14 oleic content. A similar relationship held for the three radioactive acids formed from precursor C 14palmitate as shown, in Fig. 1. It is clear from the data in Table 2 and Fig. 2 that considerably more oleic than stearic was deposited in the body fat of the boll weevil feeding on a diet of C 1~ acetate or stearic acid. Recent studies showing a direct conversion of stearate to oleate were reported by Bade (1964) for aseptic roaches, Eurycotisfloridana (Walker). Decarboxylation of the carbon atom in the first position of both precursor and product removed all radioactivity from the C14-stearic acid and the oleic acid formed from it. Our present results with boll weevil larvae suggested that the direct desaturation pathway is in operation on all long-chain fatty acids obtained from the diet and on fatty acids synthesized from acetate. Furthermore, no conversion of oleate to linoleate took place. These features were characteristic of the animal-type biosynthesis mechanism described by Erwin et al. (1964) for some protozoa and for the vertebrates, and supported an earlier observation (Lambremont, 1965) that the weevils, like other metazoans, are not capable of forming linoleic acid. The linoleate component (6.3 per cent) given in Table 3 for the boll weevil fatty acids therefore apparently arose solely from dietary linoleate.
SYNTHESIS AND METABOLIC CONVERSION OF FATTY ACIDS BY THE LARVAL BOLL WEEVIL 301
Unpublished results from research we conducted in our laboratory in cooperation with Dr. Erma Vanderzant further strengthened our belief that the weevil's linoleic acid was derived from the diet. G L C analyses of adults reared on a variety of chemically defined larval media showed that linoleic acid in the body fat only when there was a direct dietary supply, and that no conversion of oleate or metabolic rearrangement of linolenate appeared to be taking place. T h a t we found a small amount of label in the linolenate fraction was not sufficient evidence to substantiate weevil biosynthesis of this polyunsaturated acid. In our experiments with CX4-acetate-injected adult weevils, consistent labeling of linolenate might have been due to the addition of two-carbon C 14 unit to nonradioactive heptadecanoic acid a small amount of which is known to be present in this insect. This process would have yielded C xa nonadecanoic acid, which is also a trace component of the boll weevil (Table 4). Both linolenic acid and nonadecanoic acid have very similar retention times by G L C . It was evident from the small amounts of radioactivity in the linolenic acid that synthesis of linolenate (if it occurs at all) could not have accounted for the amounts (2.6 per cent in Table 3) we found in the weevil's body fat. Most, if not all, of the linolenic acid must, therefore, have come from the diet. T h e lack of synthesis of the Cls polyenoic fatty acids was significant in the light of recent work by Vanderzant & Richardson (1964) on the lipid requirements of the adult boll weevil. These workers reported that adults reared from a fat-deficient larval diet depended heavily upon the adult food for those lipid constituents needed for the synthesis of eggs. Addition of either linoleic or linolenic acids increased the n u m b e r of eggs laid and females lived longer. Oleic acid also partially replaced dietary fat, but was not as effective as the two polyunsaturated acids, and possibly spared traces of these latter acids already in the insect or its diet. T h e observations of these workers in conjunction with ours presented here, support the general view that the polyunsaturated C18 fatty acids are not formed by the tissues of insects. Acknowledgements--We thank L. D. Newsom, H. D. Richardson and Rosamond Killebrew of Louisiana State University for their cooperative support of this project. We also thank Dr. Erma S. Vanderzant of the Entomology Research Division, at College Station, Texas, for sending the adults reared on defined media, and Rhonda Frayer and Opal Colvin, also of this Division, for their technical assistance. REFERENCES BADE M. L. (1964) Biosynthesis of fatty acids in the roach Eurycotis floridana. J. Insect Physiol. 10, 333-341. CARROLLK. K. (1961) Separation of lipid classes by chromatography on Florisil. ft. Lipid Res. 2, 135-141. CONIGLIO J. G. ~ fATE D. L. (1958) The distribution and biosynthesis of palmitic and stearic acids in liver, intestine and carcass of intact normal and fasted rats. ft. Biol. Chem. 232, 361-368. EARLE N . W . , GAINES R. C. & ROUSSEL J. S. (1959) A larval diet for the boll weevil containing an acetone powder of cotton squares. J. Econ. Ent 52, 710-712. ERWIN J., HULANICKAB. & BLOCH K. (1964) Comparative aspects of unsaturated fatty acid synthesis. Comp. Biochem. Physiol. 12, 191-207. 2r
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FARQUHAR J. W., iNSUL W., ROSEN P., STOFFEL W. • AHRENS E. H. (1959) T h e analysis of fatty acid mixtures by gas liquid chromatography. Nutr. Rev. (Suppl.) 17, 1-30. JAMES A. T. (1960) Qualitative and quantitative determination of the fatty acids by gasliquid chromatography. In Methods of Biochemical Analysis (Edited by GLICK D.), Vol. 8, pp. 1-59. LAMBREMONT E. N. (1965) Biosynthesis of fatty acids in aseptically reared insects. Comp. Biochem. Physiol. 14, 419-424. LAMBREMONT E. N. & BLUM M. S. (1963) Fatty-acids of the boll weevil. Ann. Ent. Soc. Amer. 56, 612-616. LAMBREMONT E. N., BLUM M. S. & SCHRADER R. M. (1964) Storage and fatty acid composition of triglycerides during adult diapause of the boll weevil. Ann. Ent. Soc. Amer. 57, 526-532. MEAD J. F. (1960) T h e metabolism of unsaturated fatty acids. In Lipide Metabolism (Edited by BLOCH K.), pp. 41-68. John Wiley, N.Y. MEAD J. F. & HOWTON D. R. (1960) Radioisotope Studies of Fatty Acid Metabolism, pp. 141. Pergamon Press, N.Y. MEINERTZ H. & DOLE V. P. (1962) Radioassay of low activity fractions encountered in gas-liquid chromatography of long-chain fatty acids. J . Lipid Res. 3, 140-144. SEDEE P. D. J. W. (1961) Intermediary metabolism in aseptically reared blowfly larvae, CaUiphora erythrocephala (Meig). II. Biosynthesis of fatty acids and amino acids. Arch. Int. Physiol. Biochem. 69, 295-309. VANDERZANT E. S. & RICHARDSON C. D. (1964) Nutrition of the adult boll weevil: lipid requirements. J . Insect Physiol. 10, 267-272. VONK H. J. & ZANDEE D. I. (1963) Intermediary metabolism of lipids and amino acids in invertebrates. Proc. X V I Int. Congr. Zool., p. 34.