G LC Analysis of Heliothis virescens Triglycerides at Various Metamorphic Stages

RANDALL WOOD, R. D. HARLOW Medical Division I Lipid Research Laboratory, Oak Ridge Associated Universities, Oak Ridge, Tennessee 37830 and EDWARD N. LAMBREMONT 2 Nuclear Science Center, Louisiana State University, Baton Rouge, Louisiana 70803

ABSTRACT

The triglycerides of Heliothis virescens (F.) , the tobacco budworm, reared on a semidefined diet, were examined at dif- ferent stages of development. The distri- but ion of triglyceride carbon numbers and the composit ion of triglyceride fat ty acids were determined by gas-liquid chro- matographic analysis of intact triglycer- ides and methyl esters. Triglycerides of carbon number 50 decreased in the unfed a d u l t s with age; the decrease corre- sponded with a decrease in palmitic acid. Linoleic acid and carbon number 50 ex- h ib i t ed fluctuations in concentrations that were correlated with metamorphosis. The observed changes in specific triglycer- ide carbon numbers with a simultaneous change in specific triglyceride fat ty acids indicate a nonrandom fat ty acid distribu- t ion in the triglycerides of this insect.

INTRODUCTION

Insects contain a high percentage of lipid, a large amount of which is triglyceride that serves as a source of energy (1). Although the fat ty acid content of the triglyceride fraction has been analyzed for several insects (2,3), the intact triglyceride structure has not ye t been examined for any insect. This communication describes the distribution of triglyceride carbon numbers and the fat ty acid composit ion of tri- glycerides found in the tobacco budworm, Heliothis virescens (F.), during the larval to adult t ransformation, and in unfed adults of various ages.

EXPERIMENTAL PROCEDURES

Insect Material

Heliothis virescens (F.) (Lepidoptera: Noctu- idae), the tobacco budworm, was reared in indi- vidual plastic containers from the egg to adult

1Under contract with the U. S. Atomic Energy Commission.

2Oak Ridge Associated Universities Research Par- ticipant.

stage on a semidefined diet described by Berger (4). Larvae were removed from the diet and held 8 hr without food prior to extract ion of lipids to minimize dietary triglyceride present in the intestinal tract. Late-stage pupae were placed in screen-topped containers of sawdust until moths emerged. Adults were placed in screen-topped half-gallon jars and given only water in order to deplete their lipid content.

Last instar larvae, pupae, and adults were homogenized in ice-cold water, lyophilized, and the total lipids were extracted by the Folch et al method (5). Neutral lipids were separated from phospholipids by silicic acid chroma- tography (6). Triglycerides were isolated by thin-layer chromatography (TLC) and hydro- genated before analysis by gas-liquid chroma- tography (GLC). Methyl esters were prepared from the triglycerides by transesterification with 2% sulfuric acid in methanol.

Thin-Layer Chromatography

Silica Gel G adsorbent layers (250 /a ) were spread on 20 x 20 cm glass plates with a modi- fied Colab applicator (7). Chromatoplates were air-dried, activated 30 min at 110 C, and stored in a desiccator until used. Neutral lipids or total lipids were applied to the plate in a narrow band with a Rodder Streaker (Rodder Instru- ment, Los Altos, Calif.). Solvent development was carried out in hexane-diethyl ether (90:10 v/v). The triglyceride band was located by spraying the chromatoplate lightly with a 0.2% Rhodamine 6G in 95% ethanol. The triglyceride band was scraped into a fr i t ted glass funnel and eluted from the adsorbent layer with sev- eral volumes of diethyl ether.

Gas-Liquid Chromatography Methyl esters and intact triglycerides were

analyzed with an Aerograph Model 204 gas chromatograph. One side of the dual column instrument was modified for the analysis of tri- glycerides and other high molecular weight compounds. The modifications will be de- scribed in a separate publication. Triglycerides were analyzed on a 70 c m x 4 mm OD (2.5 mm ID) Pyrex column packed with 1% OV-1 on 100-120 mesh Gas Chrom Q manually tempera- ture programmed from 200-335 C at approxi-

159

160 RANDALL WOOD, R. D. HARLOW AND EDWARD N. LAMBREMONT

TABLE I

Lipid Content of Heliothis virescens at Various Metamorphic Stages

Metamorphic stage

Lip id ,% Lipid, % Number of

insects Dry wt. Wet wt. NL a PL b

Young larvae 66 Mature larvae 40 Young pupae 42 Mid-age pupae 50 Old pupae 39 Newly emerged adults 42 4 Day old adults 22 7 Day old adults 26 11 Day old adults 15 Larval diet ---

15.2 2.7 92.7 7.3 14.2 9.4 92.3 7.7 43.3 14.9 93.7 6.2 26.5 9.2 96.4 3.6 26.4 8.3 91.0 9.0 29.8 12.7 94.7 5.3 27.4 11.6 . . . . . . 15.0 6.4 . . . . .

9.4 3.0 . . . . . 2.6 0.6 76.1 23.9

aNL, neutral lipid.

bpL, phospholipid.

mately 5 C/min. Temperatures of the flash heater and detector were maintained at 325 C and 350 C. Helium carder gas, hydrogen and oxygen flow rates were 100, 50, and 300 ml/min, respectively.

Methyl esters were chromatographed on a 152 cm x 3 mm OD (1.75 mm ID) Pyrex column packed with 15% ethylene glycol succi- hate silicone polymer (EGSS-X) coated on 100-120 mesh Gas Chrom P. The column tem- p e r a t u r e was manually programmed from 125-190 C at approximately 3 C/min. Flash heater and detector temperatures were main- tained at 250 C. The flow rate of the helium carrier gas was 40-60 ml/min. Hydrogen and air flow rates were regulated to give maximum de- tector sensitivity.

2'T 2'4 MINUTES

II DAY OLD ADULTS 52

MATURE LARVAE

t 100 90

52 80

s ~ ~ so,.=,

$0 2O

,o 4 8

FIG. 1. Representative chromatograms (GLC) of intact triglycerides of Heliothis virescens larvae and adults. The comparison illustrates the changes that occur in the carbon number distribution of the tri- glycerides during development. Chromatographic conditions are described in the text.

Methyl ester and triglyceride peak areas were measured by triangulation and represent the mean of three determinations. Methyl ester values are given as uncorrected area percentages since values obtained for NIH standard methyl ester mixtures (D and F) agreed (+5%) with the known percentages. Standard triglyceride mix- tures were used for instrument calibration and the mole percentages given have been corrected for slight losses of the higher molecular weight triglycerides.

M a t e r i a l s

Purified triglycerides and methyl ester stand- ard mixtures were obtained from The Hormel Institute, Austin, Minn. Glass-distilled solvents were purchased from the Burdick and Jackson Laboratories , Inc., Muskegon, Mich. Other chemicals were reagent grade or better and were used without further purification.

RESULTS AND DISCUSSION

The percentages of total lipids, neutral lipids and phospholipids in Heliothis virescens at vari- ous stages of development are shown in Table I. The total lipid content , based on either wet weight or dry weight, shows the same trend: a maximum is reached at the early pupal stage and decreases to a minimum value in mid-age pupae. The to ta l lipids remain constant until emergence of the adult, at which time the lipid content begins to decrease with age. The neu- tral lipids of Heliothis represent approximately 93% of the total lipids and appear to be unaf- fected by metamorphosis. Although the lipid class composit ion was not quantitatively deter- mined, the neutral lipids consist primarily of

L1PIDS, VOL. 4, NO. 2

INSECT TRIGLYCERIDES

TABLE II Calculated and Determined Triglyceride Carbon

Number Distribution of Heliothis virescens at Various Stages of Development

161

Carbon number, %a Stage

examined 48 50 52 54 56 58

Young larvae 0.9 b 17.9 64.3 16.5 0.5 .... Calculated c 4.4 22.3 42.1 29.1 3.8 0.2 Mature larvae 2.6 33.6 56.6 7.2 .... Calculated 8.6 31.4 40.6 18.4 0.7 Young pupae 2.3 31.7 58.6 7.4 .... Calculated 8.1 30.0 39.6 18.2 0.7 Mid-age pupae 2.5 35.2 51.4 10.4 0.5 Calculated 8.8 31.3 39.6 18.5 1.5 Old pupae 2.0 35.8 54.8 8.3 T Calculated 8.8 30.4 39.9 19.2 1.5 Newly emerged adults 2.3 36.2 54.4 7.2 .... Calculated 9.3 32.2 39.8 1 "/.7 1.0 4 Day old adults 0.8 21.6 62.2 15.4 .... Calculated 5.5 24.7 42.0 23.8 1.8 7 Day old adults 1.2 23.3 55.3 19.9 0.2 Calculated 4.5 22.5 42.3 27.5 2.0 11 Day old adults 0.8 20.2 53.5 25.3 0.2 .... Calculated 3.1 16.5 35.4 32.5 10.8 1.5 Larval diet triglycerides 0.4 7.9 39.2 48.2 3.6 0.6 Calculated 1.2 9.4 34.6 50.6 4.3 0.1

a The carbon number represents the sum of the number of carbon atoms in the hydro- carbon chains.

b Percentages represent the mean of three determinations. c The random distribution percentages were calculated from the respective fatty acid

percentages in Table III.

tr iglycerides. Representa t ive chromatograms of triglycer-

ides isolated f rom m a t u r e larvae and 11 day old adults (Fig. 1) i l lustrate the change in carbon number dis t r ibut ion during development . Tri- glyceride carbon number dis t r ibut ion percent- a g e s ob ta ined for different deve lopmenta l stages and the diet are given in Table II along wi th calculated r andom dis t r ibut ion percent- ages. The fa t ty acid dis t r ibut ion in the tr iglycer- ides is n o n r a n d o m at all stages: de te rmined per- centages o f carbon numbers 52 and 54 are 15-20% higher and 7-10% lower, respect ively, than calculated values. The dis t r ibut ion of car- bon numbers in this insect is very narrow (48-56) at all stages of deve lopment , wi th car- bon numbers 50, 52 and 54 represent ing more than 95% of the total . Af te r the young adult stage, the percentage of carbon number 50 de- creased with age. Carbon number 54 increased but showed f luc tuat ions corresponding to meta- morph ic changes. Fol lowing the early last instar larval stage, carbon number 52 showed the least variat ion. The difference be tween the carbon n u m b e r dis t r ibut ion of dietary tr iglycerides and the insect t r iglycerides indicates the presence of_ hn active t r iglyceride synthesizing system in-

volving effher to ta l de novo synthesis, hydro l - ysis and rear rangement o f dietary triglycerides, or a combina t ion of both .

Fa t t y acid me thy l esters of tr iglycerides ranged in chain l eng th f rom C14:0 to C20:4 but C16 and C18 fa t ty acids represented more than 95% of the to ta l in all stages of develop- ment (Table III). The dietary fa t ty acid compo- sit ion (Table III) is no t ref lected in the fa t ty acid compos i t ion o f the insect triglycerides. Oleic and l inoleic acids represented 16% and 53% of the to ta l dietary fa t ty acids, whereas these same acids represented 40-45% and 7-17%, respect ively, of the tr iglycerides in Heliothis virescens. Our data are similar to those repor ted by Schaefer (8) who has repor ted that larvae of the bo l lworm, Heliothis zea (Boddie) , when reared on wheat germ diet high in l inoleic acid, conta in only small amounts of this acid in the to ta l lipids. As seen in Table III, the diet con- ta ined only traces o f palmitole ic acid bu t the insect t r iglycerides conta ined up to 10% of this fa t ty acid. Vanderzan t (9) and Schaefer ( 8 ) r e - por ted that pa lmi to le ic acid is synthesized by Heliothis zea when this acid is absent f rom the diet. Oleic and palmitole ic acid percentages showed the least var ia t ion f rom early last instar

LIPIDS, VOL. 4, No. 2

162 RANDALL WOOD, R. D. HARLOW AND EDWARD N. LAMBREMONT TABLE III

Triglyceride Fatty Acid Composition of Heliothis virescens at Various Stages of Development

Metamorphic stage

Fatty acid, %a

14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:4

Young larvaeb 0.3 31.7 2.5 Mature larvaeb 0.4 33.9 9.2 Young pupae 0.3 33.8 8.6 Mid-age pupae 0.4 34.8 8.7 Old pupae 0.5 33.9 8.6 Newly emerged adults 0.5 34.4 9.7 4 Day old adults 0.4 29.9 6.2 7 Day old adults 0.5 26.7 7.0 11 Day old adults 0.5 23.3 6.1 Diet lipids 0.7 21.3 T Diet triglycerides 0.7 18.5 T

2.1 42.5 17.0 1.5 3.1 0.8 47.6 6.7 0.6 0.8 0.9 45.8 7.9 0.8 0.7 0.8 40.3 12.0 1.3 1.7 0.6 42.5 11.5 0.7 1.6 0.7 43.8 9.9 T 1.1 0.3 46.3 14.6 0.6 1.6 0.6 46.6 16.0 1.0 1.6 0.7 42.5 16.6 1.3 9.0 1.4 16.1 52.9 6.9 0.8 1.5 18.9 51.7 6.5 2.3

a Percentages represent means of duplicate analyses. b Last larval instar.

larval stage to the adult stage. Palmit ic acid showed a decrease wi th age, and the same t rend as observed for t r ig lycedde ca rbon n u m b e r 50. Linoleic acid exh ib i t ed f luc tua t ions in concen- t ra t ions cor responding to m e t a m o r p h o s i s tha t w a s also observed for t r iglyceride carbon n u m b e r 54.

The observed changes in specific t r iglyceride carbon num ber s wi th a cor responding simulta- neous change in specific t r iglyceride f a t t y acids and the lack of agreement b e t w e e n de t e rmined and calculated carbon n u m b e r percentages indi- cate a n o n r a n d o m fa t ty acid d i s t r ibu t ion pat- tern. Adul t s (11 day old) ma in ta ined on water to dep le te their l ipid reserves (Table I) conserve a rachadonic acid (Table III); this acid may serve a vital func t ion in insects.

REFERENCES

1. Gilbert, L. I. in "Advances in Insect Physiology,"

Vol. 4, J. W. L. Beament, J. E. Treherne and V. B. Wiggleworth, Editors, Academic Press, London & New York, 1967, p. 69-211.

2. Lambremont, E. N., M. S. Blum and R. M. Schrader, Ann. Entomol. Soc. Am. 57, 526-532 (1964).

3. Nelson, D. R., A. C. Terranova and D. R. Sukkestad, Comp. Biochem. Physiol. 20, 907-917 (1967).

4. Berger, R. S., U.S. Department of Agriculture, Bulletin No. ARS-33-84, 1963.

5. Folch, J., M. Lees and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497-509 (1957).

6. Wood, R., and F. Snyder, Lipids 3, 129-135 (1968).

7. Wood, R., and F. Snyder, L Chromatog. 21, 318-319 (1966).

8. Schaefer, C. H., J. Insect PhysioL 14, 171-178 (1968).

9. Vanderzant, E. S., Ann. Entomol. Soc. Am. 61, 120 (1968).

[Received August 7, 1968]

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