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Constituents of the boll weevil, anthonomus grandis boheman—III lipids and fatty acids of subcellular particles of pupae
Comp. Biochem. Physiol., 1972, Vol. 43B, pp. 883 to 890. Pergamon Press. Printed in Great Britain
C O N S T I T U E N T S OF THE BOLL WEEVIL, A N T H O N O M U S GRANDIS BOHEMAN--III LIPIDS AND FATTY ACIDS OF SUBCELLULAR PARTICLES OF PUPAE* A. C. THOMPSON, R. D. HENSON, R. C. G U E L D N E R and P. A. H E D I N Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture, State College, Mississippi 39762 (Received 9 March 1972) Abstract--1. Phospholipids were the principal lipids in the mierosomes, nuclei and mitochondria of the boll weevil, Anthonomus grandis Boheman. 2. The major fatty acid in the phospholipids of the boll weevil was octadecadienoic acid (18:2) followed by octadecenoic acid (18:1) and hexadecanoic acid (16:0). The weight of neutral lipid varied in the subcellular particles, but the ratios of fatty acids remained the same. 3. Phosphatidyl choline and phosphatidyl ethanolamine were present in equal amounts in the mitochondria, and phosphatidyl choline predominated in the other subcellular particles of the boll weevil. 4. Triglycerides made up about 50 per cent of the neutral lipids of the nuclei, microsomes and cytoplasm. The principal neutral lipids of the cell mitochondria were diglycerides and monoglycerides. INTRODUCTION STUDIES of the phospholipids in the subcellular fractions of animal and animal parts were extensively reviewed by Fleischer & Rouser (1965), but such studies in insects have been limited to the housefly, Musca domestica L. (Crone, 1964; Khan & Hodgson, 1967; Chan, 1970) and to the tobacco horn worm, Manduca sexta (L.) (Chan & Lester, 1970). In the boll weevil, Anthonomus grandis Boheman, the omission of most lipids and lipogenic factors from the diet did not seriously affect egg hatch (Vanderzant & Richardson, 1964) but did reduce egg production by 50 per cent. Oviposition was restored by the addition of cholesterol, vitamins and polyunsaturated fatty acids, particularly the 18:2 and 18:3 acids (Earle et al., 1967). However, subsequent studies of the metabolic conversion of 14C-acetate to fatty acids by the boll weevil showed that most of the radioactivity appeared in the 16:1 and 18:1 monounsaturated acids (Lambremont, 1965). Also, later studies showed that the oleic acid of larvae, pupae and newly molted unfed adult boll weevils which had been * Presented at the spring meeting of the American Oil Chemist's Society Meeting, Houston, Texas. 883
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treated with 14C-acetate contained 60 per cent of the incorporated radioactivity ( L a m b r e m o n t et al., 1965), and the weevil also desaturated palmitic and stearic acids to the corresponding monounsaturated fatty acids. Our studies of the composition of the phospholipids of adult boll weevils (Henson et al., 1971, 1972) showed that phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) were present in nearly equal quantities in whole insects; cardiolipin and sphingomyelin were next in concentration. Also, the concentration of cardiolipin in the immature stages of the boll weevil was constant, but lower than in the adult. T h e percentage composition of sphingomyelin gradually increased in both larvae and pupae. Phospholipids are primary components of biological membranes and neutral lipids provide energy for normal cell function (Fleicher & Rouser, 1965; Green & Tzagololoff, 1966). However, the role of cardiolipin in the inner membrane of mitochondria (Parsons, 1967; Chan, 1970) has not been elucidated. MATERIALS AND METHODS lnsect material Eggs obtained from boll weevils reared in the laboratory were mechanically planted in a modified cottonseed meal larval diet (Gast & Davich, 1966) that had been mixed with agar, poured into Petri dishes and allowed to gel. The Petri dishes were then held at constant conditions for 9-11 days until pupae were available. The pupae were hand removed from the diet plates and immediately homogenized for fractionation. Preparation of subcellular fractions and lipid extraction Boll weevil pupae were homogenized in twelve times their weight of 0"05 M Tris-HC1 buffer, pH 7"3, containing 0"25 M sucrose, and 0"5 mM EDTA (ethylenediaminetetracetic acid). The homogenate was fractionated by the scheme outlined by Khan & Hodgson (1967). Each subcellular fraction was suspended in the sucrose buffer and centrifuged three times. The cross contamination of the mitochondria and microsomes was found to be less than 10 per cent (Hogboom & Schneider, 1953; Applemans et al., 1955). The lipids from each subcellular fraction were extracted with chloroform-methanol (2 : 1 v/v). The extracts were washed once with 0"05% calcium chloride (Folch et al., 1957), dried over anhydrous sodium sulfate and made to standard volume. Chromatography a. Total lipids. Silicic acid (35 g) was slurried in chloroform and poured into a glass column (2 x 25 cm). Then the total solution (10 ml) was applied to the column. The neutral lipids were eluted with chloroform (200 ml), and the phospholipids with methanol (200 ml). The recovery of phosphorus was 90 per cent. Thin-layer chromatography (TLC) of the phospholipids followed by phosphorus analysis of each component before and after column chromatography accounted for all phospholipid phosphorus. b. Neutral lipids. Florisil containing 8% water (Carroll, 1961) was slurried in cyclohexane and poured into a glass column (3 x 25 cm). The subcellular neutral lipids in cyclohexane were fractionated according to the method of Carroll (1961) except that cyclohexane was substituted for hexane. The column fractions were monitored by T L C (Cmelik, 1969). c. Phospholipids. The phospholipids were separated by two-dimensional T L C in the systems outlined by Henson et al. (1972). The T L C plates were held in a vacuum for 1 hr between development in the first and second directions. The phospholipids were detected
CONSTITUENTSOF THE BOLL WEEVIL--In
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by spraying with molybdate (Vaskousky & Kostetsky, 1968) and quantitatively removed from the plate; the phosphorus was determined by the perchlorate method of Chen et al. (1956) as modified by Mitlin (1960). d. Fatty acid methyl esters. Fatty acid methyl esters were prepared with boron trifluoridemethanol (Metcalf et al., 1966). The qualitative and quantitative estimations of the methyl esters were done with a gas-liquid chromatograph (GLC) equipped with flame ionization on a 6 ftx ] in. O.D. stainless steel column packed with 10"5% DEGA on 60/80-mesh H M D S treated gas Chrom-P. The column temperature was 190°C. RESULTS Lipid content of insects T h e polar lipids (Table 1) were present in greater concentration than the neutral lipids except in the cytoplasmic fraction, which contained considerable quantities of fat bodies and free fatty acids. T h e concentration of polar lipids (found b y weighing the nonvolatiles of the methanol eluate f r o m silicic acid TABLE 1--PERCENTAGE OF POLAR LIPIDS AND NEUTRAL LIPIDS IN THE SUBCELLULAR FRACTIONS OF BOLL WEEVIL PUPAE
Cell fraction Residue Microsomes Nuclei Mitochondria Cytoplasm
Polar lipid
Neutral lipid
27"8 68"5 72"3 62"8 17"4
72"2 31"5 27"7 37"2 82"6
chromatography) was 8.3 m g / g on a fresh weight basis. T h e concentration of phospholipids determined b y phosphorus and fatty acid analysis was 0.2 m g / g fresh weight. Boll weevil pupae contained 3 per cent phospholipid and 13 per cent neutral lipid. T h u s the phospholipids comprised 18 per cent of the total lipids b y weight and 23 per cent by phosphorus and fatty acid analysis. TABLE 2--DISTRIBUTION OF NEUTRAL LIPID IN SUBCELLULARFRACTIONOF BOLL WEEVIL PUPAE Percentage of total neutral lipids of cell fractions Lipid class Sterols Fatty acid esters Triglycerides Diglycerides Monoglycerides Free fatty acids 30
Nuclei 0"4 13-7 43"5 17"8 21"1 3"8
Mitochondria Microsomes 1 "0 3"8 14"5 24"9 38"3 17"5
1-4 5"7 43"7 15"7 10"6 23"4
Cytoplasm 0"9 -47-2 19-2 9-7 23"3
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Separation of lipid groups T h e distribution of the neutral lipids in the subcellular fractions is shown in T a b l e 2. ( T h e fatty acids esters also included hydrocarbons, but they accounted for less than 1 per cent of these fractions.) I n the nuclei microsomes, and cytoplasm, triglycerides ( T G L ) accounted for 40 per cent of the total neutral lipids. T h e distribution of the phospholipids in the subcellular fractions is shown in T a b l e 3. Phosphatidyl ethanolamine and P C comprise 60-80 per cent of the phospholipids in all fractions. Also, the distribution of these two major phospholipids approximated that observed for the adult insect (Henson et al., 1971). TABLE 3--PHosPHOLIPID CLASSES IN SUBCELLULARFRACTIONS FROM BOLL WEEVIL PUPAE
Percentage of phospholipids in total Subcellular fraction Cell wall Nuclei Mitochondria Microsomes Cytoplasm
LPC*
SPH*
LPE*
PI*
PS*
PC*
PE*
CL*
3.1 5.3 2"3 3"3 --
8.6 8"2 8"5 6"6 12'2
8.6 4.4 3"1 2-5 --
2.6 6.1 3"3 2.8 --
9-0 9-8 4"2 5.7 --
39'4 32"8 36"9 45'1 48"8
25-3 26-5 36-9 34-1 39"0
9.1 6.9 4"8 3.3 --
* LPC, lysophosphatidylcholine; SPH, sphingomyelin; LPE, lysophosphatidylethanolamine; PI, phosphatidyl inositol; PS, phosphatidyl serine ; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine; CL, cardiolipin.
Fatty acids T h e fatty acid composition of the phospholipids (Table 4) of each subcellular fraction was similar to that found in the diet (Table 5). T h e major acid in the phospholipids was 18:2, and 18:1 was next in abundance. T h e phospholipids of T A B L E 4----FATTY ACIDS IN TOTAL P H O S P H O L I P I D S OF SUBCELLULAR PARTICLES OF BOLL WEEVIL PUPAE
Percentage fatty acids in cell fractions* Cell particle Cell wall Nuclei Mitochondria Microsomes Cytoplasm
16:0
16:1
18:0
18:1
18:2
18:3
19.4 7"5 9"2 15"4 25"5
10.8 9"5 9'0 12"8 13-8
10.8 10"9 7"9 17-0 9-5
31"9 13"8 21 "2 22"5 14"8
26"9 52"9 47"4 28"1 36.4
-5'4 5"3 4"2 --
* Sphingomyelin fatty acids are excluded from this table since 1 N KOH does not hydrolyse these compounds.
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the nuclei and the mitochondria were 81"6 and 82.9 per cent unsaturated, respectively. T h e neutral lipids (Table 5) contained four major fatty acids (16:0, 16:1, 18:1 and 18:2) with 18:1 in abundance. The total micromolar composition of fatty acids found in the various neutral lipid classes is given in Table 6. T h e
TABLE 5--FATTY
ACIDS I N THE NEUTRAL L I P I D S
OF SUBCELLULAR FRACTIONS OF BOLL WEEVIL PUPAE
Percentage fatty acids in neutral lipids of cell fractions* Cell fraction
14:0
16:0
16:1
18:0
18:1
18:2
Cell wall Nuclei Mitochondria Microsomes Cytoplasm
1"5 1"8 1.0 1"4 1"0
16"4 21"2 24-2 22-1 20"1
12'3 13"2 12"5 15'6 28"2
5-6 7"0 8"1 8"8 6-2
29"8 40"6 37"9 36"5 32"0
34'5 16'0 15"9 15'4 12"2
*Pupal diet: •4:0 (2'0%); 16:0 (27"4%); 16:1 (1"9%); 18:0 (7"2%); 18:1 (20"9%); 18:2 (40"1%). TABLE 6--TOTAL
/zmoles OF
FATTY ACIDS I N THE NEUTRAL L I P I D CLASSES
OF SUBCELLULAR FRACTIONS OF BOLL WEEVIL PUPAE
/zmoles of fatty acids in neutral lipids Subcellular fraction
SE*
TGL*
DGL*
MGL*
FFA*
Microsomes Mitochondria Cytoplasm Nuclei
1"0 1"3 0"0 2"2
19-6 11"7 115"3 17"8
4"7 13"4 32"4 5"3
1"6 10-3 7"9 3.1
3-5 4"7 19"0 5"2
* SE, sterol esters; TGL, triglycerides; DGL, diglycerides; MGL, monoglycerides; FFA, free fatty acids. distribution of the fatty acids in the mitochondria fraction is given in Table 7. Oleic and palmitic acid were the principal fatty acids of the neutral lipids from cell fractions (Table 5) and, therefore, were the major acids in the neutral lipids of the mitochondria fraction. Since the methyl esters were prepared by a series of reactions which involve basic hydrolysis of the lipid, the fatty acids of the sphingomyelin were not reported in Table 4 because 1 N K O H does not hydrolyze these compounds. However, Henson et aL (1971) reported that 53 per cent of the sphingomyelin fatty acids of boll weevil pupae were 21:1 and 22:0.
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TABLE 7--FATTY ACIDS IN THE NEUTRAL LIPIDS OF THE MITOCHONDRIA FRACTION OF B O L L W E E V I L P U P A E
Percentage of fatty acids in mitochondria Fatty acids
SE
TGL
DGL
MGL
FFA
14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3
15.2 -30.7 15.3 7.6 23.0 7.6 --
8.5 3.6 21.4 14.5 8.5 27.3 16.2 --
1-4 0.7 26.4 13-4 8.9 35.8 13.4 --
3.8 1.9 23.5 11.6 9.7 23.3 16.5 9.7
--19.1 -12.7 27.6 40.6 --
DISCUSSION All subcellular fractions of the boll weevil contained both phospholipids and neutral lipids, and the preponderance of triglycerides in the cytoplasm fraction reflected the composition of the fat bodies in this fraction. However, the neutral lipids were present in the subcellular fractions of boll weevil pupae in much higher concentrations than in rat liver: Spiro & McKibben (1956) reported 2.5, 1.4 and 0.0 per cent neutral lipid from the nuclei, mitochondria and microsomes, respectively, of this tissue. This greater concentration of T G L in the microsomes and nuclei and of D G L and M G L in the mitochondria fraction of boll weevil pupae may therefore reflect a greater dependence on these glycerides for transport and energy (Beenakkers & Gilbert, 1968). However, Cmelic (1969) found no M G L or D G L in the neutral lipids of the various organs of immature termites Macrotermes goliath (Sjostedt). The subcellular fractions of boll weevil pupae each contained more PC than PE; this same relationship was reported ( C h a n & Lester, 1970) for muscle mitochondria of the tobacco hornworm. However, in house fly (Musca domestica L.), PE exceeded PC by 50 per cent in all subcellular fractions (Khan & Hodgson, 1957). However, the microsomes of boll weevil pupae (Table 3) contained more PC and PE than the other subcellular particles, which again is in contrast to the distribution in the subcellular particles of the housefly. The concentration of cardiolipin in the mitochondria and microsomes of the boll weevil was similar to that observed by Crone (1964) and Chan (1970) for the mitochondria of the housefly, but lower than that reported by Khan & Hodgson (1967). However, Khan & Hodgson (1967) used iodine as a location agent for cardiolipin (Skidmore & Entenmann, 1962), and this procedure tends to show a high level of cardiolipin since the cardiolipin irrigates with the solvent front in the T L C system along with other phosphorus-containing materials and is not distinguished by color differences. When the lipids were detected with the molybdate spray of Vaskousky & Kostetsky (1968), the cardiolipin appeared as a concise spot.
C O N S T I T U E N T S OF T H E B O L L W E E V I L - - I I I
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Little is known about the effect of the various types of fatty acids on the solubility and permeability of membranes in insects. However, unsaturation is thought to decrease the molecular packing of the repeating units and thus to allow the interstitial spacing of the overlapping nonpolar fragments to increase in the membrane, which, in turn, would determine the size of molecule that could pass through the membrane. T h e degree of unsaturation therefore exerts a physiochemical behaviour to the phospholipids that have a direct effect on the liquid crystalline characteristics to the membranes controlling the movement of molecular species through the membrane. T h e fatty acids of boll weevil pupae were generally similar to the dietary fatty acids with respect to molecular weight and degree of unsaturation. However, in the diet, 18:1 (21 per cent) and 18:2 (40 per cent) were the major unsaturated dietary fatty acids; in the pupae, 12-30 per cent of the unsaturation appeared in 16:1. T h e fatty acid specificity of the boll weevil neutral lipids was also indicated by the molar ratios of the acids. T h e y were similar in all the neutral lipids from each subcellular fraction. Acknowledgement--The authors thank O. Lindig of this laboratory for providing the insects used in this study. REFERENCES
APPLEMENS F., WATTAUX R. & DE DUVE C. (1955) Tissue fractionation studies. The association of acid phosphatase with a special class of cytoplasmic granules in rat liver. Biochem.oT. 59, 438-445. BEENAKKERSA. M. TH. & GILBERTL. I. (1968) The fatty acid composition of fat body and haemolymph lipids in Hyalophora cecropia and its relation to lipid release. ~. Insect Physiol. 14, 581-594. CARROLK. K. (1961) Separation of lipid classes by chromatography on Florisil. ft. Lipid Res. 2, 135-141. CHAN S. K. (1970) Phospholipid composition in the mitochondria of the housefly, Musea domestica: a re-examination, ft. Insect Physiol. 16, 1575-1577. CHAN S. K. & LESTERR. L. (1970) Biochemical studies on the developing thoracic muscles of the tobacco horn worm--II. Phospholipid composition in mitochondria during development. Biochim. biophys. Acta 210, 180-181. CHEN P. S., TORABARST. Y. & WARNER H. (1956) Microdetermination of phosphorus. Analyt. Chem. 28, 1756. CMELIK S. H. W. (1969) The neutral lipids from various organs of the termite Macrotermes goliath, ft. Insect Physiol. 15, 839-849. CRONE H. D. (1964) Phospholipid composition of flight muscles sarcosomes from the housefly, Musea domestica L. aT. Insect Physiol. 10, 499-507. EARL N. W., SLATTENB. & BURKSM. L. (1967) Essential fatty acids in the diet of the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae)..7- Insect Physiol. 13, 187-200. FLEICHERS. & ROUSERG. (1965) Lipids of subcellular particles, uTAOCS 42, 588-607. FOLCH J., LEES M. & SLOANE-STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissue. 3¢. biol. Chem. 226, 497-509. GASTR. T. & DAVICHT. B. (1966) Insect Colonization and Mass Production (Edited by SMITH C. N.), pp. 405-518. Academic Press, New York.
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GREEN D. E. & TZAGOLOLOFFA. (1966) Role of lipids in the structure and function of biological membranes. J. Lipid Res. 7, 587-602. HENSON R. D., THOMPSON A. C., GUELDNER R. C. & HEDIN P. A. (1971a) The phospholipid composition of the boll weevil, Anthonomus grandis Boheman. Lipids 6, 352-355. HENSON R. D., THOMPSON A. C., GUELDNER R. C. & HEDIN P. A. (1972) Constituents of the boll weevil-II. Variations in lipid content during metamorphosis. J. Insect Physiol. 18, 161-167. HOCBOOM C. H. & SCHNEIDER W. C. (1953) Intracellular distribution of e n z y m e - - X I . Glutamic dehydrogenase. J. biol. Chem. 204, 233-238. KHAN M. A. Q. & HODOSON E. (1967) Phospholipids of subcellular fractions from the housefly, Musca domestica L. J. Insect Physiol. 13, 653-664. LAMBREMONT E. N. (1965) Biosynthesis of fatty acids in aseptically reared insects. Comp. Biochem. Physiol. 14, 419-424. LAMBREMONT E. N., STEIN C. I. & BENNSTT A. F. (1965) Synthesis and metabolic conversion of fatty acids by the larval boll weevil. Comp. Biochem. Physiol. 16, 289-302. MITLIN N. (1960) Determination of nucleic acids in the testes of three cockroach species. Ann. ent. Soc. Am. 53, 491-494. METCALPE L. C., SCHMITZ A. A. & PFLKA J. R. (1966) Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. ~4nalyt. Chem. 38, 514-515. PARSONS D. F. (1957) Improvement in the procedure for the purification of mitochondrial outer and inner membrane : comparison of the outer membrane with smooth endoplasmic recticulum. In Mitochondrial Structure and Compartmentation (Edited by QuACLIAmELLO E., PAPA S., SLATF.aE. C., & TA6Ea J. M., pp. 29-70. Adriatica Editrica, Bari, Italy. SKIDMOm~ W. D. & ENTENMANN C. (1962) Two-dimensional thin-layer chromatography of rat liver phosphatides..7. Lipid Res. 3, 471-475. SP1aO M. J. & McKIBREN J. M. (1956) Lipids of liver cell fractions. J. biol. Chem. 219, 643-651. VANDEaZANT E. S. & RICHARDSON C. D. (1964) Nutrition of the adult boll weevil: Lipid requirements. `7. Insect Physiol. 10, 267-272. VASKOUSKYV. E. & KOSTETSKYE. Y. (1968) Modified spray for the detection of phospholipids on thin-layer chromatograms. `7. Lipid Res. 9, 396.
Key Word Index--Insect lipid metabolism; pupal lipids; octadecadienoic acid, hexadecanoic acid; octadecenoic acid; lipids; phospholipids; boll weevil; Anthonomus grandis.
C O N S T I T U E N T S OF THE BOLL WEEVIL, A N T H O N O M U S GRANDIS BOHEMAN--III LIPIDS AND FATTY ACIDS OF SUBCELLULAR PARTICLES OF PUPAE* A. C. THOMPSON, R. D. HENSON, R. C. G U E L D N E R and P. A. H E D I N Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture, State College, Mississippi 39762 (Received 9 March 1972) Abstract--1. Phospholipids were the principal lipids in the mierosomes, nuclei and mitochondria of the boll weevil, Anthonomus grandis Boheman. 2. The major fatty acid in the phospholipids of the boll weevil was octadecadienoic acid (18:2) followed by octadecenoic acid (18:1) and hexadecanoic acid (16:0). The weight of neutral lipid varied in the subcellular particles, but the ratios of fatty acids remained the same. 3. Phosphatidyl choline and phosphatidyl ethanolamine were present in equal amounts in the mitochondria, and phosphatidyl choline predominated in the other subcellular particles of the boll weevil. 4. Triglycerides made up about 50 per cent of the neutral lipids of the nuclei, microsomes and cytoplasm. The principal neutral lipids of the cell mitochondria were diglycerides and monoglycerides. INTRODUCTION STUDIES of the phospholipids in the subcellular fractions of animal and animal parts were extensively reviewed by Fleischer & Rouser (1965), but such studies in insects have been limited to the housefly, Musca domestica L. (Crone, 1964; Khan & Hodgson, 1967; Chan, 1970) and to the tobacco horn worm, Manduca sexta (L.) (Chan & Lester, 1970). In the boll weevil, Anthonomus grandis Boheman, the omission of most lipids and lipogenic factors from the diet did not seriously affect egg hatch (Vanderzant & Richardson, 1964) but did reduce egg production by 50 per cent. Oviposition was restored by the addition of cholesterol, vitamins and polyunsaturated fatty acids, particularly the 18:2 and 18:3 acids (Earle et al., 1967). However, subsequent studies of the metabolic conversion of 14C-acetate to fatty acids by the boll weevil showed that most of the radioactivity appeared in the 16:1 and 18:1 monounsaturated acids (Lambremont, 1965). Also, later studies showed that the oleic acid of larvae, pupae and newly molted unfed adult boll weevils which had been * Presented at the spring meeting of the American Oil Chemist's Society Meeting, Houston, Texas. 883
884
A . C . THOMPSON, R. D. HENSON, R. C. GUELDNERAND P. A. HEDIN
treated with 14C-acetate contained 60 per cent of the incorporated radioactivity ( L a m b r e m o n t et al., 1965), and the weevil also desaturated palmitic and stearic acids to the corresponding monounsaturated fatty acids. Our studies of the composition of the phospholipids of adult boll weevils (Henson et al., 1971, 1972) showed that phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) were present in nearly equal quantities in whole insects; cardiolipin and sphingomyelin were next in concentration. Also, the concentration of cardiolipin in the immature stages of the boll weevil was constant, but lower than in the adult. T h e percentage composition of sphingomyelin gradually increased in both larvae and pupae. Phospholipids are primary components of biological membranes and neutral lipids provide energy for normal cell function (Fleicher & Rouser, 1965; Green & Tzagololoff, 1966). However, the role of cardiolipin in the inner membrane of mitochondria (Parsons, 1967; Chan, 1970) has not been elucidated. MATERIALS AND METHODS lnsect material Eggs obtained from boll weevils reared in the laboratory were mechanically planted in a modified cottonseed meal larval diet (Gast & Davich, 1966) that had been mixed with agar, poured into Petri dishes and allowed to gel. The Petri dishes were then held at constant conditions for 9-11 days until pupae were available. The pupae were hand removed from the diet plates and immediately homogenized for fractionation. Preparation of subcellular fractions and lipid extraction Boll weevil pupae were homogenized in twelve times their weight of 0"05 M Tris-HC1 buffer, pH 7"3, containing 0"25 M sucrose, and 0"5 mM EDTA (ethylenediaminetetracetic acid). The homogenate was fractionated by the scheme outlined by Khan & Hodgson (1967). Each subcellular fraction was suspended in the sucrose buffer and centrifuged three times. The cross contamination of the mitochondria and microsomes was found to be less than 10 per cent (Hogboom & Schneider, 1953; Applemans et al., 1955). The lipids from each subcellular fraction were extracted with chloroform-methanol (2 : 1 v/v). The extracts were washed once with 0"05% calcium chloride (Folch et al., 1957), dried over anhydrous sodium sulfate and made to standard volume. Chromatography a. Total lipids. Silicic acid (35 g) was slurried in chloroform and poured into a glass column (2 x 25 cm). Then the total solution (10 ml) was applied to the column. The neutral lipids were eluted with chloroform (200 ml), and the phospholipids with methanol (200 ml). The recovery of phosphorus was 90 per cent. Thin-layer chromatography (TLC) of the phospholipids followed by phosphorus analysis of each component before and after column chromatography accounted for all phospholipid phosphorus. b. Neutral lipids. Florisil containing 8% water (Carroll, 1961) was slurried in cyclohexane and poured into a glass column (3 x 25 cm). The subcellular neutral lipids in cyclohexane were fractionated according to the method of Carroll (1961) except that cyclohexane was substituted for hexane. The column fractions were monitored by T L C (Cmelik, 1969). c. Phospholipids. The phospholipids were separated by two-dimensional T L C in the systems outlined by Henson et al. (1972). The T L C plates were held in a vacuum for 1 hr between development in the first and second directions. The phospholipids were detected
CONSTITUENTSOF THE BOLL WEEVIL--In
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by spraying with molybdate (Vaskousky & Kostetsky, 1968) and quantitatively removed from the plate; the phosphorus was determined by the perchlorate method of Chen et al. (1956) as modified by Mitlin (1960). d. Fatty acid methyl esters. Fatty acid methyl esters were prepared with boron trifluoridemethanol (Metcalf et al., 1966). The qualitative and quantitative estimations of the methyl esters were done with a gas-liquid chromatograph (GLC) equipped with flame ionization on a 6 ftx ] in. O.D. stainless steel column packed with 10"5% DEGA on 60/80-mesh H M D S treated gas Chrom-P. The column temperature was 190°C. RESULTS Lipid content of insects T h e polar lipids (Table 1) were present in greater concentration than the neutral lipids except in the cytoplasmic fraction, which contained considerable quantities of fat bodies and free fatty acids. T h e concentration of polar lipids (found b y weighing the nonvolatiles of the methanol eluate f r o m silicic acid TABLE 1--PERCENTAGE OF POLAR LIPIDS AND NEUTRAL LIPIDS IN THE SUBCELLULAR FRACTIONS OF BOLL WEEVIL PUPAE
Cell fraction Residue Microsomes Nuclei Mitochondria Cytoplasm
Polar lipid
Neutral lipid
27"8 68"5 72"3 62"8 17"4
72"2 31"5 27"7 37"2 82"6
chromatography) was 8.3 m g / g on a fresh weight basis. T h e concentration of phospholipids determined b y phosphorus and fatty acid analysis was 0.2 m g / g fresh weight. Boll weevil pupae contained 3 per cent phospholipid and 13 per cent neutral lipid. T h u s the phospholipids comprised 18 per cent of the total lipids b y weight and 23 per cent by phosphorus and fatty acid analysis. TABLE 2--DISTRIBUTION OF NEUTRAL LIPID IN SUBCELLULARFRACTIONOF BOLL WEEVIL PUPAE Percentage of total neutral lipids of cell fractions Lipid class Sterols Fatty acid esters Triglycerides Diglycerides Monoglycerides Free fatty acids 30
Nuclei 0"4 13-7 43"5 17"8 21"1 3"8
Mitochondria Microsomes 1 "0 3"8 14"5 24"9 38"3 17"5
1-4 5"7 43"7 15"7 10"6 23"4
Cytoplasm 0"9 -47-2 19-2 9-7 23"3
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HENSON,
R. C. GUELDNER AND P. A. HEDIN
Separation of lipid groups T h e distribution of the neutral lipids in the subcellular fractions is shown in T a b l e 2. ( T h e fatty acids esters also included hydrocarbons, but they accounted for less than 1 per cent of these fractions.) I n the nuclei microsomes, and cytoplasm, triglycerides ( T G L ) accounted for 40 per cent of the total neutral lipids. T h e distribution of the phospholipids in the subcellular fractions is shown in T a b l e 3. Phosphatidyl ethanolamine and P C comprise 60-80 per cent of the phospholipids in all fractions. Also, the distribution of these two major phospholipids approximated that observed for the adult insect (Henson et al., 1971). TABLE 3--PHosPHOLIPID CLASSES IN SUBCELLULARFRACTIONS FROM BOLL WEEVIL PUPAE
Percentage of phospholipids in total Subcellular fraction Cell wall Nuclei Mitochondria Microsomes Cytoplasm
LPC*
SPH*
LPE*
PI*
PS*
PC*
PE*
CL*
3.1 5.3 2"3 3"3 --
8.6 8"2 8"5 6"6 12'2
8.6 4.4 3"1 2-5 --
2.6 6.1 3"3 2.8 --
9-0 9-8 4"2 5.7 --
39'4 32"8 36"9 45'1 48"8
25-3 26-5 36-9 34-1 39"0
9.1 6.9 4"8 3.3 --
* LPC, lysophosphatidylcholine; SPH, sphingomyelin; LPE, lysophosphatidylethanolamine; PI, phosphatidyl inositol; PS, phosphatidyl serine ; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine; CL, cardiolipin.
Fatty acids T h e fatty acid composition of the phospholipids (Table 4) of each subcellular fraction was similar to that found in the diet (Table 5). T h e major acid in the phospholipids was 18:2, and 18:1 was next in abundance. T h e phospholipids of T A B L E 4----FATTY ACIDS IN TOTAL P H O S P H O L I P I D S OF SUBCELLULAR PARTICLES OF BOLL WEEVIL PUPAE
Percentage fatty acids in cell fractions* Cell particle Cell wall Nuclei Mitochondria Microsomes Cytoplasm
16:0
16:1
18:0
18:1
18:2
18:3
19.4 7"5 9"2 15"4 25"5
10.8 9"5 9'0 12"8 13-8
10.8 10"9 7"9 17-0 9-5
31"9 13"8 21 "2 22"5 14"8
26"9 52"9 47"4 28"1 36.4
-5'4 5"3 4"2 --
* Sphingomyelin fatty acids are excluded from this table since 1 N KOH does not hydrolyse these compounds.
887
CONSTITUENTS OF THE BOLL W E E V I L - - I l l
the nuclei and the mitochondria were 81"6 and 82.9 per cent unsaturated, respectively. T h e neutral lipids (Table 5) contained four major fatty acids (16:0, 16:1, 18:1 and 18:2) with 18:1 in abundance. The total micromolar composition of fatty acids found in the various neutral lipid classes is given in Table 6. T h e
TABLE 5--FATTY
ACIDS I N THE NEUTRAL L I P I D S
OF SUBCELLULAR FRACTIONS OF BOLL WEEVIL PUPAE
Percentage fatty acids in neutral lipids of cell fractions* Cell fraction
14:0
16:0
16:1
18:0
18:1
18:2
Cell wall Nuclei Mitochondria Microsomes Cytoplasm
1"5 1"8 1.0 1"4 1"0
16"4 21"2 24-2 22-1 20"1
12'3 13"2 12"5 15'6 28"2
5-6 7"0 8"1 8"8 6-2
29"8 40"6 37"9 36"5 32"0
34'5 16'0 15"9 15'4 12"2
*Pupal diet: •4:0 (2'0%); 16:0 (27"4%); 16:1 (1"9%); 18:0 (7"2%); 18:1 (20"9%); 18:2 (40"1%). TABLE 6--TOTAL
/zmoles OF
FATTY ACIDS I N THE NEUTRAL L I P I D CLASSES
OF SUBCELLULAR FRACTIONS OF BOLL WEEVIL PUPAE
/zmoles of fatty acids in neutral lipids Subcellular fraction
SE*
TGL*
DGL*
MGL*
FFA*
Microsomes Mitochondria Cytoplasm Nuclei
1"0 1"3 0"0 2"2
19-6 11"7 115"3 17"8
4"7 13"4 32"4 5"3
1"6 10-3 7"9 3.1
3-5 4"7 19"0 5"2
* SE, sterol esters; TGL, triglycerides; DGL, diglycerides; MGL, monoglycerides; FFA, free fatty acids. distribution of the fatty acids in the mitochondria fraction is given in Table 7. Oleic and palmitic acid were the principal fatty acids of the neutral lipids from cell fractions (Table 5) and, therefore, were the major acids in the neutral lipids of the mitochondria fraction. Since the methyl esters were prepared by a series of reactions which involve basic hydrolysis of the lipid, the fatty acids of the sphingomyelin were not reported in Table 4 because 1 N K O H does not hydrolyze these compounds. However, Henson et aL (1971) reported that 53 per cent of the sphingomyelin fatty acids of boll weevil pupae were 21:1 and 22:0.
888
A.C.
THOMPSON, R. D. HENSON, R. C. GUELDNER AND P. A. HEDIN
TABLE 7--FATTY ACIDS IN THE NEUTRAL LIPIDS OF THE MITOCHONDRIA FRACTION OF B O L L W E E V I L P U P A E
Percentage of fatty acids in mitochondria Fatty acids
SE
TGL
DGL
MGL
FFA
14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3
15.2 -30.7 15.3 7.6 23.0 7.6 --
8.5 3.6 21.4 14.5 8.5 27.3 16.2 --
1-4 0.7 26.4 13-4 8.9 35.8 13.4 --
3.8 1.9 23.5 11.6 9.7 23.3 16.5 9.7
--19.1 -12.7 27.6 40.6 --
DISCUSSION All subcellular fractions of the boll weevil contained both phospholipids and neutral lipids, and the preponderance of triglycerides in the cytoplasm fraction reflected the composition of the fat bodies in this fraction. However, the neutral lipids were present in the subcellular fractions of boll weevil pupae in much higher concentrations than in rat liver: Spiro & McKibben (1956) reported 2.5, 1.4 and 0.0 per cent neutral lipid from the nuclei, mitochondria and microsomes, respectively, of this tissue. This greater concentration of T G L in the microsomes and nuclei and of D G L and M G L in the mitochondria fraction of boll weevil pupae may therefore reflect a greater dependence on these glycerides for transport and energy (Beenakkers & Gilbert, 1968). However, Cmelic (1969) found no M G L or D G L in the neutral lipids of the various organs of immature termites Macrotermes goliath (Sjostedt). The subcellular fractions of boll weevil pupae each contained more PC than PE; this same relationship was reported ( C h a n & Lester, 1970) for muscle mitochondria of the tobacco hornworm. However, in house fly (Musca domestica L.), PE exceeded PC by 50 per cent in all subcellular fractions (Khan & Hodgson, 1957). However, the microsomes of boll weevil pupae (Table 3) contained more PC and PE than the other subcellular particles, which again is in contrast to the distribution in the subcellular particles of the housefly. The concentration of cardiolipin in the mitochondria and microsomes of the boll weevil was similar to that observed by Crone (1964) and Chan (1970) for the mitochondria of the housefly, but lower than that reported by Khan & Hodgson (1967). However, Khan & Hodgson (1967) used iodine as a location agent for cardiolipin (Skidmore & Entenmann, 1962), and this procedure tends to show a high level of cardiolipin since the cardiolipin irrigates with the solvent front in the T L C system along with other phosphorus-containing materials and is not distinguished by color differences. When the lipids were detected with the molybdate spray of Vaskousky & Kostetsky (1968), the cardiolipin appeared as a concise spot.
C O N S T I T U E N T S OF T H E B O L L W E E V I L - - I I I
889
Little is known about the effect of the various types of fatty acids on the solubility and permeability of membranes in insects. However, unsaturation is thought to decrease the molecular packing of the repeating units and thus to allow the interstitial spacing of the overlapping nonpolar fragments to increase in the membrane, which, in turn, would determine the size of molecule that could pass through the membrane. T h e degree of unsaturation therefore exerts a physiochemical behaviour to the phospholipids that have a direct effect on the liquid crystalline characteristics to the membranes controlling the movement of molecular species through the membrane. T h e fatty acids of boll weevil pupae were generally similar to the dietary fatty acids with respect to molecular weight and degree of unsaturation. However, in the diet, 18:1 (21 per cent) and 18:2 (40 per cent) were the major unsaturated dietary fatty acids; in the pupae, 12-30 per cent of the unsaturation appeared in 16:1. T h e fatty acid specificity of the boll weevil neutral lipids was also indicated by the molar ratios of the acids. T h e y were similar in all the neutral lipids from each subcellular fraction. Acknowledgement--The authors thank O. Lindig of this laboratory for providing the insects used in this study. REFERENCES
APPLEMENS F., WATTAUX R. & DE DUVE C. (1955) Tissue fractionation studies. The association of acid phosphatase with a special class of cytoplasmic granules in rat liver. Biochem.oT. 59, 438-445. BEENAKKERSA. M. TH. & GILBERTL. I. (1968) The fatty acid composition of fat body and haemolymph lipids in Hyalophora cecropia and its relation to lipid release. ~. Insect Physiol. 14, 581-594. CARROLK. K. (1961) Separation of lipid classes by chromatography on Florisil. ft. Lipid Res. 2, 135-141. CHAN S. K. (1970) Phospholipid composition in the mitochondria of the housefly, Musea domestica: a re-examination, ft. Insect Physiol. 16, 1575-1577. CHAN S. K. & LESTERR. L. (1970) Biochemical studies on the developing thoracic muscles of the tobacco horn worm--II. Phospholipid composition in mitochondria during development. Biochim. biophys. Acta 210, 180-181. CHEN P. S., TORABARST. Y. & WARNER H. (1956) Microdetermination of phosphorus. Analyt. Chem. 28, 1756. CMELIK S. H. W. (1969) The neutral lipids from various organs of the termite Macrotermes goliath, ft. Insect Physiol. 15, 839-849. CRONE H. D. (1964) Phospholipid composition of flight muscles sarcosomes from the housefly, Musea domestica L. aT. Insect Physiol. 10, 499-507. EARL N. W., SLATTENB. & BURKSM. L. (1967) Essential fatty acids in the diet of the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae)..7- Insect Physiol. 13, 187-200. FLEICHERS. & ROUSERG. (1965) Lipids of subcellular particles, uTAOCS 42, 588-607. FOLCH J., LEES M. & SLOANE-STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissue. 3¢. biol. Chem. 226, 497-509. GASTR. T. & DAVICHT. B. (1966) Insect Colonization and Mass Production (Edited by SMITH C. N.), pp. 405-518. Academic Press, New York.
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Key Word Index--Insect lipid metabolism; pupal lipids; octadecadienoic acid, hexadecanoic acid; octadecenoic acid; lipids; phospholipids; boll weevil; Anthonomus grandis.