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The lipid-class spectrum and fatty-acid content of the boll weevil egg
Comp. Biochem. Physiol., 1966, VoL 18, pp. 975 to 981. Pergamon Press Ltd. Printed in Great Britain
SHORT COMMUNICATION THE LIPID-CLASS SPECTRUM AND FATTY-ACID C O N T E N T OF T H E BOLL WEEVIL EGG* J O S E P H E. B U M G A R N E R t and E D W A R D N. L A M B R E M O N T : ~ Department of Entomology and Entomology Research Division, Agricultural Research Service, U.S.D.A., Louisiana State University, Baton Rouge, Louisiana, U.S.A. (Received 7 March 1966)
A b s t r a c t - - 1 . Lipids from eggs of the boll weevil (Anthonomus grand.is Boheman;
Coleoptera, Curculionidae) were fractionated into individual classes by a combination of silicie acid and Florisil column chromatography. Triglycerides were the predominant fraction, and accounted for more than 70 per cent of the neutral lipid matter. 2. The fatty acid content of adult males and females and eggs resembles that of the diet when dietary fat content is high. Females feeding on low fat diets deposit lipids in the egg yolk which contain principally those fatty acids that the adult can synthesize from non-lipid components. INTRODUCTION THE lipid composition of insect eggs is an area of study that has been much neglected. T h e small amounts of material with which to work has in the past confined such studies to gravimetric measurements of the total lipid content of the egg as compared with its non-lipid constituents. Only recently, with more refined analytical techniques becoming available, can the chemical composition of insectegg lipids be studied in greater detail. Babcock & Rutschky (1961) in their review pointed out that, as a general rule, the eggs of terrestrial insects are characterized by high lipid content. During embryogenesis in most species, there is a progressive decrease in fat content of the egg, suggesting that fats serve as a source of energy. T h e s e decreases are usually associated with the neutral lipid fractions, which contain fatty acids, and * A portion of this work was supported by Public Health Service Research Grant No. AI 05881-TMP from the National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, U.S.A., in cooperation with the Louisiana Agricultural Experiment Station. We are indebted to Dr. L. D. Newsom of the Department of Entomology, Louisiana State University, for his active support of this work, and to Ann Bennett and Ronda Frayer, Entomology Research Division, U.S.D.A., for technical assistance. We thank Dr. N. W. Earle of this Division for supplying eggs from the cotton flower bud diet. t Graduate Assistant, Department of Entomology, Louisiana State University. Entomologist, Entomology Research Division, Agricultural Research Service, U.S.D.A. 975
976
JOSEPH E. BUMGARNER AND EDWARD N . LAMBREMONT
not with bound lipids, such as phospholipids or unsaponifiable lipids. In some species, sequential catabolic changes in carbohydrate, then protein, and finally fat have been observed during growth of the embryo. Our interest in egg lipids in the boll weevil (Anthonomus grandis Boheman; Coleoptera, Curculionidae) was prompted by several prior reports. Vanderzant & Richardson (1964) reported that the boll weevil required lipids and lipogenic factors for optimum egg production, and that this requirement could be partially satisfied by polyunsaturated fatty acids. Addition of either linoleic or linolenic acid increased the number of eggs and the females lived longer. Earle et al. (1966) showed that boll weevil larvae required linoleic acid, and that as little as 0.8/~g of linoleic acid carried over from the parent generation through the egg was enough to satisfy the essential fatty-acid requirements of the larva. Lambremont (1965) and Lambremont et al. (1965) showed that the boll weevil could produce at best a trace of linoleic acid, and very small quantities of linolenic acid from C14-1abeled acetate. In addition, these workers suspected that the small amount of C 14 radioactivity found in the 18 carbon polyunsaturated acids from insects injected with labeled acetate was due to the formation of gamma linoleic and gamma linolenic acid. It is generally accepted that essential fatty-acid activity resides only in the alpha isomer of linoleic acid which can be formed only by plants and some protozoa (Mead, 1960; Erwin et al., 1964). The present paper summarizes our initial efforts in this field and forms a basis for more elaborate studies. We wished to determine the gross lipid-class spectrum of the boll weevil egg and the distribution of fatty acids to total fat and isolated triglycerides. We also wished to evaluate the influence of the female's dietary fatty acids upon those deposited in the egg yolk. MATERIALS AND METHODS Insect material Most of the insect eggs, adults and diet used in this study were obtained from the Boll Weevil Laboratory, State College, Mississippi, U.S.A., through the kindness of Dr. Robert Gast. The adult oviposition colony maintained at this laboratory was reared as larvae on a modification of a diet described by Vanderzant & Davich (1958) and contained soybean protein and acetone powder of cotton flower buds as the principal sources of fatty acids. The adults were maintained on a diet containing acetone powder of cotton flower buds, soybean protein and germinated cottonseed as sources of fatty acids. The eggs at the time of collection were not over 16 hr old. They were preserved for later work by placing them in a solution of chloroform : methanol (2 : 1 v/v), and were stored in this solution at 20°C. _
Preparation and analysis of total lipid extract The eggs were thoroughly macerated in the chloroform : methanol solution in which they had been stored. Broken chorions and other non-lipid matter were removed by filtration through sintered glass, and the filtrate mixed with 2()0,i~ of
L I P I D S AND FATTY ACID CONTENT OF BOLL WEEVIL EGGS
977
its volume of aqueous 0.034% magnesium chloride solution. After the two phases separated, the upper phase was discarded and the surface of the lower phase was washed carefully by adding and removing 5 ml portions of equilibrated upper phase rinse (Burchfield & Storrs, 1961). The chloroform phase was vacuum evaporated, and the lipid residue dissolved in 5 ml n-hexane and added to a 4 g silicic acid column. The neutral lipids were eluted in chloroform, and the phospholipids were recovered in a 100 ml portion of methanol. The neutral lipids were further resolved into individual lipid classes by column chromatography on Florisil ® as described by Carroll (1961). The following fractions were obtained: hydrocarbons, sterol esters, triglycerides, free sterols, diglycerides, monoglycerides and free fatty acids. Isolated classes were placed in a vacuum oven at 40°C in tared tubes and the solvents removed. The tubes were reweighed to obtain weights of the individual classes. Efficiency of separating these classes of compounds was checked by thin-layer chromatography by the double-development technique described by Lambremont et al. (1966). Lipids were extracted from adult males and females and the diet by the procedure just described. The protective paraffin coating on the adult diet pellets was discarded before extracting lipids from the diet.
Analysis of fatty acids by gas-liquid chromatography (GLC) The conditions used for GLC analysis of the fatty acids were as described by Lambremont et al. (1965). The performance of the chromatograph was checked daily by analyzing standard mixtures of fatty-acid methyl esters. Its quantitative accuracy was determined periodically by analyzing Standard Fatty Acid Methyl Ester "Mixture E" sent from the Metabolism Study Section, National Institutes of Health, Bethesda, Maryland, U.S.A., through the courtesy of Dr. William H. Goldwater. Fatty acids were identified by comparing their relative retention ratios to those of authentic standards. RESULTS T A B L E 1 - - E S T I M A T E D C O M P O S I T I O N OF TOTAL UNFRACTIONATED L I P I D S AND N~UTRAL L I P I D S OF BOLL WEEVIL EGGS
Wet weight of eggs Weight of lipids Weight of phospholipids Weight of neutral lipids
Grams
Per cent
31-22 2"39* 0"71 1'68
7"7 2"3 5'4
* Calculated from weights of neutral lipids and phospholipids. Table 1 is a summary of the gross distribution of neutral lipid and phospholipid contained in a sample of boll weevil eggs. In this sample, 7.7 per cent of the wet weight of the eggs was lipid material, with the neutral lipids representing the larger
978
JOSEPH E. BUMGARNER AND EDWARD N . LAMBREMONT
portion. Since there is a wide variability in the lipid content of insect eggs as summarized by Fast (1964), ranging from 1.5 per cent to 26.5 per cent of the wet weight of the egg, it is difficult to find a firm basis of comparison with other insects. Within any given sample from the same species, moisture differences of eggs held under differing conditions, or deposited in various types of oviposition media could significantly affect these estimates. Nevertheless, the gross lipid composition of this particular sample of boll weevil eggs falls within accepted limits. TABLE 2--NEtlTRAL LIPID CLASSES OF THE BOLL WEEVIL EGG (AVERAGE OF 3 DETERMINATIONS)
Lipid class Hydrocarbons Sterol esters Triglycerides Sterols* Diglycerides Monoglycerides~ Free fatty acids
Weight (mg) 5"8 5-8 55.4 4'0 0"6 1"6 4"9
Range 4"1-7"2 4-7-6'5 53"5-57-4 2"9-4-7 0"3-0"8 0-4"7 3-6-7"0
Composition (%) 7"4 7-4 70-9 5"1 0"7 2"0 6"2
* Sterols separated by Florisil column chromatography are known to contain some diglyceride (Carroll, 1961). I" Two of the three analyses showed no monoglycerides. Table 2 is a summary of a gravimetric analysis of the neutral lipid classes obtained from three samples of eggs. It is clear that the triglycerides are the dominant fraction which strongly suggests that they serve to store fatty acids in the yolk. Triglycerides alone account for more than 70 per cent of the neutral lipid matter. Sterols, sterol esters, hydrocarbons and free fatty acids occupy a secondary position and together account for about 20 per cent of the neutral lipids, while mono- and diglycerides make up the remainder at slightly less than 3 per cent of the total. These results compare favorably with the neutral lipid spectrum of housefly eggs, Musca domestica L., which had 84 per cent triglyceride and 12.7 per cent as sterol and sterol ester (Dutky et al., 1963). In the boll weevil egg, approximately 60 per cent of the total sterol was esterified, whereas in the house-fly egg this fraction constituted about 40 per cent. An analysis of the fatty-acid content of the triglyceride fraction obtained from adult males and females, their diet and eggs is presented in Table 3. All groups contain three major components, palmitic acid, oleic acid and linoleic acid, which together in the insects and their eggs account for about 80 per cent of the fatty acids. In the diet, the three major acids represent more than 92 per cent of the fatty acids. Short-chain acids and other minor components (which are not shown in the Table) make up the remainder. The short-chain components comprise from 10 to 13 different acids, some of which are not found consistently in every sample.
979
LIPIDS AND FATTYACID CONTENTOF BOLLWEEVILEGGS
A close correlation existed in the triglyceride-fatty-acid content of the males and females with the possible exception of stearic and palmitoleic acid, which were significantly lower in males. T h e fatty-acid spectrum of the adults reflects that of the diet, but is not an exact duplication. T h e spectrum of the eggs also resembles that of the females, but of the three principal fatty acids, oleic acid is lower, while palmitic and linoleic acids are higher in the eggs. TABLE 3nMETHYL ESTERSOF TRIGLYCERIDEFATTYACIDS(COMPOSITION~o)
Fatty acids C16 (palmitic) C16:1 (palmitoleic) C1~ (heptadecanoic) Cls (stearic) Ct8:1 (oleic) Cls:z (linoleie) Cxs:a (linolenic)
Adult male Adult female boll weevil boll weevil 27.3 2-2 0"3 3"8 26"4 30-1 Trace
27.5 3"9 0"4 6"7 25"1 30"2 Trace
Boll weevil eggs
Adult boll weevil diet (uncoated)
31.2 0"7 0"4 2-7 11-4 37"1 Trace
24.2 0.5 0-1 2"3 17"8 50"4 Trace
TABLE ~ METHYLESTERSOF TOTALFATTYACIDS OF BOLLWEEVILEGGS(COMPOSITION~/o) FROMADULTSFEEDINGON TWO DIFFERENTDIETS Total fatty acids in eggs (%) Fatty acids
Cotton flower bud diet
Germinated cotton seed diet
Cls (palmitic) Cxe:l (palmitoleic) C17 (heptadecanoic) Cls (stearic) Cls:t (oleic) Cls:z (linoleic) Cls:a (linolenic)
26.8 13"1 2"2 6"8 49"4 3"4 Trace
26.8 2"4 0-4 8"8 18"7 42"1 0'6
T o determine further the influence of total dietary fat on the pattern of fatty acids deposited in the egg, an analysis of eggs from females on a low- and a high-fat diet was made. T h e low-fat diet was prepared from constituents that had been exhaustively extracted with chloroform : methanol (2 : 1) which removed most of the lipids. Trilinolein was then added to the diet to 0.1 per cent concentration as the principal fatty-acid constitutent (Earle et al., 1966). T h e trilinolein was 9 9 + per cent pure as stated by the manufacturer, however G L C revealed 1.5 per cent palmitic and 0.2 per cent oleic acid to be present. T h e germinated cottonseed diet described earlier (see Table 3) was the high-fat diet. Analysis of total lipids in a
980
JOSEPH E. BUMGARNER AND EDWARD N. LAMBREMONT
sample of the germinated cottonseed diet showed that it contained approximately 2.0 per cent fat, a twentyfold increase over the low-fat diet. A striking difference was observed in the fatty-acid content of eggs from the two diets. This analysis differed from that in Table 3 because the total fatty acids were analyzed rather than just the triglyceride fatty acids. The results, presented in Table 4, show that in the low-fat diet linoleic acid accounts for only 3.4 per cent, whereas in the high-fat diet it represents 42.1 per cent of the total fatty acids. The principal components in the low-fat diet are saturated and mono-unsaturated 16and 18-carbon acids, which are those known to be synthesized by this insect from non-lipid constituents of the diet (Lambremont, 1965 ; Lambremont et al., 1965). Eggs from the high-fat diet reflect a combination of dietary fatty acids plus those synthesized by the females. DISCUSSION The high neutral lipid content of the egg represented predominately by triglycerides is consistent with the idea that fatty acids are stored in the yolk and are utilized for various metabolic functions during embryogenesis. It would seem logical that the fatty acids of the egg would not remain in the relatively inert triglyceride fraction, but would be released during development to more metabolically active lipid compounds. It would also be expected that the structural phospholipids of the developing larva would obtain the required complement of fatty acids from breakdown of the triglyceride fraction. The correlation between the fatty acids of adults, their eggs and the diet on which they were feeding agrees with other published observations. Blackith & Howden (1961) described the direct influence of the female's diet on the lipid content of newly hatched locusts, and also showed that wide variations could occur in the fat content in the eggs of the same species. Several factors in the boll weevil can exert an influence on the type of fatty acid available for deposition in the yolk. Because the adult can synthesize some of its own fatty acids (Lambremont, 1965), these are the ones most likely to appear in the body fat and egg yolk of females feeding on a low-fat diet. Those that the adult cannot synthesize must come directly from the diet. This situation exists in the boll weevil, which incorporates significantly high amounts of linoleic acid from a diet rich in this component, and much lower amounts when it is lower in the diet. The data in Table 3 suggest a selective deposition of linoleic acid in the egg yolk, since the egg fat has a higher linoleic content than the female's body fat. However, wide variations in this acid do exist, and even the lower quantities in eggs from the low-fat diet were sufficient for normal development of the embryo. REFERENCES BABCOCK K. L. & RUTSCHKYC. W. (1961) Lipids in insect eggs: A review with new evidence from the milkweed bug Oncopeltusfasciatus (Hemiptera, Lygaeidae). Ann. ent. Soc. Am. 54, 156-165. BLACKITHR. E. & HOWDENG. F. (1961) The food reserves of hatchling locusts. Comp. Biochem. Physiol. 3, 108-124.
L I P I D S A N D F A T T Y A C I D C O N T E N T O F B O L L W E E V I L EGGS
981
BURCHFIELDH. P. & STORRS E. E. (1961) Biochemical Applications of Gas Chromatography. Academic Press, New York and London. CARROLL K. K. (1961) Separation of lipid classes by chromatography on Florisil. 07. Lipid Res. 2, 135-141. DUTKY R. C., ROBBINSW. E., KAPLaNIS J. N. & SHORTINOT. J. (1963) The sterol esters of housefly eggs. Comp. Biochem. Physiol. 9, 251-255. EARLE N. W., SLATTENB. S. & BURKS M. L. (1966) Essential fatty acids in the diet of the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae). (Unpublished observations.) ERWIN J., HUL~ICKa B. & BLOCH K. (1964) Comparative aspects of unsaturated fatty acid synthesis. Comp. Biochem. Physiol. 12, 191-207. FAST P. G. (1964) Insect lipids: A review. Mem. ent. Soc. Canada 37, 1-50. LAMBREMONTE. N. (1965) Biosynthesis of fatty acids in aseptically reared insects. Comp. Biochem. Physiol. 14, 419-424. LAMBREMONTE. N., BUMC~'qER J. E. & BENNETT A. F. (1966) Lipid biosynthesis in the boll weevil (Coleoptera: Curculionidae): Distribution of radioactivity in the principal lipid classes synthesized from C14-1-acetate. Comp. Biochem. Physiol. (To be published.) LAMBREMONTE. N., STEIN C. I. & BENNETTA. F. (1965) Synthesis and metabolic conversion of fatty acids by the larval boll weevil. Comp. Biochem. Physiol. 16, 289-302. MEAD J. F. (1960) The metabolism of unsaturated fatty acids. In Lipide Metabolism (Edited by BLOCH K.), pp. 41-66. Wiley, New York. VANDEBZANTE. S. & DAVlCH T. B. (1958) Laboratory rearing of the boll weevil: A satisfactory larval diet and oviposition studies. 07. econ. Ent. 51, 288-291. VANDEBZ~'~TE. S. & RICHARDSONC. D. (1964) Nutrition of the adult boll weevil: Lipid requirements. 07. Insect Physiol. 10, 267-272.
SHORT COMMUNICATION THE LIPID-CLASS SPECTRUM AND FATTY-ACID C O N T E N T OF T H E BOLL WEEVIL EGG* J O S E P H E. B U M G A R N E R t and E D W A R D N. L A M B R E M O N T : ~ Department of Entomology and Entomology Research Division, Agricultural Research Service, U.S.D.A., Louisiana State University, Baton Rouge, Louisiana, U.S.A. (Received 7 March 1966)
A b s t r a c t - - 1 . Lipids from eggs of the boll weevil (Anthonomus grand.is Boheman;
Coleoptera, Curculionidae) were fractionated into individual classes by a combination of silicie acid and Florisil column chromatography. Triglycerides were the predominant fraction, and accounted for more than 70 per cent of the neutral lipid matter. 2. The fatty acid content of adult males and females and eggs resembles that of the diet when dietary fat content is high. Females feeding on low fat diets deposit lipids in the egg yolk which contain principally those fatty acids that the adult can synthesize from non-lipid components. INTRODUCTION THE lipid composition of insect eggs is an area of study that has been much neglected. T h e small amounts of material with which to work has in the past confined such studies to gravimetric measurements of the total lipid content of the egg as compared with its non-lipid constituents. Only recently, with more refined analytical techniques becoming available, can the chemical composition of insectegg lipids be studied in greater detail. Babcock & Rutschky (1961) in their review pointed out that, as a general rule, the eggs of terrestrial insects are characterized by high lipid content. During embryogenesis in most species, there is a progressive decrease in fat content of the egg, suggesting that fats serve as a source of energy. T h e s e decreases are usually associated with the neutral lipid fractions, which contain fatty acids, and * A portion of this work was supported by Public Health Service Research Grant No. AI 05881-TMP from the National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, U.S.A., in cooperation with the Louisiana Agricultural Experiment Station. We are indebted to Dr. L. D. Newsom of the Department of Entomology, Louisiana State University, for his active support of this work, and to Ann Bennett and Ronda Frayer, Entomology Research Division, U.S.D.A., for technical assistance. We thank Dr. N. W. Earle of this Division for supplying eggs from the cotton flower bud diet. t Graduate Assistant, Department of Entomology, Louisiana State University. Entomologist, Entomology Research Division, Agricultural Research Service, U.S.D.A. 975
976
JOSEPH E. BUMGARNER AND EDWARD N . LAMBREMONT
not with bound lipids, such as phospholipids or unsaponifiable lipids. In some species, sequential catabolic changes in carbohydrate, then protein, and finally fat have been observed during growth of the embryo. Our interest in egg lipids in the boll weevil (Anthonomus grandis Boheman; Coleoptera, Curculionidae) was prompted by several prior reports. Vanderzant & Richardson (1964) reported that the boll weevil required lipids and lipogenic factors for optimum egg production, and that this requirement could be partially satisfied by polyunsaturated fatty acids. Addition of either linoleic or linolenic acid increased the number of eggs and the females lived longer. Earle et al. (1966) showed that boll weevil larvae required linoleic acid, and that as little as 0.8/~g of linoleic acid carried over from the parent generation through the egg was enough to satisfy the essential fatty-acid requirements of the larva. Lambremont (1965) and Lambremont et al. (1965) showed that the boll weevil could produce at best a trace of linoleic acid, and very small quantities of linolenic acid from C14-1abeled acetate. In addition, these workers suspected that the small amount of C 14 radioactivity found in the 18 carbon polyunsaturated acids from insects injected with labeled acetate was due to the formation of gamma linoleic and gamma linolenic acid. It is generally accepted that essential fatty-acid activity resides only in the alpha isomer of linoleic acid which can be formed only by plants and some protozoa (Mead, 1960; Erwin et al., 1964). The present paper summarizes our initial efforts in this field and forms a basis for more elaborate studies. We wished to determine the gross lipid-class spectrum of the boll weevil egg and the distribution of fatty acids to total fat and isolated triglycerides. We also wished to evaluate the influence of the female's dietary fatty acids upon those deposited in the egg yolk. MATERIALS AND METHODS Insect material Most of the insect eggs, adults and diet used in this study were obtained from the Boll Weevil Laboratory, State College, Mississippi, U.S.A., through the kindness of Dr. Robert Gast. The adult oviposition colony maintained at this laboratory was reared as larvae on a modification of a diet described by Vanderzant & Davich (1958) and contained soybean protein and acetone powder of cotton flower buds as the principal sources of fatty acids. The adults were maintained on a diet containing acetone powder of cotton flower buds, soybean protein and germinated cottonseed as sources of fatty acids. The eggs at the time of collection were not over 16 hr old. They were preserved for later work by placing them in a solution of chloroform : methanol (2 : 1 v/v), and were stored in this solution at 20°C. _
Preparation and analysis of total lipid extract The eggs were thoroughly macerated in the chloroform : methanol solution in which they had been stored. Broken chorions and other non-lipid matter were removed by filtration through sintered glass, and the filtrate mixed with 2()0,i~ of
L I P I D S AND FATTY ACID CONTENT OF BOLL WEEVIL EGGS
977
its volume of aqueous 0.034% magnesium chloride solution. After the two phases separated, the upper phase was discarded and the surface of the lower phase was washed carefully by adding and removing 5 ml portions of equilibrated upper phase rinse (Burchfield & Storrs, 1961). The chloroform phase was vacuum evaporated, and the lipid residue dissolved in 5 ml n-hexane and added to a 4 g silicic acid column. The neutral lipids were eluted in chloroform, and the phospholipids were recovered in a 100 ml portion of methanol. The neutral lipids were further resolved into individual lipid classes by column chromatography on Florisil ® as described by Carroll (1961). The following fractions were obtained: hydrocarbons, sterol esters, triglycerides, free sterols, diglycerides, monoglycerides and free fatty acids. Isolated classes were placed in a vacuum oven at 40°C in tared tubes and the solvents removed. The tubes were reweighed to obtain weights of the individual classes. Efficiency of separating these classes of compounds was checked by thin-layer chromatography by the double-development technique described by Lambremont et al. (1966). Lipids were extracted from adult males and females and the diet by the procedure just described. The protective paraffin coating on the adult diet pellets was discarded before extracting lipids from the diet.
Analysis of fatty acids by gas-liquid chromatography (GLC) The conditions used for GLC analysis of the fatty acids were as described by Lambremont et al. (1965). The performance of the chromatograph was checked daily by analyzing standard mixtures of fatty-acid methyl esters. Its quantitative accuracy was determined periodically by analyzing Standard Fatty Acid Methyl Ester "Mixture E" sent from the Metabolism Study Section, National Institutes of Health, Bethesda, Maryland, U.S.A., through the courtesy of Dr. William H. Goldwater. Fatty acids were identified by comparing their relative retention ratios to those of authentic standards. RESULTS T A B L E 1 - - E S T I M A T E D C O M P O S I T I O N OF TOTAL UNFRACTIONATED L I P I D S AND N~UTRAL L I P I D S OF BOLL WEEVIL EGGS
Wet weight of eggs Weight of lipids Weight of phospholipids Weight of neutral lipids
Grams
Per cent
31-22 2"39* 0"71 1'68
7"7 2"3 5'4
* Calculated from weights of neutral lipids and phospholipids. Table 1 is a summary of the gross distribution of neutral lipid and phospholipid contained in a sample of boll weevil eggs. In this sample, 7.7 per cent of the wet weight of the eggs was lipid material, with the neutral lipids representing the larger
978
JOSEPH E. BUMGARNER AND EDWARD N . LAMBREMONT
portion. Since there is a wide variability in the lipid content of insect eggs as summarized by Fast (1964), ranging from 1.5 per cent to 26.5 per cent of the wet weight of the egg, it is difficult to find a firm basis of comparison with other insects. Within any given sample from the same species, moisture differences of eggs held under differing conditions, or deposited in various types of oviposition media could significantly affect these estimates. Nevertheless, the gross lipid composition of this particular sample of boll weevil eggs falls within accepted limits. TABLE 2--NEtlTRAL LIPID CLASSES OF THE BOLL WEEVIL EGG (AVERAGE OF 3 DETERMINATIONS)
Lipid class Hydrocarbons Sterol esters Triglycerides Sterols* Diglycerides Monoglycerides~ Free fatty acids
Weight (mg) 5"8 5-8 55.4 4'0 0"6 1"6 4"9
Range 4"1-7"2 4-7-6'5 53"5-57-4 2"9-4-7 0"3-0"8 0-4"7 3-6-7"0
Composition (%) 7"4 7-4 70-9 5"1 0"7 2"0 6"2
* Sterols separated by Florisil column chromatography are known to contain some diglyceride (Carroll, 1961). I" Two of the three analyses showed no monoglycerides. Table 2 is a summary of a gravimetric analysis of the neutral lipid classes obtained from three samples of eggs. It is clear that the triglycerides are the dominant fraction which strongly suggests that they serve to store fatty acids in the yolk. Triglycerides alone account for more than 70 per cent of the neutral lipid matter. Sterols, sterol esters, hydrocarbons and free fatty acids occupy a secondary position and together account for about 20 per cent of the neutral lipids, while mono- and diglycerides make up the remainder at slightly less than 3 per cent of the total. These results compare favorably with the neutral lipid spectrum of housefly eggs, Musca domestica L., which had 84 per cent triglyceride and 12.7 per cent as sterol and sterol ester (Dutky et al., 1963). In the boll weevil egg, approximately 60 per cent of the total sterol was esterified, whereas in the house-fly egg this fraction constituted about 40 per cent. An analysis of the fatty-acid content of the triglyceride fraction obtained from adult males and females, their diet and eggs is presented in Table 3. All groups contain three major components, palmitic acid, oleic acid and linoleic acid, which together in the insects and their eggs account for about 80 per cent of the fatty acids. In the diet, the three major acids represent more than 92 per cent of the fatty acids. Short-chain acids and other minor components (which are not shown in the Table) make up the remainder. The short-chain components comprise from 10 to 13 different acids, some of which are not found consistently in every sample.
979
LIPIDS AND FATTYACID CONTENTOF BOLLWEEVILEGGS
A close correlation existed in the triglyceride-fatty-acid content of the males and females with the possible exception of stearic and palmitoleic acid, which were significantly lower in males. T h e fatty-acid spectrum of the adults reflects that of the diet, but is not an exact duplication. T h e spectrum of the eggs also resembles that of the females, but of the three principal fatty acids, oleic acid is lower, while palmitic and linoleic acids are higher in the eggs. TABLE 3nMETHYL ESTERSOF TRIGLYCERIDEFATTYACIDS(COMPOSITION~o)
Fatty acids C16 (palmitic) C16:1 (palmitoleic) C1~ (heptadecanoic) Cls (stearic) Ct8:1 (oleic) Cls:z (linoleie) Cxs:a (linolenic)
Adult male Adult female boll weevil boll weevil 27.3 2-2 0"3 3"8 26"4 30-1 Trace
27.5 3"9 0"4 6"7 25"1 30"2 Trace
Boll weevil eggs
Adult boll weevil diet (uncoated)
31.2 0"7 0"4 2-7 11-4 37"1 Trace
24.2 0.5 0-1 2"3 17"8 50"4 Trace
TABLE ~ METHYLESTERSOF TOTALFATTYACIDS OF BOLLWEEVILEGGS(COMPOSITION~/o) FROMADULTSFEEDINGON TWO DIFFERENTDIETS Total fatty acids in eggs (%) Fatty acids
Cotton flower bud diet
Germinated cotton seed diet
Cls (palmitic) Cxe:l (palmitoleic) C17 (heptadecanoic) Cls (stearic) Cls:t (oleic) Cls:z (linoleic) Cls:a (linolenic)
26.8 13"1 2"2 6"8 49"4 3"4 Trace
26.8 2"4 0-4 8"8 18"7 42"1 0'6
T o determine further the influence of total dietary fat on the pattern of fatty acids deposited in the egg, an analysis of eggs from females on a low- and a high-fat diet was made. T h e low-fat diet was prepared from constituents that had been exhaustively extracted with chloroform : methanol (2 : 1) which removed most of the lipids. Trilinolein was then added to the diet to 0.1 per cent concentration as the principal fatty-acid constitutent (Earle et al., 1966). T h e trilinolein was 9 9 + per cent pure as stated by the manufacturer, however G L C revealed 1.5 per cent palmitic and 0.2 per cent oleic acid to be present. T h e germinated cottonseed diet described earlier (see Table 3) was the high-fat diet. Analysis of total lipids in a
980
JOSEPH E. BUMGARNER AND EDWARD N. LAMBREMONT
sample of the germinated cottonseed diet showed that it contained approximately 2.0 per cent fat, a twentyfold increase over the low-fat diet. A striking difference was observed in the fatty-acid content of eggs from the two diets. This analysis differed from that in Table 3 because the total fatty acids were analyzed rather than just the triglyceride fatty acids. The results, presented in Table 4, show that in the low-fat diet linoleic acid accounts for only 3.4 per cent, whereas in the high-fat diet it represents 42.1 per cent of the total fatty acids. The principal components in the low-fat diet are saturated and mono-unsaturated 16and 18-carbon acids, which are those known to be synthesized by this insect from non-lipid constituents of the diet (Lambremont, 1965 ; Lambremont et al., 1965). Eggs from the high-fat diet reflect a combination of dietary fatty acids plus those synthesized by the females. DISCUSSION The high neutral lipid content of the egg represented predominately by triglycerides is consistent with the idea that fatty acids are stored in the yolk and are utilized for various metabolic functions during embryogenesis. It would seem logical that the fatty acids of the egg would not remain in the relatively inert triglyceride fraction, but would be released during development to more metabolically active lipid compounds. It would also be expected that the structural phospholipids of the developing larva would obtain the required complement of fatty acids from breakdown of the triglyceride fraction. The correlation between the fatty acids of adults, their eggs and the diet on which they were feeding agrees with other published observations. Blackith & Howden (1961) described the direct influence of the female's diet on the lipid content of newly hatched locusts, and also showed that wide variations could occur in the fat content in the eggs of the same species. Several factors in the boll weevil can exert an influence on the type of fatty acid available for deposition in the yolk. Because the adult can synthesize some of its own fatty acids (Lambremont, 1965), these are the ones most likely to appear in the body fat and egg yolk of females feeding on a low-fat diet. Those that the adult cannot synthesize must come directly from the diet. This situation exists in the boll weevil, which incorporates significantly high amounts of linoleic acid from a diet rich in this component, and much lower amounts when it is lower in the diet. The data in Table 3 suggest a selective deposition of linoleic acid in the egg yolk, since the egg fat has a higher linoleic content than the female's body fat. However, wide variations in this acid do exist, and even the lower quantities in eggs from the low-fat diet were sufficient for normal development of the embryo. REFERENCES BABCOCK K. L. & RUTSCHKYC. W. (1961) Lipids in insect eggs: A review with new evidence from the milkweed bug Oncopeltusfasciatus (Hemiptera, Lygaeidae). Ann. ent. Soc. Am. 54, 156-165. BLACKITHR. E. & HOWDENG. F. (1961) The food reserves of hatchling locusts. Comp. Biochem. Physiol. 3, 108-124.
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BURCHFIELDH. P. & STORRS E. E. (1961) Biochemical Applications of Gas Chromatography. Academic Press, New York and London. CARROLL K. K. (1961) Separation of lipid classes by chromatography on Florisil. 07. Lipid Res. 2, 135-141. DUTKY R. C., ROBBINSW. E., KAPLaNIS J. N. & SHORTINOT. J. (1963) The sterol esters of housefly eggs. Comp. Biochem. Physiol. 9, 251-255. EARLE N. W., SLATTENB. S. & BURKS M. L. (1966) Essential fatty acids in the diet of the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae). (Unpublished observations.) ERWIN J., HUL~ICKa B. & BLOCH K. (1964) Comparative aspects of unsaturated fatty acid synthesis. Comp. Biochem. Physiol. 12, 191-207. FAST P. G. (1964) Insect lipids: A review. Mem. ent. Soc. Canada 37, 1-50. LAMBREMONTE. N. (1965) Biosynthesis of fatty acids in aseptically reared insects. Comp. Biochem. Physiol. 14, 419-424. LAMBREMONTE. N., BUMC~'qER J. E. & BENNETT A. F. (1966) Lipid biosynthesis in the boll weevil (Coleoptera: Curculionidae): Distribution of radioactivity in the principal lipid classes synthesized from C14-1-acetate. Comp. Biochem. Physiol. (To be published.) LAMBREMONTE. N., STEIN C. I. & BENNETTA. F. (1965) Synthesis and metabolic conversion of fatty acids by the larval boll weevil. Comp. Biochem. Physiol. 16, 289-302. MEAD J. F. (1960) The metabolism of unsaturated fatty acids. In Lipide Metabolism (Edited by BLOCH K.), pp. 41-66. Wiley, New York. VANDEBZANTE. S. & DAVlCH T. B. (1958) Laboratory rearing of the boll weevil: A satisfactory larval diet and oviposition studies. 07. econ. Ent. 51, 288-291. VANDEBZ~'~TE. S. & RICHARDSONC. D. (1964) Nutrition of the adult boll weevil: Lipid requirements. 07. Insect Physiol. 10, 267-272.