JOURNAL
OF INVERTEBRATE
Neutral
PATHOLOGY
Lipids
27, 161-169
of Aedes
(1976)
aegypti
Cells Cultured T. K. YANG,
E. McMEANS,
The Hormel Institute,
and Aedes in Vitro
L. E. ANDERSON,
University Received
of Minnesota, March
albopictus
AND
H. M. JENKIN
Austin, Minnesota55912
3, 1975
Neutral lipids were examined from spinner culture cells of two species of mosquitoes, Aedes aegypti and A. albopictus, in exponential and stationary phases of growth. The lipids were separated into constituent classes by thin-layer chromatography. The fatty acid profiles of the methyl esters of each class were determined by gas-liquid chromatography. There were changes in the fatty acid profiles and increases in the amount of polyenes in the 1,2- and l,3diglycerides, free fatty acids, and triglyceride fractions with aging of the mosquito cells. The sterol ester fraction was similar at the exponential phase of growth in both Aedes cells. Changes occurred in the fatty acid profiles, and there was a decrease in polyenes from the exponential to the stationary phase of growth.
INTRODUCTION Results from experimental studies of dipteran insects have shown that most of the species examined have a lipid composition different from those of other orders of insects and vertebrate animals. The neutral lipids of many dipterans contained a large proportion of palmitoleic acid (16:l) and had 50% or more of fatty acids with chain lengths of less than 18 carbons. Phosphatidylethanolamine was the major component of the phospholipids. These unique characteristics of the lipid composition of Diptera were usually found together (Fast, 1966; Fast and Brown, 1962). Mosquito cells grown in spinner or monolayer cultures have shown differences in the fatty acid profiles of lipids from those of insects of other orders and mammals (Jenkin et al., 1971; Luukkonen et al., 1973; Townsend et al., 1972). Buckley (1969) suggested that the Aedes albopictus and A. aegypti cell lines could be important tools for screening a&virus infections. Host cells differing in lipid profiles may provide good model systems for the study of a&viruses. Rates of lipid synthesis, chain elongation, and desaturation of fatty acids at several developmental stages of dipteran insects have been investigated (Municio et al., 1970, 1972; Fast, 1970), and it has been demonstrated that these biological processes
were functions of the developmental stage. Hayashiya and Harwood (1968) determined the fatty acid composition in various developmental stages of two strains of the mosquito Anopheles freeborni. They found the closest similarity in the lipids of adults 10 days of age. This work suggested that comparison of fatty acids should be made between insects at similar growth stages. Aedes aegypti and A. albopictus mosquito cells grown in spinner culture showed changes in the fatty acid profiles of the total lipid, total neutral lipid, and total phospholipids when the late logarithmic and stationary phases of growth were compared (McMeans et al., 1975). The fatty acid profiles of the neutral lipids were determined to locate where changes in the amounts of fatty acids were occurring which might give a better understanding of metabolism of lipids in insect cells. In this report, comparisons were made of the neutral lipid classes between two species of mosquito cells at two stages of development and between the mosquito cells and medium. MATERIALS
METHODS
Cell culture. The A. albopictus cells, Singh’s strain (1967) were obtained from Ms. F. Paul, Naval Medical Research Institute, Bethesda, Maryland. The A. aegypti cells,
161 Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
162
YANG
Singh’s strain (1967), were supplied by Dr. S. H. Hsu, Naval Medical Research Unit No. 2, Taipei, Taiwan. The procedures for growing the cells in suspension and harvesting the cells were performed according to McMeans et al. (1975). The term aging is used to denote cells which were in the exponential phase and have reached a plateau in growth, i.e., the stationary phase. Lipid analysis. The method of Makino et al. (1970) was followed for the extraction of lipids from the mosquito cells. The lipids were fractionated on silicic acid columns as described by Rouser et al. (1967). The total neutral lipids were further fractionated by thin-layer chromatography in a solvent system containing petroleum ether-diethyl ether-acetic acid (90: 15:2). Authentic standards obtained from the Lipid Preparation Laboratory, The Hormel Institute, Austin, Minnesota, were used for tentative identification of the lipid classes. The thin-layer chromatographic plates were sprayed with 2’,7’-dichlorofluoroscein and examined under ultraviolet light to locate the lipid bands (Malins and Mangold, 1960). The lipids were scraped from the plates and the bands containing the sterol and mono- and diglyceride fractions were extracted with diethyl ether. The extracted fractions of lipids were rechromatographed on a silica gel H plate in petroleum ether-diethyl ether-acetic acid (50:70: 1) as the solvent system. The positions of the lipid bands were located by the same method as described ahove and the bands scraped from the plate. The lipids in the silica gel were directly methylated. The sterols were extracted from the silica gel and quantitated on the basis of dry weight with an electronic balance (Model G, Cahn Division, Ventron Instruments Corp., Paramount, California). Preparation of methyl ester. Methyl esters were prepared by transesterification by using 5% HCl in anhydrous methanol (Stoffel et al., 1959) or 14% boron trifluoride in methanol (Applied Science Laboratories, State College, Pennsylvania) in a mixture of
ET AL.
methanol 1964).
and benzene (Morrison
and Smith,
Analysis of methyl ester. The methyl esters of the fatty acids were analyzed by gas-liquid chromatography in a Fisher-Victoreen gas chromatograph, Model 4000, at 185°C with a flame ionization detector and vibrating reed electrometer. The column consisted of S-ft x i/s-inch-i.d. (244 x 0.32 cm) aluminum tubing packed with 15% EGSS-X on Gas Chrom P, 100/120 mesh (Applied Science Laboratories, State College, Pennsylvania). Identification of the individual fatty acids was based on relative retention time. Confirmation of chain length and presence of the esters and unsaturated fatty acids were determined by hydrogenation (Ackman, 1963; Hofstetter et al., 1965).
RESULTS The quantitative results of the neutral lipid composition of leafhopper medium and the A. aegypti and A. albopictus cells at exponential and stationary phases of growth are shown in Table 1. The fatty acid profiles of the neutral lipid fractions isolated from the mosquito cells and the medium are shown in Tables 2-6. A. aegypti Cells. Fatty Acid Profiles from the Logarithmic of Growth
and Stationary
Phases
The A. aegypti cells showed an increase in the amount of sterol ester and a decrease in the amount of free fatty acid occurring in the neutral lipid classes from the exponential to the stationary phase (Table 1). The 1,2diglycerides (Table 2) of the mosquito cells showed increases in polyenes (13-23s) and an increase in the 18:2 fatty acid with aging. The 1,3-diglycerides (Table 3) also showed an increase in polyenes (7-15%), in 18:2 fatty acid, and in 20-carbon unsaturated acids with aging of the cells. The free fatty acids (Table 4) of the A. aegypti cells had an increase of polyenes (11-23%), a decrease of 18:l fatty acid, and an increase in 20-carbon unsaturated fatty acids from the exponential to sta-
NEUTRAL
Monoglyceride 1,2-Diglyceride 1,3-Diglyceride Free Fatty Acid Triglyceride Sterol Ester Free Sterol
163
CELLS
TABLE 1 of Neutral Lipid Classes of Aedes Aegypti and Aedes AIbopictus Cells in Late Exponential and Stationary Phases of Growth
Distribution Neutral lipid classes
LIPIDS OF MOSQUITO
Aedes aegypti
Leafhopper TrC f 0.0 + 0.0 15.1 f 0.1 19.2 f 0.4 56.1 f 0.2 8.6 f 0.4 0.2 0.3
Aedes albopictus
Expa
Statb
Tr 1.6 f 0.1 0.1 + 0.0 5.0 + 0.2 67.3 f 3.0 8.1 f 2.0 18.1 f 0.8
Tr 1.0 + 0.4 0.2 f 0.1 1.6 f 0.3 55.7 f 11.5 18.6 f 6.0 23.1 f 4.8
Stat
Exp
Ao.oe * 0.0
0.2d 1.8
0.1 + 0.0 3.6 f 0.1 76.0 f 0.5 8.5 f 0.9 9.8 f 0.3
1.1 * 0.1 0.1 ?: 0.0 2.2 f 0.0 46.6 f 1.1 27.4 * 1.4 22.7 f 0.8
aExponential phase of growth. bStationary phase of growth. ‘Tr, trace, less than 0.1%. dRelative percentage of fatty acid. eMean f standard error.
TABLE 2 Constituents Fatty Acids of Neutral Lipid Classes: 1,2-Diglycerides Constituent 12:oc 14:o 14:1 15:o 16:O 16:l 17:o 17:l 18:O 18:l 18:2 18:3 20:o 2O:l 20:3 20:4 20:5
22g Saturates Monoenes Polyenes
Leafhopper medium 1.3d f l.oe 4.4 + 1.3 Trf ?r 2.0 f 3.1 Tr 0.7 f 0.5 1.5 f 0.7 26.5 k 1.6 4.0 f 0.2 0.5 f 0.3 0.9 f 0.7
31.2 7.5
1.4 f 0.8 1.5 + 0.3 0.6 + 0.2 4.9 f 0.8 51.3 34.7 12.9
Aedes aegypti
Aedes albopictus
Expa
Statb
1.5 k 0.3 f 0.5 zi 0.2 Tr 23.2 + 4.2 15.3 f 3.3 0.8 f 0.1
+ 0.8 + 1.0 ?: 0.5 Tr 20.7 + 0.6 11.1 f 1.9 0.7 f 0.0
7.4 32.4 6.8 0.8 1.2
+ 0.5 ST3.4 f 2.0 -A 0.3
6.8 30.3
+ 0.1 Tr
0.9 0.7 0.9 4.3 2.4
3.3 1.8
0.5 f 0.0 2.1 f 0.5 2.5 + 0.7 31.4 49.5 12.7
‘Exponential phase of growth. bStationary phase of growth. CNumber of carbon atoms in acid: number of double bonds. dRelative percentage of fatty acid. eMean + standard error. fTr, trace, less than 0.5%. g22-series acids.
0.9 3.0 0.9
f
0.7
f 1.0 14.1 + 0.1 1.1 f 0.1 2
0.2
Exp 0.6 4.1
+ 0.2 + 0.1
1.5 + 0.1 f 0.3 f 0.1 1.0 + 0.1
24.5 13.0
8.0 38.3 4.6
+ 0.3 + 0.3
+ 0.1 0.8 ?I 0.1 0.9 2 0.1
f 0.1 f f
0.3 0.3
f 1.0
33.0 43.0 22.8
Tr 1.5 + 0.2 0.8 f 0.2 40.6 51.3 7.7
Stat f 0.2 ?r 0.3 i 0.1 Tr 16.3 + 0.4 12.1 f 2.6 0.7 + 0.1 0.9 3.4 0.7
7.1 ? 0.2 k 1.5 * 0.2 i 0.1 1.8 i 0.2 0.8 * 0.1 0.5 f 0.2 5.8 f 0.7 2.8 f 0.1
28.8 16.1 1.4
30.2 42.4 26.6
164
YANG ET AL. TABLE 3 Constituent Fatty Acids of Neutral Lipid Classes: 1,3-Diglycerides
Constituent 12:oc 14:o 14:l 15:o 16:0 16:l 17:o 17:l 18:0 18:l 18:2 18:3 20:o 2O:l 20:2 20:3 20:4 20:5 22g Saturates Monoenes Polyenes
Leafhopper medium 4.2 f 0.7 1.2 + 0.7 34.2 2 3.3 7.1 f 3.1 0.7 f 0.1 Tr 7.6 ?- 0.1 25.0 c 1.4 3.3 f 0.7
Aedes aegvpti
Expa
Aedes albopictus
Statb
0.5~ f 03 5.1 f 0.3 TI
0.5 3.6 2.4 0.7 26.9 8.7
29.3 + 1.4 9.2 + 1.0 14.1 + 2.4 30.9 f 2.8 5.2 I? 0.3
* 0.2 f 0.8 + 1.7 2 0.1 + 1.9 + 0.0
11.4 f 28.4 f 8.3 f Tr 1.7 f
Exp Trf 4.2 f 0.3 0.7 + 0.6 29.0 f 0.3 11.0 * 0.6
0.2 2.5 0.4
14.7 f 1.1 36.0 + 0.1 2.6 f 0.0
0.4
0.6 + 0.0
Stat TI 2.5 f Tr 0.5 f 22.8 + 6.1 + Tr 14.4 22.9 9.2 0.6 3.9 0.5
1.0 0.1 0.8 0.2
r 0.6 t 1.4 * 0.4 + 0.6 * 0.1 f 0.5
1.1 f 0.8
3.6 + 2.5
2.8 + 2.3 2.8 + 0.7 6.1 t 6.1 3.8 * 3.8 49.0 32.1 18.8
Tr 0.8 ?: 0.8 0.6 + 0.6
Tr 0.8 f 0.0 3.7 i- 0.5 2.3 f 0.9
0.5 f 0.0 0.6 f 0.0
2.4 t 0.7 5.5 + 1.9 7.9 f 1.2
52.6 40.1 6.6
44.8 39.5 15.1
49.2 47.0 3.7
44.1 29.5 25.6
aExponential phase of growth. bStationary phase of growth. CNumber of carbon atoms in acid: number of double bonds. dRelative percentage of fatty acid. eMean f standard error. fTr, trace, less than 0.5%. g22-series acids.
tionary phase of growth. There was an increase of polyenes (l l-21%), decreases in 18:0 and 18:l fatty acids, and increases in 18:2 and 20-carbon unsaturated acids in the triglyceride fraction (Table 5) with the aging of the cells. The sterol ester fraction (Table 6) showed some decrease in polyenes (19-13%) and in the percentage of 16:0 acid and a slight increase in 16: 1 acid with the aging of the cells. A. albopictus Cells. Fatty Acid Projles from the Logarithmic and Stationar,v Phasesof Growth
The A. albopictus cells showed a decrease in the amount of triglycerides and increases in the amounts of sterol esters and sterols with aging (Table 1). There were increases in polyenes, decreases in 16:0 and 18:l fatty
acids, and increases in 18:2 and 20-carbon unsaturated fatty acids in the 1,2- and 1,3diglycerides, free fatty acids, and triglycerides with aging of the mosquito cells (Tables 2-5). In addition, in the 1,3-diglyceride fraction there was a decrease in the amount of 16:l acid and in the free fatty acid fraction there were decreases in the amounts of 16:l and 18:0 acids from the exponential to the stationary phase. The sterol ester fraction (Table 6) had a decrease in polyenes (2014%), decreases in 16:0 and 18:2 fatty acids, and an increase in 18:l fatty acid with aging of the cells. A. aegypti and A. albopictus Cells. Comparison of Neutral Lipids
When the two Aedes species were compared, the A. aegypti cells contained a larger
NEUTRAL
LIPIDS OF MOSQUITO
165
CELLS
TABLE 4 Constituent Fatty Acids of Neutral Lipid Classes: Free Fatty Acids Constituent 12:oe 14:o 14:l 15:o 16:0 16:l 17:o 18:0 18:l 18:2 18:3 20:o 2O:l 20:2 20:3 20:4 20:5 Saturates Monoenes Polyenes
Leafhopper medium 0.5d * 0.1e 2.3 i 0.0 0.8 24.3 7.0 0.8 13.9 40.5 5.3 1.0
r 0.3 A 3.1 k 1.4 + 0.2 f 0.1 t 0.8 f 0.0 f 0.3
Tr 0.7 f 0.1 2.4 + 0.1 42.6 47.5 9.4
Aedes aegypti Expa
2.0 A 0.4 4.2 i 0.4 1.4 + 0.3
1.1 f 0.3 2.8 f 0.5 1.8 + 0.1 20.5 17.2 0.9 8.1 36.4 5.8 0.9 1.2
Aedes albopictus Statb
t 1.5 f 1.4 + 0.1 f 0.8 + 1.9 t 1.5 f 0.3 5 0.5
20.7 + 1.8 12.9 + 2.3 0.9 + 0.1 7.3 i- 1.6 25.7 f 0.3 9.9 f 1.6 2.1 f 0.9 1.5 A 0.6 Tr
0.5 f 0.6 2.3 + 0.1 1.0 i 0.4 34.6 55.4 10.5
2.9 f 1.8 5.6 i 0.2 2.7 f 0.2 36.6 40.0 23.2
Exp 0.5 * 0.2 4.2 f 0.0 2.0 k 0.1 25.7 i 0.1 13.2 f 0.1 10.2 36.8 3.9 0.6 1.1
+ 0.1 + 0.1 + 0.2 + 0.0 + 0.2
Stat 2.0 + 0.1 4.3 * 0.1 1.0 k 0.0 Trf 18.5 + 0.2 11.0 + 0.1 0.7 f. 0.1 8.1 f 0.0 27.4 i 0.2 12.2 Yi0.1 1.3 f 0.1 2.0 f 0.1 0.9 f 0.0
Tr 1.6 * 0.4 43.7 50.0 6.1
1.1 f 0.0 6.6 f 0.3 3.2 + 0.1 35.6 40.3 24.4
aExponential phase of growth. bStationary phase of growth. ‘Number of carbon atoms in acid: number of double bonds. dRelative percentage of fatty acid. eMean k standard error. fTr, trace, less than 0.5%.
percentage of free sterols at the exponential stage. Only small differences were observed between the two species at the stationary phase in the relative amounts of the lipid fractions of the neutral lipid class. The fatty acid profiles of the 1,2- and 1,3diglycerides were similar at the exponential phase of growth of the two Aedes species. In the stationary phase, the A. aegypti cells contained more 16:0 and less 18:2 acid in the 1,2-diglyceride fraction and more 16: 1 and less 18:0 and 20-carbon unsaturated acids in the 1,3-diglyceride fraction than the A. albopictur cells. The free fatty acid fraction of the A. aegypti cells, exponential phase, contained less 16:0 and more 16:l acid than the A. ulbopictus cells at the same phase, but the acid composition was similar at the stationary phase of growth of the cells. In the triglyceride fraction at the exponential phase, the A. uegypti cells had a smaller rela-
tive percentage of 16:0 and a larger percentage of 18:2 acid and in the stationary phase had smaller percentages of 18:O and 18:l acids and a slightly larger percentage of 16:l acid than the A. albopictus cells. The fatty acid profile of the sterol ester fraction was similar at the exponential stage for both mosquito cells. At the stationary phase the A. uegypti cells had more 16:0 and 16:l acids and less 18:l fatty acid in the sterol ester fraction than the A. ulbopictus cells. Lipids in Mosquito Cells Compared to Growth Medium
The calf serum in the medium provided the only exogenous source of lipids to the cells. The main components of the leafhopper medium were sterol esters, 57%; triglycerides, 19%; and free fatty acids, 15% (Table 1). The mosquito cells contained a higher proportion of triglycerides and smaller amounts of sterol
166
YANG TABLE
ET AL. 5
Constituent Fatty Acids of Neutral Lipid Classes: Triglycerides Constituent 12:oc 14:o 14:l 15:o 16:0 16:l 17:o 17:l 18:0 18:l 18:2 18:3 20:o 2O:l 20:2 20:3 20:4 20:5 22g Saturates Monoenes Polyenes
Leafhopper medium Trd
5.5 f 0.7 1.2 f 0.6 30.1 f 4.0 7.0 f 2.1 0.5 + 0.4 0.6 f 0.0 9.0 f 0.5 37.0 + 0.6 3.7 + 0.2 1.4 f. 0.5 TI
1.1 f 1.1 0.5 f 0.1 0.9 f 0.0 Tr TI 46.3 44.6 7.6
Aedes aegypti
Aedes albopictus
Exp’
Statb
1.8e + 0.5f 4.1 f 0.3 1.6 f 0.3 Tr 21.1 f 0.7 13.8 + 0.1 0.8 ?: 0.1
6.1 f 0.2 4.3 f 0.4 2.9 + 0.1 Tr 18.6 + 0.2 14.6 f 1.2 0.7 t 0.0
8.2 35.7 6.7 1.0 1.7
6.1 23.2 13.9 1.4 1.6 0.7
f 0.3 + 0.3 _+0.1 f 0.1 f 0.6
TI 2.4 f 0.4 0.9 f 0.2
?:0.0 f 0.2 _+2.0 f 0.1 f 0.1 f 0.4
Tr 4.0 f 0.4 1.7 f 0.0
37.7 51.1 11.0
37.6 41.4 21.0
Exp 1.1 f 0.1 3.9 k 0.2 1.5 25.0 13.4 0.7
* f f k
0.1 0.3 0.2 0.0
9.0 37.8 3.9 0.7 1.5
k 0.3 f 0.6 _+0.1 i: 0.1 + 0.0
Stat 3.8 f 0.0 4.2 f 0.1 1.5 f 0.1 Tr 17.1 + 0.1 11.6 f 0.1 0.7 + 0.1 8.0 28.7 12.1 1.3 2.6 0.8
?r 0.1 f 0.2 i 0.2 f 0.0 i 0.1 A 0.0
Tr 1.0 t 0.1 0.5 f 0.1
0.6 f 0.1 4.7 2 0.0 2.3 f 0.2
42.7 51.2 6.1
36.4 42.6 21.0
aExponential phase of growth. bStationary phase of growth. CNumber of carbon atoms in acid: number of double bonds. dTr, trace, less than 0.5%. eRelative percentage of fatty acid. fMean f standard error. g22-series acids.
esters and free fatty acids than the medium. The percentages of neutral lipid fractions of the Aedes cells were similar to those in other insect cells and unlike those of mammalian cells. The amount of sterol in the medium and in the A. albopictus cells in the exponential phase was approximately equal. The A. aegypti cells at both phases of growth and the A. albopictus cells in the stationary phase contained a larger percentage of sterol than the medium. I ,2- Diglycerides The fatty acid profiles of the l,Zdiglycerides of the medium and mosquito cells are shown in Table 2. The 1,Zdiglycerides of both mosquito cells had smaller amounts of 16:O fatty acid in both phases and larger
amounts of 18:2 and 20-carbon unsaturated acids in the stationary phase in comparison to the fatty acids in the growth medium. The A. afbopictm cells in the exponential phase had larger percentages of 16: 1 and 18: 1 fatty acids than the medium. 1,3-Diglycerides In the 1,3-diglycerides (Table 3) the Aedes cells had larger percentages of 18:0 at both phases of growth, and a larger amount of 18:2 fatty acid in the stationary phase than was found in the medium. The A. albopictw cells, in addition, had a larger percentage of 18:l fatty acid in the exponential phase and a smaller percentage of 16:0 acid in the stationary phase than were found in the medium.
NEUTRAL
LIPIDS
OF
MOSQUITO
167
CELLS
TABLE 6 Constituent Fatty Acids of Neutral Lipid Classes: Sterol Esters Constituent 12:oc 14:o 15:o 16:0 16:l 17:o 17:l 18:O 18:l 18:2 18:3 2o:o 2O:l 20:2 20:3 20:4 20:5 Saturates Monoenes Polyenes
Leafhopper medium 0.6d 2.6 1.4 13.5 9.8
f 0.6e t 0.8 k 0.5 + 2.2 A 2.4
1.5 1.2 20.4 30.4 4.2 1.1
f 0.3 f 0.5 f 0.9 + 5.8 f 0.5 f 0.5
Aedes aegypti
Expa
Statb
Exp
Stat
5.0 f 0.6
1.8 A 0.4 Tr 17.8 * 0.3 12.7 i 0.4 1.2 i 0.0
2.0 + 0.5 Tr 8.2 + 1.4 10.6 f 0.8 1.6 k 0.1
3.9 zk0.1 42.5 f 0.9 12.9 f 0.2 1.7 * 0.1 Tr
4.8 + 0.2 57.1 f 2.0 9.0 f 0.2 0.7 i 0.1 1.0 + 0.1 0.8 + 0.1 Tr 0.7 + 0.0 2.1 + 0.3 1.1 + 0.1 17.6 68.5 13.6
1.8 + 0.2 Trf
16.2 f 1.4 12.9 + 2.6 1.4 f 0.3
12.1 f 0.0 18.2 + 1.3 1.8 f 0.6
3.3 44.4 11.5 1.0 1.0
5.0 f 0.5 42.5 f 0.6 9.1 f 1.8 0.8 f 0.8 0.6 f 0.1 Tr 1.5 f 1.5 Tr 1.9 f 0.3 Tr 24.5 60.7 13.3
f 1.5 f 1.8 + 0.7 f 0.9 f 1.0
0.9 f 0.9
1.9 * 0.6 8.6 f 0.6 1.8 + 0.0 20.4 31.7 47.8
Aedes albopictus
1.4 f 0.4 2.9 2 0.3 2.6 f 1.6 23.7 57.3 19.4
0.5 f 0.1 3.4 + 0.1 1.3 f 0.5 24.7 55.2 19.8
aExponential phase of growth. bStationary phase of growth. CNumber of carbon atoms in acid: number of double bonds. dRelative percentage of fatty acid. eMean i standard error. fTr, trace, less than 0.5%.
the stationary phase than the medium. The The free fatty acid profiles of A. aegypti A. albopictus cells had larger amounts of and A. albopictus (Table 4) contained less 16: 1 acid in both phases of growth and larger 18:O acid and more 16:l acid in both phases, amounts of 18:2 and 20-carbon unsaturated and less 18:l and more 18:2 and 20-carbon acids and smaller amounts of 16:O and 18:l unsaturated acids in the stationary phase fatty acids in the stationary phase than the than were observed in the leafhopper me- medium. dium. The A. albopictus cells in the sta- Sterol Esters tionary phase had less 16:0 acid than the meIn comparison to the medium, the A. dium. aegypti and A. albopictw cells, exponential Triglycerides and stationary phases, had smaller amounts of 18:2 and 20-carbon unsaturated fatty acids Compared to the medium, the two mosand larger amounts of 18:l fatty acid (Table quito cells contained more 16:l fatty acid in 6). The A. aegypti cells, stationary phase, the triglyceride fraction (Table 5) at the exhad a larger percentage of 16: 1 acid than the ponential and stationary phases. The A. medium. aegypti cells had smaller amounts of 16:0 acid and larger amounts of 16:l and 18:2 DISCUSSION acids in both phases of growth and smaller amounts of 18:0 and 18:l acids and larger The variations in composition of the conamounts of 20-carbon unsaturated acids in stituents of the neutral lipids of the two Free Fatty Acids
168
YANG
species are not large enough to use as a taxonomic tool. lmportant aspects of the metabolic activity of lipids in insects are the rates and synthesis of fatty acids by insects at different stages of development (Municio et al., 1972; Fast, 1970). All insects have a definite requirement for sterols (Clayton, 1964; Vaughn et al., 1971). Some insects either cannot synthesize polyunsaturated 18-carbon acids or synthesize these acids at a rate sufficient to meet their growth requirements (Stephen and Gilbert, 1969; Chippendale et al., 1964). Van Handel and Lum (1961) reported that linoleic and linolenic acids could be synthesized to a limited extent by Aedes sollicitans. Fast (1970) lists species requiring a dietary source of polyunsaturated 18carbon fatty acids for successful metamorphosis from larvae to adults and for effective reproduction. Yurkiewicz and Bhalla (1969) studied the lipids of the mutant bronze Aedes aegypti and the wild-type A. aegypti to detect alterations in lipid metabolism which might account for the failure of the eggs of the mutant bronze to complete its embryonic development. The mutant bronze mosquito differed from the wild-type A. aegypti in that it possessed more saturated fatty acids and less unsaturated acids in a number of neutral lipid and phospholipid fractions. The mosquito cells grown in spinner culture showed increases in the amount of unsaturation of acids and chain length of fatty acids with aging of cells in the free fatty acids and diglycerides. In the triglyceride fraction, both Aedes cells showed more unsaturation of the fatty acids, and the A. albopictus cells also showed chain elongation in the stationary phase in comparison to the exponential phase of growth. In A. aegypti cells there was a slight decrease in average chain length of the fatty acids, and in the A. albopictus cells there was some increase in chain length of the acids in the sterol ester fraction with aging. In both mosquito cells the amount of unsaturation of acids was about the same or showed a slight decrease in the sterol ester fraction from the exponential
r-El
”
AL.
to the stationary phase of growth. Percentage increases in the free sterols and sterol esters in the stationary phases of the mosquito cells would indicate that the growth medium provided an ample supply of sterols for synthesis. A significant amount of the fatty acids synthesized by both mitochondria and microsomes is incorporated into triglycerides, phospholipids, cholesterol, and cholesterol esters (Aeberhard and Menkes, 1968). As the A. albopictus cells aged there was a decrease in the percentage of triglycerides and increases in the amounts of sterols and sterol esters. These changes in the lipid composition of the mosquito cell would indicate that different metabolic rates or pathways occurred in the hydrolysis to free fatty acids and synthesis of the lipid components. There was a tendency for greater change with aging in the fatty acid profiles of the A. albopictus cells than was observed in A. aegypti cells. These studies can now be used as a basis for designing radioisotope experiments to determine turnover rates of specific lipid fractions which may play an important role in the normal and arbovirus infected mosquito cells. ACKNOWLEDGMENTS The authors thank A. Conway for her excellent technical assistance. This study was supported in part by Public Health Service Research Grant No. HL 08214 from the Program Projects Branch, Extramural Programs, National Heart and Lung Institute, and by The Hormel Foundation.
REFERENCES R. G. 1963. Structural correlation of unsaturated fatty acid esters through graphical comparison of gas-liquid chromatographic retention times on a polyester substrate. J. Amer. Oil Chem. Sot.. 42, 558-564. AEBERHARD, E., AND MENKES, J. H. 1968. Biosynthesis of long chain fatty acids by subcellular particles of mature brain. J. Biol. Chem., 243,38343840. BUCKLEY, S. M. 1969. Susceptibility of the Aedes albopictur and A. aegypti cell lines to infection with arboviruses. Proc. Sot. Exp. Biol. Med.. 131,625-630. CHIPPENDALE, G. M., BECK, S. D., AND STRONG, F. M. 1964. Methyl linolenate as an essential nutrient for ACKMAN,
NEUTRAL
LIPIDS
OF
MOSQUITO
CELLS
169
tained from the logarithmic and stationary phases of the cabbage looper, Trichoplusia ni (Hubner). Nature growth. Lipids, 10,99m104. (London), 204,7 lo-7 11. CLAYTON, R. B. 1964. The utilization of sterols by MORRISON, W. R., AND SMITH, L. M. 1964. Preparation insects./. LipidRes., $3-19. of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res.. 5, FAST, P. G. 1966. A comparative study of the phospholipids and fatty acids of some insects. Lipids, 1, 600-608. 209-215. MUNICIO, A. M., ODRIOZOLA, J. M., AND PINERIO, A. FAST, P. G. 1970. Insect lipids. In “Progress in the 1970. Biochemistry of the development of the insect Chemistry of Fats and Other Lipids” (R. T. Holman, Ceratifis capirata. In vitro biosynthesis of fatty acids ed.), Vol. 11, pp. 181-242. Pergamon Press, New r from “C-acetate during metamorphosis. Camp. Biochem. Physiol., 31,387-395. York. FAST, P. G., AND BROWN, A. W. A. 1962. Lipids of MUNICIO. A. M., ODRIOZOLA, J. M., PINERIO, A., AND DDT-resistant and susceptible larvae of Aedes RIBERA, A. 1972. In vitro elongation and desaturation aegypti. Ann. Entomol. Sot. Amer., 55,663-672. of fatty acids during development of insects. Biochim. HAYASHIYA, K., AND HARWOOD, R. F. 1968. Fatty acids Biophys. Acta. 280,248-257. of the mosquito Anopheles freeborni. Ann. Entomol. ROUSER. G., KRITCHEVSKY, G., SIMON, G., AND NELSON, G. J. 1967. Quantitative analysis of brain and Sot. Amer., 61,278-280. HOFSTETTER, H. H., SEN, N., AND HOLMAN, R. T. 1965. spinach leaf lipids employing silicic acid column Characterization of unsaturated fatty acids by chromatography and acetone for elution of glycolipgas-liquid chromatography. J. Amer. Oil Chem. Sot.. ids. Lipids, 2,37-40. 42,537T540. SINGH, K. R. P. 1967. Cell cultures derived from larvae JENKIN, H., TOWNSEND, D., MAKINO, S., AND YANG. of Aedes albopicrus (Skuse) and Aedes aegypti (L.) T. K. 1971. Comparative lipid analysis of A&es Curr. Sci., 36,506&508. aegypti and monkey kidney cells (MK-2) cultivated in STEPHEN, W. F., JR., AND GILBERT, L. I. 1969. Fatty vitro. In “Current Topics in Microbiology and Immuacid biosynthesis in the silkmoth, Hyalophara cenology” (E. Weiss, ed.), Vol. 55, pp. 97-102. Springercropia. J. Insect Physiol.. 15,1833-1854. Verlag, New York. STOFFEL, W., CHU, F., AND AHRENS, E. G., JR. 1959. LUUKKONEN, A., BRUMMER-KORVENKONTIO, M., AND Analysis of long-chain fatty acids by gas-liquid RENKONEN, 0. 1973. Lipids of cultured mosquito cells chromatography. Anal. Chem., 31,3077308. (Aedes albopictw) comparison with cultured mamTOWNSEND, D., JENKIN, H. M., AND YANG, T. K. 1972. malian fibroblasts (BHK 21 cells). Biochim. Biophys. Lipid analysis of Aedes aegypfi cells cultivated in Aria. 3%,256-261. vitro. Biochim. Biophys. Acta, 260,20-25. MAKINO, S., JENKIN, H. M., Yu, H. M., AND VAN HANDEL, E., AND LUM, P. T. M. 1961. Sex as reguTOWNSEND, D. 1970. Lipid composition of Chlamydia lator of triglyceride metabolism in the mosquito. psittaci grown in monkey kidney cells in defined meScience, 134, 1979m1980. dium. J. Bacterial., 103,62-70. VAUGHK, J. L., LOULOUDES, S. J., AND DOUGHERTY, K. MALINS, D. C., AND MANGOLD, H. K. 1960. Analysis of 1971. The uptake of free and serum-bound sterols by complex lipid mixtures by thin-layer chromatography insect cells in vifro. In “Current Topics in Miand complementary methods. J. Amer. Oil Chem. crobiology and Immunology” (E. Weiss, ed.), Vol. 55, Sot., 31,576-578. pp. 92-97, Springer-Verlag, New York. MCMEANS, E.. YANG, T. K., ANDERSON, L. E., AND YURKIEWICZ, W. J., AND BHALLA. S. C. 1969. Lipids of JENKIN, H. M. 1975. Comparison of the lipid composithe mutant bronze mosquito, Aedes aegypri (L.). tion of Aedes aegypti and Aedes albopictus cells obEntomol. News, 80, 177-184.
OF INVERTEBRATE
Neutral
PATHOLOGY
Lipids
27, 161-169
of Aedes
(1976)
aegypti
Cells Cultured T. K. YANG,
E. McMEANS,
The Hormel Institute,
and Aedes in Vitro
L. E. ANDERSON,
University Received
of Minnesota, March
albopictus
AND
H. M. JENKIN
Austin, Minnesota55912
3, 1975
Neutral lipids were examined from spinner culture cells of two species of mosquitoes, Aedes aegypti and A. albopictus, in exponential and stationary phases of growth. The lipids were separated into constituent classes by thin-layer chromatography. The fatty acid profiles of the methyl esters of each class were determined by gas-liquid chromatography. There were changes in the fatty acid profiles and increases in the amount of polyenes in the 1,2- and l,3diglycerides, free fatty acids, and triglyceride fractions with aging of the mosquito cells. The sterol ester fraction was similar at the exponential phase of growth in both Aedes cells. Changes occurred in the fatty acid profiles, and there was a decrease in polyenes from the exponential to the stationary phase of growth.
INTRODUCTION Results from experimental studies of dipteran insects have shown that most of the species examined have a lipid composition different from those of other orders of insects and vertebrate animals. The neutral lipids of many dipterans contained a large proportion of palmitoleic acid (16:l) and had 50% or more of fatty acids with chain lengths of less than 18 carbons. Phosphatidylethanolamine was the major component of the phospholipids. These unique characteristics of the lipid composition of Diptera were usually found together (Fast, 1966; Fast and Brown, 1962). Mosquito cells grown in spinner or monolayer cultures have shown differences in the fatty acid profiles of lipids from those of insects of other orders and mammals (Jenkin et al., 1971; Luukkonen et al., 1973; Townsend et al., 1972). Buckley (1969) suggested that the Aedes albopictus and A. aegypti cell lines could be important tools for screening a&virus infections. Host cells differing in lipid profiles may provide good model systems for the study of a&viruses. Rates of lipid synthesis, chain elongation, and desaturation of fatty acids at several developmental stages of dipteran insects have been investigated (Municio et al., 1970, 1972; Fast, 1970), and it has been demonstrated that these biological processes
were functions of the developmental stage. Hayashiya and Harwood (1968) determined the fatty acid composition in various developmental stages of two strains of the mosquito Anopheles freeborni. They found the closest similarity in the lipids of adults 10 days of age. This work suggested that comparison of fatty acids should be made between insects at similar growth stages. Aedes aegypti and A. albopictus mosquito cells grown in spinner culture showed changes in the fatty acid profiles of the total lipid, total neutral lipid, and total phospholipids when the late logarithmic and stationary phases of growth were compared (McMeans et al., 1975). The fatty acid profiles of the neutral lipids were determined to locate where changes in the amounts of fatty acids were occurring which might give a better understanding of metabolism of lipids in insect cells. In this report, comparisons were made of the neutral lipid classes between two species of mosquito cells at two stages of development and between the mosquito cells and medium. MATERIALS
METHODS
Cell culture. The A. albopictus cells, Singh’s strain (1967) were obtained from Ms. F. Paul, Naval Medical Research Institute, Bethesda, Maryland. The A. aegypti cells,
161 Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
162
YANG
Singh’s strain (1967), were supplied by Dr. S. H. Hsu, Naval Medical Research Unit No. 2, Taipei, Taiwan. The procedures for growing the cells in suspension and harvesting the cells were performed according to McMeans et al. (1975). The term aging is used to denote cells which were in the exponential phase and have reached a plateau in growth, i.e., the stationary phase. Lipid analysis. The method of Makino et al. (1970) was followed for the extraction of lipids from the mosquito cells. The lipids were fractionated on silicic acid columns as described by Rouser et al. (1967). The total neutral lipids were further fractionated by thin-layer chromatography in a solvent system containing petroleum ether-diethyl ether-acetic acid (90: 15:2). Authentic standards obtained from the Lipid Preparation Laboratory, The Hormel Institute, Austin, Minnesota, were used for tentative identification of the lipid classes. The thin-layer chromatographic plates were sprayed with 2’,7’-dichlorofluoroscein and examined under ultraviolet light to locate the lipid bands (Malins and Mangold, 1960). The lipids were scraped from the plates and the bands containing the sterol and mono- and diglyceride fractions were extracted with diethyl ether. The extracted fractions of lipids were rechromatographed on a silica gel H plate in petroleum ether-diethyl ether-acetic acid (50:70: 1) as the solvent system. The positions of the lipid bands were located by the same method as described ahove and the bands scraped from the plate. The lipids in the silica gel were directly methylated. The sterols were extracted from the silica gel and quantitated on the basis of dry weight with an electronic balance (Model G, Cahn Division, Ventron Instruments Corp., Paramount, California). Preparation of methyl ester. Methyl esters were prepared by transesterification by using 5% HCl in anhydrous methanol (Stoffel et al., 1959) or 14% boron trifluoride in methanol (Applied Science Laboratories, State College, Pennsylvania) in a mixture of
ET AL.
methanol 1964).
and benzene (Morrison
and Smith,
Analysis of methyl ester. The methyl esters of the fatty acids were analyzed by gas-liquid chromatography in a Fisher-Victoreen gas chromatograph, Model 4000, at 185°C with a flame ionization detector and vibrating reed electrometer. The column consisted of S-ft x i/s-inch-i.d. (244 x 0.32 cm) aluminum tubing packed with 15% EGSS-X on Gas Chrom P, 100/120 mesh (Applied Science Laboratories, State College, Pennsylvania). Identification of the individual fatty acids was based on relative retention time. Confirmation of chain length and presence of the esters and unsaturated fatty acids were determined by hydrogenation (Ackman, 1963; Hofstetter et al., 1965).
RESULTS The quantitative results of the neutral lipid composition of leafhopper medium and the A. aegypti and A. albopictus cells at exponential and stationary phases of growth are shown in Table 1. The fatty acid profiles of the neutral lipid fractions isolated from the mosquito cells and the medium are shown in Tables 2-6. A. aegypti Cells. Fatty Acid Profiles from the Logarithmic of Growth
and Stationary
Phases
The A. aegypti cells showed an increase in the amount of sterol ester and a decrease in the amount of free fatty acid occurring in the neutral lipid classes from the exponential to the stationary phase (Table 1). The 1,2diglycerides (Table 2) of the mosquito cells showed increases in polyenes (13-23s) and an increase in the 18:2 fatty acid with aging. The 1,3-diglycerides (Table 3) also showed an increase in polyenes (7-15%), in 18:2 fatty acid, and in 20-carbon unsaturated acids with aging of the cells. The free fatty acids (Table 4) of the A. aegypti cells had an increase of polyenes (11-23%), a decrease of 18:l fatty acid, and an increase in 20-carbon unsaturated fatty acids from the exponential to sta-
NEUTRAL
Monoglyceride 1,2-Diglyceride 1,3-Diglyceride Free Fatty Acid Triglyceride Sterol Ester Free Sterol
163
CELLS
TABLE 1 of Neutral Lipid Classes of Aedes Aegypti and Aedes AIbopictus Cells in Late Exponential and Stationary Phases of Growth
Distribution Neutral lipid classes
LIPIDS OF MOSQUITO
Aedes aegypti
Leafhopper TrC f 0.0 + 0.0 15.1 f 0.1 19.2 f 0.4 56.1 f 0.2 8.6 f 0.4 0.2 0.3
Aedes albopictus
Expa
Statb
Tr 1.6 f 0.1 0.1 + 0.0 5.0 + 0.2 67.3 f 3.0 8.1 f 2.0 18.1 f 0.8
Tr 1.0 + 0.4 0.2 f 0.1 1.6 f 0.3 55.7 f 11.5 18.6 f 6.0 23.1 f 4.8
Stat
Exp
Ao.oe * 0.0
0.2d 1.8
0.1 + 0.0 3.6 f 0.1 76.0 f 0.5 8.5 f 0.9 9.8 f 0.3
1.1 * 0.1 0.1 ?: 0.0 2.2 f 0.0 46.6 f 1.1 27.4 * 1.4 22.7 f 0.8
aExponential phase of growth. bStationary phase of growth. ‘Tr, trace, less than 0.1%. dRelative percentage of fatty acid. eMean f standard error.
TABLE 2 Constituents Fatty Acids of Neutral Lipid Classes: 1,2-Diglycerides Constituent 12:oc 14:o 14:1 15:o 16:O 16:l 17:o 17:l 18:O 18:l 18:2 18:3 20:o 2O:l 20:3 20:4 20:5
22g Saturates Monoenes Polyenes
Leafhopper medium 1.3d f l.oe 4.4 + 1.3 Trf ?r 2.0 f 3.1 Tr 0.7 f 0.5 1.5 f 0.7 26.5 k 1.6 4.0 f 0.2 0.5 f 0.3 0.9 f 0.7
31.2 7.5
1.4 f 0.8 1.5 + 0.3 0.6 + 0.2 4.9 f 0.8 51.3 34.7 12.9
Aedes aegypti
Aedes albopictus
Expa
Statb
1.5 k 0.3 f 0.5 zi 0.2 Tr 23.2 + 4.2 15.3 f 3.3 0.8 f 0.1
+ 0.8 + 1.0 ?: 0.5 Tr 20.7 + 0.6 11.1 f 1.9 0.7 f 0.0
7.4 32.4 6.8 0.8 1.2
+ 0.5 ST3.4 f 2.0 -A 0.3
6.8 30.3
+ 0.1 Tr
0.9 0.7 0.9 4.3 2.4
3.3 1.8
0.5 f 0.0 2.1 f 0.5 2.5 + 0.7 31.4 49.5 12.7
‘Exponential phase of growth. bStationary phase of growth. CNumber of carbon atoms in acid: number of double bonds. dRelative percentage of fatty acid. eMean + standard error. fTr, trace, less than 0.5%. g22-series acids.
0.9 3.0 0.9
f
0.7
f 1.0 14.1 + 0.1 1.1 f 0.1 2
0.2
Exp 0.6 4.1
+ 0.2 + 0.1
1.5 + 0.1 f 0.3 f 0.1 1.0 + 0.1
24.5 13.0
8.0 38.3 4.6
+ 0.3 + 0.3
+ 0.1 0.8 ?I 0.1 0.9 2 0.1
f 0.1 f f
0.3 0.3
f 1.0
33.0 43.0 22.8
Tr 1.5 + 0.2 0.8 f 0.2 40.6 51.3 7.7
Stat f 0.2 ?r 0.3 i 0.1 Tr 16.3 + 0.4 12.1 f 2.6 0.7 + 0.1 0.9 3.4 0.7
7.1 ? 0.2 k 1.5 * 0.2 i 0.1 1.8 i 0.2 0.8 * 0.1 0.5 f 0.2 5.8 f 0.7 2.8 f 0.1
28.8 16.1 1.4
30.2 42.4 26.6
164
YANG ET AL. TABLE 3 Constituent Fatty Acids of Neutral Lipid Classes: 1,3-Diglycerides
Constituent 12:oc 14:o 14:l 15:o 16:0 16:l 17:o 17:l 18:0 18:l 18:2 18:3 20:o 2O:l 20:2 20:3 20:4 20:5 22g Saturates Monoenes Polyenes
Leafhopper medium 4.2 f 0.7 1.2 + 0.7 34.2 2 3.3 7.1 f 3.1 0.7 f 0.1 Tr 7.6 ?- 0.1 25.0 c 1.4 3.3 f 0.7
Aedes aegvpti
Expa
Aedes albopictus
Statb
0.5~ f 03 5.1 f 0.3 TI
0.5 3.6 2.4 0.7 26.9 8.7
29.3 + 1.4 9.2 + 1.0 14.1 + 2.4 30.9 f 2.8 5.2 I? 0.3
* 0.2 f 0.8 + 1.7 2 0.1 + 1.9 + 0.0
11.4 f 28.4 f 8.3 f Tr 1.7 f
Exp Trf 4.2 f 0.3 0.7 + 0.6 29.0 f 0.3 11.0 * 0.6
0.2 2.5 0.4
14.7 f 1.1 36.0 + 0.1 2.6 f 0.0
0.4
0.6 + 0.0
Stat TI 2.5 f Tr 0.5 f 22.8 + 6.1 + Tr 14.4 22.9 9.2 0.6 3.9 0.5
1.0 0.1 0.8 0.2
r 0.6 t 1.4 * 0.4 + 0.6 * 0.1 f 0.5
1.1 f 0.8
3.6 + 2.5
2.8 + 2.3 2.8 + 0.7 6.1 t 6.1 3.8 * 3.8 49.0 32.1 18.8
Tr 0.8 ?: 0.8 0.6 + 0.6
Tr 0.8 f 0.0 3.7 i- 0.5 2.3 f 0.9
0.5 f 0.0 0.6 f 0.0
2.4 t 0.7 5.5 + 1.9 7.9 f 1.2
52.6 40.1 6.6
44.8 39.5 15.1
49.2 47.0 3.7
44.1 29.5 25.6
aExponential phase of growth. bStationary phase of growth. CNumber of carbon atoms in acid: number of double bonds. dRelative percentage of fatty acid. eMean f standard error. fTr, trace, less than 0.5%. g22-series acids.
tionary phase of growth. There was an increase of polyenes (l l-21%), decreases in 18:0 and 18:l fatty acids, and increases in 18:2 and 20-carbon unsaturated acids in the triglyceride fraction (Table 5) with the aging of the cells. The sterol ester fraction (Table 6) showed some decrease in polyenes (19-13%) and in the percentage of 16:0 acid and a slight increase in 16: 1 acid with the aging of the cells. A. albopictus Cells. Fatty Acid Projles from the Logarithmic and Stationar,v Phasesof Growth
The A. albopictus cells showed a decrease in the amount of triglycerides and increases in the amounts of sterol esters and sterols with aging (Table 1). There were increases in polyenes, decreases in 16:0 and 18:l fatty
acids, and increases in 18:2 and 20-carbon unsaturated fatty acids in the 1,2- and 1,3diglycerides, free fatty acids, and triglycerides with aging of the mosquito cells (Tables 2-5). In addition, in the 1,3-diglyceride fraction there was a decrease in the amount of 16:l acid and in the free fatty acid fraction there were decreases in the amounts of 16:l and 18:0 acids from the exponential to the stationary phase. The sterol ester fraction (Table 6) had a decrease in polyenes (2014%), decreases in 16:0 and 18:2 fatty acids, and an increase in 18:l fatty acid with aging of the cells. A. aegypti and A. albopictus Cells. Comparison of Neutral Lipids
When the two Aedes species were compared, the A. aegypti cells contained a larger
NEUTRAL
LIPIDS OF MOSQUITO
165
CELLS
TABLE 4 Constituent Fatty Acids of Neutral Lipid Classes: Free Fatty Acids Constituent 12:oe 14:o 14:l 15:o 16:0 16:l 17:o 18:0 18:l 18:2 18:3 20:o 2O:l 20:2 20:3 20:4 20:5 Saturates Monoenes Polyenes
Leafhopper medium 0.5d * 0.1e 2.3 i 0.0 0.8 24.3 7.0 0.8 13.9 40.5 5.3 1.0
r 0.3 A 3.1 k 1.4 + 0.2 f 0.1 t 0.8 f 0.0 f 0.3
Tr 0.7 f 0.1 2.4 + 0.1 42.6 47.5 9.4
Aedes aegypti Expa
2.0 A 0.4 4.2 i 0.4 1.4 + 0.3
1.1 f 0.3 2.8 f 0.5 1.8 + 0.1 20.5 17.2 0.9 8.1 36.4 5.8 0.9 1.2
Aedes albopictus Statb
t 1.5 f 1.4 + 0.1 f 0.8 + 1.9 t 1.5 f 0.3 5 0.5
20.7 + 1.8 12.9 + 2.3 0.9 + 0.1 7.3 i- 1.6 25.7 f 0.3 9.9 f 1.6 2.1 f 0.9 1.5 A 0.6 Tr
0.5 f 0.6 2.3 + 0.1 1.0 i 0.4 34.6 55.4 10.5
2.9 f 1.8 5.6 i 0.2 2.7 f 0.2 36.6 40.0 23.2
Exp 0.5 * 0.2 4.2 f 0.0 2.0 k 0.1 25.7 i 0.1 13.2 f 0.1 10.2 36.8 3.9 0.6 1.1
+ 0.1 + 0.1 + 0.2 + 0.0 + 0.2
Stat 2.0 + 0.1 4.3 * 0.1 1.0 k 0.0 Trf 18.5 + 0.2 11.0 + 0.1 0.7 f. 0.1 8.1 f 0.0 27.4 i 0.2 12.2 Yi0.1 1.3 f 0.1 2.0 f 0.1 0.9 f 0.0
Tr 1.6 * 0.4 43.7 50.0 6.1
1.1 f 0.0 6.6 f 0.3 3.2 + 0.1 35.6 40.3 24.4
aExponential phase of growth. bStationary phase of growth. ‘Number of carbon atoms in acid: number of double bonds. dRelative percentage of fatty acid. eMean k standard error. fTr, trace, less than 0.5%.
percentage of free sterols at the exponential stage. Only small differences were observed between the two species at the stationary phase in the relative amounts of the lipid fractions of the neutral lipid class. The fatty acid profiles of the 1,2- and 1,3diglycerides were similar at the exponential phase of growth of the two Aedes species. In the stationary phase, the A. aegypti cells contained more 16:0 and less 18:2 acid in the 1,2-diglyceride fraction and more 16: 1 and less 18:0 and 20-carbon unsaturated acids in the 1,3-diglyceride fraction than the A. albopictur cells. The free fatty acid fraction of the A. aegypti cells, exponential phase, contained less 16:0 and more 16:l acid than the A. ulbopictus cells at the same phase, but the acid composition was similar at the stationary phase of growth of the cells. In the triglyceride fraction at the exponential phase, the A. uegypti cells had a smaller rela-
tive percentage of 16:0 and a larger percentage of 18:2 acid and in the stationary phase had smaller percentages of 18:O and 18:l acids and a slightly larger percentage of 16:l acid than the A. albopictus cells. The fatty acid profile of the sterol ester fraction was similar at the exponential stage for both mosquito cells. At the stationary phase the A. uegypti cells had more 16:0 and 16:l acids and less 18:l fatty acid in the sterol ester fraction than the A. ulbopictus cells. Lipids in Mosquito Cells Compared to Growth Medium
The calf serum in the medium provided the only exogenous source of lipids to the cells. The main components of the leafhopper medium were sterol esters, 57%; triglycerides, 19%; and free fatty acids, 15% (Table 1). The mosquito cells contained a higher proportion of triglycerides and smaller amounts of sterol
166
YANG TABLE
ET AL. 5
Constituent Fatty Acids of Neutral Lipid Classes: Triglycerides Constituent 12:oc 14:o 14:l 15:o 16:0 16:l 17:o 17:l 18:0 18:l 18:2 18:3 20:o 2O:l 20:2 20:3 20:4 20:5 22g Saturates Monoenes Polyenes
Leafhopper medium Trd
5.5 f 0.7 1.2 f 0.6 30.1 f 4.0 7.0 f 2.1 0.5 + 0.4 0.6 f 0.0 9.0 f 0.5 37.0 + 0.6 3.7 + 0.2 1.4 f. 0.5 TI
1.1 f 1.1 0.5 f 0.1 0.9 f 0.0 Tr TI 46.3 44.6 7.6
Aedes aegypti
Aedes albopictus
Exp’
Statb
1.8e + 0.5f 4.1 f 0.3 1.6 f 0.3 Tr 21.1 f 0.7 13.8 + 0.1 0.8 ?: 0.1
6.1 f 0.2 4.3 f 0.4 2.9 + 0.1 Tr 18.6 + 0.2 14.6 f 1.2 0.7 t 0.0
8.2 35.7 6.7 1.0 1.7
6.1 23.2 13.9 1.4 1.6 0.7
f 0.3 + 0.3 _+0.1 f 0.1 f 0.6
TI 2.4 f 0.4 0.9 f 0.2
?:0.0 f 0.2 _+2.0 f 0.1 f 0.1 f 0.4
Tr 4.0 f 0.4 1.7 f 0.0
37.7 51.1 11.0
37.6 41.4 21.0
Exp 1.1 f 0.1 3.9 k 0.2 1.5 25.0 13.4 0.7
* f f k
0.1 0.3 0.2 0.0
9.0 37.8 3.9 0.7 1.5
k 0.3 f 0.6 _+0.1 i: 0.1 + 0.0
Stat 3.8 f 0.0 4.2 f 0.1 1.5 f 0.1 Tr 17.1 + 0.1 11.6 f 0.1 0.7 + 0.1 8.0 28.7 12.1 1.3 2.6 0.8
?r 0.1 f 0.2 i 0.2 f 0.0 i 0.1 A 0.0
Tr 1.0 t 0.1 0.5 f 0.1
0.6 f 0.1 4.7 2 0.0 2.3 f 0.2
42.7 51.2 6.1
36.4 42.6 21.0
aExponential phase of growth. bStationary phase of growth. CNumber of carbon atoms in acid: number of double bonds. dTr, trace, less than 0.5%. eRelative percentage of fatty acid. fMean f standard error. g22-series acids.
esters and free fatty acids than the medium. The percentages of neutral lipid fractions of the Aedes cells were similar to those in other insect cells and unlike those of mammalian cells. The amount of sterol in the medium and in the A. albopictus cells in the exponential phase was approximately equal. The A. aegypti cells at both phases of growth and the A. albopictus cells in the stationary phase contained a larger percentage of sterol than the medium. I ,2- Diglycerides The fatty acid profiles of the l,Zdiglycerides of the medium and mosquito cells are shown in Table 2. The 1,Zdiglycerides of both mosquito cells had smaller amounts of 16:O fatty acid in both phases and larger
amounts of 18:2 and 20-carbon unsaturated acids in the stationary phase in comparison to the fatty acids in the growth medium. The A. afbopictm cells in the exponential phase had larger percentages of 16: 1 and 18: 1 fatty acids than the medium. 1,3-Diglycerides In the 1,3-diglycerides (Table 3) the Aedes cells had larger percentages of 18:0 at both phases of growth, and a larger amount of 18:2 fatty acid in the stationary phase than was found in the medium. The A. albopictw cells, in addition, had a larger percentage of 18:l fatty acid in the exponential phase and a smaller percentage of 16:0 acid in the stationary phase than were found in the medium.
NEUTRAL
LIPIDS
OF
MOSQUITO
167
CELLS
TABLE 6 Constituent Fatty Acids of Neutral Lipid Classes: Sterol Esters Constituent 12:oc 14:o 15:o 16:0 16:l 17:o 17:l 18:O 18:l 18:2 18:3 2o:o 2O:l 20:2 20:3 20:4 20:5 Saturates Monoenes Polyenes
Leafhopper medium 0.6d 2.6 1.4 13.5 9.8
f 0.6e t 0.8 k 0.5 + 2.2 A 2.4
1.5 1.2 20.4 30.4 4.2 1.1
f 0.3 f 0.5 f 0.9 + 5.8 f 0.5 f 0.5
Aedes aegypti
Expa
Statb
Exp
Stat
5.0 f 0.6
1.8 A 0.4 Tr 17.8 * 0.3 12.7 i 0.4 1.2 i 0.0
2.0 + 0.5 Tr 8.2 + 1.4 10.6 f 0.8 1.6 k 0.1
3.9 zk0.1 42.5 f 0.9 12.9 f 0.2 1.7 * 0.1 Tr
4.8 + 0.2 57.1 f 2.0 9.0 f 0.2 0.7 i 0.1 1.0 + 0.1 0.8 + 0.1 Tr 0.7 + 0.0 2.1 + 0.3 1.1 + 0.1 17.6 68.5 13.6
1.8 + 0.2 Trf
16.2 f 1.4 12.9 + 2.6 1.4 f 0.3
12.1 f 0.0 18.2 + 1.3 1.8 f 0.6
3.3 44.4 11.5 1.0 1.0
5.0 f 0.5 42.5 f 0.6 9.1 f 1.8 0.8 f 0.8 0.6 f 0.1 Tr 1.5 f 1.5 Tr 1.9 f 0.3 Tr 24.5 60.7 13.3
f 1.5 f 1.8 + 0.7 f 0.9 f 1.0
0.9 f 0.9
1.9 * 0.6 8.6 f 0.6 1.8 + 0.0 20.4 31.7 47.8
Aedes albopictus
1.4 f 0.4 2.9 2 0.3 2.6 f 1.6 23.7 57.3 19.4
0.5 f 0.1 3.4 + 0.1 1.3 f 0.5 24.7 55.2 19.8
aExponential phase of growth. bStationary phase of growth. CNumber of carbon atoms in acid: number of double bonds. dRelative percentage of fatty acid. eMean i standard error. fTr, trace, less than 0.5%.
the stationary phase than the medium. The The free fatty acid profiles of A. aegypti A. albopictus cells had larger amounts of and A. albopictus (Table 4) contained less 16: 1 acid in both phases of growth and larger 18:O acid and more 16:l acid in both phases, amounts of 18:2 and 20-carbon unsaturated and less 18:l and more 18:2 and 20-carbon acids and smaller amounts of 16:O and 18:l unsaturated acids in the stationary phase fatty acids in the stationary phase than the than were observed in the leafhopper me- medium. dium. The A. albopictus cells in the sta- Sterol Esters tionary phase had less 16:0 acid than the meIn comparison to the medium, the A. dium. aegypti and A. albopictw cells, exponential Triglycerides and stationary phases, had smaller amounts of 18:2 and 20-carbon unsaturated fatty acids Compared to the medium, the two mosand larger amounts of 18:l fatty acid (Table quito cells contained more 16:l fatty acid in 6). The A. aegypti cells, stationary phase, the triglyceride fraction (Table 5) at the exhad a larger percentage of 16: 1 acid than the ponential and stationary phases. The A. medium. aegypti cells had smaller amounts of 16:0 acid and larger amounts of 16:l and 18:2 DISCUSSION acids in both phases of growth and smaller amounts of 18:0 and 18:l acids and larger The variations in composition of the conamounts of 20-carbon unsaturated acids in stituents of the neutral lipids of the two Free Fatty Acids
168
YANG
species are not large enough to use as a taxonomic tool. lmportant aspects of the metabolic activity of lipids in insects are the rates and synthesis of fatty acids by insects at different stages of development (Municio et al., 1972; Fast, 1970). All insects have a definite requirement for sterols (Clayton, 1964; Vaughn et al., 1971). Some insects either cannot synthesize polyunsaturated 18-carbon acids or synthesize these acids at a rate sufficient to meet their growth requirements (Stephen and Gilbert, 1969; Chippendale et al., 1964). Van Handel and Lum (1961) reported that linoleic and linolenic acids could be synthesized to a limited extent by Aedes sollicitans. Fast (1970) lists species requiring a dietary source of polyunsaturated 18carbon fatty acids for successful metamorphosis from larvae to adults and for effective reproduction. Yurkiewicz and Bhalla (1969) studied the lipids of the mutant bronze Aedes aegypti and the wild-type A. aegypti to detect alterations in lipid metabolism which might account for the failure of the eggs of the mutant bronze to complete its embryonic development. The mutant bronze mosquito differed from the wild-type A. aegypti in that it possessed more saturated fatty acids and less unsaturated acids in a number of neutral lipid and phospholipid fractions. The mosquito cells grown in spinner culture showed increases in the amount of unsaturation of acids and chain length of fatty acids with aging of cells in the free fatty acids and diglycerides. In the triglyceride fraction, both Aedes cells showed more unsaturation of the fatty acids, and the A. albopictus cells also showed chain elongation in the stationary phase in comparison to the exponential phase of growth. In A. aegypti cells there was a slight decrease in average chain length of the fatty acids, and in the A. albopictus cells there was some increase in chain length of the acids in the sterol ester fraction with aging. In both mosquito cells the amount of unsaturation of acids was about the same or showed a slight decrease in the sterol ester fraction from the exponential
r-El
”
AL.
to the stationary phase of growth. Percentage increases in the free sterols and sterol esters in the stationary phases of the mosquito cells would indicate that the growth medium provided an ample supply of sterols for synthesis. A significant amount of the fatty acids synthesized by both mitochondria and microsomes is incorporated into triglycerides, phospholipids, cholesterol, and cholesterol esters (Aeberhard and Menkes, 1968). As the A. albopictus cells aged there was a decrease in the percentage of triglycerides and increases in the amounts of sterols and sterol esters. These changes in the lipid composition of the mosquito cell would indicate that different metabolic rates or pathways occurred in the hydrolysis to free fatty acids and synthesis of the lipid components. There was a tendency for greater change with aging in the fatty acid profiles of the A. albopictus cells than was observed in A. aegypti cells. These studies can now be used as a basis for designing radioisotope experiments to determine turnover rates of specific lipid fractions which may play an important role in the normal and arbovirus infected mosquito cells. ACKNOWLEDGMENTS The authors thank A. Conway for her excellent technical assistance. This study was supported in part by Public Health Service Research Grant No. HL 08214 from the Program Projects Branch, Extramural Programs, National Heart and Lung Institute, and by The Hormel Foundation.
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