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Synthesis of energy for overwintering in natural populations of the mosquito Culex tarsalis
Comp. Biochem. Physiol., 1970, Vol. 35, pp. 503 to 506. PergamonPress. Printed in Great Britain
SHORT COMMUNICATION SYNTHESIS OF ENERGY FOR O V E R W I N T E R I N G IN NATURAL P O P U L A T I O N S OF THE M O S Q U I T O CULEX TARSALIS C H A R L E S H. S C H A E F E R * and R O B E R T K. W A S H I N O t University of California (Received 5 ffanuary 1970)
Abstract--1. Overwintering Culex tarsalis adults synthesized lipids during October and early November 1968 in the Sacramento Valley of California. Approximately 78 per cent of the total lipids are triglycerides, which are the main store of energy for overwintering. 2. About 15 per cent of the total lipids separated with the free fatty acid fraction. 3. The fatty acids of the triglyceride fraction are almost entirely composed of compounds commonly encountered in insect tissues, while five unknown compounds account for over 15 per cent of the free fatty acid fraction. INTRODUCTION C U L E X T A R S A L I S , the vector of encephalitis in California, overwinters as in-
seminated, adult females. Previous work has indicated that a large increase and decrease in lipid content of adults occurs during the fall and winter (Schaefer & Washino, 1969). A study was conducted to (1) provide information on shortterm changes in the amount of lipids in adults during the overwintering period, (2) to determine the lipid class distribution and (3) to determine the fatty acid content of the major lipid classes. MATERIALS AND METHODS Overwintering Culex tarsalis adults were collected in Sutter, Yolo and Solano Counties of California, as previously described (Schaefer & Washino, 1969). Collection of adults was discontinued after January 1969 because of initiation of blood feeding. Extraction and determination of total lipids were also as described in the work above. The lipid classes of the largest sample (22 November 1968) were separated by column chromatography on Florisil as described by Carroll (1961). In addition, the lipid classes of all of the samples were compared following separation by thin-layer chromatography on silica-gel layers; the solvent system was n-hexane-ethyl ether-acetic acid (90 : 10 : 1 v/v) and detection was by iodine-vapor uptake. The triglyceride fraction of the largest sample was saponified and the fatty acids were esterified and analyzed by gas-liquid chromatography as previously described (Schaefer & Washino, 1969). In addition, the free fatty acid fraction from the same sample was also analyzed in the same manner. * Mosquito Control Research Laboratory, 5545 East Shields Avenue, Fresno, California, 93727. t Department of Entomology, Davis, California, 95616. 503
504
CHARLES H . SCHAEFER AND ROBERT K . W A S H I N O
RESULTS AND DISCUSSION T h e lipid content of C. tarsalis adults at the various collection dates is shown in T a b l e 1. While the n u m b e r of adults in some samples was relatively small, due to collection difficulty, there is a consistent trend in fresh weight and in lipid content. I t is apparent that lipid synthesis occurred under field conditions in October and that the m a x i m u m lipid content was reached by early N o v e m b e r , after which lipid content steadily declined. I n an approximate 1-month period (October to N o v e m b e r ) , the lipid content m o r e than doubled. T h e s e results confirm the earlier report of changes in lipids of overwintering C. tarsalis. Culex tarsalis AI)ULTS DURING THE 1 9 6 8 - - 1 9 6 9 x,VINTERING PERIOD
TABLE 1 - - T H E
L I P I D CONTENT OF
OVER-
Collection date
No.
Fresh wt. (mg/adult)
Total lipids (rag/adult)
Lipids (%) (wet x~t.)
2 October 1968 10 October 1968 22 October 1968 5 November 1968 22 November 1968 27 November 1968 4 December 1968 13 December 1968 19 December 1968 7 January 1969 17 January 1969 30 January 1969
7 7 33 22 102 20 16 56 50 54 23 8
1.30 1.92 2.11 2'46 2.24 2.33 2.56 2"13 2-04 1.91 1 91 1"89
0.2(/ 0.32 0'37 0.46 0-34 0-34 0-35 t).28 0.28 0.22 0.18 0'18
15'1 16-5 17"5 18-6 15"4 14"5 13 "9 13"3 13-7 11 "8 9.7 9"3
T h e sample of 22 N o v e m b e r 1968 contained 35.2 m g of total lipids which was large enough to allow gravimetric determination of the fractions separated by column c h r o m a t o g r a p h y (Table 2). Approximately 93 per cent of the total lipids were present in two fractions: triglycerides 77.6 per cent and free fatty acids r['ABLE 2 - - L I P I D
CLASS ANALYSES OF TOTAL LIPIDS FROM
Culex tarsalis
ADULTS COLLECTED
22 NOVEMBER1968 Fraction 1. 2. 3. 4. 5. 6. 7.
Lipid class
Eluant
Lipids (mg)
% of total
Hydrocarbons Sterol esters Triglycerides Sterols Diglycerides Monoglycerides Free fatty acids
n-Hexane 5% ether in hexane 15% ether in hexane 25% ether in hexane 50% ether in hexane 2% methanol in ether 4% acetic acid in ether
0-58 0"79 29.15 0'50 0.57 0'28 5.68
1.54 2.10 77.63 1.33 1.52 0.74 15" 12
SYNTHESIS OF ENERGY FOR OVERWINTRRING CULEX TARSALIS
505
15'1 per cent. T h e lipids from the other adult samples were not large enough to allow separation and gravimetric measurement; however, visual comparison of the classes separated by thin-layer chromatography revealed that all samples had lipid class distributions generally similar to that of the 22 November 1968 sample. T h e January 1969 samples showed diminished amounts of triglycerides in relation to the free fatty acid fractions, in comparison to the November or December 1968 samples. This indicates that the free fatty acid fraction is utilized to a much lesser extent than triglycerides. Based on evidence given in this paper, triglycerides are the main energy reserve for C. tarsalis as is reported for another mosquito, Aedes sollicitans (Van Handel, 1965). W h e n the triglyceride fraction of the 22 November 1968 sample was saponified, 27.7 mg of fatty acids were obtained. Esterification of the latter yielded 28.3 mg of the methyl esters. Esterification of the free fatty acid fraction (5.7 mg) of the same sample yielded only 3.2 mg of methyl esters; this low yield will be discussed later. T h e fatty acid composition of the triglyceride and free fatty acid fractions of the 22 November 1968 sample is shown in Table 3. T h e triglyceride fraction has a fatty acid composition similar to that previously reported for that of the total lipids of C. tarsalis (Schaefer & Washino, 1969), except that unknown compounds are T A B L E 3 - - P E R C E N T A G E COMPOSITION OF THE FATTY ACIDS FROM THE TRIGLYCERIDE AND FREE FATTY ACID FRACTIONS OF THE 2 2 NOVEMBER 1 9 6 8 SAMPLE OF Culex tar$ali$ LIPIDS
Compound* C 12:0 C 12:1 C 14:0 C 14:1 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2 plus unknown No. 1 Unknown No. 3 C 18:3 Unknown No. 2 Unknown No. 4 Unknown No. 5 Unknown No. 6
Triglyceride fraction
Free fatty acid fraction
Relative retention time
1-0 0"4 5'2 8'0 17'6 47'1 0'7 18'7
0'3 Tracer 1-6 2-2 12"0 30'2 2'4 24'2
------1"00 1"24
6"3 0"8 4 "4 2.2 3"5 2'2 7"6
1'61 2-03 2"24 2.65 3"88 4"71 5"33
1'4 N.D.:~ Trace Trace N.D. Trace N.D.
* The first number refers to the number of carbons, the second to the number of double bonds. t Trace is < 0"2 per cent. N.D. is not detected.
506
CHARLESH.
SCHAEFER AND ROBERT K . WASHINO
almost absent. It is apparent that most of the unknown materials are in the free fatty acid fraction. The relative retention times for the unknowns and those for known C 18 compounds are shown in Table 3. It is likely that unknown No. 3 is C 20:1, as it has an identical retention time and since it was previously demonstrated that hydrogenation of the fatty acids yielded a small amount of C 20:0 (Schaefer & Washino, 1969). The low yield of methyl esters for the free fatty acid fraction may be due to several factors: (1) the esterification efficiency of the BFa-methanol reagent with the unknowns may be less than for the identified fatty acids and/or (2) the free fatty acid fraction obtained by column chromatography of the total lipids may contain non-fatty acid components. Thus, C. tarsalis overwintering adults synthesize triglycerides as their main store of energy for overwintering during October and early November. The source and composition of the lipid precursor(s) is presently under investigation. REFERENCES CARROLLK. K. (1961) Separation of lipid classes by chromatography on florisil, ft. Lipid Research 2, 135-141. SCHAEFERC. H. & WASHINOR. K. (1969) Changes in the composition of lipids and fatty acids in adult Culex tarsalis and Anopheles freeborni during the overwintering period, ft. Insect Physiol. 15, 395-402. VANHANDELE. (1965) The obese mosquito. J. Physiol., Lond. 181,478-486. Key Word Index--Culex tarsalis; mosquito; lipids; triglycerides; fatty acids.
SHORT COMMUNICATION SYNTHESIS OF ENERGY FOR O V E R W I N T E R I N G IN NATURAL P O P U L A T I O N S OF THE M O S Q U I T O CULEX TARSALIS C H A R L E S H. S C H A E F E R * and R O B E R T K. W A S H I N O t University of California (Received 5 ffanuary 1970)
Abstract--1. Overwintering Culex tarsalis adults synthesized lipids during October and early November 1968 in the Sacramento Valley of California. Approximately 78 per cent of the total lipids are triglycerides, which are the main store of energy for overwintering. 2. About 15 per cent of the total lipids separated with the free fatty acid fraction. 3. The fatty acids of the triglyceride fraction are almost entirely composed of compounds commonly encountered in insect tissues, while five unknown compounds account for over 15 per cent of the free fatty acid fraction. INTRODUCTION C U L E X T A R S A L I S , the vector of encephalitis in California, overwinters as in-
seminated, adult females. Previous work has indicated that a large increase and decrease in lipid content of adults occurs during the fall and winter (Schaefer & Washino, 1969). A study was conducted to (1) provide information on shortterm changes in the amount of lipids in adults during the overwintering period, (2) to determine the lipid class distribution and (3) to determine the fatty acid content of the major lipid classes. MATERIALS AND METHODS Overwintering Culex tarsalis adults were collected in Sutter, Yolo and Solano Counties of California, as previously described (Schaefer & Washino, 1969). Collection of adults was discontinued after January 1969 because of initiation of blood feeding. Extraction and determination of total lipids were also as described in the work above. The lipid classes of the largest sample (22 November 1968) were separated by column chromatography on Florisil as described by Carroll (1961). In addition, the lipid classes of all of the samples were compared following separation by thin-layer chromatography on silica-gel layers; the solvent system was n-hexane-ethyl ether-acetic acid (90 : 10 : 1 v/v) and detection was by iodine-vapor uptake. The triglyceride fraction of the largest sample was saponified and the fatty acids were esterified and analyzed by gas-liquid chromatography as previously described (Schaefer & Washino, 1969). In addition, the free fatty acid fraction from the same sample was also analyzed in the same manner. * Mosquito Control Research Laboratory, 5545 East Shields Avenue, Fresno, California, 93727. t Department of Entomology, Davis, California, 95616. 503
504
CHARLES H . SCHAEFER AND ROBERT K . W A S H I N O
RESULTS AND DISCUSSION T h e lipid content of C. tarsalis adults at the various collection dates is shown in T a b l e 1. While the n u m b e r of adults in some samples was relatively small, due to collection difficulty, there is a consistent trend in fresh weight and in lipid content. I t is apparent that lipid synthesis occurred under field conditions in October and that the m a x i m u m lipid content was reached by early N o v e m b e r , after which lipid content steadily declined. I n an approximate 1-month period (October to N o v e m b e r ) , the lipid content m o r e than doubled. T h e s e results confirm the earlier report of changes in lipids of overwintering C. tarsalis. Culex tarsalis AI)ULTS DURING THE 1 9 6 8 - - 1 9 6 9 x,VINTERING PERIOD
TABLE 1 - - T H E
L I P I D CONTENT OF
OVER-
Collection date
No.
Fresh wt. (mg/adult)
Total lipids (rag/adult)
Lipids (%) (wet x~t.)
2 October 1968 10 October 1968 22 October 1968 5 November 1968 22 November 1968 27 November 1968 4 December 1968 13 December 1968 19 December 1968 7 January 1969 17 January 1969 30 January 1969
7 7 33 22 102 20 16 56 50 54 23 8
1.30 1.92 2.11 2'46 2.24 2.33 2.56 2"13 2-04 1.91 1 91 1"89
0.2(/ 0.32 0'37 0.46 0-34 0-34 0-35 t).28 0.28 0.22 0.18 0'18
15'1 16-5 17"5 18-6 15"4 14"5 13 "9 13"3 13-7 11 "8 9.7 9"3
T h e sample of 22 N o v e m b e r 1968 contained 35.2 m g of total lipids which was large enough to allow gravimetric determination of the fractions separated by column c h r o m a t o g r a p h y (Table 2). Approximately 93 per cent of the total lipids were present in two fractions: triglycerides 77.6 per cent and free fatty acids r['ABLE 2 - - L I P I D
CLASS ANALYSES OF TOTAL LIPIDS FROM
Culex tarsalis
ADULTS COLLECTED
22 NOVEMBER1968 Fraction 1. 2. 3. 4. 5. 6. 7.
Lipid class
Eluant
Lipids (mg)
% of total
Hydrocarbons Sterol esters Triglycerides Sterols Diglycerides Monoglycerides Free fatty acids
n-Hexane 5% ether in hexane 15% ether in hexane 25% ether in hexane 50% ether in hexane 2% methanol in ether 4% acetic acid in ether
0-58 0"79 29.15 0'50 0.57 0'28 5.68
1.54 2.10 77.63 1.33 1.52 0.74 15" 12
SYNTHESIS OF ENERGY FOR OVERWINTRRING CULEX TARSALIS
505
15'1 per cent. T h e lipids from the other adult samples were not large enough to allow separation and gravimetric measurement; however, visual comparison of the classes separated by thin-layer chromatography revealed that all samples had lipid class distributions generally similar to that of the 22 November 1968 sample. T h e January 1969 samples showed diminished amounts of triglycerides in relation to the free fatty acid fractions, in comparison to the November or December 1968 samples. This indicates that the free fatty acid fraction is utilized to a much lesser extent than triglycerides. Based on evidence given in this paper, triglycerides are the main energy reserve for C. tarsalis as is reported for another mosquito, Aedes sollicitans (Van Handel, 1965). W h e n the triglyceride fraction of the 22 November 1968 sample was saponified, 27.7 mg of fatty acids were obtained. Esterification of the latter yielded 28.3 mg of the methyl esters. Esterification of the free fatty acid fraction (5.7 mg) of the same sample yielded only 3.2 mg of methyl esters; this low yield will be discussed later. T h e fatty acid composition of the triglyceride and free fatty acid fractions of the 22 November 1968 sample is shown in Table 3. T h e triglyceride fraction has a fatty acid composition similar to that previously reported for that of the total lipids of C. tarsalis (Schaefer & Washino, 1969), except that unknown compounds are T A B L E 3 - - P E R C E N T A G E COMPOSITION OF THE FATTY ACIDS FROM THE TRIGLYCERIDE AND FREE FATTY ACID FRACTIONS OF THE 2 2 NOVEMBER 1 9 6 8 SAMPLE OF Culex tar$ali$ LIPIDS
Compound* C 12:0 C 12:1 C 14:0 C 14:1 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2 plus unknown No. 1 Unknown No. 3 C 18:3 Unknown No. 2 Unknown No. 4 Unknown No. 5 Unknown No. 6
Triglyceride fraction
Free fatty acid fraction
Relative retention time
1-0 0"4 5'2 8'0 17'6 47'1 0'7 18'7
0'3 Tracer 1-6 2-2 12"0 30'2 2'4 24'2
------1"00 1"24
6"3 0"8 4 "4 2.2 3"5 2'2 7"6
1'61 2-03 2"24 2.65 3"88 4"71 5"33
1'4 N.D.:~ Trace Trace N.D. Trace N.D.
* The first number refers to the number of carbons, the second to the number of double bonds. t Trace is < 0"2 per cent. N.D. is not detected.
506
CHARLESH.
SCHAEFER AND ROBERT K . WASHINO
almost absent. It is apparent that most of the unknown materials are in the free fatty acid fraction. The relative retention times for the unknowns and those for known C 18 compounds are shown in Table 3. It is likely that unknown No. 3 is C 20:1, as it has an identical retention time and since it was previously demonstrated that hydrogenation of the fatty acids yielded a small amount of C 20:0 (Schaefer & Washino, 1969). The low yield of methyl esters for the free fatty acid fraction may be due to several factors: (1) the esterification efficiency of the BFa-methanol reagent with the unknowns may be less than for the identified fatty acids and/or (2) the free fatty acid fraction obtained by column chromatography of the total lipids may contain non-fatty acid components. Thus, C. tarsalis overwintering adults synthesize triglycerides as their main store of energy for overwintering during October and early November. The source and composition of the lipid precursor(s) is presently under investigation. REFERENCES CARROLLK. K. (1961) Separation of lipid classes by chromatography on florisil, ft. Lipid Research 2, 135-141. SCHAEFERC. H. & WASHINOR. K. (1969) Changes in the composition of lipids and fatty acids in adult Culex tarsalis and Anopheles freeborni during the overwintering period, ft. Insect Physiol. 15, 395-402. VANHANDELE. (1965) The obese mosquito. J. Physiol., Lond. 181,478-486. Key Word Index--Culex tarsalis; mosquito; lipids; triglycerides; fatty acids.