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Lipid composition of the developing larval fat body of Phormia regina
J. InsectPhysiol.,1967, Vol. 13, pp. 889 to 898. Pepnon
Prus Ltd.
Printed in Great Britain
LIPID COMPOSITION OF THE DEVELOPING LARVAL FAT BODY OF PHORMIA REGINA L. T. WIMER and R. H. LUMB University of South Carolina, Department of Biology, Columbia, South Carolina (Received 22 August 1966 ; mvised 23 Nouember 1966)
Abstract-The lipid composition of the larval fat body of Phmia mgina was analysed at different periods during third&star development. Lipid represented 21.8 per cent of the fat-body dry weight in the early third instar, whereas in the mature larva it constituted 52.5 per cent. Fractionation of the lipid into triglyceride, partial glycerides (includes free fatty acids), and phospholipid was accomplished using column and thin-layer chromatography. Triglyceride was found as the predominant lipid class accounting for almost 90 per cent of the total lipid of the prepupal fat body. Fatty-acid analysis of the three lipid fractions, employing gas-liquid chromatography, revealed the presence of three major (palmitoleic, oleic, and palmitic) and three minor (myristic, steak, and myristoleic) fatty acids. The major fatty acids constituted 81.6 to 964 per cent of the total fatty acids and in general palmitoleic acid predominated. Phospholipid exhibited a greater percentage of palmitoleic and oleic acids than did triglyceride with the exception of the prepupal fat body. The change in the pattern of distribution of fatty acids in the prepupa resulted from a decline in the percentage of pahnitoleic and oleic acids of phospholipid with a corresponding increase in the percentage of palmitic acid. The distribution of fatty acids in triglyceride did not change appreciably during development. INTRODUCTION
DIPTEROUS larvae are known to accumulate lipids during development (YUILL and CRAIG, 1937; LEVINSONand SILVERMAN,1954; PEARINCOTT, 1960), and the chemical nature of the larval lipids has been reported for several species (BIEBER et al., 1961; FASTand BROWN,1962; BARLOW,1965 ; MIURAet al., 1965 ; TAKATA and HARWOOD, 1964). The major site of lipid storage has long been considered to be the larval fat body although only recently was its exact composition determined (WIMER,1%2; SRIDH~UU and BHAT,1965 ; WLODAWER and BAR&KA, 1965). The pattern of accumulation of fat-body reserves may well be under hormonal control. However, before a study of the control mechanisms is feasible, a determination of the chemical constituents of the fat body is first necessary in order to reveal metabolic changes associated with growth and development. MATERIALS
AND METHODS
The atock culture of PM re@ruzMe&n waa acquired from the strain maintained at the Department of Biology, Johns Hopkins University. 889
890
L. T.
WIMER
AND
R. H. LUMB
Culture techniques
The adults were housed in a screen wire cage and fed crystalline sucrose and water. Pork liver was placed in the cage to stimulate egg production (BRUSTand FRAENKJZL,1955). The eggs were collected over a 3 hr period and processed following the procedure of HILL et al. (1947). The eggs, when maintained at 28.5 of:O*S”C, began to hatch about 14 hr after the end of the collecting period and were allowed to hatch over a 3 hr period. By weighing the eggs before and after the hatching period, it was possible to estimate the total number of eggs placed in the culture bottles and the number that hatched. This was essential to minimize variations in larval growth due to population size. The culture medium was the same as the ‘basic diet’ of BRUSTand FRAENKEL (1955) for Phormiu except that dried brewer’s yeast was substituted for the vitamin solution. The bottles of larvae were covered with fine mesh cloth. On the second day coarse sawdust was placed in each bottle to prevent excessive desiccation of the medium (HILL et al., 1947). Larval growth was measured from the mean time of hatching ( + 1.5 hr) to the prepupal stage (Fig. 1). Ten larvae of almost uniform size were removed at selected times from each culture, weighed, and discarded. The stage at puparium formation is called the prepupa.
6
2
3
4
5
6
PP
7
Days
FIG. 1. Growth curve of larvaldevelopment. 1,Ecdysie to second instar; -, to third instar. PP-prepupa.
Determination of percentqe
ecdyeis
of water
The percentage of water was determined for the whole larva and the fat body. Fat bodies were dissected in insect Ringer’s solution (EPHRUSSIand BEADLE,1936). The dry weight was obtained by freezing with solid carbon dioxide and drying in vacua to a constant weight. The same procedure was used to obtain fat bodies for
lipid extraction except they were stored at - 20°C until used.
LIPID COMPOSITION OF LARVAL FAT BODY OF PHORbUA
REGINA
891
Lipid extract&m and separation of lipid classes (Procedwe A) (1957). This extraction method was according to Procedure 5 of ENTENMAN Lyophilixed fat bodies were homogenixed in 2 ml of 10% trichloroacetic acid (TCA) at 0°C using an all-glass homogenizer. The homogenate was centrifuged at 15,OOOgand 0°C for 30 min. The residue was washed once with 2 ml of cold 10% TCA and then 2 ml of cold distilled water. Lipids were extracted twice from the remaining residue with 6 ml of absolute ethanol-diethyl ether (3 : 1) at 60°C for 1 hr, and the residuewaswashed oncewith mlof ether. The combined lipid fraction was evaporatedto drynessin wucuo,takenup immediatelyin benzene, and centrifuged to remove traces of protein. The benzene fraction was evaporated in dryness in sacua and weighed. All weighings were made with a Mettler Balance Model MS. Lipid classes were separated by silicic acid column chromatography according to the procedure of CHANGand SWIZUY (1963). Lipids were eluted with: (I) 150 ml of 20% benzene in hexane (hydrocarbons and sterol esters); (II) 300 ml of 60% benxene in hexane collected in 20 ml samples (triglyceride); (III) 250 ml of chloroform (partial glycerides, free fatty acids, and sterols) ; and (IV) 300 ml of methanol (phospholipids). Monitoring of the samples on thin-layer chromatography (TLC) revealed incomplete separationof fractions II and III. To separate these two fractions more completely, the latter few samples of fraction II and fraction III were combined and rechromatographed as before. When rechromatographed, the latter samples of fraction II again contained some components found in fraction III, so these samples were combined with fraction III. However, rechromatographing did facilitate more complete separation. The four fractions were evaporated to dryness and weighed. Lipid extmctbn and separation of &id ckzsses (Procedtie B) Lipids were extracted and washed using the procedure of FOLCHet al. (1957). Lyophilized fat bodies were homogenized in chloroform-methanol (2 : 1) and extracted three times yielding a total of 20 ml. This fraction was washed with 4 ml of Folch salt solution. An aliquot of the lipid fraction was evaporated to dryness and weighed. The remainder was used for separation of lipid classes. Lipid classes were separated employing thin-layer chromatography. Plates coated with silica-gel H were washed with methanol-concentracted HCI (9 : 1) before being used (BANDERATH, 1964). Hexane-diethyl ether-acetic acid (90 : 10 : 1) waa used to isolate the triglycerides. The triglyceride area was stained lightly with iodine vapour and removed from the plate. The partial glyceride fraction (monoand diglycerides, free fatty acids, and sterol) was separated from phospholipid by placing the same plate in diethyl ether. Lipids were eluted from the silica gel by washing twice with 8 ml of chloroform-methanol (2 : 1) and once with methanol. An aliquot of each fraction was evaporated to dryness and weighed. Gas-&aid chromatography (GLC) procedure fat fatty-acid analyses The fatty-acid methylation procedure was carried out according to the method of MORGANet al. (1963). A Barber-Coleman Model 10 gas chromatograph was
892
L. T. WIMER AND R. H. LUMB
used for GLC. The stationary phase support was acid-washed Chromosorb W 30160 mesh which was coated with diethylene glycol succinate. Argon served as the carrier gas. The columns were maintained at 19O”C, the detector cells at 205°C and the flash heater at 225°C. The chromatograms were analysed by the triangular approximation method employing an IBM 7040 computer, which was also utilized for calculation of relative retention volumes. Fatty acids were identified by comparison with standards and by use of relative retention volumes. RESULTS
Larval growth During growth the larvae increased in weight from 0.06 mg at hatching to 50.1 mg just prior to day 6 (Fig. 1). The first ecdysis occurred about 40 hr after hatching, and the weight of the early second-instar larva was O-9 mg. The duration of second&star development was 37 hr, and the weight of the early third instar was about 9.0 mg. The largest increase in weight occurred during third-in&r development which was completed in slightly over 4 days (about 7.3 days for entire larval development). The early third-instar larva contained 82.8 per cent water with the value decreasing to 69.4 per cent in the prepupa. The decrease in weight of the larva prior to pupation (Fig. 1) resulted from a loss of water. The percentage of water in the fat body was somewhat less than that of the whole larva ranging from 46.9 to 58.5 per cent. The higher percentages were associated with the early and late thirdinstar larvae. The fat body is known to be a storage organ, and it represented an increasing proportion of the total body weight in the developing third-instar larva. In the early third instar the fat body accounted for 16.1 per cent of the total body weight (dry wt/dry wt), whereas in the prepupa it constituted 38.3 per cent. Lipi’d composition of the fat body Lipids were extracted from fat bodies at five (Procedure B) or six periods (Procedure A) during third-instar development (Table 1). The two extraction procedures showed inconclusive results. The percentage of lipid for two of the periods was comparable (Table 1, LP5 and PP), while the chloroform-methanol extraction showed slightly higher total lipid for three periods (LP2, LP3 and LP4). A comparison of the two fractionation procedures shows that TLC consistently yielded a higher percentage of triglyceride than did the column technique. This was attributed to the incomplete separation of the triglyceride from the partial glyceride fraction when using column chromatography and also to the lower percentage recovery from the column. The consistently higher percentage of phospholipid obtained from column chromatography was attributed to the presence of a small amount of neutral lipid as revealed by monitoring. Thin-layer chromatography is considered more accurate than column chromatography because of the distinct separation of the three fractions and because of the more consistent values for percentage recovery.
LIPID COMPOSITION OF LARVAL FAT BODY OF PHORMIA
REGINA
893
The column technique revealed the presence of about 1 per cent sterol ester and hydrocarbon, and this fraction is not included in the percentage recovered (Table 1). Only traces of sterol were found in the partial glyceride fractions. TABLS ~-COMPARISON OF ETHANOL-DIBTHYL SILICIC
ACID
COLUMN CHROMAToGRAPHY EXTRMXION
Time after ecdysis to third instar (hr)
ETHRR EXTRACTION OF FAT-BODY LIPIDS AND
FRACTIONATION WITH
CHLORoFORM-METHANOL
AND THIN-LAYER CHROMATOGRAPHY FRACITONATION
Fat body (mg dry wt.)
Total lipid (% dry wt.)
Triglyceride (% total lipid)
Partial glycerides (% total lipid)
Phosphohpid (% total lipid)
Amount recovered (%)
Ethanoldiethyl 0.66 (128)t 12 (LPI)* 1.47 (82) 28 (LP2) 2.53 (44) 44 (LP3) 3.31 (40) 60 (LP4) 4.09 (38) 84 (LPS) Prepupa (PP) 4.81 (26)
ether extraction and silicic acid column fractionation 33.2 90.6 18.0 21.8 39.4 17.9 88.5 19.5 33.7 51.1 10.4 95.2 21.7 41.3 63.1 11.4 96.0 15-2 47.6 69.4 8.1 92.6 9.9 52.5 74.6 11.5 101.3 9.1 43.4 80.7
Chloroform-methanol 1.59 (47) 28 (LP2) 2.42 (36) 44 (LP3) 3.75 (28) 60 (LP4) 4.31 (24) 84 (LPS) Prepupa (PP) 5.82 (20)
extraction and thin-layer chromatography fractionation 16.4 103.3 13.8 39.5 72.8 8.5 97.7 7.3 49.0 81.9 4.8 96.7 5-8 52.6 86.1 5.8 101.3 7.0 52.3 88.5 5.9 99.3 4.5 44.6 88.9
* (LPl)-larval period 1, etc. t (128)-number of fat bodies analysed; fat bodies were divided into almost equal numbers and duplicate analyses made.
During development, lipid constituted an increasing proportion of the total fat body (Table 1) except just prior to pupation when the percentage decreased. The percentage decrease did not result from a net decline in lipid (Table 2) but rather from a proportional increase in protein (WIMER,unpublished observation). Triglyceride is the major storage form of lipid, and it showed a proportional increase during development (Table 1). The increasing proportion of triglyceride resulted in a concomitant decrease in the percentage of partial glycerides and phospholipid. In spite of this decrease, however, a net synthesis of partial glycerides and phospholipid did occur (Table 2). Fatty-acid analysis of da$mnt &id classes Fatty acids were analysed only on the duplicate fractions separated by TLC (Table 3). Palmitoleic (C16:l) and ok (C18:l) were found to be the major unsaturated acids and palmitic (C16:O) the major saturate. These three fatty acids accounted for 816-964 per cent of the total (lowest in triglyceride of LP5 and
894
L. T. WIM~RANDR. H. LUMB
highest in phospholipid of LPS). Myristic (Cl&O), myristoleic (Clkl), and stearic (Cl&O) are considered minor components with traces of lauric (C12:O) and linoleic (C18:2) acids. TARLE 2-CONTRNT
OF TOTAL LIPID AND DIFFRRRNT LIPID CLASSES OF THR FAT BODY DURING THIRD-INSTAR DRVRLOPMRNT OF PhO?WIkl YC&KJ
Time
after
ecdysis
to
third instar (hr) 28 (~~2)t 44 (LP3) 60 (LP4) a4 (LP5) Prepupa (PP)
Partial
Fat body (m:Yy
Total lipid (:%Y
1.59 2.42 3.75 4.31 5.82
Triglyceride * (mglfst body)
glycerides
O-46 0.97 l-70 l-99 2.31
0.09 o-09 0.11 O-16 o-12
0.63 1.19 l-97 2.25 2.59
Phospholipid (:%Y
(Zzt
0.10 0.10 o-10 0.13 0.15
* Data from thin-layer chromatography. 7 (LP2)-larval period 2, etc. TABLE3--FA’I-TY-ACID
COMPOSITION OF FAT-BODY TRIGLYCRRIDE, PARTIAL GLYCRRIDBS, AND
PHOBPHOLIPID DURING THIRD-INBTAR DRVELOPMRNT OF &?T7Ih
C16:l
cla:l
C16:O
C14:o
W$?h
cla:o
C14:l
Others*
28 hrt (LP~): Triglyceride Partial glycerides Phospholipid
31.6 39-a 38-o
26.4 29.3 29.3
25.9 20.7 24.0
6.8 4.4 3.9
3.2 1.3 0.9
2.7 2.3 1.0
3.4 22 2.9
44 hr (LP3) Triglyceride Partial glycerides Phospholipid
30.6 33.7 42.5
29-a 31-4 35.4
24.1 26.1 la-5
5.6 3.4 1.3
3-O 1.4 0.1
2.5 1.5 0.4
44 2-5 1.8
60 hr (LP4) Triglyceride Partial glycerides Phospholipid
32.3 36.6 37.5
27.6 32.3 38.2
25.6 21.2 19.4
5.4 3-6 1.4
3.6 l-5 0.5
2.1 2.2 o-4
3.4 2.6 2.6
84 hr (LPS) Triglyceride Partial glycerides Phospholipid
27.7 37.1 37-5
30.7 29.1 39-2
23.2 21.6 20.1
7.0 3.2 0.8
4-7 2.0 0.3
2.6 1.8 0.3
41 5.2 1.8
Prepupa (PP) Triglyceride Partial glycerides Phospholipid
32.9 27.4 30.2
29.1 27.2 28.7
27.4 30.7 2a5
43 3.3 2*3
2.6 4-a O-5
l-3 0.9 1.9
2.4 5.7 7.9
* Laurie, linoleic, and several unidentified fatty acids. t Hours sfter ecdysis to third instar. $ (LP2)---larval period 2, etc.
LIPID COMPOSITION OF LARVAL FAT BODY OF PHORMIA
REGINA
895
Several general statements can be made concerning the pattern of distribution to ofthefottyacidainthethreefrrctionawirthinagivenperioduldrrlaowith on the change in distribution of the fatty.acids during development : (1) the of fatty acids usually was palmitoleic>oleic ,palmitic >myristic >stearic > myriatoleic; (2) phospholipid and partial glyceride more frequently exhibited a greater percentage of pahnitoleic and oleic acids than did triglyceride with the exception of the prepupal stage when the pattern was reversed; the change in the distribution of fatty acids in the prepupal stage resulted chiefly from a decline in the percentage of pahnitoleic and oleic acids in the phospholipid and partial glyceride fractions; in the phospholipid fraction oleic acid showed an increasing trend until the prepupal stage; (3) palmitic, myristic, steak, and myristoleic acids were usually higher in triglyceride than in phospholipid or partial glycerides; however, a striking change in the distribution of palmitic acid occurred in the prepupa as a result of its increase in both the phospholipid and partial glyceride fractions.
DISCUSSION Studies on the lipid composition of insects have been confined primarily to whole animals, and some of these studies will be included for comparative purposes. Emphasis will be placed on the order Diptera although a cursory account of members of other insect orders will be given. According to FAST(1964) the average lipid content of larvae is about 30 per cent whereas in the adult the value is about 20 per cent (dry-weight basis). Since the fat body is considered as the major site of lipid storage, it is expected that its lipid content would be higher. This principle held true for the adult fat body of Phomtia regiruE(ORR, 1964) which contained over 51 per cent lipid (dry weight). In the larval fat body of Photrrda mgzku the lipid content reached a high of 52 per cent (Table 1, LPS). SRIDHARAand BHAT (1%5) have presented a comprehensive study on the lipid composition of different silkworm tissues and found that the fat body contained 32 per cent lipid while the whole larva contained only 17 per cent (dry weight). In addition, the silkworm fat body contained 59 per cent of the total lipid in the larva. Triglyceride has been found as the predominant lipid class in the fat body (Table 1) as well as in whole animals. Employing column chromatography, values of greater than 80 per cent triglyceride (percentage of total lipid) were found for aduh aphids (STRONG, 1963), adult diapausing boll weevils (LAMBRRMONT et al., 1964), and larvae of the greater wax moth (YOUNG, 1964). SRILMRA and BRAT (1965) found only 43 per cent triglyceride (including free fatty acids) in whole larvae of Bomlyx mot-i, whereas the isolated fat body yielded about 62 per cent. The opposite condition was found for phospholipid with the higher percentage in whole animals and lower percentage in the fat body (SRIDHARAand BRAT, 1965; W~ODA~ERand BAR&KA, 1%5; compare Table 1 with BIBPERet al., 1961). In fact the predominant lipid class in the intestine and silk gland of the silkworm was phospholipid (SRIDHARAand BUT, 1965).
896
L. T. WIMERANDR. H.
LIJMB
The percentage of triglyceride in the fat body is directly correlated with the stage of development (Table 1). In the early third-in&u larva, prior to the period of rapid accumulation of lipid, the fat body would be expected to contain a greater proportion of non-storage lipids, i.e. lipid which constitutes structural components such as the phospholipid of membranes. During subsequent growth and development the storage lipids (triglyceride) increased at a faster rate than did the structural lipids (phospholipid) resulting in a proportional increase in the former and a concomitant decrease in the latter. In addition, WIMER (unpublished observation) observed a decline in the percentage of fat-body protein associated with the proportional decrease in phospholipid. In a recent review of insect lipids, FAST (1964) noted that relatively complete fatty-acid analyses are available for only about forty-five species of insects. All of these studies were on lipids obtained from whole animals, and with the exception of the paper by FAST and BROWN(1962), th e analyses were made on total lipid. Since the review by FAST(1964) several reports have appeared on lipids of individual tissues and also on the fatty-acid composition of different lipid classes (LAMBREMONTet al., 1964; FAST, 1965 ; SRIDHARAand BHAT, 1965 ; W~ODAWER and Btisx~, 1965). The principal fatty acids in insect lipids as listed by FAST (1965) are myristic, myristoleic, palmitic, palmitoleic, stearic, oleic, linoleic, and linolenic. The quantitative distribution of these acids varies for different insect orders as pointed out originally by BARLOW(1963, 1964). Two basic differences noted by BARLOW (1963) were the high percentage of palmitoleic acid in Diptera and the high percentage of myristic acid in Aphidoidea. The high precentage of myristic acid in aphids was also reported by STRONG (1963). BARLOW(1964) has suggested a possible relationship between the high pahnitoleic acid content of the lipid of A&u u#nis (Diptera : Sarcophagidae) and its apparent absence of a requirement for polyunsaturated fatty acids. FAST (1%5) has shown recently that the high percentage of palmitoleic acid is not characteristic of all Diptera, since only a small percentage was found in two species of Cecidomyiidae. The quantitative distribution of fatty acids in the fat body of Phormkz regina (Table 3) agrees well with the distribution of fatty acids in other Diptera (VAN HANDEL and LUM, 1961; FAST and BROWN, 1962; BARLOW,1963; MIX et al., 1965). The only basic difference is the trace amount of linoleic acid as compared with larger amounts found by BARLOW(1964) for several different genera. The fatty-acid composition of whole Calltphora erythrocephala larvae (MIURA et al., 1965) is very similar to results presented here for Phormia regina (Calliphoridae). Two other reports on the fatty-acid composition of the insect fat body are the works of SRIDHARAand BHAT (1965) on the silkworm, Bombyx mori, and W~ODAWERand BARAI&KA(1965) on wax-moth larvae, GaUeria me&mella. Since both of these studies are on Lepidoptera, their results are at variance with those obtained for Phonmia fat body. Apparently the present investigation is the only report on the fatty-acid composition of different lipid classes during development, although FAST and BROWN
LIPID COMWSITION OF LARVALFAT BODY OF PHOIUUIA RJZINA
897
(1962) detcrmined the fatty-acid composition of neutral lipid and cephalin for a single larval stage of As&s wgypti. In both Fkwmak rspiaa and A&s tzqppti phospholipid exhibited a higher percentage of palmitoleic and oleic than did neutral lipid. Another striking feature of the results for Pkomda is the change in the quantitative distribution of pahnitoleic, oleic, and palmitic acids of the phospholipids from LP5 to the prepupa. These changes could result from the utilization or synthesis of specific phospholipid fatty acids. Since phospholipid is presumably involved in the synthesis and secretion of protein (HOKIN and HOKIN, 1956; HENDLBR,1958), and since protein synthesis increases just prior to pupation (BUTIZRWORTH et al., 1965 ; WIMER,unpublished observation), the conclusion is tentatively proposed that the change in the distribution of fatty acids in phospholipid is related to an increase in protein synthesis. The distribution of fatty acids in triglyceride did not change appreciably during development. Ac~Zedg~t-The authors express their appreciation critically reading the manuscript.
to Dr. J. M. HERR for
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HOKIN L. E. and HOKINM. R. (1956) The actions of pancreozymin in pancreas t&es and
the r61eof phoepholipida in enzyme secretion. y. Physiol., Land. 132,442+53. LAMBREMONT E. N., BLUMM. S., and SCHRADER R. M. (1964) Storage and fatty acid composition of triglycerides during adult diapauseof the boll weevil. Ann. ent. Sot. Am. 57, 526-532. LEVINSON 2. H. and SILVBRMAN P. H. (1954) Studies on the lipids of Muscu tic&u (Macq.) during growth and metamorphosis. Biochem.J. !58,294-297. MIURAK., VONKH. J., ZANDEJI D. I., and Houx N. W. H. (1965) Biosynthesisof unsaturated and saturatedfatty acids in asepticallyreared larvae of CaUa”horaetythroce#h& (Meig.). Archs int. Physiol. Biochim. 73,65-72. MORGANT. E., HANAHAN D. J., and EKHOLM J. (1963) A rapid method for deacyIation of phoapholipida and neutral lipids. Fed. Proc. 22,419. ORRC. W. M. (1964) The influence of nutritional and hormonal factors on the chemistry of the fat body, blood, and ovaries of the blowfly Phormiu regina Meig. _7.InsecctPhysioi. 10,103-119. PEARINCOTT J. V. (1960) Changes in the lipid content during growth and metamorphosis of the housefly, Musca domestica Linnaeus. g. ceil. con@. Physiol. 55, 167-174. F~NDERATHK. (1964) Thin-ZuyerChromatography. Academic Press, New York. SRIDIURAS. and BUT J. V. (1965) Lipid composition of the silkworm Bombyx mori L. r. Insect Physiol. 11,449-%2. STRONGF. E. (1963) Studies on lipids in Bornehomopterous insects. HiZgardia 34,43-61. TAKATAN. and HARWOODR. F. (1964) Fatty acid composition during postembryonic development of the moaquito C&x tarsalis. Ann. ent. Sot. Am. 57, 749-753. VAN HANDELE. and LUM P. T. M. (1961) Sex aa a regulator of triglyceride metabolism. Scietzce,N. Y. 134,1979-1980. WIMBRL. T. (1962) Biochemical changes associated with growth and development of the larval blowfly, Phormiu regina Meigen. Ph.D. Thesis, University of Virginia. WLODAWERP. and BAR&U J. (1965) Lipolytic activity of the fat body of the wax moth larvae--I. Fatty acid composition of the fat body and of the haemolymph lipids and release of free fatty acid8 from the fat body during incubation. Acta b&him. $01. 12, 23-37. YOUNG R. G. (1964) Lipids of the larva of the greater wax moth, Galleria melkme~%. Ann. ent. Sot. Am. 57,321-324. YUILLJ. S. and CRAIGR. (1937) The nutrition of flesh fly larvae,LucZa sericata (Meig.)-II. The development of fat. J. exp. Zool. 75, 169-178.
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LIPID COMPOSITION OF THE DEVELOPING LARVAL FAT BODY OF PHORMIA REGINA L. T. WIMER and R. H. LUMB University of South Carolina, Department of Biology, Columbia, South Carolina (Received 22 August 1966 ; mvised 23 Nouember 1966)
Abstract-The lipid composition of the larval fat body of Phmia mgina was analysed at different periods during third&star development. Lipid represented 21.8 per cent of the fat-body dry weight in the early third instar, whereas in the mature larva it constituted 52.5 per cent. Fractionation of the lipid into triglyceride, partial glycerides (includes free fatty acids), and phospholipid was accomplished using column and thin-layer chromatography. Triglyceride was found as the predominant lipid class accounting for almost 90 per cent of the total lipid of the prepupal fat body. Fatty-acid analysis of the three lipid fractions, employing gas-liquid chromatography, revealed the presence of three major (palmitoleic, oleic, and palmitic) and three minor (myristic, steak, and myristoleic) fatty acids. The major fatty acids constituted 81.6 to 964 per cent of the total fatty acids and in general palmitoleic acid predominated. Phospholipid exhibited a greater percentage of palmitoleic and oleic acids than did triglyceride with the exception of the prepupal fat body. The change in the pattern of distribution of fatty acids in the prepupa resulted from a decline in the percentage of pahnitoleic and oleic acids of phospholipid with a corresponding increase in the percentage of palmitic acid. The distribution of fatty acids in triglyceride did not change appreciably during development. INTRODUCTION
DIPTEROUS larvae are known to accumulate lipids during development (YUILL and CRAIG, 1937; LEVINSONand SILVERMAN,1954; PEARINCOTT, 1960), and the chemical nature of the larval lipids has been reported for several species (BIEBER et al., 1961; FASTand BROWN,1962; BARLOW,1965 ; MIURAet al., 1965 ; TAKATA and HARWOOD, 1964). The major site of lipid storage has long been considered to be the larval fat body although only recently was its exact composition determined (WIMER,1%2; SRIDH~UU and BHAT,1965 ; WLODAWER and BAR&KA, 1965). The pattern of accumulation of fat-body reserves may well be under hormonal control. However, before a study of the control mechanisms is feasible, a determination of the chemical constituents of the fat body is first necessary in order to reveal metabolic changes associated with growth and development. MATERIALS
AND METHODS
The atock culture of PM re@ruzMe&n waa acquired from the strain maintained at the Department of Biology, Johns Hopkins University. 889
890
L. T.
WIMER
AND
R. H. LUMB
Culture techniques
The adults were housed in a screen wire cage and fed crystalline sucrose and water. Pork liver was placed in the cage to stimulate egg production (BRUSTand FRAENKJZL,1955). The eggs were collected over a 3 hr period and processed following the procedure of HILL et al. (1947). The eggs, when maintained at 28.5 of:O*S”C, began to hatch about 14 hr after the end of the collecting period and were allowed to hatch over a 3 hr period. By weighing the eggs before and after the hatching period, it was possible to estimate the total number of eggs placed in the culture bottles and the number that hatched. This was essential to minimize variations in larval growth due to population size. The culture medium was the same as the ‘basic diet’ of BRUSTand FRAENKEL (1955) for Phormiu except that dried brewer’s yeast was substituted for the vitamin solution. The bottles of larvae were covered with fine mesh cloth. On the second day coarse sawdust was placed in each bottle to prevent excessive desiccation of the medium (HILL et al., 1947). Larval growth was measured from the mean time of hatching ( + 1.5 hr) to the prepupal stage (Fig. 1). Ten larvae of almost uniform size were removed at selected times from each culture, weighed, and discarded. The stage at puparium formation is called the prepupa.
6
2
3
4
5
6
PP
7
Days
FIG. 1. Growth curve of larvaldevelopment. 1,Ecdysie to second instar; -, to third instar. PP-prepupa.
Determination of percentqe
ecdyeis
of water
The percentage of water was determined for the whole larva and the fat body. Fat bodies were dissected in insect Ringer’s solution (EPHRUSSIand BEADLE,1936). The dry weight was obtained by freezing with solid carbon dioxide and drying in vacua to a constant weight. The same procedure was used to obtain fat bodies for
lipid extraction except they were stored at - 20°C until used.
LIPID COMPOSITION OF LARVAL FAT BODY OF PHORbUA
REGINA
891
Lipid extract&m and separation of lipid classes (Procedwe A) (1957). This extraction method was according to Procedure 5 of ENTENMAN Lyophilixed fat bodies were homogenixed in 2 ml of 10% trichloroacetic acid (TCA) at 0°C using an all-glass homogenizer. The homogenate was centrifuged at 15,OOOgand 0°C for 30 min. The residue was washed once with 2 ml of cold 10% TCA and then 2 ml of cold distilled water. Lipids were extracted twice from the remaining residue with 6 ml of absolute ethanol-diethyl ether (3 : 1) at 60°C for 1 hr, and the residuewaswashed oncewith mlof ether. The combined lipid fraction was evaporatedto drynessin wucuo,takenup immediatelyin benzene, and centrifuged to remove traces of protein. The benzene fraction was evaporated in dryness in sacua and weighed. All weighings were made with a Mettler Balance Model MS. Lipid classes were separated by silicic acid column chromatography according to the procedure of CHANGand SWIZUY (1963). Lipids were eluted with: (I) 150 ml of 20% benzene in hexane (hydrocarbons and sterol esters); (II) 300 ml of 60% benxene in hexane collected in 20 ml samples (triglyceride); (III) 250 ml of chloroform (partial glycerides, free fatty acids, and sterols) ; and (IV) 300 ml of methanol (phospholipids). Monitoring of the samples on thin-layer chromatography (TLC) revealed incomplete separationof fractions II and III. To separate these two fractions more completely, the latter few samples of fraction II and fraction III were combined and rechromatographed as before. When rechromatographed, the latter samples of fraction II again contained some components found in fraction III, so these samples were combined with fraction III. However, rechromatographing did facilitate more complete separation. The four fractions were evaporated to dryness and weighed. Lipid extmctbn and separation of &id ckzsses (Procedtie B) Lipids were extracted and washed using the procedure of FOLCHet al. (1957). Lyophilized fat bodies were homogenized in chloroform-methanol (2 : 1) and extracted three times yielding a total of 20 ml. This fraction was washed with 4 ml of Folch salt solution. An aliquot of the lipid fraction was evaporated to dryness and weighed. The remainder was used for separation of lipid classes. Lipid classes were separated employing thin-layer chromatography. Plates coated with silica-gel H were washed with methanol-concentracted HCI (9 : 1) before being used (BANDERATH, 1964). Hexane-diethyl ether-acetic acid (90 : 10 : 1) waa used to isolate the triglycerides. The triglyceride area was stained lightly with iodine vapour and removed from the plate. The partial glyceride fraction (monoand diglycerides, free fatty acids, and sterol) was separated from phospholipid by placing the same plate in diethyl ether. Lipids were eluted from the silica gel by washing twice with 8 ml of chloroform-methanol (2 : 1) and once with methanol. An aliquot of each fraction was evaporated to dryness and weighed. Gas-&aid chromatography (GLC) procedure fat fatty-acid analyses The fatty-acid methylation procedure was carried out according to the method of MORGANet al. (1963). A Barber-Coleman Model 10 gas chromatograph was
892
L. T. WIMER AND R. H. LUMB
used for GLC. The stationary phase support was acid-washed Chromosorb W 30160 mesh which was coated with diethylene glycol succinate. Argon served as the carrier gas. The columns were maintained at 19O”C, the detector cells at 205°C and the flash heater at 225°C. The chromatograms were analysed by the triangular approximation method employing an IBM 7040 computer, which was also utilized for calculation of relative retention volumes. Fatty acids were identified by comparison with standards and by use of relative retention volumes. RESULTS
Larval growth During growth the larvae increased in weight from 0.06 mg at hatching to 50.1 mg just prior to day 6 (Fig. 1). The first ecdysis occurred about 40 hr after hatching, and the weight of the early second-instar larva was O-9 mg. The duration of second&star development was 37 hr, and the weight of the early third instar was about 9.0 mg. The largest increase in weight occurred during third-in&r development which was completed in slightly over 4 days (about 7.3 days for entire larval development). The early third-instar larva contained 82.8 per cent water with the value decreasing to 69.4 per cent in the prepupa. The decrease in weight of the larva prior to pupation (Fig. 1) resulted from a loss of water. The percentage of water in the fat body was somewhat less than that of the whole larva ranging from 46.9 to 58.5 per cent. The higher percentages were associated with the early and late thirdinstar larvae. The fat body is known to be a storage organ, and it represented an increasing proportion of the total body weight in the developing third-instar larva. In the early third instar the fat body accounted for 16.1 per cent of the total body weight (dry wt/dry wt), whereas in the prepupa it constituted 38.3 per cent. Lipi’d composition of the fat body Lipids were extracted from fat bodies at five (Procedure B) or six periods (Procedure A) during third-instar development (Table 1). The two extraction procedures showed inconclusive results. The percentage of lipid for two of the periods was comparable (Table 1, LP5 and PP), while the chloroform-methanol extraction showed slightly higher total lipid for three periods (LP2, LP3 and LP4). A comparison of the two fractionation procedures shows that TLC consistently yielded a higher percentage of triglyceride than did the column technique. This was attributed to the incomplete separation of the triglyceride from the partial glyceride fraction when using column chromatography and also to the lower percentage recovery from the column. The consistently higher percentage of phospholipid obtained from column chromatography was attributed to the presence of a small amount of neutral lipid as revealed by monitoring. Thin-layer chromatography is considered more accurate than column chromatography because of the distinct separation of the three fractions and because of the more consistent values for percentage recovery.
LIPID COMPOSITION OF LARVAL FAT BODY OF PHORMIA
REGINA
893
The column technique revealed the presence of about 1 per cent sterol ester and hydrocarbon, and this fraction is not included in the percentage recovered (Table 1). Only traces of sterol were found in the partial glyceride fractions. TABLS ~-COMPARISON OF ETHANOL-DIBTHYL SILICIC
ACID
COLUMN CHROMAToGRAPHY EXTRMXION
Time after ecdysis to third instar (hr)
ETHRR EXTRACTION OF FAT-BODY LIPIDS AND
FRACTIONATION WITH
CHLORoFORM-METHANOL
AND THIN-LAYER CHROMATOGRAPHY FRACITONATION
Fat body (mg dry wt.)
Total lipid (% dry wt.)
Triglyceride (% total lipid)
Partial glycerides (% total lipid)
Phosphohpid (% total lipid)
Amount recovered (%)
Ethanoldiethyl 0.66 (128)t 12 (LPI)* 1.47 (82) 28 (LP2) 2.53 (44) 44 (LP3) 3.31 (40) 60 (LP4) 4.09 (38) 84 (LPS) Prepupa (PP) 4.81 (26)
ether extraction and silicic acid column fractionation 33.2 90.6 18.0 21.8 39.4 17.9 88.5 19.5 33.7 51.1 10.4 95.2 21.7 41.3 63.1 11.4 96.0 15-2 47.6 69.4 8.1 92.6 9.9 52.5 74.6 11.5 101.3 9.1 43.4 80.7
Chloroform-methanol 1.59 (47) 28 (LP2) 2.42 (36) 44 (LP3) 3.75 (28) 60 (LP4) 4.31 (24) 84 (LPS) Prepupa (PP) 5.82 (20)
extraction and thin-layer chromatography fractionation 16.4 103.3 13.8 39.5 72.8 8.5 97.7 7.3 49.0 81.9 4.8 96.7 5-8 52.6 86.1 5.8 101.3 7.0 52.3 88.5 5.9 99.3 4.5 44.6 88.9
* (LPl)-larval period 1, etc. t (128)-number of fat bodies analysed; fat bodies were divided into almost equal numbers and duplicate analyses made.
During development, lipid constituted an increasing proportion of the total fat body (Table 1) except just prior to pupation when the percentage decreased. The percentage decrease did not result from a net decline in lipid (Table 2) but rather from a proportional increase in protein (WIMER,unpublished observation). Triglyceride is the major storage form of lipid, and it showed a proportional increase during development (Table 1). The increasing proportion of triglyceride resulted in a concomitant decrease in the percentage of partial glycerides and phospholipid. In spite of this decrease, however, a net synthesis of partial glycerides and phospholipid did occur (Table 2). Fatty-acid analysis of da$mnt &id classes Fatty acids were analysed only on the duplicate fractions separated by TLC (Table 3). Palmitoleic (C16:l) and ok (C18:l) were found to be the major unsaturated acids and palmitic (C16:O) the major saturate. These three fatty acids accounted for 816-964 per cent of the total (lowest in triglyceride of LP5 and
894
L. T. WIM~RANDR. H. LUMB
highest in phospholipid of LPS). Myristic (Cl&O), myristoleic (Clkl), and stearic (Cl&O) are considered minor components with traces of lauric (C12:O) and linoleic (C18:2) acids. TARLE 2-CONTRNT
OF TOTAL LIPID AND DIFFRRRNT LIPID CLASSES OF THR FAT BODY DURING THIRD-INSTAR DRVRLOPMRNT OF PhO?WIkl YC&KJ
Time
after
ecdysis
to
third instar (hr) 28 (~~2)t 44 (LP3) 60 (LP4) a4 (LP5) Prepupa (PP)
Partial
Fat body (m:Yy
Total lipid (:%Y
1.59 2.42 3.75 4.31 5.82
Triglyceride * (mglfst body)
glycerides
O-46 0.97 l-70 l-99 2.31
0.09 o-09 0.11 O-16 o-12
0.63 1.19 l-97 2.25 2.59
Phospholipid (:%Y
(Zzt
0.10 0.10 o-10 0.13 0.15
* Data from thin-layer chromatography. 7 (LP2)-larval period 2, etc. TABLE3--FA’I-TY-ACID
COMPOSITION OF FAT-BODY TRIGLYCRRIDE, PARTIAL GLYCRRIDBS, AND
PHOBPHOLIPID DURING THIRD-INBTAR DRVELOPMRNT OF &?T7Ih
C16:l
cla:l
C16:O
C14:o
W$?h
cla:o
C14:l
Others*
28 hrt (LP~): Triglyceride Partial glycerides Phospholipid
31.6 39-a 38-o
26.4 29.3 29.3
25.9 20.7 24.0
6.8 4.4 3.9
3.2 1.3 0.9
2.7 2.3 1.0
3.4 22 2.9
44 hr (LP3) Triglyceride Partial glycerides Phospholipid
30.6 33.7 42.5
29-a 31-4 35.4
24.1 26.1 la-5
5.6 3.4 1.3
3-O 1.4 0.1
2.5 1.5 0.4
44 2-5 1.8
60 hr (LP4) Triglyceride Partial glycerides Phospholipid
32.3 36.6 37.5
27.6 32.3 38.2
25.6 21.2 19.4
5.4 3-6 1.4
3.6 l-5 0.5
2.1 2.2 o-4
3.4 2.6 2.6
84 hr (LPS) Triglyceride Partial glycerides Phospholipid
27.7 37.1 37-5
30.7 29.1 39-2
23.2 21.6 20.1
7.0 3.2 0.8
4-7 2.0 0.3
2.6 1.8 0.3
41 5.2 1.8
Prepupa (PP) Triglyceride Partial glycerides Phospholipid
32.9 27.4 30.2
29.1 27.2 28.7
27.4 30.7 2a5
43 3.3 2*3
2.6 4-a O-5
l-3 0.9 1.9
2.4 5.7 7.9
* Laurie, linoleic, and several unidentified fatty acids. t Hours sfter ecdysis to third instar. $ (LP2)---larval period 2, etc.
LIPID COMPOSITION OF LARVAL FAT BODY OF PHORMIA
REGINA
895
Several general statements can be made concerning the pattern of distribution to ofthefottyacidainthethreefrrctionawirthinagivenperioduldrrlaowith on the change in distribution of the fatty.acids during development : (1) the of fatty acids usually was palmitoleic>oleic ,palmitic >myristic >stearic > myriatoleic; (2) phospholipid and partial glyceride more frequently exhibited a greater percentage of pahnitoleic and oleic acids than did triglyceride with the exception of the prepupal stage when the pattern was reversed; the change in the distribution of fatty acids in the prepupal stage resulted chiefly from a decline in the percentage of pahnitoleic and oleic acids in the phospholipid and partial glyceride fractions; in the phospholipid fraction oleic acid showed an increasing trend until the prepupal stage; (3) palmitic, myristic, steak, and myristoleic acids were usually higher in triglyceride than in phospholipid or partial glycerides; however, a striking change in the distribution of palmitic acid occurred in the prepupa as a result of its increase in both the phospholipid and partial glyceride fractions.
DISCUSSION Studies on the lipid composition of insects have been confined primarily to whole animals, and some of these studies will be included for comparative purposes. Emphasis will be placed on the order Diptera although a cursory account of members of other insect orders will be given. According to FAST(1964) the average lipid content of larvae is about 30 per cent whereas in the adult the value is about 20 per cent (dry-weight basis). Since the fat body is considered as the major site of lipid storage, it is expected that its lipid content would be higher. This principle held true for the adult fat body of Phomtia regiruE(ORR, 1964) which contained over 51 per cent lipid (dry weight). In the larval fat body of Photrrda mgzku the lipid content reached a high of 52 per cent (Table 1, LPS). SRIDHARAand BHAT (1%5) have presented a comprehensive study on the lipid composition of different silkworm tissues and found that the fat body contained 32 per cent lipid while the whole larva contained only 17 per cent (dry weight). In addition, the silkworm fat body contained 59 per cent of the total lipid in the larva. Triglyceride has been found as the predominant lipid class in the fat body (Table 1) as well as in whole animals. Employing column chromatography, values of greater than 80 per cent triglyceride (percentage of total lipid) were found for aduh aphids (STRONG, 1963), adult diapausing boll weevils (LAMBRRMONT et al., 1964), and larvae of the greater wax moth (YOUNG, 1964). SRILMRA and BRAT (1965) found only 43 per cent triglyceride (including free fatty acids) in whole larvae of Bomlyx mot-i, whereas the isolated fat body yielded about 62 per cent. The opposite condition was found for phospholipid with the higher percentage in whole animals and lower percentage in the fat body (SRIDHARAand BRAT, 1965; W~ODA~ERand BAR&KA, 1%5; compare Table 1 with BIBPERet al., 1961). In fact the predominant lipid class in the intestine and silk gland of the silkworm was phospholipid (SRIDHARAand BUT, 1965).
896
L. T. WIMERANDR. H.
LIJMB
The percentage of triglyceride in the fat body is directly correlated with the stage of development (Table 1). In the early third-in&u larva, prior to the period of rapid accumulation of lipid, the fat body would be expected to contain a greater proportion of non-storage lipids, i.e. lipid which constitutes structural components such as the phospholipid of membranes. During subsequent growth and development the storage lipids (triglyceride) increased at a faster rate than did the structural lipids (phospholipid) resulting in a proportional increase in the former and a concomitant decrease in the latter. In addition, WIMER (unpublished observation) observed a decline in the percentage of fat-body protein associated with the proportional decrease in phospholipid. In a recent review of insect lipids, FAST (1964) noted that relatively complete fatty-acid analyses are available for only about forty-five species of insects. All of these studies were on lipids obtained from whole animals, and with the exception of the paper by FAST and BROWN(1962), th e analyses were made on total lipid. Since the review by FAST(1964) several reports have appeared on lipids of individual tissues and also on the fatty-acid composition of different lipid classes (LAMBREMONTet al., 1964; FAST, 1965 ; SRIDHARAand BHAT, 1965 ; W~ODAWER and Btisx~, 1965). The principal fatty acids in insect lipids as listed by FAST (1965) are myristic, myristoleic, palmitic, palmitoleic, stearic, oleic, linoleic, and linolenic. The quantitative distribution of these acids varies for different insect orders as pointed out originally by BARLOW(1963, 1964). Two basic differences noted by BARLOW (1963) were the high percentage of palmitoleic acid in Diptera and the high percentage of myristic acid in Aphidoidea. The high precentage of myristic acid in aphids was also reported by STRONG (1963). BARLOW(1964) has suggested a possible relationship between the high pahnitoleic acid content of the lipid of A&u u#nis (Diptera : Sarcophagidae) and its apparent absence of a requirement for polyunsaturated fatty acids. FAST (1%5) has shown recently that the high percentage of palmitoleic acid is not characteristic of all Diptera, since only a small percentage was found in two species of Cecidomyiidae. The quantitative distribution of fatty acids in the fat body of Phormkz regina (Table 3) agrees well with the distribution of fatty acids in other Diptera (VAN HANDEL and LUM, 1961; FAST and BROWN, 1962; BARLOW,1963; MIX et al., 1965). The only basic difference is the trace amount of linoleic acid as compared with larger amounts found by BARLOW(1964) for several different genera. The fatty-acid composition of whole Calltphora erythrocephala larvae (MIURA et al., 1965) is very similar to results presented here for Phormia regina (Calliphoridae). Two other reports on the fatty-acid composition of the insect fat body are the works of SRIDHARAand BHAT (1965) on the silkworm, Bombyx mori, and W~ODAWERand BARAI&KA(1965) on wax-moth larvae, GaUeria me&mella. Since both of these studies are on Lepidoptera, their results are at variance with those obtained for Phonmia fat body. Apparently the present investigation is the only report on the fatty-acid composition of different lipid classes during development, although FAST and BROWN
LIPID COMWSITION OF LARVALFAT BODY OF PHOIUUIA RJZINA
897
(1962) detcrmined the fatty-acid composition of neutral lipid and cephalin for a single larval stage of As&s wgypti. In both Fkwmak rspiaa and A&s tzqppti phospholipid exhibited a higher percentage of palmitoleic and oleic than did neutral lipid. Another striking feature of the results for Pkomda is the change in the quantitative distribution of pahnitoleic, oleic, and palmitic acids of the phospholipids from LP5 to the prepupa. These changes could result from the utilization or synthesis of specific phospholipid fatty acids. Since phospholipid is presumably involved in the synthesis and secretion of protein (HOKIN and HOKIN, 1956; HENDLBR,1958), and since protein synthesis increases just prior to pupation (BUTIZRWORTH et al., 1965 ; WIMER,unpublished observation), the conclusion is tentatively proposed that the change in the distribution of fatty acids in phospholipid is related to an increase in protein synthesis. The distribution of fatty acids in triglyceride did not change appreciably during development. Ac~Zedg~t-The authors express their appreciation critically reading the manuscript.
to Dr. J. M. HERR for
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HOKIN L. E. and HOKINM. R. (1956) The actions of pancreozymin in pancreas t&es and
the r61eof phoepholipida in enzyme secretion. y. Physiol., Land. 132,442+53. LAMBREMONT E. N., BLUMM. S., and SCHRADER R. M. (1964) Storage and fatty acid composition of triglycerides during adult diapauseof the boll weevil. Ann. ent. Sot. Am. 57, 526-532. LEVINSON 2. H. and SILVBRMAN P. H. (1954) Studies on the lipids of Muscu tic&u (Macq.) during growth and metamorphosis. Biochem.J. !58,294-297. MIURAK., VONKH. J., ZANDEJI D. I., and Houx N. W. H. (1965) Biosynthesisof unsaturated and saturatedfatty acids in asepticallyreared larvae of CaUa”horaetythroce#h& (Meig.). Archs int. Physiol. Biochim. 73,65-72. MORGANT. E., HANAHAN D. J., and EKHOLM J. (1963) A rapid method for deacyIation of phoapholipida and neutral lipids. Fed. Proc. 22,419. ORRC. W. M. (1964) The influence of nutritional and hormonal factors on the chemistry of the fat body, blood, and ovaries of the blowfly Phormiu regina Meig. _7.InsecctPhysioi. 10,103-119. PEARINCOTT J. V. (1960) Changes in the lipid content during growth and metamorphosis of the housefly, Musca domestica Linnaeus. g. ceil. con@. Physiol. 55, 167-174. F~NDERATHK. (1964) Thin-ZuyerChromatography. Academic Press, New York. SRIDIURAS. and BUT J. V. (1965) Lipid composition of the silkworm Bombyx mori L. r. Insect Physiol. 11,449-%2. STRONGF. E. (1963) Studies on lipids in Bornehomopterous insects. HiZgardia 34,43-61. TAKATAN. and HARWOODR. F. (1964) Fatty acid composition during postembryonic development of the moaquito C&x tarsalis. Ann. ent. Sot. Am. 57, 749-753. VAN HANDELE. and LUM P. T. M. (1961) Sex aa a regulator of triglyceride metabolism. Scietzce,N. Y. 134,1979-1980. WIMBRL. T. (1962) Biochemical changes associated with growth and development of the larval blowfly, Phormiu regina Meigen. Ph.D. Thesis, University of Virginia. WLODAWERP. and BAR&U J. (1965) Lipolytic activity of the fat body of the wax moth larvae--I. Fatty acid composition of the fat body and of the haemolymph lipids and release of free fatty acid8 from the fat body during incubation. Acta b&him. $01. 12, 23-37. YOUNG R. G. (1964) Lipids of the larva of the greater wax moth, Galleria melkme~%. Ann. ent. Sot. Am. 57,321-324. YUILLJ. S. and CRAIGR. (1937) The nutrition of flesh fly larvae,LucZa sericata (Meig.)-II. The development of fat. J. exp. Zool. 75, 169-178.