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Lipid utilization in adult Pieris brassicae with special reference to the rôle of linolenic acid
J. Insect Physiol., 1974, Vol. 20, pp. 1257 to 1269. Pergamon Press. tinted
in Great Brituin
LIPID UTILIZATION IN ADULT PIERIS BRASSICAE WITH SPECIAL REFERENCE TO THE ROLE OF LINOLENIC ACID SEPPO TURUNEN Department of Physiological Zoology, University of Helsinki, 00100 Helsinki 10, Finland (First received 2 October 1973 ; revised 19 October 1973) Abstract-Fatty acids of adult Pieris brassicae and the incorporation of dietary linolenic acid-lJ4C into adult (and egg) lipids were analysed 1 and 9 days after ecdysis. Females grown on a leaf diet retained palmitic, palmitoleic, and oleic acids but lost linoleic and linolenic acids during adult life, while males utilized their fatty acids more evenly. On an artificial diet both sexes retained pahnitic acid but utilized palmitoleic and oleic acids. In both cases females laid eggs with a high palmitic and oleic acid content. Analysis of thorax flight muscles (artificial diet) revealed that 67.9% of the lipids in l-day females and 83.6% in 9-day females was phospholipid (PL). During adult life linolenic acid increased in thorax neutral lipids (NL) from 14.6 to 20.0% in females and from 18.5 to 30.0% in males. Males incorporated more linolenic acid-1-14C especially in fat body and flight muscle PL than females. The majority of this was recovered from phosphatidyl cholines (PTC) in l-day adults whereas in 9-day adults phosphatidyl ethanolamines (PTE) and another compound, most likely cardiolipin, contained more label (29.0% in PTC, 33.1% in PTE, 34.9% in cardiolipin, and 2.0% in sphingomyelin in the thorax of females). The females also incorporated the label into egg lipids (42.2% in PL, 57.8% in NL). There was recovered from PTC 54.5% of the label in egg PL. Most of the label in thorax NL was found to be in free fatty acids (FFA). The label disappeared from triglycerides during adult life and tended to accumulate in FFA (82.7% in 9-day females) while in diglycerides the label did not vary during adult life (17.2% in 9-day post-emergence females). PTC apparently is a fairly labile PL type which is utilized in muscle whereas PTE and cardiolipin may be more structural in function and accumulated more label from linolenic acid with increasing adult age. Linolenic acid, then, essentially is a structural fatty acid and its r61e appears to be mainly in the structures of flight muscle membranes and organelles. INTRODUCTION
TOWARDSTHE adult stage lipid metabolism in Lepidoptera concerns the synthesis of adult structures (i.e. DUTKOWSKI and ZIAJKA,1972) and, in many species, the preservation of reserve lipid to be utilized for energy in flight muscles (DOMROESE and GILBERT,1964; STEVENSON,1968; SACKTOR,1970). The development of eggs also requires deposition of lipid by the female. In view of the common use 1257
1258
SEPPO
TURUNEN
of artificial diets for rearing Lepidoptera it is necessary that the fatty acid requirements of the adult stage are understood, especially as regards the utilization of the essential fatty acids. Artificial diets often have a lipid content that differs from the insect’s natural source of food. It has been shown that this may bring about an altered tissue fatty acid content which is reflected in increased syntheses of some fatty acids to levels not ordinarily found in the species (SCHAEFER,1968 ; GRAUand TERRIERE, 1971; TURUNEN, 1973a). The diet commonly used for rearing Pie~is brassicue provides notably less linolenic acid than a diet of cabbage leaves (TURUNEN,1973a). Linolenic acid, as an essential fatty acid, is a constituent of egg lipids and also must have an important rale in adult flight muscles, as it is known to be concentrated especially in phospholipids when its dietary availability is low (TURUNEN, 1973b). A study was therefore undertaken to determine what are the fatty acid requirements of adult Pieris on an artificial as well as on a natural diet, and what specifically is the r61e of linolenic acid in adult and egg lipids. It was interesting also, with respect to an understanding of lipid utilization in Pieris, to find out whether there are sexual differences in fat metabolism in adults as has recently been reported in the histology of Pied fat body (KARLINSKYand POULAERT,1971). MATERIALS AND METHODS Experimental animals Pieris brassicae were from a laboratory-reared stock maintained under controlled conditions as described previously (TURUNEN, 1973a, b). The diapause stock was reared on cabbage (Bras&z oleracea var. ace$zaZu). The larvae pupated and entered diapause under conditions simulating autumn. They were thereafter kept at + 3 to + 4°C and a relative humidity of 65 to 70%. After 6 months the Eight months from the beginning of fatty acids of pupae were determined. diapause the pupae were brought to +9’C for 24 hr, then to + 17°C for another 24 hr, and finally to the insectary (+ 23”C, 65% r.h., 18 hr day length). The adults emerged after 10 to 11 days in the insectary. For the non-diapausing stock the pupal stage under the conditions used lasts from lOi to 11 days. Before the pupal-adult ecdysis the pupae were transferred to a cage (JUNNIKKALA,1966) illuminated with both artificial and direct sunlight to assure optimal flying activity and copulation during the flying period of 4 to 5 hr daily. Except for that the lights were turned off and the window shaded, leaving only the normal illumination, whereby all flying activity ceased. Adults referred to as l-day adults were sacrificed immediately after the pupaladult ecdysis or within 24 hr from ecdysis, while 9-day adults ranged from 8 to 10 days after ecdysis. Thus 9-day adults had flown for approximately 40 hr while l-day adults essentially had not flown before analysis. The adults had access to a 12% solution of honey during the flying period. In l-day females ovaries are still rather undeveloped and ova are barely visible. The first eggs are laid about 4 days after ecdysis.
LIPID UTILIZATIONIN ADULTPIERIS
1259
BRASSICAE
Analytical procedures and labelled diets All procedures and methods of analysis have been described (TURUNEN, 1972, 1973b). Visualization of phospholipids on thin-layer chromatograms was accomplished by a specific phospholipid spray, the action of which is based on metallic copper and ammonium molybdate in a sulphuric acid solution (GOSWAMI and FREY, 1971). Identification of radioactive phospholipid fractions required rechromatography with unlabelled standards and the use of several different solvent systems (c$ STAHL, 1969). The labelled diet contained 50 $Zi l*C (linolenic acid-lJ*C) per 568 g diet. Larvae were placed on the radioactive diet immediately after ecdysis to the fifth instar. RESULTS Fatty acids of the diapause stock Table 1 shows the fatty acid composition of diapausing pupae and l-day and g-day post-emergent adults. The dietary fatty acid composition has been reported earlier (TURUNEN, 1973a). In pupae the major fatty acid in neutral (NL) and TABLE ~--RELATIVE PERCENTAGE FATTYACIDCOMPOSITION OF P.brassicaeREARED ONCABBAGE Stage
16:0
16:l
l&O
l&l
18:2
18:3
Female NL* Diapause pupae 1 -day adult 9-day adult
14.5 12.4 20.5
6.9 5.3 5.1
1.1 1.1 2.9
33.0 33.3 39.1
9.0 10.0 7.8
35.5 38.0 24.6
Female PLt Diapause pupae 1 -day adult 9-day adult
8.9 11.0 16.1
2.9 2.3 2.8
12.5 6.6 5.6
22.6 22.1 19.2
21.2 14.9 14.7
31.9 43.1 41.6
Male NL Diapause pupae 1 -day adult 9-day adult
14.1 13.7 13.4
7.0 6.3 2.6
1.1 1.9 4.1
30.7 27,2 26.4
9.2 9.9 11.3
38.0 41 *o 42.2
Male PL Diapause pupae 1 -day adult 9-day adult
9.8 10.2 7.8
3.2 2.3 1.2
13.8 8-l 6.2
21.1 19.5 22.4
20.3 14.9 17.4
31.8 45.0 45.0
Eggs NL PL
20.9 13.2
4.2 5.9
2.7 6.0
40.9 36.8
8.9 17.6
22.4 20.5
* Neutral lipids. t Phospholipids. Each value is an average of three determinations, each consisting of the pooled tissues of 6 to 9 animals.
1260
SSPPO TImmEN
phospholipids (PL) is linolenate which is, exceptionally, more concentrated in NL during diapause (355% in females, 38.0% in males). The fatty acid patterns of diapausing pupae appear alike in both sexes. At the time of adult ecdysis the relative amount of linolenic acid has increased in both sexes but more notably so in PL (from 31.9 to 43-l% in females and from 3 l-8 to 45.0% in males), perhaps at the expense of other fatty acids which have been consumed. One-day females appear to have a higher concentration of oleate in NL than l-day males (33.3 vs. 27*2%), having perhaps synthesized this acid while the relative amount of oleate has actually decreased in males. An interesting aspect is an abrupt decrease in the PL stearate content from 12.5% in diapausing female pupae to 6.6% in l-day adults. Corresponding values in males were 13.8 and S*l%. There was a s~~t~eous fall in the PL linoleate content from 21.2 to 14.9% in females and from 20.3 to 14.9% in males. After 9 days of adult life the fatty acid patterns were rather dissimilar in the two sexes (Table 1). During this period the females laid eggs but the actual percentage of fertile females was not determined. The females accumulated palmitic acid (20.5% in NL, 16.1% in PL) while no such trend was shown by the males (13.4% in NL, 7.8% in PL). The females also retained more palmitoleic acid in NL than males (5-l vs. 2.6%). Th ere was a substantial increase in NL oleic acid in the females (39.1%) but none in the males (26.4%). NIore remarkable, however, was the distribution of the polyunsaturated acids. In females both linoleic and linolenic acids decreased during adult life in the neutral fraction (7.8 and 24.6% linoleate and linolenate, respectively) while practically no change was found in males (11.3 and 42*2%, respectively). Thus in Pieris reared on a cabbage diet the females tend to retain more palmitic, palmitoleic, and oleic acids and lose linoleic and linolenic acids during adult life, whereas males appear to utilize their fatty acids rather more evenly. However, as is discussed later, this difference may also be explained in terms of lipid synthesis which may occur at different rates in males and females. In Table 1 the fatty acid composition of eggs of the diapause stock has been shown. A characteristic feature is a comparatively high degree of saturation if compared with adult tissues. Oleic acid is the prevalent fatty acid in both NL and PL (40.9 and 36.8%, respectively). The content of palmitic acid is also high (20.9 and 13.2% in NL and PL, respectively). Egg NL contains almost the same amount of linolenic acid as 9-day females (22.4% in eggs vs. 24.6% in adults) while in egg PL the relative amount of linolenic acid is only half of that in g-day females (205 vs. 41.6%). Fatty acids of adults reared on an artzjicial diet When the data above are compared with values from adults reared diet as larvae, both similarities and differences in the utilization are revealed (Table 2). The metabolism of the 16 carbon acids fairly similar in both sexes. Both retain a high level of palmitic days after the pupal-adult ecdysis (29.2% in females, 26-O% in
on an artificial of fatty acids apparently is acid in NL 9 males), while
LIPID UTILIZATIONIN ADULTPIERIS TABLE ~-RELATIVE
1261
ERASSICAE
PERCENTAGE FATTY ACID COMPOSITIONOF P. brussicae REAREDON AN ARTIFICIALDIET
Stage
18:0
18:l
18~2
18:3
13.3 6.1
2.1 3.9
47.6 44.8
14.3 6.5
7.2 9.5
10.2 8.7
4.7 3.1
7.4 8.0
28.7 28.8
26.4 28.4
22.6 23.0
Male NL 1 -day adult 9-day adult
16.7 26.0
13.9 5.3
2.1 4.2
46.3 40.5
14.0 10.4
7.0 13.6
Male PL 1 -day adult 9-day adult
10.3 7.3
4.8 1.8
7.2 7.2
28.3 25.8
26.5 31.5
22.9 26.4
Eggs NL PL
18.7 11.5
8.4 5.3
4.2 10.7
45.7 30.1
13.5 22.0
9.5 20.6
16:0
16:l
Female NL 1 -day adult 9-day adult
15.5 29.2
Female PL 1 -day adult 9-day adult
Legend as in Table
1.
on the leaf diet this was encountered only in the females. TOn the artificial diet both sexes utilized palmitoleic acid during adult life. Thus l-day females had 13.3% palmitoleic acid in NL but g-day females contained only 6.1%. In males the corresponding values were 13.9 and 5*3o/o. Both sexes utilized oleic acid during adult life, although this was more readily demonstrated in males (46.3% in l-day and 4O*5o/o in g-day males). The low content of linolenic acid allows little variation during adult life but some accumulation of this acid was noted in the males, analogously to the situation on the leaf diet. By comparison the fatty acid content of eggs on the two diets showed some notable similarities. In both cases the egg oleic and palmitic acid contents were high, and in egg PL the concentration of linolenic acid, quite remarkably, was the same on both diets. The overall uniformity of the PL fatty acid pattern in the two cases suggests that the PL fatty acid content is not allowed to vary as much as the neutral lipid fatty acids for normal embryonic development. Fatty acids of the thorax To find out whether the flight and leg muscle fatty acids would give relevant data on the utilization of fatty acids by adult Pieris, further analyses were made from thorax lipids. Thoraces without appendages and the haemolymph carefully removed, mostly contain muscle tissue. Preliminary data had shown that NL in the thorax contained relatively small concentrations of triglycerides, a major lipid
1262
SEPPO TLlRUNEN
fraction in most tissues, but instead had high levels of free fatty acids. Analysis of NL of similarly prepared thoraces might suggest what fatty acids are utilized in muscle during adult life. Phospholipids, on the other hand, are expected to be mainly structural in function, although they do have a r61e in intracellular energy metabolism and possibly also in fatty acid transport. Table 3 shows the fatty acid composition of thoraces from l- and 9-day adults (artificial diet). On the whole the content of oleic acid was lower in muscle than in whole animal extracts (39.4% in NL of l-day females, 36.7% in NL of l-day males). There was also less palmitoleic acid but more polyunsaturated acids than in whole animals. Thus the content of linolenic acid in NL of l-day females was 14.6% and in males 18*5o/o, and in PL, respectively, 26.6 and 27.5%. After 9 days relative changes in PL fatty acids were slight with the exception of a rise in linoleic acid (29.7% in l-day and 37.1% in 9-day females). In the neutral fraction oleic acid decreased from 39.4 to 32.6% in females and from 36.7 to 23.5% in males. The relative amount of linolenic acid increased during the same period from 14.6 to 20.0% in females and from 18.5 to 30.0% in males. Palmitoleic acid, on the other hand, decreased to less than half the value of l-day adults (from 9.1 to 4.1% in females). Oleic and palmitoleic acids thus appeared to be utilized while linolenic and linoleic acids were retained during adult life. TABLE
3-RELATIVE
PERCENTAGE
FATTY ACID COMPOSITION (ARTIFICIAL DIRT)
OF THORACES
OF P. brassicae
16:0
16:l
18:O
18:l
18:2
l&3
Female NL 1 -day adult 9-day adult
16.1 21.0
9.1 4.1
4.4 5.6
39.4 32.6
16.4 16.7
14.6 20.0
Female PL 1 -day adult 9-day adult
9.2 6.9
3.7 2.0
5.5 4.4
25.3 24.6
29.7 37.1
26.6 25.0
Male NL 1 -day adult 9-day adult
13.7 13.6
7.3 3.7
4.3 4.7
36.7 23.5
195 24.5
18.5 30.0
Male PL 1 -day adult 9-day adult
8.9 5.4
3.8 1.9
6.1 4.9
23.8 22.3
29.9 35.9
27.5 29.6
Stage
Thoraces were thoroughly rinsed in insect Ringer to remove all haemolymph. Myristic acid has not been included in the list although its relative proportion increases up to 3% of all NL fatty acids in 9-day adults. Each value is an average of three determinations. 12 to 15 thoraces were used in one determination.
Incorporation of linohic
acid into adult lipid-s
Linolenic acid is accumulated during adult life in the males but less so in the females (artificial diet). It is mainly concentrated in PL on the artificial diet
LIPID UTILIZATION IN ADULTPIERIS
1263
BRASSICAE
while no such distinction can be made concerning Pieris reared on a leaf diet. In order to examine the function of linolenic acid more closely the larvae were given labelled linolenic acid as a dietary constituent and its distribution was then followed in adult and egg lipids. Table 4 gives the specific activities (counts/min per mg lipid) of adult lipids as well as the relative amounts of NL and PL from the weight of total lipids. PL in all cases retained more label than NL. The males, moreover, retained more label in the fat body and thorax than the females. The specific activity of male fat body PL was 30,995 counts/min and that of females 24,733 counts/n& in l-day adults. In the thorax of l-day adults the specific activity of PL was 26,765 counts/min in males and 22,668 counts/min in females. In 9-day adults the corresponding values were 30,546 and 25,677 counts/min. The specific activity of egg lipids (l-2 days after laying) was lower than in maternal tissues, perhaps due in part to accumulation of carotenoids which are lipid soluble and thus increase the weight of the lipid fraction. TABLE ~-SPECIFIC ACTIVITIES OF ADULTLIPIDS (counts/mm DIETARYLINOLRNICACID-l-14c
per mg lipid) INCORPORATING
o/0 from weight of total lipid Tissue
Total no. of animals
NL
PL
NL
PL
32.3 27.3
Whole animal 1 -day female 1 -day male
20 (3) 20 (3)
11,103 10,003
17,430 17,769
67,7 72.7
Fat body 1 -day female 1-day male
26 (2) 26 (2)
9466 9941
24,733 30,995
92.6
7.4
92.9
7.1
15,454 16,507 17,299 18,086
22,668 25,677 26,765 30,546
32.1 16.4 16.3 135
67.9 83.6 83.7 86.5
15,479
77.1
22.9
Thorax 1 -day 9-day 1 -day g-day Eggs
27 44 33 49
female female male male ca.
(3) (4) (3) (4)
2000(2)
6575
The number in parentheses indicates the number of determinations. The specific activities are averages of the indicated number of determinations.
From Table 4 the relative amounts of NL and PL can also be seen. Fat body lipids were mostly neutral (92.6% in l-day adult females). In whole animal extracts 67.7% of the lipids in females and 72.7% in males were neutral. In the thorax, quite to the contrary, most of the lipid was phospholipid. Curiously in l-day females more of the lipid was found to be neutral than in l-day males (31.1
1264
SEPPO TIJRUNEN
VS.
16.4%). In 9-day adult males 13*5o/o of the lipids in the thorax were neutral and 865% polar. Corresponding values in females were 16.4 and 83.6%. The distribution of linolenic acid in major PL types in adult tissues is shown in Table 5. In l-day adults phosphatidyl cholines (PTC) retained most of the activity. Thus in the fat body of males 47.9% of the label was recovered from PTC, 40.4% in phosphatidyl ethanolamines (PTE), and 11.6% in a less polar type, tentatively identified as cardiolipin. Traces of activity were found in sphingomyelin and another fraction corresponding to phosphatidyl serines. In the thorax 37.8% of the activity in males was found in PTC, 36.2% in PTE, 21.7% in cardiolipin and 4.3% in an unidentified fraction migrating on the front of PTE. Nine days after emergence of the adults more label was recovered from PTE and especially from cardiolipin. Thus in the thorax of 9-day males 33.9% was recovered from cardiolipin, 32.1% from PTE, and 29.6% from PTC, with some activity in sphingomyelin and the unidentified fraction. Few sexual differences were detected in connexion with any of the tissues studied. In the eggs a somewhat different pattern of labelling is seen (Table 5). PTC retained 54.S”h of the activity while only 38*1o/o was recovered from PTE and 7.4% from cardiolipin. Apparently PTC constitute a relatively greater part of
TABLE
5--RELATIVE
PERCENTAGE
DISTRIBUTION ADULT
OF
LABEL
FROM
LINOLENIC
ACID-~-~~~
PTE
Unknownt
Cardiolipin
nd nd
36.4 39.3
tr tr
21.9 20.9
46.8 47.9
tr tr
41.5 40.4
nd nd
11.7 11.6
;r2
37.8 29.0 35.7 29.6
tr tr tr tr
36.2 33.1 33.0 32.1
4.3 1.0 3.1 2.2
21.7 34.9 28.2 33.9
nd
54.5
nd
38.1
tr
Sphingomyelin
PTC
Whole animal 1 -day female 1 -day male
ndf nd
41.7 39.8
Fat body 1 -day female 1 -day male
tr tr
Tissue
Thorax 1 -day 9-day l-day 9-day Eggs
female female male male
IN
PHOSPHOLIPIDS
:r0
Unknown*
* The R, value corresponds to that of phosphatidyl serines. t Migrates immediately ahead of PTE in the.solvent system used. 1 Not detected. Details on the number of animals and determinations appear in Table 4. minimum of three TLC analyses were made from each sample.
7.4
A
LIPID UTILIZATION IN ADULTPIERIS
1265
BRASSICAE
PL in eggs than in adult tissues, possibly having a rBle as a reserve lipid type accumulating linolenic acid which is utilized in embryogenesis. It is noteworthy in this connexion that although only 22+9o/oof egg lipids were PL, 42.2% of the label from linolenic acid in egg lipids was recovered from PL. Neutral lipids also incorporated notable quantities of the label (Table 6) but in most tissues the bulk of this was found in triglycerides (TGL). In l-day whole animal extracts 87*Oo/oof the label was recovered from TGL in females. Diglycerides (DGL), free fatty acids (FFA), and sterol esters all incorporated about equal amounts, in the order of 4%. In the fat body approximately 94% of the label in both sexes was found in TGL. Even more distinct was the concentration of the label in TGL in the eggs where 97.0% was recovered from this fraction, with 3.0% in DGL. TABLET-RELATIVE PERCENTAGEDISTRIBUTION IN ADULT
Tissue
MGL
OPLABELFROMLINOLENIC
NEUTRAL
ACID-~-~*C
LIPIDS
DGL
FFA
TGL
SE
Whole animal 1 -day female
l-2
3.9
3.7
87.0
4.2
Fat body 1 -day female 1 -day male
tr tr
l-5 1.4
1.9 1.6
93.8 94.1
2.8 2,8
tr tr tr tr
16.7 17.2 16.1 22.1
64.4 82-7 66.0 77.9
18.9 1?9 tr
tr tr tr tr
tr
3-o
tr
97.0
tr
Thorax 1 -day 9-day i-day 9&y Eggs
fern&e female male male
MGL, Monoglycerides; DGL, diglycerides; TGL, triglycerides; FFA, free fatty acids; SE, sterol esters. Details on the number of animals and determinations appear in Table 4. A minims of three TLC analyses were made from each sample of thorax lipids, two from other samples.
The labelling pattern was rather different in the thorax (Table 6). A major portion of the activity was found in FFA. One-day females retained 64.4% and males 66.0% of the label in FFA, with approximately 16% in DGL and 18% in TGL in both sexes. After 9 days more label had been incorporated into FFA (82.7% in females, 77*9o/o in males) or lost from TGL which had only trace amounts of the label. DGL still contained considerable amounts of activity after 9 days (17.2% in females, 22.1% in males).
1266
SEPPO
~RUNRN
DISCUSSION
From the data presented above some suggestions may now be made to explain the utilization of lipid by the adult Pieris. As in many insects, the eggs of Pieris require lipids for the processes of embryogenesis (ALLAIS et al., 1964; MARTIN, 1969). The relatively high percentage of PL in the eggs implies utilization of PL, in addition to cellular structures, for purposes of energy. The high percentage of label from linolenic acid in PTC in egg PL would suggest an increased incorporation of PTC into egg lipids by the female. Utilization of PTC as a possible energy source in embryonic development is supported by the work of ALLAIS et al. (1964) who reported a decrease in PTC in the eggs of Locusta migratoriu at the time of blastokinesis. Furthermore, PTC may also be a rather labile PL type in the adult thorax, as was shown by a relatively greater loss of label from PTC as compared to PTE during adult life when linolenic acid was increasingly utilized in the synthesis of cardiolipin. In addition to the utilization of lipid in eggs, lipids are also oxidized during adult life in flight muscles in Lepidoptera (DOMROESEand GILBERT, 1964; STEVENSON,1968, 1972) and in locusts and cockroaches (BEENAKKERS,1965; SACKTOR,1965) whereas little or no capacity in this respect has been reported in Diptera (SACKTOR,1955; CHILDRESSet al., 1967). During the first 10 days after emergence the fat body is greatly reduced in Pieris as evidently lipid is consumed for energy. According to the present results there appears to be sexual dimorphism in the utilization of lipid by Pieris. If we exclude the possibility of synthesis of the polyunsaturated fatty acids by the adult, the relative increase in the content of linolenic and linoleic acids in the male but not in the female during adult life indicates either selective incorporation of these acids into eggs by the female, increased synthesis of other fatty acids (oleic and palmitic) by the female, or a somewhat greater rate of lipid utilization in the male. Sexual dimorphism concerning the overall greater quantity of lipid in the males of several Lepidoptera has been reported (review by GILBERT, 1967). Though the present results show an increase in the content of palmitate in both sexes on the artificial diet this is in no way intended to imply that palmitate is not rapidly metabolized in adults. On the contrary, preliminary results indicate that the biological half life of palmitate is fairly short but its synthesis more than balances the loss in adults. The role of linolenic acid in previous developmental stages of Pieris has been discussed recently (TURUNEN, 1973b). Both sexes incorporate linolenic acid at about equal rates during larval and early pupal stages and little evidence is found of sexual differences in the labelling pattern in larval, pupal, and adult phospholipids when whole animal extracts are analysed. The specific activity of PL in the fat body of l-day adults, however, is greater in the males. This holds also for the thorax lipids of both l- and 9-day adult males. The thorax of males then, besides having more PL, also had more label from linolenic acid in the PL than females. Apparently the quantitative aspects of lipid metabolism in flight muscle show sexual dimorphism in Pieris, whereas the role of linolenic acid in muscle PL appears to be much the same in both sexes.
LIPII) UTILrZATION M ADULT
HERIS
BRASSICAE
1267
The labelling pattern in flight muscle PL deserves some more comment, however. Nine days after emergence there has been a shift of label from PTC to PTE and cardiolipin, suggesting that PTC may have been utilized by adults while the two other fractions appear to be mainly structural in function, perhaps constituents of mitochon~ia (CRONE, 1964; THOMASand GILBERT, 1967b). The remarkable increase in the label in thorax cardiolipin toward later adult life suggests synthesis of this lipid in adults and after pupal-adult ecdysis especially in females. It is interesting that in another Lepidopteran, Hyalophoru cecro$&, fatty acids of cardiolipin were found to consist mostly of linolenate (THOMASand GILBERT, 1967b). Quantitatively cardiolipin in pieris appears to be less important than PTC or PTE even in the thorax, but may be a major PL type with respect to the content of linolenic acid. In eggs and in adult fat body but especially in earlier developmental stages its occurrence is less conspicuous. Referring to Tables 1 and 2 the availability of linolenic acid in different diets may result in alterations in fatty acid metabolism that are reflected also in the adult stage. Pieris that had grown on a cabbage diet and been maintained in diapause for 8 months (roughly the situation that would occur in nature in Finland) show more sexual differences in the adult fatty acid pattern than Pieris reared on an artificial diet. Diapause in itself did not appear to create specific demands for any particular fatty acid, except that the saturated 18 carbon acid, stearate, was markedly reduced in diapausing pupae if compared to fifth instar larvae (TURUNEN, 1973a). The shortage of linolenic acid in the artificial diet brings about an increased synthesis of palmitoleic acid and is also reflected in a low linolenic and a high palmitoleic acid content in egg NL on the artificial medium. Females reared on cabbage lay eggs with a higher linolenic and a lower palmitoleic acid content. The neutral lipids apparently are important both as reserve lipids and as a means of lipid transport. TGL, which constitutes a major portion of the neutral fraction, incorporated most of the label from linolenic acid when whole animal, fat body, or egg lipids were considered. Analyses of thorax NL revealed, however, that in muscle most of linolenic acid in this fraction is free, with the rest being incorporated in TGL and DGL. In some insects DGL have been reported to be the major lipid type transported in the haemolymph to sites of utilization (CHINO and GILBERT, 1965) whereas in other species both free fatty acids and TGL have been described {COOK and EDDINGTON, 1967; WLO~~~ and LAGWINSKA,1967; STEVENSON,1972). It is also possible that PL function as specific transport compounds for some fatty acids (THOMAS and GILBERT, 1967). The fact that most of the label from linolenic acid in flight muscle NL was in FFA and the observation by STEVENSON(1972) that the turnover rate of FFA is high during flight suggests that FFA are an important form of lipid utilization in the muscles of Pie&. Judging from the present study the function of linolenic acid may not be of impo~~ce in energy yielding processes primarily, but instead as a constituent of flight muscle membranes and organelles which in turn are essential in energy metabolism in the adult.
1268
SEX’POTURUNIXN
Ae~owle~~~-~~e author wishes to thank Professor SYKK~ P~OXEN of the University of Helsinki for generously allowing him to use the facilities of his laboratory. REFERENCES J. P., BEAGERARD J., ETIENNEJ., and POLONOVSKI J. (1964) Nature et evolution des lipides au tours de l’embryogenese de Locusta migratoria. J. Insect Physiol. 10,753-772. BEENAKKE~ A. M. (1965) Transport of fatty acids in Locusta migratoria during sustained flight. J, Insect Physiol. II, 879-888. CHILDRE~~C. C., SACKTORB., and TRAYNORD. R. (1967) Function of car&tine in the fatty acid oxidase-deficient insect flight muscle. J. biol. Chem. 242, 754-760. CHINO H. and GILBERT L. I. (1965) Lipid release and transport in insects. Biochim. biophys. Acta 98, 94-110. COOKB. J. and E~DINCTONL. C. (1967) The release of triglycerides and free fatty acids from the fat body of the cockroach, Periplaneta americana. J, Insect Physiol. 13, 13611372. CRONE H. D. (1964) Phospholipid composition of flight muscle sarcosomes from the housefly, MusEa domestica. J. Insect Physiol. 10, 499-507. DOMROWXK. A. and GILBERT L. I. (1964) The rBle of lipid in adult development and flight-muscle metabolism in ~yal~hora cecropia. J. ezp. Biol. 41, 573-590. DUTKOWSKIA. B. and ZIAJKAB. (1972) Synthesis and degradation of glycerides in fat body of normal and ovariotectomized females of Galleria mellonella. J. Insect Physiol. 18, 1351-1367. GILBERT L. I. (1967) Lipid metabolism and function in insects. A&. Insect Physiol. 4, 69-211. GOSWAMIS. K. and FREY C, F. (1971) Spray detection of phospholipids on thin-layer chromatograms. J. Lipid Ref. 12, 509-510. GRAU P. A. and T~WUERE L. C. (1971) Fatty acid profile of the cabbage looper, Trichoplusia si, and the effect of diet and rearing conditions. J. Insect Physiol. 17, 1637-1649. JUNNIKKAL+A E, (1966) Effect of Braconid parasitization on the nitrogen metabolism of Pieris brassicae L. Ann. Acad. Sci. fem. (A)4, 100, l-83. KARLINSKYA. and POULAERTJ. (1971) Evolution du tissu adipeux au tours de la vie imaginale de Pieris brassicae L. (Lepidopdre). Bull. Sot. zool. Fr. 96, 453-466. MARTIN J. S. (1969) Lipid composition of fat body and its contribution to the maturing oocytes in Pyrrhocoris apterus. J. Insect Physiol. 15, 1025-1045. SACKTOR B. (1955) Cell structure and the metabolism of insect flight muscle. r. biophys. mocha. Cytol. 1, 29-46. SACKTOR B. (1965) Energetics and respiratory metabolism of muscular contraction. In The Physiology of Insects (Ed. by ROCWTEINM.) 2,483-580. Academic Press, New York. SACKTORB. (1970) Regulation of intermediary metabolism, with special reference to the control mechanisms in insect flight muscle. Adv. Insect Physiol. 7, 267-347. SCHAEFZRC, H, (1968) The relationship of the fatty acid composition of Heliothis zea larva to that of its diet. J. Insect Physiol. 14, 171-178. STAHL E. (1969) Thin-layer Chromatography, 2nd ed. Springer, Berlin. STEVENSON E. (1968) The camitine-independent oxidation of pahnitate plus malate by moth flight-muscle ~tochon~ia. Biochem. J. 110, 105-110. STEVENSONE. (1972) Haemolymph lipids and fat body lipases of the southern armyworm moth. J_ Insect Physiol. 18, 1751-1756. THOMASK. K. and GILBERT L. I. (1967a) In witro studies on the release and transport of phospholipids. J. Insect Physiol. 13, 963-980. THOMASK, K. and GILBERT L. I. (196715) Phospholipid synthesis during flight muscle development in the American silkmoth, Hyalophora cecropia. Camp. Biochem. Physiol. 21, 279-290. ALLAIS
LIPID UTILIZATION IN ADULT PIERIS ERASSICAE
1269
TURUNENS. (1972) Fatty acids of the fat body of Pieris bmssicae L. during metamorphosis; effect of a low concentration of a chlorinated insecticide. Am. Ad. Sci. fem. (A)4, 193, l-7. TURUNBN S. (1973a) Utilization of fatty acids by Pieris brassicae reared on artificial and natural diets. J. Insect Physiol. 19, 1999-2009. TURUNEN S. (1973b) R61e of labelled dietary fatty acids and acetate in phospholipids during the metamorphosis of Pieris bra&cue. J. Insect Physiol. 19, 2327-2340. WLODAMR P. and LAGWINSKA E. (1967) Uptake and release of lipids by the isolated fat body of the wazmoth larva. J. Insect Physiol. 13, 319-331.
in Great Brituin
LIPID UTILIZATION IN ADULT PIERIS BRASSICAE WITH SPECIAL REFERENCE TO THE ROLE OF LINOLENIC ACID SEPPO TURUNEN Department of Physiological Zoology, University of Helsinki, 00100 Helsinki 10, Finland (First received 2 October 1973 ; revised 19 October 1973) Abstract-Fatty acids of adult Pieris brassicae and the incorporation of dietary linolenic acid-lJ4C into adult (and egg) lipids were analysed 1 and 9 days after ecdysis. Females grown on a leaf diet retained palmitic, palmitoleic, and oleic acids but lost linoleic and linolenic acids during adult life, while males utilized their fatty acids more evenly. On an artificial diet both sexes retained pahnitic acid but utilized palmitoleic and oleic acids. In both cases females laid eggs with a high palmitic and oleic acid content. Analysis of thorax flight muscles (artificial diet) revealed that 67.9% of the lipids in l-day females and 83.6% in 9-day females was phospholipid (PL). During adult life linolenic acid increased in thorax neutral lipids (NL) from 14.6 to 20.0% in females and from 18.5 to 30.0% in males. Males incorporated more linolenic acid-1-14C especially in fat body and flight muscle PL than females. The majority of this was recovered from phosphatidyl cholines (PTC) in l-day adults whereas in 9-day adults phosphatidyl ethanolamines (PTE) and another compound, most likely cardiolipin, contained more label (29.0% in PTC, 33.1% in PTE, 34.9% in cardiolipin, and 2.0% in sphingomyelin in the thorax of females). The females also incorporated the label into egg lipids (42.2% in PL, 57.8% in NL). There was recovered from PTC 54.5% of the label in egg PL. Most of the label in thorax NL was found to be in free fatty acids (FFA). The label disappeared from triglycerides during adult life and tended to accumulate in FFA (82.7% in 9-day females) while in diglycerides the label did not vary during adult life (17.2% in 9-day post-emergence females). PTC apparently is a fairly labile PL type which is utilized in muscle whereas PTE and cardiolipin may be more structural in function and accumulated more label from linolenic acid with increasing adult age. Linolenic acid, then, essentially is a structural fatty acid and its r61e appears to be mainly in the structures of flight muscle membranes and organelles. INTRODUCTION
TOWARDSTHE adult stage lipid metabolism in Lepidoptera concerns the synthesis of adult structures (i.e. DUTKOWSKI and ZIAJKA,1972) and, in many species, the preservation of reserve lipid to be utilized for energy in flight muscles (DOMROESE and GILBERT,1964; STEVENSON,1968; SACKTOR,1970). The development of eggs also requires deposition of lipid by the female. In view of the common use 1257
1258
SEPPO
TURUNEN
of artificial diets for rearing Lepidoptera it is necessary that the fatty acid requirements of the adult stage are understood, especially as regards the utilization of the essential fatty acids. Artificial diets often have a lipid content that differs from the insect’s natural source of food. It has been shown that this may bring about an altered tissue fatty acid content which is reflected in increased syntheses of some fatty acids to levels not ordinarily found in the species (SCHAEFER,1968 ; GRAUand TERRIERE, 1971; TURUNEN, 1973a). The diet commonly used for rearing Pie~is brassicue provides notably less linolenic acid than a diet of cabbage leaves (TURUNEN,1973a). Linolenic acid, as an essential fatty acid, is a constituent of egg lipids and also must have an important rale in adult flight muscles, as it is known to be concentrated especially in phospholipids when its dietary availability is low (TURUNEN, 1973b). A study was therefore undertaken to determine what are the fatty acid requirements of adult Pieris on an artificial as well as on a natural diet, and what specifically is the r61e of linolenic acid in adult and egg lipids. It was interesting also, with respect to an understanding of lipid utilization in Pieris, to find out whether there are sexual differences in fat metabolism in adults as has recently been reported in the histology of Pied fat body (KARLINSKYand POULAERT,1971). MATERIALS AND METHODS Experimental animals Pieris brassicae were from a laboratory-reared stock maintained under controlled conditions as described previously (TURUNEN, 1973a, b). The diapause stock was reared on cabbage (Bras&z oleracea var. ace$zaZu). The larvae pupated and entered diapause under conditions simulating autumn. They were thereafter kept at + 3 to + 4°C and a relative humidity of 65 to 70%. After 6 months the Eight months from the beginning of fatty acids of pupae were determined. diapause the pupae were brought to +9’C for 24 hr, then to + 17°C for another 24 hr, and finally to the insectary (+ 23”C, 65% r.h., 18 hr day length). The adults emerged after 10 to 11 days in the insectary. For the non-diapausing stock the pupal stage under the conditions used lasts from lOi to 11 days. Before the pupal-adult ecdysis the pupae were transferred to a cage (JUNNIKKALA,1966) illuminated with both artificial and direct sunlight to assure optimal flying activity and copulation during the flying period of 4 to 5 hr daily. Except for that the lights were turned off and the window shaded, leaving only the normal illumination, whereby all flying activity ceased. Adults referred to as l-day adults were sacrificed immediately after the pupaladult ecdysis or within 24 hr from ecdysis, while 9-day adults ranged from 8 to 10 days after ecdysis. Thus 9-day adults had flown for approximately 40 hr while l-day adults essentially had not flown before analysis. The adults had access to a 12% solution of honey during the flying period. In l-day females ovaries are still rather undeveloped and ova are barely visible. The first eggs are laid about 4 days after ecdysis.
LIPID UTILIZATIONIN ADULTPIERIS
1259
BRASSICAE
Analytical procedures and labelled diets All procedures and methods of analysis have been described (TURUNEN, 1972, 1973b). Visualization of phospholipids on thin-layer chromatograms was accomplished by a specific phospholipid spray, the action of which is based on metallic copper and ammonium molybdate in a sulphuric acid solution (GOSWAMI and FREY, 1971). Identification of radioactive phospholipid fractions required rechromatography with unlabelled standards and the use of several different solvent systems (c$ STAHL, 1969). The labelled diet contained 50 $Zi l*C (linolenic acid-lJ*C) per 568 g diet. Larvae were placed on the radioactive diet immediately after ecdysis to the fifth instar. RESULTS Fatty acids of the diapause stock Table 1 shows the fatty acid composition of diapausing pupae and l-day and g-day post-emergent adults. The dietary fatty acid composition has been reported earlier (TURUNEN, 1973a). In pupae the major fatty acid in neutral (NL) and TABLE ~--RELATIVE PERCENTAGE FATTYACIDCOMPOSITION OF P.brassicaeREARED ONCABBAGE Stage
16:0
16:l
l&O
l&l
18:2
18:3
Female NL* Diapause pupae 1 -day adult 9-day adult
14.5 12.4 20.5
6.9 5.3 5.1
1.1 1.1 2.9
33.0 33.3 39.1
9.0 10.0 7.8
35.5 38.0 24.6
Female PLt Diapause pupae 1 -day adult 9-day adult
8.9 11.0 16.1
2.9 2.3 2.8
12.5 6.6 5.6
22.6 22.1 19.2
21.2 14.9 14.7
31.9 43.1 41.6
Male NL Diapause pupae 1 -day adult 9-day adult
14.1 13.7 13.4
7.0 6.3 2.6
1.1 1.9 4.1
30.7 27,2 26.4
9.2 9.9 11.3
38.0 41 *o 42.2
Male PL Diapause pupae 1 -day adult 9-day adult
9.8 10.2 7.8
3.2 2.3 1.2
13.8 8-l 6.2
21.1 19.5 22.4
20.3 14.9 17.4
31.8 45.0 45.0
Eggs NL PL
20.9 13.2
4.2 5.9
2.7 6.0
40.9 36.8
8.9 17.6
22.4 20.5
* Neutral lipids. t Phospholipids. Each value is an average of three determinations, each consisting of the pooled tissues of 6 to 9 animals.
1260
SSPPO TImmEN
phospholipids (PL) is linolenate which is, exceptionally, more concentrated in NL during diapause (355% in females, 38.0% in males). The fatty acid patterns of diapausing pupae appear alike in both sexes. At the time of adult ecdysis the relative amount of linolenic acid has increased in both sexes but more notably so in PL (from 31.9 to 43-l% in females and from 3 l-8 to 45.0% in males), perhaps at the expense of other fatty acids which have been consumed. One-day females appear to have a higher concentration of oleate in NL than l-day males (33.3 vs. 27*2%), having perhaps synthesized this acid while the relative amount of oleate has actually decreased in males. An interesting aspect is an abrupt decrease in the PL stearate content from 12.5% in diapausing female pupae to 6.6% in l-day adults. Corresponding values in males were 13.8 and S*l%. There was a s~~t~eous fall in the PL linoleate content from 21.2 to 14.9% in females and from 20.3 to 14.9% in males. After 9 days of adult life the fatty acid patterns were rather dissimilar in the two sexes (Table 1). During this period the females laid eggs but the actual percentage of fertile females was not determined. The females accumulated palmitic acid (20.5% in NL, 16.1% in PL) while no such trend was shown by the males (13.4% in NL, 7.8% in PL). The females also retained more palmitoleic acid in NL than males (5-l vs. 2.6%). Th ere was a substantial increase in NL oleic acid in the females (39.1%) but none in the males (26.4%). NIore remarkable, however, was the distribution of the polyunsaturated acids. In females both linoleic and linolenic acids decreased during adult life in the neutral fraction (7.8 and 24.6% linoleate and linolenate, respectively) while practically no change was found in males (11.3 and 42*2%, respectively). Thus in Pieris reared on a cabbage diet the females tend to retain more palmitic, palmitoleic, and oleic acids and lose linoleic and linolenic acids during adult life, whereas males appear to utilize their fatty acids rather more evenly. However, as is discussed later, this difference may also be explained in terms of lipid synthesis which may occur at different rates in males and females. In Table 1 the fatty acid composition of eggs of the diapause stock has been shown. A characteristic feature is a comparatively high degree of saturation if compared with adult tissues. Oleic acid is the prevalent fatty acid in both NL and PL (40.9 and 36.8%, respectively). The content of palmitic acid is also high (20.9 and 13.2% in NL and PL, respectively). Egg NL contains almost the same amount of linolenic acid as 9-day females (22.4% in eggs vs. 24.6% in adults) while in egg PL the relative amount of linolenic acid is only half of that in g-day females (205 vs. 41.6%). Fatty acids of adults reared on an artzjicial diet When the data above are compared with values from adults reared diet as larvae, both similarities and differences in the utilization are revealed (Table 2). The metabolism of the 16 carbon acids fairly similar in both sexes. Both retain a high level of palmitic days after the pupal-adult ecdysis (29.2% in females, 26-O% in
on an artificial of fatty acids apparently is acid in NL 9 males), while
LIPID UTILIZATIONIN ADULTPIERIS TABLE ~-RELATIVE
1261
ERASSICAE
PERCENTAGE FATTY ACID COMPOSITIONOF P. brussicae REAREDON AN ARTIFICIALDIET
Stage
18:0
18:l
18~2
18:3
13.3 6.1
2.1 3.9
47.6 44.8
14.3 6.5
7.2 9.5
10.2 8.7
4.7 3.1
7.4 8.0
28.7 28.8
26.4 28.4
22.6 23.0
Male NL 1 -day adult 9-day adult
16.7 26.0
13.9 5.3
2.1 4.2
46.3 40.5
14.0 10.4
7.0 13.6
Male PL 1 -day adult 9-day adult
10.3 7.3
4.8 1.8
7.2 7.2
28.3 25.8
26.5 31.5
22.9 26.4
Eggs NL PL
18.7 11.5
8.4 5.3
4.2 10.7
45.7 30.1
13.5 22.0
9.5 20.6
16:0
16:l
Female NL 1 -day adult 9-day adult
15.5 29.2
Female PL 1 -day adult 9-day adult
Legend as in Table
1.
on the leaf diet this was encountered only in the females. TOn the artificial diet both sexes utilized palmitoleic acid during adult life. Thus l-day females had 13.3% palmitoleic acid in NL but g-day females contained only 6.1%. In males the corresponding values were 13.9 and 5*3o/o. Both sexes utilized oleic acid during adult life, although this was more readily demonstrated in males (46.3% in l-day and 4O*5o/o in g-day males). The low content of linolenic acid allows little variation during adult life but some accumulation of this acid was noted in the males, analogously to the situation on the leaf diet. By comparison the fatty acid content of eggs on the two diets showed some notable similarities. In both cases the egg oleic and palmitic acid contents were high, and in egg PL the concentration of linolenic acid, quite remarkably, was the same on both diets. The overall uniformity of the PL fatty acid pattern in the two cases suggests that the PL fatty acid content is not allowed to vary as much as the neutral lipid fatty acids for normal embryonic development. Fatty acids of the thorax To find out whether the flight and leg muscle fatty acids would give relevant data on the utilization of fatty acids by adult Pieris, further analyses were made from thorax lipids. Thoraces without appendages and the haemolymph carefully removed, mostly contain muscle tissue. Preliminary data had shown that NL in the thorax contained relatively small concentrations of triglycerides, a major lipid
1262
SEPPO TLlRUNEN
fraction in most tissues, but instead had high levels of free fatty acids. Analysis of NL of similarly prepared thoraces might suggest what fatty acids are utilized in muscle during adult life. Phospholipids, on the other hand, are expected to be mainly structural in function, although they do have a r61e in intracellular energy metabolism and possibly also in fatty acid transport. Table 3 shows the fatty acid composition of thoraces from l- and 9-day adults (artificial diet). On the whole the content of oleic acid was lower in muscle than in whole animal extracts (39.4% in NL of l-day females, 36.7% in NL of l-day males). There was also less palmitoleic acid but more polyunsaturated acids than in whole animals. Thus the content of linolenic acid in NL of l-day females was 14.6% and in males 18*5o/o, and in PL, respectively, 26.6 and 27.5%. After 9 days relative changes in PL fatty acids were slight with the exception of a rise in linoleic acid (29.7% in l-day and 37.1% in 9-day females). In the neutral fraction oleic acid decreased from 39.4 to 32.6% in females and from 36.7 to 23.5% in males. The relative amount of linolenic acid increased during the same period from 14.6 to 20.0% in females and from 18.5 to 30.0% in males. Palmitoleic acid, on the other hand, decreased to less than half the value of l-day adults (from 9.1 to 4.1% in females). Oleic and palmitoleic acids thus appeared to be utilized while linolenic and linoleic acids were retained during adult life. TABLE
3-RELATIVE
PERCENTAGE
FATTY ACID COMPOSITION (ARTIFICIAL DIRT)
OF THORACES
OF P. brassicae
16:0
16:l
18:O
18:l
18:2
l&3
Female NL 1 -day adult 9-day adult
16.1 21.0
9.1 4.1
4.4 5.6
39.4 32.6
16.4 16.7
14.6 20.0
Female PL 1 -day adult 9-day adult
9.2 6.9
3.7 2.0
5.5 4.4
25.3 24.6
29.7 37.1
26.6 25.0
Male NL 1 -day adult 9-day adult
13.7 13.6
7.3 3.7
4.3 4.7
36.7 23.5
195 24.5
18.5 30.0
Male PL 1 -day adult 9-day adult
8.9 5.4
3.8 1.9
6.1 4.9
23.8 22.3
29.9 35.9
27.5 29.6
Stage
Thoraces were thoroughly rinsed in insect Ringer to remove all haemolymph. Myristic acid has not been included in the list although its relative proportion increases up to 3% of all NL fatty acids in 9-day adults. Each value is an average of three determinations. 12 to 15 thoraces were used in one determination.
Incorporation of linohic
acid into adult lipid-s
Linolenic acid is accumulated during adult life in the males but less so in the females (artificial diet). It is mainly concentrated in PL on the artificial diet
LIPID UTILIZATION IN ADULTPIERIS
1263
BRASSICAE
while no such distinction can be made concerning Pieris reared on a leaf diet. In order to examine the function of linolenic acid more closely the larvae were given labelled linolenic acid as a dietary constituent and its distribution was then followed in adult and egg lipids. Table 4 gives the specific activities (counts/min per mg lipid) of adult lipids as well as the relative amounts of NL and PL from the weight of total lipids. PL in all cases retained more label than NL. The males, moreover, retained more label in the fat body and thorax than the females. The specific activity of male fat body PL was 30,995 counts/min and that of females 24,733 counts/n& in l-day adults. In the thorax of l-day adults the specific activity of PL was 26,765 counts/min in males and 22,668 counts/min in females. In 9-day adults the corresponding values were 30,546 and 25,677 counts/min. The specific activity of egg lipids (l-2 days after laying) was lower than in maternal tissues, perhaps due in part to accumulation of carotenoids which are lipid soluble and thus increase the weight of the lipid fraction. TABLE ~-SPECIFIC ACTIVITIES OF ADULTLIPIDS (counts/mm DIETARYLINOLRNICACID-l-14c
per mg lipid) INCORPORATING
o/0 from weight of total lipid Tissue
Total no. of animals
NL
PL
NL
PL
32.3 27.3
Whole animal 1 -day female 1 -day male
20 (3) 20 (3)
11,103 10,003
17,430 17,769
67,7 72.7
Fat body 1 -day female 1-day male
26 (2) 26 (2)
9466 9941
24,733 30,995
92.6
7.4
92.9
7.1
15,454 16,507 17,299 18,086
22,668 25,677 26,765 30,546
32.1 16.4 16.3 135
67.9 83.6 83.7 86.5
15,479
77.1
22.9
Thorax 1 -day 9-day 1 -day g-day Eggs
27 44 33 49
female female male male ca.
(3) (4) (3) (4)
2000(2)
6575
The number in parentheses indicates the number of determinations. The specific activities are averages of the indicated number of determinations.
From Table 4 the relative amounts of NL and PL can also be seen. Fat body lipids were mostly neutral (92.6% in l-day adult females). In whole animal extracts 67.7% of the lipids in females and 72.7% in males were neutral. In the thorax, quite to the contrary, most of the lipid was phospholipid. Curiously in l-day females more of the lipid was found to be neutral than in l-day males (31.1
1264
SEPPO TIJRUNEN
VS.
16.4%). In 9-day adult males 13*5o/o of the lipids in the thorax were neutral and 865% polar. Corresponding values in females were 16.4 and 83.6%. The distribution of linolenic acid in major PL types in adult tissues is shown in Table 5. In l-day adults phosphatidyl cholines (PTC) retained most of the activity. Thus in the fat body of males 47.9% of the label was recovered from PTC, 40.4% in phosphatidyl ethanolamines (PTE), and 11.6% in a less polar type, tentatively identified as cardiolipin. Traces of activity were found in sphingomyelin and another fraction corresponding to phosphatidyl serines. In the thorax 37.8% of the activity in males was found in PTC, 36.2% in PTE, 21.7% in cardiolipin and 4.3% in an unidentified fraction migrating on the front of PTE. Nine days after emergence of the adults more label was recovered from PTE and especially from cardiolipin. Thus in the thorax of 9-day males 33.9% was recovered from cardiolipin, 32.1% from PTE, and 29.6% from PTC, with some activity in sphingomyelin and the unidentified fraction. Few sexual differences were detected in connexion with any of the tissues studied. In the eggs a somewhat different pattern of labelling is seen (Table 5). PTC retained 54.S”h of the activity while only 38*1o/o was recovered from PTE and 7.4% from cardiolipin. Apparently PTC constitute a relatively greater part of
TABLE
5--RELATIVE
PERCENTAGE
DISTRIBUTION ADULT
OF
LABEL
FROM
LINOLENIC
ACID-~-~~~
PTE
Unknownt
Cardiolipin
nd nd
36.4 39.3
tr tr
21.9 20.9
46.8 47.9
tr tr
41.5 40.4
nd nd
11.7 11.6
;r2
37.8 29.0 35.7 29.6
tr tr tr tr
36.2 33.1 33.0 32.1
4.3 1.0 3.1 2.2
21.7 34.9 28.2 33.9
nd
54.5
nd
38.1
tr
Sphingomyelin
PTC
Whole animal 1 -day female 1 -day male
ndf nd
41.7 39.8
Fat body 1 -day female 1 -day male
tr tr
Tissue
Thorax 1 -day 9-day l-day 9-day Eggs
female female male male
IN
PHOSPHOLIPIDS
:r0
Unknown*
* The R, value corresponds to that of phosphatidyl serines. t Migrates immediately ahead of PTE in the.solvent system used. 1 Not detected. Details on the number of animals and determinations appear in Table 4. minimum of three TLC analyses were made from each sample.
7.4
A
LIPID UTILIZATION IN ADULTPIERIS
1265
BRASSICAE
PL in eggs than in adult tissues, possibly having a rBle as a reserve lipid type accumulating linolenic acid which is utilized in embryogenesis. It is noteworthy in this connexion that although only 22+9o/oof egg lipids were PL, 42.2% of the label from linolenic acid in egg lipids was recovered from PL. Neutral lipids also incorporated notable quantities of the label (Table 6) but in most tissues the bulk of this was found in triglycerides (TGL). In l-day whole animal extracts 87*Oo/oof the label was recovered from TGL in females. Diglycerides (DGL), free fatty acids (FFA), and sterol esters all incorporated about equal amounts, in the order of 4%. In the fat body approximately 94% of the label in both sexes was found in TGL. Even more distinct was the concentration of the label in TGL in the eggs where 97.0% was recovered from this fraction, with 3.0% in DGL. TABLET-RELATIVE PERCENTAGEDISTRIBUTION IN ADULT
Tissue
MGL
OPLABELFROMLINOLENIC
NEUTRAL
ACID-~-~*C
LIPIDS
DGL
FFA
TGL
SE
Whole animal 1 -day female
l-2
3.9
3.7
87.0
4.2
Fat body 1 -day female 1 -day male
tr tr
l-5 1.4
1.9 1.6
93.8 94.1
2.8 2,8
tr tr tr tr
16.7 17.2 16.1 22.1
64.4 82-7 66.0 77.9
18.9 1?9 tr
tr tr tr tr
tr
3-o
tr
97.0
tr
Thorax 1 -day 9-day i-day 9&y Eggs
fern&e female male male
MGL, Monoglycerides; DGL, diglycerides; TGL, triglycerides; FFA, free fatty acids; SE, sterol esters. Details on the number of animals and determinations appear in Table 4. A minims of three TLC analyses were made from each sample of thorax lipids, two from other samples.
The labelling pattern was rather different in the thorax (Table 6). A major portion of the activity was found in FFA. One-day females retained 64.4% and males 66.0% of the label in FFA, with approximately 16% in DGL and 18% in TGL in both sexes. After 9 days more label had been incorporated into FFA (82.7% in females, 77*9o/o in males) or lost from TGL which had only trace amounts of the label. DGL still contained considerable amounts of activity after 9 days (17.2% in females, 22.1% in males).
1266
SEPPO
~RUNRN
DISCUSSION
From the data presented above some suggestions may now be made to explain the utilization of lipid by the adult Pieris. As in many insects, the eggs of Pieris require lipids for the processes of embryogenesis (ALLAIS et al., 1964; MARTIN, 1969). The relatively high percentage of PL in the eggs implies utilization of PL, in addition to cellular structures, for purposes of energy. The high percentage of label from linolenic acid in PTC in egg PL would suggest an increased incorporation of PTC into egg lipids by the female. Utilization of PTC as a possible energy source in embryonic development is supported by the work of ALLAIS et al. (1964) who reported a decrease in PTC in the eggs of Locusta migratoriu at the time of blastokinesis. Furthermore, PTC may also be a rather labile PL type in the adult thorax, as was shown by a relatively greater loss of label from PTC as compared to PTE during adult life when linolenic acid was increasingly utilized in the synthesis of cardiolipin. In addition to the utilization of lipid in eggs, lipids are also oxidized during adult life in flight muscles in Lepidoptera (DOMROESEand GILBERT, 1964; STEVENSON,1968, 1972) and in locusts and cockroaches (BEENAKKERS,1965; SACKTOR,1965) whereas little or no capacity in this respect has been reported in Diptera (SACKTOR,1955; CHILDRESSet al., 1967). During the first 10 days after emergence the fat body is greatly reduced in Pieris as evidently lipid is consumed for energy. According to the present results there appears to be sexual dimorphism in the utilization of lipid by Pieris. If we exclude the possibility of synthesis of the polyunsaturated fatty acids by the adult, the relative increase in the content of linolenic and linoleic acids in the male but not in the female during adult life indicates either selective incorporation of these acids into eggs by the female, increased synthesis of other fatty acids (oleic and palmitic) by the female, or a somewhat greater rate of lipid utilization in the male. Sexual dimorphism concerning the overall greater quantity of lipid in the males of several Lepidoptera has been reported (review by GILBERT, 1967). Though the present results show an increase in the content of palmitate in both sexes on the artificial diet this is in no way intended to imply that palmitate is not rapidly metabolized in adults. On the contrary, preliminary results indicate that the biological half life of palmitate is fairly short but its synthesis more than balances the loss in adults. The role of linolenic acid in previous developmental stages of Pieris has been discussed recently (TURUNEN, 1973b). Both sexes incorporate linolenic acid at about equal rates during larval and early pupal stages and little evidence is found of sexual differences in the labelling pattern in larval, pupal, and adult phospholipids when whole animal extracts are analysed. The specific activity of PL in the fat body of l-day adults, however, is greater in the males. This holds also for the thorax lipids of both l- and 9-day adult males. The thorax of males then, besides having more PL, also had more label from linolenic acid in the PL than females. Apparently the quantitative aspects of lipid metabolism in flight muscle show sexual dimorphism in Pieris, whereas the role of linolenic acid in muscle PL appears to be much the same in both sexes.
LIPII) UTILrZATION M ADULT
HERIS
BRASSICAE
1267
The labelling pattern in flight muscle PL deserves some more comment, however. Nine days after emergence there has been a shift of label from PTC to PTE and cardiolipin, suggesting that PTC may have been utilized by adults while the two other fractions appear to be mainly structural in function, perhaps constituents of mitochon~ia (CRONE, 1964; THOMASand GILBERT, 1967b). The remarkable increase in the label in thorax cardiolipin toward later adult life suggests synthesis of this lipid in adults and after pupal-adult ecdysis especially in females. It is interesting that in another Lepidopteran, Hyalophoru cecro$&, fatty acids of cardiolipin were found to consist mostly of linolenate (THOMASand GILBERT, 1967b). Quantitatively cardiolipin in pieris appears to be less important than PTC or PTE even in the thorax, but may be a major PL type with respect to the content of linolenic acid. In eggs and in adult fat body but especially in earlier developmental stages its occurrence is less conspicuous. Referring to Tables 1 and 2 the availability of linolenic acid in different diets may result in alterations in fatty acid metabolism that are reflected also in the adult stage. Pieris that had grown on a cabbage diet and been maintained in diapause for 8 months (roughly the situation that would occur in nature in Finland) show more sexual differences in the adult fatty acid pattern than Pieris reared on an artificial diet. Diapause in itself did not appear to create specific demands for any particular fatty acid, except that the saturated 18 carbon acid, stearate, was markedly reduced in diapausing pupae if compared to fifth instar larvae (TURUNEN, 1973a). The shortage of linolenic acid in the artificial diet brings about an increased synthesis of palmitoleic acid and is also reflected in a low linolenic and a high palmitoleic acid content in egg NL on the artificial medium. Females reared on cabbage lay eggs with a higher linolenic and a lower palmitoleic acid content. The neutral lipids apparently are important both as reserve lipids and as a means of lipid transport. TGL, which constitutes a major portion of the neutral fraction, incorporated most of the label from linolenic acid when whole animal, fat body, or egg lipids were considered. Analyses of thorax NL revealed, however, that in muscle most of linolenic acid in this fraction is free, with the rest being incorporated in TGL and DGL. In some insects DGL have been reported to be the major lipid type transported in the haemolymph to sites of utilization (CHINO and GILBERT, 1965) whereas in other species both free fatty acids and TGL have been described {COOK and EDDINGTON, 1967; WLO~~~ and LAGWINSKA,1967; STEVENSON,1972). It is also possible that PL function as specific transport compounds for some fatty acids (THOMAS and GILBERT, 1967). The fact that most of the label from linolenic acid in flight muscle NL was in FFA and the observation by STEVENSON(1972) that the turnover rate of FFA is high during flight suggests that FFA are an important form of lipid utilization in the muscles of Pie&. Judging from the present study the function of linolenic acid may not be of impo~~ce in energy yielding processes primarily, but instead as a constituent of flight muscle membranes and organelles which in turn are essential in energy metabolism in the adult.
1268
SEX’POTURUNIXN
Ae~owle~~~-~~e author wishes to thank Professor SYKK~ P~OXEN of the University of Helsinki for generously allowing him to use the facilities of his laboratory. REFERENCES J. P., BEAGERARD J., ETIENNEJ., and POLONOVSKI J. (1964) Nature et evolution des lipides au tours de l’embryogenese de Locusta migratoria. J. Insect Physiol. 10,753-772. BEENAKKE~ A. M. (1965) Transport of fatty acids in Locusta migratoria during sustained flight. J, Insect Physiol. II, 879-888. CHILDRE~~C. C., SACKTORB., and TRAYNORD. R. (1967) Function of car&tine in the fatty acid oxidase-deficient insect flight muscle. J. biol. Chem. 242, 754-760. CHINO H. and GILBERT L. I. (1965) Lipid release and transport in insects. Biochim. biophys. Acta 98, 94-110. COOKB. J. and E~DINCTONL. C. (1967) The release of triglycerides and free fatty acids from the fat body of the cockroach, Periplaneta americana. J, Insect Physiol. 13, 13611372. CRONE H. D. (1964) Phospholipid composition of flight muscle sarcosomes from the housefly, MusEa domestica. J. Insect Physiol. 10, 499-507. DOMROWXK. A. and GILBERT L. I. (1964) The rBle of lipid in adult development and flight-muscle metabolism in ~yal~hora cecropia. J. ezp. Biol. 41, 573-590. DUTKOWSKIA. B. and ZIAJKAB. (1972) Synthesis and degradation of glycerides in fat body of normal and ovariotectomized females of Galleria mellonella. J. Insect Physiol. 18, 1351-1367. GILBERT L. I. (1967) Lipid metabolism and function in insects. A&. Insect Physiol. 4, 69-211. GOSWAMIS. K. and FREY C, F. (1971) Spray detection of phospholipids on thin-layer chromatograms. J. Lipid Ref. 12, 509-510. GRAU P. A. and T~WUERE L. C. (1971) Fatty acid profile of the cabbage looper, Trichoplusia si, and the effect of diet and rearing conditions. J. Insect Physiol. 17, 1637-1649. JUNNIKKAL+A E, (1966) Effect of Braconid parasitization on the nitrogen metabolism of Pieris brassicae L. Ann. Acad. Sci. fem. (A)4, 100, l-83. KARLINSKYA. and POULAERTJ. (1971) Evolution du tissu adipeux au tours de la vie imaginale de Pieris brassicae L. (Lepidopdre). Bull. Sot. zool. Fr. 96, 453-466. MARTIN J. S. (1969) Lipid composition of fat body and its contribution to the maturing oocytes in Pyrrhocoris apterus. J. Insect Physiol. 15, 1025-1045. SACKTOR B. (1955) Cell structure and the metabolism of insect flight muscle. r. biophys. mocha. Cytol. 1, 29-46. SACKTOR B. (1965) Energetics and respiratory metabolism of muscular contraction. In The Physiology of Insects (Ed. by ROCWTEINM.) 2,483-580. Academic Press, New York. SACKTORB. (1970) Regulation of intermediary metabolism, with special reference to the control mechanisms in insect flight muscle. Adv. Insect Physiol. 7, 267-347. SCHAEFZRC, H, (1968) The relationship of the fatty acid composition of Heliothis zea larva to that of its diet. J. Insect Physiol. 14, 171-178. STAHL E. (1969) Thin-layer Chromatography, 2nd ed. Springer, Berlin. STEVENSON E. (1968) The camitine-independent oxidation of pahnitate plus malate by moth flight-muscle ~tochon~ia. Biochem. J. 110, 105-110. STEVENSONE. (1972) Haemolymph lipids and fat body lipases of the southern armyworm moth. J_ Insect Physiol. 18, 1751-1756. THOMASK. K. and GILBERT L. I. (1967a) In witro studies on the release and transport of phospholipids. J. Insect Physiol. 13, 963-980. THOMASK, K. and GILBERT L. I. (196715) Phospholipid synthesis during flight muscle development in the American silkmoth, Hyalophora cecropia. Camp. Biochem. Physiol. 21, 279-290. ALLAIS
LIPID UTILIZATION IN ADULT PIERIS ERASSICAE
1269
TURUNENS. (1972) Fatty acids of the fat body of Pieris bmssicae L. during metamorphosis; effect of a low concentration of a chlorinated insecticide. Am. Ad. Sci. fem. (A)4, 193, l-7. TURUNBN S. (1973a) Utilization of fatty acids by Pieris brassicae reared on artificial and natural diets. J. Insect Physiol. 19, 1999-2009. TURUNEN S. (1973b) R61e of labelled dietary fatty acids and acetate in phospholipids during the metamorphosis of Pieris bra&cue. J. Insect Physiol. 19, 2327-2340. WLODAMR P. and LAGWINSKA E. (1967) Uptake and release of lipids by the isolated fat body of the wazmoth larva. J. Insect Physiol. 13, 319-331.