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Rôle of labelled dietary fatty acids and acetate in phospholipids during the metamorphosis of Pieris brassicae
7. Insect Physiol., 1973, Vol. 19, pp. 2327 to 2340. Pergamon Press. Printed in Great Britain
ROLE OF LABELLED DIETARY FATTY ACIDS AND ACETATE IN PHOSPHOLIPIDS DURING THE METAMORPHOSIS OF PIERIS BRAS5’ICAE SEPPO
TURUNEN
Department of Physiological Zoology, University of Helsinki, 00100 Helsinki 10, Finland (Received 2 May 1973) Abstract-The incorporation of labelled dietary palmitic, linoleic, and linolenic acids into neutral (NL) and phospholipids (PL) during the metamorphosis of Pie& brassicae was studied, and the ability of the fat body to incorporate acetate into PL determined. Thirty-three per cent of total lipid in early fifth instar larvae (minus haemolymph) is PL, while the corresponding value in female 4-day pupae is 13.0 per cent and in the fat body of 4-day pupae 6.3 per cent. Incorporation of label into PL was studied more closely and in all cases the label was recovered The from phosphatidylcholine (PTC) and phosphatidylethanolamine (PTE). label from palmitate was also found in sphingomyelin and possibly phosphatidylserine. Specific activity of PL in the case of palmitic and linolenic acids was greatest in late fifth instar larvae. In early fifth instar larvae on palmitic acid-l -14C 39.0 per cent of label was in PTC, 52.8 per cent in PTE, and 2-O per cent in sphingomyelin. In late fifth instar 45.0 per cent was in PTC, 45.5 per cent in PTE, and 6.5 per cent in sphingomyelin, while in 4-day female pupae 45.2 per cent was in PTC, 41.3 per cent in PTE, and 13.5 per cent in sphingomyelin. The label from linolenic acid only varied a little from early fifth instar to 4-day pupae, 51.8 per cent being in PTC and 48.2 per cent in PTE in early fifth instar larvae. The label from linoleic acid is incorporated in fat body PL almost exclusively in PTC and PTE, 55.8 and 43.2 per cent respectively in 4-day female pupae. Injected acetate is distributed after 1 hr between PTC (58.6 per cent), PTE (24.4 per cent), and sphingomyelin (17.0 per cent). It was concluded that the polyunsaturated acids are proportionately more common in PTE than in other PL types, and that the fatty acids of sphingomyelin are mainly those that the insect is capable of synthesizing from acetate. Palmitic acid is desaturated by Pieris to palmitoleic acid and the latter possibly utilized in PTE to compensate for a deficiency of linolenic acid in the artificial diet. No saturation of linoleic or linolenic acid was found. The rates of PL and NL synthesis during development and the r6le of the investigated fatty acids in the biosynthesis of PL are discussed. INTRODUCTION
THE FUNCTION of phospholipids as membrane constituents has been well established. Numerous other rbles have also been assigned to these lipids, including a Ale as mediators in active transport (HOKIN and HOKIN, 1961) and mitochondrial electron transfer (GREEN and FLEISCHER, 1963). The phospholipid fatty acid composition of the lepidopterous species Pieris brassicae appears to be rather 2327
2328
SEPPO TURUNEN
sensitive to low concentrations of chlorinated insecticides, compounds that are lipid soluble and probably interfere with several of the functions of biological membranes (ELA et al., 1970; OMER et al., 1971; TURUNEN, 1972). In some Lepidoptera phospholipids have been shown to accumulate the polyunsaturated fatty acids, linoleic and linolenic, suggesting a r81e for these acids in membrane structures (TERRIERE and GRAU, 1972; TURUNEN, 1973). In contrast to the comparatively stable fatty acid composition of triglycerides the fatty acid pattern of phospholipids exhibits some interesting changes in PieGs during metamorphosis, including a notable increase in the content of linolenic acid prior to pupation and a high content of palmitoleic acid in larvae grown on a diet low in linolenic acid (TURUNEN, 1972). However, the role of fatty acids in the various PL types has remained largely unknown in insects. Incorporation of the non-fatty acid components of phosphatidic esters has been studied recently in some insect species (THOMAS and GILBERT, 1967; BLANKENSHIPand MILLER, 1971; DWIVEDY (PTC), phosphaand BRIDGES, 1973), and the synthesis of phosphatidylcholine tidylethanolamine (PTE), and several minor PL types has been observed. While care must be taken in interpreting the significance of the incorporation of labelled fatty acids into PL, as it is not always clear whether biosynthesis is taking place (GILBERT, 1967), studies using labelled fatty acids in diet in connexion with quantitative measurement of PL give valuable data on the role of specific fatty acids in phosphatidic esters. The purpose of the present study was to determine the rates of neutral and phospholipid synthesis in P. hsicae and correlate this with the incorporation of dietary fatty acids into the various PL types. Further, it was attempted to establish whether the essential fatty acids are specifically utilized in some PL types and, whether, in light of an obvious deficiency of one of these acids, the r81e of palmitoleic acid in Pieris could be explained in more detail. This paper describes the result of these experiments. MATERIALS
AND METHODS
Expehmental animals The insects used in this study were from a laboratory-reared stock of Pie& brassicae grown on an artificial diet as previously described (TURUNEN, 1972). The larvae were reared from the first to the third instar in 100 ml glass dishes containing the appropriate diet. The dishes were capped with filter paper and held upside down at 65% humidity and a temperature of 23°C. At third instar the larvae were transferred to 250 ml dishes. This was repeated at the beginning of the fifth instar. The age and sex of Pievis was determined as described earlier (TURUNEN, 1973) using the larval-pupal ecdysis as the starting point in counting pupal age and the pupal-adult ecdysis in counting adult age. Larvae referred to as early fifth instar had not been feeding more than a few hours after the fourth to fifth ecdysis. Late fifth instar larvae are near the end of the feeding period and would attach on the rearing dishes for pupation within 24 hr.
FATTYACIDSANDACETATEIN LIPIDS DURINGM.ETAMORPHOSIS OF PIERIS
2329
Reagents and radiopurity of isotopes All chemicals were reagent grade unless otherwise noted. Chloroform and methanol used for solvents in TLC were distilled before use. Standards for identification of phospholipids were phosphatidylcholine (Sigma Chemical Co.), phosphatidylethanolamine, phosphatidylserine, and sphingomyelin (Fluka A.G.) Radioisotopes used were sodium acetate-1-“C, palmitic acid-1-l*C, linoleic acid-lJ4C, and linolenic acid-lJ4C, all purchased from the Radiochemical Centre (England). The purity of all fatty acids was tested by methylating an aliquot of the total lipid fraction of the radioactive diets immediately after cooling of the diet (see below), and GLC of the fatty acid methyl esters. The chromatograph (Perkin-Elmer F 11) was provided with an eluent splitter which directed nine-tenths of the flow to a counter for radioactivity. The activity of peaks appearing from the column was recorded with a minimum of delay by an integrator (Berthold, Metrawatt A.G.) and, by matching both charts, the activity of each peak could be counted. The columns were prepacked diethyleneglycol succinate (20%) on HMDS Chromosorb W (mesh SO/loo). H e1ium was used as the carrier gas. The oven temperature was 185°C. The data sheets accompanying the isotopes indicated a purity of over 99 per cent for palmitic acid, 98 per cent for linoleic acid, and 97 per cent for linolenic acid. GLC of methylated acids as described above confirmed these figures. The purity of acetate-l J*C was checked by TLC. No detectable impurities were found. It could be important that the purity of labelled fatty acids included in the diet be checked after preparation of the diet, especially if this involves temperatures much higher than ambient and a free access of oxygen. The polyunsaturated acids, for example, could be partially saturated under these conditions. Preparation of radioactive diets The basic diet used in this work is one described by DAVID and GARDINER (1966). Linseed oil (1 ml/379 g diet) is added to the diet to make up for a deficiency of linolenic acid (TURUNEN, 1973). The diet is prepared in a water-bath at 65°C with a rotator providing even mixing of all constituents. The labelled acids were added to the diet in a benzene solution slightly prior to pouring the diet in glass dishes. After 2 min satisfactory mixing of the labelled fatty acids was achieved throughout the diet as shown by counting the radioactivity from several dishes. Injection of acetate-l-T Four-day female pupae were injected (5 ~1) with a Hamilton 10 ~“1 syringe ventrally through the intersegmental membrane between the two most anterior abdominal segments. The concentration of the acetate solution was adjusted so that each 5 ,ul gave O-125 &i of 14C. After 1 or 2 hr the pupae were killed in liquid nitrogen and stored at - 25°C until analysed.
SEPPO TURUNF.N
2330 Chromatography
and radioassay
of lipids
The animals were killed in liquid nitrogen, fat bodies dissected, and rinsed in cold insect Ringer, or, when whole animals were analysed, the intestine and haemolymph were removed in Ringer in order to avoid contamination from dietary label. The pupae were similarly washed in Ringer to remove the meconium and haemolymph. The technique has been described earlier (TURUNEN, 1973). Total lipids were extracted in chloroform-methanol (2 : 1) according to the method of FOLCH et al. (1957). The total lipids were further fractionated into neutral and polar lipids as described earlier (TURUNEN, 1972). The fractions were taken to tared test-tubes, evaporated to dryness under nitrogen, weighed, and a known volume of hexane added. An aliquot of this was transferred to liquid scintillation vials containing 10 ml of scintillation fluid (Omnifluor, NEN). The rest of the lipids were analysed further by TLC. Glass plates covered with O-250 mm of silica gel-G (Merck) were divided into seven lanes 16 mm wide separated by 10 mm lanes, and the lipid samples applied in a narrow band across the 16 mm lanes. Polar lipids were developed with appropriate standards in chloroform-methanol-water (65 : 25 : 4) for an average of 1 hr 45 min in tanks lined with filter paper. Visualization was done by spraying the standard lanes with 0.2% 2’,7’-dichlorofluorescein in 96o/0 ethanol and viewing the plates in 254 nm U.V. light. Rechromatography of pooled PL fractions with a standard was used to confirm the identity of the radioactive PL fractions. An assay of radioactivity was performed by scanning each lane with an apparatus including a Geiger counter and a recording system (Berthold, Metrawatt A.G.). Each lane was scanned throughout its length whereby all active spots were registered and could then be marked on the plate, scraped into scintillation vials if necessary, or analysed further after extraction from the gel. The liquid scintillator used was a Wallac Decem-NLT314 Liquid Scintillation Counter (Turku, Finland) with an efficiency of 90 per cent for 1% activity. This apparatus has an external standard count method to determine counting efficiency. RESULTS In the stages studied the relative percentage PL content was greatest in larvae (Table 1). Early fifth instar larvae contained 33.0% PL and 67.0% NL in total lipid reflecting, probably, the small volume of the fat body before the intense feeding period during the fifth instar. With increasing volume of the fat body NL is accumulated as the reserve fat (77.5 per cent in late fifth instar) while the relative amount of PL falls. In 4-day pupae the lipids are only 13.0 per cent polar, 87.0 per cent neutral. The polar phospholipids are especially scarce in the fat body, 6.3 per cent in 4-day pupae, 6-O per cent in 3-day adults, showing that an important function for this tissue is the deposition of reserve lipid to be utilized in the The rise in the content of NL is reflected in the building of adult structures. overall increase of tissue lipid as shown by Table 1. Early fifth instar larvae, for example, have only 16 per cent of the total combined NL and PL that can be
FATTY ACIDSAND ACETATEIN LIPIDS DURINGMETAMORPHOSIS OF
PIERIS
2331
extracted from 4-day pupae. The size of the fat body is probably greatest at about the time of pupation and is notably diminished toward the adult stage. TABLE I-RRLATIYE PER CENT COMPOSITION OF TOTALLIPID IN P. brassicae DURINGMETAMORPHOSIS
Stage Early fifth * Late fifth* 4-day pupae* Males Females 4-day pupae? Fat body 3-day adultst Fat body
Lipid extracted per animal (mg)
No. of experiments
Total no. of animals
7 9
85 68
33.0 22~5
67.0 77.5
1.4 8.6
8 7
64 47
14.6 13.0
85.4 87.0
8.3 8.8
8
.79
6.3
93.7
5.9
4
40
6.0
94-o
1.9
* All tissues except haemolymph extracted. -1_Sexes were not separated.
and intestine (meconium in pupae) were
Fig. I shows the amount of radioactivity (counts/min per mg lipid) in the three labelled diets containing palmitoleic acid-lJ4C (diet l), linoleic acid-l-14C (diet Z), and linolenic acid-1-14C (diet 3). Diets 1 and 3 contained 50 &i 14C per 568 g diet, or about 16,000 countsjmin per mg lipid. In diet 2 the concentration of label was 100 ,uCi per 568 g diet. The data reveal that almost 90 per cent of the label from diets 1 and 3, and almost 80 per cent from diet 2, is incorporated into larval lipid. There is an oversupply of linoleate in this diet as has been noted previously (TURUNEN, 1973) and the larvae control the amount of this acid taken from the diet. Palmitic and linolenic acids are taken up approximately in proportion to their amount in the diet. GLC of methylated larval and pupal lipids showed that Pieris desaturates much of palmitic acid to palmitoleic acid and also slightly to oleic acid. Linoleic and linolenic acids are not metabolized to any other l&carbon analogues, and all activity in larval and pupal lipids after methylation is recovered from the same acid that was added in the diet. The specific activities of tissue PL and NL are given in Table 2. There is little loss of specific activity due to the breakdown of any of the three acids during the developmental stages studied. Incorporation of palmitic/palmitoleic acid is prominent during the fifth instar. into both PL and NL. The label is retained in NL but gradually lost from PL. Thus the specific activity of PL in early fifth instar is 14,825 counts/min per mg lipid, in late fifth instar 17,320 counts/min and in 4-day male pupae 14,480 counts/min. Corresponding activities in NL are 13,480, 17,110, and 18,080 counts/min.
SEPPO TURUNEN
2332
0 palmitic
linolenic
FIG. 1. Concentration of labelled fatty acids in diets and frass of fifth instar larvae (counts/min per mg lipid). Values above frass columns denote percentage from total dietary radioactivity present in frass. TABLE ~-SPECIFIC ACTIVITIES OF TISSUELIPIDSOF P. bvassicae REARED ON DIETSCONTAINING LABELLED FATTYACIDS(COuntS/min per mg LIPID) Linoleic acid
Palmitic acid
Linolenic acid
-
PL
NL
13,480 (5) 17,110 (4)
15,360 (4) 20,080 (6)
9705 (4) 11,405 (4)
18,080 (4) 16,910 (3)
18,240 (4) 17,760 (3)
12,475 (4) 11,625 (3)
Stage
PL
NL
Early fifth* Late fifth* 4-day pupae* Males Females 4-day pupae Fat body (males) Fat body (females) 3-day adults Fat body (males) Fat body (females)
14,825 (6) 17,320 (4) 14,480 (4) 13,650 (4)
PL
NL
37,420 (4)
25,890 (4)
43,410 (4)
28,370 (4)
42,600 (3)
30,670 (3) 27,870 (3)
* Lipids from all tissues except haemolymph and intestine (meconium in pupae) were extracted. Figures in parentheses indicate the number of experiments. The technique involved in the determination of specific activities is described in the Materials and Methods section. Tissue controls gave an average of 26 counts/min per mg lipid.
FATTY
ACIDS AND
ACETATE
IN LIPIDS DURING
METAMORPHOSIS
2333
OF PIERIS
Similarly on a diet containing linolenic acid an increase in the specific activity of PL from early fifth instar (15,360 counts/min) to late fifth instar (20,080 counts/min) is noted, with a slight decrease in 4-day pupae (18,240 counts/min in males). The NL show only small changes in specific activity during development (9705 counts/min in early fifth instar, 11,405 countsjmin in late fifth instar, 12,475 counts/min in 4-day male pupae). As previously noted the fat body PL content is rather low in Pieris. Incorporation of linoleic acid into fat body lipids was studied in pupae and adults reared on a labelled diet from the beginning of the fifth instar. The dietary concentration of the label was twice that of the two other diets to make up for a lower PL content. Fat body PL incorporated distinctly more of the label than NL (per mg lipid). In 4-day pupae the females, moreover, accumulated more label than the males (43,410 vs. 37,420 counts/min in PL and 28,370 vs. 25,890 counts/min in NL). In 3-day adult males the specific activity of NL is higher than in male pupae, while that of adult females does not differ from the pupal value. The total per cent distribution of the label between NL and PL during metamorphosis indicates a gradual shift of label from PL to NL toward the pupal stage (Table 3). While 35.8 per cent of the activity from palmitic/palmitoleic acid is in TABLE
~-PER
CENT DISTRIBUTION OFLABEL INNEUTRAL (NL) AND PHOSPHOLIPIDS P. bYUSSiCUe REARED ON DIETS CONTAINING LABELLBD FATTY ACIDS
Pahnitic acid
Linolenic acid
PL
NL
77.2
43.8 32.7
56.2 67.3
87.9 89.2
20.0 18.5
80.0 81.5
NL
Stage
PL
Early fifth* Late fifth*
3.58
64.2
22.8 12.1 10.8
4-day pupae * Males Females 4-day pupae Fat body (males) Fat body (females) 3-day adults Fat body (females)
Linoleic acid
(PL) IN
PL
NL
8.8
91.2
9.3
90.7
8.9
91.1
* Lipids from all tissues except haemolymph and intestine (meconium in pupae) were extracted.
PL in early fifth instar larvae, only 10.8 per cent is found in the PL of 4-day pupae. Early fifth instar larvae again retain 43.8 per cent of the label from linolenic acid in PL, while 32.7 per cent is in PL in late fifth instar larvae and 18.5 per cent in 4-day pupae. No similar tendency is found in the distribution of the label from
SEPPO TURUNEN
2334
linoleic acid in the fat body lipids of pupae and adults. In both 4-day pupae and 3-day adults approximately 9 per cent of the label is in PL. Distribution of label among phospholipid classes Five types of PL were distinguished in Pieris. Of these phosphatidylcholine (PTC) and phosphatidylethanolamine (PTE) account for the bulk of PL. Sphingomyelin is far less common but nevertheless regularly present. Two additional bands corresponding to lysophosphatidylcholine and phosphatidylserine were tentatively identified by TLC. No gravimetric measurements of the individual fractions were attempted. Table 4 gives the distribution of activity among the PL types of Pieris reared on a diet containing palmitic acid-l J4C. There is a striking tendency toward increased incorporation of the label into sphingomyelin from early fifth instar larvae (2.0 per cent) to 4-day pupae (135 per cent in females). The label is gradually TABLE ~-PER
Stage Early fifth Late fifth 4-day pupae Males Females
CENTDISTRIBUTION OF LABELAMONGPHOSPHOLIPIDS OF P. bvassicae REARED ON A DIETCONTAINING PALMITICACID-l -14C No. of experiments *
Total no. of animals
3 5
26 30
5 4
28 24
Sphingomyelin
PTC
Unknownt
PTE
2.0 6.5
39.0 45.0
6.2
52.8
3.0
45.5
13.1 13.5
48.3 45.2
Trace Trace
38.6 41.3
* Each sample was chromatographed a minimum of three times. The average per cent distributions of all analyses have been listed. -I_The Rf value equals that of phosphatidylserine. The technique used in the determination of activities by TLC is described in the Materials and Methods section. Compare legend of Table 2.
lost from PTE (52.8 per cent in early fifth instar, 41.3 per cent in female and 38.6 per cent in male 4-day pupae) but a parallel increase in activity is noted in PTC (39.0 per cent in early fifth instar, 45.2 per cent in female and 48.3 per cent in male 4-day pupae). Early fifth instar larvae apparently incorporate the label into phosphatidylserine (6.2 per cent), but only trace amounts of label can be found in this fraction in 4-day pupae. The distribution of label from linolenic acid, in clear distinction to palmitic/ palmitoleic acid, is rather uniform (Table 5). Only two PL types incorporate the label. PTC at all stages contained more label than PTE. In early fifth instar larvae 51.8 per cent of the label was in PTC and 48.2 per cent in PTE. Corresponding values for late fifth instar larvae were 55.3 and 44.7 per cent. No sexual differences in 4-day pupae were found in the labelling pattern, while some were obvious in the case of palmitic/palmitoleic acid. Referring to Table 2 it appears
FATTY ACIDS AND ACETATE IN LIPIDS DURING METAMORPHOSISOF PIERIS
233.5
that from early to late fifth instar -the metabolism of linolenic acid in PL is perhaps more diversified than in pupae, and that the labelling pattern in 4-day pupae roughly simulates that of late fifth instar larvae. TABLE S---PER CENT DISTRIBUTION OF LABEL AMONG PHOSPHOLIPIDS OF P. brassicae REARED ON A DIET CONTAINING LINOLENIC ACID-1 -14c
Stage Early fifth Late fifth 4-day pupae Males Females
No. of experiments*
Total no. of animals
Sphingomyelin
3 3
59 38
Not detected Not detected
51.8 55.3
48.2 44.7
4 3
36 23
Not detected Not detected
53.8 53.4
46.2 46.6
PTC
PTE
* Legend as in Table 4.
Incorporation of linoleic acid into fat body PL in pupae and adults is shown in Table 6. The labelling pattern follows closely that of linolenic acid. PTC containing over half of the activity in both sexes in 4-day pupae (55.8 per cent in females, 57.3 per cent in males), but interestingly 3-day adult males retain more of the label in PTE (50.2 per cent) while in adult females the distribution of the label resembles that of pupae. Somewhat over 1 per cent of the label was recovered from sphingomyelin in both pupae and adults. TABLE 6-PER
CENT DISTRIBUTION OF LABEL AMONG PHOSPHOLIPIDS IN THE FAT BODY OF
P. brassicae RRARRDON A DIET CONTAINING LINOLEIC ACID-~J~C
Stage 4-day pupae Males Females 3-day adults Males Females * Legend
No. of experiments *
Total no. of animals
Sphingomyelin
3 3
20 20
1.6 1.0
57.3 55.8
41.1 43.2
2 3
18 31
1.4 1.2
48.4 56.6
50.2 42.2
PTC
PTE
as in Table 4.
The incorporation of injected acetate-l J4C into fat body PL reveals a somewhat different pattern than those discussed above, showing similarities with the distribution of label from the lbcarbon acids. After 1 hr 58.6 per cent of the label in females was recovered from PTC and only 24.4 per cent in PTE, while 17.0 per cent was in sphingomyelin (Table 7). Within 2 hr additonal label was incorporated into PTC (63.1 per cent) while some was lost from sphingomyelin. Assuming that
2336
SEPPO
TURUNEN
the polyunsaturated la-carbon fatty acids are poorly synthesized from acetate by Pieris, some interesting conclusions can be proposed. PTE apparently accumulate proportionately more of the polyunsaturated acids than other PL types and the polyunsaturated acids, furthermore, may constitute a major portion of the fatty acids of PTE. PTC contains mostly those acids that the fat body is capable of synthesizing from acetate, i.e. palmitate/palmitoleate, stearate, and oleate, all known to be constituents of PL in Pieris. Oleate and palmitate are generally the major products of fatty acid synthesis from acetate in insects, and apparently are utilized by Pieris in PTC to a far greater extent than in PTE. Sphingomyelin is quantitatively a minor PL type in Pien’s, and if we consider the relatively large proportion of label from acetate in sphingomyelin as well as the data on the incorporation of linoleate and linolenate, it appears safe to suggest that the fatty acid moiety of this PL type mostly consists of those acids synthesized from acetate. TABLE 7-PER
Time of incorporation
w
CENT DISTRIBUTION OF LABEL FROM INJECTEDACETATE-1-14c IN PHOSPHOLIPIDS OF THE FAT BODY OF 4-day FEMALE PUPAE OF P. braStiCaf?
No. of experiments*
Total no. of animals
Sphingomyelin
PTC
PTE
4 2
26 10
17-o 13-l
58.6 63.1
24.4 23.8
1 2
* Legend as in Table 4. DISCUSSION
The biosynthesis of TGL and PL are thought to proceed along common pathways through a key intermediate, phosphatidic acid (KENNEDY, 1963). The latter can yield a diglyceride to be later converted to triglycerides, phosphatidylcholines, Label from injected acetate is readily incorporated or phosphatidylethanolamines. into both TGL and PL in Pieris, indicating the synthesis of fatty acids and incorporation of the synthesized acids into these lipids. Although larvae, being metabolically more active than pupae, synthesize more lipid, it is also expected that pupae incorporate the label from both acetate and fatty acids into lipids required for adult structures (c$ STEPHEN and GILBERT, 1969). A number of papers dealing with the PL of insects have demonstrated the presence of the same general types of PL in insects as in vertebrates (HACK et al., 1962; Gilbert, 1967). PTC appears to be the major PL type in Hymenoptera, Lepidoptera, and Orthoptera (BRIDGES, 1972), while in Diptera PTE predominates (BIEBER et al., 1961). Sphingomyelin has been found to be almost always of a minor occurrence in insects, but nevertheless its presence has been detected in some Lepidoptera, Coleoptera, and Orthoptera, while its absence has been established in some Diptera (BRIDGES, 1972). It is of interest that where sphingomyelin adults
has been found (CHOJNACKI
in Lepidoptera,
and KORZYBSKI,
its concentration
has been high in pupae and
1962; SRIDHARA and BHAT,
1962).
The
PL
of
FATTY
ACIDS AND
ACETATE
IN LIPIDS DURING
METAMORPHOSIS
OF PIERIS
2337
P. brassicae are composed of at least PTC, PTE, and sphingomyelin. The presence of lysophosphatidylcholine and phosphatidylserine has also been tentatively identified, although they are both of a minor occurrence in this species. The relative amount of PL as a percentage of total lipid in Pieris is highest in larvae before the enhanced TGL synthesis during the fifth instar. The decrease is almost linear from early fifth instar to 4-day pupae (Table 1). This is in agreement with the results of SRIDHARAand BHAT (1965) who report that 23 per cent of larval lipid in Bombyx mori is PL whereas only 18-19 per cent of pupal lipid is PL. These authors also report a low concentration of PL in the fat body of Bombyx (3 per cent), as has now been shown to be the case in Pieris. In contrast to the gradual decrease in the relative PL content from larvae to pupae in whole animal extracts, the relative fat body content of PL in Pieris apparently remains on a static level in pupae and adults. The total extractable PL, as well as TGL, however, increases during the fifth instar. Late fifth instar larvae have 2.5 times more PL than early fifth instar larvae, while the corresponding value is over seven times for NL, showing intense synthesis of both lipid types during the last larval stage. The low PL content of the fat body of pupae and adults suggests that PL is utilized preferentially for structures such as ovaries and eggs (cj. ALLAISet al., 1964) where some PL types may have a rhle in supplying energy for embryonic processes (GILBERT, 1967), or they may be employed as structural lipid in flight muscles (CRONE, 1964). The present results have supported the view that both linoleic and linolenic acid are utilized in PL in a greater proportion than NL. Moreover, it is apparent that their presence in PL in Pieris is restricted mainly to PTE and PTC. Characteristically the pattern of labelling from linolenic acid changes little from the beginning of the fifth instar to Cday pupae when the whole animal without haemolymph is considered, with the possible exception of slightly more linolenic acid being required in PTE in early fifth instar larvae than in later stages. A somewhat similar pattern is found in the case of linoleic acid. No sexual differences are indicated by the present data with respect to linoleic or linolenic acid in pupae, but in the fat body of adults the distribution of linoleic acid in PTE and PTC varies between the sexes. Two presumptive explanations may be proposed to account for these differences. Firstly, it is possible that the change in the labelling has occurred only in the adult male which incorporates more linoleic acid in PTE or has more PTE as such in the fat body than do pupae. It is.a.lso conceivable that the adult female may utilize more linoleic acid or PTE, for example in the structures of ovaries and eggs, showing as a decrease in the fat body content of PTE. The requirements for PTE in the adult could result from its riYe in flight muscle sarcosomes, as shown at least in lMusca domestica (Crone, 1964) or, as already discussed, from the accumulation of PTE in egg lipids in the female (ALLAIS et al., 1964). The incorporation of the polyunsaturated fatty acids into PTE merits further comment. The fact that the label from acetate is retained chiefly in PTC in the fat body of pupae, with only a minor portion being recovered from PTE, would
2338
SE~O TIJRUNEN
suggest, bearing in mind that acetate most likely is not incorporated into linoleic or linolenic acid (cz ZEBE and MCSHAN, 1959; SRIDHARA and BHAT, 1964; LAMBREMONT,1965), that the polyunsaturated acids are preferentially utilized in the synthesis of PTE, possibly making up a majority of the fatty acids in PTE. This situation would be comparable with that of some vertebrate tissues (I&ODES, 1958). It appears thus that PTE performs a diversity of functions in insects and that the polyunsatured fatty acids are specifically utilized in this PL type. The special role of the polyunsaturated acids is also emphasized by the fact that in Pieris, at least, neither of them can be synthesized from the other, nor can they be saturated to form oleic or stearic acid. In contrast to the observed stability of the labelling pattern of linoleic and linolenic acid, the metabolism of palmitic acid is more diversified in Pieris. Free palmitic acid is desaturated to pahnitoleic acid and also lengthened to form oleic acid (cJ STEPHENand GILBERT, 1969). Palmitic/palmitoleic acids are utilized preferentially in PTE in larvae, while toward the pupal stage more of these acids are incorporated into PTC and also sphingomyelin. Whether this is evidence of sphingomyelin synthesis in late fifth instar larvae and particularly in pupae cannot be decided from the present data, but would not seem impossible, considering the simultaneous increase in the quantity of PL. The synthesis of palmitoleic acid by F’ieris on the artificial diet used here has been noted previously and may be a response of larvae, growing on a diet deficient in linolenic acid (c$ SCHAEFER, 1968; GRAU and TERRIERE,1971; TURUNEN, 1973). As previously shown it is in fifth instar larvae that the amount of palmitoleic acid reaches a maximum in the fat body PL of Pieris, decreasing thereafter toward the adult stage (TURUNEN, 1972). It is interesting that the label from palmitate/palmitoleate is prominent in PTE in early fifth instar larvae, and also that linolenic acid apparently is required in PTE especially during early fifth instar. Possibly palmitoleic acid partially replaces the function of linolenic acid in PTE in larvae unable to acquire enough of linolenic acid from the diet, whereas after pupation the requirement for linolenic acid can be more easily met with. It should be remembered that much linolenic and linoleic acid are lost at each ecdysis (personal data), thus effecting a continuous loss and sustaining a requirement for these acids especially in larvae. The functions of pahnitoleic acid may reach beyond that of substituting a dietary deficiency of another fatty acid. Interestingly, the content of palmitoleic acid is high in some overwintering lepidopterous larvae (BRACKENand HARRIS,1969). We may theorize that the requirement for available unsaturated acids rises higher during the period of overwintering and an increased synthesis of palmitoleic acid is resorted to, as apparently most insects are unable to synthezise the polyunsaturated acids. Referring to the role of these acids in the PL of Pieris, it would be interesting to know whether the high titre of palmitoleic acid is used in PTE in these overwintering species. Acknowledgement-The author wishes to thank Professor Sy~utii PESONEN(University of Helsinki) for generously allowing him to use the facilities of his laboratory.
FATTY ACIDSAND ACETATEIN LIPIDS DURINGMETAMORPHOSIS OF
PIERZS
2339
REFERENCES ALLAIS J. P., BERGERARD J., ETIENNE J., and POLONOVSKI J. (1964) Nature et evolution des lipides au tours de l’embryogeneae de Locusta migratoria. J. Insect Physiol. 10,753-772. BIEBER L. L., BROOKESV. J., CHELDELINV. H., and NEWBURGHR. W. (1961) The isolation of methylcholine containing phospholipid from Phormia regina larvae. Biochem. biophys. Res. Commun. 6, 237-240. BLANKENSHIPJ. W. and MILLER G. J. (1971) In Go synthesis of phosphatidyl choline and ethanolamine in larvae of the dried-fruit moth. r. § Physiol. 17, 2061-2067. BRACKENC. K. and HARRISP. (1969) High palmitoleic acid in Lepidoptera. Nature, Lond. 224, 84-85. BRIDGESR. G. (1972) Choline metabolism in insects. Adv. Insect Physiol. 9, 51-110. CHOJNACKIT. and KORZYBSKIT. (1962) Biosynthesis of phospholipids in insects-III. Incorporation of a2P-orthophosphate into phospholipids of Arctia caia moths. Acta biochim. pal. 9, 95-110. CRONE H. D. (1964) Phospholipid composition of flight muscle sarcosomes from the housefly, Musca domestica L. 3. Insect Physiol. 10,499-507. DAVID W. A. L. and GARDINERB. 0. C. (1966) Rearing Pieris brassicae (L.) on semisynthetic diets with and without cabbage. Bull. ent. Res. 56, 581-593. DWIVEDYA. K. and BRIDGESR. G. (1973) The effect of dietary changes on the phospholipid composition of the haemolymph lipoproteins of larvae of the housefly, Musca domestica. J. Insect Physiol. 19, 559-576. ELA R., CHEFURKAW., and ROBINSONJ. R. (1970) In viva glucose metabolism in the normal and poisoned cockroach, Periplaneta americana. J. Insect Physiol. 16, 2137-2156. FOLCH J., LEES M., and SLOANE-STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. r. biol. Chem. 226,497-509. GILBERT L. I. (1967) Lipid metabolism and function in insects. Adv. Insect Physiol. 4, 69-211. GRAUP. A. and TERRIEREL. C. (1971) Fatty acid profile of the cabbage looper, Trichoplusia ni, and the effect of diet and rearing conditions. y. Insect Physiol. 17, 1637-1649. GREEN D. E. and FLEISCHERS. (1963) The role of lipids in mitochondrial electron transfer and oxidative phosphorylation. Biochim. biophys. Acta 70, 5.54-582. HACK M. H., GUSSINA. E., and LOWE M. E. (1962) Comparative lipid biochemistry-I. Phosphatides of invertebrates (Porifera to Chordata). Comp. Biochem. Physiol. 5, 217-221. HOKIN L. E. and HOKINM. R. (1961) Diglyceride kinase and phosphatidic acid phosphatase in erythrocyte membranes. Nature, Lond. 189, 836-837. KENNEDYE. P. (1963) The biosynthesis of complex lipids. Proc. 5th int. Congr. Biochem. 7, 113-133. LAMBREMONTE. N. (1965) Biosynthesis of fatty acids in aseptically reared insects. Comp. Biochem. Physiol. 14, 419-424. OMERV. V. and ECOBICHOND. J. (1971) The acute effect of some chlorinated hydrocarbon insecticides on the acetylcholine content of rat brain. Can. j(. Physiol. Pharmac. 49, 79-83. RHODESD. N. (1958) Phospholipids-5. The effect of cod-liver oil in the diet on the composition of hen’s egg phospholipids. Biochem. J. 68, 380-384. SCHAEFERC. H. (1968) The relationship of the fatty acid composition of Heliothis zea larva to that of its diet. r. Insect Physiol. 14, 171-178. SRIDHARA S. and BHATJ. V. (1962) Phospholipids of the silkworm, Bombyx mori. Curr. Sci. 31, 240. SRIDHARAS. and BHAT J. V. (1964) Incorporation of 1 -l*C-acetate into the lipids of the silkworm, Bombyx mori L. Biochem. J. 91, 120-123.
2340
SSPPO TURUNEN
SRIDHARAS. and BHAT J. V. (1965) Lipid composition of the silkworm, Bombyx mori L. . J, Insect Physiol. 11, 449-462. STEPHENW. F., Jr. and GILBERT L. I. (1969) Fatty acid biosynthesis in the silkmoth, Hyalophora cecropia. J, Insect Physiol. 15, 1833-1854. TEFXRIERE L. C. and GRAU P. A. (1972) Dietary requirements and tissue levels of fatty acids in three Noctuidae. 3. Insect Physiol. 18, 633-647. THOMAS K. K. and GILBERTL. I. (1967) In vitro studies on the release and transport of phospholipids. y. Insect Physiol. 13, 963-980. TURUNENS. (1972) Fatty acids of the fat body of Pieris brassicae L. during metamorphosis ; effect of a low concentration of a chlorinated insecticide. Ann. Acad. Sci. fenn. (A) 4, 193, l-7. TURUNENS. (1973) Utilization of fatty acids by Pieris brassicae L. reared on artificial and natural diets. r. Insect Physiol. 19, 1999-2009. ZEBE E. and MCSHAN W. H. (1959) Incorporation of r4C-acetate into long chain fatty acids by the fat body of Pqodenia eridania. Biochim. biophys. Acta 31, 513-518.
ROLE OF LABELLED DIETARY FATTY ACIDS AND ACETATE IN PHOSPHOLIPIDS DURING THE METAMORPHOSIS OF PIERIS BRAS5’ICAE SEPPO
TURUNEN
Department of Physiological Zoology, University of Helsinki, 00100 Helsinki 10, Finland (Received 2 May 1973) Abstract-The incorporation of labelled dietary palmitic, linoleic, and linolenic acids into neutral (NL) and phospholipids (PL) during the metamorphosis of Pie& brassicae was studied, and the ability of the fat body to incorporate acetate into PL determined. Thirty-three per cent of total lipid in early fifth instar larvae (minus haemolymph) is PL, while the corresponding value in female 4-day pupae is 13.0 per cent and in the fat body of 4-day pupae 6.3 per cent. Incorporation of label into PL was studied more closely and in all cases the label was recovered The from phosphatidylcholine (PTC) and phosphatidylethanolamine (PTE). label from palmitate was also found in sphingomyelin and possibly phosphatidylserine. Specific activity of PL in the case of palmitic and linolenic acids was greatest in late fifth instar larvae. In early fifth instar larvae on palmitic acid-l -14C 39.0 per cent of label was in PTC, 52.8 per cent in PTE, and 2-O per cent in sphingomyelin. In late fifth instar 45.0 per cent was in PTC, 45.5 per cent in PTE, and 6.5 per cent in sphingomyelin, while in 4-day female pupae 45.2 per cent was in PTC, 41.3 per cent in PTE, and 13.5 per cent in sphingomyelin. The label from linolenic acid only varied a little from early fifth instar to 4-day pupae, 51.8 per cent being in PTC and 48.2 per cent in PTE in early fifth instar larvae. The label from linoleic acid is incorporated in fat body PL almost exclusively in PTC and PTE, 55.8 and 43.2 per cent respectively in 4-day female pupae. Injected acetate is distributed after 1 hr between PTC (58.6 per cent), PTE (24.4 per cent), and sphingomyelin (17.0 per cent). It was concluded that the polyunsaturated acids are proportionately more common in PTE than in other PL types, and that the fatty acids of sphingomyelin are mainly those that the insect is capable of synthesizing from acetate. Palmitic acid is desaturated by Pieris to palmitoleic acid and the latter possibly utilized in PTE to compensate for a deficiency of linolenic acid in the artificial diet. No saturation of linoleic or linolenic acid was found. The rates of PL and NL synthesis during development and the r6le of the investigated fatty acids in the biosynthesis of PL are discussed. INTRODUCTION
THE FUNCTION of phospholipids as membrane constituents has been well established. Numerous other rbles have also been assigned to these lipids, including a Ale as mediators in active transport (HOKIN and HOKIN, 1961) and mitochondrial electron transfer (GREEN and FLEISCHER, 1963). The phospholipid fatty acid composition of the lepidopterous species Pieris brassicae appears to be rather 2327
2328
SEPPO TURUNEN
sensitive to low concentrations of chlorinated insecticides, compounds that are lipid soluble and probably interfere with several of the functions of biological membranes (ELA et al., 1970; OMER et al., 1971; TURUNEN, 1972). In some Lepidoptera phospholipids have been shown to accumulate the polyunsaturated fatty acids, linoleic and linolenic, suggesting a r81e for these acids in membrane structures (TERRIERE and GRAU, 1972; TURUNEN, 1973). In contrast to the comparatively stable fatty acid composition of triglycerides the fatty acid pattern of phospholipids exhibits some interesting changes in PieGs during metamorphosis, including a notable increase in the content of linolenic acid prior to pupation and a high content of palmitoleic acid in larvae grown on a diet low in linolenic acid (TURUNEN, 1972). However, the role of fatty acids in the various PL types has remained largely unknown in insects. Incorporation of the non-fatty acid components of phosphatidic esters has been studied recently in some insect species (THOMAS and GILBERT, 1967; BLANKENSHIPand MILLER, 1971; DWIVEDY (PTC), phosphaand BRIDGES, 1973), and the synthesis of phosphatidylcholine tidylethanolamine (PTE), and several minor PL types has been observed. While care must be taken in interpreting the significance of the incorporation of labelled fatty acids into PL, as it is not always clear whether biosynthesis is taking place (GILBERT, 1967), studies using labelled fatty acids in diet in connexion with quantitative measurement of PL give valuable data on the role of specific fatty acids in phosphatidic esters. The purpose of the present study was to determine the rates of neutral and phospholipid synthesis in P. hsicae and correlate this with the incorporation of dietary fatty acids into the various PL types. Further, it was attempted to establish whether the essential fatty acids are specifically utilized in some PL types and, whether, in light of an obvious deficiency of one of these acids, the r81e of palmitoleic acid in Pieris could be explained in more detail. This paper describes the result of these experiments. MATERIALS
AND METHODS
Expehmental animals The insects used in this study were from a laboratory-reared stock of Pie& brassicae grown on an artificial diet as previously described (TURUNEN, 1972). The larvae were reared from the first to the third instar in 100 ml glass dishes containing the appropriate diet. The dishes were capped with filter paper and held upside down at 65% humidity and a temperature of 23°C. At third instar the larvae were transferred to 250 ml dishes. This was repeated at the beginning of the fifth instar. The age and sex of Pievis was determined as described earlier (TURUNEN, 1973) using the larval-pupal ecdysis as the starting point in counting pupal age and the pupal-adult ecdysis in counting adult age. Larvae referred to as early fifth instar had not been feeding more than a few hours after the fourth to fifth ecdysis. Late fifth instar larvae are near the end of the feeding period and would attach on the rearing dishes for pupation within 24 hr.
FATTYACIDSANDACETATEIN LIPIDS DURINGM.ETAMORPHOSIS OF PIERIS
2329
Reagents and radiopurity of isotopes All chemicals were reagent grade unless otherwise noted. Chloroform and methanol used for solvents in TLC were distilled before use. Standards for identification of phospholipids were phosphatidylcholine (Sigma Chemical Co.), phosphatidylethanolamine, phosphatidylserine, and sphingomyelin (Fluka A.G.) Radioisotopes used were sodium acetate-1-“C, palmitic acid-1-l*C, linoleic acid-lJ4C, and linolenic acid-lJ4C, all purchased from the Radiochemical Centre (England). The purity of all fatty acids was tested by methylating an aliquot of the total lipid fraction of the radioactive diets immediately after cooling of the diet (see below), and GLC of the fatty acid methyl esters. The chromatograph (Perkin-Elmer F 11) was provided with an eluent splitter which directed nine-tenths of the flow to a counter for radioactivity. The activity of peaks appearing from the column was recorded with a minimum of delay by an integrator (Berthold, Metrawatt A.G.) and, by matching both charts, the activity of each peak could be counted. The columns were prepacked diethyleneglycol succinate (20%) on HMDS Chromosorb W (mesh SO/loo). H e1ium was used as the carrier gas. The oven temperature was 185°C. The data sheets accompanying the isotopes indicated a purity of over 99 per cent for palmitic acid, 98 per cent for linoleic acid, and 97 per cent for linolenic acid. GLC of methylated acids as described above confirmed these figures. The purity of acetate-l J*C was checked by TLC. No detectable impurities were found. It could be important that the purity of labelled fatty acids included in the diet be checked after preparation of the diet, especially if this involves temperatures much higher than ambient and a free access of oxygen. The polyunsaturated acids, for example, could be partially saturated under these conditions. Preparation of radioactive diets The basic diet used in this work is one described by DAVID and GARDINER (1966). Linseed oil (1 ml/379 g diet) is added to the diet to make up for a deficiency of linolenic acid (TURUNEN, 1973). The diet is prepared in a water-bath at 65°C with a rotator providing even mixing of all constituents. The labelled acids were added to the diet in a benzene solution slightly prior to pouring the diet in glass dishes. After 2 min satisfactory mixing of the labelled fatty acids was achieved throughout the diet as shown by counting the radioactivity from several dishes. Injection of acetate-l-T Four-day female pupae were injected (5 ~1) with a Hamilton 10 ~“1 syringe ventrally through the intersegmental membrane between the two most anterior abdominal segments. The concentration of the acetate solution was adjusted so that each 5 ,ul gave O-125 &i of 14C. After 1 or 2 hr the pupae were killed in liquid nitrogen and stored at - 25°C until analysed.
SEPPO TURUNF.N
2330 Chromatography
and radioassay
of lipids
The animals were killed in liquid nitrogen, fat bodies dissected, and rinsed in cold insect Ringer, or, when whole animals were analysed, the intestine and haemolymph were removed in Ringer in order to avoid contamination from dietary label. The pupae were similarly washed in Ringer to remove the meconium and haemolymph. The technique has been described earlier (TURUNEN, 1973). Total lipids were extracted in chloroform-methanol (2 : 1) according to the method of FOLCH et al. (1957). The total lipids were further fractionated into neutral and polar lipids as described earlier (TURUNEN, 1972). The fractions were taken to tared test-tubes, evaporated to dryness under nitrogen, weighed, and a known volume of hexane added. An aliquot of this was transferred to liquid scintillation vials containing 10 ml of scintillation fluid (Omnifluor, NEN). The rest of the lipids were analysed further by TLC. Glass plates covered with O-250 mm of silica gel-G (Merck) were divided into seven lanes 16 mm wide separated by 10 mm lanes, and the lipid samples applied in a narrow band across the 16 mm lanes. Polar lipids were developed with appropriate standards in chloroform-methanol-water (65 : 25 : 4) for an average of 1 hr 45 min in tanks lined with filter paper. Visualization was done by spraying the standard lanes with 0.2% 2’,7’-dichlorofluorescein in 96o/0 ethanol and viewing the plates in 254 nm U.V. light. Rechromatography of pooled PL fractions with a standard was used to confirm the identity of the radioactive PL fractions. An assay of radioactivity was performed by scanning each lane with an apparatus including a Geiger counter and a recording system (Berthold, Metrawatt A.G.). Each lane was scanned throughout its length whereby all active spots were registered and could then be marked on the plate, scraped into scintillation vials if necessary, or analysed further after extraction from the gel. The liquid scintillator used was a Wallac Decem-NLT314 Liquid Scintillation Counter (Turku, Finland) with an efficiency of 90 per cent for 1% activity. This apparatus has an external standard count method to determine counting efficiency. RESULTS In the stages studied the relative percentage PL content was greatest in larvae (Table 1). Early fifth instar larvae contained 33.0% PL and 67.0% NL in total lipid reflecting, probably, the small volume of the fat body before the intense feeding period during the fifth instar. With increasing volume of the fat body NL is accumulated as the reserve fat (77.5 per cent in late fifth instar) while the relative amount of PL falls. In 4-day pupae the lipids are only 13.0 per cent polar, 87.0 per cent neutral. The polar phospholipids are especially scarce in the fat body, 6.3 per cent in 4-day pupae, 6-O per cent in 3-day adults, showing that an important function for this tissue is the deposition of reserve lipid to be utilized in the The rise in the content of NL is reflected in the building of adult structures. overall increase of tissue lipid as shown by Table 1. Early fifth instar larvae, for example, have only 16 per cent of the total combined NL and PL that can be
FATTY ACIDSAND ACETATEIN LIPIDS DURINGMETAMORPHOSIS OF
PIERIS
2331
extracted from 4-day pupae. The size of the fat body is probably greatest at about the time of pupation and is notably diminished toward the adult stage. TABLE I-RRLATIYE PER CENT COMPOSITION OF TOTALLIPID IN P. brassicae DURINGMETAMORPHOSIS
Stage Early fifth * Late fifth* 4-day pupae* Males Females 4-day pupae? Fat body 3-day adultst Fat body
Lipid extracted per animal (mg)
No. of experiments
Total no. of animals
7 9
85 68
33.0 22~5
67.0 77.5
1.4 8.6
8 7
64 47
14.6 13.0
85.4 87.0
8.3 8.8
8
.79
6.3
93.7
5.9
4
40
6.0
94-o
1.9
* All tissues except haemolymph extracted. -1_Sexes were not separated.
and intestine (meconium in pupae) were
Fig. I shows the amount of radioactivity (counts/min per mg lipid) in the three labelled diets containing palmitoleic acid-lJ4C (diet l), linoleic acid-l-14C (diet Z), and linolenic acid-1-14C (diet 3). Diets 1 and 3 contained 50 &i 14C per 568 g diet, or about 16,000 countsjmin per mg lipid. In diet 2 the concentration of label was 100 ,uCi per 568 g diet. The data reveal that almost 90 per cent of the label from diets 1 and 3, and almost 80 per cent from diet 2, is incorporated into larval lipid. There is an oversupply of linoleate in this diet as has been noted previously (TURUNEN, 1973) and the larvae control the amount of this acid taken from the diet. Palmitic and linolenic acids are taken up approximately in proportion to their amount in the diet. GLC of methylated larval and pupal lipids showed that Pieris desaturates much of palmitic acid to palmitoleic acid and also slightly to oleic acid. Linoleic and linolenic acids are not metabolized to any other l&carbon analogues, and all activity in larval and pupal lipids after methylation is recovered from the same acid that was added in the diet. The specific activities of tissue PL and NL are given in Table 2. There is little loss of specific activity due to the breakdown of any of the three acids during the developmental stages studied. Incorporation of palmitic/palmitoleic acid is prominent during the fifth instar. into both PL and NL. The label is retained in NL but gradually lost from PL. Thus the specific activity of PL in early fifth instar is 14,825 counts/min per mg lipid, in late fifth instar 17,320 counts/min and in 4-day male pupae 14,480 counts/min. Corresponding activities in NL are 13,480, 17,110, and 18,080 counts/min.
SEPPO TURUNEN
2332
0 palmitic
linolenic
FIG. 1. Concentration of labelled fatty acids in diets and frass of fifth instar larvae (counts/min per mg lipid). Values above frass columns denote percentage from total dietary radioactivity present in frass. TABLE ~-SPECIFIC ACTIVITIES OF TISSUELIPIDSOF P. bvassicae REARED ON DIETSCONTAINING LABELLED FATTYACIDS(COuntS/min per mg LIPID) Linoleic acid
Palmitic acid
Linolenic acid
-
PL
NL
13,480 (5) 17,110 (4)
15,360 (4) 20,080 (6)
9705 (4) 11,405 (4)
18,080 (4) 16,910 (3)
18,240 (4) 17,760 (3)
12,475 (4) 11,625 (3)
Stage
PL
NL
Early fifth* Late fifth* 4-day pupae* Males Females 4-day pupae Fat body (males) Fat body (females) 3-day adults Fat body (males) Fat body (females)
14,825 (6) 17,320 (4) 14,480 (4) 13,650 (4)
PL
NL
37,420 (4)
25,890 (4)
43,410 (4)
28,370 (4)
42,600 (3)
30,670 (3) 27,870 (3)
* Lipids from all tissues except haemolymph and intestine (meconium in pupae) were extracted. Figures in parentheses indicate the number of experiments. The technique involved in the determination of specific activities is described in the Materials and Methods section. Tissue controls gave an average of 26 counts/min per mg lipid.
FATTY
ACIDS AND
ACETATE
IN LIPIDS DURING
METAMORPHOSIS
2333
OF PIERIS
Similarly on a diet containing linolenic acid an increase in the specific activity of PL from early fifth instar (15,360 counts/min) to late fifth instar (20,080 counts/min) is noted, with a slight decrease in 4-day pupae (18,240 counts/min in males). The NL show only small changes in specific activity during development (9705 counts/min in early fifth instar, 11,405 countsjmin in late fifth instar, 12,475 counts/min in 4-day male pupae). As previously noted the fat body PL content is rather low in Pieris. Incorporation of linoleic acid into fat body lipids was studied in pupae and adults reared on a labelled diet from the beginning of the fifth instar. The dietary concentration of the label was twice that of the two other diets to make up for a lower PL content. Fat body PL incorporated distinctly more of the label than NL (per mg lipid). In 4-day pupae the females, moreover, accumulated more label than the males (43,410 vs. 37,420 counts/min in PL and 28,370 vs. 25,890 counts/min in NL). In 3-day adult males the specific activity of NL is higher than in male pupae, while that of adult females does not differ from the pupal value. The total per cent distribution of the label between NL and PL during metamorphosis indicates a gradual shift of label from PL to NL toward the pupal stage (Table 3). While 35.8 per cent of the activity from palmitic/palmitoleic acid is in TABLE
~-PER
CENT DISTRIBUTION OFLABEL INNEUTRAL (NL) AND PHOSPHOLIPIDS P. bYUSSiCUe REARED ON DIETS CONTAINING LABELLBD FATTY ACIDS
Pahnitic acid
Linolenic acid
PL
NL
77.2
43.8 32.7
56.2 67.3
87.9 89.2
20.0 18.5
80.0 81.5
NL
Stage
PL
Early fifth* Late fifth*
3.58
64.2
22.8 12.1 10.8
4-day pupae * Males Females 4-day pupae Fat body (males) Fat body (females) 3-day adults Fat body (females)
Linoleic acid
(PL) IN
PL
NL
8.8
91.2
9.3
90.7
8.9
91.1
* Lipids from all tissues except haemolymph and intestine (meconium in pupae) were extracted.
PL in early fifth instar larvae, only 10.8 per cent is found in the PL of 4-day pupae. Early fifth instar larvae again retain 43.8 per cent of the label from linolenic acid in PL, while 32.7 per cent is in PL in late fifth instar larvae and 18.5 per cent in 4-day pupae. No similar tendency is found in the distribution of the label from
SEPPO TURUNEN
2334
linoleic acid in the fat body lipids of pupae and adults. In both 4-day pupae and 3-day adults approximately 9 per cent of the label is in PL. Distribution of label among phospholipid classes Five types of PL were distinguished in Pieris. Of these phosphatidylcholine (PTC) and phosphatidylethanolamine (PTE) account for the bulk of PL. Sphingomyelin is far less common but nevertheless regularly present. Two additional bands corresponding to lysophosphatidylcholine and phosphatidylserine were tentatively identified by TLC. No gravimetric measurements of the individual fractions were attempted. Table 4 gives the distribution of activity among the PL types of Pieris reared on a diet containing palmitic acid-l J4C. There is a striking tendency toward increased incorporation of the label into sphingomyelin from early fifth instar larvae (2.0 per cent) to 4-day pupae (135 per cent in females). The label is gradually TABLE ~-PER
Stage Early fifth Late fifth 4-day pupae Males Females
CENTDISTRIBUTION OF LABELAMONGPHOSPHOLIPIDS OF P. bvassicae REARED ON A DIETCONTAINING PALMITICACID-l -14C No. of experiments *
Total no. of animals
3 5
26 30
5 4
28 24
Sphingomyelin
PTC
Unknownt
PTE
2.0 6.5
39.0 45.0
6.2
52.8
3.0
45.5
13.1 13.5
48.3 45.2
Trace Trace
38.6 41.3
* Each sample was chromatographed a minimum of three times. The average per cent distributions of all analyses have been listed. -I_The Rf value equals that of phosphatidylserine. The technique used in the determination of activities by TLC is described in the Materials and Methods section. Compare legend of Table 2.
lost from PTE (52.8 per cent in early fifth instar, 41.3 per cent in female and 38.6 per cent in male 4-day pupae) but a parallel increase in activity is noted in PTC (39.0 per cent in early fifth instar, 45.2 per cent in female and 48.3 per cent in male 4-day pupae). Early fifth instar larvae apparently incorporate the label into phosphatidylserine (6.2 per cent), but only trace amounts of label can be found in this fraction in 4-day pupae. The distribution of label from linolenic acid, in clear distinction to palmitic/ palmitoleic acid, is rather uniform (Table 5). Only two PL types incorporate the label. PTC at all stages contained more label than PTE. In early fifth instar larvae 51.8 per cent of the label was in PTC and 48.2 per cent in PTE. Corresponding values for late fifth instar larvae were 55.3 and 44.7 per cent. No sexual differences in 4-day pupae were found in the labelling pattern, while some were obvious in the case of palmitic/palmitoleic acid. Referring to Table 2 it appears
FATTY ACIDS AND ACETATE IN LIPIDS DURING METAMORPHOSISOF PIERIS
233.5
that from early to late fifth instar -the metabolism of linolenic acid in PL is perhaps more diversified than in pupae, and that the labelling pattern in 4-day pupae roughly simulates that of late fifth instar larvae. TABLE S---PER CENT DISTRIBUTION OF LABEL AMONG PHOSPHOLIPIDS OF P. brassicae REARED ON A DIET CONTAINING LINOLENIC ACID-1 -14c
Stage Early fifth Late fifth 4-day pupae Males Females
No. of experiments*
Total no. of animals
Sphingomyelin
3 3
59 38
Not detected Not detected
51.8 55.3
48.2 44.7
4 3
36 23
Not detected Not detected
53.8 53.4
46.2 46.6
PTC
PTE
* Legend as in Table 4.
Incorporation of linoleic acid into fat body PL in pupae and adults is shown in Table 6. The labelling pattern follows closely that of linolenic acid. PTC containing over half of the activity in both sexes in 4-day pupae (55.8 per cent in females, 57.3 per cent in males), but interestingly 3-day adult males retain more of the label in PTE (50.2 per cent) while in adult females the distribution of the label resembles that of pupae. Somewhat over 1 per cent of the label was recovered from sphingomyelin in both pupae and adults. TABLE 6-PER
CENT DISTRIBUTION OF LABEL AMONG PHOSPHOLIPIDS IN THE FAT BODY OF
P. brassicae RRARRDON A DIET CONTAINING LINOLEIC ACID-~J~C
Stage 4-day pupae Males Females 3-day adults Males Females * Legend
No. of experiments *
Total no. of animals
Sphingomyelin
3 3
20 20
1.6 1.0
57.3 55.8
41.1 43.2
2 3
18 31
1.4 1.2
48.4 56.6
50.2 42.2
PTC
PTE
as in Table 4.
The incorporation of injected acetate-l J4C into fat body PL reveals a somewhat different pattern than those discussed above, showing similarities with the distribution of label from the lbcarbon acids. After 1 hr 58.6 per cent of the label in females was recovered from PTC and only 24.4 per cent in PTE, while 17.0 per cent was in sphingomyelin (Table 7). Within 2 hr additonal label was incorporated into PTC (63.1 per cent) while some was lost from sphingomyelin. Assuming that
2336
SEPPO
TURUNEN
the polyunsaturated la-carbon fatty acids are poorly synthesized from acetate by Pieris, some interesting conclusions can be proposed. PTE apparently accumulate proportionately more of the polyunsaturated acids than other PL types and the polyunsaturated acids, furthermore, may constitute a major portion of the fatty acids of PTE. PTC contains mostly those acids that the fat body is capable of synthesizing from acetate, i.e. palmitate/palmitoleate, stearate, and oleate, all known to be constituents of PL in Pieris. Oleate and palmitate are generally the major products of fatty acid synthesis from acetate in insects, and apparently are utilized by Pieris in PTC to a far greater extent than in PTE. Sphingomyelin is quantitatively a minor PL type in Pien’s, and if we consider the relatively large proportion of label from acetate in sphingomyelin as well as the data on the incorporation of linoleate and linolenate, it appears safe to suggest that the fatty acid moiety of this PL type mostly consists of those acids synthesized from acetate. TABLE 7-PER
Time of incorporation
w
CENT DISTRIBUTION OF LABEL FROM INJECTEDACETATE-1-14c IN PHOSPHOLIPIDS OF THE FAT BODY OF 4-day FEMALE PUPAE OF P. braStiCaf?
No. of experiments*
Total no. of animals
Sphingomyelin
PTC
PTE
4 2
26 10
17-o 13-l
58.6 63.1
24.4 23.8
1 2
* Legend as in Table 4. DISCUSSION
The biosynthesis of TGL and PL are thought to proceed along common pathways through a key intermediate, phosphatidic acid (KENNEDY, 1963). The latter can yield a diglyceride to be later converted to triglycerides, phosphatidylcholines, Label from injected acetate is readily incorporated or phosphatidylethanolamines. into both TGL and PL in Pieris, indicating the synthesis of fatty acids and incorporation of the synthesized acids into these lipids. Although larvae, being metabolically more active than pupae, synthesize more lipid, it is also expected that pupae incorporate the label from both acetate and fatty acids into lipids required for adult structures (c$ STEPHEN and GILBERT, 1969). A number of papers dealing with the PL of insects have demonstrated the presence of the same general types of PL in insects as in vertebrates (HACK et al., 1962; Gilbert, 1967). PTC appears to be the major PL type in Hymenoptera, Lepidoptera, and Orthoptera (BRIDGES, 1972), while in Diptera PTE predominates (BIEBER et al., 1961). Sphingomyelin has been found to be almost always of a minor occurrence in insects, but nevertheless its presence has been detected in some Lepidoptera, Coleoptera, and Orthoptera, while its absence has been established in some Diptera (BRIDGES, 1972). It is of interest that where sphingomyelin adults
has been found (CHOJNACKI
in Lepidoptera,
and KORZYBSKI,
its concentration
has been high in pupae and
1962; SRIDHARA and BHAT,
1962).
The
PL
of
FATTY
ACIDS AND
ACETATE
IN LIPIDS DURING
METAMORPHOSIS
OF PIERIS
2337
P. brassicae are composed of at least PTC, PTE, and sphingomyelin. The presence of lysophosphatidylcholine and phosphatidylserine has also been tentatively identified, although they are both of a minor occurrence in this species. The relative amount of PL as a percentage of total lipid in Pieris is highest in larvae before the enhanced TGL synthesis during the fifth instar. The decrease is almost linear from early fifth instar to 4-day pupae (Table 1). This is in agreement with the results of SRIDHARAand BHAT (1965) who report that 23 per cent of larval lipid in Bombyx mori is PL whereas only 18-19 per cent of pupal lipid is PL. These authors also report a low concentration of PL in the fat body of Bombyx (3 per cent), as has now been shown to be the case in Pieris. In contrast to the gradual decrease in the relative PL content from larvae to pupae in whole animal extracts, the relative fat body content of PL in Pieris apparently remains on a static level in pupae and adults. The total extractable PL, as well as TGL, however, increases during the fifth instar. Late fifth instar larvae have 2.5 times more PL than early fifth instar larvae, while the corresponding value is over seven times for NL, showing intense synthesis of both lipid types during the last larval stage. The low PL content of the fat body of pupae and adults suggests that PL is utilized preferentially for structures such as ovaries and eggs (cj. ALLAISet al., 1964) where some PL types may have a rhle in supplying energy for embryonic processes (GILBERT, 1967), or they may be employed as structural lipid in flight muscles (CRONE, 1964). The present results have supported the view that both linoleic and linolenic acid are utilized in PL in a greater proportion than NL. Moreover, it is apparent that their presence in PL in Pieris is restricted mainly to PTE and PTC. Characteristically the pattern of labelling from linolenic acid changes little from the beginning of the fifth instar to Cday pupae when the whole animal without haemolymph is considered, with the possible exception of slightly more linolenic acid being required in PTE in early fifth instar larvae than in later stages. A somewhat similar pattern is found in the case of linoleic acid. No sexual differences are indicated by the present data with respect to linoleic or linolenic acid in pupae, but in the fat body of adults the distribution of linoleic acid in PTE and PTC varies between the sexes. Two presumptive explanations may be proposed to account for these differences. Firstly, it is possible that the change in the labelling has occurred only in the adult male which incorporates more linoleic acid in PTE or has more PTE as such in the fat body than do pupae. It is.a.lso conceivable that the adult female may utilize more linoleic acid or PTE, for example in the structures of ovaries and eggs, showing as a decrease in the fat body content of PTE. The requirements for PTE in the adult could result from its riYe in flight muscle sarcosomes, as shown at least in lMusca domestica (Crone, 1964) or, as already discussed, from the accumulation of PTE in egg lipids in the female (ALLAIS et al., 1964). The incorporation of the polyunsaturated fatty acids into PTE merits further comment. The fact that the label from acetate is retained chiefly in PTC in the fat body of pupae, with only a minor portion being recovered from PTE, would
2338
SE~O TIJRUNEN
suggest, bearing in mind that acetate most likely is not incorporated into linoleic or linolenic acid (cz ZEBE and MCSHAN, 1959; SRIDHARA and BHAT, 1964; LAMBREMONT,1965), that the polyunsaturated acids are preferentially utilized in the synthesis of PTE, possibly making up a majority of the fatty acids in PTE. This situation would be comparable with that of some vertebrate tissues (I&ODES, 1958). It appears thus that PTE performs a diversity of functions in insects and that the polyunsatured fatty acids are specifically utilized in this PL type. The special role of the polyunsaturated acids is also emphasized by the fact that in Pieris, at least, neither of them can be synthesized from the other, nor can they be saturated to form oleic or stearic acid. In contrast to the observed stability of the labelling pattern of linoleic and linolenic acid, the metabolism of palmitic acid is more diversified in Pieris. Free palmitic acid is desaturated to pahnitoleic acid and also lengthened to form oleic acid (cJ STEPHENand GILBERT, 1969). Palmitic/palmitoleic acids are utilized preferentially in PTE in larvae, while toward the pupal stage more of these acids are incorporated into PTC and also sphingomyelin. Whether this is evidence of sphingomyelin synthesis in late fifth instar larvae and particularly in pupae cannot be decided from the present data, but would not seem impossible, considering the simultaneous increase in the quantity of PL. The synthesis of palmitoleic acid by F’ieris on the artificial diet used here has been noted previously and may be a response of larvae, growing on a diet deficient in linolenic acid (c$ SCHAEFER, 1968; GRAU and TERRIERE,1971; TURUNEN, 1973). As previously shown it is in fifth instar larvae that the amount of palmitoleic acid reaches a maximum in the fat body PL of Pieris, decreasing thereafter toward the adult stage (TURUNEN, 1972). It is interesting that the label from palmitate/palmitoleate is prominent in PTE in early fifth instar larvae, and also that linolenic acid apparently is required in PTE especially during early fifth instar. Possibly palmitoleic acid partially replaces the function of linolenic acid in PTE in larvae unable to acquire enough of linolenic acid from the diet, whereas after pupation the requirement for linolenic acid can be more easily met with. It should be remembered that much linolenic and linoleic acid are lost at each ecdysis (personal data), thus effecting a continuous loss and sustaining a requirement for these acids especially in larvae. The functions of pahnitoleic acid may reach beyond that of substituting a dietary deficiency of another fatty acid. Interestingly, the content of palmitoleic acid is high in some overwintering lepidopterous larvae (BRACKENand HARRIS,1969). We may theorize that the requirement for available unsaturated acids rises higher during the period of overwintering and an increased synthesis of palmitoleic acid is resorted to, as apparently most insects are unable to synthezise the polyunsaturated acids. Referring to the role of these acids in the PL of Pieris, it would be interesting to know whether the high titre of palmitoleic acid is used in PTE in these overwintering species. Acknowledgement-The author wishes to thank Professor Sy~utii PESONEN(University of Helsinki) for generously allowing him to use the facilities of his laboratory.
FATTY ACIDSAND ACETATEIN LIPIDS DURINGMETAMORPHOSIS OF
PIERZS
2339
REFERENCES ALLAIS J. P., BERGERARD J., ETIENNE J., and POLONOVSKI J. (1964) Nature et evolution des lipides au tours de l’embryogeneae de Locusta migratoria. J. Insect Physiol. 10,753-772. BIEBER L. L., BROOKESV. J., CHELDELINV. H., and NEWBURGHR. W. (1961) The isolation of methylcholine containing phospholipid from Phormia regina larvae. Biochem. biophys. Res. Commun. 6, 237-240. BLANKENSHIPJ. W. and MILLER G. J. (1971) In Go synthesis of phosphatidyl choline and ethanolamine in larvae of the dried-fruit moth. r. § Physiol. 17, 2061-2067. BRACKENC. K. and HARRISP. (1969) High palmitoleic acid in Lepidoptera. Nature, Lond. 224, 84-85. BRIDGESR. G. (1972) Choline metabolism in insects. Adv. Insect Physiol. 9, 51-110. CHOJNACKIT. and KORZYBSKIT. (1962) Biosynthesis of phospholipids in insects-III. Incorporation of a2P-orthophosphate into phospholipids of Arctia caia moths. Acta biochim. pal. 9, 95-110. CRONE H. D. (1964) Phospholipid composition of flight muscle sarcosomes from the housefly, Musca domestica L. 3. Insect Physiol. 10,499-507. DAVID W. A. L. and GARDINERB. 0. C. (1966) Rearing Pieris brassicae (L.) on semisynthetic diets with and without cabbage. Bull. ent. Res. 56, 581-593. DWIVEDYA. K. and BRIDGESR. G. (1973) The effect of dietary changes on the phospholipid composition of the haemolymph lipoproteins of larvae of the housefly, Musca domestica. J. Insect Physiol. 19, 559-576. ELA R., CHEFURKAW., and ROBINSONJ. R. (1970) In viva glucose metabolism in the normal and poisoned cockroach, Periplaneta americana. J. Insect Physiol. 16, 2137-2156. FOLCH J., LEES M., and SLOANE-STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. r. biol. Chem. 226,497-509. GILBERT L. I. (1967) Lipid metabolism and function in insects. Adv. Insect Physiol. 4, 69-211. GRAUP. A. and TERRIEREL. C. (1971) Fatty acid profile of the cabbage looper, Trichoplusia ni, and the effect of diet and rearing conditions. y. Insect Physiol. 17, 1637-1649. GREEN D. E. and FLEISCHERS. (1963) The role of lipids in mitochondrial electron transfer and oxidative phosphorylation. Biochim. biophys. Acta 70, 5.54-582. HACK M. H., GUSSINA. E., and LOWE M. E. (1962) Comparative lipid biochemistry-I. Phosphatides of invertebrates (Porifera to Chordata). Comp. Biochem. Physiol. 5, 217-221. HOKIN L. E. and HOKINM. R. (1961) Diglyceride kinase and phosphatidic acid phosphatase in erythrocyte membranes. Nature, Lond. 189, 836-837. KENNEDYE. P. (1963) The biosynthesis of complex lipids. Proc. 5th int. Congr. Biochem. 7, 113-133. LAMBREMONTE. N. (1965) Biosynthesis of fatty acids in aseptically reared insects. Comp. Biochem. Physiol. 14, 419-424. OMERV. V. and ECOBICHOND. J. (1971) The acute effect of some chlorinated hydrocarbon insecticides on the acetylcholine content of rat brain. Can. j(. Physiol. Pharmac. 49, 79-83. RHODESD. N. (1958) Phospholipids-5. The effect of cod-liver oil in the diet on the composition of hen’s egg phospholipids. Biochem. J. 68, 380-384. SCHAEFERC. H. (1968) The relationship of the fatty acid composition of Heliothis zea larva to that of its diet. r. Insect Physiol. 14, 171-178. SRIDHARA S. and BHATJ. V. (1962) Phospholipids of the silkworm, Bombyx mori. Curr. Sci. 31, 240. SRIDHARAS. and BHAT J. V. (1964) Incorporation of 1 -l*C-acetate into the lipids of the silkworm, Bombyx mori L. Biochem. J. 91, 120-123.
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SSPPO TURUNEN
SRIDHARAS. and BHAT J. V. (1965) Lipid composition of the silkworm, Bombyx mori L. . J, Insect Physiol. 11, 449-462. STEPHENW. F., Jr. and GILBERT L. I. (1969) Fatty acid biosynthesis in the silkmoth, Hyalophora cecropia. J, Insect Physiol. 15, 1833-1854. TEFXRIERE L. C. and GRAU P. A. (1972) Dietary requirements and tissue levels of fatty acids in three Noctuidae. 3. Insect Physiol. 18, 633-647. THOMAS K. K. and GILBERTL. I. (1967) In vitro studies on the release and transport of phospholipids. y. Insect Physiol. 13, 963-980. TURUNENS. (1972) Fatty acids of the fat body of Pieris brassicae L. during metamorphosis ; effect of a low concentration of a chlorinated insecticide. Ann. Acad. Sci. fenn. (A) 4, 193, l-7. TURUNENS. (1973) Utilization of fatty acids by Pieris brassicae L. reared on artificial and natural diets. r. Insect Physiol. 19, 1999-2009. ZEBE E. and MCSHAN W. H. (1959) Incorporation of r4C-acetate into long chain fatty acids by the fat body of Pqodenia eridania. Biochim. biophys. Acta 31, 513-518.