Fatty-acid esters of lutein in Pieris brassicae fed on natural and artificial diets

Insect Biochem., 1975, Vol. 5, pp. 861 to 875. Pergamon Press. Printed in Great Britain

FATTY-ACID ESTERS OF L U T E I N IN P I E R I S B R A S S I C A E FED ON N A T U R A L AND A R T I F I C I A L D I E T S HARTMUT KAYSER Abteilung fiir Biologie I, Universit~it Ulna, D-7900 Ulm/Do., Germany (Received 7 April 1975; revised 23 April 1975) AbstractmThe fatty-acid esters of lutein in Pieris brassicae pupae and adults were studied after rearing on a natural and an artificial diet. The reliability of the indirect method applied was confirmed by mass spectrometry. Lutein occurs as diesters and 3'-monoesters (no 3-isomers were found) with the same fatty acids on both diets: palmitic (16 : 0), palmitoleie (16 : 1), stearic (18 : 0), oleic (18 : 1), linoleic (18 : 2), and linolenic acid (18 : 3). On the natural diet linolenate was the most prominent ester followed by oleate. The relative composition of the diesters was similar to that of the monoesters. The monoester compositions differed only slightly between pupae and adults, and between 1-day adult males and females. During adult life of females no change in the monoester composition was stated. On the artificial diet oleate was the main monoester; linolenate was low. During adult life a strong increase in lutein diesters and a decrease in monoesters and free lutein was found on both diets. In 1-day and 14-day males more lutein is esterified than in the corresponding females. The tendency to diester formation is more pronounced in the male, too. The results are discussed in respect to the dietary fatty-acid supply and sexual dimorphism of lipid utilization in Pieris. INTRODUCTION CAROTENOIDS m a y undergo several metabolic reactions in the insect body. One of these is the degradation of the carotene molecule to retinal for photoreception. On the other hand the intact carotenoid can be modified b y introduction of oxygen functions or by other oxidative reactions (KAYSER, 1975b in prep.). I n most cases the chromophoric system and, consequently, the colour of the pigment has changed b y these modifications. T h i s fact is utilized in some insects, which m a y occur in various colour types; probably, hormonal factors are involved in those p h e n o m e n a (cf. FUZFAU-BRAESCH, 1972; HARASHIMA, 1972; I~YSER, 1974). Furthermore, the unchanged carotenoid m a y be the subject of metabolic reactions leading to fattyacid esters for example. Carotenoid esters are very c o m m o n in insects (cf. KAYSER, 1975a, 1975b), and m a y indicate interesting features of the fatty-acid metabolism. Recently, an indirect way of analysis has been developed involving the combination of different thin-layer methods for the identification of the fatty-acid moiety of whole carotenoid esters in Aglias urticae (KAYSER, 1975a). T h i s method is now applied to lutein esters occurring in Pieris brassicae (KAYsER, 1974). Since this insect is generally reared on a semi-synthetic diet (DAVID and GARDINER, 1966) in our laboratory, a comparative study of the esters synthesized on a natural and an 861

HARTMUT I~AYSER

862

artificial diet was of interest in respect to the relevance of carotenoid supply and the nature of the fatty acids bound to the pigment. MATERIALS AND METHODS

Animals The general maintenance and the origin of P. brassicae has been described previously (KAYSER, 1974; KAYSERand ANGERSBACH,1975). Most of the work has been done with the French stock (I). For the study of the pupae the English stock (II) has been used, too. The larvae were reared on green leaves of cabbage (Brassica oleracea var. gongylodes), or on a semi-synthetic diet according to DAVID and GARDINER(1966). In this diet cabbage powder and wheat germ as ingredients served as a source for carotenoids. Adults were killed either within 24 hr after pupal-adult ecdysis (1-day adults), or after a 2 week period in the butterfly cage for copulation and egg-laying (14-day adults). The 1-day adults were kept quiet without feeding. In the females the ovaries were still undeveloped. In the 14-day adults parts of the wings had been lost during flight in the cage.

Analytical procedures The analysis of carotenoids and carotenoid esters was performed in the same way as reported in detail previously (KaYsER, 1975a). TLC-separations were done by partition (silica gel-G), adsorption (CaCOs/MgO), reversed-phase partition (paraffine impregnated cellulose), and argentation chromatography (AgNO3 in the adsorbent or solvent). The mass spectra were taken at 70 eV (some also at 12 eV) and 210°C on a Varian CH 5 mass spectrometer. Quantitative measurements were done by spectral photometry and by densitometric scanning.

RESULTS I t h a d b e e n s h o w n (KAYSER, 1974) that in the p u p a e of Pieris brassicae a b o u t one fifth of total lutein occurs as a monoester. L a t e r on the examination of adults of b o t h sexes revealed an identical fraction at this stage. F u r t h e r m o r e , a lutein diester could be demonstrated, w h i c h was present only in trace a m o u n t s in the pupal stage (cf. Fig. 1).

Lutein esters of pupae and adults reared on cabbage (1) Monoesters. As the two h y d r o x y l g r o u p s of lutein are chemically different,

2 m

3

J

4

m

m

5

g

A

B

FIa. 1. Silica gel-G chromatogram of carotenoid extracts from pupae of Aglais urticae (A) and adults of Pieris brassicae (B). 1, B-carotene; 2, lutein diester; 3, lutein 3-monoester; 4, lutein 3'-monoester; 5, free lutein.

F A T T Y - A C I D ESTERS OF L U T E I N I N PIERI$ BRASSICAE

863

two isomeric monoesters are possible: esterification of the hydroxyl at C-3 of the ~-ionon ring (3-ester), or esterification of the allylic hydroxyl at C-3' of the e-ring (Y-ester). Both isomeric esters could be established in the pupae of Aglais urticae (KAYssR, 1975a). Since the allylic OH-group is less polar than the other, the 3'-esters behave more polar and, therefore, can be distinguished on the chromatogram. Co-chromatography of the Pieris extract with that from Aglais on silica gel showed similar behaviour of the Pieris monoester fraction to the 3'-esters of Aglais. A fraction corresponding to the less polar 3-isomers could never be demonstrated in Pieris. The chemistry of the Pieris ester indicated its allylic nature, too, i.e. there was no ether formation in acidic alcohols, but only a substitution of the ester group by the more stable ether group (cf. KAYSER, 1975a). The lutein monoester fraction from Pieris was acetylated and studied by reversed-phase chromatography on paraffine impregnated cellulose. For comparison the acetates of the known lutein monoesters from Tagetes flowers (myristate 14 : 0, palmitate 16 : 0, stearate 18 : 0), and of the recently identified monoesters from Aglais pupae (linoleate 18 : 2, linolenate 18 : 3 ; cf. KAYSER, 1975a) were used. The Pieris monoester splits into five zones, one of them can be separated into two zones by multiple development (Fig. 2). The Pieris fraction (P1) migrates like

m

m

I

m

m

I N

m m

m

m

m

m ~ m m

A

m

B

Im ~

~mmm

m

m

~

C

D

A

E

FIG. 2. Reversed-phase partition chromatogram of acetylated lutein monoesters from: (A) Tagetes (from bottom to top: stearate, palmitate, myristate), (C) Pieris, cabbage reared (stearate, palmitate, oleate, linoleate/palmitoleate, linolenate, unknown ester), (B) mixture of (A) and (C), (D) Pieris, artificial diet reared, (E) Aglais (linoleate, linolenate). the Stearate from Tagetes, fraction (P2a) like the palmitate, fraction (P2b) runs above the latter, fraction (Pz) is more polar than the myristate, but is similar to the linoleate from Aglais, fraction (P4) migrates with the linolenate, fraction (Ps) is more polar than all esters used. These data indicate, that in the lutein monoester fraction of Pieris both saturated (Px, P2~) and unsaturated (P2b, P3, P4) fatty acids are bound. This can be clearly demonstrated by two-dimensional reversed-phase partition argentation chromatography, as has been shown in the case of the Aglais

864

HARTMUT

KAYSER

esters (KAISER, 1975a). As seen in Fig. 3 the Pieris fractions (P1) and (P~a) do not migrate in the Ag+ ions containing solvent in contrast to the fractions (P2b)--(Ps), which run as sharp and concentrated zones owing to the formation of polar silver complexes. T h e fatty acids of these esters must therefore be unsaturated. Quite unexpected fraction (P3) separated into two esters, one of which behaved similar to (P~b)" T h e degree of unsaturation was established by argentation adsorption chromato-

1

z..........t

,::':::::~

;I

I

l r

2.

FIG. 3. Two-dimensional chromatogram of acetylated lutein monoesters of Pieris reared on cabbage. First run: reversed-phase partition; second run: argentation partition (AgNO8 in the solvent). Punctuated zones indicate the positions after the first run. For identification of the esters see legend of Fig. 2, C. The dashed line represents the silver front as a result of solvent demixing.

m

I m

gore

A

/ m

B

C

Fzo. 4. Argentation adsorption chromatogram on silica gel-G of acetylated lutein monoesters from: (A) Aesculus, (B) Aglals, (C) Pierls.

865

F A T T Y - A C I D ESTERS O F L U T E I N I N PIERIS BRASSICAE

graphy, using the linoleate (18:2) and linolenate (18:3) from Aglais, and the palmitate (16 : 0) and linolenate from Aesculus autumn leaves (cf. K~YSER, 1975a) as references. The chromatogram shows (Fig. 4), that part of the P~r/~ esters is similarly less adsorbed like the palmitate, indicating saturated fatty acids (fraction Ax), a weak, diffuse zone behaves like the linoleate with two double bonds (fraction A2), the main part of the Pieris esters, however, is identical to the linolenates from .4glais and Aesculus, supporting the presence of this trienoic acid (fraction Aa). A smaU amount of the Pieris ester is still stronger adsorbed than the linolenate (fraction A~). In order to correlate the fractions (A) with the fractions (P) the isolated zones (P2)--(Pn) (P~ yielded a too less amount) were re-chromatographed in the argentation adsorption system. Thereby fraction (Pa), containing a saturated (Pan) and a 86~

(4) o IK

50CD

30-

o~g

10-

I

,ll

,

II

i31~g

,I

(a/

50-

•o,~

,.~

30-

10I

Ill

i

,

100~

~g

cg

,

~,

~

|

i

(2)

A

50-

i u.

30-

~o,

__I

lt,

,,f, ,~ ,,? ~ X:[ :[ k , , I 5~

700

OO0

I

r

I

~I

J

I

800

m/.

Fxo. 5. Mass spectra (70 eV, 210°C) of acetylated lutein monoesters from Pieris. (2): oleate-acetate (Mx), palmitate-acetate(M~), (3): linoleate-acetate (Mx), palmitoleate-acetate(M~), (4): linolenate-aeetate. FA, fatty acid.

~o

866

HARTMUT KAYSER

unsaturated (Pg.b) fatty acid, yielded (A1), (Pa) splitted into (A1) and (As) , (P4) was identical to (A3) and (Ps) appeared as the most polar zone (A4). It is evident, that fraction (Pzb) must be an ester with a monoenoic acid. Since the introduction of a double bond into a fatty-acid aliphatic chain is equivalent to a reduction of the chain by two CH2-groups (cf. KaYSER, 1975a), the fatty acids of the lutein monoesters of Pieris can be identified as followed: (P1)-18 : 0, (P2~)--16 : 0, (P2b)--18 : 1, (Ps)--18 : 2, (P4)--18 : 3, (Ps)--unknown. In order to check up the reliability of the indirect method of fatty-acid analysis, the fractions (P2)--(Ps) were isolated in a preparative scale and subjected to mass spectrometry. The amount of fraction (P1) was insufficient. The mass spectrum of the total fraction (P~) exhibited two molecular ions at m/e 874(M1) and 848(M2) , in deed (Fig. 5). Loss of the allylie fatty acids from both esters yielded identical ions at m/e 592 (base peak). The mass differences are 874 (M1)- 592 = 282 m.u. and 848(M2)- 592 = 256 m.u., respectively. These differences exactly agree with the postulated 18 : 1 and 16 : 0 fatty acids. Both molecular ions are characterized by the loss of acetic acid (60 m.u.) from the acetylated fl-ionon ring, and of toluene (92 m.u.) and xylene (106 m.u.) from the polyene chain of the carotenoid (ScHwI~TER et al., 1965): m/e 814(M1--60), 788(M~--60), 782(M~--92), 768(Mx--106), 756(M2--92), and 742(M2--106). Multiple fragmentations, which are typical for carotenoid mass spectra (ENZELL et al., 1969), yielded peaks at m/e 722(M~--60--92), 708(Mx--60--106 ), 696(M~--60--92), 682(M2--60--106 ). After the elimination of the fatty acids (FA) the spectra of both esters are identical: m/e 532(M--FA--60), 500(M--FA--92), 486(M--FA-106), 440(M--FA--60--92), 426(M--FA--60--a06). The favoured elimination of the fatty acid over the acetic acid is caused by the allylic position of the fatty acid (cf. BUDZIKIEWICZet al., 1970). This is a mass speetrometrical evidence for the 3'-isomers. From the intensity ratio of the two molecular peaks the 18 : 1-ester accounts for 60~o and the 16: 0-ester for 4 0 ~ of the total fraction (P2). As derived from the argentation partition chromatogram fraction (P3) must be a mixture of two esters, too. As seen in Fig. 5 the molecular ions appear at m/e 872(M1) and 846(M~). Fragmentations analogous to fraction (P2) were found: m/e 812(M1--60), 786(M2--60 ), 780(M~--92), 766(M1--106), 754(M2--92), 740(M2--106), 720(M1--60--92), 706(M1--60--106), 694(Mz--60--92), 680(M~-60--106). The ion at m/e 592 (81,6~ of the base peak at m/e 55) results from elimination of the fatty acids from both molecular ions. In the lower mass region the spectra of the fraction (Pc) and (P3) are identical. The mass differences of 872 (M1)--592 = 280 m.u. and of 846(M2)--592 = 254 m.u. correspond to a 18 : 2 and a 16 : 1 fatty acid, respectively. According to the peak heights of the molecular ions the 18 : 2-ester takes about 61 ~ of the mixture. The monoester fraction (P4) revealed a single ester by mass spectrometry with a molecular ion at m/e 870(M). All typical fragment ions were observed: m/e 810(M--60), 778(M--92), 764(Mm106), 718(M--60--92), 704(M--60--106). Loss of the fatty acid results in a peak at m/e 592 (86,1 ~ of the base peak at m/e

FATTY-ACID ESTERS OF L U T E I N I N PIERIS BRASSICAE

867

91), indicating a mass difference of 870(M)--592 = 278 m.u. which correspond to the expected 18 : 3 fatty acid. From fraction (P4) no satisfactory mass spectrum was obtained. The direct analysis of the carotenoid esters by mass spectrometry completely agrees with the information obtained by the indirect method of reversed-phase partition combined with argentation chromatography. Accordingly, the following fatty acids are esterified with the Y-hydroxyl of lutein in Pieris: palmitic (16 : 0), palmitoleic (16 : 1), stearic (18 : 0), oleic (18 : 1), linoleic (18 : 2), linolenic (18 : 3), and a still unknown fatty acid, which is more polar than the trienoic one. Because of the known specificity of the argentation method not only to the number of double bonds, but also to their position and geometry (cf. MoRRis, 1966), the identity of the unsaturated fatty acids with the upper mentioned ones may be accepted. Quantitative measurements were carried out by densitometry of reversedphase partition chromatograms of the monoester fraction on paraffine impregnated cellulose. In the case of the pupae both insect stocks were studied separately, and measurements were done before and after the acetylation of the monoester T A B L E 1 - - P E R C E N T A G E FATTY-ACID COMPOSITION OF LUTEIN MONOESTERS FROM REARED ON CABBAGE 18:0

Stage

18:1 16:0

18:2 16:1

18:3

unknowa

Pupae § Stock I

(13~

27,7Z1,1

12,2~_0,4

42,0Z0,8

14,4~1,0

2,5~0,2

26,9~1,1

11,6±1,!

43,0±0,6

15,9~0,7

native (8)

3,6~0,5

31,6~0,7

13,5~0,5

~0,0!0,6

I|~3~0,7

acetyl,(6)

4,7~0,3

31,5~I,0

12,7~0,3

38,4~0,3

12,8~0,8

l-day male(5)

3,6±0,2

40,3~0,9

I|,24£0,3

35,5±0,9

9,5~0,5

l-day female

4p0~0,5

37,9~213

15,6~l~5

36,~],8

6,94~0,9

lj6

37m9

13,3

37,8

9,4

2~2

36~7

12,3

39,6

9,3

native

acctyl,(9)

3,5~0,3 ~

Stock II

Adults

(5) l-day female

(2) 14-day female (2)

Three days a f t e r

larval-pupal

ecdysis

Number of determinations using the same extract

P. brassicae

868

H A R T M U T KAYSER

fraction. As shown in Table 1 the data on the acetates agree well with those on the native esters. Therefore, the monoesters were generally acetylated, since their purification was more successful in this form. The pupal monoesters of the two different stocks exhibit no marked differences in their relative fatty acid composition, minor variations may be related to experimental conditions. Fraction (P1) is the less one, whereas fraction (P4) predominates (Fig. 6). Basing on the ratios for the components of the mixed fractions as calculated from the mass spectra, the following sequence of increasing relative amounts of the fatty acids is obtained: 18 : 0< 16 : 1< 18 : 2< 16 : 0 < u n k n o w n < 18 : 1 4 1 8 : 3 In the study of the adult monoesters newly emerged males and females (1-day adults) were used. The data in Table 1 show a general agreement in both sexes. The little differences may represent normal biological variation. If compared with the pupae fraction (P~), containing palmitic and oleic acid, is increased by about 30 ~o, the fractions (P~) with linolenic acid and (Ps) are a little reduced. During adult life after emergence an enhanced utilization of fatty acids is to be expected especially in the female for the synthesis of egg lipids. Therefore the lutein monoesters of females after having laid eggs (14-day adults) have been investigated, but no differences to 1-day adults were observed (Table 1). This means, that a selective utilization of single fatty acids of the monoesters does not occur to a measurable amount in the female adult.

(2) Diesters. The lutein diester fraction from pupae and adults has been identified by co-chromatography with the lutein diester fraction from Aglais (KAXsER, 1975a) on silica gel-G, by its spectral properties, and by its saponification to lutein via monoesters. On paraffine impregnated cellulose the diester fraction separated into about

LO ~O

G f

FIG. 6. Densitometer scan of a reversed-phase partition chromatogram of lutein monoesters from Pieris reared on cabbage. The peaks correspond to (from left to right) stearate(P1), palmitate/oleate(P~), palmitoleate/linoleate(P3), linolenate(P4), unknown ester(Ps). The arrow on the abscissa indicates the direction of chromatography.

FATTY-ACID

F,S T E I ~

OF LUTEIN

IN PIERIS BRASSICAE

869

m

i

n

n

n

/

H

i

u

n - - - -

A

B

C

mn

D

FIG. 7. Reversed-phase partition chromatogram (3 x developed) of lutein diesters from: (A) Aglais, (B) Pieris, cabbage reared, (C) Tagetes, (D) Pieris, artificial diet reared, fifteen different zones (Fig. 7). The diesters from pupae and from male and female adults were identical. Because of the great number of zones the diester fraction seemed to be too complex for an analysis of each ester. Therefore the diesters were saponified to a mixture of 3- and 3'-monoesters, which after acetylation yielded a uniform fraction on silica gel. The reversed-phase partition chromatogram of these monoester acetates showed six zones, which corresponded to the fractions (P1)--(Ps) of the native monoesters. Also the two-dimensional argentation partition chromatogram revealed exactly the same ester pattern (cf. Fig. 3) as was obtained from the native ones. Accordingly, the fatty acids of the diesters and the monoesters are identical. The densitometer scan of the diester-derived monoesters was very similar to that of the native monoesters (cf. Fig. 6), demonstrating fairly equal relative amounts of the seven fatty acids in monoesters and diesters.

Lutein ester formation during adult life Since it had been shown, that the fatty-acid patterns of the lutein monoesters and diesters do not exhibit any quantitative changes during adult life after emergence, the process of lutein ester formation on the whole was studied to get information on the physiological function of carotenoid esters and the fatty acids bound to them. Male and female adults were extracted soon after emergence (1-day adults), and after reproduction (14-day adults). Table 2 summarizes the data. The total carotenoid content was higher in males than in females at every stage. During adult life a loss of 3,4/~g carotenoid was stated in both sexes; females lost more flcarotene than males. Concerning 1-day adults most of the total lutein was found in

870

HARTMUT KAYSER TABLE 2--CAROTENOID COMPOSITION OF ADULT P. brassicae REARED ON CABBAGE Total

Stage §

We~ w e i g h t per

adult

carotenoid per adult

(=~)

fl-carotene

total lutein

luteln diester

lutein monoester

free lutein

(pg)

(z)

(Z)

(Pg)

(z)=

(~)

(z)~

(Pg)

(Z)~

l-day male

157,9

~,4

22,6

77,4

0,4

6,5

4,0

61,2

2,1

32,3

14-day male

80,3

4,9

27,7

72,3

I,I

32,1

1,5

41,3

1,0

26,5

I-day female

157,1

7,6

31,4

68,6

0,3

6,5

2,1

42,0

2,7

51,5

]4-day female

87,6

4,2

34,1

65,9

0,7 25,3

0,9

31,0

1,2

43,7

§

were

48-149 a d u l t s

used

~t

each

stage

Percentage of total !uteln

the monoester fraction in the male (61.2~), but in the free-lutein fraction in the female (51.5~o); both sexes contained only small amounts of diesters (6"5%). Adult life is characterized by a decrease of free lutein and lutein monoester, corresponding to an increase of the lutein diester fraction (Fig. 8). This tendency of esterification is more pronounced in the male than in the female, but the relative dominance of monoesters in the male and free lutein in the female is maintained in

L

LME

LDE

I DAY

MALE

14-DAY

MALE

C

FIG. 8. Densitometer scans taken from photographs of silica gel-G chromatograms of carotenoid extracts from Pieris, showing the strong increase in the lutein diester fraction (LDE) during emerged adult life. (LME) lutein monoester, (L) free lutein, (C) ]~-carotene.

FATTY-ACID EST/~I~ OF L U T E I N I N PIERIS BRASSICAE

871

14-day adults. Since the lutein diester fraction increases not only by percentage but also in its absolute amount, this increase can not be explained by a selective loss of free lutein and monoesters during adult life. In this case the diesters would arise to 12.5 ~ only. Therefore, a strong metabolic shift must be developed in the adults.

Lutein esters of adults reared on the artificial diet In both sexes from the artificial diet fl-carotene, lutein diester, lutein monoester, and free lutein were detected as in the cabbage-fed ones. Zeaxanthin which is only a trace pigment in cabbage fed animals (cf. KAYsEa, 1974) was present to a higher extent (ca. 1 0 ~ of lutein). (1) Monoesters. The chromatographic behaviour of the monoester fraction on silica gel-G exhibited the allylic nature of the ester, as it was found in the case of the cabbage-fed animals, too. After acetylation it separated into five zones on paraffine impregnated cellulose (Fig. 2). Comparison with the monoester-acetates from cabbage-fed insects showed the identity of these zones with the fractions (P1), (P2a), (P2b), (Pa), and (P4). Two-dimensional argentation partition chromatography revealed an ester pattern corresponding to (P1)--(P4), too: the saturated fatty acids 18 : 0 and 16 : 0, the monoenoic acids 18 : 1 and 16 : 1, and the polyenoic acids 18 : 2 and 18 : 3. Accordingly, on the two diets identical lutein monoesters are synthesized except of fraction (Ps) with the unknown fatty acid, which was only present in cabbage-fed animals. A densitogram of the separated monoesters from 1-day adults reared on the artificial diet is presented in Fig. 9. The quantitative data are summarized in Table 3. No difference was stated between males and females in the percentage amounts of the different esters. The 18 : 1 fatty acid is the most prominent one, contrasting to the very low amount of the 18 : 3 acid, which was high in cabbage fed insects. (2) Diesters. The lutein diester fraction from adults reared on the artificial

q¢

O

i

I

I

I

I

"~

FIQ. 9. Densitometer scan of a reversed-phase partition chromatogram of lutein monoesters from Pieris reared on an artificial diet. The peaks correspond to (from left to right) stearate(P1), palmitate(P2a), oleate(P~b), palmitoleateflinoleate(Ps) , linolenate(P4). The arrow on the abscissa indicates the direction of chromatography.

872 TABLE

HARTMUT 3~PERCENTAGE

KAYgER

F A T T Y A C I D C O M P O S I T I O N O1~ L U T E I N M O N O E S T E R S F R O M

e. brassicae

REARED O N T H E A R T I F I C I A L D I E T 18:0

Stage

(6) ~

I-day

male

l-day

female

|

i

3,1~0,4 ~

(4) 4,9~0,3

18:|

18:2

16t0

16:1

65,3~1,3

24,2~0,9

7,5+0,5

63,1~0,6

24,7Z0,I

7,2Z0,6

N u u b e r of d e t e r m i n a t i o n s

using

the

some

18:3

extract

Mean value z S.E.H.

diet yielded only few zones by reversed-phase partition chromatography (Fig. 7). Since this fraction was obtained only in trace amounts no further work on its fatty acids could be done.

Lutein ester formation during adult life From artificial-diet reared animals the whole ester fractions besides free lutein and/3-carotene were measured in 1-day and 14-day males and females, respectively. The results are presented in Table 4. If compared with the cabbage-fed ones a higher relative amount of/3-carotene and a lower rate of lutein esterification was established in both sexes. In agreement with the adults from cabbage free lutein and lutein monoester was reduced, and the diester content raised during adult life. Generally, male and female adults demonstrated qualitatively the same metabolic trends on both feeding conditions during their life after emergence. DISCUSSION In addition to previous work (KAYSER, 1975a) it has been shown, that the fatty acid composition of carotenoid esters can be successfully analysed by indirect TABLE 4---CAROTENOID

COMPOSITION OF ADULT

B-carotel~e

Stage CX)

l-day male

total lute~n (z)

P. brassicae

REARED O N T H E A R T I F I C I A L D I E T

luteg.n

luteln

die~ter

monocster

(g) ~

(~)~

free lutaln

(z) ~

2g,O

72,0

2j8

36,0

61,2

15,1"

g4,9

30,2

23,5

~6,7

female

26,3

73,7

tr~c

30,0

70,0

14-day female

12,3

87,7

19,2

12,2

65,7

14-day male l-day

Pcreont~Jge of t o t a l

]utein

FATTY-ACID ESTEI~ OF L U T E I N I N PIERI$ B R A S S I C A E

873

methods from very little material. Even complicated mixtures of esters with saturated and unsaturated fatty acids can be separated by the combination of reversed-phase partition and argentation chromatography, the latter of which can be applied as partition or adsorption method. In the present study all esters, which were available in sufficient amounts, were subjected to mass spectrometry, and full coincidence was found with the data obtained by the indirect method, which only needs some authentic esters as references for chromatography (cf. KaYSER, 1975a). In Pieris diet supplied lutein is bound to a series of fatty acids to form monoand diesters, as it has been found in pupae of Aglais urticae (KAYSER,1975a). But in contrast to Aglais, where both isomeric monoester types of lutein were found, in Pieris only the allylic 3'-esters are synthesized. This may be a result of enzyme specificity, leading to a reaction with the allylic hydroxyl of free lutein only. Both on the cabbage diet and the artificial diet the same saturated and unsaturated fatty acids are bound to lutein during the development of Pieris. One still unknown unsaturated acid, which is more polar than linolenic acid, was only present after cabbage feeding. Except of this acid the identified ones exactly agree with the recent data of TURUNEN(1972, 1973a) on the fatty acids of Pieris, studying neutral and phospholipids by gas chromatography. After cabbage feeding linolenic and oleic acid are the main carotenoid-bound fatty acids. The other unsaturated ones, linoleic and palmitoleic acid, are only incorporated at a low rate. The great number of fatty acids in the lutein esters of Pieris contrasts to the only two ones, linoleic and linolenic acid, found in Aglais (KAYsER, 1975a). Insects can not synthesize polyenoic acids (cf. GILBERT, 1967), and therefore they must be supplied by the diet. TURUNEN(1973a) showed, that the high uptake of linolenic acid in Pieris is related to the high level in the food (about 50 ~o in Brassica oleracea). Owing to the deficiency of linolenic acid in the artificial diet (cf. TURUNEN, 1973a) only low amounts of this acid were established in these lutein esters, but high levels of oleic acid. According to TURLrNEN(1973b) this one is selectively accumulated from the diet and synthesized from palmitic acid along with palmitoleic acid, and may substitute for linolenic acid. Generally, the quantitative analysis of the lutein ester fatty acids revealed differences in the lipid metabolism as a result of the fatty-acid composition of the diet, and matched closely with the data on other lipid classes obtained by TURUNEN (1974). The quantitative fatty-acid composition of the lutein monoesters fits best with that of the neutral lipids; the phospholipids differ in their composition especially in the case of the artificial diet. In Fig. 10 the data of the present work are compared with those of TURUNEN (1974), using 1-day males in both cases. The coincidence is striking, suggesting similar physiological r61es for neutral lipids and carotenoid esters. As shown for the neutral lipids (TIJRImEN, 1972, 1973b) the fatty-acid composition of the lutein esters exhibit no marked changes during the development of Pieris. Only a small increase in palmitate/oleate and a decrease in linolenate and the unknown ester was stated in newly emerged adults without significant sexual differences. From the present data these changes can not be

874

HARTMUT KAYSER

ARTIFICIALDIET

18:0

18:1 16:0

18:0

18:1 18:2 18:3 16:0 16=1

ii

18:2 18:3 16=1 CABBAGE DIET

FIo. 10. Comparison of the percentage fatty acid amounts in lutein monoesters (LME) and neutral lipids (NL) in Pieris adults (1-day males) after rearing on a natural and an artificial diet. Data on NL are based on TURUNEN(1974). In LME (cabbage diet) calculation was done without the unknown ester. decided to result from a synthesis, or a utilization, or a shift of preferred fatty acids within lipid classes. According to TURUNEN (1974) during the adult stage of Pieris the fatty-acid composition changes in the female but not in the male. Contrasting to that, no selective utilization of fatty acids could be found within the lutein monoesters during two weeks of female life. Possibly, these compounds are not related to the formation of egg material. Yet, marked changes in the lutein esters are established in connection with sexual dimorphism, if the rates of lutein ester formation are viewed. On both diets less lutein is esterified in the female than in the male. In artificial-diet reared adults this rate is much lower despite of the very low carotenoid content ( < 0.5/~g per adult). This may be explained by the preferred accumulation of lutein in the integument (and eggs in the female), where lutein esters remain low (K~¥sER, 1974). Similar in both sexes free lutein and its monoesters are converted to diesters during adult life. This shift is more pronounced in the male than in the female, and may be related to lipid utilization during flight. In the thoracic muscles of Pieris most of the neutral lipids consist of free fatty acids, which accumulate during adult life (TuRUNEN, 1974). Owing to its higher flying activity more fatty

FATTY-ACID ESTERS OF LUTEIN I N PIERIS BRAS$ICAE

875

acids are expected to be stored in the male and, as a consequence, m a y be bound to lutein. I n the female, however, besides the lower accumulation in the thorax part of the fatty acids are lost with the eggs. T h i s m i g h t be a possible explanation for the sexual differences in lutein ester formation during the adult life of Pieris. Acknowledgement--I wish to thank Mrs. B. ZORN for technical assistance and Dr PRox and Mr K6STEI~(Dr K. THOMAE Ltd., Biberach) for running the mass spectra. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 87).

REFERENCES ]]UDZIKIEWICZ H., BRZEZINKAH., and JOHANNES B. (1970) Zur Photosynthese grtiner Pflanzen, 2. Mitt.: Massenspektro skopisehe Untersuchungen an Carotinoiden. Mh. Chem. 1019 579-609. DAVID W. A. L. and GARDINER B. O. C. (1966) Rearing Pieris brassicae (L.) on semisynthetic diets with and without cabbage. Bull. ent Res. 56, 581-593. ENZELL C. R., FRANCISG. W., and LIAAEN-JENSENS. (1969) Mass spectrometric studies of carotenoids. 2. A survey of fragmentation reactions. Acta Chem. scand. 239 727-750. FUZEAU-BRAESCH S. (1972) Pigments and colour changes. A. Rev. Ent. 17, 403-424. GILBERT L. I. (1967) Lipid metabolism and function in insects. Adv. Insect Physiol. 49 69-211. HARASHIMAK., OHNO T., SAWACHIKAT., HIDAKAT., and OHNISHI E. (1972) Carotenoids in orange pupae of the swallowtail, Papilio xuthus. Insect Biochem. 29 29-48. KAYSERH. (1974) Die Rolle der Carotinoide und des Gallenfarbstoffs bei der Farbmodifikation der Puppen yon Pieris brassicae. J. Insect Physiol. 209 89-103. KAYSER H. (1975a) The use of argentation chromatography for the analysis of fatty-acid esters of polyenes: the structure of carotenoid esters of Aglais urticae (Lepidoptera, Insecta). Z. Naturf. 30C9 369-378. KAYSER H. (1975b) Isolation of 3-hydroxy-J316-carotene-3'-one from two moth species, Actias seleue and Cerura vinula, jT. comp. Physiol. (B). In press. KAYSERH. and ANCERSBACHD. (1975) Dose effects in light-controlled pupal melanization in Pieris brassicae specificities to spectral ranges. J. Insect Physiol. 219 589-594. MoRRIs L. J. (1966) Separations of lipids by silver ion chromatography. J. Lipid Res. 79 717-732. SCHWlETER U., BOLLICER H. R., CHOPARD-DIT-J~.ANL. H., ENGI~RT G., KOFLER M., K6NIC A., PLANTAV. C., ROE~G R., VETTERW., and ISLERO. (1965) Synthesen in der Carotinoid-Reihe. 19. Mitteilung: Physikalische Eigenschaften der Carotine. Chimia 199 294-302. TORUNEN S. (1972) Fatty acids of the fat body of Pieris brassicae L. during metamorphosis; effect of low concentration of a chlorinated insecticide. Ann. Acad. Sci. fenn. (A) 49 193, 1-7. TtmUNEN S. (1973a) Utilization of fatty acids by Pieris brassieae reared on artificial and natural diets. 3t. Insect Physiol. 19, 1999-2009. TURUNEN S. (1973b) R61e of labelled dietary fatty acids and acetate in phospholipids during metamorphosis of Pieris brassicae. ~t. Insect Physiol. 19, 2327-2340. q'~RUNEN S. (1974) Lipid utilization in adult Pieris brassicae with special reference to the r61e of linolenic acid. J. Insect Physiol. 209 1257-1269.