Comp. Biochem. Physiol., 1976, Vol. 54B, pp. 227 to 230. Pergamon Press. Printed in Great Britain
ON THE FUNCTION OF CYCLOPROPANE FATTY ACIDS IN MILLIPEDES (DIPLOPODA) R. C. H. M. OUDEJANS,D. J. VAN DER HORST, F. A. OPMEER AND W. J. TIELEMAN Laboratory of Chemical Animal Physiology, State University of Utrecht, Padualaan 8, Utrecht, The Netherlands
(Received 27 May 1975) Abstract--1. Localization of cyclopropane fatty acids in the millipede Graphidostreptus tumuliporus is not restricted to the eggs and one particular organ, but bulk amounts are found in all tissues. 2. There is a correlation between the process of egg-ripening and the synthesis of cyclopropane fatty acids. 3. In the lipid structures of the female diplopod, cyclopropane fatty acids are not preferred to monoenoic ones, but are considered to mimic them due to their nearly identical physicochemical properties. 4. The function of cyclopropane fatty acids in millipedes is related to maintaining of the lipid structures of the vulnerable early larval stages in a desiccating arid environment, requiring a minimum susceptibility to oxygen.
INTRODUCTION
sizing capacity of individual organs was obtained through the use of radiolabelled acetate.
T ~ SPECIFICcyclopropane fatty acid formation operative in millipedes leading to an accumulation of cyclic acids (Oudejans et al., 1971a, b; van der Horst MATERIALS AND METHODS et al., 1972, 1973) has stressed the need to obtain an Millipedes of the species Graphidostreptus tumuliporus understanding of the function of cyclopropane fatty (Karsch) (Diplopoda: Spirostreptida) were supplied by the acids in millipede physiology. Institut Fondamental d'Afrique Noire (Dakar, Senegal) by From studies on bacterial lipids it is known that courtesy of Dr. R. Roy. the physicochemical properties of the phospholipids The animals were injected ventrolaterally, each with 10 do not change upon replacement of the monoenoic /~Ci sodium-[1-14C]-acetate (sp. act. 2 mCi/mmole; New aliphatic chains by cyclopropane ones (Cullen et al., England Nuclear). Incubation in a perspex container at 1971). Therefore the obvious preference of cyclic room temperature was ended after 96 hr by freezing the acids, despite their energetically expensive biosynthe- animals at -26°C. Five adult females were selected and dissected. At first sis, suggests some distinct advantage. sight the anatomy of the animal seems rather simple: a The cyclopropane fatty acids in certain plant structural lipids reportedly are involved in drought toler- rigid multisegmental tube with a longitudinal intestinal tract and the remaining cavity occupied by fat body, ovarance and cold hardiness (Bervaes et al., 1972; Kuiper ies, eggs and haemolymph. Individual organs or tissues, & Stuiver, 1972). In seeds and seedlings of mallow however, were not easily to be separated. Ultimately, five species bulk amounts of cyclic fatty acids are de- fractions were isolated: head (including the apodal anterior posited in the storage lipids (Yano et al., 1972). Ob- segments), exoskeleton, fat body (including the ovaries), viously these acids function in the development of eggs and gut. From all fractions lipids were extracted with chlorothe plant embryo. Within the phylum Arthropoda, the occurrence of form-methanol and purified over Sephadex G-25 (Terner cyclopropane fatty acids is restricted to the diplopod et al., 1970). Samples of the purified lipids were separated order Spirostreptida (van der Horst et al., 1972; Zan- by preparative thin-layer chromatography on silicagel G (Merck) into individual lipid classes, using the solvent sysdee et al., 1975). The strict limitation of these acids tems of Freeman & West (1966). Upon spraying with a to female individuals and in addition the accumu- 0'005~o aqueous solution of Rhodamine 6-G, lipid classes lation in the egg yolk material (van der Horst et al., were marked under u.v. light. Phospholipids were re1972) may be indicative of a similar function in the covered from the silica with chloroform-methanol 1:1 development of larval stages. (v/v), and other lipid classes with chloroform. The phospholipids and the triglycerides were saponified One of the accessible starting points to check the validity of this hypothesis is to study the localization with methanolic KOH and fatty acid fractions were isoof cyclopropane acids over the female millipede body, lated. These fatty acid fractions were methylated with diaas one would expect the site of cyclic acid production zomethane as well as the free fatty acid fractions, whereafter pure fatty acid methyl esters were obtained by column to be one particular organ or tissue, for instance the chromatography over silicic acid (Mallinckrodt, 100 mesh), ovary, from which the synthesized cyclic acids are using hexane~liethylether 95:5 (v/v) as the eluens. Fatty transported to the oocytes. acid compositions were determined by gas chromaSupplementary, some indication of the lipid synthe- tography (Oudejans et al., 1971a, b). 227
228
R. C. H. M. OUDEJANS,D. J. VAN DER HORST, F. A. OPMEERAND ~¢'. J. TIIiLEMAN Table 1. Weight and radioactivity of the isolated tissues from five female G. tumulipor,,~ Tissue
fresh wt
i!pids
g
mg
dpm per mg
[~ of radloacti,±ty in total 2ipids
0varies/fat body
13.8
517
11255
:;5~9
Exoskeleton
23.8
416
13661
>~,O
Eggs
13.3
1761
1204
Gut
13.9
153
14170
I~6
30
14085
66.4
2877
Head
Total
13.:
15~ 2.6
100.0
From the distribution of the radioactivity over the lipid classes (Table 2) it is obvious that triglycerides, free fatty acids and hydrocarbons are the most actively synthesized products. Particularly the hydrocarbon fraction is remarkable: in only some 4~o of the total lipid weight over 22~ of the total radiolabel is incorporated. This phenomenon has previously led to studies on the hydrocarbon metabolism (Oudejans, 1972b; Oudejans & Zandee, 1973). Concerning the isolated tissues, in the exoskeleton the labelling of phospholipids and sterols is in accordance with the high turn-over of these structural lipids. The substantial labelling of the hydrocarbons, however, is generated by another phenomenon: the loss of hydrocarbons, due to a continuous abrasion of the epicuticle, requires an intensive production. In the gut triglycerides are hardly present. Probably free fatty acids, resulting from hydrolase activity, are transported to the exoskeleton and particularly to the fat body, where they are esterified to glycerides. The fat body and ovaries are providing the egg with glycerides rather than free fatty acids, as holds for other transport forms of fatty acids (sterol esters: 39-4~,,,). The high radioactivity of hydrocarbons in the fat body--apart from the fatty acid containing lipids--points to a synthesis of these components in this organ as well, though it is widely distant from the site of ultimate use. This organ may be the center for the production of hydrocarbons in behalf of the egg capsules. Valid partial data for the fatty acid composition of the tissues, focused on the cyclopropane fatty acids
Radioactivities of all fractions were measured in tolueneOmnifluor (New England Nuclear) with a Packard liquid scintillation counter, Model 2420. RESULTS
Quantitatively, only two of the female tissues under study are rich in lipids: the ovaries with the fat body o/ (18~ of the total lipids) and the exoskeleton (14. 5/o of the total lipids). An exceptional position is occupied by the eggs: these are extremely rich in lipids (61'2~o of the total lipids) (Table 1). The radioactivity recovered in the lipids is mainly localized in the fat body and the exoskeleton, while the eggs are only relatively low-labelled. In fact, the presence of radiolabel in the egg lipids is due only to an incorporation of freshly synthesized lipid components transported from other organs to immature eggs. Concerning the lipid class composition of these tissues (Table 2), it is apparent that in the exoskeleton structural lipids are major components. Apart from phospholipids and sterols, hydrocarbons are important structures, as these components are indispensable in the epicuticular water balance properties (Oudejans, 1972a, b). In the gut free fatty acids are dominating lipids, probably originating from intestinal lipase activity. The major part of all lipid classes is recovered in the eggs. The egg lipids are responsible for the dominating amount of triglycerides in the total animal (62%).
Table 2. Proportional lipid class distribution and lipid class radioactivity in female G. tumuliporus Tissue
Hydrocarbons
Sterol esters
Triglycerides
I+
II +
I
II
I
Ovaries/ Fat body
14.4
31o2
19.4
10.7
16o 9
40.6
17~I
7~9
Exoskeleton
22,2
45.7
I0~I
24.8
9~9
24.9
23.9
78,2
Eggs
II
Sterols I
II
Free fatty acids
Phospholipids
I
II
l
18~I
50.4
23.9
15~2
15.0
21~9
30.0
46~8
II
53.9
6.4
58.6
59o4
72.2
32~0
31~0
9,2
48.0
4.4
37~6
17,8
Gut
6.1
11.3
9~7
20.7
0.7
1.7
26~6
0
20.1
21o5
5.0
15~9
Head
3.4
5.4
2.2
4.4
0.3
0.8
1.4
4°7
0~8
1.8
3.5
4.3
Total animal
4.1
22.2
2,8
2.5
62,0
43.6
5°0
2.0
11.3
21.2
14.8
8,8
+ Proportional weight (1) and radioactivity
(If)
229
Function of cyclopropane fatty acids Table 3. Distribution of cyclopropane fatty acids and their monoenoic precursors in phospholipid (PL), triglyceride fiG), and free fatty acid (FFA) fractions from female G. tumuliporus Fatty acid
0varies/Fat body
Exoekeleton
TG
TG
PL
FFA
FL
Eggs FFA
TG
PL
Gut FFA
TG
PL
Head FFA
FFA
17:0 cyclopropane
1.8
1.9
3.2
3.2
2.8
3.5
2.2
3.0
4.4
2.8
1.6
4.5
4.8
18:0 cyclopropane
19.0
21.7
24,7
16.0
18.2
21,6
26,6
33,1
22.0
14.6
12.6
29.5
~5~8
19:0 oyclopropane
70.7
6.1
13.5
9.0
6,0
10°5
11.9
14.9
14,1
9~0
3,5
10.7
5.~
total
31.5
29.7
41.4
28.2
27.0
35.6
40.7
51.0
40.5
26.4
17,7
44.7
25.9
16:1
2,1
1.4
2.4
1.6
1.9
1.0
2.1
1.1
1.8
1.1
1.0
1.0
17:1
2.2
1.8
2,5
1.1
1.0
1.7
2.1
1.8
0.4
1o4
0.3
1.4
18:1
16o7
18.5
21,1
15.4
20.3
22.9
19.2
18.3
1.4
5.2
14.0
17.8
3~7
other monoenoic
3.3
1.7
2,5
4.6
3.1
2.9
4.5
1.5
4.3
3.4
5.I
2~7
5,2
total
26.1
26.0
22.8
27.6
28.9
24.8
26.8
4,0
11.3
19.6
25.4
7.7
22.5
52.9
24.0
23.7
32.8
27,1
26.3
27.5
32.8
33.0
58.4
35.1
36.5
36.1
3.2
8.5
2.6
6.8
6.8
8.9
4.6
7.9
14.0
11.2
9.0
8.8
9.2
6.0
10.4
9.5
4.4
9.5
3.1
2.1
3.4
0.1
3.8
13.9
0.9
5.2
Batu~&ted straight-chain saturated
branched-chain
polyenoio
of Graphidostreptus tumuliporus are depicted in Table 3. The data obtained on the sterol ester fractions are not included due to the minute quantities and low radioactivities of their fatty acids. In the head only the amount of purified free fatty acids was sufficient to obtain reliable gas chromatographic data. Since cyclopropane fatty acids are synthesized from monoenoic ones, e.g. 18:1 ~ 19.'0 cyclopropane (van der Horst et al., 1973), the relevant monounsaturated fatty acids are inserted separately. All other fatty acids are ranked according to typical structural features. DISCUSSION
It is obvious that there is not only an impressive biosynthesis of cyclopropane fatty acids in the female body to meet the needs of the eggs, but this holds also for the synthesis of all other lipid structures: the lipid spectrum of the total female organism is largely clouded by that of the eggs (Tables 1 and 2). The distribution of cyclopropane fatty acids appears not to be restricted to one distinct organ: all tissues investigated contain extremely high levels of these cyclic acids, although the major quantum is localized in the egg lipids (Table 3). Two alternative explanations for this unexpected observation may be offered: Firstly, the non-specific occurrence of cyclopropane acids could point to some benefit of these acids in the physiology of the female millipede. This seems less probable, as in juvenile females---which carry no mature eggs--we have never found cyclic acids. Since cyclopropane fatty acids are absent in male animals as well (van der Horst et al., 1972), there is hardly any evidence for the validity of the assumption that cyclopropane fatty acids are required for the survival or environmental physiology of the species. Secondly, the distribution of all three cyclopropane fatty acids over the various organs and tissues of the
adult female diplopod may be attributed to a lack of discrimination between monoenoic and cyclic fatty acids in the female system due to their rather identical physicochemical properties. The fact is that there is an obvious correlation between egg ripening and cyclopropane fatty acid synthesis. Initiation and synchronization of both processes may quite well be regulated by a single factor, most likely a hormonal substance. Once cyclic acids are formed on behalf of the eggs, cyclopropane and monoenoic fatty acids seem to compete anff--on account of their mimic properties ----exchange in the female lipids, so the rather homogenous distribution of cyclic acids in the female animal is not a result of any preference of cyclopropane acids over monoenoic ones. The function of cyclic acids is, therefore, related to the development of the eggs. The cyclopropane fatty acids of the egg yolk triglycerides will serve for membrane lipid synthesis in the early larval stages and may be indispensable to survive the arid environment, in which spirostreptid millipedes are found. In this destructive climate, the maintaining of lipid structures requires a minimum susceptibility to oxygen, which--in this particular case--is an advantage of cyclic acids over monoenoic ones. A second, though less prominent function in eggs may be a protection against harmful penetration of microorganisms, since antimicrobial substances are absent in the egg capsules of millipedes (Eisner et al., 1970). REFERENCES BERVAES J. C. A. M., KUIPER P. J, C. & KYLIN A. (1972)
Conversion of digalactosyl diglyceride (extra long carbon chain conjugates) into monogalactosyl diglyceride of pine needle chloroplasts upon dehardening. Physiologia Pl. 27, 231-235. CULLENJ., PHILLIPSM. C. & SHIPLEYG. G. (1971) The effects of temperature on the composition and physical
230
R.C.H.M.
OUDEJANS,D. J. VAN DER HORST, F. A. OPMEER AND W..|. TIEI.EMAN
properties of the lipids of Pseudomonas fluorescens. Biochem. J. 125, 733-742. EISNER T., ZAHLER S. A., CARREL J. E., BROWN D. J. & LONES G. W. (1970) Absence of antimicrobial substances in the egg capsules of millipedes. Nature, Lond. 225, 661. FrEEMaN C. P. & WEST D. (1966) Complete separation of lipid classes on a single thin-layer plate. J. Lipid Res. 7, 324~327. KUIPER P. J. C. & STUIWR B. (1972) Cyclopropane fatty acids in relation to earliness in spring and drought tolerance in plants. Plt. Physiol., Lancaster 49, 307-309. OUDEJANS R. C. H. M. (1972a) Composition of the saturated hydrocarbons from males, females, and eggs of the millipede, Graphidostreptus tumuliporus. J. Insect Physiol. 18, 857-863. OUDEJANS R. C. H. M. (1972b) Hydrocarbons in the millipede Graphidostreptus tumuliporus (Karsch) (Myriapoda: Diplopoda)--I. In vivo incorporation of 14C-labelled precursors into the hydrocarbon fraction. Comp. Biochem. Physiol. 42B, 15 22. OUDEJANS R. C. H. M., VAN DER HORST D. J. & VAN DON6EN J. P. C. M. (1971b) Isolation and identification of cyclopropane fatty acids from the millipede Graphidostreptus tumuliporus (Karsch) (Myriapoda: Diplopoda). Biochemistry 10, 4938~,941. OUDEJANS R. C. H. M., VAN DER HORST D. J. & ZANDEE D. I. (1971a) Fatty acid composition of the millipede Graphidostreptus tumuliporus (Karsch) (Myriapoda: Diplopoda). Comp. Biochem. Physiol. 40B, 1-6.
OUDEJANS R. C. H. M. & ZANDEE D. I. (19731 The bio,,ynthesis of the hydrocarbons in males and females of the millipede Graphidostreptus tumuliporus. J. l ,scct Ph) siol. 19, 2245-2253. TERNER C., SZABO E. I. & SMITH N. L. {1970) Separation of gangliosides, corticosteroids and water-soluble nonlipids from lipid extracts by Sephadex columns. J. Chromat. 47, 15 19. VAN DER HORST D. J., OUDEJANS R. C. H. M., PI.U¢.; A. G. & VAN HARMELEN H. J. M. (1973) Biosynthesis of cyclopropane fatty acids in the millipede Graphidostreptus tumuliporus (Karsch) (Diplopoda: SpirostreptidaL Comp. Biochem. Physiol. 46B, 395-404. VAN DER HORST D. J., OUDEJANS R C. H. M. & ZA~DEI: D. I. (1972) Occurrence of cyclopropane fatty acids in females and eggs of the millipede Graphidostreptus tumuliporus (Karsch) (Myriapoda: Diplopoda), as contrasted by their absence in the males. Comp. Biochem. Physiol. 41B, 417-423. YANO I., MORRIS L. J., NICHOLS B. W. & JAMESA. T. (1972) The distribution of cyclopropane and cyclopropene fatly acids in higher plants (Malvaceae). Lipids 7, 30- 34. ZANDEE D. 1., VAN DER HORST D. J. & OUDEJANS R. C. H. M. {1975) The occurrence, biosynthesis and physiological significance of cyclopropane fatty acids in some millipedes. Proceedings XXVIth International Congress of Physiological Sciences, New Delhi 1974 (In press).
ON THE FUNCTION OF CYCLOPROPANE FATTY ACIDS IN MILLIPEDES (DIPLOPODA) R. C. H. M. OUDEJANS,D. J. VAN DER HORST, F. A. OPMEER AND W. J. TIELEMAN Laboratory of Chemical Animal Physiology, State University of Utrecht, Padualaan 8, Utrecht, The Netherlands
(Received 27 May 1975) Abstract--1. Localization of cyclopropane fatty acids in the millipede Graphidostreptus tumuliporus is not restricted to the eggs and one particular organ, but bulk amounts are found in all tissues. 2. There is a correlation between the process of egg-ripening and the synthesis of cyclopropane fatty acids. 3. In the lipid structures of the female diplopod, cyclopropane fatty acids are not preferred to monoenoic ones, but are considered to mimic them due to their nearly identical physicochemical properties. 4. The function of cyclopropane fatty acids in millipedes is related to maintaining of the lipid structures of the vulnerable early larval stages in a desiccating arid environment, requiring a minimum susceptibility to oxygen.
INTRODUCTION
sizing capacity of individual organs was obtained through the use of radiolabelled acetate.
T ~ SPECIFICcyclopropane fatty acid formation operative in millipedes leading to an accumulation of cyclic acids (Oudejans et al., 1971a, b; van der Horst MATERIALS AND METHODS et al., 1972, 1973) has stressed the need to obtain an Millipedes of the species Graphidostreptus tumuliporus understanding of the function of cyclopropane fatty (Karsch) (Diplopoda: Spirostreptida) were supplied by the acids in millipede physiology. Institut Fondamental d'Afrique Noire (Dakar, Senegal) by From studies on bacterial lipids it is known that courtesy of Dr. R. Roy. the physicochemical properties of the phospholipids The animals were injected ventrolaterally, each with 10 do not change upon replacement of the monoenoic /~Ci sodium-[1-14C]-acetate (sp. act. 2 mCi/mmole; New aliphatic chains by cyclopropane ones (Cullen et al., England Nuclear). Incubation in a perspex container at 1971). Therefore the obvious preference of cyclic room temperature was ended after 96 hr by freezing the acids, despite their energetically expensive biosynthe- animals at -26°C. Five adult females were selected and dissected. At first sis, suggests some distinct advantage. sight the anatomy of the animal seems rather simple: a The cyclopropane fatty acids in certain plant structural lipids reportedly are involved in drought toler- rigid multisegmental tube with a longitudinal intestinal tract and the remaining cavity occupied by fat body, ovarance and cold hardiness (Bervaes et al., 1972; Kuiper ies, eggs and haemolymph. Individual organs or tissues, & Stuiver, 1972). In seeds and seedlings of mallow however, were not easily to be separated. Ultimately, five species bulk amounts of cyclic fatty acids are de- fractions were isolated: head (including the apodal anterior posited in the storage lipids (Yano et al., 1972). Ob- segments), exoskeleton, fat body (including the ovaries), viously these acids function in the development of eggs and gut. From all fractions lipids were extracted with chlorothe plant embryo. Within the phylum Arthropoda, the occurrence of form-methanol and purified over Sephadex G-25 (Terner cyclopropane fatty acids is restricted to the diplopod et al., 1970). Samples of the purified lipids were separated order Spirostreptida (van der Horst et al., 1972; Zan- by preparative thin-layer chromatography on silicagel G (Merck) into individual lipid classes, using the solvent sysdee et al., 1975). The strict limitation of these acids tems of Freeman & West (1966). Upon spraying with a to female individuals and in addition the accumu- 0'005~o aqueous solution of Rhodamine 6-G, lipid classes lation in the egg yolk material (van der Horst et al., were marked under u.v. light. Phospholipids were re1972) may be indicative of a similar function in the covered from the silica with chloroform-methanol 1:1 development of larval stages. (v/v), and other lipid classes with chloroform. The phospholipids and the triglycerides were saponified One of the accessible starting points to check the validity of this hypothesis is to study the localization with methanolic KOH and fatty acid fractions were isoof cyclopropane acids over the female millipede body, lated. These fatty acid fractions were methylated with diaas one would expect the site of cyclic acid production zomethane as well as the free fatty acid fractions, whereafter pure fatty acid methyl esters were obtained by column to be one particular organ or tissue, for instance the chromatography over silicic acid (Mallinckrodt, 100 mesh), ovary, from which the synthesized cyclic acids are using hexane~liethylether 95:5 (v/v) as the eluens. Fatty transported to the oocytes. acid compositions were determined by gas chromaSupplementary, some indication of the lipid synthe- tography (Oudejans et al., 1971a, b). 227
228
R. C. H. M. OUDEJANS,D. J. VAN DER HORST, F. A. OPMEERAND ~¢'. J. TIIiLEMAN Table 1. Weight and radioactivity of the isolated tissues from five female G. tumulipor,,~ Tissue
fresh wt
i!pids
g
mg
dpm per mg
[~ of radloacti,±ty in total 2ipids
0varies/fat body
13.8
517
11255
:;5~9
Exoskeleton
23.8
416
13661
>~,O
Eggs
13.3
1761
1204
Gut
13.9
153
14170
I~6
30
14085
66.4
2877
Head
Total
13.:
15~ 2.6
100.0
From the distribution of the radioactivity over the lipid classes (Table 2) it is obvious that triglycerides, free fatty acids and hydrocarbons are the most actively synthesized products. Particularly the hydrocarbon fraction is remarkable: in only some 4~o of the total lipid weight over 22~ of the total radiolabel is incorporated. This phenomenon has previously led to studies on the hydrocarbon metabolism (Oudejans, 1972b; Oudejans & Zandee, 1973). Concerning the isolated tissues, in the exoskeleton the labelling of phospholipids and sterols is in accordance with the high turn-over of these structural lipids. The substantial labelling of the hydrocarbons, however, is generated by another phenomenon: the loss of hydrocarbons, due to a continuous abrasion of the epicuticle, requires an intensive production. In the gut triglycerides are hardly present. Probably free fatty acids, resulting from hydrolase activity, are transported to the exoskeleton and particularly to the fat body, where they are esterified to glycerides. The fat body and ovaries are providing the egg with glycerides rather than free fatty acids, as holds for other transport forms of fatty acids (sterol esters: 39-4~,,,). The high radioactivity of hydrocarbons in the fat body--apart from the fatty acid containing lipids--points to a synthesis of these components in this organ as well, though it is widely distant from the site of ultimate use. This organ may be the center for the production of hydrocarbons in behalf of the egg capsules. Valid partial data for the fatty acid composition of the tissues, focused on the cyclopropane fatty acids
Radioactivities of all fractions were measured in tolueneOmnifluor (New England Nuclear) with a Packard liquid scintillation counter, Model 2420. RESULTS
Quantitatively, only two of the female tissues under study are rich in lipids: the ovaries with the fat body o/ (18~ of the total lipids) and the exoskeleton (14. 5/o of the total lipids). An exceptional position is occupied by the eggs: these are extremely rich in lipids (61'2~o of the total lipids) (Table 1). The radioactivity recovered in the lipids is mainly localized in the fat body and the exoskeleton, while the eggs are only relatively low-labelled. In fact, the presence of radiolabel in the egg lipids is due only to an incorporation of freshly synthesized lipid components transported from other organs to immature eggs. Concerning the lipid class composition of these tissues (Table 2), it is apparent that in the exoskeleton structural lipids are major components. Apart from phospholipids and sterols, hydrocarbons are important structures, as these components are indispensable in the epicuticular water balance properties (Oudejans, 1972a, b). In the gut free fatty acids are dominating lipids, probably originating from intestinal lipase activity. The major part of all lipid classes is recovered in the eggs. The egg lipids are responsible for the dominating amount of triglycerides in the total animal (62%).
Table 2. Proportional lipid class distribution and lipid class radioactivity in female G. tumuliporus Tissue
Hydrocarbons
Sterol esters
Triglycerides
I+
II +
I
II
I
Ovaries/ Fat body
14.4
31o2
19.4
10.7
16o 9
40.6
17~I
7~9
Exoskeleton
22,2
45.7
I0~I
24.8
9~9
24.9
23.9
78,2
Eggs
II
Sterols I
II
Free fatty acids
Phospholipids
I
II
l
18~I
50.4
23.9
15~2
15.0
21~9
30.0
46~8
II
53.9
6.4
58.6
59o4
72.2
32~0
31~0
9,2
48.0
4.4
37~6
17,8
Gut
6.1
11.3
9~7
20.7
0.7
1.7
26~6
0
20.1
21o5
5.0
15~9
Head
3.4
5.4
2.2
4.4
0.3
0.8
1.4
4°7
0~8
1.8
3.5
4.3
Total animal
4.1
22.2
2,8
2.5
62,0
43.6
5°0
2.0
11.3
21.2
14.8
8,8
+ Proportional weight (1) and radioactivity
(If)
229
Function of cyclopropane fatty acids Table 3. Distribution of cyclopropane fatty acids and their monoenoic precursors in phospholipid (PL), triglyceride fiG), and free fatty acid (FFA) fractions from female G. tumuliporus Fatty acid
0varies/Fat body
Exoekeleton
TG
TG
PL
FFA
FL
Eggs FFA
TG
PL
Gut FFA
TG
PL
Head FFA
FFA
17:0 cyclopropane
1.8
1.9
3.2
3.2
2.8
3.5
2.2
3.0
4.4
2.8
1.6
4.5
4.8
18:0 cyclopropane
19.0
21.7
24,7
16.0
18.2
21,6
26,6
33,1
22.0
14.6
12.6
29.5
~5~8
19:0 oyclopropane
70.7
6.1
13.5
9.0
6,0
10°5
11.9
14.9
14,1
9~0
3,5
10.7
5.~
total
31.5
29.7
41.4
28.2
27.0
35.6
40.7
51.0
40.5
26.4
17,7
44.7
25.9
16:1
2,1
1.4
2.4
1.6
1.9
1.0
2.1
1.1
1.8
1.1
1.0
1.0
17:1
2.2
1.8
2,5
1.1
1.0
1.7
2.1
1.8
0.4
1o4
0.3
1.4
18:1
16o7
18.5
21,1
15.4
20.3
22.9
19.2
18.3
1.4
5.2
14.0
17.8
3~7
other monoenoic
3.3
1.7
2,5
4.6
3.1
2.9
4.5
1.5
4.3
3.4
5.I
2~7
5,2
total
26.1
26.0
22.8
27.6
28.9
24.8
26.8
4,0
11.3
19.6
25.4
7.7
22.5
52.9
24.0
23.7
32.8
27,1
26.3
27.5
32.8
33.0
58.4
35.1
36.5
36.1
3.2
8.5
2.6
6.8
6.8
8.9
4.6
7.9
14.0
11.2
9.0
8.8
9.2
6.0
10.4
9.5
4.4
9.5
3.1
2.1
3.4
0.1
3.8
13.9
0.9
5.2
Batu~&ted straight-chain saturated
branched-chain
polyenoio
of Graphidostreptus tumuliporus are depicted in Table 3. The data obtained on the sterol ester fractions are not included due to the minute quantities and low radioactivities of their fatty acids. In the head only the amount of purified free fatty acids was sufficient to obtain reliable gas chromatographic data. Since cyclopropane fatty acids are synthesized from monoenoic ones, e.g. 18:1 ~ 19.'0 cyclopropane (van der Horst et al., 1973), the relevant monounsaturated fatty acids are inserted separately. All other fatty acids are ranked according to typical structural features. DISCUSSION
It is obvious that there is not only an impressive biosynthesis of cyclopropane fatty acids in the female body to meet the needs of the eggs, but this holds also for the synthesis of all other lipid structures: the lipid spectrum of the total female organism is largely clouded by that of the eggs (Tables 1 and 2). The distribution of cyclopropane fatty acids appears not to be restricted to one distinct organ: all tissues investigated contain extremely high levels of these cyclic acids, although the major quantum is localized in the egg lipids (Table 3). Two alternative explanations for this unexpected observation may be offered: Firstly, the non-specific occurrence of cyclopropane acids could point to some benefit of these acids in the physiology of the female millipede. This seems less probable, as in juvenile females---which carry no mature eggs--we have never found cyclic acids. Since cyclopropane fatty acids are absent in male animals as well (van der Horst et al., 1972), there is hardly any evidence for the validity of the assumption that cyclopropane fatty acids are required for the survival or environmental physiology of the species. Secondly, the distribution of all three cyclopropane fatty acids over the various organs and tissues of the
adult female diplopod may be attributed to a lack of discrimination between monoenoic and cyclic fatty acids in the female system due to their rather identical physicochemical properties. The fact is that there is an obvious correlation between egg ripening and cyclopropane fatty acid synthesis. Initiation and synchronization of both processes may quite well be regulated by a single factor, most likely a hormonal substance. Once cyclic acids are formed on behalf of the eggs, cyclopropane and monoenoic fatty acids seem to compete anff--on account of their mimic properties ----exchange in the female lipids, so the rather homogenous distribution of cyclic acids in the female animal is not a result of any preference of cyclopropane acids over monoenoic ones. The function of cyclic acids is, therefore, related to the development of the eggs. The cyclopropane fatty acids of the egg yolk triglycerides will serve for membrane lipid synthesis in the early larval stages and may be indispensable to survive the arid environment, in which spirostreptid millipedes are found. In this destructive climate, the maintaining of lipid structures requires a minimum susceptibility to oxygen, which--in this particular case--is an advantage of cyclic acids over monoenoic ones. A second, though less prominent function in eggs may be a protection against harmful penetration of microorganisms, since antimicrobial substances are absent in the egg capsules of millipedes (Eisner et al., 1970). REFERENCES BERVAES J. C. A. M., KUIPER P. J, C. & KYLIN A. (1972)
Conversion of digalactosyl diglyceride (extra long carbon chain conjugates) into monogalactosyl diglyceride of pine needle chloroplasts upon dehardening. Physiologia Pl. 27, 231-235. CULLENJ., PHILLIPSM. C. & SHIPLEYG. G. (1971) The effects of temperature on the composition and physical
230
R.C.H.M.
OUDEJANS,D. J. VAN DER HORST, F. A. OPMEER AND W..|. TIEI.EMAN
properties of the lipids of Pseudomonas fluorescens. Biochem. J. 125, 733-742. EISNER T., ZAHLER S. A., CARREL J. E., BROWN D. J. & LONES G. W. (1970) Absence of antimicrobial substances in the egg capsules of millipedes. Nature, Lond. 225, 661. FrEEMaN C. P. & WEST D. (1966) Complete separation of lipid classes on a single thin-layer plate. J. Lipid Res. 7, 324~327. KUIPER P. J. C. & STUIWR B. (1972) Cyclopropane fatty acids in relation to earliness in spring and drought tolerance in plants. Plt. Physiol., Lancaster 49, 307-309. OUDEJANS R. C. H. M. (1972a) Composition of the saturated hydrocarbons from males, females, and eggs of the millipede, Graphidostreptus tumuliporus. J. Insect Physiol. 18, 857-863. OUDEJANS R. C. H. M. (1972b) Hydrocarbons in the millipede Graphidostreptus tumuliporus (Karsch) (Myriapoda: Diplopoda)--I. In vivo incorporation of 14C-labelled precursors into the hydrocarbon fraction. Comp. Biochem. Physiol. 42B, 15 22. OUDEJANS R. C. H. M., VAN DER HORST D. J. & VAN DON6EN J. P. C. M. (1971b) Isolation and identification of cyclopropane fatty acids from the millipede Graphidostreptus tumuliporus (Karsch) (Myriapoda: Diplopoda). Biochemistry 10, 4938~,941. OUDEJANS R. C. H. M., VAN DER HORST D. J. & ZANDEE D. I. (1971a) Fatty acid composition of the millipede Graphidostreptus tumuliporus (Karsch) (Myriapoda: Diplopoda). Comp. Biochem. Physiol. 40B, 1-6.
OUDEJANS R. C. H. M. & ZANDEE D. I. (19731 The bio,,ynthesis of the hydrocarbons in males and females of the millipede Graphidostreptus tumuliporus. J. l ,scct Ph) siol. 19, 2245-2253. TERNER C., SZABO E. I. & SMITH N. L. {1970) Separation of gangliosides, corticosteroids and water-soluble nonlipids from lipid extracts by Sephadex columns. J. Chromat. 47, 15 19. VAN DER HORST D. J., OUDEJANS R. C. H. M., PI.U¢.; A. G. & VAN HARMELEN H. J. M. (1973) Biosynthesis of cyclopropane fatty acids in the millipede Graphidostreptus tumuliporus (Karsch) (Diplopoda: SpirostreptidaL Comp. Biochem. Physiol. 46B, 395-404. VAN DER HORST D. J., OUDEJANS R C. H. M. & ZA~DEI: D. I. (1972) Occurrence of cyclopropane fatty acids in females and eggs of the millipede Graphidostreptus tumuliporus (Karsch) (Myriapoda: Diplopoda), as contrasted by their absence in the males. Comp. Biochem. Physiol. 41B, 417-423. YANO I., MORRIS L. J., NICHOLS B. W. & JAMESA. T. (1972) The distribution of cyclopropane and cyclopropene fatly acids in higher plants (Malvaceae). Lipids 7, 30- 34. ZANDEE D. 1., VAN DER HORST D. J. & OUDEJANS R. C. H. M. {1975) The occurrence, biosynthesis and physiological significance of cyclopropane fatty acids in some millipedes. Proceedings XXVIth International Congress of Physiological Sciences, New Delhi 1974 (In press).
