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Biochemistry of the development of the insect ceratitis capitata. Changes of the positional distribution of fatty acids in diacylethanolamine phosphoglycerides
BIOCHIMICA
ET BIOPHYSICA
ACTA
527
BBA 55849
BIOCHEMISTRY CAPITATA.
OF THE
DEVELOPMENT
CHANGES OF THE POSITIONAL
ACIDS IN DIACYLETHANOLAMINE
J. M. FERNANDEZ Depavtment
(Received
SOUSA,
of Biochemistry
November
A. M. MUNICIO
Faculty
of Sciences,
OF THE INSECT CERATITIS DISTRIBUTION
OF FATTY
PHOSPHOGLYCERIDES
AND
A. RIBERA
University
of Ma&id,
Madrid
(Spa&)
zoth, rgyo)
SUMMARY
The positional distribution of fatty acids in diacylethanolamine phosphoglycerides from the Dipterous Ceratitis cafiitata has been investigated in the egg, larval, pharate adult and adult stages of development. The patterns of distribution confirm the general rule that in animal phospholipids unsaturated acids are mainly located at the z-position while saturated ones are situated at the I-position. In spite of that some differences in the distribution have been found during metamorphosis. The unsaturated fatty acid 18: 107 is only present in eggs and acylates exclusively at the r-position whereas the isomer 18:rog is incorporated into both positions. A net tendency to increase the unsaturation degree during metamorphosis in ethanolamine phosphoglycerides has been shown.
INTRODUCTION
The positional distribution of fatty acids in various phospholipids has been investigated by several workers making use of the specificity of snake venom phospholipase A1-7. The distribution of fatty acids over the different positions of glycerol in phospholipids is not random. Saturated and polyunsaturated fatty acids occupy determined structural positions in the phospholipids of animalsa. As a general rule saturated acids predominate in position I of animal phospholipids, polyunsaturated acids in position 2. The mono-unsaturated acid distribution appears to be indistinct. Nevertheless, definite relationships between these chemical structures and the biological functionality of phospholipids have not yet been obtained. However, the finding of differences in the composition of the fatty acids of phospholipids of different tissues@and the fact that dietary fatty acids exert changes on the phospholipid fatty acidslylo-13 suggest a certain relationship between chemical structure and particular metabolic events. Behind the obvious dramatic morphological changes that occur during .insect Biochinz.Biophys.
Acta,
231 (1971). 527-534
metamorphosis
are very fundamental
biochemical
processes
obligatorily
dependent
on hormones. Induction of developmental changes implies the acquisition of metamorphic competence by target cells that change the rate of synthesis of some molecules (nucleic
acids, proteins,
phospholipids)
and the membrane
permeability.
The
biochemistry of development and metamorphosis of holometabclous Diptera has been previously studied 14-16 from the points of view of their lipid composition and metabolic regulation. As maturation complexity
and differentiation
one can expect
membrane
of cells is accompanied systems
changes which may be accompanied by concomitant make-up in order to serve newly acquired functions.
by a greater structural
to undergo
extensive
The present paper reports the positional distribution of fatty carbon of the diglyceride moiety of ethanolamine glycerophosphatides developmental
stages of the insect Ce~~atitis capitatn.
MATERIAL
METHODS
AND
structural
alterations in their pbospholipid acids on each from different
Refeference materials Fatty acid methyl esters were obtained
from Applied
Science Lab. (State Col-
lege, Pa., U.S.A.) and Sigma Chemical Co. (St. Louis, MO., U.S.A.). Phosphatidylethanolamine, bran extract, Type V; from Sigma Chemical Co., was purified by thinlayer chromatography on Kieselgel H (Merck) plates using chloroform-methanol--water (65: 25 :4, by vol.) as the solvent system. C~otnl~s adamznltteus venom containing phospholipase A (EC x.1.1.4) was purchased from Koch-Light Lab. Ltd., and washed three times with cold ether in order to remove any lipid contaminant. All solvents were glass-distilled. Rearing
of insects Culturing of Celfatitis
capitata
was carried out at 25" and 70-75%
relative humi-
dity. Eggs were collected at 12-24 h. Larvae were reared 2-3 days before larval-pupal apolysis and were fed on a diet made up of sucrose (174 g), Brewer’s yeast (rr6 g), 1”/0 HCl(r3 ml), bran (275 g), water (7~0 ml), @US Nipagin and Nipasol(1.4 g of each one) at a rate of 4000 larvae per IOO g of diet. Pharate adults were taken 5 days after larval-pupal apolysis. Newly emerged adults were starved for I-Z days and collected. Insects were killed by immersing them in liquid nitrogen immediately after collection and stored under N, at -30’ until required. Further processing of this material was always done within the next 5 days, thereby substantially avoiding a possible alteration of lipids. of lipids Stock material (70 g) was homogenised in water (200 ml) and the homogenate filtered through two leads of muslin. Extraction of lipids was carried out according to the fundamental procedure of BLIGH AKD DYER 17.The lipid extract was washed with one fourth of its volume of 0.73 o/O NaCl solution, and the organic phase separated and dried over sodium sulphate. Total lipids were stored at -30’ in chloroform solution under nitrogen until needed.
Isolatiovz
Biockim. Biophys. Acta, 231
(1971)527-534
POSITZOXAL
DISTRIBUTION
529
OF FATTY ACIDS
li$d classes Separation of polar lipids was accomplished by silicic acid column chromatography (zoo@ mg total lipids in chloroform solution per g column wt.). Elution of neutral lipids was readily accomplished by passing anhydrous chloroform until thin-layer chromatography monitoring of the effiuent gave a negative test. This was followed by methanol which eluted all the remaining polar lipids. Phosphatidylethanolamine is the major lipid of the phospholipid fraction and accounts for so--600/ of it according to the developmental stage of the insectl5. Phosphatidylcholine account for 20--35~/~of the phospholipid fraction. Minor components are phosphatidylserine, pllosphatidyIglycero1, polyglycerol phosphatides, and lyso-derivatives.
fractionationin
PhosphatidylethanohPPzi?zes r-mm-thick Kieselgel H chromatoplates (20 cmx40 cm) were used to isolate phosphatidylethanolamines from polar lipids. 80-roo mg of polar lipids were applied on the plates as a continous band and developed in chloroform-methanol-water (65 : 25 :4, by vol.) as the solvent system. Phosphatidylethanolamines run free of other lipids at approximately half the length of the plate as a homogeneous band, lndentification of phosphatidylethanolamine was done by comparison with a standard sample and through the positive reaction by spraying with the ninhydrin and DITTMER~~ reagents. Bands were located by successive spraying of the latcra.1 sides of the plates with r”,b I, in methanol and the DITTMER reagent. The pl~osphatidyl~thanolam~ne bands were scraped off, and the phos~ho~ipid was recovered from the silicic acid by successive extractions with IO vol. of chlorofor_m-methanol {z : I, I :I and I : g, by vol.) and finally methanol. Phosphatidylethanolamines isolated in this way appeared as a single spot on the two-dimensional system, chloroform-methanol-water (65 : zg : 4, by vol.) and n-butanol-acetic acid-water (60: zo:zo, by vol.). Absence of ethanolamine plasmalogens was confirmed by the method of OWENS”~.
Fatty acid analysis Fatty acid methyl esters from phosphatidylethanoIamines were prepared by the ~ETCALFE~O methanolysis procedure. Analyses of methyl esters were performed on a Perkin-Elmer Fzo FE gas chromatograph equipped with a flame ionization detector. The columns were of stainless steel tubing, jo m long and o.zj mm internal diameter, with a DEGS coating. Other performance details and identification methods were as previously describedIe. The analytical figures quoted represent an average of five samples. Hydrolysis by $hoqbholipase A Phosphatidylethanolamines (20 mg) were dissolved in freshly distilIed ether (5 ml) and to this solution Crotak adamanteus venom (4 mg) in 0.1 M borate buffer (0.5 ml, pH 7.2) and 5 mM CaCI, (0.5 ml) were addedzl. The mixture was vigorously shaken at 28” for 1.10 h in a NBS Gyrotory shaker. The progress of the enzymatic reaction was followed by thin-layer chromatography on micro slides coated with silica gel. The reaction was stopped by the addition of methanol (IO ml) to the reaction Biochinz.Biophys.
Acta,
231 (1971) 527-534
mixture
after which the solvent
was evaporated
and lysophosphatidylethanolamines {z : I, by vol.) plates
and separated
(20 x 20
developing RESULTS
using
cm)
to dryness.
The obtained
by thin-layer
chromatography
chloroform-methanol-water
acias
on 630 p Kieselge!
(6s : 25 : 4: by vol.)
H
as the
system.
AliD
DISCUSSION
The recognition that amino glycerophosphatides participate metabolic functions of biological systems9,“* suggests the existence bution
fatty
were taken into solution by chloroform-met&no!
of fatty
acids on each carbon of the diglyceride
bution will contribute the estabhshment
to the definition
moiety.
of the chemically
of a certain relationship
between
in structural and of selected distri-
This positiona!
distinct molecular
these structures
and
and the develop-
mental stages during the metamorphosis of insects. The overall fatty acid composition of phosphatidylethanolamines lipids from the four developmental
distri-
species
and polar
stages studied in the insect C. cap&a
is recorded
in Table 1. From these data there appears a number of differences in the composition of total fatty acids from phosphatidylethanolamine during the different stages. Paimitoieic
acid accounts
ethanolamines
for the 420/6 of the total
fatty
acids from phosphatidyi-
in eggs, 4-5 times more than in any of the other stages. The percentages
of individual C,, fatty acids in eggs are lower than in the later stages of development, and it is worthy to note the very low levels or the nearly complete absence of pclyunsaturated
C,, (18 : z,18 : 3). Another
characteristic
ethanolamines from eggs is the presence in the other developmental stages.
TABLE
I
FATTY ACID DURING
of the fatty acids of phosphatidyiof low levels of C12and C,,I fatty acids, absent
COMPOSITION
DEVELOPMENT
Fatty acid
OF OF
12:0 12:1 14:0 I#:1
16:o 16:1w7 16:mg 1710
LCW?JlZe PL
PE
PL
I.4
-
-
-
0.7 I.3
-
T8.2
26.7
22.0
42.1 -
35.4
12.3 tr
27.3
24.8
tr -
POLAR
Ad&s
PE
-PE
33.8 8.8 tr
PL
0.79
4.1 tr
5.0
29.4
35.X
24.4
-
17.3 2.7 tr
14.4 I.9
-
tr
2.3
0.76
as (% monoenes/Ioo)
Biochim. Biophys.
(Pi)
PL
0.3 -_
32.3 14.9 0.6
26.1
22.7
10.9
17.3
5-3 tr 30.0 14.2
5.4
5.0
-
0.8
26.3 14.1 2.8
::i
0.2
tr
0.7
1.0
Acta, ‘231 (1971)
0.90
0.57
+~(~/f dienes/roo) 527-534
0.82
C.80
3.’
tr 29.8 20.0 5.9 0.8
29.6 21.3 -
tr 0.6
Unsaturation degree (d /mole) *
LIPIDS
tr
I.0
tr
tr 3.8 7.’
1.9
1x:2
TOTAL
Phavate adults
tr
0.8
tr
6.5
Is:3 20:Cl 20: 1 22:0
tr
tr
2.7
18:ro7 18: 10~9
AND
0.9
“.4 17.2
x8:0
(PE)
PHOSPHATII,YLETHAiXOLAMINE
CU+hZ
-%S PE
* Expressed
c.
0.98
3.4 0.9
tr
1 .oo -
+3(%
trienes/Ioc).
POSITIONAL
DISTRIBUTION
53r
OF FATTY ACIDS
The main characteristic of the distribution of fatty acids in phosphatidylethanolamines from larvae, pharate adults and adults of C. ca&tata is the increase of unsaturated C,, fatty acids while the levels of palmitoleic acid are notably reduced. In terms of the molar percentages of the different unsaturated fatty acids there is a net tendency to increase the unsaturation degree during metamorphosis in both polar lipids and phosphatidylethanolamines (Table I). This unsaturation tendency is clearly shown in the larval ontogenesis and in the development of the pharate adult to its emergence; the initiation of the pharate adult not only does not imply the progress of unsaturation but a minor regression of it. The fatty acids from ethanolamine glycerophosphatides follow the same pattern of relative proportion of unsaturated fatty acids during development as the total fatty acids from polar classes. In this relation palmitoleic acid contributes to the unsaturation only during the development of the pharate adult, whereas unsaturated C,, fatty acids contribute to the net tendency to unsaturation during metamorphosis in both transition eggs-larvae and pharate adult-adult. The increasing levels of 18 :z and 18 :3 during larval ontogenesis counterbalance the percentage decrease of 16 :I clearly shown in this stage. In the evaluation of the different 18:I isomers, it is of interest to note that the isomer 18 :107 is only present in phosphatidylethanolamines from eggs and disappears almost completely in the following stages of development. At present it is not possible to define in precise terms the meaning of the restricted presence of this singular fatty acid in eggs of C. cappitata. Nevertheless, this fact might be related to the recently described observation on the presence of cis-vaccenyl acetate in the ejaculatory bulb of adult male Drosophila melanoga.steF. The selective presence of vaccenic acid in eggs would seem to confirm the suggestion of BRIEGER AND BUTTERWORTHY" on the implication of vaccenyl acetate in some aspect of reproduction. The comparison of the abundance of particular unsaturated fatty acids in ethanolamine glycerophosphatides and in total polar fatty acids is shown in Table II. In it can be seen the change of unsaturated fatty acids in ethanolamine glycerophosphatides in relation to the content of the same particular fatty acid present in the total polar fatty acids along the developmental stages. There are two types of changes of fatty acids according to the number of chain carbon atoms. The relative abundance of 16 :10~7shows the highest level in eggs and much lower ratios in larval, pharate adult and adult stage; the minimum ratio is exhibited by the larval stage. On the contrary, eggs exhibit the minimum ratios in every one of the 18C unsaturated fatty acids and these values are notably increased in the other developmental stages. These data are in agreement with the previous result+ showing that the fatty acid distriTABLE II CHANGES
DURING
DEVELOPMENTAL
PHATIDYLETHANOLAMINES
Fatty
acid
16:x07
STAGES
(PE)
AND
TOTAL
OF
UNSATURATED
POLAR
LIPIDS
FATTY
ACID
RATIO
BETWEEN
PHOS-
(PL)
PEjPL J%W
Larvae
Pharate
adults
Adults
I.2
0.4 -
0.6 -
0.6
18 : 107 r8:mg 18:~
0.8
I.4
1.1
I.0
0.8
I.2
I.0
I.0
18:3
-
I.4
1.7
I.7
0.9
Biochim.
Biophys.
Acta,
231 (1971)
527-534
bution in different
classes of lipids from C. c@tata
is not dependent
on the unsatura-
tion degree of the fatty acids but on the carbon chain length. The distribution of fatty acids in the I- and z-positions
of the ethanolamine
glycerophosphatides
is shown in Figs, XFL~.
from eggs. larvae, pharate
In each developmental Con. The other fatty unsaturated
fatty
stage palmitic
and stearic acids were only found at the I-PO%
acids are distributed
acids amount
adults and ad&s
in both I- and a-positions.
a-linked
poly-
to more than 70 %.
Fig. I. Fatty acid distribution in diacylethanolamine Black bars, position I ; striped bars, position 1.
Fig. 2. Fatty acid distribution in diacylethanolamine Black bars, position I ; striped bars, position 2.
phosphoglycerides
phosphoglycerides
from C.
caphxta
eggs.
from C. capitata larvae.
POSITIONAL
DISTRIBUTION
OF FATTY ACIDS
533
Fig. 3. Fatty acid distribution in diacylethanolamine phosphoglycerides adults. Black bars, position I ; striped bars, position 2.
Fig. 4. Fatty acid distribution in diacylethanolamine Black bars, position I ; striped bars, position 2.
phosphoglycerides
from C. capitata pharate
from C. capitata adults.
This pattern of distribution shown by ethanolamine glycerophosphatides from C. capdata confirms the general rule that in animal phospholipids, unsaturated acids are mainly located at the z-position while saturated ones are situated at the r-position. Nevertheless, some significant differences can be stated with respect to the distribution pattern of unsaturated fatty acids during metamorphosis. The particular fatty acid, 18 : 1~7, acylates exclusively the r-position of phosphatidyl-ethanolamine from eggs, whereas the isomer 18 : 10.19 is incorporated, without apparent discrimination, into Biochim.
Biophys. Acta, 231
(1971) 527-534
both positions and the double bond fatty acid, 18: 2, is asymmetricaEly distributed into the z-position of the glycerol moiety. This pattern of distribution exhibited by eggs is not maintained throcgh the other studied developmental stages. Thus, the similarity of d~st~ib~t~o~lshown by 18 : rwg in eggs is changed in larvae, pharate ad&s and adults in which the z-position of phosphatidyl-ethanolamines contains most of this acid; on the contrary, the exclusi.ve presence of 18 : 2 in the z-position in eggs is not kept in the following stages of deve!opment of the insect in which this fatty acid is shared by both positions. ACKNOWLEDGEMENT
This work was supported by a gran t from the “Fond0 Naciona3 para ei DesajrrolIo de la lnvestigacicin Cientifica”. REFERENCES L. M. G. VAN GOLDEUZD L, L.M.V~~H DEENEPI‘,B~oc~z~~z. Biophys. Acta, 1%~ (1966) 496. L. Xi. G. VAX GOLDE, V. TOMASI AND L. L. M. VAN DEENEP~, Chem. Pbzys. Lipids, I (1907) ~82. H. RROCKERHOFB, R. J. HOYLE AKD P. C. H~ANG, Biochim. Bici-phys. Acta, x44 (1967) 541. KANKUR~ AND S. XUJIXA, J.BiocWe% Tokyo, 61 (1967) 732. 4 I-1. ORUYAXA, 'I?. I
2
L. ;?x. G. VAN GOLDE, W. A. PIETERSOX AND L. L. M. VAX DEIX-EX:B~OCB~~?Z.Gio_Phys.;I&, 152 (1968) 84. 6 A. KUKSIS W. C. BECKENRIDGE, L.~~'uAI ASD 0. STBCHNYK, J. Lipid, Res. 10(196g) 25. 7 L. MARAI AND A. KUKSIS, J. Lipid Res., 10 (1969) 141. s H. BROCKEXHOPF AND R. G. ACKMAN, J.Lipid Res., 8 (r967) 6Gr. Vol. 3. BE3A Librarp, E!sevier,Amster9 G. x3. ilxsxi~ .4ND J. N. Hs-~+~THoRNE,~~~ff~~~o~,~~~~s, dam, 1964. F. D. COLLINS, B&hem. J,, 88 (1966) 319. IO H. W.SPRECWER,I?I.J.DUTTON,F.D.GUNSTONE,P.T.SYKES AXD R.T. HoLMAs,L~$~~s,z II I2
(rg67) 122. J.Irwoco, S.M.
HO~KIXS,D.J.MCTYTOSH,H.SHFEHANAX~U
K.L.LYMxN,L~;~~~~,z
(1967)
479.
‘3 J, A. BALIXT, D. A. BEELER, D. H. TREBLE A?;D H. L. SPITZER, J. Lipid Res., 8 (1967) 486. Pkysiol., 36 (1970) 27~. I‘+ M. A. MADARIAGA, A. M. Mu~xcro AS\TDA. RIBERA, Gov+. B&hem 15 M. P. CASTILL&, R. E. CSTAI,_&N,A. &I.b~WNIcI0 .&ND A. SUBREZ, Cfl?@.$.BiOCb%.PhysiOl.,38
(I971) 109. 16 A.M.blr-s~cro, J_M_ODRIO~OL~ASD A.PIAEIRo,Go~~.B~ocJZT~~. Physiol., 37 (1970) 3817. 17 E. G. BLIGN ANI~ W. J. DYER, Can. _i.B&hem. Physiol., 37 (1955)) 9r1. IS J, C. DITTMER, $7"Lipzd Ales., j (1964) r26. 19 K. OWENS,~~~~~~~TFZ.J., TOO (1966) 354. 20 i. E. METCBLFE AND A. A. SCHMITZ, AnuZ. Cfze??&, 33 (rg6r) 363. ZI L. M. G. Vais GOLDE AND L. L. 161. VrxN DEESEN, Chem. Plzys.Lipids1 I (1967) 157. PFogfess illi i'he 22 L. L. 41. VAX BEEKEN, in R. T. HOL~US, P~~s~~o~~~~ds in ~,~o~~e$~~~~~~s, Chwwist~y of Fats a& OBer Lipids, Vol. 8, Pergamon Press,London, 1965. 23 C. BRIEGER AND F. M. BUTTERWORTII, Scieuzce, i67('970) 1262. Biochim.
Bio@ys.
Acta,
232 (1971)
527-531
ET BIOPHYSICA
ACTA
527
BBA 55849
BIOCHEMISTRY CAPITATA.
OF THE
DEVELOPMENT
CHANGES OF THE POSITIONAL
ACIDS IN DIACYLETHANOLAMINE
J. M. FERNANDEZ Depavtment
(Received
SOUSA,
of Biochemistry
November
A. M. MUNICIO
Faculty
of Sciences,
OF THE INSECT CERATITIS DISTRIBUTION
OF FATTY
PHOSPHOGLYCERIDES
AND
A. RIBERA
University
of Ma&id,
Madrid
(Spa&)
zoth, rgyo)
SUMMARY
The positional distribution of fatty acids in diacylethanolamine phosphoglycerides from the Dipterous Ceratitis cafiitata has been investigated in the egg, larval, pharate adult and adult stages of development. The patterns of distribution confirm the general rule that in animal phospholipids unsaturated acids are mainly located at the z-position while saturated ones are situated at the I-position. In spite of that some differences in the distribution have been found during metamorphosis. The unsaturated fatty acid 18: 107 is only present in eggs and acylates exclusively at the r-position whereas the isomer 18:rog is incorporated into both positions. A net tendency to increase the unsaturation degree during metamorphosis in ethanolamine phosphoglycerides has been shown.
INTRODUCTION
The positional distribution of fatty acids in various phospholipids has been investigated by several workers making use of the specificity of snake venom phospholipase A1-7. The distribution of fatty acids over the different positions of glycerol in phospholipids is not random. Saturated and polyunsaturated fatty acids occupy determined structural positions in the phospholipids of animalsa. As a general rule saturated acids predominate in position I of animal phospholipids, polyunsaturated acids in position 2. The mono-unsaturated acid distribution appears to be indistinct. Nevertheless, definite relationships between these chemical structures and the biological functionality of phospholipids have not yet been obtained. However, the finding of differences in the composition of the fatty acids of phospholipids of different tissues@and the fact that dietary fatty acids exert changes on the phospholipid fatty acidslylo-13 suggest a certain relationship between chemical structure and particular metabolic events. Behind the obvious dramatic morphological changes that occur during .insect Biochinz.Biophys.
Acta,
231 (1971). 527-534
metamorphosis
are very fundamental
biochemical
processes
obligatorily
dependent
on hormones. Induction of developmental changes implies the acquisition of metamorphic competence by target cells that change the rate of synthesis of some molecules (nucleic
acids, proteins,
phospholipids)
and the membrane
permeability.
The
biochemistry of development and metamorphosis of holometabclous Diptera has been previously studied 14-16 from the points of view of their lipid composition and metabolic regulation. As maturation complexity
and differentiation
one can expect
membrane
of cells is accompanied systems
changes which may be accompanied by concomitant make-up in order to serve newly acquired functions.
by a greater structural
to undergo
extensive
The present paper reports the positional distribution of fatty carbon of the diglyceride moiety of ethanolamine glycerophosphatides developmental
stages of the insect Ce~~atitis capitatn.
MATERIAL
METHODS
AND
structural
alterations in their pbospholipid acids on each from different
Refeference materials Fatty acid methyl esters were obtained
from Applied
Science Lab. (State Col-
lege, Pa., U.S.A.) and Sigma Chemical Co. (St. Louis, MO., U.S.A.). Phosphatidylethanolamine, bran extract, Type V; from Sigma Chemical Co., was purified by thinlayer chromatography on Kieselgel H (Merck) plates using chloroform-methanol--water (65: 25 :4, by vol.) as the solvent system. C~otnl~s adamznltteus venom containing phospholipase A (EC x.1.1.4) was purchased from Koch-Light Lab. Ltd., and washed three times with cold ether in order to remove any lipid contaminant. All solvents were glass-distilled. Rearing
of insects Culturing of Celfatitis
capitata
was carried out at 25" and 70-75%
relative humi-
dity. Eggs were collected at 12-24 h. Larvae were reared 2-3 days before larval-pupal apolysis and were fed on a diet made up of sucrose (174 g), Brewer’s yeast (rr6 g), 1”/0 HCl(r3 ml), bran (275 g), water (7~0 ml), @US Nipagin and Nipasol(1.4 g of each one) at a rate of 4000 larvae per IOO g of diet. Pharate adults were taken 5 days after larval-pupal apolysis. Newly emerged adults were starved for I-Z days and collected. Insects were killed by immersing them in liquid nitrogen immediately after collection and stored under N, at -30’ until required. Further processing of this material was always done within the next 5 days, thereby substantially avoiding a possible alteration of lipids. of lipids Stock material (70 g) was homogenised in water (200 ml) and the homogenate filtered through two leads of muslin. Extraction of lipids was carried out according to the fundamental procedure of BLIGH AKD DYER 17.The lipid extract was washed with one fourth of its volume of 0.73 o/O NaCl solution, and the organic phase separated and dried over sodium sulphate. Total lipids were stored at -30’ in chloroform solution under nitrogen until needed.
Isolatiovz
Biockim. Biophys. Acta, 231
(1971)527-534
POSITZOXAL
DISTRIBUTION
529
OF FATTY ACIDS
li$d classes Separation of polar lipids was accomplished by silicic acid column chromatography (zoo@ mg total lipids in chloroform solution per g column wt.). Elution of neutral lipids was readily accomplished by passing anhydrous chloroform until thin-layer chromatography monitoring of the effiuent gave a negative test. This was followed by methanol which eluted all the remaining polar lipids. Phosphatidylethanolamine is the major lipid of the phospholipid fraction and accounts for so--600/ of it according to the developmental stage of the insectl5. Phosphatidylcholine account for 20--35~/~of the phospholipid fraction. Minor components are phosphatidylserine, pllosphatidyIglycero1, polyglycerol phosphatides, and lyso-derivatives.
fractionationin
PhosphatidylethanohPPzi?zes r-mm-thick Kieselgel H chromatoplates (20 cmx40 cm) were used to isolate phosphatidylethanolamines from polar lipids. 80-roo mg of polar lipids were applied on the plates as a continous band and developed in chloroform-methanol-water (65 : 25 :4, by vol.) as the solvent system. Phosphatidylethanolamines run free of other lipids at approximately half the length of the plate as a homogeneous band, lndentification of phosphatidylethanolamine was done by comparison with a standard sample and through the positive reaction by spraying with the ninhydrin and DITTMER~~ reagents. Bands were located by successive spraying of the latcra.1 sides of the plates with r”,b I, in methanol and the DITTMER reagent. The pl~osphatidyl~thanolam~ne bands were scraped off, and the phos~ho~ipid was recovered from the silicic acid by successive extractions with IO vol. of chlorofor_m-methanol {z : I, I :I and I : g, by vol.) and finally methanol. Phosphatidylethanolamines isolated in this way appeared as a single spot on the two-dimensional system, chloroform-methanol-water (65 : zg : 4, by vol.) and n-butanol-acetic acid-water (60: zo:zo, by vol.). Absence of ethanolamine plasmalogens was confirmed by the method of OWENS”~.
Fatty acid analysis Fatty acid methyl esters from phosphatidylethanoIamines were prepared by the ~ETCALFE~O methanolysis procedure. Analyses of methyl esters were performed on a Perkin-Elmer Fzo FE gas chromatograph equipped with a flame ionization detector. The columns were of stainless steel tubing, jo m long and o.zj mm internal diameter, with a DEGS coating. Other performance details and identification methods were as previously describedIe. The analytical figures quoted represent an average of five samples. Hydrolysis by $hoqbholipase A Phosphatidylethanolamines (20 mg) were dissolved in freshly distilIed ether (5 ml) and to this solution Crotak adamanteus venom (4 mg) in 0.1 M borate buffer (0.5 ml, pH 7.2) and 5 mM CaCI, (0.5 ml) were addedzl. The mixture was vigorously shaken at 28” for 1.10 h in a NBS Gyrotory shaker. The progress of the enzymatic reaction was followed by thin-layer chromatography on micro slides coated with silica gel. The reaction was stopped by the addition of methanol (IO ml) to the reaction Biochinz.Biophys.
Acta,
231 (1971) 527-534
mixture
after which the solvent
was evaporated
and lysophosphatidylethanolamines {z : I, by vol.) plates
and separated
(20 x 20
developing RESULTS
using
cm)
to dryness.
The obtained
by thin-layer
chromatography
chloroform-methanol-water
acias
on 630 p Kieselge!
(6s : 25 : 4: by vol.)
H
as the
system.
AliD
DISCUSSION
The recognition that amino glycerophosphatides participate metabolic functions of biological systems9,“* suggests the existence bution
fatty
were taken into solution by chloroform-met&no!
of fatty
acids on each carbon of the diglyceride
bution will contribute the estabhshment
to the definition
moiety.
of the chemically
of a certain relationship
between
in structural and of selected distri-
This positiona!
distinct molecular
these structures
and
and the develop-
mental stages during the metamorphosis of insects. The overall fatty acid composition of phosphatidylethanolamines lipids from the four developmental
distri-
species
and polar
stages studied in the insect C. cap&a
is recorded
in Table 1. From these data there appears a number of differences in the composition of total fatty acids from phosphatidylethanolamine during the different stages. Paimitoieic
acid accounts
ethanolamines
for the 420/6 of the total
fatty
acids from phosphatidyi-
in eggs, 4-5 times more than in any of the other stages. The percentages
of individual C,, fatty acids in eggs are lower than in the later stages of development, and it is worthy to note the very low levels or the nearly complete absence of pclyunsaturated
C,, (18 : z,18 : 3). Another
characteristic
ethanolamines from eggs is the presence in the other developmental stages.
TABLE
I
FATTY ACID DURING
of the fatty acids of phosphatidyiof low levels of C12and C,,I fatty acids, absent
COMPOSITION
DEVELOPMENT
Fatty acid
OF OF
12:0 12:1 14:0 I#:1
16:o 16:1w7 16:mg 1710
LCW?JlZe PL
PE
PL
I.4
-
-
-
0.7 I.3
-
T8.2
26.7
22.0
42.1 -
35.4
12.3 tr
27.3
24.8
tr -
POLAR
Ad&s
PE
-PE
33.8 8.8 tr
PL
0.79
4.1 tr
5.0
29.4
35.X
24.4
-
17.3 2.7 tr
14.4 I.9
-
tr
2.3
0.76
as (% monoenes/Ioo)
Biochim. Biophys.
(Pi)
PL
0.3 -_
32.3 14.9 0.6
26.1
22.7
10.9
17.3
5-3 tr 30.0 14.2
5.4
5.0
-
0.8
26.3 14.1 2.8
::i
0.2
tr
0.7
1.0
Acta, ‘231 (1971)
0.90
0.57
+~(~/f dienes/roo) 527-534
0.82
C.80
3.’
tr 29.8 20.0 5.9 0.8
29.6 21.3 -
tr 0.6
Unsaturation degree (d /mole) *
LIPIDS
tr
I.0
tr
tr 3.8 7.’
1.9
1x:2
TOTAL
Phavate adults
tr
0.8
tr
6.5
Is:3 20:Cl 20: 1 22:0
tr
tr
2.7
18:ro7 18: 10~9
AND
0.9
“.4 17.2
x8:0
(PE)
PHOSPHATII,YLETHAiXOLAMINE
CU+hZ
-%S PE
* Expressed
c.
0.98
3.4 0.9
tr
1 .oo -
+3(%
trienes/Ioc).
POSITIONAL
DISTRIBUTION
53r
OF FATTY ACIDS
The main characteristic of the distribution of fatty acids in phosphatidylethanolamines from larvae, pharate adults and adults of C. ca&tata is the increase of unsaturated C,, fatty acids while the levels of palmitoleic acid are notably reduced. In terms of the molar percentages of the different unsaturated fatty acids there is a net tendency to increase the unsaturation degree during metamorphosis in both polar lipids and phosphatidylethanolamines (Table I). This unsaturation tendency is clearly shown in the larval ontogenesis and in the development of the pharate adult to its emergence; the initiation of the pharate adult not only does not imply the progress of unsaturation but a minor regression of it. The fatty acids from ethanolamine glycerophosphatides follow the same pattern of relative proportion of unsaturated fatty acids during development as the total fatty acids from polar classes. In this relation palmitoleic acid contributes to the unsaturation only during the development of the pharate adult, whereas unsaturated C,, fatty acids contribute to the net tendency to unsaturation during metamorphosis in both transition eggs-larvae and pharate adult-adult. The increasing levels of 18 :z and 18 :3 during larval ontogenesis counterbalance the percentage decrease of 16 :I clearly shown in this stage. In the evaluation of the different 18:I isomers, it is of interest to note that the isomer 18 :107 is only present in phosphatidylethanolamines from eggs and disappears almost completely in the following stages of development. At present it is not possible to define in precise terms the meaning of the restricted presence of this singular fatty acid in eggs of C. cappitata. Nevertheless, this fact might be related to the recently described observation on the presence of cis-vaccenyl acetate in the ejaculatory bulb of adult male Drosophila melanoga.steF. The selective presence of vaccenic acid in eggs would seem to confirm the suggestion of BRIEGER AND BUTTERWORTHY" on the implication of vaccenyl acetate in some aspect of reproduction. The comparison of the abundance of particular unsaturated fatty acids in ethanolamine glycerophosphatides and in total polar fatty acids is shown in Table II. In it can be seen the change of unsaturated fatty acids in ethanolamine glycerophosphatides in relation to the content of the same particular fatty acid present in the total polar fatty acids along the developmental stages. There are two types of changes of fatty acids according to the number of chain carbon atoms. The relative abundance of 16 :10~7shows the highest level in eggs and much lower ratios in larval, pharate adult and adult stage; the minimum ratio is exhibited by the larval stage. On the contrary, eggs exhibit the minimum ratios in every one of the 18C unsaturated fatty acids and these values are notably increased in the other developmental stages. These data are in agreement with the previous result+ showing that the fatty acid distriTABLE II CHANGES
DURING
DEVELOPMENTAL
PHATIDYLETHANOLAMINES
Fatty
acid
16:x07
STAGES
(PE)
AND
TOTAL
OF
UNSATURATED
POLAR
LIPIDS
FATTY
ACID
RATIO
BETWEEN
PHOS-
(PL)
PEjPL J%W
Larvae
Pharate
adults
Adults
I.2
0.4 -
0.6 -
0.6
18 : 107 r8:mg 18:~
0.8
I.4
1.1
I.0
0.8
I.2
I.0
I.0
18:3
-
I.4
1.7
I.7
0.9
Biochim.
Biophys.
Acta,
231 (1971)
527-534
bution in different
classes of lipids from C. c@tata
is not dependent
on the unsatura-
tion degree of the fatty acids but on the carbon chain length. The distribution of fatty acids in the I- and z-positions
of the ethanolamine
glycerophosphatides
is shown in Figs, XFL~.
from eggs. larvae, pharate
In each developmental Con. The other fatty unsaturated
fatty
stage palmitic
and stearic acids were only found at the I-PO%
acids are distributed
acids amount
adults and ad&s
in both I- and a-positions.
a-linked
poly-
to more than 70 %.
Fig. I. Fatty acid distribution in diacylethanolamine Black bars, position I ; striped bars, position 1.
Fig. 2. Fatty acid distribution in diacylethanolamine Black bars, position I ; striped bars, position 2.
phosphoglycerides
phosphoglycerides
from C.
caphxta
eggs.
from C. capitata larvae.
POSITIONAL
DISTRIBUTION
OF FATTY ACIDS
533
Fig. 3. Fatty acid distribution in diacylethanolamine phosphoglycerides adults. Black bars, position I ; striped bars, position 2.
Fig. 4. Fatty acid distribution in diacylethanolamine Black bars, position I ; striped bars, position 2.
phosphoglycerides
from C. capitata pharate
from C. capitata adults.
This pattern of distribution shown by ethanolamine glycerophosphatides from C. capdata confirms the general rule that in animal phospholipids, unsaturated acids are mainly located at the z-position while saturated ones are situated at the r-position. Nevertheless, some significant differences can be stated with respect to the distribution pattern of unsaturated fatty acids during metamorphosis. The particular fatty acid, 18 : 1~7, acylates exclusively the r-position of phosphatidyl-ethanolamine from eggs, whereas the isomer 18 : 10.19 is incorporated, without apparent discrimination, into Biochim.
Biophys. Acta, 231
(1971) 527-534
both positions and the double bond fatty acid, 18: 2, is asymmetricaEly distributed into the z-position of the glycerol moiety. This pattern of distribution exhibited by eggs is not maintained throcgh the other studied developmental stages. Thus, the similarity of d~st~ib~t~o~lshown by 18 : rwg in eggs is changed in larvae, pharate ad&s and adults in which the z-position of phosphatidyl-ethanolamines contains most of this acid; on the contrary, the exclusi.ve presence of 18 : 2 in the z-position in eggs is not kept in the following stages of deve!opment of the insect in which this fatty acid is shared by both positions. ACKNOWLEDGEMENT
This work was supported by a gran t from the “Fond0 Naciona3 para ei DesajrrolIo de la lnvestigacicin Cientifica”. REFERENCES L. M. G. VAN GOLDEUZD L, L.M.V~~H DEENEPI‘,B~oc~z~~z. Biophys. Acta, 1%~ (1966) 496. L. Xi. G. VAX GOLDE, V. TOMASI AND L. L. M. VAN DEENEP~, Chem. Pbzys. Lipids, I (1907) ~82. H. RROCKERHOFB, R. J. HOYLE AKD P. C. H~ANG, Biochim. Bici-phys. Acta, x44 (1967) 541. KANKUR~ AND S. XUJIXA, J.BiocWe% Tokyo, 61 (1967) 732. 4 I-1. ORUYAXA, 'I?. I
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Acta,
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527-531