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Fatty acid composition of Turbatrix aceti and its culture medium
Comp. Bioehem. Physiol., 1972, Vol. 41B, pp. 89 to 98. Pergamon Press. Printed in Great Britain
FATTY ACID C O M P O S I T I O N OF T U R B A T R I X A C E T I A N D ITS CULTURE MEDIUM* L. R. K R U S B E R G Department of Botany, University of Maryland, College Park, Maryland 20742
(Received 21 June 1971) Abstract--1. Turbatrix aceti propagated axenieally at 30°C contained about 24 per cent lipid on a dry weight basis of which slightly over one-half was fatty acids. 2. Of the 31 fatty acids identified oleie plus vaccenic acids, present in equal quantities, accounted for 25-34 per cent of the total fatty acids, and stearie plus linoleic accounted for another 24 per cent. 3. About 65 per cent of the total amount of fatty acids was unsaturated. 4. Beef liver extract in the medium was the major source of dietary fatty acids of which only two, 16:1c°7 and 18:3 ~°s, were present in sufficient quantities to account for the amounts of these fatty acids occurring in the nematodes; the major fatty acids of liver extract were palmitic, stearie, oleic and linoleic. INTRODUCTION THE TOTAL lipid and fatty acid contents of several nematodes have been examined (Beames, 1965; Sivapalan & Jenkins, 1966; Krusberg, 1967). M a n y of the same fatty acids occur in the various free-living and plant-parasitic nematodes investigated thus far, although the total and relative amounts of individual fatty acids may differ widely among nematode species. In only one study (Krusberg, 1967) were the kinds of unsaturated fatty acids extracted from nematodes determined. For metabolic studies it is desirable to use a nematode which can be propagated easily and rapidly in relatively large quantities axenically on defined medium. The propagation of the vinegar eelworm, Turbatrix aceti (Mueller) Peters, meets these requirements except that the culture medium is undefined. The kinds and quantities of particular metabolites in the medium can be determined, however, so that any changes will be due to only the nematodes and not to other organisms. Panagrellus redivivus can be propagated similarly, although for the lipid studies of Sivapalan & Jenkins (1966) this nematode was reared with microorganisms. None of the nematodes investigated by Krusberg (1967) have yet been propagated axenically. With the interest in lipid metabolism in T. aceti it was decided that the major fatty acids in this nematode should be identified and their relative quantities * Scientific Article No. A1700, Contribution No. 4461 of the Maryland Agricultural Experiment Station. This investigation was supported in part by U.S. Public Health Service Grant 5 RO1 AI-03808. 89
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measured. T h e fatty acid content of the m e d i u m components before nematode propagation and the m e d i u m after nematode propagation were also investigated in order to determine any possible contribution of m e d i u m fatty acids to nematode fatty acids. W i t h these data it should then be possible to p e r f o r m comparative studies of fatty acid metabolism in T. aceti and certain plant-parasitic nematodes. MATERIALS AND M E T H O D S T. aceti was propagated on the medium described previously (Cole & Krusberg, 1968) for 21 days at 30°C. Nematodes were washed until free of medium components by repeated cycles of centrifugation at 300 g and suspension in chilled distilled water. Nematodes suspended in 10 ml of distilled water were then disrupted by a single pass through a French pressure cell, the homogenate was frozen and then lyophilized. All calculations were subsequently based on the weight (200-500 mg) of batches of lyophilized nematode homogenate. Total lipid from lyophilized nematode homogenate was extracted after the method of Folch et al. (1957). The total lipid was saponified (Cole & Krusberg, 1968), the fatty acids extracted and methylated with BC13-methanol reagent (Applied Science Lab., State College, Pa.). When interest was solely with fatty acids (as with certain harvests of nematodes, lyophilized beef liver extract, spent medium, or the soy peptone + yeast extract) the material was saponified directly, and the fatty acids then extracted and methylated. Fatty acid methyl esters were passed through a column of Woelm acid alumina, grade 1, deactivated with 1"5% water (Alupharm Chemicals, New Orleans, La.), using hexane followed by hexane: benzene (1 : 1, v/v) as the eluting solvents. The methods used to identify and quantitatively measure fatty acid esters were essentially those used previously (Krusberg, 1967). Both analytical and preparative gas-liquid chromatography (GLC) of fatty acid esters were with stabilized diethylene glycol succinate (DEGS, Analabs, Inc., North Haven, Conn.) or SE-30 silicone gum (Applied Science Lab.). Individual fatty acid esters collected by GLC were finally purified by passing through a column of Woelm alumina as described earlier. Methods used for thin-layer chromatography (AgNOs-silica gel TLC), hydrogenation of fatty acid esters, and locating double bond positions in unsaturated fatty acid esters were described previously (Krusberg, 1967). Infrarared spectra of samples in 1"5 mm KBr pellets were obtained with a Perkin-Elmer Model 257 Grating Spectrophotometer equipped with a 4X beam condenser. Mass spectra were obtained with an LKB Model 9000 Mass Spectrometer. Nuclear magnetic resonance (NMR) spectra were obtained with a Varian A-60A NMR Spectrometer. RESULTS T h e total lipid content of T. aceti ranged f r o m 20.2 to 28.7 per cent on a dry weight basis, averaging 24-0 per cent for nematodes f r o m the various harvests. T o t a l fatty acids accounted for 11-0-16.8 per cent of the dry weight of nematodes, averaging 14.0 per cent or slightly over one-half of the total lipid. A G L C c h r o m a t o g r a m of the fatty acid esters from T. aceti is seen in Fig. 1. Examination of the c h r o m a t o g r a m reveals that the major portion of the fatty acids in this nematode contain eighteen to twenty carbons and that unsaturated fatty acids clearly predominate over saturated fatty acids. T h e 10- to 17-carbon fatty acids were all saturated with the exception of some 16:1. T h e fatty acids iso-15:0 and iso-17:0 were isolated and their structures confirmed b y infrared and mass spectrometer analyses. T h e N M R spectrum for iso-15:0 confirmed the presence
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of the terminal gem-dimethyl configuration. The fatty acids iso-11:0 and iso-13:0 were only tentatively identified since they occurred in such small amounts as to be difficult to isolate in quantities sufficient for further analyses. These assignments were made because: (1) iso-11:0 and iso-13:0 had the same relationship on GLC to 10:0 and 12:0 as did iso-15:0 and iso-17:0 to 14:0 and 16:0, respectively, (2) on AgNO3-silica gel T L C they moved with the saturated fatty acid esters, and (3) they were not affected by hydrogenation. Iso-14:0, iso-16:0 and iso-18:0 were identified on the basis of their relationships to 14:0, 16:0 and 18:0, respectively, on GLC and comparison of G L C retention characteristics with authentic iso fatty acid methyl esters purchased from (Applied Science Lab.). These iso acid esters also moved with the saturated fatty acid esters on AgNO3-silica gel T L C and were unaffected by hydrogenation. Several additional minor fatty acids were noted in preparations from T. aceti especially when large quantities of fatty acid ester fractions separated by AgNO3silica gel T L C were passed through the gas chromatograph. No attempts were made to identify these fatty acids. A total of 31 fatty acids were identified in extracts of T. aceti (Table 1). About 34 per cent of the total fatty acids in this nematode were saturated, with 16:0 and 18:0 predominating. From 29 to 36 per cent of the total fatty acids were monounsaturated, with the two isomers of 18:1 accounting for 91 per cent of the monounsaturates. The 18:1 fraction was composed of equal quantities of oleic and cis-vaecenic acids with a trace of the a13 form present; the combined fraction made up around 2-5 per cent of the dry weight of nematodes. The 16:1 fraction contained 85 per cent the a9 form and 15 per cent the a l l form, whereas 20:1 contained 2 per cent the a9 form, 24 per cent the al 1 form and 74 per cent the a13 form. Polyunsaturated fatty acids accounted for 26 to 39 per cent of the total nematode fatty acids, of which almost one-third was linoleic acid. Only single forms of 18:2, 20:2, 20:3 and 20:5 were detected. 7-Linolenic acid (18:3 ~G) accounted for 85 per cent and a-linolenic acid (18:3 ~8) for 15 per cent of the total 18:3 occurring in T. aceti, whereas arachidonic acid and its isomer 8,11,14,17-eicosatetraenoic acid were present in about equal amounts. Using authentic standards (Hormel Institute, Austin, Minn.) it was found that y-18:3 would elute on GLC (DEGS column) just before ~-18:3, just as 20:4 ~6 elutes before 20:4 ~3. The nematode 18:3 fraction from AgNOa-silica gel T L C also separated on GLC into two components corresponding to ~-18:3 and a-18:3. Peak areas indicated relative amounts to be 85 and 15 per cent, respectively, the same as was determined by analysis of the oxidative degradation products of the combined 18:3 fraction. Unsaturated fatty acids from nematodes harvested 18 months apart contained the same percentage composition of isomers. The fatty acid composition of the beef liver extract and soy peptone + yeast extract, the primary constituents of the culture medium for T. aceti, is seen in Table 2. Three different preparations of liver extract were analyzed and the positions of the double bonds in the unsaturated fatty acids isolated were determined. The total amounts of liver extract processed were from 60 to 200 ml, although the
FATTYACIDCOMPOSITIONOF TURBATRIX ACETI
93
TABLE 1--FATTY ACIDSOF T. aceti (mg/g dry wt.) Fatty acid
Experiment 1 * mg
10:0 iso-11:0
12:0 iso-13:0 iso-14:0
14:0 iso-15:0
15:0 iso-16:0
16:0 iso-17:0
17:0 iso-18:0
18:0 20:0 16:1 ~v ~5 18:1 ~9 ~ ~0s 20:1 ~n ~8 ~0~ 18:2°~ 20:2°A 18:3°~ ~n 20:3 °~ 20:4¢°e ~o3 20:5 °~n Total
% of total
Trace Trace 0"11 0"03 Trace 2'20 5"59 Trace Trace 8"39 2"50 Trace 3.58 16.54 0.55 0.26 0"05 15"81 15"81 Trace 0"06 0"72 2"24 12"31 1"30 2"22 0"36 9"33 5"27 4"97 10"07 120-27
Experiment 2 t
0"1 0"02 1"8 4-6 7"0 2'1 3.0 13.7 0.4 0.2 0"04 13"1 13"1 0.05 0.6 1"8 12"0 1"1 1"8 0"3 7"7 4"4 4"1 8"3 101"3
* Average from four replications,
mg Trace Trace 0-51 0"26 Trace 4"74 7"32 Trace Trace 16"28 4-60 Trace 1.81 22.23 0-73 0.58 0"10 26"18 26"18 Trace 0.10 1"17 3"56 15"80 1"16 2"89 0"47 8"36 3"18 4"16 6"28 158-65
% of total
0"3 0"16 3"0 4"6 10"2 2"9 1"1 14-0 0.4 0"36 0"02 16"5 16"5 0"02 0"7 2"2 10"0 0"7 1"8 0"3 5"3 2"0 2"6 3"9 99"56
t Average from three replications.
values for fatty acids presented in Table 2 are for 5 ml of extract, the amount used in the medium per 100-ml culture. Similarly, the fatty acids were extracted from 3 times the amount of soy peptone + yeast extract used to prepare a culture, and then the values for each fatty acid were adjusted, as seen in Table 2, to the amounts added per 100 ml of medium. Because of the small quantities of fatty acids obtained from these plant products it was not possible to determine the double bond positions in the unsaturated fatty acids found, and it was assumed that they were the c o m m o n ones found in plants, i.e. myristoleic, palmitoleic, oleic and linoleic acids.
L. R. KRUSBERG
94 '-['ABLE 2 - - F A T T Y
Fatty acid
ACID COMPOSITION OF BEEF LIVER EXTRACT AND SOY PEPTONE "~ YEAST EXTRACT ( ~ g ) *
Liver extract
Soy peptone + Preparation 1 Preparation 2 Preparation 3 yeast extract
12:0 14:0 16:0 iso-18 :0 18:0 20:0 14:V°~ 16:1 ~7 18:1 o~9 ~7 20:1 o~ 18:2 ~°e 20:2 ~6 18:3 `03 20:3 ~°e 20:4 °~
30 750 50 2260 10 Trace 70 84 3 Trace 1110 Trace 50 460 590
Total
6250
50 710
Trace 60 660
800
1210
20 140 950 30 Trace 370 Trace 170 130 280
10 120 840 30 Trace 20 20 10
3650
2980
30 70 90 10 30 20 20 90 60 90
510
* Calculated for quantities used to propagate one 100 -ml culture of T. aceti. T h e fatty acid composition of the beef liver extract from one preparation to another was variable as seen from the data in Table 2, and furthermore fewer fatty acids were present than in the nematode. In general, the major fatty acids were palmitic, stearic, oleic and linoleic. T h e 18:1 fraction consisted of 97 per cent oleie acid and 3 per cent vaccenie acid. Variations in the amounts of stearic and linoleic acids among preparations was most noticeable. Several fatty acids, such as 20:0, 20:3 and 20:4, were present in some preparations, but were not detected in others. T h e soy peptone + yeast extract was a minor contributor of fatty acids to the medium although it contained at least ten different fatty acids. A balance sheet for fatty acids of T. aceti, those available in the medium and those remaining in the medium following propagation of nematodes, is seen in Table 3. T h e values in column 3 do not represent amounts actually available from any particular batch of medium, because the greatest values for liver extract fatty acids were selected from among the three preparations seen in Table 2, and were added to the values for fatty acids from the soy peptone + yeast extract to arrive at the figures in this column. Comparing the values in column 3 with those in column 4 it is obvious that T. aceti must be removing fatty acids from the medium, and the approximate quantities of each removed are seen in column 5 (minus values). As seen in column 5 (plus values) a few fatty acids which were absent from the original medium appear in the medium following propagation of nematodes; these fatty
95
FATTY ACID COMPOSITION OF TURBATRIX ACETI T A B L E 3 - - F A T T Y ACID CONTENT (IN ~ g ) OF T. aceti REPRESENTING ONE CULTURE, AND OF THE MEDIUM BEFORE AND AFTER NEMATODE PROPAGATION
Fatty acid
10:0 iso- l1:0 12:0 iso-13 :0 iso-14 :0 14:0 iso-15 :0 15:0 iso-16 :0 16:0 iso-17 :0 17:0 iso-18 :0 18:0 20:0 14:1 ~ 16:1 ojT ~05
18:1 o~ COT m5
20:1 ~u ~O9
CoT
18:2 ~6
20:2~e 18:3 ~6 ~o3
20:3 ~°e 20:4 ~6 ¢03
20:5 ~a Total
1
2
3
4
5
Experiment 1 (nematodes) *
Experiment 2 (nematodes) *
Total available in medium
Total remaining in medium
Difference, medium
Trace Trace 24 Trace Trace 457 1164 Trace Trace 1742 519 Trace 748 3437 113
Trace Trace 109 Trace Trace 1012 1575 Trace Trace 3481 984
55 10 3285 3285 Trace 12 150 462 2561 271 463 75 1940 1094 1034 2099
125 22 5623 5623 Trace 21 249 766 3390 252 621 101 1800 685 893 1357
25,000
33,981
Trace 29
17
-12
126
86 30
-40 +30
832
244
-588
12 2285 29 41 234
660 14 12 61
-12 -1625 -15 -29 -173
1006 29
387
-619
Trace
15
+ 15
1192 24
154 11
-1038 -13
166 457 589
3 67 62 35 76
-163 -390 -527 +35 +76
7051
1934
-5088
Trace
385 4750 157
* Dry wt. of nematodes: Experiment 1,207"2 mg; Experiment 2, 214"4 mg acids must have been contributed by the nematodes. It is equally obvious comparing the values in column 5 with those in columns 1 and 2 (T. a c a / f a t t y acids) that the nematodes must be synthesizing a major portion of most of their fatty acids except perhaps 16:1 ~ and 18:3 ~s, which they remove from the medium in sufficient quantities to more than account for the amounts of these two fatty acids they contain.
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DISCUSSION These findings for the total lipid content of T. aceti (24 per cent) were considerably lower than the 41% reported by Comenga Gerpe (1955) and slightly lower than the 29°/0 reported by Barret et al. (1971). These discrepancies could be due to several factors, such as, variation in populations of nematodes and rearing under xenie rather than axenic conditions on different media and under different conditions. This study demonstrates that T. aceti contains a rich variety of fatty acids many of which are also found in other free-living and plant-parasitic nematodes (Sivapalan & Jenkins, 1966; Krusberg, 1967). Although the 18:1 fatty acids in T. aceti are found in greatest abundance, just as in the nematodes previously examined (Krusberg, 1967), they are not nearly as prominent (3-6 per cent of the dry weight of T. aceti) as in certain of the other nematodes (about 19 per cent of the dry weight in three species, Krusberg, 1967). The lower amount of 18:1 is entirely due to less vaccenic (18:1 ~7) as the oleic acid level in T. aceti is about the same as in the two Ditylenchus spp. investigated (Krusberg, 1967). Stearic acid, however, is far more abundant in T. aceti than in any of the other nematodes investigated. It is interesting that the 18:3 in T. aceti is mostly ~-linolenic acid, with only a small amount of the more common a-linolenic acid, as suggested by Rothstein & Gotz (1968). ~-Linolenic acid has not been reported from nematodes of other species. Although the total fatty acids varied between the nematodes analyzed in the two experiments (Table 1) the percentage composition of the individual fatty acids were similar, usually varying by less than 4 per cent on a total fatty acid basis. These variations could be due to factors, such as, differences in diet and the relative proportions of nematodes in different stages of development in the populations examined. The kinds of fatty acids occurring in the culture medium components, that is, the liver extract and soy peptone + yeast extract, were fewer than those in T. aceti. The degree of variation in the fatty acid content among preparations of liver extract was unexpected. This variation in liver extract fatty acids was not reflected in nematode fatty acids; eight samples of T. aceti propagated on six different preparations of liver extract over a period of more than two years all had quite similar fatty acid patterns by GLC analyses. Thus the fatty acid content of the nematode is largely controlled by the nematode rather than by the diet. In this respect it is interesting that 14:1~5 and 18:3~3 found in the medium components were largely removed from the medium by the nematodes, but little or none of these acids was detected in the nematodes. As mentioned earlier T. aceti must synthesize most of its component fatty acids as implied by the data presented in Table 3. The fatty acids remaining in the medium after propagation of nematodes could come from three sources: (1) those not ingested by the nematode, (2) those discharged by the nematodes, and (3) those appearing from autolysis of nematodes which died. The latter two factors should be considered in studies of fatty acid metabolism in T. aceti.
FATTY ACID COMPOSITION OF T U R B A T R I X A C E T I
97
T. aceti is propagated on a very rich medium, consisting of soy peptone, yeast extract, liver extract and acetic acid. The dry matter in the quantity of these components used to propagate a 100-ml culture of T. aceti is approximately 9.5 g, whereas the yield in nematode dry matter is around 200 mg, or slightly more than 2 per cent. Hence, comparing yield with input these cultures are rather inefficient. A study of the biosynthesis of fatty acids by T. aceti was reported by Rothstein & Gotz (1968). Nematodes were propagated for 2-3 weeks in the presence of 14C-labeled acetate or one of several 14C-labeled fatty acids. On the basis of 1'C label found in both the methyl and carboxyl ends of oleic, linoleic and 8,11,14eicosatrienoic acids they stated that T. aceti was capable of de novo synthesis of polyunsaturated fatty acids. In addition, varying amounts of label were found in every fatty acid examined regardless of the labeled substrate used. These authors further proposed pathways for biosynthesis of fatty acids in T. aceti based on these labeling patterns. Because of the long periods of culturing used it is likely that the label from substrates became redistributed in many metabolites in the nematodes and therefore the proposed fatty acid biosynthesis pathways may be irrelevant. These pathways should be examined by analysis of nematodes incubated in the presence of labeled substrates and sampled at different intervals to ascertain the path of the label in detail. At least three discrepancies exist between my findings and those of Rothstein & Gotz (1968): (1) they state that oleic acid is the most prevalent fatty acid in T. aceti based on analysis of oxidative degradation products of the acid. My studies have shown that the 18:1 fraction ia s 1:1 mixture of oleic and vaccenic acids with a trace of 18:1°5. (2) They claim that 14:1 is present in T. aceti. I was unable to detect 14:1 in T. aceti; the compound occupying that position on GLC chromatograms is really iso-15:0. (3) Two compounds actually occupy the position they claim for 16:1 on GLC. The 16:1 contributes a minor share of this peak, the major portion being iso-17:0. Rothstein & Gotz (1968) demonstrated that T. aceti can incorporate label from substrate acetate and fatty acids into nematode fatty acids. Pathways of fatty acid synthesis and demonstration of possible "essential" fatty acids for T. aceti nutrition, however, must await more critical investigations. Acknowledgements--Mass spectra were kindly prepared by R. C. Dutky and S. R. Dutky, Insect Physiology Laboratory, Entomology Division, U.S.D.A., Beltsville, Maryland, and NMR spectra by S. R. Dutky. REFERENCES
BARRETTJ . , WARD C. W. • FAIRBAIRND. (1971) Lipid metabolism in the free-living nematodes Panagrellus redivivus and Turbatrix aceti. Comp. Biochem. Physiol. 38B, 279-284. BEAMESC. G. (1965) Neutral lipids of Ascaris lumbricoides with special reference to the esterified fatty acids. Expl Parasitol. 16, 291-299. COLE R. J. & KRUSBERGL. R. (1968) Sterol metabolism in Turbatrix aeeti. Life Sci. 7, 713-724. COVmNOAGERPEi . (1955) Biochemical observations on the anguillules of vinegar. R. esp. Fisiol. 11, 181-186. 4
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FOLCH J., LEES M. & SLOANE-STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues, ft. biol. Chem. 226, 497-509. KRUSBER~ L. R. (1967) Analyses of total lipids and fatty acids of plant-parasitic nematodes and host tissues. Comp. Biochem. Physiol. 21, 83-90. ROTHSTEIN ~I. & GOTZ P. (1968) Biosynthesis of fatty acids in the free-living nematode, Turbatrix aceti. Archs Biochem. Biophys. 126, 131-140. SIVAPALANP. & JENKINSW. R. (1966) Phospholipid and long-chain fatty acid composition of the nematode Panagrellus redivivus. Proc. Helminth. Soe. Wash. 33, 149-157. Key Word Index--Turbatrix aceti; nematode; fatty acids; lipid.
FATTY ACID C O M P O S I T I O N OF T U R B A T R I X A C E T I A N D ITS CULTURE MEDIUM* L. R. K R U S B E R G Department of Botany, University of Maryland, College Park, Maryland 20742
(Received 21 June 1971) Abstract--1. Turbatrix aceti propagated axenieally at 30°C contained about 24 per cent lipid on a dry weight basis of which slightly over one-half was fatty acids. 2. Of the 31 fatty acids identified oleie plus vaccenic acids, present in equal quantities, accounted for 25-34 per cent of the total fatty acids, and stearie plus linoleic accounted for another 24 per cent. 3. About 65 per cent of the total amount of fatty acids was unsaturated. 4. Beef liver extract in the medium was the major source of dietary fatty acids of which only two, 16:1c°7 and 18:3 ~°s, were present in sufficient quantities to account for the amounts of these fatty acids occurring in the nematodes; the major fatty acids of liver extract were palmitic, stearie, oleic and linoleic. INTRODUCTION THE TOTAL lipid and fatty acid contents of several nematodes have been examined (Beames, 1965; Sivapalan & Jenkins, 1966; Krusberg, 1967). M a n y of the same fatty acids occur in the various free-living and plant-parasitic nematodes investigated thus far, although the total and relative amounts of individual fatty acids may differ widely among nematode species. In only one study (Krusberg, 1967) were the kinds of unsaturated fatty acids extracted from nematodes determined. For metabolic studies it is desirable to use a nematode which can be propagated easily and rapidly in relatively large quantities axenically on defined medium. The propagation of the vinegar eelworm, Turbatrix aceti (Mueller) Peters, meets these requirements except that the culture medium is undefined. The kinds and quantities of particular metabolites in the medium can be determined, however, so that any changes will be due to only the nematodes and not to other organisms. Panagrellus redivivus can be propagated similarly, although for the lipid studies of Sivapalan & Jenkins (1966) this nematode was reared with microorganisms. None of the nematodes investigated by Krusberg (1967) have yet been propagated axenically. With the interest in lipid metabolism in T. aceti it was decided that the major fatty acids in this nematode should be identified and their relative quantities * Scientific Article No. A1700, Contribution No. 4461 of the Maryland Agricultural Experiment Station. This investigation was supported in part by U.S. Public Health Service Grant 5 RO1 AI-03808. 89
90
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measured. T h e fatty acid content of the m e d i u m components before nematode propagation and the m e d i u m after nematode propagation were also investigated in order to determine any possible contribution of m e d i u m fatty acids to nematode fatty acids. W i t h these data it should then be possible to p e r f o r m comparative studies of fatty acid metabolism in T. aceti and certain plant-parasitic nematodes. MATERIALS AND M E T H O D S T. aceti was propagated on the medium described previously (Cole & Krusberg, 1968) for 21 days at 30°C. Nematodes were washed until free of medium components by repeated cycles of centrifugation at 300 g and suspension in chilled distilled water. Nematodes suspended in 10 ml of distilled water were then disrupted by a single pass through a French pressure cell, the homogenate was frozen and then lyophilized. All calculations were subsequently based on the weight (200-500 mg) of batches of lyophilized nematode homogenate. Total lipid from lyophilized nematode homogenate was extracted after the method of Folch et al. (1957). The total lipid was saponified (Cole & Krusberg, 1968), the fatty acids extracted and methylated with BC13-methanol reagent (Applied Science Lab., State College, Pa.). When interest was solely with fatty acids (as with certain harvests of nematodes, lyophilized beef liver extract, spent medium, or the soy peptone + yeast extract) the material was saponified directly, and the fatty acids then extracted and methylated. Fatty acid methyl esters were passed through a column of Woelm acid alumina, grade 1, deactivated with 1"5% water (Alupharm Chemicals, New Orleans, La.), using hexane followed by hexane: benzene (1 : 1, v/v) as the eluting solvents. The methods used to identify and quantitatively measure fatty acid esters were essentially those used previously (Krusberg, 1967). Both analytical and preparative gas-liquid chromatography (GLC) of fatty acid esters were with stabilized diethylene glycol succinate (DEGS, Analabs, Inc., North Haven, Conn.) or SE-30 silicone gum (Applied Science Lab.). Individual fatty acid esters collected by GLC were finally purified by passing through a column of Woelm alumina as described earlier. Methods used for thin-layer chromatography (AgNOs-silica gel TLC), hydrogenation of fatty acid esters, and locating double bond positions in unsaturated fatty acid esters were described previously (Krusberg, 1967). Infrarared spectra of samples in 1"5 mm KBr pellets were obtained with a Perkin-Elmer Model 257 Grating Spectrophotometer equipped with a 4X beam condenser. Mass spectra were obtained with an LKB Model 9000 Mass Spectrometer. Nuclear magnetic resonance (NMR) spectra were obtained with a Varian A-60A NMR Spectrometer. RESULTS T h e total lipid content of T. aceti ranged f r o m 20.2 to 28.7 per cent on a dry weight basis, averaging 24-0 per cent for nematodes f r o m the various harvests. T o t a l fatty acids accounted for 11-0-16.8 per cent of the dry weight of nematodes, averaging 14.0 per cent or slightly over one-half of the total lipid. A G L C c h r o m a t o g r a m of the fatty acid esters from T. aceti is seen in Fig. 1. Examination of the c h r o m a t o g r a m reveals that the major portion of the fatty acids in this nematode contain eighteen to twenty carbons and that unsaturated fatty acids clearly predominate over saturated fatty acids. T h e 10- to 17-carbon fatty acids were all saturated with the exception of some 16:1. T h e fatty acids iso-15:0 and iso-17:0 were isolated and their structures confirmed b y infrared and mass spectrometer analyses. T h e N M R spectrum for iso-15:0 confirmed the presence
FATTY
ACID
COMPOSITION
OF
I
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91
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L.R. KRUSBERG
of the terminal gem-dimethyl configuration. The fatty acids iso-11:0 and iso-13:0 were only tentatively identified since they occurred in such small amounts as to be difficult to isolate in quantities sufficient for further analyses. These assignments were made because: (1) iso-11:0 and iso-13:0 had the same relationship on GLC to 10:0 and 12:0 as did iso-15:0 and iso-17:0 to 14:0 and 16:0, respectively, (2) on AgNO3-silica gel T L C they moved with the saturated fatty acid esters, and (3) they were not affected by hydrogenation. Iso-14:0, iso-16:0 and iso-18:0 were identified on the basis of their relationships to 14:0, 16:0 and 18:0, respectively, on GLC and comparison of G L C retention characteristics with authentic iso fatty acid methyl esters purchased from (Applied Science Lab.). These iso acid esters also moved with the saturated fatty acid esters on AgNO3-silica gel T L C and were unaffected by hydrogenation. Several additional minor fatty acids were noted in preparations from T. aceti especially when large quantities of fatty acid ester fractions separated by AgNO3silica gel T L C were passed through the gas chromatograph. No attempts were made to identify these fatty acids. A total of 31 fatty acids were identified in extracts of T. aceti (Table 1). About 34 per cent of the total fatty acids in this nematode were saturated, with 16:0 and 18:0 predominating. From 29 to 36 per cent of the total fatty acids were monounsaturated, with the two isomers of 18:1 accounting for 91 per cent of the monounsaturates. The 18:1 fraction was composed of equal quantities of oleic and cis-vaecenic acids with a trace of the a13 form present; the combined fraction made up around 2-5 per cent of the dry weight of nematodes. The 16:1 fraction contained 85 per cent the a9 form and 15 per cent the a l l form, whereas 20:1 contained 2 per cent the a9 form, 24 per cent the al 1 form and 74 per cent the a13 form. Polyunsaturated fatty acids accounted for 26 to 39 per cent of the total nematode fatty acids, of which almost one-third was linoleic acid. Only single forms of 18:2, 20:2, 20:3 and 20:5 were detected. 7-Linolenic acid (18:3 ~G) accounted for 85 per cent and a-linolenic acid (18:3 ~8) for 15 per cent of the total 18:3 occurring in T. aceti, whereas arachidonic acid and its isomer 8,11,14,17-eicosatetraenoic acid were present in about equal amounts. Using authentic standards (Hormel Institute, Austin, Minn.) it was found that y-18:3 would elute on GLC (DEGS column) just before ~-18:3, just as 20:4 ~6 elutes before 20:4 ~3. The nematode 18:3 fraction from AgNOa-silica gel T L C also separated on GLC into two components corresponding to ~-18:3 and a-18:3. Peak areas indicated relative amounts to be 85 and 15 per cent, respectively, the same as was determined by analysis of the oxidative degradation products of the combined 18:3 fraction. Unsaturated fatty acids from nematodes harvested 18 months apart contained the same percentage composition of isomers. The fatty acid composition of the beef liver extract and soy peptone + yeast extract, the primary constituents of the culture medium for T. aceti, is seen in Table 2. Three different preparations of liver extract were analyzed and the positions of the double bonds in the unsaturated fatty acids isolated were determined. The total amounts of liver extract processed were from 60 to 200 ml, although the
FATTYACIDCOMPOSITIONOF TURBATRIX ACETI
93
TABLE 1--FATTY ACIDSOF T. aceti (mg/g dry wt.) Fatty acid
Experiment 1 * mg
10:0 iso-11:0
12:0 iso-13:0 iso-14:0
14:0 iso-15:0
15:0 iso-16:0
16:0 iso-17:0
17:0 iso-18:0
18:0 20:0 16:1 ~v ~5 18:1 ~9 ~ ~0s 20:1 ~n ~8 ~0~ 18:2°~ 20:2°A 18:3°~ ~n 20:3 °~ 20:4¢°e ~o3 20:5 °~n Total
% of total
Trace Trace 0"11 0"03 Trace 2'20 5"59 Trace Trace 8"39 2"50 Trace 3.58 16.54 0.55 0.26 0"05 15"81 15"81 Trace 0"06 0"72 2"24 12"31 1"30 2"22 0"36 9"33 5"27 4"97 10"07 120-27
Experiment 2 t
0"1 0"02 1"8 4-6 7"0 2'1 3.0 13.7 0.4 0.2 0"04 13"1 13"1 0.05 0.6 1"8 12"0 1"1 1"8 0"3 7"7 4"4 4"1 8"3 101"3
* Average from four replications,
mg Trace Trace 0-51 0"26 Trace 4"74 7"32 Trace Trace 16"28 4-60 Trace 1.81 22.23 0-73 0.58 0"10 26"18 26"18 Trace 0.10 1"17 3"56 15"80 1"16 2"89 0"47 8"36 3"18 4"16 6"28 158-65
% of total
0"3 0"16 3"0 4"6 10"2 2"9 1"1 14-0 0.4 0"36 0"02 16"5 16"5 0"02 0"7 2"2 10"0 0"7 1"8 0"3 5"3 2"0 2"6 3"9 99"56
t Average from three replications.
values for fatty acids presented in Table 2 are for 5 ml of extract, the amount used in the medium per 100-ml culture. Similarly, the fatty acids were extracted from 3 times the amount of soy peptone + yeast extract used to prepare a culture, and then the values for each fatty acid were adjusted, as seen in Table 2, to the amounts added per 100 ml of medium. Because of the small quantities of fatty acids obtained from these plant products it was not possible to determine the double bond positions in the unsaturated fatty acids found, and it was assumed that they were the c o m m o n ones found in plants, i.e. myristoleic, palmitoleic, oleic and linoleic acids.
L. R. KRUSBERG
94 '-['ABLE 2 - - F A T T Y
Fatty acid
ACID COMPOSITION OF BEEF LIVER EXTRACT AND SOY PEPTONE "~ YEAST EXTRACT ( ~ g ) *
Liver extract
Soy peptone + Preparation 1 Preparation 2 Preparation 3 yeast extract
12:0 14:0 16:0 iso-18 :0 18:0 20:0 14:V°~ 16:1 ~7 18:1 o~9 ~7 20:1 o~ 18:2 ~°e 20:2 ~6 18:3 `03 20:3 ~°e 20:4 °~
30 750 50 2260 10 Trace 70 84 3 Trace 1110 Trace 50 460 590
Total
6250
50 710
Trace 60 660
800
1210
20 140 950 30 Trace 370 Trace 170 130 280
10 120 840 30 Trace 20 20 10
3650
2980
30 70 90 10 30 20 20 90 60 90
510
* Calculated for quantities used to propagate one 100 -ml culture of T. aceti. T h e fatty acid composition of the beef liver extract from one preparation to another was variable as seen from the data in Table 2, and furthermore fewer fatty acids were present than in the nematode. In general, the major fatty acids were palmitic, stearic, oleic and linoleic. T h e 18:1 fraction consisted of 97 per cent oleie acid and 3 per cent vaccenie acid. Variations in the amounts of stearic and linoleic acids among preparations was most noticeable. Several fatty acids, such as 20:0, 20:3 and 20:4, were present in some preparations, but were not detected in others. T h e soy peptone + yeast extract was a minor contributor of fatty acids to the medium although it contained at least ten different fatty acids. A balance sheet for fatty acids of T. aceti, those available in the medium and those remaining in the medium following propagation of nematodes, is seen in Table 3. T h e values in column 3 do not represent amounts actually available from any particular batch of medium, because the greatest values for liver extract fatty acids were selected from among the three preparations seen in Table 2, and were added to the values for fatty acids from the soy peptone + yeast extract to arrive at the figures in this column. Comparing the values in column 3 with those in column 4 it is obvious that T. aceti must be removing fatty acids from the medium, and the approximate quantities of each removed are seen in column 5 (minus values). As seen in column 5 (plus values) a few fatty acids which were absent from the original medium appear in the medium following propagation of nematodes; these fatty
95
FATTY ACID COMPOSITION OF TURBATRIX ACETI T A B L E 3 - - F A T T Y ACID CONTENT (IN ~ g ) OF T. aceti REPRESENTING ONE CULTURE, AND OF THE MEDIUM BEFORE AND AFTER NEMATODE PROPAGATION
Fatty acid
10:0 iso- l1:0 12:0 iso-13 :0 iso-14 :0 14:0 iso-15 :0 15:0 iso-16 :0 16:0 iso-17 :0 17:0 iso-18 :0 18:0 20:0 14:1 ~ 16:1 ojT ~05
18:1 o~ COT m5
20:1 ~u ~O9
CoT
18:2 ~6
20:2~e 18:3 ~6 ~o3
20:3 ~°e 20:4 ~6 ¢03
20:5 ~a Total
1
2
3
4
5
Experiment 1 (nematodes) *
Experiment 2 (nematodes) *
Total available in medium
Total remaining in medium
Difference, medium
Trace Trace 24 Trace Trace 457 1164 Trace Trace 1742 519 Trace 748 3437 113
Trace Trace 109 Trace Trace 1012 1575 Trace Trace 3481 984
55 10 3285 3285 Trace 12 150 462 2561 271 463 75 1940 1094 1034 2099
125 22 5623 5623 Trace 21 249 766 3390 252 621 101 1800 685 893 1357
25,000
33,981
Trace 29
17
-12
126
86 30
-40 +30
832
244
-588
12 2285 29 41 234
660 14 12 61
-12 -1625 -15 -29 -173
1006 29
387
-619
Trace
15
+ 15
1192 24
154 11
-1038 -13
166 457 589
3 67 62 35 76
-163 -390 -527 +35 +76
7051
1934
-5088
Trace
385 4750 157
* Dry wt. of nematodes: Experiment 1,207"2 mg; Experiment 2, 214"4 mg acids must have been contributed by the nematodes. It is equally obvious comparing the values in column 5 with those in columns 1 and 2 (T. a c a / f a t t y acids) that the nematodes must be synthesizing a major portion of most of their fatty acids except perhaps 16:1 ~ and 18:3 ~s, which they remove from the medium in sufficient quantities to more than account for the amounts of these two fatty acids they contain.
96
L . R . KRUSBERG
DISCUSSION These findings for the total lipid content of T. aceti (24 per cent) were considerably lower than the 41% reported by Comenga Gerpe (1955) and slightly lower than the 29°/0 reported by Barret et al. (1971). These discrepancies could be due to several factors, such as, variation in populations of nematodes and rearing under xenie rather than axenic conditions on different media and under different conditions. This study demonstrates that T. aceti contains a rich variety of fatty acids many of which are also found in other free-living and plant-parasitic nematodes (Sivapalan & Jenkins, 1966; Krusberg, 1967). Although the 18:1 fatty acids in T. aceti are found in greatest abundance, just as in the nematodes previously examined (Krusberg, 1967), they are not nearly as prominent (3-6 per cent of the dry weight of T. aceti) as in certain of the other nematodes (about 19 per cent of the dry weight in three species, Krusberg, 1967). The lower amount of 18:1 is entirely due to less vaccenic (18:1 ~7) as the oleic acid level in T. aceti is about the same as in the two Ditylenchus spp. investigated (Krusberg, 1967). Stearic acid, however, is far more abundant in T. aceti than in any of the other nematodes investigated. It is interesting that the 18:3 in T. aceti is mostly ~-linolenic acid, with only a small amount of the more common a-linolenic acid, as suggested by Rothstein & Gotz (1968). ~-Linolenic acid has not been reported from nematodes of other species. Although the total fatty acids varied between the nematodes analyzed in the two experiments (Table 1) the percentage composition of the individual fatty acids were similar, usually varying by less than 4 per cent on a total fatty acid basis. These variations could be due to factors, such as, differences in diet and the relative proportions of nematodes in different stages of development in the populations examined. The kinds of fatty acids occurring in the culture medium components, that is, the liver extract and soy peptone + yeast extract, were fewer than those in T. aceti. The degree of variation in the fatty acid content among preparations of liver extract was unexpected. This variation in liver extract fatty acids was not reflected in nematode fatty acids; eight samples of T. aceti propagated on six different preparations of liver extract over a period of more than two years all had quite similar fatty acid patterns by GLC analyses. Thus the fatty acid content of the nematode is largely controlled by the nematode rather than by the diet. In this respect it is interesting that 14:1~5 and 18:3~3 found in the medium components were largely removed from the medium by the nematodes, but little or none of these acids was detected in the nematodes. As mentioned earlier T. aceti must synthesize most of its component fatty acids as implied by the data presented in Table 3. The fatty acids remaining in the medium after propagation of nematodes could come from three sources: (1) those not ingested by the nematode, (2) those discharged by the nematodes, and (3) those appearing from autolysis of nematodes which died. The latter two factors should be considered in studies of fatty acid metabolism in T. aceti.
FATTY ACID COMPOSITION OF T U R B A T R I X A C E T I
97
T. aceti is propagated on a very rich medium, consisting of soy peptone, yeast extract, liver extract and acetic acid. The dry matter in the quantity of these components used to propagate a 100-ml culture of T. aceti is approximately 9.5 g, whereas the yield in nematode dry matter is around 200 mg, or slightly more than 2 per cent. Hence, comparing yield with input these cultures are rather inefficient. A study of the biosynthesis of fatty acids by T. aceti was reported by Rothstein & Gotz (1968). Nematodes were propagated for 2-3 weeks in the presence of 14C-labeled acetate or one of several 14C-labeled fatty acids. On the basis of 1'C label found in both the methyl and carboxyl ends of oleic, linoleic and 8,11,14eicosatrienoic acids they stated that T. aceti was capable of de novo synthesis of polyunsaturated fatty acids. In addition, varying amounts of label were found in every fatty acid examined regardless of the labeled substrate used. These authors further proposed pathways for biosynthesis of fatty acids in T. aceti based on these labeling patterns. Because of the long periods of culturing used it is likely that the label from substrates became redistributed in many metabolites in the nematodes and therefore the proposed fatty acid biosynthesis pathways may be irrelevant. These pathways should be examined by analysis of nematodes incubated in the presence of labeled substrates and sampled at different intervals to ascertain the path of the label in detail. At least three discrepancies exist between my findings and those of Rothstein & Gotz (1968): (1) they state that oleic acid is the most prevalent fatty acid in T. aceti based on analysis of oxidative degradation products of the acid. My studies have shown that the 18:1 fraction ia s 1:1 mixture of oleic and vaccenic acids with a trace of 18:1°5. (2) They claim that 14:1 is present in T. aceti. I was unable to detect 14:1 in T. aceti; the compound occupying that position on GLC chromatograms is really iso-15:0. (3) Two compounds actually occupy the position they claim for 16:1 on GLC. The 16:1 contributes a minor share of this peak, the major portion being iso-17:0. Rothstein & Gotz (1968) demonstrated that T. aceti can incorporate label from substrate acetate and fatty acids into nematode fatty acids. Pathways of fatty acid synthesis and demonstration of possible "essential" fatty acids for T. aceti nutrition, however, must await more critical investigations. Acknowledgements--Mass spectra were kindly prepared by R. C. Dutky and S. R. Dutky, Insect Physiology Laboratory, Entomology Division, U.S.D.A., Beltsville, Maryland, and NMR spectra by S. R. Dutky. REFERENCES
BARRETTJ . , WARD C. W. • FAIRBAIRND. (1971) Lipid metabolism in the free-living nematodes Panagrellus redivivus and Turbatrix aceti. Comp. Biochem. Physiol. 38B, 279-284. BEAMESC. G. (1965) Neutral lipids of Ascaris lumbricoides with special reference to the esterified fatty acids. Expl Parasitol. 16, 291-299. COLE R. J. & KRUSBERGL. R. (1968) Sterol metabolism in Turbatrix aeeti. Life Sci. 7, 713-724. COVmNOAGERPEi . (1955) Biochemical observations on the anguillules of vinegar. R. esp. Fisiol. 11, 181-186. 4
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FOLCH J., LEES M. & SLOANE-STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues, ft. biol. Chem. 226, 497-509. KRUSBER~ L. R. (1967) Analyses of total lipids and fatty acids of plant-parasitic nematodes and host tissues. Comp. Biochem. Physiol. 21, 83-90. ROTHSTEIN ~I. & GOTZ P. (1968) Biosynthesis of fatty acids in the free-living nematode, Turbatrix aceti. Archs Biochem. Biophys. 126, 131-140. SIVAPALANP. & JENKINSW. R. (1966) Phospholipid and long-chain fatty acid composition of the nematode Panagrellus redivivus. Proc. Helminth. Soe. Wash. 33, 149-157. Key Word Index--Turbatrix aceti; nematode; fatty acids; lipid.