Organic Acids of Ditylenchus triformis and Turbatrix aceti 1

JESSICA M. CASTILLO AND L. R. KRUSBERG 2

Abstract: Pyruvic acid, lactic acid and several tricarboxylic acid cycle acids were extracted from Ditylenchus tri]ormis and Turbatrix aceti and identified. Fumaric acid was predominant in both nematodes. Small amounts of malic and ,~-ketoglutaric acids and intermediate quantities of lactic, citric, succinic, and pyruvic acids occurred in D. trilormis. In T. aceti citric, lactic, and a-ketoglutaric acids were less abundant than succinic, malic and pyruvic acids. Only traces of aconitic and oxalacetic acids occurred in both nematodes. All the organic acids detected accounted for only about one per cent of the dry weight of nematodes of the two species. Key Words: Tricarboxylic acid cycle acids.

The tricarboxylic acid cycle (TCA-cycle) obligate aerobe which thrives in fermenting has been studied in only a few species of cultures. nematodes, mostly animal parasites. Evi- dence for the presence of this cycle in nema- MATERIALS AND METHODS todes has been obtained by various means, Ditylenchus triformis originally isolated such as: detecting enzyme activities in nem- from soil and maintained in laboratory cul- atode preparations (4, 5, 6, 9, 14), deter- ture for 10 years was cultured on the fungus mining TCA-cycle acids in nematodes (3, Pyrenochaeta terrestris growing on potato 22), testing the ability of intact nematodes dextrose agar in 26 C. Four- to six-week-old or nematode homogenates to utilize different cultures were harvested by modified Baer- TCA-cycle intermediates (5, 12, 21), using mann funnel. Turbatrix aceti was propagated inhibitors of specific enzyme systems and axenically in liquid medium containing 3% respiratory metabolism in nematodes (5, 13, yeast extract, 4% soy peptone, 2% (v/v) 14), and determining incorporation of 14C glacial acetic acid and 5% (v/v) fresh beef from labelled substrates into TCA-cycle in- liver extract. Cultures were incubated at 30 C termediates in nematodes (7, 14). Succinic and harvested after 3-4 weeks. Around 4-6 was the major organic acid in Ascaris lum- ml of wet packed D. trilormis per 100 petri bricoides whereas pyruvic, lactic, oxalacetic plates and 10-20 ml of T. aceti per 5-10 and ~-ketoglutaric acids occurred in small flasks (100 ml of medium/flask) were ob- amounts (22). tained at each harvest. Nematodes were sus- The purpose of this study was to determine whether organic acids of the TCA cycle pended in 5-10 ml of distilled water and dis- and some metabolically-related acids occur rupted with a cold French pressure cell. The in the two nematodes, Ditylenchus triformis homogenates were immediately frozen at -5 (Hirschmann and SasseD, a fungus-feeder, C and lyophilized. and Turbatrix aceti (Muller), a free-living EXTRACTION OF NEMATODES AND PREPA- RATION OF ESTERS OF ORGANIC ACIDS: Ly- Received for publication 29 January 1971. ophilized homogenate (300 to 800 mg) was 1 Scientific Article No. A1675, Contribution No. 4425, of the Maryland Agricultural Experiment Station. Adapted rehomogenized in boiling 80% ethanol and from a Ph.D. dissertation by the senior author. Supported the homogenate filtered. The filtrate was in part by NE-34 Northeast Regional Nematode Project and U. S. Public Health Service Grant No. 5 ROI A103808. evaporated to dryness at 40 C and the residue 2 Graduate Assistant and Professor, Department of Botany, University of Maryland, College Park 20742. The senior extracted with chloroform followed by water author's present address: Department of Biology, Min- danao State University, Lanao del Sur, Philippines. to obtain the lipids and the water-soluble sub- 284 ORGANIC Acres OF D. triJormis AND T. aceti • Castillo, Krusberg 285

stances, respectively. The water-soluble frac- EXTRACTION OF KETO ACIDS, PREPARATION tion, containing the organic acids, was con- OF 2,4-DINITROPHENYLHYDRAZONES AND centrated to about one ml at 40 C and then TLC OF HYDRAZONES: Keto acids were ex- acidified to pH 2 with concentrated sulfuric tracted from lyophilized nematode tissues acid (10). The acidified solution was mixed with 1-5 ml of 5% perchloric acid. One to thoroughly with acid-washed silicic acid (0.5 two ml of 0.5% 2,4-dinitrophenylhydrazine ml/g) and packed into a glass column (30 x in 2N HCI was added to the filtrate and the 1 cm), from which the organic acids were hydrazones formed were extracted with ethyl eluted with about 400 ml of diethyl ether. acetate and aqueous 10% sodium carbonate The eluate was evaporated to dryness at 25 C (11). and the residue was subsequently dissolved Hydrazones of keto acids were dissolved in in 1-2 ml of diethyl ether containing 10% ethyl acetate and spotted on TLC plates methanol. The acids were methylated with coated with cellulose powder 250 ~ thick. diazomethane prepared from N-methyl-N- Chromatograms were developed with n- nitroso-p-toluene sulfonamide (20). The solu- butanol/ethanol/0.5M NH4OH (7:1:2, tion containing the esters was concentrated v/v/v) at 25 C. Rf values of hydrazones of with a stream of dry nitrogen and dried over nematode keto acids were compared with anhydrous sodium sulfate. Methyl esters of those of known keto acids. Spraying de- commercial acids were also prepared in this veloped plates with 2% NaOH in 90% ethanol way. Samples were chromatographed im- caused the hydrazones to change from a yel- mediately or stored at -5 C. low to red-brown confirming their identities GAS CHROMATOGRAPHY (GLC) OF ESTERS: (1). Cellulose containing individual hydra- Methyl esters of acids were analyzed using zones was scraped from unsprayed plates and a Chromalab Model A-110 Gas Chromato- the hydrazones were eluted with 0.1M potas- graph with an argon fl-ionization detector. sium phosphate buffer at pH 7.4 for quanti- Silanized glass columns (183 cm × 3.4 ram) tation. Optical densities of solutions were were packed with Chromosorb W (80-100 measured at 365 nm (11). Standard concen- mesh) coated with 15% diethylene glycol tration curves were prepared with chromato- succinate (DEGS). Argon carrier gas was graphed authentic acid hydrazones and were maintained at 30 psig and a flow rate of 27 used to quantitate the nematode acid hydra- ml/min. zones. Samples were chromatographed isother- Hydrazones were recrystallized from cold mally at 135 C to separate the low-boiling ethyl acetate solution by addition of hexane, esters, and 185 C to separate the high-boiling and crystals were then utilized for melting esters (19). Temperature programming, point determinations and for infrared spectral when used, was from 70 C to 185 C at the analyses. rate of 5 C/min. The relative retention times on GLC of the unknown methyl esters of RESULTS nematode acids were compared to retention Organic acids identified from both D. tri- times of known esters, both individually and [ormis and T aceti were pyruvic, succinic, in mixtures. Methyl esters were quantitated lactic, fumaric, malie, a-ketoglutaric, aconitic, by triangulation of peaks on chromatograms. citric and oxalacetic acids. Individual esters were trapped from the gas Hydrazones of pyruvic, a-ketoglutaric and chromatograph and analyzed by infrared oxalacetie acids were separated and identi- spectrophotometry. fied by TLC. Two spots were obtained for 286 Journal of Nematology, Vol. 3, No. 3, July 1971

TABLE 1. Organic acids isolated from Ditylenchus abundant, and succinic, malic and pyruvic triformis, in/zmoles/g dry wt. of nematodes. acids were intermediate in abundance. Experiment Number Aconitic and oxalacetic acids occurred in Acid 1 2 3 4 Mean amounts too small to measure in the quanti- ties of nematodes available although the for- Lactic 4.7 3.6 4.4 2.9 3.9 mer was tentatively identified by GLC in the Fumaric 33.7 37.7 33.1 34.8 34.8 methylated acids and the latter by its hydra- Succinic 7.8 6.5 6.6 7.9 7.2 zone on TLC. Isocitric acid was not detected Malic 1.2 0.9 0.9 1.3 1.1 in nematode preparations. Citric 3.7 3.0 3.7 3.9 3.6 Pyruvic 9.3 11.0 8.4 9.4 DISCUSSION c~-Ketoglutaric 0.5 0.7 0.6 0.6 The total organic acids recovered consti- tuted about 0.8% of the dry wt. of D. triformis pyruvic acid and were designated as pyruvate and 1.2% of T. aceti. These findings are con- hydrazone-1 (Rf 0.78) and pyruvate hydra- sistent with the general observation that or- zone-2 (Rf 0.57). a-Ketoglutarate hydrazone ganic acids occur in low quantities in animal had an Rt of 0.13 and oxalacetate hydrazone tissues (8). an Rf of 0.17. Melting points and infrared The presence of these acids supports the spectra of the keto acid hydrazones from evidence based on studies of enzymes from nematodes agreed with those for the corre- D. triformis (9) and T. aceti (5) that the TCA sponding authentic keto acid hydrazones. cycle is operative in these nematodes. Ells Furnaric acid was the predominant organic and Read (5), however, suggested that a typi- acid (57% of the total organic acids) in D. cal TCA cycle does not occur in T. aceti triform& (Table 1). Small quantities of malic based (i) on the limited ability of some nema- and a-ketoglutaric acids and intermediate tode preparations to carry out reactions of quantities of lactic, succinic, citric and pyru- this cycle, (ii) on the failure of some inter- vic acids were also detected. In T. aceti mediates to stimulate respiration in homog- fumaric acid was also the predominant or- enates and in intact nematodes, and (iii) on ganic acid (65% of the total organic acids) the failure to detect cytochrome oxidase. In (Table 2), occurring in almost twice the contrast, D. trilormis preparations possessed concentration found in D. tri]ormis. Citric, several active TCA-cycle enzymes as well as lactic and a-ketoglutaric acids were least active cytochrome oxidase (9). Studies with the animal parasite, Ascaris TABLE 2. Organic acids isolated from Turbatrix lumbricoides, have shown succinic acid to be aceti, in ~moles/g dry wt. of nematodes. the major acidic component of the perienteric fluid (3, 22) and muscles (22). Pyruvic, lac- Experiment Number tic, oxalacetic and a-ketoglutaric acids were Acid 1 2 3 4 Mean detected in small amounts, and several other Lactic 1.0 0.8 0.2 0.2 0.6 organic acids occurred only in traces. Al- Fumaric 68.2 77.6 71.0 54.6 67.9 though an operative TCA cycle was indicated Succinic 8.5 9.2 11.4 24.7 13.4 in the muscles of Ascaris (14), the cycle must Malic 8.3 22.2 9.9 7.1 11.9 be only of minor importance since its metab- Citric 3.0 1.4 3.5 3.0 2.7 olism is essentially anaerobic. Carbon diox- Pyruvic 8.5 7.4 5.7 7.2 ide fixation and subsequent dismutation re- c~-Ketoglutaric 0.6 0.4 0.3 0.4 actions between carboxylic acids at different ORGANIC ACIDS OF D. tri/ormis AND T. aceti . Castillo, Krusberg 287

oxidation levels may explain the reason for 4. COSTELLO, L. C., AND S. GROLLMAN. 1959. Studies on the reactions of the Krebs cycle the kind of oxidative metabolism occurring in in Strongyloides papillosus infective larvae. Ascaris (14). Bueding et al. (2) suggested Exp. Parasitol. 8: 83-89. that a reversal of the succinoxidase reaction 5. ELLS, H. A., AND C. P. READ. 1961. Physi- in Ascaris may be an anaerobic adaptation ology of the vinegar eel, Turbatrix aceti (Nematoda). 1. Observation on respiratory and may account for the accumulation of metabolism. Biol. Bull. 120:326-335. succinic acid. Ells and Read (5) suggested 6. GOLDBER6, E. 1957. Studies on the inter- that the same reaction may occur in T. aceti. mediary metabolism of Trichinella spiralis. Exp. Parasitol. 6:367-382. The accumulation of fumarate in T. aceti 7. Hmn, W. F., ANt) E. C. DOUGHERTY. 1966. and D. trilormis may be due to inhibition of Evidence for the tricarboxylic acid cycle in fumarate reductase under the aerobic condi- Panagrellus redivivus and changes observed under varying conditions of culture. Nema- tions of the nematode cultures. Oxygen has tologica 12:93. (Abstr.) been found to inhibit this enzyme in certain 8. KREBS, H. A., ANn J. M. LOWENSTEIN. 1960. animal parasites (15). If anaerobic incuba- The tricarboxylic acid cycle. Vol. 1, p. 129- 203. ln: D. M. Greenberg (ed.) Chemical tion of nematodes caused a decrease in fuma- pathways of metabolism. Academic Press, rate and an increase in succinate the presence New York, 2nd edition. of a fumarate reductase resembling that in 9. KRUSBERG,L.R. 1960. Hydrolytic and res- piratory enzymes of species of Ditylenchus animal parasites would be suggested. and Pratylenchus. Phytopathology 50:9-22. Isocitrate lyase and malate synthetase, two 10. KUKmS, A., AND P. PmORESCm. 1967. Isola- enzymes of the glyoxylate pathway, were de- tion of Krebs cycle acids from tissues for gas chromatography. Anal. Biochem. 19: tected in Caenorhabditis briggsae (16), and 468--480. in T. aceti, Panagrellus redivivus and Rhab- 11. KUN, E., ANt M. GARCIA-HERNANI~EZ. 1957. Identification and quantitative determina- ditis anomala (17, 18). Glyoxylic acid was tion of keto acids by paper chromatography. not detected in T. aceti or D. trilormis in this Biochim. Biophys. Acta 23 : 181-186. study, although the pathway may be func- 12. MASSEY,V., AND W. P. ROGERS. 1949. The tdcarboxylic acid cycle in nematode para- tioning in these nematodes. The glyoxylate sites. Nature 163:909. pathway is probably operative in T. aceti, 13. MASSEY,V., AND W. P. ROGERS. 1951. Con- ditions affecting the action of fluoroacetate since this nematode thrives in fermenting on the metabolism of nematode parasites media containing relatively high levels of and vertebrate animals. Aust. J. Sci. Res. B4:561-574. acetic acid, a precursor of the two-carbon 14. OYA, H., G. KIKUCHI, T. BANDO, AND H. compounds metabolized in this pathway (17, HAYASI-n. 1965. Muscle tricarboxylic acid 18). The glyoxylate pathway has not been cycle in Ascaris lumbricoides var suis. Exp. Parasitol. 17:229-240. investigated in D. triformis. 15. PRmHARD, R.K. 1970. Mode of action of the anthelminthic thiabendazole in Haemon- LITERATURE CITED chus contortus. Nature 228:684-685. 16. ROTHSTErN, M., AND HELEN MAYOH. 1964. 1. BLOCK, R. J., E. L. DURRUM, AND G. ZWEIG. Nematode biochemistry. IV. On isocitrate 1958. A manual of paper chromatography lyase in Caenorhabditis briggsae. Arch. and paper electrophoresis. Academic Press, BiDchem. Biophys. 108:134-142. New York, 2nd ed. 710 p. 17. ROTHSTEIN, M., AND HELEN MAYOH. 1965. 2. BUED1NG, E., N. ENTNER, AND E. FARBER. Nematode biochemistry. VII. Presence of 1955. Dissociation of succinoxidase systems isocitrate lyase in Panagrellus redivivus, of Ascaris lurnbricoides and of rat kidney. Turbatrix aceti, and Rhabditis anomala. Biochim. Biophys. Acta 18:305-306. Comp. Biochem. Physiol. 16:361-365. 3. BUEDING,E., AND G. W. FARRAR. 1956. Iden- 18. ROTHSTEIN,M.,ANDH. MAYOH. 1966. Nem- tification of succinic acid as a constituent of atode biochemistry. VIII. Malate synthe- the perienteric fluid of Ascaris lumbricoides. tase. Comp. Biochem. Physiol. 17:1181- Exp. Parasitol. 5:345-349. 1188. 288 Journal o/ Nematology, Vol. 3, No. 3, July 1971

19. RUMSEY,T. S., C. H. NOLLER, J. C. BURNS, D. 21. SCHWABE, C.W. 1957. Observation on the KALB, C. L. RHYKERD, AND D. L. HILL. respiration of free-living and parasitic Nip- 1964. Gas chromatographic resolution of postrongylus muris larvae. Amer. J. Hy- pyruvic, lactic, glyoxylic, oxalic, malonic, giene. 65:325-327. fumaric, malic, alpha-ketoglutaric, and citric 22. UENO, Y., H. OVA, AND T. BANDO. 1960. A acids in forage and rumen fluid. J. Dairy method for the separation of tricarboxylic Sci. 47:1418-1421. 20. SCHLENK, H., AND JOANNE L. GELLERMAN. cycle intermediates by celite-column chro- 1960. Esterification of fatty acids with matography and its application to the tissues diazomethane on a small scale. Anal. Chem. of Ascaris lumbricoides var. suis. J. Bio- 32:1412-1414. chem. 47:771-776.