Comp. Biochem. Physiol. Vol. 86B, No. 1, pp. 103-107, 1987 0305-0491/87 $3.00+0.00 Printed in Great Britain Pergamon Journals Ltd

STEROL METABOLISM IN THE PANAGRELLUS REDIVIVUS, TURBATRIX ACET1 AND

DAVID J. CHITWOOD, WILLIAM R. LUSBY and THOMAS A. SALT* Insect and Hormone Laboratory, Agricultural Research Service, USDA, Building 467, BARC-E, Beltsville, MD 20705, USA and *Department of Botany, University of Maryland, College Park, MD 20742, USA

(Received 24 March 1986)

Abstract--1. Panagrellus redivivus, and Caenorhabditis elegans were sterilely propagated in semidefined media containing sitosterol or cholesterol, and sterols were isolated and identified by capillary gas-liquid chromatography-mass spectrometry. 2. Each species was capable of removal of the C-24 ethyl substituent of sitosterol and production of 4x -methylsterols. 3. Other modifications of the sterol nucleus varied among the species, as only T. aceti and C. elegans introduced AT-and A~14)-bonds significantly. 4. P. redivivus and, to a much lesser extent, 7". aceti reduced AS-bonds to produce substantial quantities of cholestanol and 4~-methylcholestanol.

INTRODUCTION positions of plant-parasitic nematodes with their hosts has indicated that these parasites are probably Unlike higher plants and vertebrates, nematodes are capable of phytosterol dealkylation and nuclear incapable of synthesizing sterols de novo and con- saturation (Chitwood et al., 1985; Cole and Krus- sequently possess a nutritional requirement for sterol berg, 1967; Orcutt et al., 1978). In addition, the (Bolla et al., 1972; Cole and Krusberg, 1968; Comley free-living nematode Turbatrix aceti converts radio- and Jaffe, 1981; Dutky et aL, 1967; Hieb and labelled sitosterol (24~-ethylcholest-5-en-3fl-ol) to Rothstein, 1968). Many structurally diverse sterols radiolabelled 7-dehydrocholesterol (Cole and Krus- satisfy this requirement, including several plant berg, 1968). The purpose of the present investigation sterols (Bolla et al., 1972; Chitwood et al., 1986; was to further characterize sterol metabolism in Dutky et al., 1967; Hieb and Rothstein, 1968). Plant this organism and another free-living nematode, sterols, or phytosterols, unlike most common Panagrellus redivivus, by capillary gas chromatog- sterols, generally contain alkyl (i.e. methyl, methyl- raphy-mass spectrometry (GC-MS). Additionally, ene, ethyl, ethylidene) substituents at C-24 of their because Lu et al. (1977) reported maximum re- side chains (Nes and McKean, 1977). production of T. aceti to occur at a lower sterol Interest in nematode sterol biochemistry has been concentration than that used in our previous experi- stimulated by the discovery that several compounds ments with C. elegans, we have replicated some of our with potent nematicidal activity towards various work with C. elegans at a lower concentration of plant- and animal-parasitic and free-living nematodes dietary sterol (10/~g/ml vs 25#g/ml) to facilitate (Bottjer et al., 1984, 1985; Douvres et al., 1980; intergeneric comparison. Feldmesser et al., 1976; Lozano et al., 1984) disrupt sterol metabolic pathways in Caenorhabditis elegans (Chitwood et al., 1984; Lozano et al., 1984, 1985b). This free-living nematode removes the C-24 MATERIALS AND METHODS alkyl substituents of many structurally diverse phytosterols, the major metabolites are usually Dietary sterols 7-dehydrocholesterol (cholesta-5,7-dien-3/~-ol), chol- Sitosterol contained no apparent impurities as determined esterol (cholest-5-en-3fl-ol) and lathosterol (cholest- by thin-layer chromatography (TLC) but contained 1.5% 7-en-3fl-ol) (Chitwood et al., 1984; Lozano et al., campesterol (24~x-methylcholest-5-en-3~-ol) by gas-liquid 1985a). In certain cases, however, other sterols com- chromatography (GLC). No impurities were detected in prise major proportions of the total sterol pool dietary cholesterol by either TLC or GLC. The dietary (Lozano et al., 1985a). In addition, C. elegans cholesterol was supplemented with [4-t4C]cbolesterol, which attaches a single methyl group to the nucleus of the had been obtained from Amersham Corp. (Arlington Heights, IL, USA) and purified via column chromatography sterol molecule to produce significant quantities of to exceed 99% purity by GLC and TLC, and was used at 4~t-methylcholest-8(14)-en-3fl-ol (Chitwood et al., a specific activity of 0.40 Ci/mol. 1983). This direct nuclear methylation pathway has not been reported to occur in any other organism. Nematode culture Sterol metabolism in other nematode species is less P. redivivus, T. aceti and C. elegans were propagated well understood. Comparison of the sterol corn- axenically at 22°C in an aqueous medium containing 103 104 DAVID J. CmrwooD et al.

30mg/ml soy peptone, 30mg/ml yeast extract, 10mg/ml Table 1. Sterol content of Panagrellus redivivus, Turbatrix aceti and dextrose, 0.5 mg/ml hemoglobin, 0.5 #l/ml Tween 80 and Caenorhabditis elegans propagated in media containing 10#g/ml 10/t g/ml sterol, with yeast extract, dextrose and hemoglobin sitosterol or cholesterol,,expressed as percentage of nematode dry extracted previously to remove endogenous sterols (Chit- weight wood et al., 1984). In addition, the medium for T. aceti contained 70 mM acetic acid. Living nematodes from late supplemented sterol logarithmic phase cultures were isolated by centrifugation, sltosterol cholesterol flotation on 40% sucrose solution, and repeated rinsing with distilled water. P. redivivus 0.16 0.12

T. aceti 0.12 0.13 Sterol analysis ~. ele~ans 0.I0 * Lipids were extracted from duplicate samples of 200-400 mg lyophilized nematodes by homogenization in a Ten-Broeck tissue grinder 3 times with chloroform/ *Experiment not performed. methanol (2:1) and subsequent partition against 0.85% NaCI (Folch et al., 1957). Lipid extracts were separated on RESULTS columns of silica gel 60 (70-230 mesh, E. Merck, Darm- stadt, FRG) into neutral lipids (eluted with chloroform) and Gravimetric quantification indicated that the polar lipids (eluted with methanol). The neutral lipids were nematode lipid extracts comprised 12.1% to 15.6%, saponified as described previously (Lozano et al., 1984) and 16.0% to 20.6% and 15.8% to 17.8% of the dry fractionated on 5.0g columns (I.0cm i.d.) of Florisil weight of P. redivivus, T. aceti, and C. elegans, (60-100 mesh, J. T. Baker, Philipsburg, NJ, USA) deacti- vated with 70 H20. Compounds were eluted with 50ml respectively. Sterol content, measured by GLC, var- hexane, 50 ml 1% diethyl ether in hexane (v/v), 50 ml and ied from 0.10% to 0.16% of nematode dry weight 100 ml portions of 5% ether, 50 ml of 20% ether, and 50 ml (Table 1). of ether. The 4g-methylsterols were in the second 5% ether All sterols isolated from nematodes were identical fraction and 4-desmethylsterols were in the 200 ether to authentic reference compounds (except for five fraction. All column chromatographic separations were subsequently described compounds for which we monitored by TLC on Anasil H plates (Analabs, North lacked reference material) by GLC relative retention Haven, CT, USA) developed with hexane/ether/acetic acid times (RRTs, Table 2) of both free sterols and steryl (80:20: 1). Sterols were quantified and tentatively identified acetate derivatives, argentation TLC, and GC-MS. by GLC and analyzed further by acetylation overnight in pyridine/acetic anhydride (3:1) at room temperature. The steryl acetates were purified on Florisil columns similar to Table 2. Gas-liquid chromatographic relative retention times of those described previously and were eluted with 5% diethyl sterols from Panagrellus redivivus, Turbatrix aceti or Caenorhabditis ether in hexane. The acetates were analyzed further by elegans, expressed relative to cholesterol. GLC was performed isothermally on a DB-I fused silica capillary column argentation column chromatography (Chitwood et al., (14m x 0.32 mm i.d., 0.25,um film) and on a packed glass column 1985), argentation TLC (Chitwood et al., 1984), GLC, UV (2m x 2mm i.d.) containing 2.0% OV-17 stationary phase spectroscopy, and GC-MS. Sterol DB-I 0V-17 Instrumentation GLC of sterols and steryl acetates was performed iso- Cholesta-5,7,g(lll-trienol 0.98 1.06 thermally with a Shimadzu model GC-9A gas chro- Cholest-8(14)-enol o.g9 1.02 matograph fitted with a 14m x 0.32 mm i.d. J & W DB-1 Cholesterol 1.00 1.00 fused silica capillary column (0.25/am film) and connected to a Shimadzu model C-R3A recording integrator, as well Cholestanol 1.02 1.02 as a Varian model 3700 instrument equipped with a packed Cholest-8(9)-enol 1.05 1.05 glass column (2m × 2mm i.d.) containing 2.0% OV-17 Desmosterol 1.08 1.20 liquid phase and connected to a Shimadzu C-R 1B recording integrator. Flame ionization detectors were used for quan- 7-Dehydrocholesterol 1.10 1.16 titative analyses; for radioactivity determinations, we Lathosterol 1.12 1.17 trapped the effluent from a Varian 3700 gas chromatograph Cholesta-8(g),24-dienol 1.15 1.27 fitted with a thermal conductivity detector and a 9.8 m x 0.75 mm i.d. Supelco SPB-1 glass capillary column Cholesta-5,7,24-trienol 1.21 1.40 (1.0 #m film). Compounds from the GLC effluent (as well Cholesta-7,24-dienol 1.23 1.42 as all column chromatographic fractions) were radio- analyzed in a xylene-based scintillation fluid (ScintiLene, Campesterol 1.30 1.32 Fisher Scientific, Fairlawn, NJ, USA) and counted in a Fucosterol 1.60 1.72 Beckman LS 5801 liquid scintillation system, with quench in Sitosterol 1.60 1.63 each sample measured with the H-number technique. Cap- illary GC-MS of steryl acetates with a DB-1 column and Stigmastanol 1.63 1.65 UV spectroscopy were performed as described previously 4a-Methyl cholest-8(14)-enol 1.18 1.15 (Lozano et aL, 1984). (I.15)* (I.13) In a control experiment to detect possible oxidative or 4~-Methylcholestanol 1.20 1.15 other modifications of the dietary sterol in the aqueous (1.17) (1.13) medium that could be mistaken for endogenous nematode 4a-Methylcholesta-8(14),24-dienol 1.29 1.41 metabolism, 200ml of 7'. aceti medium containing (1.26) (1.37) [~4C]cholesterol was not inoculated with nematodes during 4a-Methylcholest-7-enol 1.31 1.35 one of the experiments, and sterols were isolated by (1.27) (1.32) partition against CHC13/MeOH (2:1) at the time of the nematode harvest and subsequently purified and analyzed *Values in parentheses are those of steryl acetate derivatives relative by GLC similarly to nematode sterols. to cholesteryl acetate. Nematode sterol metabolism 105

The RRTs of the free sterols vs cholesterol were Table 4. Relativepercentages of sterols from Panagrellus redivivus identical to the RRTs of the acetates vs cholesteryl and Turbatrix aceti propagated in media containing 10pg/ml acetate, except for the four 4ct-methylsterols, which cholesterol possessed their characteristic decrease in RRT as steryl acetates (Patterson, 1971). In addition, UV Recovered sterol P. redlvlvus T. aceti

spectra of 7-dehydrocholesteryl acetate (2m~x at 272, Cholesterol 69.8 39.0 282, and 294 nm) and cholesta-5,7,9(11)-trienyl ace- tate (2rex at 311,325, and 340 nm) were characteristic Cholestanol 26.6 1.9 of compounds with A 5'7- and AS'7'9°l)-bonds, re- 7-Dehydrocholesterol * 38.4 spectively. Lathosterol 0.4 5.9 We lacked authentic standards of cholesta-8(9),24- Cholest-8(14)-enol * 0.5 dienol, cholesta-5,7,24-trienol, cholesta-7,24-dienol Cholest-B(9)-enol * 0.4 and 4~t-methylcholesta-8(14),24-dienol, but the re- spective compounds from nematodes in our present Cholest-5,7,9(|1)-trienol * D.6 experiments were identical by GLC and GC-MS to 4a-Methylcholest-8(14)-enol * 7.1 the same compounds isolated previously by us from 4a-Methylcholestanol 3.2 4.7

C. elegans (Chitwood et al., 1984; Lozano et al., 4a-Methylcholest-7-enol * 1.5 1984). Although not previously isolated from nema- todes, cholest-8(9)-enyl acetate migrated during ar- *Not detected. gentation chromatography between stanyl and ALsteryl acetates, had GLC RRTs as expected (Itoh et al., 1982), and possessed a mass spectrum [m/z and consistent with a literature spectrum (Galli and (relative intensity): 428 (molecular ion M +, 89%), Maroni, 1967). 413 (M-CH 3, 18%), 368 (M4SH3COOH, 5%), 353 Regardless of the dietary sterol, the major sterols (M-CH3COOH-CH3, 16%), 315 (M-side chain, of P. redivivus were cholesterol, cholestanol, and 9%), 255 (M--CH3COOH-side chain, 23%), 229 4~t-methylcholestanol (Tables 3 and 4). In addition, (M-CH3 COOH-side chain-C2H2, 37%), 213 P. redivivus from sitosterol-supplemented media (M-CH3COOH-side chain-C3H6, 46%), and 55 contained significant quantities of sitosterol and (C4H7, 100%)] identical to that in the Incos library desmosterol (cholesta-5,24-dien-3fl-ol). Excluding

Table 3. Relative percentages of sterols from Panagrellus redivivu& Turbatrix aceti and Caenorhabditis elegans propagated in media containing 10/~g/ml sitosterol. (Sitosterolcontained 1.5% campesterolas an impurity)

Recovered sterol ~. redivivus ~. aceti C. ele~ans

Sitosterol 6.7 4.5 49.6

Stigmastanol 0.1 * Trace a

Cholesterol 61.7 18.8 9.4

Cholestanol 20.3 1.1 Trace

7-Dehydrocholesterol * 42.9 27.7

Lathosterol 0.4 9.4 3.3

Cholest-8(14)-enol * 0.4 Trace

Cholest°8(9)-enol * 0.5 *

Desmosterol 6.1 0.9 *

Cholesta-7,24-dienol * 0.2 Trace

Cholesta-8(9),24-dienol * 0.1 *

Cholesta-5,7,24-trienol * 0.2 Trace

Cholesta-S,7,9(11)-trienol * 1.5 1.2

Campesterol 0.4 0.1 1.0

Fucosterol O.t 0.3 Trace

4a-Methylcholest-8(14)-enol Trace 10.4 7.6

4a-Methylcholestanol 4.2 6.3 *

4=-Methylcholest-7°enol * 2.4 0.2

4a-Methylcholest-8(14),24-dienol Trace * *

aTrace is defined as less than 0.05% of total sterol. *Not detected. 106 DAVID J. CHITWOOD et aL dietary sitosterol, C. elegans contained 7-dehydro- 1985a). Propagation of nematodes in media contain- cholesterol, cholesterol, 4~-methylcholest-8(14)-enol ing A~-sterol reductase inhibitors has indicated and lathosterol as its major sterols (Table 3). T. aceti that fucosterol (24-cis-ethylidenecholest-5-en-3[J-oi), generally contained the same sterols found in the cholesta-5,7,24-trienol, desmosterol, and cholesta- other two species, with 7-dehydrocholesterol as the 7,24-dienol are key intermediates in the conversion of major metabolite (Tables 3 and 4). sitosterol to 7-dehydrocholesterol, cholesterol, and In experiments with [t4C]cholesterol, all sterols lathosterol by C. elegans (Chitwood et al., 1984; from P. redivivus and T. aceti were radiolabelled with Lozano et al., 1984). Desmosterol was similarly approximately the same specific activity as the dietary identified as a sitosterol metabolite in 7". aceti incu- cholesterol, except for those for which quantities were bated with the A:4-sterol reductase inhibitor tri- insufficient for accurate measurement: these included parinol succinate (Cole and Krusberg, 1968). In our cholest-8(14)-enol, cholest-8(9)-enol and cholesta- present experiments, detection of small quantities of 5,7,9(ll)-trienol from T. aceti and lathosterol from P. fucosterol, cholesta-5,7,24-trienol and cholesta-7,24- redivivus. Polar lipid fractions contained less than 1% dienol in sitosterol-fed T. aceti and fucosterol and of the radioactivity in the total lipid fractions and desmosterol in sitosterol-fed P. redivivus indicates were not analyzed further. Separation into neutral that these compounds are potential intermediates in and polar lipid fractions prior to saponification was pathways for sitosterol dealkylation and subsequent necessary to minimize subsequent occurrence of non- metabolism in these species. steroidal compounds (as determined by GC-MS) in Of substantial interest is the 4-methylation path- nematode 4-methylsterol fractions with GLC RRTs way, which is unique to nematodes and was pre- similar to sterols. viously demonstrated only in C. elegans (Chitwood In the control experiment in which T. aceti medium et al., 1983). C. elegans produces predominantly containing radiolabelled cholesterol was incubated 4~-methylcholest-8(14)-enol and small amounts of without nematodes, 99.4% of the recovered radio- 4~-methylcholest-7-enol by this pathway, whereas labelled material behaved identically to cholesterol the 4-methylsterol of P. redivivus consists nearly during column chromatography, TLC, and prepara- entirely of 4~-methylcholestanol. The 4-methylation tive GLC. During analytical GLC, trace quantities pathway in T. aceti shares characteristics of both (less than 0.1% of cholesterol) of several unidentified species, in that As(~4)-, A°-, and AT-4~-methylsterols compounds were present with DB-1 RRTs of 1.05, are produced. P. redivivus is characterized further by 1.10, 1.12, 1.16, 1.22, 1.32, 1.35, 1.60 and 1.69; production of large amounts of cholestanol; T. aceti however, their small relative proportion precluded is again intermediate between P. redivivus and C. OV-17 analysis. Because liquid scintillation counting elegans in this respect. P. redivivus differs from the of trapped GLC effluent indicated that none of these other two species in that it does not introduce trace components were radiolabelled, they were not AT-bonds to any great extent, as the major nondietary characterized further. sterol of C. elegans and T. aceti, 7-dehydrochol- esterol, was not detected in P. redivivus. DISCUSSION Taxonomically, T. aceti and P. redivivus are members of the family Cephalobidae in the order The lipid contents of the three nematode species , and C. elegans is a member of the family (12.1% to 20.6% of dry weight) are similar to Rhabditidae in the same order (Nicholas, 1984). It is corresponding values (15.4% to 19.8%) reported interesting that such large differences in sterol metab- previously in the same species propagated axenically olism exist among such taxonomically related, bio- (Chitwood and Krusberg, 1981; Chitwood et al., logically similar nematodes. Additional examination 1984; Hutzell and Krusberg, 1982; Lozano et al., of other nematode genera may reveal further sterol 1985a; Willett and Downey, 1974). The sterol con- metabolic variations among these organisms. tents of the three species (0.10% to 0.16%) are also As in our previous work with C. elegans (Chitwood similar to corresponding values (0.09% to 0.13%) in et al., 1984), the fact that the sterols of T. aceti and our previous investigations with C. elegans (Chit- P. redivivus fed [J4C]cholesterol contained approxi- wood et al., 1984; Lozano et al., 1984) and the 0.15% mately the same specific activity as the dietary choles- value for T. aceti propagated in a medium containing terol (where practical to measure) indicates that the 12.5-13.0/~g/ml sterol (Cole and Krusberg, 1968). sterols were produced by nematodes by metabolism The latter authors noted that sterol content of T. of the dietary cholesterol rather than by de novo aceti in such a medium was several times that of biosynthesis or by concentration of some non- nematodes from a medium containing only radiolabelled media contaminant. In the control ex- 2.5-3.0/~g/ml sterol. We observed no difference in periment with aged T. aceti medium, the lack of sterol content between C. elegans in our present detection of significant quantities of putative sterols experiments with 10/~g/ml sitosterol and that in our with GLC RRTs different from cholesterol as well as previous studies with 25 #g/ml sitosterol (Chitwood the failure to detect any radiolabelled sterols other et al., 1984; Lozano et al., 1984). than cholesterol are further evidence that the com- The production of substantial quantities of pounds detected in the present investigation are true 24-desalkylsterols (e.g., cholesterol, cholestanol, metabolites and not oxidative products or other 7-dehydrocholesterol) by each species cultured in artifacts. sitosterol-supplemented medium indicates that each Difficulty in axenic propagation of plant-parasitic species is capable of C-24 dealkylation, as previously nematodes has limited investigation of sterol metabo- demonstrated in T. aceti (Cole and Krusberg, 1968) lism in these to comparisons of sterol com- and C. elegans (Chitwood et aL, 1984; Lozano et al., positions of host and parasite. Such comparisons Nematode sterol metabolism 107 have indicated that phytoparasitic nematodes possi- Cole R. J. and Krusberg L. R. (1967) Sterol compositions bly dealkylate phytosterols obtained from their hosts of the nematodes Ditylenchus triformis and Ditylenchus (Chitwood et al., 1986). Because C. elegans is capable dipsaci, and host tissues. Exp. Parasitol. 21, 232-239. Cole R. J. and Krusberg L. R. (1968) Sterol metabolism in of C-24 dealkylation, many of our recent in- Turbatrix aceti. Life Sci. 7, 713-724. vestigations have employed this species as a model Comley J. C. W. and Jaffe J. J. (1981) Isoprenoid bio- organism for investigation of sterol metabolism in synthesis in adult Brugia pahangi and Dirofilaria immitis. plant-parasitic nematodes. However, 7-dehydrochol- J. Parasitol. 67, 609-616. esterol, generally the major sterol of C. elegans, has Douvres F. W., Thompson M. J. and Robbins W. E. (1980) not yet been detected in any plant-parasitic nema- In vitro cultivation of Ostertagia ostertagi, the medium tode; moreover, C. elegans does not produce the large stomach worm of cattle. II. Effect of insect-growth- quantities of stanols detected in many plant parasites disrupting amines and amides on development. Vet. (Chitwood et al., 1985; Orcutt et aL, 1978; Svoboda Parasitol. 7, 195-205. Dutky S. R., Robbins W. E. and Thompson J. V. (1967) The and Rebois, 1977). Because production of stanols, demonstration of sterols as requirements for the growth, lack of formation of significant quantities of 7-de- development and reproduction of the DD-136 nematode. hydrocholesterol and existence of C-24 dealkylation Nematologica 13, 140. capability are three characteristics shared by most Feldmesser J., Thompson M. J., Robbins W. E. and plant-parasitic nematodes and P. redivivus, the latter Sponaugle R. P. 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