Archives of Insect Biochemistry and Physiology 29:1-9 (1995) 7328

Supplied by U. S. Dept. of Agric., National Center for Agricultural Utilization Research, Peoria, IL

Developmental Differences in the Sterol Composition of SoJenopsis invicta Amadou S. Ba, De-An Guo, Robert A. Norton, Sherman A. Phillips, Jr., and W. David Nes Department of Plant and Soil Science (A.S.B., S.A.P.J, and Department of Chemistry and Biochemistry (D.-A.G., W.D.N.J, Texas Tech University, Lubbock, Texas; and Mycotoxin Research Unit, U.S. Department of Agriculture/ARS, Peoria, IlIinois (R.A.N.J

Twenty-six sterols were isolated from eggs, larvae, workers, and queens of the red imported fire ant, Solenopsis invicta Buren. They were identified by chro­ matographic (TLC, CLC, and HPLC) and spectral methods (MS and lH-NMR). Queens possessed the most varied sterol composition (24 sterols were detected). The major sterols from queens were the doubly bioalkylated 24a-ethyl cholest­ 5- and 7-en-3I3-ols whereas the major sterol from the other developmental stages was , a sterol which lacks a C-24 alkyl group. From fourth instar larvae were isolated two yeasts, Candida parapsilosis and Yarrowia lipolytica. Both yeasts were found to synthesize similar sterols, primarily and zymosterol (90% of the sterol mixture). A minor sterol (approximately 12% of the total sterol mixture) deteded in eggs, larvae, and workers was 24-methyl cholesta-5,22E-dien-3/3-01 (). Brassicasterol may have originated from ergosterol produced by the fungal endosymbiotes. The amount of sterol in each developmental stage was as follows: approximately 24 Jlg sterol/queen, 3 Jlg sterol/worker, 2 Jlg steroillarvae, and 0.02 Jlg sterol/egg. The sterol com­ position of the red imported fire ant differed from that of leaf-cutting ants pre­ viously investigated where 24-methyl sterols of ectosymbiotic fungal origin were the major sterols detected in soldiers and workers. © 1995 Wiley·Liss, Inc.

Key words: Solenopsis invicta, ants, fungi, sitosterol, ergosterol, cholesterol

INTRODUCTION The Hymenoptera is an advanced group of insects that require a di· etary source of sterol to support development and reproduction (Svoboda et al., 1994a). Many insects contain large amounts of cholesterol, which

Acknowledgments: This study was supported by a grant from the Texas Tech University Biotech­ nology Institute mini-grant program to W.D.N. and from the Texas State Line-Item for Fire Ant Research to SAP.

Received October 3,1994; accepted November 30,1994.

Address reprint requests to Dr. W. David Nes, Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409.

© 1995 Wiley-Liss, Inc. 2 Ba et al. they obtain from metabolism of 24-alkyl sterols synthesized by host plants (Svoboda and Chitwood, 1992), or from artificial diets rich in cholesterol (Ritter, 1984). Several years ago, we observed a correlation between in­ creasing cholesterol content in animal systems and the development of the nervous system (Nes and Nes, 1980). Therefore, it was of interest for us to learn recently that some phytophagous hymenopteran may use 24­ alkyl sterols as cholesterol surrogates (Ritter et aI., 1982; Maurer et aI., 1992). For instance, the leaf-cutting ant, Atta cephalotes isthmicola, an in­ sect that cultivates fungi (which synthesize ergosterol) for food (Ritter et aI., 1982), accumulates 24-methyl sterols in the brain and whole ant. No cholesterol could be found in the ant, providing the first evidence of a functional nervous system in an animal entirely lacking cholesterol. A sec­ ond leaf-cutting ant, Acromyrmex octospinosus, was found to accumulate 24-methyl sterols (Maurer et aI., 1992), suggesting that ants might utilize fungal symbionts for their source of nutritional sterol generally. Alterna­ tively, Svoboda and Lusby (1986) discovered that the Allegheny mound ants and the red imported fire ants (pupae and workers) feeding on an omnivorous diet possessed significant amounts of cholesterol and vary­ ing amounts of , e.g., sitosterol and campesterol. The purpose of this study was twofold: to establish the sterol composition of the red imported fire ant at different developmental stages and to determine whether the red imported fire ant sterol composition from field collected colonies contained significant levels of ergosterol or phytosterols. The re­ sults show the red imported fire ant queens possess the most varied sterol composition of any insect studied to date, and that they differ from leaf-cut­ ting ants in their ability to accumulate phytosterols during development. They are more like aphids, e.g., Shizaphis graminum (Campell and Nes, 1983), which also possess symbiotes (Houk and Griffiths, 1980; Douglas, 1988), in their ability to accumulate sitosterol (24a-ethyl cholesterol) and cholesterol.

MATERIALS AND METHODS Solenopsis invicta Buren The ants were collected in Taylor county, Texas, during the months of Sep­ tember 1993 and April 1994. The study site is a range land habitat at the southeastern edge of lake Kirby near Abilene. Colonies from the first collec­ tion were maintained at room temperature for 4 months and were fed a ster­ ile diet of boiled eggs mixed with soybean oil. The colonies from the second trip were maintained in boxes free of food and analyzed for sterol within 5 days of collection. Endosymbiotic fungi were isolated from fourth instar larvae of the red im­ ported fire ant. The meconium, a compact pellet in the larval gut, was re­ moved surgically and washed in sterile distilled water and placed on acidified yeast-malt extract agar. Two different yeasts were identified from the plates using the established methods (Van der walt and Yarrow, 1984), Candida parapsilosis and Yarrowia lipolytica. The two strains were grown to stationary phase growth in shake culture, as described (Nes et aI., 1993). Sterol Composition of S. invicta 3 Sterol Analysis Sterols were isolated from ants and yeasts by saponifying the whole or­ ganism in an aqueous solution of 10% KOH in MeOH containing 10% water at reflux for 30 min, then extracting the neutral lipid with diethyl ether, and chromatographing the resulting non-saponifiable lipid fraction using TLC and HPLC to obtain pure sterol fractions for further analysis. GLC \vas performed using a 3% SE-30 packed column (Xu et al., 1988) and HPLC was performed using a Zorbax ODS Cwreversed phase column connected to an ISCO vari­ able wavelength detector set at 205 nm (Xu et al., 1988). GC-MS was per­ formed on HPLC sterol fractions using a Table-top HP model provided by R.A. Norton at the USDA laboratory and lH-NMR was performed on a Bruker AF-200 NMR spectrometer at the Texas Tech University. Sterols were identi­ fied by their rates of movement in TLC, GLC, and HPLC, expressed as Rf values (TLC) or retention times (RRT in GLC and a c in HPLC) relative to cholesterol (RRTc), and by comparison of the mass spectra (compare Rahier and Benveniste, 1989) of samples eluted form HPLC with those of authentic specimens available to us. To confirm the double bond positions and 24-alkyl sterol stereochemistry in 24-ethyl sterols, we obtained a lH-NMR spectrum of a sterol fraction eluted from HPLC.

RESULTS Red imported fire ants collected from the field were analyzed for sterols. Queens were found to possess the most varied sterol composition of the four developmental stages examined. The chromatographic and spectral proper­ ties of the 24 sterols isolated from queens are shown in Table 1 (and illus­ trated in Fig. 1). The most unusual sterol that accumulated in the queen was 24a-ethyl cholest-7-en-3p-ol. (cholest-7-en-3p-ol), a 24-desalkyl sterol is the usual 7-ene sterol isolated from insects (Svoboda et al., 1994a; Kircher, 1982). We confirmed the position of the double bond and configura­ tion of the 24-ethyl group after elution of the sterol from an HPLC fraction containing sitosterol (Kalinowska et al., 1990) and that of a mixture of 5-ene and 7-ene (Fig. 2). Several trace (::;;0.1%, the limit of detection using GLC, compare Xu et al., 1988) sterols with substitution at C-4 were detected, e.g., 24(28)-methylene parkeol and 24(28)-methylene . The isolation of , 24-methylene cycloartanol and , which also possess C-4 methyl group(S) at C-4, is of special interest since insects are thought to be unable to metabolize these sterol intermediates to cholesterol (Corio-Costet et al., 1989). Table 2 shows the sterol composition of eggs, larvae, workers, and queens of red imported fire ants analyzed shortly after their collection from the field. The eggs, larvae, and workers possessed similar sterol composition, with cholesterol predominating the sterol mixture whereas the queens possessed a different sterol profile with 24-ethyl sterols predominating the sterol mix­ ture Assuming the ants we collected were feeding on plant material that was available in the natural habitat (no decaying animals were obvious in the collection site that might provide a major source of cholesterol) and as­ suming further that all plants synthesize mainly 24-ethyl sterols (Nes and 4 Ba et aI.

TABLE 1. Chromatographic and Spectral Properties of Sterols From Red Imported Fire Ant· TLC GLC HPLC Sterol (R l) (RRTc) (ex,) MS (M- and other diagnostic ions)' Cholesta-5,22-dienol 0.18 0.92 0.82 384 369 366 351 300 255 Cholesterol 0.18 1.00 1.00 386 371 368 353 301 255 Cholest-7-enol 0.16 1.10 1.09 386 371 368 353 273 255 Cholestanol 0.18 1.03 1.11 388 373 370 355 331 262 Ergosterol 0.18 1.21 0.76 396 381 378 363 337 271 Brassicasterol 0.18 1.12 0.85 398 383 380 300 271 255 Ergosta-5,23-dienol 0.18 1.26 0.95 398 383 365 339 314 271 Campesterol 0.18 1.29 1.13 400 385 382 367 315 289 Ergost-7-enol 0.16 1.42 1.23 400 385 379 314 273 255 0.18 1.65 1.00 412 397 369 314 299 281 Avenasterol 0.16 1.72 1.03 412 397 368 314 299 271 0.18 1.40 1.10 412 397 394 351 300 271 Stigmasta-7,22- 0.16 1.54 1.20 412 397 379 300 271 255 dienol Stigmast-22-enol 0.18 1.44 1.22 414 399 396 353 300 273 Sitosterol 0.18 1.60 1.18 414 399 396 354 329 303 Stigmast-7-enol 0.16 1.76 1.29 414 399 381 314 273 255 Stigmastanol 0.18 1.64 1.30 416 401 359 314 299 .,-- Obtusifoliol 0.25 1.48 0.92 426 411 383 327 285-" 245 24-Methylene 0.25 1.64 0.97 412 397 379 328 313 285 lophenol 4ex-Methy124-methy- 0.25 1.49 0.97 412 397 383 327 313 285 lenecholest-8-enol Citrastadienol 0.25 2.15 1.13 426 411 368 328 313 285 Cycloartenol 0.29 1.84 1.04 426 411 408 393 365 339 Cycloartanol 0.29 1.88 1.14 428 413 410 393 367 341 24-Methylene 0.29 1.88 1.07 440 425 422 411 379 300 lanosterol 24-Methylene 0.29 2.03 1.07 440 425 407 397 341 313 parkeol 24-Methvlene 0.29 2.10 1.12 440 425 422 407 379 353 cyclo;rtanol ·TLC was developed in benzene/ether (85:15). GLC was performed on 3'7c SE-30 glass packed column at 245°C isothermally. HPLC was operated on Zorbax ODS column eluted with metha- nol at 1.00 ml/min at room temperature. The UV detector was set at 205 nm. aMS fragmentation pattern of sterols; only significant ions (~1O% abundance) are given be- tween m/z 255 and 440 amu.

Nes, 1980), then some or all of the ant cholesterol present in the four ant stages may have been formed in situ by C-24 dealkylation. At no develop­ mental stage was 4,4-dimethyl (including pentacyclic triterpenoids) or 4­ monomethyl sterols in a significant steady-state concentration in the ant. When the ants were maintained on a cholesterol-supplemented diet (eggs/soybean oil), the sterol profile of the workers (only stage studied) was similar to the sterol composition of the workers shown in Table 2, except that the former contained a slightly higher content of cholesterol (approximately 80% of the total sterols). Brassicasterol and ergosterol were detected in the red imported fire ants at Sterol Composition of S. invicta 5

10 ~ 17N A B D M E H

I

Fig. 1. Structures (side chain and nucleus) of sterols identified in this study.

several stages of development (approximately 10-12% of the total sterols). Both sterols possess ,622-24-methyl groups. This side chain grouping is un­ common in the constitution (although brassicasterol may occur in Brassica plants) (Nes and Mckean, 1977), but frequently is introduced into sterols by fungi (Patterson, 1994), suggesting that the fungal endosymbionts might supply some endogenous sterol for the ants. Therefore, we analyzed the sterols of. yeast obtained from the midgut. The major sterols (approxi­ mately 90% of the total sterols) from both yeast strains (see Materials and Methods) were ergosterol (M'" 396, RRTCf 1.26 on 3% 5£-30) and zymosterol (M'" 384, RRTCf 1.13 on 3% 5£-30).

DISCUSSION Sterols are used by insects, as they are in vertebrates, as essential mem­ brane inserts, in reproduction, as precursors of hormones, and as pre­ cursors of defensive secretions (Kircher, 1982). The differences in the structures 6 Ba et al.

CI) en c: o c. en CI) ex: ...o ti CI) iii c

1 '6 '7 8 91011 Time (min.)

6:0 5:5 5:0 4.5 o(ppm) 1.2 1:1 1:0 .9 .8 .6 .5 o(ppm)

Fig. 2. l H-NMR spectra (bottom two spectra) and GLC chromatogram (top panel) of a sterol sample that was eluted from the HPLC in the region of cholesterol and cholest-7-en-3p-ol. and compositions of sterols in insects (review by Svoboda et al., 1994a), nota­ bly involving the extent to which C-24 is alkylated, suggests insects might have evolved specific requirements for the type and amount of 24-alkyl and 24-desalkyl sterols which may be utilized functionally. Svoboda et al. con­ cluded that in certain orders, an early branching of the phylogenetic tree oc­ curred, resulting in more primitive species that dealkylate and more advanced species that do not, although there may be exceptions to the hypothesis (Campbell and Nes, 1983). As we now show, different ant groups (e.g., A. cephalotes isthmicola and A. octospinosus) which were thought to be similar in having the advanced sterol trait-lack of C-24 dealkylation, may contain gen­ era (e.g., 5. invicta) that possess the trait, suggesting the trait may not be characteristic of primitiveness. From the sterol composition data of the eggs, larvae, workers, and queens, it would appear that during the early growth and development period phy­ tosterols were likely accumulated from host plants and actively dealkylated to cholesterol. Fungal ergosterol may have provided some of the sterol in the sterol mixture (e.g., of brassicasteroD, but it was unlikely that ergosterol was a strong source of 24-alkyl sterol for cholesterol production. This follows from Sterol Composition of S. invicta 7

TABLE 2. Sterol Composition of Red Imported Fire Ant at Different Developmental Stages" Sterol Structure' Egg Larva Worker Queen Cholesta-5,22-dienol lA 0.8 N.D. 0.7 N.D. Cholesterol lB 44.8 50.3 55.3 21.7 Cholest-7-enol 2B 4.0 9.3 1.0 7.4 Cholestanol 3B N.D. N.D. N.D. 2.8 Ergosterol 4D 0.8 1.2 0.5 N.D. Brassicasterol 10 12.5 9.1 11.2 3.6 Ergosta-5,23-dienol lE 0.7 3.2 N.D. 2.3 Campesterol lG 12.5 8.2 10.6 5.6 Ergost-7-enol 2G 0.6 1.0 N.D. 2.8 Isofucosterol 1I 3.4 1.2 4.3 tr Avenasterol 21 0.5 1.3 N.D. 2.4 Stigmasterol lH 0.8 1.4 3.4 4.0 Stigmasta-7,22-dienol 2H N.D. N.D. N.D. tr Stigmast-22-enol 3H N.D. N.D. N.D. tr Sitosterol lJ 17.4 10.1 12.8 32.8 Stigmast-7-enol 2J 1.1 3.1 0.2 11.9 Stigmastanol 3J N.D. N.D. N.D. 2.7 Obtusifoliol 5F N.D. N.D. N.D. tr 24-Methylenelophenol 6F N.D. N.D. N.D. tr 4a-MethyI24-methylene 7F N.D. N.D. N.D. tr cholest-8-enol Citrastadienol 61 N.D. 0.6 N.D. tr Cycloartenol 8B N.D. N.D. N.D. tr Cycloartanol 8C N.D. N.D. N.D. tr 24-Methylenelanosterol 9F N.D. N.D. N.D. tr 24-Methyleneparkeol 10F N.D. N.D. N.D. tr 24-Methylenecycloartanol 8F N.D. N.D. N.D. tr Total sterol (f,lg/part) 0.02 2.44 3.18 24.17 Number examined/ 80,000/0.55 1,400/0.75 5,800/10.3 420/3.74 Fr. wt. (g) "As percent total sterol; tr, trace; N.D., not detected. 'Structures of sterols are shown in Figure 2. Because of confusions in the revised IUPAC sys- tem for naming and numbering the sterol structure, we continue to use the conventional "Nes" system (Parker and Nes, 1992).

our observation that ergosterol fed to Manduca sexia produces signific~nt amounts of cholesta-5,7-dien-3p-ol (Svoboda et al., 1994b). The red imported fire ant may reduce the ,:l7-bond of ergosterol, but fails to do so with stigmast­ 7-en-3p-ol, which accumulates in the queen. Many of the unusual 4,4-dim­ ethyl and 4-monomethyl sterols detected in the queens were obviously of plant origin (Nes and Nes, 1980). The sterol profile of eggs was different from the queens whereas larvae and workers possessed similar sterol composition to eggs. The reason for the differences in sterol content is not clear. Neverthe­ less, there appears to be dealkylation being performed by the ants, as well as selective accumulation and transfer of sterol from one developmental stage to the next. Studies in progress with radiolabeled sterols and side chain me­ tabolism inhibitors are planned to shed light on the status and function of sterols in S. invicia. 8 Ba et al. LITERATURE CITED Campbell BC, Nes WD (1983): A reappraisal of sterol biosynthesis and metabolism in aphids. J Insect Physiol 29:149-156.

Corio-Costet MF, Charlet Benveniste P, Hoffman J (1989): Metabolism of dietary t.8-sterols and 9~, 19-cyclopropyl sterols by LOCllsta migratoria. Arch Insect Biochem Physiol11:47-62.

Douglas AE (1988): On the source of sterols in the green peach aphid, Myzus persicae, reared on holidic diets. J Insect PhysioI34:403-408.

Houk EJ, Griffiths GW (1980): Intracellular symbiotes of the Homoptera. Annu Rev Ent 25:161-187.

Kalinowska M, Nes WR, Crumley FG, Nes WD (1990): Stereochemical differences in the ana­ tomical distribution of C-24 alkylated sterols in Kalanchoe diagremontiana. Phytochemis­ try 29:3427-3434.

Kircher HW (1982): Sterols in insects. In Dupont JP (ed): Cholesterol Systems in Insects and Animals. Boca Raton: CRC Press, pp 1-50.

Maurer P, Debieu D, Malosse C, Leroux P, Riba G (1992): Sterols and symbiosis in the leaf­ cutting ant Acromyrex octospinous (Reich) (Hymenoptera, Forrnicidae:Attinil. Arch Insect Bio­ chern PhysioI20:13-21.

Nes WD, Janssen GG, Crumley FG, Kalinowska M, Akihisa T (1993): The structural require­ ments of sterols for membrane function in Saccharomyces cerevisiae. Arch Biochem Bio­ phys 300:724-733.

Nes WR, Mckean ML (1977): Biochemistry of and Other Isopentenoids. Baltimore: University Park Press.

Nes WR, Nes WD (1980): Lipids in Evolution. New York: Plenum Press.

Parker SR, Nes WD (1994): Regulation of sterol biosynthesis and its phylogenetic implica­ tions. In Nes WD, Parish EJ, Trzaskos JM (eds): Regulation of Isopentenoid Metabolism. Washington DC: American Chemical Society Press, pp 110-145.

Patterson GW (1994): Phylogenetic distribution of sterols. In Nes WD (ed): Isopentenoids and Other Natural Produces: Evolution and Function. Washington DC: American Chemical So­ ciety Press, pp 90-108.

Rahier A, Benveniste P (1989): Mass spectral identification of phytosterols. In Nes WD, Parish EJ (eds): Analysis of Sterols and Other Biologically Significant Steroids. New York: Aca­ demic Press, pp 223-250.

Ritter KS (1984): Unusual aspects of the sterol biochemistry of insects. In Nes WD, Fuller G, Tsai L (eds): Isopentenoids in Plants: Biochemistry and Function. New York, Dekker, pp 389-400.

Ritter KS, Weiss BA, Norrbom AL, Nes WR (1982): Identification of t.5,7-24-methylene- and 24-methyl sterols in the brain and whole body of Atta cephalotes isthmicola. Comp Biochem Physiol B 71:345-349.

Svoboda JA, Chitwood DJ (192): Inhibition of sterol metabolism in insects and nematodes. In Nes WD, Parish EJ, Trzaskos JM (eds): Regulation of Isopentenoid Metabolism. Washing­ ton DC: American Chemical Society Press, pp 203-218.

Svoboda JA, Lusby WR (1986): Sterols of phytophagous and omnivorous species of Hy­ menoptera. Arch Insect Biochem PhysioI 3:13-18. Sterol Composition of S. invicta 9 Svoboda JA, Feldlaufer MF, Weirich GW (1994a): Evolutionary aspects of steroid utilization by insects. In Nes WD (ed): Isopentenoids and Other Natural Produces: Evolution and Func­ tion. Washington DC: American Chemical Society Press, pp 129-139.

Svoboda JA, Ross SA, Nes WD (1994b): Comparative studies of metabolism of 4-desmethyl, 4-monomethyl and 4,4-dimethyl sterols in Manduca sexta Lipids. (In press).

Van der walt JP, Yarrow D (1984): Methods for the isolation, maintenance, classification, and identification of yeasts. In Kreger-van RN (ed): The Yeasts: A Taxonomic Study, 3rd ed. Amsterdam: Elsevier, pp 45-104.

Xu S, Norton RA, Crumley FG, Nes WD (1988): Comparison of the chromatographic proper­ ties of sterols, select additional steroids and triterpenoids: Gravity flow liquid chromatog­ raphy, thin layer chromatography, gas-liquid chromatography and high performance liquid chromatography. JChromatogr 452:377-389.