Archives of Insect Biochemistry and Physiology 29:l-9 (1 995) Developmental Differences in the Sterol Composition of Solenopsis 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.), and Department of Chemistry and Biochemistry (D.-A.G., W.D.N.), Texas Tech University, Lubbock, Texas; and Mycotoxin Research Unit, U S . Department of AgriculturelARS, Peoria, Illinois (R.A.N.) Twenty-six sterols were isolated from eggs, larvae, workers, and queens of the red imported fire ant, Solenopsis invicta Buren. They were identified by chromatographic (TLC, GLC, and HPLC) and spectral methods (MS and 'H-NMR). Queens possessed the most varied sterol composition (24 sterols were detected). The major sterols from queens were the douhly bioalkylated 24a-ethyl cholest5- and 7-en-3P-01~whereas the major sterol from the other developmental stages was cholesterol, 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 ergosterol and zymosterol (90% of the sterol mixture). A minor sterol (approximately 1 2 % of the total sterol mixture) detected in eggs, larvae, and workers was 24-methyl cholesta-5,22E-dien-3~-ol (brassicasterol). Brassicasterol may have originated from ergosterol produced by the fungal endosymbiotes. The amount of sterol in each developmental stage was as follows: approximately 24 pg steroI/queen, 3 pg sterol/worker, 2 pg sterol/larvae, and 0.02 pg sterol/egg. The sterol composition of the red imported fire ant differed from that of leaf-cutting ants previously investigated where 24-methyl sterols of ectosymbiotic fungal origin were the major sterols detected in soldiers and workers. o 1995 Wile\,-Liss, Inc. Key words: Solenopsis invicta, ants, fungi, sitosterol, ergosterol, cholesterol INTRODUCTION The Hymenoptera is an advanced group of insects that require a dietary 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 Biotechnology Institute mini-grant program to W.D.N. and from the Texas State Line-Item for Fire Ant Research to S.A.P. 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. 0 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 increasing 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 24alkyl sterols as cholesterol surrogates (Ritter et al., 1982; Maurer et al., 1992). For instance, the leaf-cutting ant, Attu cephalotes isthrnicola, an insect that cultivates fungi (which synthesize ergosterol) for food (Ritter et al., 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 second leaf-cutting ant, Acrurnyrmex octospinusus, was found to accumulate 24-methyl sterols (Maurer et al., 1992), suggesting that ants might utilize fungal symbionts for their source of nutritional sterol generally. Alternatively, 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 varying amounts of phytosterols, 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 results 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-cutting ants in their ability to accumulate phytosterols during development. They are more like aphids, e.g., Shizaphis graminurn (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 September 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 collection were maintained at room temperature for 4 months and were fed a sterile 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 imported fire ant. The meconium, a compact pellet in the larval gut, was removed 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, 19841, Candidu puvupsilosis and Yavrowiu lipolyticu. The two strains were grown to stationary phase growth in shake culture, as described (Nes et al., 1993). Sterol Composition of S. invicta 3 Sterol Analysis Sterols were isolated from ants and yeasts by saponifying the whole organism 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-saponifiablelipid fraction using TLC and HPLC to obtain pure sterol fractions for further analysis. GLC was performed using a 3% SE-30 packed column (Xu et al., 1988) and HPLC was performed using a Zorbax ODS C18-reversedphase column connected to an ISCO variable wavelength detector set at 205 nm (Xu et al., 1988). GC-MS was performed on HPLC sterol fractions using a Table-top HP model provided by R.A. Norton at the USDA laboratory and 'H-NMR was performed on a Bruker AF-200 NMR spectrometer at the Texas Tech University. Sterols were identified by their rates of movement in TLC, GLC, and HPLC, expressed as Rf values (TLC) or retention times (RRT in GLC and a, in HPLC) relative to cholesterol (RRT,), and by comparison of the mass spectra (compare Rahier and Benveniste, 1989) of samples eluted form HI'LC 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 'H-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 properties of the 24 sterols isolated from queens are shown in Table 1 (and illustrated in Fig. 1).The most unusual sterol that accumulated in the queen was 24a-ethyl cholest-7-en-3P-01. Lathosterol (cholest-7-en-3~-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 configuration 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 cholesterols (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)-methyleneparkeol and 24(28)-methylenelanosterol. The isolation of cycloartenol, 24-methylene cycloartanol and obtusifoliol, 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 mixture. 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 assuming further that all plants synthesize mainly 24-ethyl sterols (Nes and 4 Baet al. TABLE 1. Chromatographic and Spectral Properties of Sterols From Red Imported Fire Antr TLC Sterol Cholesta-5,22-dienol Cholesterol Cholest-7-en01 Cholestanol Ergosterol Brassicasterol Ergosta-5,23-dienol Campesteroi Ergost-7-en01 Isofurosterol Avenasterol Stigmasterol Stigmasta-7,22dienol Stigmast-22-enol Sitosterol Stigmast-7-en01 Stigmastanol Obtusifoliol 24-Meth ylene lophenol 4a-Methyl24-methylenecholest-8-en01 Citrastadienol Cycloartenol Cycloartanol 24-Methylene lanosterol 24-Methylene parkeol 24-Methylene cycloartanol GLC HPLC (R3 (RRTJ (G) MS (M’ and other diagnostic ions)” 0.18 0.18 0.16 0.18 0.18 0.18 0.18 0.18 0.16 0.18 0.16 0.18 0.16 0.92 1.00 1.10 1.03 1.21 1.12 1.26 1.29 1.42 1.65 1.72 1.40 1.54 0.82 384 386I 1.@ 1.09 386 1.11 388 0.76 396 0.85 398 0.95 398 1.13 400 1.23 400 412 1.oo 412 1.03 412 1.10 1.20 412 0.18 0.18 0.16 0.18 0.25 0.25 1.44 1.60 1.76 1.64 1.48 1.64 1.22 1.18 1.29 1.30 0.92 0.97 0.25 1.49 0.25 0.29 0.29 0.29 369 371 371 373 381 383 383 385 385 397 397 397 397 366 368 368 370 378 380 365 382 379 369 368 394 379 351 353 353 355 363 300 339 367 314 314 314 351 300 300 301 273 331 337 271 314 315 273 299 299 300 271 255 262 271 255 271 289 255 281 271 271 255 414 414 414 416 426 412 399 399 399 401 411 397 396 396 381 359 383 379 353 354 314 314 327 328 300 329 273 299 285 313 273 303 255 255 245 285 0.97 412 397 383 327 313 285 2.15 1.84 1.88 1.88 1.13 1.04 1.14 1.07 426 426 428 440 411 411 413 425 368 408 410 422 328 393 393 411 313 365 367 379 285 339 341 300 0.29 2.03 1.07 440 425 407 397 341 313 0.29 2.10 1.12 440 425 422 407 379 353 255 2.55 *TLC was developed in benzene/ether (85:15). GLC was performed on 3% SE-30 glass packed column at 245°C isothermally. HPLC was operated on Zorbax ODS column eluted with methanol at 1.00 ml/min at room temperature. The UV detector was set at 205 nm. “MS fragmentation pattern of sterols; only significant ions (210% abundance) are given between 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 developmental stage was 4,4-dimethyl (including pentacyclic triterpenoids) or 4monomethyl 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. invicfa 5 8 7 H 5 @ @ 9 HO 10 2 V - Y 17N A y”y B I Fig. 1. C D & H J 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 A2’-24-methyl groups. This side chain grouping is uncommon in the phytosterol 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 (approximately 90% of the total sterols) from both yeast strains (see Materials and Methods) were ergosterol (M’ 396, RRT,, 1.26 on 3% SE-30) and zymosterol (M’ 384, RRT,, 1.13 on 3% SE-30). DISCUSSION Sterols are used by insects, as they are in vertebrates, as essential membrane inserts, in reproduction, as precursors of steroid hormones, and as precursors of defensive secretions (Kircher, 1982).The differences in the structures 6 Ba et al. i I d - 1 6 7 8 91011 Time (min.) i LJ.---f\6.0 1.2 1.1 1.0 .9 .8 .7 .6 .5 5.5 5.0 " 4.5 6 (PPW " Fig. 2. 'H-NMR spectra (bottom t w o spectra) and CLC 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), notably 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. concluded that in certain orders, an early branching of the phylogenetic tree occurred, 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 genera (e.g., s. 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 phytosterols 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 brassicasterol), 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 Cholesta-5,ZZ-dienol Cholesterol Cholest-7-en01 Cholestanol Ergosterol Brassicasterol Ergosta-5,23-dienol Campesterol Ergost-7-en01 Isofucosterol Avenasterol Stigmasterol Stigmasta-7,ZZ-dienol Stigmast-22-en01 Sitosterol Stigmast-7-en01 Stigmastanol Obtusifoliol 24-Methylenelophenol 4a-Methyl24-methylene cholest-8-en01 Citrastadienol Cycloartenol Cycloartanol 24-Methylenelanosterol 24-Methyleneparkeol 24-Methylenecycloartanol Total sterol @@part) Number examined/ Fr. wt. (g) Structure” Era Larva Worker 1A 1B 2B 3B 4D 1D 1E 1G 2G 11 21 1H 2H 3H 1J 2J 3J 5F 6F 7F 0.8 44.8 4.0 N.D. 0.8 12.5 0.7 12.5 0.6 3.4 0.5 0.8 N.D. N.D. 17.4 1.1 N.D. N.D. N.D. N.D. N.D. 50.3 9.3 N.D. 1.2 9.1 3.2 8.2 0.7 55.3 1.o N.D. 0.5 11.2 N.D. 10.6 N.D. 4.3 N.D. 3.4 N.D. N.D. 12.8 0.2 N.D. N.D. N.D. N.D. 61 8B 8C 9F 1OF 8F N.D. N.D. N.D. N.D. N.D. N.D. 0.02 80,000/0.55 0.6 N.D. N.D. N.D. N.D. N.D. 2.44 1,400/0.75 1.o 1.2 1.3 1.4 N.D. N.D. 10.1 3.1 N.D. N.D. N.D. N.D. Queen N.D. 21.7 7.4 2.8 N.D. 3.6 2.3 5.6 2.8 tr 2.4 4.0 tr tr 32.8 11.9 2.7 tr tr tr N.D. tr N.D. tr N.D. tr N.D. tr N.D. tr N.D. tr 24.17 3.18 5,800/ 10.3 420/3.74 ‘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 system 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 Maiduca sexta produces significant amounts of cholesta-5,7-dien-3P-o1 (Svoboda et al., 1994b). The red imported fire ant may reduce the A7-bond of ergosterol, but fails to do so with stigmast7-en-3P-01, which accumulates in the queen. Many of the unusual 4,4-dimethyl 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. Nevertheless, 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 metabolism inhibitors are planned to shed light on the status and function of sterols in S . invicta. 8 Ba et al. LITERATURE CITED Campbell BC, Nes WD (1983): A reappraisal of sterol biosynthesis and metabolism in aphids. J Insect Physiol29:149-156. 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