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Composition of Sojenopsis Invicta Amadou S 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 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 12% of the total sterol mixture) deteded in eggs, larvae, and workers was 24-methyl cholesta-5,22E-dien-3/3-01 (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 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 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 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. Lathosterol (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 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)-methylene parkeol and 24(28)-methylene lanosterol. 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 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
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