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Developmental differences in the sterol composition of Solenopsis invicta.

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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.
Corio-Costet MF, Charlet Benveniste P, Hoffman J (1989): Metabolism of dietary A*-sterols
and 9p, 19-cyclopropyl sterols by Locusta migratoria. Arch Insect Biochem Physiol 11:47-62.
Douglas AE (1988): On the source of sterols in the green peach aphid, Myzus persicae, reared
on holidic diets. J Insect Physiol34:403-408.
Houk EJ, Griffiths GW (1980):lntracellular symbiotes of the Homoptera. Annu Rev Ent 25:161-187.
Kalinowska M, Nes WR, Crumley FG, Nes WD (1990): Stereochemical differences in the anatomical distribution of C-24 alkylated sterols in Kalanchoe diagremontiana. Phytochemistry 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 leafcutting ant Acvomyvex octospinous (Reich) (Hymenoptera, F0rmicidae:Attini). Arch Insect Biochem Physiol20:13-21.
Nes WD, Janssen GG, Crumley FG, Kalinowska M, Akihisa T (1993): The structural requirements of sterols for membrane function in Saccharomyces cerevisiae. Arch Biochem Biophys 300~724-733.
Nes WR, Mckean ML (1977): Biochemistry of Steroids 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 implications. 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 Society 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: Academic 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.
and
Ritter KS, Weiss BA, Norrbom AL, Nes WR (1982): Identification of A55’7-24-methylene24-methyl sterols in the brain and whole body of Attu cepkulotes zsthmicola. 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. Washington DC: American Chemical Society Press, pp 203-218.
Svoboda ]A, Lusby WR (1986): Sterols of phytophagous and omnivorous species of Hymenoptera. Arch Insect Biochem Physiol3:13-18.
Sterol Composition of S. invicfa
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 Function. 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.
X u S, Norton RA, Crumley FG, Nes WD (1988): Comparison of the chromatographic proper-
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