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Metabolism of dietary ╬Ф8-sterols and 9╬▓ 19-cyclopropyl sterols by Locusta migratoria.

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Archives of Insect Biochemistry and Physiology 1 1 :47-62 (1 989)
Metabolism of Dietary A*-Sterols and 9p,
19-Cyclopropyl Sterols by Locusta migratoria
Marie E Corio-Costet, Maurice Charlet, Pierre Benveniste, and Jules Hoffman
Laboratoire de Biochimie V6g6tale (M.F.C.-C., l? B.) and Laboratoire de Biologie Gkne'rale (M.C.,
1.H.), Unit6 Associie au Centre National de la Recherche Scientifique, Strasbourg, France
Adult female locusts were reared on wheat seedlings (experimental wheat)
containing more than 97% of 9,lO-cyclopropyl sterols and A'-sterols and less
than 2% of A5-sterols.These insects showed a dramatic decrease in their cholesterol, cholestanol and A7-cholestanolcontent comparedwith control insects.
These changes were more dramatic for steryl esters than for free sterols. Similar results were observed in eggs laid by the insects fed on experimental wheat.
The decrease in A5-, A'- and A7-sterolsin insects reared on experimental wheat
was compensated by a markedaccumulationof A8-sterolsand 9P,19-cyclopropyl
sterols in the free sterol fraction and especially in the steryl ester fractions.
The ecdysteroid content of eggs laid by experimental female insects was reduced
by up to 80% compared with controls. These and other results suggest that
the dietary 9P,19-cyclopropyl sterols and A8-sterols cannot be used by Locusta
in place of A5-sterolsfor ecdysteroid biosynthesis. To give support to this hypothesis, the experimental wheat was supplemented with various sterols before
being presented to the insects immediately after the first egg laying. When
cholesterol, sitosterol, or cholestanol were used as supplements, there was a
complete recovery of the ecdysteroid titer in the eggs, a disappearance of
9p,l9-cyclopropyl sterols, and a restoration of the cholesterol content in both
the animals and the eggs. Stigmasterol, stigmastanol, and ergosterol were much
less efficient in reversing the effects of the experimental wheat. As expected,
9p,l9-cyclopropyl sterols were totally ineffective. These results are discussed
in the light of our information on the role played by sterols in insect development
and on the structural features required by the sterols to fulfill their role.
Key words: Trificumsativum, fenpropimorph,steryl esters, ecdysteroids, 24-methyl pollinastanol
Acknowledgments: This study was supported by the Centre National de la Recherche Scientifique
(grant ATP No. 1885: Relation Plantes-lnsectes). I thank B. Bastian for typing the manuscript.
Received October 11,1988; accepted April 21,1989.
Address reprint requests to Pierre Benveniste, Laboratoire de Biochimie Vegetate, tnstitut de
Botanique, 28 rue Coethe, 67083 Strasbourg, France.
0 1989 Alan R. Liss, Inc.
Corio-Costet et al.
Insects are unable to synthesize de novo sterols and require an exogenous
source of sterols to complete their development [l]. As in most eukaryotic
organisms, sterols are membrane components. In addition, in insects they serve
as precursors for ecdysteroids, which are hormones controlling developmental and reproductive processes [1,2]. It is well established that the phytophagous locust Locustu migrutoriu uses phytosterols such as sitosterol, stigmasterol,
and campesterol as a source of cholesterol, which in turn is metabolized to
ecdysone [1,2]. These typical plant sterols have been shown to be dealkylated,
probably in the midgut, and a pathway has been proposed for this dealkylation
in the case of sitosterol, involving successive desaturation of the 24-28 bond,
epoxidation of the
double bond, and, finally, acid-catalyzed opening
of the epoxide and cleavage of the 24-28 bond [2-41. As yet, the enzymes postulated to be involved in this pathway have never been characterized.
The structural requirements for sterols to be used in insect development
have received great attention. As early as the 1960s a vast array of analogs of
cholesterol were tested as potential cholesterol sparers on Dermestes vulpinus,
a carnivorous beetle [5,6]. From these studies it was concluded that the prerequisites for optimal activity were 1)a generally planar nuclear structure; 2) a
sterol skeleton of the cholestane type with no double bond, one double bond
located at A5 or A7, or two conjugated double bonds (A5&r7);and 3) a P-oriented
3-hydroxy group [5]. Recently, a study performed on a phytophagous insect,
HeIiothis zeu, has given results consistent with the preceding conclusion except
that A5t7-sterolswere shown to inhibit growth of larvae [7]. Development of
Drosophilu melunoguster larvae, which is normal when the larvae are reared on
Sacchuromyces cerevisiue, was shown to be blocked when they are reared on sterol mutant strains of yeast containing cholestane derivatives (erg-6)or As-sterols
(erg-2) in place of ergosterol [8,9]. From these studies a detailed picture of the
structural needs for a sterol to meet the sterol requirements of several insects
is emerging and has been discussed recently [2,10]. However, there are not
studies dealing with the use of 9pI19-cyclopropyl sterols as sterol supplements
to trigger insect development. The question is pertinent since 1)9P,19-cyclopropyl
sterols are ubiquitously found in the plant kingdom and may accumulate in
various physiological circumstances [11,121;2) several studies have shown that
9P,19-cyclopropyl sterols, although structurally very different from normally
occurring A5-sterols,may be used by various organisms in place of the A5-sterols
[13-161. We have shown that long-term treatment of maize or wheat seedlings
with low concentrationsof the systemic fungicides tridemorph or fenpropimorph
leads to an almost complete replacement in these plants of the A5-sterols by
9P,19-cyclopropyl sterols (more than 90% of total sterols) and As-sterols (less
than 10%of total sterols) [17-221. L. migrutoriu were reared on such experimental wheat of defined sterol composition. The treated insects showed a dramatic decrease in their cholesterol content when reared on experimental wheat,
and the females layed eggs with an ecdysteroid concentration reduced by up
to SO%, compared with controls [23]. Also, the ecdysteroid titers in the
hemolymph of fifth instar larvae of Locustu reared on experimental wheat were
Sterol Metabolism in f. migrittoria
drastically reduced [24]. The severe reduction of the ecdysteroid content in
eggs laid by females reared on experimental wheat was associated with a series
of developmental arrests and/or abnormalities [23,25].
In the present study we have developed some aspects of the preceding work
and focused our attention on the fate of the atypical dietary sterols in insects.
The main results of these studies are: 1)9P,19-cyclopropylsterols and As-sterols
are absorbed by the insects; 2) they are extensively esterified; and 3) the defect
in cholesterol formation resulting from the presence of atypical sterols in the
diet leads to a strong decrease in ecdysteroid content of the eggs (this, however, can be reversed by supplementation with sterols of appropriate structure).
Plant Material and Insects
Wheat (Triticum sutivum) caryopses were purchased locally. For some experiments the variety Etoile de Choisy (R.A.G.T., Rodez, France) was used. The
caryopses were allowed to germinate in vermiculite, and the seedlings were
watered daily with a solution of fenpropimorphin water (5mgfliter). The plants
were presented to the insects after 14 days. An aliquot fraction was analyzed
to determine the sterol profile. In some experiments the treated wheat was
supplemented by exogenous sterols by spraying the wheat with a solution of
sterols in dichloromethane (3.3 mM). L. migrutoriu migrutorioides were reared
in phase greguriu at a day temperature of 28-30°C, falling to 25°C at night. Electric lights inside the cages allowed temperature gradients up to 38°C. Relative
humidity was around 70%.The light/darkcycle was 12:12. Females were reared
on experimental wheat immediately after emergence and were kept on this
diet until the first egg laying.
Chemicals and Dietary Sterols
Fenpropimorph (4-[3-(4-butylphenyl)-2-methyl]
propyl-2,6-dimethylmorpholine) was a gift of Dr. H. Pommer (BASF, Limburgerhof, F.R.G.). The
cyclopropyl sterols* used as standards were extracted from maize or wheat
seedlings treated with tridemorph or fenpropimorph [17-201. The dietary sterols used in these experiments were examined by GC-MS: cholesterol (Sigma
Chemical Company, St. Louis, MO; 99% pure); 5a-cholestan-3P-ol (AldrichChimie, Strasbourg, France; 99% pure); ergosterol (Aldrich, 98% pure); stig*The systematic names for the sterols used in this study are as follows: cholesterol (7), cholest5-en-3p-ol;cholestanol (2), 5a-cholestan-3P-01; desmosterol ( 3 ) ,choIesta-5,24-dien-3P-ol; 7dehydrocholesterol ( 4 ,cholesta-5,7-dien-3P-o1; lathosterol (5), 5a-cholest-7-en-3p-ol;
24-methylene cholesterol (6),ergosta-5,24(28)-dien-3P-ol; 24-methyl cholesterol (7), 24-methylcholest-5-en-3p-ol; 24-methyl cholestanol (8), 24-methyl-5a-cholestan-3P-ol; stigmasterol (9),
(24S)-24-ethyI-choIesta-5,E-22-dien-3P-ol; sitosterol(77),(24R)-24-ethyl-choIest-5-en-3P-ol;isofucosterol (72), stigmasta-5,Z-24(28)-dien-3P-ol; pollinastanol (75),14a-methyl-9P,19-cyclo-5acholestan-3P-01; 24-methyl pollinastanol(78), 14a,24-dimethyl-9~-19-cyclo-5a-cholestan-3~-ol;
24-ethylpollinastanol(20),14a-methyl,24-ethyl-9P,19-cyclo-5a-cholestan-3P-ol; cycloeucalenol
(27), 4,14a-dimethyl-9~,19-cyclo-5a-ergost-24(28)-en-3~-ol;24-dihydro-cycloeucalenol(22),
31-norcyclobranol(23), 4,14a-dimethyl-9P,19cyclo-5a-ergost-24-en-3P-ol;
stigmastanol (24), 5a-stigmastan-3P-01.
Corio-Costet et al.
masterol (Fluka AG, Buchs, Switzerland; 95.5% pure); sitosterol (Fluka AG;
81% sitosterol, 11%isofucosterol, 6% campesterol, and 2% stigmasterol);
stigmastanol (Fluka AG) was in fact shown to contain 70% 24-ethyl-5acholestan-3P-01and 30% 24-methyl-5a-cholestan-3~-ol.
Isolation and Identification of Sterols and Steryl Esters
An identical procedure was applied to the roots and leaves from wheat seedlings and to the insects and their eggs. The tissues were harvested, frozen,
and lyophilized; they were homogenized in the presence of dichloromethanemethanol (2:l). After evaporation of the solvent, the residue was saponified
using KOH (6% w/v) in methanol. This step was omitted when steryl esters
were recovered. The unsaponifiable material was extracted three times with
hexane, and the pooled extracts were dried. The residue was chromatographed
on Merck HF 254 TLC plates (0.2mm) with dichloromethane as the developing solvent (two runs). The bands of 4,4-dimethyl-sterols, 4a-methyl-sterols,
and 4-desmethyl-sterols were scraped off and each type eluted. The three classes
of compounds were acetylated at room temperature for 14 h using a mixture
of pyridine-acetic anhydride-toluene (1:2:2).The crude acetates were purified
by TLC using dichloromethane as the developing solvent. The acetates were
analyzed by GC using an FID and a glass capillary column (30 m x 0.25 mm)
coated with OV-1 (J & W Scientific, Folsom, CA). The temperature program
included a fast rise from 60 to 240°C (30°C min-l) and then a slow rise from
240°C to 280°C (2°C mir -I). An internal standard of cholesterolwas used. GC-MS
was carried out at an ionizing energy of 70 eV. The separations were performed
on a capillary column (25m x 0.25 mm) coated with SE 30 (Spiral,Dijon, France).
Most mass spectra have been given previously [19]. PMR**spectroscopy was
carried out in CDC13solution on a Bmcker 200 MHz spectometer (Wissembourg,
France). The spectra of the products considered in this paper have been detailed
elsewhere [17,18,20].
Extraction and Separation of Ecdysteroids
Eggs and animals were homogenized in ethanol-water (60:40), heated for
60°C for 15min, and centrifuged at 800g for 10 min. The supernatant was dried
under reduced pressure or under NZ. The dried extracts were dissolved in
ethanol-water. Ecdysteroids were separated by TLC on precoated silica gel 60F
254 plates (Merck) in a chloroform-ethanol (80:20) solvent system (two runs).
The products were eluted by 5 mm bands with ethanol-water (95:5), except
for the three bands close to the origin which were eluted with the more polar
solvent ethanol-water (60:40). Aliquots of the polar fractions were submitted
to enzymatic hydrolysis for 18 h at 37°C in 50 mM acetate buffer (pH 5.3) that
contained semipurified p-glucuronidase (5,000U/ml) from Helix pomutiu (Sigma
No. G0751). This enzyme also contained sulfatase and phosphatase activities.
After hydrolysis, the free ecdysteroids were extracted with chloroform-ethanol
**Abbreviations used: ACAT = acyl CoenzymeA:cholesterol acyltransferase; FID = flame ionization detector; CCMS = gas chromatography-mass spectrometry; PMR = proton nuclear
magnetic resonance spectroscopy; RIA = radioimmunoassay; RRT = retention time relative
to cholesterol.
Sterol Metabolism in 1. migratoria
(80:20) [26]. The molecules comigrating with 2-deoxyecdysone and ecdysone
standards were eluted with ethanol-water (95:5) and subjected to RIA
The dried extracts were suspended in 0.1 M citrate buffer (pH 6.2), and
[23,24-3H]ecdysone (9,000 cpm, 40 CUnmol [l Ci = 37 GBl]) was added to
each sample (radiolabeledecdysone was generously provided by Prof. P. Karlson,
Marburg, F.R.G.). This solution was dialyzed against an antiecdysone (oximethyroglobulin) antibody diluted 1:4,000 (antibody generously provided by Dr.
Reum, Dietzenbach, F.R.G.) and referred to as black [27]. The technique was
devised by De Reggi et al. [28]. Under these conditions, the concentration
of ecdysone required for 50% inhibition of [23,24-3H]ecdysone binding
was 10 nM. Cross reactions between 2-deoxyecdysone, ecdysone, and 20hydroxyecdysone were 1:l:O. 05.
Total (Free and Esterified) Sterol Composition of Insects Reared on
Experimental Wheat
In the experimental plants used in the present work, the 9P,19-cyclopropyl
sterols represented more than 90% of the total sterols and consisted essentially of 24-methyl pollinastanol, cycloeucalenol, dihydro-cycloeucalenol, and
31-norcyclobranol.The A'-sterols, primarily 24-ethyl-5a-cholest-8-en-3p-01
represented 5%of the total sterols. The A5-sterols
(sitosterol, stigmasterol, campesterol) were less than 2%. No cholesterol was
detected in either control or experimental plants. These results are in agreement with published data [20]. The sterol content in the treated females was
analyzed after their first egg laying. As shown in Figure 1, the sterol (free
and esterified) content was altered in animals reared on experimental wheat.
The major result is that experimental females had a decreased content in cholesterol (1) and 5a-cholestan-3P-01 (2) and high titers of A8-sterols and
9~,19-cyclopropylsterols, which were undetectable in controls. As-Sterols were
essentially in the form of 5a-cholest-8-en-3P-01 (14), with trace amounts of
(16) and 24-ethyl-5a-cholest-8-en3P-01
9P, 19-cyclopropyl sterols were mainly represented by 24-methyl pollinastanol
(18); in addition, small amounts of pollinastanol (15) were detectable. Separate determinations in the gut (which contained wheat) and the carcass of the
insects showed no major differences in the sterol profiles (namely, as regards
the ratio of 24-methyl pollinastanol to cholesterol), indicating that these profiles were not significantly affected by the sterols present in the gut.
Composition of Free Sterols and Steryl Esters
The analyses described above were performed after saponification of the
sterol extract. We were, therefore, unable to estimate the contribution of the
steryl esters in both experimental and control insects. This is a crucial point,
since steryl esters are quantitatively important and metabolically very active
Corio-Costet et al.
Fig. 1. GC profiles of Locusta migratoria. A: Sterol composition of insects reared on control
wheat. B: Sterol composition of insects reared on experimental wheat. All the sterols were
acetates of cholesterol (I), cholestanol (2), desmosterol (31, 7-dehydrocholesterol (4,lathosterol (3,24-methylene cholesterol (6), 24-methyl cholesterol (3,24-methyl cholestanol (8),
stigmasterol (9), 24-methyl-5a-cholest-7-en-3P-ol (IO),sitosterol (II), isofucosterol (72),24ethyl-5a-cholest-7-en-3P-ol (I3),5a-cholest-8-en-3P-oI(14, pollinastanol (75),24-methyl-5~~cholest-8-en-3P-ol (I6), 24-methyl pollinastanol (I8), 24-ethyl-5a-cholest-8-en-3P-ol (I9),
24-ethyl pollinastanol (20).The sterols were analyzed after saponification of the extract.
in other organisms, such as yeast or vertebrates [29]. Therefore, in the following experiments the steryl esters were separated from the free sterols, and
both fractions were studied separately. Analyses performed on whole animals
and on their eggs are presented in Table 1. From Table 1it is clear that 1)there
is an accumulationof 9p,l9-cyclopropyl sterols (15,18) and of As-sterols (14,16,29)
at the expense of A5- and A7-sterolsin experimental insects (these atypical sterols are present in free and esterified forms);2) whereas most of A5- and As-sterols
are not alkylated at C-24, 9p, 19-cyclopropyl sterols are essentially 24-methyl
sterols (this feature was observed in both free and esterified sterols); 3 ) in general, whole animals are richer in esters than are eggs; and 4) esters are much
poorer in A5- and A7-sterolsand richer in cyclopropyl and A'-sterols than are
Sterol Metabolism in f. rnigraforia
TABLE 1. Free and Esterified Sterols in Locustu migraforia (Female adults and eggs) Reared
With Normal (Column A) and Exuerimental (Column B) Wheat
Free Steryl Free Steryl Free Steryl Free Steryl
sterols esters sterols esters sterols esters sterols esters
(Ad ( ' 4 2 )
(A31 (A41 (Bl) (82) (B3) (B4)
81a 56
81.5 57.5
Cholesterol ( 1 )
4 14
_ _ - 20
1.6 1.8
4.5 3
Lathosterol (5)
_ _ - 2
24-Methylene cholesterol (6)
- - _ 0.4
1.2 1.4
5.5 3.5
0.7 0.7
Stigmasterol (9)
24-Methyl pollinastanol(Z8)
- _ - 20
1.2 0.4
8 13
13 22
0.5 31-Norcyclobranol(Z3)
94 78
0.5 7
4 14
_ _ - 20
1.5 2
7.5 3
Cyclopropyl sterols
0.5 57
Free sterols
Stervl esters
Sterols (pg)/insect or egg
1,230 600
1.3 1,000 750
21.2 36
0.5 7
2.5 4
7.5 0.45
"Values are percentages of total sterols.
bThe values in this row are the sums of the values for 21,22, and 23.
the free forms (Table 1, columns B1 and B2). Esters from whole animals are
also poorer in As-sterols (Table 1, colums B1 and B2). In conclusion, the changes
in sterol content of experimental animals or eggs are stronger in the ester than
in the free sterol fraction. Previous analyses performed on the hemolymph of
females reared on experimental and normal wheat have shown that the major
difference between the two was a 90% reduction of the titer of cholesterol and
an accumulation of 5a-cholest-8-en-3P-01 and of 24-methyl pollinastanol(18)
in the experimental hemolymph [23]. The present studies show an addition
that steryl esters are absent in the blood from control animals and present in
trace amounts in experimental animals.
Ecdysteroid Composition in Eggs of Experimental Females
According to previous data [23], the experimental diet also induces a reduction in the ecdysteroid titer in the eggs. Females reared on normal diet to the
Corio-Costetet al.
first egg laying were given exclusively experimental diet for five successive
gonotrophic cycles. The ecdysteroid content declined rapidly; during the
last cycle, less than 25% of the normal ecdysteroid content per egg was
present. Larvae reared on experimental wheat also contained strongly reduced titers of ecdysteroids [24]. These drastic effects did not result from
the fungicide (fenpropimorph) used to alter the sterol content of wheat:
Indeed, injecting fenpropimorph into the insects, or feeding them wheat
coated with the fungicide but with a normal sterol composition, did not
affect their sterol or ecdysteroid profiles [23]. The severe reduction of the
ecdysteroid content in oocytes [25], in eggs [23], and in larvae [24] was
shown to be associated with a series of developmental arrests and/or abnormalities [23-251.
Effect of supplementationof experimental wheat with exogenous sterols. The
above results suggested that the decline in ecdysteroids in eggs from insects reared on experimental wheat was due to a lack of precursors for
ecdysteroid biosynthesis in the insects. In support of this hypothesis, the
following experiment was devised: Females were reared on experimental
wheat immediately after emergence and were kept on this diet until the
first egg laying. The eggs were collected, and their ecdysteroid titer was
determined. During the following gonotrophic cycles, the insects were reared
on experimental wheat that was supplemented by spraying the seedlings
with a solution of various sterols in water-ethanol (4:l). The eggs laid during the subsequent cycles were collected, and the ecdysteroids were measured by RIA.
Effect of supplementation on ecdysteroid content of eggs. The results of an
initial series of experiments (Table 2) show that addition of cholesterol to
the experimental wheat restored the normal ecdysteroid level after two additional gonotrophic cycles (i.e., 7-8 days). The addition of sitosterol led
to similar effects but there was not a total recovery of the ecdysteroid titer.
In the case of supplementation with stigmasterol, the recovery was still lower.
In a second series of experiments, the effect of the classical A5-sterols was
compared with that of other sterols absent from the plant diet. Cholestanol
and stigmastanol were chosen to examine whether the absence of double
bonds in the sterol skeleton has an effect on recovery of the ecdysteroid
titer; ergosterol was used because hz7-sterols are considered to play an important role during ecdysteroid biosynthesis [1,2]; cyclopropyl sterols were
used because they were expected to be ineffective. Whereas cholesterol,
5a-cholestanol, and sitosterol led to complete recovery of ecdysteroid content,
stigmasterol and ergosterol were much less efficient (Table 3). Finally, females
were not able to produce a second egg pod when reared on experimental
wheat supplemented with either 5a-stigmastan-3P-o1(24)or 9P, 19-cyclopropyl
sterols after the first egg laying; this suggests that these two sterols are
totally unable to restore the ecdysteroid titer.
Effect of supplementation on sterol content of eggs and insects. The eggs
used for ecdysteroid analysis were also used for the determination of sterol
composition. After the last egg laying, the insects were killed and their sterol
contents were measured. The results (Table 4) indicate that 1) supplemen-
Sterol Metabolism in L. migraforia
TABLE 2. Ecdysteroids in Eggs Laid by Female Adults of Locusta migratoria Reared
on Normal (Column A) Wheat and on Experimental Wheat Supplemented With Exogenous
Sterols (Column B)
RIA measuremenp
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented with
cholesterol after the first egg laying
Third egg pod; experimental wheat supplemented with
cholesterol after the first egg laying
Fourth egg pod; experimental wheat supplemented with
cholesterol after the first egg laying
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented with
sitoserol after the first egg laying
Third egg pod; experimental wheat supplemented with
sitosterol after the first egg laying
Fourth egg pod; experimental wheat supplemented with
sitosterol after the first egg laying
cirst egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented with
stigmasterol after the first egg laying
Third egg pod; experimental wheat supplemented with
stigmasterol after the first egg laying
Fourth egg pod; experimental wheat supplemented with
stigmasterol after the first egg laying
1.57 i 0.07
1.68 0.04
1.70 * 0.05
1.54 * 0.08
0.42 i 0.06
1.38 0.18
1.57 i 0.22
1.57 0.07
1.68 i 0.01
0.70 & 0.11
1.12 i 0.20
1.70 i 0.05
1.26 i 0.22
1.54 f 0.08
1.57 i 0.07
1.68 0.04
0.46 i 0.08
0.94 i 0.10
1.70 * 0.05
1.54 * 0.08
* 0.09
0.84 i 0.08
1.02 i 0.10
“RIA measurements are expressed in nmole equivalents of synthetic ecdysone per egg. Each
value represents the mean of at least four separate measurements.
tation with cholesterol leads to an increase in the cholesterol content of
eggs, while the titer of cyclopropyl sterols decreases; 2) supplementation
with cholestanol leads to effects similar to those of cholesterol, except that
there is an accumulation of A’-sterols; 3 ) sitosterol is almost as efficient as
cholesterol in increasing A5-sterol titer and in decreasing cyclopropyl sterols;
4)ergosterol is much less active than the three preceding sterols; 5) stigmasterol has almost no effect on either the A5-sterol or the cyclopropyl sterol composition; and 6) as stigmastanol (24) and cyclopropyl sterol addition do not
permit a second egg laying, it is expected that these sterols do not lead to
either recovery of cholesterol or decrease of cyclopropyl sterols. The sterol content of female insects has also been determined before and after supplementation with exogenous sterols. The results (Table 5) are essentially parallel to
those obtained with eggs: 1)cholesterol and sitosterol lead to recovery of the
cholesterol content and to a decrease in the cyclopropyl sterol and A*-sterol
content; 2) cholestanol leads to a decrease in cyclopropyl sterol and As-sterol
content (in addition, there is an accumulation of this stanol in insects); and 3)
stigmasteroland stigmastanol seem to have intermediary effects. In both cases
the titer of cyclopropyl sterols decreases significantly, although the level of
Corio-Costet et al.
TABLE 3. Ecdysteroids in Eggs Laid by Locusta migratoria Reared on Experimental Wheat
Sumlemented With Exoeenous Sterols
Ecdysteroid content"
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented with
cholesterol after the first egg laying
Third egg pod; experimental wheat supplemented with
cholesterol after the first egg laying
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented with
sitosterol after the first egg laying
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented with
stigmasterol after the first egg laying
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented with
cholestanol after the first egg laying
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented with
ergosterol after the first egg laying
Third egg pod; experimental wheat supplemented with
ergosterol after the first egg laying
"Percentage of ecdysteroid present in L. rnigratwia reared on normal wheat. In these experiments the
ecdysteroid content in control eggs was 2.85 t 0.15 nmol equivalent of synthetic ecdysone per egg.
TABLE 4. Sterol Composition of Eggs From Insects Reared on Experimental Wheat
Supplemented With Sterols
Sterol supplements
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented
after the first egg laying
Third egg pod; experimental wheat supplemented
after the first egg laying
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented
after first egg laying
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented
after first egg laying
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented
after first egg laying
First egg pod; experimental wheat nonsupplemented
Second egg pod; experimental wheat supplemented
after first egg laying
Third egg pod; experimental wheat supplemented
after first egg laying
Sterol Metabolismin L. migratoria
TABLE 5. Sterol Compositionof Female Insects Reared on Experimental Wheat
nonsupplemented and Supplemented With Sterols
Sterol supplements
Experimental wheat nonsupplemented
Experimental wheat supplemented with Cholesterol”
Cyclopropyl sterolsb
34 33.5 4.5
”Theinsects were sacrificed after the last egg laying.
bThe insects were sacrificed 4 days after supplementation.
cholesterol remains comparable to that of experimental insects that were
not supplemented. In both cases significant amounts of the untransformed
sterols used for supplementation are recovered. In the case of complementation with stigmastanol, the accumulation of stanols is remarkable (Table 6).
Ergosterols do not lead to either recovery of cholesterol or decrease in cyclopropyl sterols. Finally, cyclopropyl sterols have no effect on the cholesterol
TABLE 6. Detailed Sterol Composition of Female Insects Reared on Experimental Wheat
Supplemented With Cholesterol (Column A), Stigmasterol (Column B), and
Stigmastanol (Column C)
Cholesterol (1)
Lathosterol (5)
24-Methyl Cholesterol (7)
Stigmasterol (9)
24-Methyl pollinastanol(18)
24-Ethyl pollinastanol(20)
Total sterols
(mg/g dry weight)
Percent of total sterols
Determination of sterol content was done after saponification of the extract.
Cholesterol + 5a-cholestan-3~-01.
Corio-Costet et al.
Insects reared on experimental wheat have a modified sterol profile in comparison to control insects. The present results indicate that the 9p,19-cyclopropyl
sterols found in the experimental insects are mainly methylated at position
C-24, whereas As- and A5-sterols are essentially dealkylated at C-24. This
shows that, in contrast to As-sterols, 9p, 19-cyclopropylsterols are not substrates
of the sterol-C-24-dealkylating enzymatic complex. It may be that the
dealkylation process is hampered by the presence of the C-14 methyl group
in 9~,19-cyclopropylsterols. Also, some conformational differences between
cyclopropyl sterols and A5-sterolscould be invoked, although the conformation
of cycloartenol in powder state and in solution has recently been shown to be
planar [30,31]. The fact that As-sterols are dealkylated is consistent with recent
data showing that A7- and A'-sterols are metabolized in this manner [7].
Previous studies have shown that insects readily esterify sterols [2]. Our
results are in agreement with these data, since we find in both control and experimental animals that steryl esters represent 30 to 40% of total sterols. In contrast, the steryl ester content of the eggs is much lower (less than 10%). A
remarkable feature is that in both eggs and whole animals the steryl ester fraction is richer in 9p,19-cyclopropylsterols and A'-sterols and poorer in A5-sterols
than the free sterol fraction (Table 1).These results lead to the following
1. The presence of a large amount of steryl esters indicates that an enzyme
catalyzing esterification is present in the insects. This enzyme could be ACAT,
since ACAT activity has been found in the fat bod of Heliothis zed [32].
Interestingly, this enzyme seems to be nonspecific for A -sterols, since the ester
fraction contains more cyclopropyl sterols than A5-sterols. However, it has been
shown in H . zea that the percentage of esterification of sitosterol and stigmasterol was much smaller than that of cholesterol and other C-27 sterols [7].
2. The relatively low level of A5-steryl esters could be consistent with the
fact that free A5-sterols would be both metabolically active and vital to membranes. In contrast, the high level of cyclopropyl sterols and of C-24 alkylated
sterols in the esterified fractions suggest that these sterols may not be important as structural components of cells or as ecdysone precursors or could even
be toxic. In this last assumption, the esterification reaction could be considered as an elimination or even a detoxification reaction. Such an interpretation does not exclude the possibility that steryl esters could be storage forms,
the hydrolysis of which would provide the insects with free sterols to serve as
structural components of cells during periods of rapid growth [2,32,33]. As
these molecular species are nearly absent from the hemolymph, in which free
sterols are bound to a protein complex [34], it is unlikely that they could play
a role in sterol transport in contrast to the case of vertebrates, in which esters
constitute the bulk of the circulating sterols [29].
As shown in our previous study, the ecdysteroid content of eggs laid by
experimental female insects is reduced by up to 80% compared with controls
Sterol Metabolism in 1. migraforia
[23]. The reduction of the ecdysteroid content in eggs is associated with a series
of developmental alterations [23-251. These and other results suggest that dietary
9p,l9-cyclopropyl sterols and As-sterols cannot be used by the insects in place
of A5-sterols for ecdysteroid biosynthesis. To give support to this hypothesis,
the experimental wheat was supplemented with various sterols before being
presented to the insects immediately after the first egg laying. Cholesterol,
cholestanol, and sitosterol led to almost complete recovery of the ecdysteroid
titer in the eggs. In addition, these sterols led to restoration of the cholesterol
content and to a marked decrease in 9P,19-cyclopropyl sterols in both female
insects and eggs (Tables 4,and 5). Correlatively, embryonic development was
also restored. These results are in agreement with our assumption. They show
that the developmental modifications are essentially due to a sterol and/or
ecdysteroid defect in animals and their eggs and that this defect does not lead
to irreversible physiological effects. In addition, the very large and rapid recovery of cholesterol content as well as the decrease of cyclopropyl sterol content
in both animals and eggs show that the turnover of these sterols is rather high.
For instance, in the case of sitosterol supplementation, the cholesterol content of eggs increases twofold whereas that of 9p,19-cyclopropyl sterols is reduced
by almost 80% after 4 days. The efficiency of cholestanol in restoring embryonic
development is surprising, since in this case the cholesterol content is slightly
increased in eggs and is not modified in whole animals, while large amounts
of cholestanol are incorporated in both cases. However, the bioavailability of
this sterol is attested to by the significant decrease in the 9P,19-cyclopropyl
sterol content (twofold in both cases) and by the increase in the ecdysteroid
titer. To explain these data, one can suggest that cholestanol is acceptable as a
structural component of membranes but can also be metabolized to cholesterol and to ecdysteroids. Examples of metabolism of A'- to A5-sterols are very
scarce [35]. In the case of the larvae of H . zeu, it was shown that cholestanol
was able to support the growth of the larvae but was only poorly metabolized
[36]. Previously, cholestanol was also shown to support the growth of the locust
Schistocercu greguriu [37]. Stigmasterol is less efficient than the preceding sterols as a supplement (Tables 2,3). In particular, stigmasterol was shown to be
ineffective in changing the content in A5-sterolsand 9P,19-cyclopropyl sterols
in both eggs (Table4)and whole insects (Table 5). In the latter, a large amount
of stigmasterol was incorporated. Therefore, our results show that stigmasterol was probably absorbed by the insects but was not metabolized. In particular, stigmasterol seemed not to be dealkylated. Our results are in agreement
with data obtained previously on L. rnigrutoriu and S. greguriu reared with a
synthetic food supplemented with stigmasterol [6,37], but are in contrast to
data from the literature showing that stigmasterol can serve as a sterol supplement in phytophagous insects and is dealkylated to cholesterol via cholesta5,22,24-trien-3P-01[37,38].
To explain this discrepancy, we suggest that the dealkylating enzymatic system of locusts is more specific than that of other insects and cannot accommodate the rather rigid conformation of the stigmasterol side chain conferred
by the E-A22 double bond. Another possibility would be the absence of the
enzymatic system capable of reducing the A22 double bond in cholesta-
Corio-Costet et al.
5,22,24-trien-3P-ol. Stigmastanoldoes not affect either the ecdysone or the sterol content. This result does not agree with literature data obtained on Tenebrio
molitor [39]. According to our results this sterol seems to be dealkylated to
some extent (Table 5), but the cholestanol produced is probably not sufficient
to affect ecdysone synthesis and cyclopropyl sterol turnover. The results with
ergosterol supplementation show that this sterol seems to increase the
ecdysteroid titers partially and to allow further laying of eggs, but does not
produce any change in sterol content in either eggs or whole animals and is
not even incorporated in the animals. Finally, 9P, 19-cyclopropylsterols are ineffective in increasing the content of A5-sterols in both eggs (Table 4) and whole
insects (Table 5).
The biological model described here shows that a deficiency in normal sterols and an accumulation of sterols that insects cannot use for normal ecdysteroid
biosynthesis can be obtained experimentally. This deficiency can be totally or
partially compensated by supplementationof diet with exogenous sterols. Therefore, the structural requirements for sterols to fulfill their roles in insect development can be determined with confidence. The present study gives evidence
that 9P,19-cyclopropyl sterols, which are usually present in small amounts in
higher plants but may accumulate in large amounts under certain physiological circumstances [11,12] or after treatment with some fungicides [17-201, cannot replace dietary A5-sterolsas ecdysteroid precursors. An unresolved question
is whether A'-sterols, which also accumulate in experimental insects, can be
used as precursors of ecdysteroids or as A5-sterolsubstitute in membranes. To
answer this question it would be necessary to use 24-ethyl-5a-cholest-8-en-3P-01
as a supplement in the experimental wheat and to see whether the normal
ecdysteroid titer is recovered in the blood or in the eggs.
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cyclopropyl, metabolico, dietary, locusta, migratoria, sterol
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