Metabolism of dietary ╬Ф8-sterols and 9╬▓ 19-cyclopropyl sterols by Locusta migratoria.код для вставкиСкачать
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. 48 Corio-Costet et al. INTRODUCTION 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 . 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 . 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 . Also, the ecdysteroid titers in the hemolymph of fifth instar larvae of Locustu reared on experimental wheat were Sterol Metabolism in f. migrittoria 49 drastically reduced . 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). MATERIALS AND METHODS 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), 4,14a-dimethyl-9~,19-cyclo-5a-ergostan-3~-ol; 31-norcyclobranol(23), 4,14a-dimethyl-9P,19cyclo-5a-ergost-24-en-3P-ol; stigmastanol (24), 5a-stigmastan-3P-01. 50 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 . 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 51 (80:20) . The molecules comigrating with 2-deoxyecdysone and ecdysone standards were eluted with ethanol-water (95:5) and subjected to RIA measurements. Radioimmunoassay 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 . The technique was devised by De Reggi et al. . 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. RESULTS 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 and 24methyl-5a-cholest-8-en-3~-ol, 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 . 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 24-methyl-5a-cholest-8-en-3~-ol (16) and 24-ethyl-5a-cholest-8-en3P-01 (19). 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 52 Corio-Costet et al. , 19 A 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 . 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 53 TABLE 1. Free and Esterified Sterols in Locustu migraforia (Female adults and eggs) Reared With Normal (Column A) and Exuerimental (Column B) Wheat A B Insects Eggs Insects Eggs 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) PDesmethylsterols 81a 56 81.5 57.5 40 13.7 Cholesterol ( 1 ) 4 14 3 14.5 0.5 7.2 Cholestanol(2) 5a-Cholest-8-en-3P-o1(14) _ _ - 20 9.2 1.6 1.8 2 4.5 3 12 Lathosterol (5) _ _ - 2 3.8 Pollinastanol(15) 24-Methylene cholesterol (6) - - _ 0.4 1.2 1.4 24-Methylcholesterol(7) 4 5.5 3.5 5 0.3 24-Methy1-5a-cholest-8-ene-3P-o1(16) 1 3 2 0.7 0.7 Stigmasterol (9) 24-Methyl pollinastanol(Z8) - _ - 20 37.5 1.2 0.4 8 13 7 11 Sitosterol(11) 1.6 24-Ethy1-5a-cholest-8-en-3P-o1(19)24-Methyl-5a-cholest-7-en-3~-ol(10) 3 4a-Methylsterols Cycloeucalenol(21) 13 22 24-Dihydro-cycloeucalenol(21) Trb 0.5 31-Norcyclobranol(Z3) A5-Sterols 94 78 43 16.5 94 73.5 0.5 7 A'-Sterols 4 14 3 14.5 _ _ - 20 11 A8-Sterols 1.5 2 A7-Sterols 2 7.5 3 12 Cyclopropyl sterols 35 63.5 0.5 57 Free sterols 67 90 43 Stervl esters 33 9 Sterols (pg)/insect or egg 1,230 600 13 1.3 1,000 750 37 0.5 2 2 4 13.5 7 3 1 1.5 1.5 0.3 2 1 27 28 6.5 2 1 1 - 21.2 36 41 23 0.5 7 2.5 4 1 2 54 65 94.5 5.5 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 . 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 , the experimental diet also induces a reduction in the ecdysteroid titer in the eggs. Females reared on normal diet to the 54 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 . 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 . The severe reduction of the ecdysteroid content in oocytes , in eggs , and in larvae  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 55 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 A B Cholesterol 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 Sitosterol 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 Stigmasterol 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 * 1.60 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 1.31 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 56 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 34 68 97 56 108 46 56 41 95 63 81 49 "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 Cholesterol 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 Cholestanol First egg pod; experimental wheat nonsupplemented Second egg pod; experimental wheat supplemented after first egg laying Sitosterol First egg pod; experimental wheat nonsupplemented Second egg pod; experimental wheat supplemented after first egg laying Stigmasterol First egg pod; experimental wheat nonsupplemented Second egg pod; experimental wheat supplemented after first egg laying Ergosterol 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 As A7 Cyclopropy1 sterols 49 80 6 4 7 2 38 14 92 2 1 5 45 60 17 4 3 6 45 19 36 76 10 10 2 4 52 10 48 50 14 9 2 4 36 38 28 50 4 20 2 2 66 38 46 6 4 44 A5 A' Sterol Metabolismin L. migratoria 57 TABLE 5. Sterol Compositionof Female Insects Reared on Experimental Wheat nonsupplemented and Supplemented With Sterols Sterol supplements A5 A’ Experimental wheat nonsupplemented Experimental wheat supplemented with Cholesterol” Cholestanol“ Sitosterol” Stigmasterol” Ergosterol” Stigmastanolb Cyclopropyl sterolsb 32 3 16 tr 82 tr 3634 1 68 8 2 65 2 30 34 33.5 4.5 4 38 A7 Cyclopropy1 sterols 1.5 tr 4 2 2 2 2 2 47.5 18 25 22 31 66 26 56 A’ ”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 content. TABLE 6. Detailed Sterol Composition of Female Insects Reared on Experimental Wheat Supplemented With Cholesterol (Column A), Stigmasterol (Column B), and Stigmastanol (Column C) A Cholesterol (1) 5a-Cholestan-3P-01(2) 5a-Cholest-8-en-3P-o1(14) Lathosterol (5) Pollinastanol(15) 24-Methyl Cholesterol (7) Ergosta-5,24(28)-dien-3P-01(6) 24-Methy1-5a-cholest-8-en-3P-o1(16) Stigmasterol (9) 24-Methyl-5a-cholestan-3P-o1(8) 24-Ethyl-5a-cholestan-3P-01 24-Methyl pollinastanol(18) Sitosterol(11) 24-Ethyl-5a-cholest-8-en-3P-01(19) 24-Ethyl pollinastanol(20) Total sterols (mg/g dry weight) 85 5 0.5 0.5 0.5 0.5 - Percent of total sterols B C 29 - 2 2 1.5 2 - 30 - - 7.0 0.5 0.5 - 29 4 4.3 - 0.5 3.0 Determination of sterol content was done after saponification of the extract. Cholesterol + 5a-cholestan-3~-01. 30 9 3.5 2 3.5 1.5 - 10.5 14 22.5 2.5 1 - - 58 Corio-Costet et al. DISCUSSION 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 . Previous studies have shown that insects readily esterify sterols . 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 considerations. 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 . 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 . 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 , 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 . Y 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 59 . 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 . 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 . Previously, cholestanol was also shown to support the growth of the locust Schistocercu greguriu . 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- 60 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 . 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. 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