Archives of Insect Biochemistry and Physiology 1:281-296 (1984) Metabolism of A '-, A5-,and A7-Sterols by Larvae of Heliothis zea Karla S . R i t t e r Department of Biological Sciences, Drexel University, Philadelphia Heliothis zea was reared on artificial diets containing A5-sterols (cholesterol, campesterol, or sitosterol), A'-sterols (lathosterol, epifungisterol, or spinasterol), or A'-sterols (cholestanol, epicoprostanol, carnpestanol, or sitostanol) in order to determine how different dietary sterols affect the type of sterols present in the tissues of the late-sixth-instar larva. Although all of the dietary sterols (except epicoprostanol) supported the growth of the larvae, not all of the sterols were metabolized to the same end products. I n each case, at least 80% of the sterols in the tissues of the larvae retained the same nucleus as that of the dietary sterol, indicating that H. zea carries out very little metabolism of ring B of A5-, A7-, and A'-sterols. The larvae dealkylated the A'-, A'-, and A'-alkylsterols to 24desalkylsterols, but a greater percentage of the A5-alkylsterols were metabolized in this manner. The sterols present as free sterols i n the larva were also present as esterifed sterols which accounted for 2-4% of the total sterols. Therefore, the sterol composition of the tissues of H. zea can be altered by varying the dietary sterols. Key words: Heliothis zea, cholesterol, campesterol, sitosterol, lathosterol, epifungisterol, spinasterol, cholestanol, epicoprostanol, campestanol, sitostanol INTRODUCTION Heliothis zea is similar to other insects in that exogenous sterols are essential for its growth and maturation. Although cholesterol* is a satisfactory dietary *The systematic names for the sterols used in this study are as follows: cholesterol, cholest5-en-3P-01; campesterol, 24a-methylcholest-5-en-3~-ol; sitosterol, 24a-ethylcholest-5-en-3P-ol; lathosterol, 501-cholest-7-en-30-01; epifungisterol, 24a-methyl-5a-cholest-7-en-3~-01; spinasterol, 24a-ethyl-5a-cholesta-7,22-trans-dien-3~-ol; cholestanol, 5c~-cholestan-3/3-01; epicoprostanol, 5P-cholestan-3a-ol; campestanol, 24a-methyl-5a-cholestan-3P-ol; sitostanol, 24a-ethyl5a-cholestan-3P-ol. Acknowledgments: I thank Mr Frederick A. Burton for his assistance in purifying the sitostanol and Mrs Josephine R. Landrey for performing the mass spectroscopy and obtaining some of the 'H-NMR spectra on the 360-MHz instrument at the National Institutes of Healthsupported Middle Atlantic regional facility for 'H-NMR at the University of Pennsylvania. This study was supported by NSF grant PCM-8206541. Received February 27,1984; accepted April 9,1984. Address reprint requests to Karla S. Ritter, Department of Biological Sciences, Drexel University, Philadelphia, PA 19104. 0 1984 Alan R. Liss, Inc. 282 Ritter sterol for this lepidopteran [l], various A5-sterols, as well as A'- and A7sterols, can also support the development of the larva to the adult stage, although the growth rate of the insect may be affected . Since the degree of alkylation of the side chain of a dietary sterol, and the position of double bonds in the molecule, can affect the growth of H . m, this suggests that the structure of the sterol may affect the efficacy with which it is utilized by this animal. For example, the structure of the molecule may affect its rate of absorption andlor its ability to function as a precursor for ecdysteroids and as a structural component of membranes. It is known that the structural requirements of other insects for sterols may vary depending upon the species [3,4]. For instance, cholesterol fulfills the cellular requirements for sterol in many insects, when it is obtained directly in the diet or derived from other sterols such as phytosterols; however, other species such as Drosophilu pachea [S], Epiluchnn vurivestis , and A f f a cephalofes isthrnicoZu  may use unusual sterols as their principal tissue sterols (ie, A7-, A'-, and A5!'-sterols, respectively); not only do Oncopeltus fasciatus , Dysdercus fusciatus , Trogodema grunariurn [lo], and Apis mellifem [ll]dealkylate little if any C28and C29dietary sterol to 24-desalkylsterol, but also the first two species have been shown to contain the C28 ecdysteroid,makisterone A [3,9]. In the case of H . zed, Svoboda et a1 demonstrated that this insect can dealkylate the C-24 alkyl groups of sitosterol  and stigmasterol , and we described cholesterol as the principal free and esterified sterol in this insect (when it was reared on an undefined diet containing wheat germ and corn oil in addition to cholesterol );however, the metabolic products from other dietary sterols in this organism have not been studied. For example, the metabolic fates of A'- and A7-sterols, with or without alkyl groups at C24, are unknown. If H. zeu metabolizes different dietary sterols to different end products andlor if it uses them unchanged in membranes, then the resulting alterations in its sterol composition might have important physiological and ecological consequences. Not only might a change in sterol composition affect the physiological characteristics of membranes (eg, fluidity), but also it might affect the development of certain parasites (eg, parasitoid insects) which depend on the sterols of their host to complete their own development . Therefore, the purpose of this investigation was to study the sterol composition of sixth-instar larvae of H. zed, which had been reared on diets supplemented with various A5-, A7-, and A'-sterols, in order to compare and contrast the metabolic fates of these different dietary sterols in this insect. MATERIALS AND METHODS H . zea Larvae of H. zeu were reared individually on artificial diets, which contained no plant material but were supplemented with different sterols (15 mgllOO ml diet) as described previously [1,2]. Metabolism of Sterols by H. zed 283 Dietary Sterols The dietary sterols used in these experiments were: cholesterol (J.T. Baker Chemical Co, Phillipsburg, NJ); campesterol, sitosterol, and cholestanol (Applied Science Laboratories, Inc, State College, PA); campestanol, lathosterol, epifungisterol (7-campesterol), and spinasterol (Research Plus Steroid Laboratories, Denville, NJ); epicoprostanol and sitostanol (stigmastanol) (Sigma Chemical Company, St Louis, MO). Each sterol was recrystallized, as necessary, from ethanol and examined analytically by GLC,* RPLC, UV, MS and 'H-NMR and was shown to be at least 98% pure, with the exception of the sitostanol. This latter sterol was found to be a mixture of sitostanol (65%), campestanol (30%), and cholestanol (5%). Therefore, for some experiments, the sitostanol was isolated from this mixture using a Perkin-Elmer Series 1 liquid chromatograph, equipped with a variable wavelength UV detector and a Perkin-Elmer preparative CI8 column, at 30"C, a mobile phase of 100% acetonitrile, and a flow rate of 10 mllmin. Fifty milligrams of the mixture was injected at a time in 2 ml of isopropanol and 20-ml fractions were collected. Because the mixture was composed of stanols, no peaks were observed at 205 nm using this procedure; however, when the acetonitrile was evaporated and each fraction resuspended in cyclohexane and analyzed using a PerkinElmer Sigma 3B gas chromatograph, equipped with a 3% QF-1 column at 230-235"C, the sterol in each fraction was identified. Those fractions that contained >98% sitostanol were pooled, dried under nitrogen, and used in the diets. Isolation of Sterols and Steryl Esters Late-sixth-instar larvae were collected after they had consumed different dietary sterols, and to ensure that no unabsorbed dietary sterols were extracted from the larvae, each larva was pinned open, after a dorsal incision had carefully been made through its integument, and the fore-, mid-, and hindgut gently removed. The cadavers, minus the intestines, were then frozen at -20°C until they were extracted. Each group of cadavers was homogenized in acetone using a Sorvall Omnimixer and the sterols obtained by continuous extraction with acetone in a Soxhlet extractor for 24 hr. In experiments to determine what percentage of the tissue sterol was esterified, the sterols and steryl esters were separated by TLC on Silica-Gel G using a solvent system of benzene-ethyl acetate (9:l vlv), the bands corresponding to the 4-desmethylsterol region, 4,4-dimethylsterol region, and steryl ester region collected and the compounds eluted with anhydrous ether. In all cases, the TLC plates were protected from light and the recovery of sterols made as rapidly as possible to protect against their oxidation. The steryl esters were then saponified overnight at 60°C in *Abbreviations: acyl coenzyme A: cholesterol acyltransferase = ACAT; gas-liquid chrornatography = CLC; mass spectroscopy = MS; proton nuclear magnetic resonance spectroscopy = 'H-NMR; retention time relative to cholesterol = RRT; reversed-phase liquid chrornatography = RPLC; tetrarnethylsilane = Si(CH& = TMS; thin-layer chromatography = TLC; ultraviolet spectroscopy = UV. 284 Ritter 5% KOH in 90% ETOH. Otherwise, the acetone extracts were first saponified, the neutral lipids extracted with ether, and the sterols isolated using TLC as above. In one set of experiments, to test the purity of the dietary ingredients, all of the dietary components (175.4 g), with the exception of the H20 and sterols, were also extracted and processed as above to test for the presence of contaminating sterols. In addition, one ingredient, the agar (Difco, Detroit, MI) (305.6 g), was treated similarly by itself to check for trace amounts of sterols. Identification of Sterols The sterols isolated from the larvae were characterized and quantitated by GLC, RPLC, LJV, MS, andlor 'H-NMR. GLC was carried out at 225-235°C using a 1%XE-60 column, 0.75% SE-30 column, andlor 3% QF-1 column; rates of movement of compounds were expressed as retention times relative to cholesterol. Analytical RPLC was carried out using a Perkin-Elmer Series 3B liquid chromatograph, equipped with a variable wavelength detector and stop-flow capabilities, a Zorbax ODS (C,,) column at 45"C, a mobile phase of acetonitrile-isopropanol (80:20 vlv) and a flow rate of 2 mllmin. The k' for cholesterol ([v,-V,]/V,; V, = the retention volume of the sterol and V, = the void volume) was used to calculate the at's (k' for test sterollk' for cholesterol) of the peaks. Ultraviolet-absorption spectra were obtained by scanning the individual peaks from RPLC between 190 and 300 nm. MS was performed via direct probe on a model 4000 Finnigan instrument with electron impact ionization at 70 eV. 'H-NMR was performed at 360 MHz at ambient temperature on a Bruker instrument, model WH360, in CDC13 with Si(CH3)4as the usual internal standard. In these studies, only those peaks that constituted >1-2% of the sample were considered to be significant (ie, distinguishable from background). All experiments were done in duplicate and when & values are reported they denote the actual range of the results. RESULTS Purity of Dietary Ingredients No 4-desmethyl- or 4,4dimethylsterols were detected in the corresponding bands from TLC of the acetone extracts of the dietary ingredients (minus sterol and H20), or the agar, when they were examined by GLC, RPLC, and MS analysis. Metabolism of A5-Sterols Cholesterol. The control larvae, which were fed cholesterol as their sole dietary sterol, contained only one peak by GLC and this peak had an RRT similar to that of cholesterol (Table 1).RPLC at 205 nm showed one major peak which also had an ac similar to cholesterol (Table 2). One minor peak, with an ac of 0.74, was also present in the usual sterol region (ie, a, >0.55 and <1.30). Although the position of this peak indicated that it could be a 1.30 f 0.03 * 0.01 1.30 f 0.04 1.00 f 0.01 Campesterol Cholesterol 1.00 1.58 & 0.07 0.08 1.57 Sitosterol Dietary sterol RRT of dietary sterol XE-60 QF-1 + + + 1.61 f 0.01 1.01 0.01 1.33 0.01 1.01 f 0.01 1.01 0.01 RRT 14 & 4 86 & 4 27 & 2 73 & 2 100 4+3 96 f 3 15 It 3 84 f 3 100 Percent of esterified sterol QF-1 * 0.01 RRT 1.65 1.02 1.35 1.02 1.01 Sterols isolated from larvae Percent of free sterol XE-60 TABLE 1. GLC Analvsis of A’-Sterols and Their Metabolites in H . zed Larvae 14 86 26 74 100 Percent of free sterol P U 2 -. ul 3 $ 286 Ritter TABLE 2. RPLC Analysis at 205 nm of A5-and A'-Sterols and Their Metabolites in H. zeu Larvae Dietary sterol (Ye of dietary sterol Sterols isolated from larvae Percent of (YC free sterol Sitosterol 1.24 5 0.04 Campesterol 1.12 f 0.03 Cholesterol Spinasterol 1.00 0.01 1.09 f 0.03 Epifungisterol 1.12 f 0.01 1.26 1.01 1.11 1.01 1.00 0.01 1.09 f 0.02 1.01 f 0.02a 1.13 Lathosterol 1.04 1.03" * 0.03 * lola 11 89 26 74 100 39 f 6 60 f 6 68 32 100 "This was a broad peak which indicated that more than one peak was present. sterol which was more polar than cholesterol (eg, a standard of 7-dehydrocholesterol had an ac of 0.77), the UV-absorption spectrum of this peak gave a spectrum characteristic of end absorption (similar to the scan of the peak with cq of loo), indicating an absence of conjugated double bonds. Since GLC, MS (the M+ value was 386), and 'H-NMR (Table 3) indicated that cholesterol was the only sterol present, this evidence suggested that the minor peak might not be a sterol. Campesterol. Larvae fed campesterol were capable of demethylating and converting this sterol to cholesterol because about 73% of the sterol recovered from the cadavers had an RRT similar to cholesterol by GLC analysis; the RRT of the rest of the recovered sterol was similar to that of a standard of campesterol (Table 1).These results were confirmed by RPLC analysis (Table 2). Sitosterol. Large quantities of dietary sitosterol were also metabolized by the sixth instar. Approximately 86% of the sterol recovered from the larvae appeared to be cholesterol by GLC and RPLC analysis; the rest of the recovered sterol appeared to be unmetabolized dietary sitosterol (Tables 1,2). Metabolism of A'-Sterols Cholestanol. The larvae fed cholestanol contained this sterol as their principal, but not sole, tissue sterol. Although only one peak was observed when the recovered sterols were analyzed by GLC on the XE-60 column, two peaks were clearly resolved on the QF-1 column (Table 4). The major peak (about 84% of the sterol) had an RRT similar to a standard of cholestanol and the rest of the sterol had an RRT similar to cholesterol (Tables 1,4).When 20 p g of this sample was analyzed by analytical RPLC at 205 nm, only one peak was observed. This peak had an ac similar to that of a 20-pg injection of cholesterol (ie, 1-00) but an area of only about 10% of the standard. This indicated that most of the sample represented stanols because saturated sterols are not detected by UV-spectroscopy. 'H-NMR confirmed the presence of cholestanol as the principal tissue sterol and cholesterol as the minor one (Table 3). Although the presence of cholesterol in the sample was not t d d d S S Multiplicityb - - 0.68 1.01 0.91 0.86 0.87 A - 0.68 1.01 0.91 0.86 0.87 B Cholesterol - 0.53 0.79 0.91 0.86 0.87 A - - 0.68 1.01 0.92 0.87 0.87 - - - 0.65 0.80 0.90 0.86 0.87 0.54 0.80 0.92 0.87 0.87 Lathosterol B Minor Major sterol sterol - 0.68 1.01 0.89d 0.86d 0.86d - - 0.65 0.80 0.89 0.86 0.86 Type of dietary sterol‘ Cholestanol B Minor Major A sterol sterol - 0.54 0.80 0.92 0.78 0.86 0.81 Epifungisterol A aSi(CH3)4which was in CDC13. b~ = Singlet, d = doublet (J = 6 Hz), t = triplet (J = 7 Hz). ‘“A” represents spectrum of dietary sterol and “B” represents spectrum of sterol@)isolated from larvae. dHidden under corresponding peaks in other sterol. C-28 C-29 C-18 C-19 c-21 C-26, 27 Chemical shift in ppm (from TMF) of ‘H at: TABLE 3. ‘H-NMRSpectra of Dietary Sterols and the Sterols Isolated from H . zea Larvae 0.81 - 0.56 0.80 1.03 0.80 0.85 A - 0.80 0.55 0.79 1.02 0.81 0.84 - - 0.53 0.80 0.91 0.86 0.86 Spinasterol B Minor Major sterol sterol 4 -. 0 : 3 1.00 1.28 1.56 1.07 2 0.02" 1.39 k 0.02b 1.66 f 0.02' "5%of sample. b30%of sample. '65% of sample. + 1.00 1.28 1.55 Cholestanol, + campestanol, sitostanol 1.00 f 0.01 1.08 2 0.03 * 0.01 1.00 Cholestanol 1.30 2 0.02 1.00 f 0.01 1.39 2 0.02 1.30 f 0.02 1.57 1.02 Campestanol * 0.02 RRT 1.56 f 0.02 1.65 RRT of dietary sterol XE-60 QF-1 Sitostanol Dietary sterol TABLE 4. GLC Analysis of A'-Sterols and Their Metabolites in H. zea L w a e 40 22 38 100 53 f 3 46 3 63 37 - - - 44 57 - - Percent of esterified sterol ** RRT 1.65 1.09 1.00 1.38 f 0.02 1.09 0.02 1.01 0.01 1.08 0.02 1.00 f 0.01 1.00 1.09 1.38 1.65 Sterols isolated from larvae Percent of free sterol XE-60 QF-1 65 34 1 51 k 4 42 f 5 7+2 8421 16 f 1 <1 38 21 41 Percent of free sterol Metabolism of Sterols by H. zed 289 detected by MS, the M+ at 388, and the similarity of the fragmentation pattern to that of a standard of cholestanol, confirmed that cholestanol was the major sterol recovered. Campestanol. The presence of a saturated ring B in dietary campestanol did not prevent the demethylation of this sterol; however, less of this dieta sterol was dealkylated by the sixth instar than was the corresponding A sterol, campesterol. (The MS s ectrum of the dietary campestanol revealed that its M+ value was 402; the H-NMR spectrum of this stanol is shown in Table 5.) By GLC analysis, approximately one half of the dietary campestanol was recovered unmetabolized by the larvae (Table 4) whereas only about one quarter of the dietary campesterol was unmetabolized (Table 1).That portion of dietary campestanol which was metabolized was converted to 24-desmethylsterol. The principal 24-desmethylsterol recovered had an RRT similar to cholestanol and was clearly resolved, on the QF-1 column, from another minor peak; this latter peak had the same RRT as cholesterol and represented only 7% of the total sterol (Table 4). No peak that corresponded to campesterol was detected. The results from RPLC, after analyzing the sterols recovered from larvae fed campestanol at 205 nm, were similar to those obtained from larvae fed cholestanol-that is, only a small peak with ac similar to cholesterol was observed, indicating that the rest of the sterol in the sample consisted of stanols. These RPLC studies substantiated the evidence that the principal sterols in the larvae fed campestanol consisted of stanols. Sitostanol. Dietary sitostanol was partially dealkylated to 24-desalkylsterol just as campestanol had been. (The MS spectrum of the dietary sitostanol revealed that its M+ value was 416; the 'H-NMR spectrum of this stanol is shown in Table 5.) By GLC analysis, 34% of the recovered sterol was cholestanol and about 1%was cholesterol; the rest of the sterol in the tissues of the larvae was identified as sitostanol by its RRT on the QF-1 column (Table 4). Only a small peak for cholesterol was observed by RPLC analysis at 205 nm, indicating that, again, most of the sterol in the larval tissues was stanol. Mixture of sitostanol, campestanol, and cholestanol. This dietary sterol, shown by GLC to be a mixture of 65% sitostanol, 30% campestanol, and 5% r P TABLE 5. 'H-NMR Spectra of the Dietary Ao-Sterols Chemical shift in ppm (from TMSa bf 1~ at) C-18 C-19 c-21 C-26,27 C-28 C-29 MultipIicityb S S d d d t Dietary A'-sterol Cholestanol Campestanol Sitostanol 0.65 0.80 0.90 0.86 0.87 0.65 0.80 0.90 0.77 0.85 0.80 0.65 0.79 0.90 0.81 0.83 - 0.84 - aSi(CH3)4which was in CDC13. s = Singlet, d = doublet (J= 6 Hz), t = triplet (J = 7 Hz). - 290 Ritter cholestanol (Table 4), was fed to the larvae to determine if any of these sterols were preferentially utilized by H. zeu, since all three of these sterols had been shown to be absorbed and partially metabolized by the larvae (see above). GLC analysis indicated that about 41% of the sterol recovered from the larvae fed this mixture was sitostanol and about 21% of the sterol recovered was campestanol (Table 4); therefore, assuming no difference in the rates of absorption, about 62% of the sitostanol fraction and 73% of the campestanol fraction remained unmetabolized. Once again, cholestanol was the principal 24-desalkylsterol present; less than 1%of the recovered sterol was cholesterol as determined by GLC on the QF-1 column (Table 4) and by RPLC analysis at 205 nm. Epicoprostanol. When 5&cholestan-3a-o1 (epicoprostanol) was used in place of 5a-cholestan-3/3-01 (cholestanol) in the diet, no larvae molted, although they ate the diet, produced frass, and lived for as long as 2 weeks. Their rate of mortality was similar to that of larvae fed diet which was not supplemented with sterol (Fig. 1).In contrast, the mortality of larvae fed diet supplemented with cholestanol was very low and similar to that of larvae fed 5 10 15 20 TIME (DAYS) Fig. 1. Mortality of larvae on diets which were supplemented with epicoprostanol (O), no and cholesterol (A). sterol (O),cholestanol (0) Metabolism of Sterols by H. zed 291 diet supplemented with cholesterol (Fig. 1).These results indicated that a 3a-hydroxyl group (instead of 30) andlor a 5fLhydrogen (instead of 5a) prevents the normal utilization of sterols in H.zea. Metabolism of A7-Sterols Lathosterol. The fate of this dietary sterol was similar to that of cholestanol in that it was utilized as the major tissue sterol in the larvae. About 82% of the recovered sterol from the larvae had an RRT similar to that of a standard of lathosterol (Table 6). The remainder of the sterol had an RRT similar to that of cholesterol. No peak for cholestanol was observed on the QF-1 column. The broadness of the single peak observed by RPLC analysis (Table 2) indicated that two sterols (eg, cholesterol and lathosterol) were present. (A mixture of standards of cholesterol and lathosterol also gave a single broad peak.) 'H-NMR confirmed that about 80% of the recovered sterol had a spectrum similar to a standard of lathosterol and 20% had a spectrum similar to cholesterol (Table 3). The mass spectrum with an M+ of 386 substantiated the evidence that only mono-unsaturated 24-desalkylsterols were present. Epifungisterol. GLC and RPLC indicated that H. zeu can demethylate this sterol just as it can campesterol and campestanol; however, a large amount (about 70%) of the recovered sterol represented unmetabolized epifungisterol (Tables 2,6). Sixteen percent of the recovered sterol was the 24-desmethylsterol lathosterol, and the rest was cholesterol. These latter two sterols were not separated by RPLC but were resolved by GLC (Tables 2,6). No cholestanol, campestanol, or campesterol was detected. Spinasterol. This dietary sterol, which differs from all of the other sterols in this study because of the presence of a A22-bond,was dealkylated just as sitosterol and sitostanol were. By GLC analysis, the principal tissue sterol had an RRT similar to lathosterol, and 34% of the recovered sterol had an RRT which indicated that it was unmetabolized spinasterol (Table 6). Less than 10% of the remaining sterol had the same RRT as cholesterol, and no dihydrospinasterol was detected (the RRT of a standard of this molecule was 1.73 on the QF-1 column). Although the lathosterol and cholesterol peaks were not separated by RPLC, the 24-desalkylsterol peak was resolved from the spinasterol peak using this technique (Table 2). The UV-spectrum of each of these peaks was characteristic of end absorption, indicating an absence of metabolites with conjugated double bonds. The presence of lathosterol as the principal metabolite of spinasterol in the tissues was confirmed by 'HNMR; although the spinasterol was also detected by this technique, the *HNMR spectrum did not indicate that cholesterol was present (Table 3). Summary of the Metabolism Studies The results of the metabolism of dietary sterols by H. z summarized in Table 7. e are ~ averaged and Esterification of Dietary Sterols The amount of sterol which was esterified in larvae fed different dietary sterols was small and averaged between 2% and 4%. Those sterols detected 1.56 f 0.02 1.47 f 0.02 1.12 f 0.01 Epifungisterol Lathosterol SE-30 1.14 It 0.02 1.45 f 0.03 1.59 f 0.05 XE-60 OF-1 1.14 f 0.01 1.45 k 0.03 1.50 f 0.02 RRT of dietary sterol Spinasterol Dietary sterol 1.57 f 0.02 1.11f 0.02 1.00 f 0.01 1.46 1.13 1.02 1.12 1.01 RRT SE-30 37 63 <1 74 15 12 84 16 Percent of free sterol TABLE 6. GLC Analysis of A7-Sterols and Their Metabolites in H . zea Larvae 1.57 f 0.05 1.16 f 0.01 1.02 f 0.01 1.44 f 0.02 1.12 f 0.01 1.01 0.01 1.15 f 0.02 1.00 f 0.01 23 68 9 70 17 13 79 22 4 f l 39 f 2 51 f 3 78 & 5 19 f 6 3+2 Sterols isolated from larvae XE-60 Percent of Percent esteriof free fied RRT sterol sterol 1.49 f 0.01 1.13 f 0.01 1.01 f 0.01 1.48 1.14 1.01 1.15 1.00 RRT QF-1 + 34 f 7 4 5+4 70 15 15 78 22 62 Percent of free sterol A5-24a-ethyl A5-24a-methyl A' Dietary sterol Sitosterol Campesterol Cholesterol Spinasterol Epifungisterol Lathosterol 13.4 9.7 42.2 A7 - - 34*7 *2 - 70 Epifungisterol Spinasterol 15.9 40 34,l 42 f 5 84+1 38 51 4 22 1 - M*1 3.9 15.5 62.7 - +3 - * + Lathosterol 63 i- 5 16 5 1 81 k 3 100 Cholestanol Campestanol Sitostanol + 27 2 - - 4.7 45.2 Cholesterol 86+4 73 f 2 - Campesterol Sitosterol 14 +_ 4 Percent of free sterols isolated from larvae 13.7 Weight of larva extracted (g) A7,"-24a-ethyl A'-24a-methyl Sitostanol 24a-ethyl Campestanol 24a-methyl Cholestanol Sitostanol (65%) (as above) + Campestanol(30%) + Cholestanol ( 5%) Structural differences from 5acholestan-3@-01 TABLE 7. Summary of the Free Tissue Sterols Isolated from H. zeu Larvae Fed Different Dietary Sterols + Cholesterol 5+4 13 i- 2 19 3 <1 1 7+2 16 i- 1 Cholesterol W bn h) 3 h( 0 r: U s 4 x 8 9 e U 3 a a4 294 Ritter as free sterols in larvae were also found in the esterified form; sitosterol and spinasterol, though, appeared to be esterified in exceptionally small amounts (Tables 1,4,6). DISCUSSION This is the first qualitative and quantitative study of the tissue sterols of H. zeu larvae fed diets containing different A'-, A5-, and A7-sterols.The results indicate that H . zea carries out very little metabolism of ring B of those dietary Po-, A5-, and A7-sterols destined to become incorporated into membranes and esterified, because after the larvae consumed the different diets ring B of their principal tissue sterols was the same as that of their dietary sterol. That is, sitostanol and campestanol were dealkylated and converted to cholestanol; sitosterol and campesterol to cholesterol; and spinasterol and epifungisterol to lathosterol. Although H. zeu is capable of dealkylating A'-, A7-, and A5-alkylsterols, since more of the latter type of sterol is metabolized to 24-desalkylsterol by the late sixth instar, the larva may dealkylate A5-sterols more efficiently than those with a double bond at the 7 position or no double bonds (the stanols). These studies indicate that the intestinal cells of H.zeu can absorb A'-, A7-, and A5-sterolswith or without a 24-alkyl group; however, whether the absorption rates are similar is not known. The fact that alkylated sterols were found at all in the larvae, from which the intestines (and therefore all unabsorbed dietary sterols) had been removed prior to analysis, indicates that some alkylated sterols may be transported out of the intestine without being metabolized first. It is not known whether these sterols may be dealkylated later at some other location, such as in the fat body. Since esters of each type of free sterol were also found in the tissues of H. zeu, this indicates that the larva produces enzyme(s) for the esterification of sterols with differences not only in the side chain but also in ring B of the nucleus. Since we have found that ACAT activity is present not only in the microsomes of the intestine but also in the microsomes of the fat body of H. zeu , and since Tavani et a1  have shown that ACAT from rat liver microsomes may esterify a number of sterols (eg, cholestanol and lathosterol) in addition to cholesterol, ACAT may at least in part be responsible for the esterification of the different tissue sterols found in H. zed. Interestingly, the 24-ethylsterolsrsitosterol and spinasterol, were found in the smallest amounts in the esterified form (1%and 4%, respectively, of the total esterified sterols) which is similar to the results of Tavani et a1 [%I, who showed that the percentage esterification of sitosterol and stigmasterol by ACAT from rat liver was significantly less than with other sterols, such as lathosterol and cholestanol. There was evidence that the larvae convert A7- and A'-sterols to cholesterol, because (1)minor quantities of cholesterol were present in varying amounts when the insects were fed these sterols, and (2) cholesterol was not found to be a contaminant of the dietary ingredients. Although some of this cholesterol may have been derived from maternal sterols, the contribution of transovarially derived sterols to the sterol pool of the sixth instar is probably quite small and doesn't explain why the amounts of cholesterol found in Metabolism of Sterols by H. zea 295 these experiments varied from < 1% to 19%. (The weight of a neonate larvae is about 0.1 mg, which is less than 0.03% of that of a later-sixth-instar larva or a pupa; therefore, assuming that the percentage of sterol in the tissues does not change, the maternal sterols represent <0.03% of the sterols in the mature larva.) The absence of sitosterol (andlor stigmasterol) from the tissues of larvae fed sitostanol and spinasterol, and the absence of campesterol in the tissues of larvae fed campestanol and epifungisterol, indicate that if A'and A7-sterols are metabolized to A5-sterols, the introduction of the double bond at the 5 position occurs after the dealkylation of the side chain. Interestingly, more desalkylsterol than alkylsterol appeared to be converted to cholesterol. If larvae do introduce a A5 bond into A7-desalkylsterols, it is not apparent whether the desaturation occurs before or after the reduction at the 7 position, because neither 7-deh drocholesterol nor cholestanol was detected as a metabolite of dietary A -sterols. These results indicate that final proof of the metabolism of A7- and Ao-sterols to cholesterol in H. zea must await further studies using dietary sterols which have been radiolabeled. Although it is possible that at least minor amounts of cholesterol may be essential to larvae so that they can carry out some critical metabolic function(s), the results corroborate the results of others, such as Clark and Bloch and Thompson et a1 , and indicate that insects may function normally, at least for one generation, with sterols other than A5-sterols as their principal membrane sterols and that they can store these molecules as steryl esters. The fact that the sterol composition of H. zeu can be changed, with the only immediately obvious effect being a change in rate of growth, is unusual, but understandable, considering that some insects (eg, D. pucheu  and E. vurivestis ) may contain sterols other than cholesterol and yet function normally. Since the sterol composition of H. zeu can be changed under laboratory conditions by varying the type of sterol in the diet, similar changes in the sterol composition of larvae might also occur naturally in the field because some host plants, such as alfalfa , contain unusual sterols such as A7sterols as their dominant sterols, instead of A5-sterols (eg, corn ). Therefore, the sterol compositions of different populations of H. zeu may differ in the field when they feed on plants (eg, corn vs alfalfa) with different sterol compositions. This change in the sterol composition of the larva may affect not only the physiology of cellular membranes, but also the development of those parasites which derive their sterols from their host (eg, hymenopterous and dipterous parasites). Consequently, the physiological as well as ecological effects of changes in the sterol composition of H . zeu should be investigated. Y [ln LITERATURE CITED 1. Ritter KS, Nes WR: The effects of cholesterol on the development of Heliothis z a . J Insect Physiol27, 175 (1981). 2. Ritter KS, Nes WR: The effects of the structure of sterols on the development of Heliothis zea. J Insect Physiol27, 419 (1981). 3. Svoboda JA, Thompson MJ, Robbins WE, Kaplanis JN: Insect steroid metabolism. Lipids 13, 742 (1978). 296 Ritter 4. Kircher HW:Sterols and insects. In: Cholesterol Systems in Insects and Animals. Dupont J, ed. CRC Press, Boca Raton, FL, pp 1-50 (1982). 5. Goodnight KC, Kircher HW: Metabolism of lathosterol by Drosophila pacheu. 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