Esterification of cholesterol and cholestanol in the whole body tissues and frass of Heliothis zea.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 7:237-247 (1988) Esterification of Cholesterol and Cholestanol in the Whole Body, Tissues, and Frass of Heliothis zed Arun Kuthiala and Karla S. Ritter Department of Bioscience and Biotecknology, Drexel University, Philadelphia, Pennsylvania The quantity of free and esterified sterols in the whole body, intestine, hernolymph, fat body, and frass of 6th-instar larvae of H. zea, fed cholesterol or cholestanol, was measured in order to determine if there was a difference in the utilization of these two molecules. The principal sterol in the tissues of the larvae was cholestanol or cholesterol, when they were fed diet containing these two molecules, respectively; there was little, if any, metabolism of dietary cholestanol t o cholesterol. There was little or no difference in the amount of total sterol in the whole body, tissues, or frass of larvae fed the two different diets, indicating that the absence of a A5-bond in cholestanol does not prevent the uptake or distribution of this sterol to various tissues. However, the relative percentage of steryl ester was significantly higher in prepupae reared on a diet containing cholestanol instead of cholesterol (6-7-, 4-,13-,4, and 2-fold increase, for the whole body, intestine, hernolymph, fat body, and frass, respectively). The average percentage of total sterol that was esterified in the tissues was greater in the fat body (10.8 k 15.4 and 44.2 f 12.3%, respectively, for larvae fed cholesterol and cholestanol) than in the hernolymph (0.5 f 0.1 and 6.3 f 0.8%) and intestine (1.2 f 0.1 and 4.7 k 1.1%).The percentage of sterol that was esterified in the frass of larvae was large (26.9 k 3.7 and 48.2 2 0.5%, respectively, for larvae fed cholesterol and cholestanol). Therefore, the fact that larvae of H. zea fed cholestanol, instead of cholesterol, contain this saturated molecule as their principal tissue sterol and preferentially esterify it may explain, at least in part, why their rate of growth on cholestanol is slower than on cholesterol. Key words: [4-'4C]cholestanol, [4-'4C]cholesterol, steryl ester, corn earworm Acknowledgments: We thank Mrs. J.R. Landrey for her assistance in preparing the [414C]cholestanoland Dr. J.T. Billheimer for reading the manuscript. This study was supported in part by National Science Foundation grant DCB-8502578. Received January6,1988; accepted February 25,1988. Dr. Kuthiala is now at the Department of Entomology, University of Missouri, Columbia, M O 65211. Address reprint requests to Dr. Karla S. Ritter, Department of Bioscience and Biotechnology, Drexel University, Philadelphia, PA 19104. 0 1988 Alan R. Liss, Inc. 238 Kuthiala and Ritter INTRODUCTION All of the members of Insecta that have been studied, including Heliothis zeu, require exogenous sterols for their development and reproduction [l]. H. zeu can utilize a variety of sterols as tissue sterols [2,3]; however, the rate of growth of the larva varies with the degree of alkylation and unsaturation of the molecule [4,5]. For example, when H. zeu is fed an artificial diet containing cholestanol instead of cholesterol, the larva grows more slowly . Cholesterol is the principal tissue sterol in larvae fed cholesterol, whereas cholestanol is the principal one in larvae fed cholestanol . This suggests that although both cholesterol and cholestanol are absorbed and transported to various sites for utilization (e.g., as components of membranes) andlor storage (e.g., as steryl esters), cholestanol is used less efficiently. Interestingly, previous studies in this laboratory have shown that, in vitro, there is a 2- to 3-fold increase in the esterification of cholestanol, by ACAT*, in the microsomes of the intestine, fat body, and ovary of H . zeu, when compared with that of cholesterol . If cholestanol is preferentially esterified in vivo and stored, instead of being used for membrane synthesis, this fact might help to explain the difference in the rate of growth of larvae on these two sterols. The purpose of this study was to compare the quantities of free and esterified sterols in the whole body, intestine, hemolymph, fat body, and frass of 6th-instar larvae of H. zeu fed cholesterol versus cholestanol in order to determine if there is a difference in the utilization of these two sterols in the various tissues. MATERIALS AND METHODS H . zea Larvae of H . zeu were reared, as described previously [4,5], on artificial media containing cholesterol or cholestanol (20 mgllOO ml). The medium containing cholesterol (J. T. Baker Chemical Co., Phillipsburg, NJ), recrystallized from ethanol, also included either [4-14C]cholesterol(Amersham, Arlington Heights, IL; 55 mCilmol), so that the final specific activity of the cholesterol was 192 pcilmmol, or [7-3H(N)]cholesterol(New England Nuclear, Boston, MA; 37 Cilmmol), so that the final specific activity of the cholesterol was 2.58 mCilmmo1. The medium containing cholestanol (Sigma Chemical Co., St. Louis, MO) also included [4-14C]cholestanol, so that the final specific activity of the cholestanol was 128 or 129 pCilmmol. The [414C]cholestanolwas prepared by the hydrogenation of [4-14C]cholesterol. The cholesterol, cholestanol, and radiolabeled cholestanol were shown to be 297% pure by GLC, RP-HPLC, MS and 'H-NMR (these procedures have been described previously 121). In RP-HPLC, the dietary cholestanol was *Abbreviations: ac = k' for test sterollk' for cholesterol; ACAT = acyl Coenzyme A:cholesterol acyl transferase; CLC = gas-liquid chromatography; 'H-NMR = proton nuclear magnetic resonance spectroscopy; k' = V , - V a 0 ; MS = mass spectrometry; RP-HPLC = reversedphase high-performance liquid chromatography; TLC = thin-layer chromatography; Vo = void volume; V, = retention volume of sterol. Esterification of Sterols 239 suspended in isopropanol and 1-ml fractions were collected from a Zorbax ODS (CIS) column (at 45°C with a mobile phase of 80% acetonitrile and 20% isopropanol at 2 mllmin) and counted. The percentage of the radioactivity associated with those fractions corresponding to cholesterol (a,of 1.00) and cholestanol (acof 1.30) was 1-3% and 97-99%, respectively. Some larvae were derived from a colony of H . zed that had been maintained on a diet containing pinto beans, wheat germ, and torula yeast  for 18 to 23 generations (population A). Other larvae were from another population, which had been maintained on a diet containing wheat germ and corn oil  for 11 to 26 generations (population B). (Note: In both cases, the amount of maternal sterol present in the larvae was probably quite small because when neonates were fed diet that lacked sterol, they remained in the first instar and did not molt until they were provided with exogenous sterol .) Collection of Prepupae, Tissues, and Frass Whole prepupae of H . zeu (1day after they had ceased feeding) as well as hemolymph, fat body, intestines, and frass were collected in order to determine their sterol content. Hemolymph was collected after puncturing an abdominal proleg with a needle. Fat body was gently removed with forceps from larvae (after they had been opened dorsally and the hemolymph had been washed from the hemocoel with cold distilled water) and blotted dry with filter paper. In some experiments, after the removal of the fat bodies, the intestines were also isolated from the larvae. The lumen of each gut was washed with distilled water, introduced via a syringe, and the tissue was blotted dry with filter paper. Frass was collected from rearing vials and lyophilized. Quantitation of Sterol in Prepupae, Tissues, and Frass Method 1. In some experiments, where the total quantity of radioactivity in the sample was measured, the fresh tissues (less than 150 mg) from individual prepupae were solubilized in 1 ml of Protosol@(New England Nuclear, Boston, MA). Hemolymph was always added directly to Protosol@ in a tared vial, in order to prevent melanization of the blood, and then weighed; the fat body and intestinal tissue were weighed and then incubated in the Protosol@at 55°C until they were visibly digested. After 12-24 h at ambient temperature, the alkaline solution was neutralized with 70 p1 of acetic acid to prevent chemiluminescence, suspended in 20 ml of scintillation cocktail (Ready SolveTM El', Beckman Instruments, Fullerton, CA), and counted in an LS 7500 scintillation counter (Beckman Instruments). The total amount of sterol per 100 mg of tissue was then determined after adjusting for background, quench, and the amount of unlabeled sterol present. Method 2. In other experiments, where the quantity of radioactivity in the free sterol versus the esterified sterol fraction was measured, whole prepupae as well as tissues and frass from several larvae were pooled and stored at -20°C until the sterols and steryl esters were extracted with methanolchloroform with a modified Bligh-Dyer technique  or with acetone in a 240 Kuthiala and Ritter Soxhlet apparatus. The intestines, hemolymph, and fat body were homogenized with a tissue grinder of 2-ml capacity; whole prepupae were homogenized in a Sowall@Omni-Mixer (Dupont Co., Newtown, CT). These samples were extracted with methanol-chloroform (2:l) for 20 min at 60°C; the extraction was repeated twice with fresh solvent (methanol:chloroform:water, 2:l:O.S).Frass was extracted for 24 h with acetone in a Soxhlet apparatus. The free sterols in the extracts were separated from the steryl esters through TLC on polysilicic-acid-gel impregnated glass fiber sheets (Gelman Sciences Inc., Ann Arbor, MI) with a mobile phase of benzene and ethyl acetate (9:l) andlor hexane, diethyl ether, and acetic acid (90:lO:l).The spots on the plates were visualized with phosphomolybdic acid and those areas with Rf values corresponding to the standards of cholesterol and cholesteryl oleate carefully noted. The different fractions of the sample were placed directly in scintillation cocktail and counted. After the counts were corrected for background and quench, the quantity of 4-desmethylsterol vs. steryl ester in each sample was calculated. Method 3. In some experiments, RP-HPLC was used to determine the type and quantity of radioactive sterols in the saponified steryl ester andlor 4desmethylsterol regions (separated by preparatory TLC on Silica Gel G with a mobile phase of benzene and ethyl acetate [9:1] and then eluted from the silica with diethyl ether). Samples were saponified overnight at 60°C in 5% KOH in 90% ETOH. In order to confirm that any radioactivity in the saponified steryl ester region actually was associated with sterol, this fraction was rechromatographed on glass fiber TLC plates, as described earlier, and the quantity of radioactivity in the free sterol region, vs. other regions, was determined. For RP-HPLC, 20 p1 of sample in isopropanol was used and 1ml fractions were collected (from a Zorbax ODS [C,] column at 45°C with a mobile phase of 80% acetonitrile and 20% isopropanol at 2 mllmin) and counted. Method 4. In one experiment, GLC was used to determine the type and amount of free sterol vs. esterified sterol in the prepupae. In this case, the free sterols and steryl esters in the methanol-chloroform extract were separated by preparatory TLC (on Silica Gel G, with a mobile phase of hexane, diethyl ether, and acetic acid [90:10:1]); the areas corresponding to the 4desmethylsterol and steryl ester regions collected and extracted with diethyl ether; and the steryl ester band saponified. The sterols were then characterized and quantitated by GLC with a QF-1 column, as described previously . RESULTS Prepupae Larvae of H . zeu were fed diet containing cholesterol or cholestanol, and the quantity of free and esterified sterol associated with the prepupae was determined. There was no difference in the total amount of sterol between cholesterol- and cholestanol-fed larvae (26 f 9 and 26 pgl100 mg wet weight, respectively) (Table 1).In both populations A and B, the relative percentage of steryl ester in the larvae fed cholestanol (7.3 and 61.7%, respectively) was 7.40 ND - +g +h - 82.0 (37,734) 37.7 (3,721) 86.9 (4,640) 92.9 (10,370) 90.3 (5,545) N D ~ 7.9 (3,638) 60.7 (5,996) 6.8 (364) 0.7 (75) 1.7 (103) ND 10.1 (4,630) 1.6 (154) 6.3 (334) 6.5 (722) 8.0 (491) ND % of Total dpm' (number of dpm) in region corresponding to 4-DesmethylSteryl sterols esters Other aThe specific activity of [414C]cholesterol was 192 pCilmmol. m e specific activity of [414C]cholestanol was 129 pCilmmol. '25 pl from 1 ml of extract (for population A only). dNot determined. eDetermined by GLC (by method 4). ff Values represent the S.D. Qhe specific activity of [7-3H]cholesterolwas 2.58 mCilmmo1. hThe specific activity of [4-'4C]cholestanol was 128 pCilmmol. (8) ND 1.52 6.21 5 1.43 Prepupae - + 5 1.24 + - + 3 1.19 Prepupae (A) - - + 3 (g) Number of larvae Type of sterol in diet Choles- Cholesterola tanolb Sample Weight of sample ND ND 31e Avg: 26 f sf 26 16 32 Total sterol (rg1100 mg of Sample) 38.3 91.2 98.7 f 0.6 92.7 98.6 98.2 99.3 61.7 8.8 1.4 1.3 f 0.6 7.3 1.8 0.7 % of Total sterol Free Esterified TABLE 1. Amount of Sterol and Steryl Ester (Determined by Method 2) in Larvae of Two Populations (A and B) of H . zea Fed Diet Containing Cholesterol or Cholestanol 242 Kuthiala and Ritter approximately 6- to 7-fold that of larvae fed cholesterol (1.3 f 0.6 and 8.8%, respectively) (Table 1). Cholesterol (a, of 1.00; RRT of 1.00) was the only sterol in the larvae fed the diet containing cholesterol, when methods 3 and 4 were used to characterize the molecules. It is not known whether a small radioactive peak in the RP-HPLC sterol region (i.e., 1.4% of the recovered dpm), with an a, of 0.69, also corresponds to a sterol. In contrast, 95 to 99% of the sterol in larvae fed the diet containing cholestanol was cholestanol (a, of 1.30; RRT of 1.08); the remaining sterol eluted with cholesterol (a,of 1.00; RRT of 1.00). Intestine The average amount of total sterol in the intestines of the larvae of population A fed cholesterol was slightly less (52 & 4 and 29 f 7 pgll00 mg wet weight for methods 1and 2, respectively) than in intestines from larvae fed cholestanol (68 f 4 and 62 & 2 pg1100 mg for methods 1and 2, respectively) (Fig. 1,Table 2). The average relative percentage of steryl ester in the intestine of larvae fed cholestanol (4.7 f 1.1%) was approximately 4 times that of the steryl ester in the intestine from larvae fed cholesterol (1.2 f 0.1%) (Table 2). Hemolymph The average amount of total sterol in the hemolymph of larvae of population A fed cholesterol was similar (39 f 2 and 33 +_ 1pgll00 mg wet weight for methods 1 and 2, respectively) to that of hemolymph from larvae fed cholestanol (36 f 2 and 24 f 3 pgll00 mg for methods 1and 2, respectively) (Fig. 1, Table 3). The average relative percentage of steryl ester in the hemolymph of larvae fed cholestanol(6.3 f O.80/,)was approximately 13 times that of hemolymph from larvae fed cholesterol (0.5 i 0.1%) (Table 3). Fat Body The average amount of total sterol in the fat body of larvae of population A fed cholesterol was similar (57 f 5 and 43 & 22 pg1100 mg wet weight for methods 1and 2, respectively) to that of fat body from larvae fed cholestanol (85 36 and 78 i 2 pgll00 mg for methods 1 and 2, respectively) (Fig. 1, Table 4). The average relative percentage of steryl ester in the fat body of larvae fed cholestanol (44.2 f 12.3%) was approximately 4 times that of fat body from larvae fed cholesterol (10.8 & 15.4%) (Table 4). When method 3 was used to characterize the molecules, 94% of the sterol in larvae fed the diet containing cholestanol was cholestanol (a, of 1.30); the remaining sterol eluted with cholesterol (a, of 1.00). Frass There was slightly less total sterol in the frass of larvae of population A, fed the diet containing cholesterol, than in the frass of larvae fed cholestanol (32 + 2 versus 39 3 &lo0 mg dry weight, respectively, as determined by method 2) (Table 5). The average relative percentage of steryl ester in the frass of larvae fed cholestanol(48.2 i 0.5%) was approximately twice that of Esterification of Sterols Intestine Hemolymph 243 Fat Body T I2Ol 100 Intestine Hemolymph Fat Body Fig. 1. Quantity of sterol in various tissues from larvae of H. zea (population A) fed [4''C]cholesterol (A) or [414C]cholestanol (6).Total sterol ( (determined I ) by measuring the amount of radioactivity associated with the solubilized tissues through method 1). Free sterol (0) and steryl ester (M) (determined by measuring the amount' of radioactivity associated with these fractions separated by TLC through method 2). (Bars represent S.D. of the mean for the fat body and the actual range of data for the intestine and hernolymph.) larvae fed cholesterol (26.9 & 3.7%) (Table 5). When the steryl esters from the frass of larvae fed cholesterol or cholestanol were saponified and analyzed by TLC by method 3, 96.6% (1,602 dpm) and 90.3% (1,295 dpm), respectively, of the radioactivity was associated with the 4-desmethylsterol region; the remainder was associated with the other regions. Cholesterol (ac of 1.00) was the only sterol present in the steryl esters of frass from larvae fed the diet containing cholesterol, and cholestanol (acof 1.30) was the only sterol present in the steryl esters of frass from larvae fed the diet containing cholestanol, when method 3 was used to characterize the molecules. 244 Kuthiala and Ritter TABLE 2. Amount of Sterol and Steryl Ester (Determined by Method 2) in the Intestine of Larvae of H. zea (Population A) Fed Diet Containing Cholesterol or Cholestanol Weight of intestine (mg) Type of sterol in diet Choles- Cholesterola tanoIb 50 + - 60 + - % of Total dpmC (number of dpm) in region corresponding to 4-Desmethyl- Steryl sterols esters Other 96.4 (3,975) 96.3 (1,454) 1.1 (49) 1.0 (15) Total sterol ( p g l lmg ~~ of intestine) 2.4 (99) 2.7 (41) % of Total sterol Free Esterified 36 98.8 1.2 22 98.9 1.1 Avg: 29 rt 7d 98.9 rt 0.1 1.2 f 0.1 25 48 + - 88.8 (720) 92.1 (1,529) + - 5.4 (44) 3.4 (57) 5.8 (47) 4.5 60 94.2 5.8 64 96.4 3.6 95.3 1.1 4.7 (75) Avg: 1.1 "The specific activity of [4-14C]cholesterol was 192 pciimmol. bThe specific activity of [4-14C]cholestanolwas 129 pCi/mmol. 'In 50 or 100 p1 from 0.5 ml of extract. df Values represent the actual range of the results. TABLE 3. Amount of Sterol and Steryl Ester (Determined by Method 2) in the Hemolymph of Larvae of H . zea (Population A) Fed Diet Containing Cholesterol or Cholestanol Weight of Type of sterol in diet 70of Total dpm' (number of dpm) in region corresponding to Total sterol hemolymph Choles- Choles- 4-Desmethyl- Steryl (pg1100 mg (mg) terola tanolb sterols esters Other of hemolymph) 195 + - 241 + - 98.1 (6,827) 98.1 (8,973) 0.4 (26) 0.4 (41) 1.5 (103) 1.4 (132) - + 270 - + 91.7 (2,692) 90.2 (2,703) 5.3 (157) 6.9 (207) 2.9 (86) 2.9 (86) Avg: Free Esterified 32 99.6 0.4 34 99.5 0.5 Avg: 33 f Id 212 % of Total sterol 99.6 & 0.1 0.5 f 0.1 26 94.5 5.5 21 92.9 7.1 93.7 rt 0.8 "The specific activity of [4-14C]cholesterolwas 192 pcilmmol. bThe specific activity of [4-14C]cholestanolwas 129 pCilmmo1. 'In 50 p1 from 0.5 ml of extract. d + Values represent the actual range of the results. DISCUSSION The ability of insects to utilize cholestanol as a dietary sterol varies with the species. Cholestanol will support growth as well as cholesterol does in some insects, whereas in others it is used less effectively or not at all [lo]. - - + + - - - 85 95 61 69 72 46.7 (1,289) 61.2 (1,896) 45.6 (1,322) 97.2 (1,986) 96.5 (2,994) 69.1 (5,205) 43.7 (1,204) 26.7 (828) 53.3 (1,544) 1.3 (26) 2.5 (79) 27.7 (2,089) % of Total dpm' (number of dpm) in region correspondingto Steryl 4DesmethYlsterols esters 'The specific activity of [4-14C]cholesterolwas 192 pcilrnmol. %e specific activity of [4-14C]cholestanolwas 129 pcilmmol. 'In 50 pl from 0.5 ml of extract. d + Values represent the S.D. + + + - + Type of sterol in diet CholesCholesterol' tanolb 64 Weight of fat body (mg) 9.6 (265) 12.1 (375) 1.1 (32) 1.6 (32) 0.9 (28) 3.1 (237) Other Avg: 55.8 78+2 69.6 51.7 89.1 46.1 * 22d 71.4 97.4 98.7 12.3 * 15.4 44.2 k 12.3 53.9 30.4 48.3 10.8 k 15.4 28.6 2.6 1.3 Esterified YO of Total sterol Free 78 77 80 Avg: 43 69 33 28 of sample) (PLg/100 mg Total sterol TABLE 4. Amount of Sterol and Steryl Ester (Determined by Method 2) in the Fat Body of Larvae of H. zea (Population A) Fed Diet Containing Cholesterol or Cholestanol 246 Kuthiala and Ritter TABLE 5. Amount of Sterol and Steryl Ester (Determined by Method 2) in the Frass of Larvae of H. zeu (PopulationA) Fed Diet Containing Cholesterol or Cholestanol Ezght of frass Type of sterol in diet (g) Cholesterola 0.5 + - 1.0 + - % of Total dpmC (number of dpm) in region corresponding to Choles- 4-Desmethyl- Steryl esters tanolb sterols 65.6 (3,602) 62.8 (5,743) 19.9 (1,090) 27.5 (2,516) Total sterol (llg1100 mg Other of frass) 14.6 (799) 9.7 (887) 34 76.8 23.2 30 69.5 30.5 Avg: 32 0.5 - + 1.0 - + 43.2 (1,171) 47.1 (2,845) 40.9 (1,110) 43.0 (2,598) 15.9 (432) 9.9 (596) Avg: % of Total sterol Free Esterified 2d 73.2 3.7 26.9 k 3.7 36 51.3 48.7 42 52.3 47.7 51.8 f 0.5 48.2 & 0.5 "The specific activity of [4-14C]cholesterolwas 192 pCilmmol. bThe specific activity of [4-14C]cholestanolwas 129 pcilmmol. '25 pl from 1ml of extract. d + Values represent the actual range of the results. Previous studies have shown that the larva of H. zed develops more slowly when fed a diet containing cholestanol instead of cholesterol  and contains cholestanol as its principal tissue sterol instead of cholesterol . In the present study, because there was little or no difference in the amount of total cholesterol vs. cholestanol in the whole body, intestine, hemolymph, fat body, and frass of prepupae fed the two different diets, the absence of a A5bond in cholestanol does not prevent the uptake or distribution of this sterol to various tissues. The RP-HPLC studies indicated that there was little, if any, metabolism of dietary cholestanol to cholesterol. Although 1-6% of the radiolabeled tissue sterols had an ac corresponding to cholesterol, 1-3% of the radiolabeled dietary cholestanol also had an a, of 1.00. Because no other sterols were detected in the dietary cholestanol, when it was analyzed by GLC, MS and 'H-NMR, it is not clear whether the small amount of tissue sterol that eluted in the cholesterol region was a contaminant or a metabolite of the dietary stanol. The relative percentage of steryl ester in prepupae was significantly higher when they were reared on a diet containing cholestanol instead of cholesterol (4-, 13-, 4-, and 2-fold increases for the intestine, hemolymph, fat body, and frass, respectively). Although the relative percentage of steryl ester in the whole body of the larvae was greater in population B than in population A, there was still a 6-7-fold increase in the relative percentage of cholestanol that was esterified, as compared with that of cholesterol, in these larvae. Therefore, cholesterol appeared to be used in the free form preferentially (e.g., for membrane synthesis), whereas cholestanol appeared to be esterified (e.g., for storage). The results of these in vivo studies are in agreement with Esterification of Sterols 247 those of previous in vitro studies, which demonstrated that there was an increase (2- to 3-fold) in the esterification of cholestanol by ACAT in microsomes of the intestine and fat body of H. zeu, as compared with cholesterol PI * The average percentage of the total sterol that was esterified in the tissues was greater in the fat body (10.8 k 15.4 and 44.2 12.3% for larvae fed cholesterol and cholestanol, respectively) than in the hemolymph (0.5 0.1 and 6.3 k 0.8%) and intestine (1.2 :& 0.1 and 4.7 1.lY0). This finding suggests that in the 6th-instar larva, sterols are transported in the hemolymph of H. zed primarily in the free form, as they are in other insects [ll], and that the fat body is a major storage site for steryl esters. It is not clear, however, why such a high percentage of the total sterol in the frass of the prepupae was esterified (26.9 and 48.2% for larvae fed cholesterol and cholestanol, respectively). Because the larva of H. zea fed cholestanol instead of cholesterol contains this molecule as its principal tissue sterol and preferentially esterifies it, this may explain, at least in part, why its rate of growth on a diet containing cholestanol is slower than on a diet containing cholesterol. However, other factors that also might affect the growth and development of the larva, such the rate of utilization of cholestanol relative to cholesterol for the synthesis of membranes and ecdysteroids, remain to be investigated. LITERATURE CITED 1. Svoboda JA, Thompson MJ: Steroids. In: Comprehensive Insect Physiology Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, New York, Vol. 10, pp 137-175 (1985). 2. Ritter KS: Metabolism of A'-, A5-, and A7-sterols by larvae of Heliothis zea. Arch Insect Biochem Physiol 1, 281 (1984). 3. Ritter KS: Utilization of A5j7- and As-sterols by larvae of Heliothis zeu. Arch Insect Biochem Physiol3, 349 (1986). 4. Ritter KS, Nes WR: The effects of cholesterol on the development of Heliothis zea. J Insect PhysiolZ7, 175 (1981). 5. Ritter KS, Nes WR: The effects of the structure of sterols on the development of Heliothis zeu. J Insect Physiol27, 419 (1981). 6. Macauley SK, Billheimer JT, Ritter KS: Sterol substrate specificity of acyl coenzyme A: Cholesterol acyltransferase from the corn earworm, Heliothis zea. J Lipid Res 27, 64 (1986). 7. Nace HR:An improved hydrogenation of cholesterol to cholestanol. J Am Chem SOC73, 2379 (1951). 8. Burton RL: Mass rearing the corn earworm in the laboratory. US Department of Agriculture Presentation Paper ARS 33-134,8 pp (1969). 9. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol37, 911 (1959). 10. Kircher HW, Gray MA: Cholestanol-cholesterol utilization by axenic Drosophila melanogasteu. J Insect Physiol24, 555 (1978). 11. Chino H, Kitazawa K: Diacylglycerol-carrying lipoprotein of hemolymph of the locust and some other insects. J Lipid Res 22, 1042 (1981).