Archives of insect Biochemistry and Physiology 3:349-362 (1986) Utilization of A5,'- and A8-Sterols by Larvae of Heliothis zea Karla S . Ritter Department of Biological Sciences, Drexel University, Philadelphia, Pennsylvania Larvae from two populations of Heliothis zea were reared on artificial diets containing various sterols, which supported suboptimal growth, and their tissue sterols were characterized in order to determine how these dietary sterols are utilized by this insect. The sterols studied included A5r7-sterols(7dehydrocholesterol or ergosterol), A8-sterols (lanosterol andlor 2Cdihydrolanosterol), and a A5-sterol (4,4-dimethylcholesterol). Although larvae did not develop on 4,4dimethylcholesterol, those fed primarily A8-4,4,14trimethylsterols developed to the third instar. When the latter sterols were spared with cholesterol, the larvae reached the sixth instar and contained 4,4,14trimethylsterols as well as cholesterol in their tissues. When larvae were fed 7dehydrocholesterol, < 1% of the larvae from one population developed to the sixth instar and these larvae contained 7-dehydrocholesterol as their principal sterol. The other larvae successfully completed their larval stage when they were transferred from the diet containing 7-dehydrocholesterol (or no sterol) to a diet containing cholesterol within at least 9 days. The sterol composition of larvae transferred from a diet containing cholesterol t o a diet containing 7dehydrocholesterol, after they had reached 60% of their final weight, was 54% cholesterol and 46% 7-dehydrocholesterol. The major sterol isolated from the tissues of the larvae fed ergosterol was also 7-dehydrocholesterol. Therefore, although the larva of H. zea can dealkylate and saturate the side chain of the A5,7,22-24fhnethylsteroI,it carries out little metabolism of the B ring of the nucleus. These studies demonstrate that, when A5r7- or A8-sterols are the principal sterols in the diet of H. zea, they are absorbed and incorporated into i t s tissues, although they slow the rate of growth and may prevent complete development of the larva. Key words: Heliothis zed, cholesterol, 7-dehydrocholesterol, 24-dihydrolanosterol, 4,4dimethylcholesterol, ergosterol, lanosterol Acknowlegments: I thank Mrs. J.R. Landrey for obtaining the MS and 'H-NMR sDectra and Dr. W.D. Rosenzweig for reading the manuscript. This stidy was partially supporied by NSF grant PCM-8206541. Received August 30,1985; accepted November 13,1985. Address reprint requests to K.S. Ritter, Department of Biological Sciences, Drexel University, Philadelphia, PA 19104. 0 1986 Alan R. Liss, inc. 350 Ritter INTRODUCTION Unlike many organisms, insects require an exogenous source of sterols in order to complete their growth and maturation. As in other animals, insects use the sterols as structural components of membranes and as precursors for hormones (eg, ecdysteroids) [l].Different species of insects, however, have different structural requirements for their sterols. For example, Heliofhis zeu can develop to the adult stage using a variety of A'-, A5-, and A7-sterols as dietary sterols, although the A7-sterols significantly retard the rate of growth of the larva [2,3]. In contrast, the fruit fly Drosophilu pucheu requires A7-sterols as dietary sterols and cannot use A5- or A'-sterols at all . Although the larva of H. zed can mature on a variety of dietary sterols, it does not always metabolize them to the same products. For example, after dealkylation of A'-, A5-, and A7-24-alkylsterols,it carries out little metabolism of the B ring of the resulting 24-desalkylsterols . Therefore, the principal type of sterol in the tissues of H. zed can be altered by varying the type of sterol fed to the larva. That is, depending on whether the larva is fed A5-, A'-, or A7-sterols, the larva will contain primarily cholesterol, cholestanol, or lathosterol, respectively. Since the larva apparently can use a variety of sterols as structural components of membranes, it is of interest to explore why some other sterols (eg, A5, 7-sterols)support only minimal or no growth of larvae . Therefore, the purpose of this investigation was to study the sterol composition of larvae, which had been reared on diets containing various sterols that do not support normal growth (ie, A5T7- and As-sterols) in order to compare and contrast the metabolic fates of these different dietary sterols in this insect. MATERIALS AND METHODS H. zea Neonate larvae of H. zed were reared individually on artificial diets, which lacked supplementation with plant materials but were supplemented with various sterols (15 mgllOO ml diet except where noted), as described previously . In some experiments, the growth of larvae and the metabolites of different dietary sterols were compared in two different populations of H. zeu. Larvae representing one of these populations (A) were kindly supplied by Dr. Wayne Brooks (North Carolina State University, Raleigh) and the larvae in the other population (B) by Dr. Norman Leppla (USDA, ARS, Gainesville, Florida); each group of stock insects was maintained through at least 20 generations. Dietary Sterols The dietary sterols used in these experiments were: cholesterol* (J.T.Baker Chemical Co, Phillipsburg, NJ); 7-dehydrocholesterol (Sigma Chemical Co, *The systematic names for the sterols used in this study are as follows: agnosterol, 4,4,14atrimethyl-5a-cholesta-7,9(11),24-trien-3~-0l; cholesterol, cholest-5-en-3P-ol; 7-dehydrocholesterol, cholesta-5,7-dien-3P-ol; 24-dihydroagnosterol, 4,4,14a-trimethyl-5a-cholesta-7,9(ll)-dien3P-01; 24dihydrolanosterol, lanost-8(9)-en-3P-ol; 4,4-dimethylcholesterol, 4a,4P-dimethylcholest-5-en-3P-01; ergosterol, ergosta-5,7,22-trien-3P-ol; lanosterol, lanosta-8(9),24-dien-3P-ol. Utilization of Sterols by H. zea 351 St. Louis, MO), ergosterol (ICN Pharmaceuticals, Inc., Cleveland, OH), 4,4dimethylcholesterol (a gift from the late Dr. H.W. Kircher, University of Arizona), "lanosterol" and 24-dihydrolanosterol(Mann Research Labs, New York, NY). Each sterol was recrystallized as necessary from ethanol, examined by GLC, ? RPLC, U V S , MS, and 'H-NMR and shown to be at least 98% pure with the exception of the "lanosterol." This latter sterol was found to be a mixture of lanosterol, 24-dihydrolanosterol, agnosterol, and 24-dihydroagnosterol, as reported by others . In GLC analysis, the 24-dihydrolanosterol plus 24-dihydroagnosterol represented 39% of the sample, and lanosterol plus agnosterol represented 61% of the sample; in RPLC analysis at 205 nm, agnosterol and dihydroagnosterol represented < 8% of the sample (ie, approximately 92% of the sterol was As-sterol). The spectra for lanosterol and 24-dihydrolanosterol from MS and 'H-NMR agreed with those reported by others [7l. Isolation of Sterols Late sixth-instar larvae were collected and extracted in order to determine the sterol composition of their tissues after they had consumed different dietary sterols. After the intestine of each larva was removed (in order to remove any unabsorbed dietary sterol), the cadavers were stored at -20°C. The various groups of cadavers were homogenized in acetone using a Sorvall Omnimixer and the sterols obtained by continuous extraction with acetone in a Soxhlet extractor for 24 h. The acetone extracts were saponified over night at 60°C in 5% KOH in 90% ETOH and the 4,4,14-trimethylsterols andl or 4-desmethysterols separated from the other neutral lipids by TLC on Silica Gel G using a solvent system of benzene and ethyl acetate (9:l). The different fractions were collected and the compounds eluted with anhydrous ether. These procedures were carried out under darkened conditions and the recoveries made as rapidly as possible to prevent degradation of the sterols. Similar extraction and purification techniques were used to isolate sterols from the diet and frass of the larvae. Identification of Sterols The sterols isolated from the larvae, diet, and frass were characterized by: GLC between 225°C and 235°C on 3% QF-1, 0.75% SE-30, andlor 1%XE-60 columns, using a Perkin-Elmer Sigma 3B gas chromatograph; analytical RPLC at 45°C on a Zorbax ODS (CIS)column with a mobile phase of acetonitrileisopropanol (80:20) and a flow rate of 2 mllmin, using a Perkin-Elmer Series 3B liquid chromatograph equipped with a variable wavelength detector; U V S by stopping the flow of mobile phase during RPLC and scanning the individ- +Abbreviations: ethanol = ETOH; gas-liquid chromatography = GLC; V, - V o N o(where V, is the retention volume of the sterol and Vo is the void volume) = k'; k' for test sterol/k' for cholesterol = aC; proton nuclear magnetic resonance spectroscopy = 'H-NMR; mass spectrometry = MS; retention time relative to cholesterol = RRT; reversed-plase liquid chromatography = RPLC; side chain = SC; singlet = s; doublet = d; thin-layer chromatography = TLC; ultraviolet spectroscopy = UVS. 352 Ritter ual peaks between 190 and 300 nm; MS via direct probe on a Model 4000 Finnigan instrument with electron impact ionization at 70 eV; andlor ‘HNMR at 360 MHz at ambient temperature on a Bruker instrument, model WH360, in CDC13 with Si(CH3)4as the usual internal standard. Separation of individual sterols in mixtures was carried out by preparative RPLC at 30°C on a Perkin-Elmer preparative C18 column, with a mobile phase of acetonitrile and a flow rate of 10 mllmin, using a Perkin-Elmer Series 1 liquid chromatograph. For all experiments, when f values are indicated, they represent the actual range of the results. RESULTS Growth on ”Lanosterol” ”Lanosterol” was fed to the two populations of larvae in order to determine whether this mixture of 4,4,14-trimethylsterols would support the development of the larvae. After 14 days, one population (A) had completed 13 -t 2% of the 295 molts that were possible (five per larva), and the other population (B) had completed 39-t 5% of its 185 possible molts. The maximum instar reached by either group was the third (Table 1). Growth on ”Lanosterol” Spared With Cholesterol Diets containing different quantities of cholesterol, in addition to lanosterol,” were fed to larvae (A) to ascertain whether the 4-desmethylsterol would spare the 4,4,14-trimethylsterols.Larvae fed diet containing cholesterol (2.5 mgllOO ml diet) in addition to ”lanosterol” (15 mgllOO ml diet) reached a maximum of the fourth instar in 14 days; in contrast, control larvae fed a diet containing cholesterol alone (2.5 mgllOO ml diet) reached only the second instar. Larvae fed “lanosterol” diet, which contained twice as much cholesterol (5 mgllOO ml diet), molted as many as five times, reaching the sixth instar in 14 days; in contrast, control larvae fed a diet containing cholesterol alone (5 mgllOO ml diet), molted up to three times, reaching only the fourth instar in 14 days. When the amount of cholesterol in the ”lanosterol” diet was increased to 10 or 15 mgllOO ml of diet, a greater percentage of larvae reached the sixth instar in 14 days; similarly, control larvae fed a diet containing only cholesterol (10 or 15 mgllOO ml diet) reached the sixth instar in 14 days but in fewer numbers (Table 1). Utilization of ”Lanosterol” Spared With Cholesterol Studies were carried out to investigate whether larvae can metabolize 4,4,14-trimethylsterols, because they developed as far as the third instar when fed these sterols in the absence of other sterols. Since the intestine of the sixth-instar larva can be removed more easily than that of the third instar larva, in these studies the larvae (A as well as B) were fed ”lanosterol” diet spared with cholesterol (5 mgllOO ml diet) to allow them to reach the sixth instar (Table 1).Then the sterols in the diet, frass, and tissues were identified and the relative amounts of the different sterols in each sample determined. Utilization of Sterols by H. zea 353 TABLE 1. Effect of Various Sterols on the Development of Larvae of H. zeu Dietary sterol None None "Lanosterol" "Lanosterol" "Lanosterol" and cholesterol Cholesterol "Lanosterol" and cholesterol "Lanosterol" and cholesterol Cholesterol "Lanosterol" and cholesterol Cholesterol "Lanosterol" and cholesterol Cholesterol 24-Dihydrolanosterol 4,4-Dimethylcholesterol ~ Amount of sterol in diet (mg1100 ml) Population Total no. of molts possible" Average YO of molts completed by day 14b Maximum instar reached by day 14 A B A B 205 260 295 185 0 0 13 f 2 39 5 1 1 3 3 A A 170 195 30 f 4 2*2 4 A 1,095 B A 1,045 255 A A 155 305 94 5 64 f 15 A A A 135 710 165 94 76 15 A 115 0 0 0 15 15 15 2.5 2.5 15 5 15 5 5 15 10 10 15 15 15 15 15 * *3 60 * 8 14 & 7 67 * *3 + 13 "Five molts per larva. bWhen error values are listed, each number is the average of two experiments range of the results. 2 6 6 4 6 6 6 6 3 1 the actual Sterol content of the "lanosterol"-cholesterol diet. One hundred grams of this diet were removed from unused rearing vials, the sterols reisolated, and five peaks detected by RPLC. The crcs of 0.65 0.02, 0.82 k 0.02, 0.93 k 0.02, 1.00 0.02, and 1.18 k 0.02 indicated that agnosterol, lanosterol, 24dihydroagnosterol, cholesterol, and 24-dihydrolanosterol were present, respectively. The relative areas of these peaks at 205 nm were 3, 58, 1, 16, and 22%, respectively. (Note: because of differences in extinction coefficients, the areas from RPLC do not necessarily reflect the true relative concentrations of the sterols in the mixture.) The UV spectra of agnosterol and 24-dihydroagnosterol revealed that the ,,A of both these sterols were 237, 243, and 252 nm, confirming the presence of heterocyclic conjugated double bonds, whereas the spectra from lanosterol, cholesterol, and dihydrolanosterol were characteristic of end absorption indicating an absence of conjugated double bonds. Three peaks were observed by GLC on XE-60 and SE-30. The RRTs of these peaks indicated that 24% of the reisolated dietary sterol was cholesterol (RRT of 1.00 on XE-60 and SE-30), 29% 24-dihydrolanosterol and 24dihydroagnosterol (RRT of 1.29 0.01 on XE-60; 1.51 on SE-30), and 47% lanosterol and agnosterol (RRT of 1.53 k 0.02 on XE-60; 1.65 on SE-30; Table 2). * * * 354 Ritter TABLE 2. Percentage of Sterols, by GLC, in Diet, Frass, and Larvae of Two Populations (A and B) of H. zed fed "Lanosterol" Plus Cholesterol (15 mg 5 mgll00 ml Diet, Respectively) + Source of sterols Diet Frass (A) Frass (B) Larvae (A) Larvae (B) Weight of sample (8) 100 100 20 42 10 Percentage of sterol isolated from sample 24-Dihydrolanosterol Lanosterol Cholesand and terola 24-dih ydroagno~terol~ agnosterol' 24 27 25 59 64 29 28 29 22 24 47 45 46 19 12 aRRT of cholesterol: XE 60, 1-00; SE-30, 1.00. bRRT of 24-dihydrolanosterol and 24-dihydroagnosterol: XE 60, 1.29; SE-30, 1.51. 'RRT of lanosterol and agnosterol: XE 60, 1.53; SE-30, 1.65. Sterol content of "lanosterol"-cholesterol frass. One hundred grams of frass from the larvae A were collected from the rearing vials and the sterols isolated and examined by RPLC. Five peaks with acs of 0.66, 0.83, 0.95, 1.00, and 1.19 indicated that agnosterol, lanosterol, 24-dihydroagnosterol, cholesterol, and 24-dihydrolanosterol were present, respectively; the relative areas of these peaks at 205 nm were 3, 57, 1, 20, and 19%, respectively. The UV spectra of agnosterol and 24-dihydroagnosterol revealed that the A, of both of these sterols were 237, 243, and 252 nm, confirming the presence of heterocyclic conjugated double bonds, whereas the spectra from lanosterol, cholesterol, and 24-dihydrolanosterol were characteristic of end absorption indicating an absence of conjugated double bonds. Three peaks were observed by GLC on XE-60 and SE-30. The relative areas and RRTs of the three peaks observed indicated that 27% of the sterol was cholesterol (RRT of 1.00 on XE-60 and SE-30), 28% 24-dihydrolanosterol andlor 24-dihydroagnosterol (RRT of 1.28 0.02 on XE-60; 1.50 on SE-30), and 45% lanosterol andlor agnosterol (RRT of 1.54 0.01 on XE-60; 1.64 on SE-30). Similar relative amounts of these sterols were isolated from the frass of the other larvae (BTable 2). Sterol content of "lanosterol"-cholesterol larvae. The sterols extracted from the tissues (minus the intestine) of 42 g larvae A as well as 10 g of larvae B produced five peaks by RPLC with acs of 0.65, 0.83, 0.94, 1.00, and 1.18, which indicated that agnosterol, lanosterol, 24-dihydroagnosterol, cholesterol, and 24-dihydrolanosterol were present, respectively. The relative areas of these peaks from sterols isolated from larvae A at 205 nm were 1, 31,1,49, and 17%, respectively. The corresponding relative areas of peaks from sterols isolated from larvae B were 1, 25, 1, 49, and 24%, respectively. The UV spectra of agnosterol and 24-dihydroagnosterol revealed that the A,, of both of these sterols were 237, 243, and 252 nm, confirming the presence of heterocyclic conjugated double bonds, whereas the spectra from lanosterol, cholesterol, and 24-dihydrolanosterol were characteristic of end absorption indicating an absence of conjugated double bonds. Three peaks were observed for both samples by GLC on XE-60 and SE-30. The RRTs of these peaks for sterols from larvae A indicated that 59% was cholesterol (RRT of Utilization of Sterols by H. zea 355 1.01 f 0.01 on XE-60 and SE-30), 22% 24-dihydrolanosterol andlor 24-dihydroagnosterol (RRT of 1.31 k 0.02 on XE-60; 1.50 on SE-30), and 19% lanosterol andlor agnosterol (RRT of 1.56 f 0.02 on XE-60; 1.64 on SE-30). Three similar peaks were observed for larvae B, and the relative percentages of these peaks were 64,24, and 12%, respectively (Table 2). Growth on 24-Dihydrolanosterol 24-Dihydrolanosterol was fed to larvae A to determine how this 4,4,14trimethylsterol, alone, affected the development of the larvae. The results were similar to those obtained for "lanosterol"; that is, after 14 days, the maximum instar reached was the third (Table 1). Growth on 4,4-Dirnethylcholesterol 4,4-Dimethylcholestero1 was fed to larvae A to determine whether the absence of the 14-methyl group in this sterol, and the presence of a double bond at the 5 position instead of the 8 position, would affect the development of larvae fed this sterol. No larvae molted to the second instar, although they ate the diet, produced frass, and lived for as long as 13 days (Table 1). Growth on 7-Dehydrocholesterol When larvae A were fed 7-dehydrocholesterol, no larvae molted, although they ate the diet, produced frass, and lived for as long as 15 days. In contrast, when larvae from the second population of H. zed (B) were fed 7-dehydrocholesterol, they completed 24% of their larval molts in 14 days; however, less than 1%of the larvae reached the sixth instar in this time interval (Table 3). Utilization of 7-Dehydrocholesterol Sterols were isolated from 1.9 g of larvae B, which eventually reached the late sixth instar after feeding on diet containing 7-dehydrocholesterol, and were identified by comparing their RRTs (on QF-l), acs, and UV spectra to those of standards. 7-Dehydrocholesterol represented 76% (by GLC) of the tissue sterols (RRT of 1.11k 0.02; ac of 0.75 0.01; ,A, 271, 282, and 293 nm) and cholesterol represented the remaining 24% (RRT of 1.00 f 0.01; ac of 1.00 0.02; UV spectrum characteristic of end absorption; Table 4). TABLE 3. Effect of A5r7-Sterolson the Development of Larvae of H. zeu Dietary sterol 7-Dehydrocholesterol 7-Dehydrocholesterol Ergosterol Ergostero1 Amount of sterol in diet (mg1100 ml) Population 15 15 15 15 A B A B Total no. of molts possiblea 360 1,290 165 150 Average % of molts completed by day 14b Maximum instar reached by day 14 o+o 1 6 4 5 24 f 3 38 & 5 49 f 11 aFive molts per larva. bEach value is the average of two experiments f the actual range of the results. 356 Ritter TABLE 4. Percentage of Sterols, by GLC, in Diet, Frass, and Larvae of Two Populations of H . zea Fed A5,'-Sterols Exogenous sterol 7-Dehydrocholesterol 7-Dehydrocholesterol Cholesterol and then 7dehydrocholesterold Ergosterol Ergosterol Ergosterol Ergosterol Ergosterol Ergosterol Type of sample Diet Weight of sample (g) 50 Percentage of sterol isolated from sample 7-DehydroCholesterola cholesterolb Ergosterol' 0 100 0 Larvae (B) 1.9 24 76 0 Larvae (B) 6.9 54 46 0 252 100 2.7 8.1 18.7 5.1 0 0 17 15 16 13 0 0 73 61 54 60 100 Diet Frass (A) Larvae (A) Larvae (8) Larvae (BY Larvae (B)f 100 10 23 30 27 aRRT of cholesterol: XE-60, 1.00 f 0.01; QF-1, 1.00 f 0.01. bRRT of 7-dehydrocholesterol: XE-60, 1.22 0.02; QF-1, 1.13 0.01. 'RRT of ergosterol: XE-60, 1.31 f 0.02, QF-1, 1.23 f 0.02. dThe larvae were fed diet containing cholesterol until they molted into the fifth instar (ie, had reached 60% of their final weight), and then they were fed diet containing 7-dehydrocholesterol until the late sixth instar. '20 f 1days old. *40 f 1days old. + + Growth on Diet Containing Cholesterol Before and After Incubation on Diet Containing 7-Dehydrocholesterol Larvae (A and B) were allowed to develop to the early second, third, fourth, and fifth instars on diet containing cholesterol and then were transferred to diet containing only 7-dehydrocholesterol in order to determine whether the poor growth of neonate larvae (both A and B) on 7-dehydrocholesterol was characteristic only of the first instar or whether the growth of all instars would be retarded after they were exposed to this sterol. Also, the reverse experiments were carried out using first-instar larvae that had been maintained on 7-dehydrocholesterol for various amounts of time (ie, 3-9 days, which corresponded to the chronological age of the cholesterol larvae transferred to 7-dehydrocholesteral in the second through fifth instars) in order to determine whether normal growth would resume after they were transferred to diet containing cholesterol. Similar studies were carried out using larvae incubated on diets that lacked sterol instead of containing 7dehydrocholesterol. The results after 9 days of incubation on the final diets are summarized in Table 5. The growth of both populations of larvae slowed when the larvae were transferred from the cholesterol diet to 7-dehydrocholesteral diet, especially as newly molted second, third, and fourth instars; similar results were obtained when the larvae were transferred to diet containing no sterol during these instars. Interestingly, those first-instar larvae of corresponding Utilization of Sterols by H. zed 357 TABLE 5. Effect of Dietary Cholesterol on Growth of Two Populations (A and B) of Larvae Before and After Incubation on Diet Containing 7-Dehydrocholesterolor No Sterol Sterol (W mgilOO ml diet) in Average instar of larvae 9 days after transfer from initial diet to final diet (instar transferred to final diet) _2nd_ _3rd~ _4th _ _5th A B A B A B A B Initial diet Final diet Cholesterol Cholesterol Cholesterol 7-Dehydrocholesterol None 7-Dehydrocholesterol Cholesterol 3.0 2.9 3.8 4.0 5.1 4.9 5.4 5.9 lsta _ lsta_ ~ lsta _ lsta _ 1.0 2.3 1.0 1.5 1.0 1.0 1.0 1.4 4.7 4.9 3.3 4.6 4.3 4.0 3.4 3.8 None Cholesterol 1.0 4.9 Cholesterol 7-Dehydrocholesterol 7-Dehydrocholesterol None None 6.0 2.7 5.1 3.6 1.0 4.4 5.8 3.7 1.0 3.3 6.0 4.1 1.0 4.4 6.0 5.1 1.0 3.0 6.0 5.5 1.0 3.2 6.0 5.9 1.0 3.0 6.0 6.0 1.0 2.5 aAlthough these larvae were in the first instar, they were the same age as those in the columns above them. chronological age, incubated on diets containing 7-dehydrocholesterol or lacking sterol for 3-9 days (ie, the chronological age of normal larvae chosen in the second through fifth instars), began to develop when transferred to diet containing cholesterol; individuals in these diferent groups subsequently reached the pupal stage after 16 or more days of incubation on the cholesterol diet (control larvae reared continuously on cholesteral began to pupate in 15 days). Utilization of 7-DehydrocholesterolAfter Incubation on Cholesterol Larvae B were reared on diet containing cholesterol until apolysis at the end of the fifth instar (at which time they weighed an average of 0.19 k 0.02 g or 60% of their final weight) and then were transferred to diet containing 7-dehydrocholesterol.This preincubation of the larvae on cholesterol allowed the larvae to develop to the late sixth instar, which was easily dissected. Identification of the tissue sterols from 6.9 g of these larvae was made by comparing the RRTs, acs, and UV spectra of the sterols to those of standards. The relative amounts of the sterols in the tissues were determined by GLC analysis. Fifty-four percent of the sterol of these larvae was cholesterol (RRT of 1.00 +_ 0.01; cyc of 0.99 f O.Ol), and 46% was 7-dehydrocholesterol (RRT of 1.13 k 0.02; a, of 0.76 +_ 0.01; A,, of 271, 282, 293 nm; Table 4). Growth on Ergosterol Larvae A that were fed diet containing ergosterol completed 38% of their 165 total larval molts and developed as far as the fourth instar after 14 days, and the other larvae (B) completed 49% of their 150 molts after 14 days and developed as far as the fifth instar (Table 3). ~ 358 Ritter Utilization of Ergosterol Sterol content of ergosterol diet. When the sterol was isolated from 252 g of ergosterol diet and its RRT (on QF-1), a,, and UV spectrum compared to those of standards, ergosterol was the only sterol detected (RRT on QF-1 of 1.23 f 0.02; a, of 0.70 k 0.02; A,, of 271, 282, and 293 nm; Table 4). Sterol content of ergosterol frass. When the sterols were isolated from 100 g of frass collected from the larvae A reared on ergosterol diet, ergosterol (RRT of 1.23 0.01 on QF-1; a, of 0.70 f 0.01; A,, of 271, 282, and 293 nm) was the only sterol detected (Table 4). The fragmentation pattern of this sterol in MS was consistent with that of a standard of ergosterol: M+ = mle 396, 42%; M+-H20-CH3 = 363, 50%; Mf-H20-SC = 253, 83%; M+-H20SC-C3H6 = 211, 100%. Sterol content of ergosterol larvae. The metabolites (>3%) of dietary ergosterol were characterized from both populations of larvae (A and B) as well as from those specific individuals in B who reached the sixth instar in 20 f 1 days and those who reached the sixth instar in 40 f 1 days in order to determine whether there were differences in degree of metabolism of ergosterol in larvae requiring different lengths of time to reach the sixth instar (Table 4). The relative amounts of the sterols in the tissues were determined by GLC analysis. The sterol composition of 2.7 g of larvae A fed ergosterol was: 17% cholesterol (RRT of 1.01 f 0.01 on XE-60 and QF-1; a, of 0.99 f O.Ol), 73% 7-dehydrocholesterol (RRT of 1.22 0.02 on XE-60 and 1.13 f 0.01 on QF-1; a, of 0.76 f O.Ol), and 10% ergosterol (RRT of 1.31 f 0.02 on XE-60 and 1.23 0.01 on QF-1; a, of 0.70 f 0.01). The character of UV absorption of each of the latter two peaks indicated that both sterols had a conjugated double bond system and so could be A537-sterols(A, 271, 282, and 293 nm), whereas the UV spectrum for the first sterol was characteristic of end absorption, indicating an absence of conjugated double bonds. Similar evidence from GLC, RPLC, and UV analyses indicated that the sterol composition of 8.1 g of larvae B fed ergosterol was 15% cholesterol, 61% 7dehydrocholesterol, and 23% ergosterol. Likewise, the sterol composition of those larvae B (18.7 g) fed ergosterol and reaching the late sixth instar in 20 days was 16% cholesterol, 54% 7dehydrocholesterol, and 30% ergosterol. When the individual sterols, from these mixtures of sterols, were separated using preparative RPLC, not only did GLC analysis of the individual peaks confirm the above identifications but the M+ and fragmentation pattern of each sterol in MS was consistent with the presence of cholesterol (M+ = mle 386, 100%; M+-85 = 301, 75%; Mf-C7H11 = 275, 63%; M+-SC = 273, 50%; M+-SC-H20 = 255, 63%), 7dehydrocholesterol (M+ = 384, 100%; Mf-CH3 = 369,l8%; M+-H20-CH3= 351, 88%; M+-H,O-C,H, = 325, 75%; Mf-H20-SC = 253, 69%; M+-C3H*-SC = 227, 44%), and ergosterol (M+ = 396) . The presence of these three sterols was confirmed by 'H-NMR. The chemical shift of 'H in ppm from Si(CH& at various carbons in cholesterol was C-18,0.68 (s);C-26 and 27,036 and 0.87 (d, J =6 Hz); C-21, 0.91 (d, J = 6 Hz); C-19, 1.01 (s). The 'H-NMR spectrum for the 7-dehydrocholesterol peak was C-18, 0.62 (s); C-26 and 27, 0.86 and 0.87 (d, J = 6 Hz); C-21, 0.94 (d, J = 6 Hz); C-19, 0.95 (s). The 'H- Utilization of Sterols by H. zea 359 NMR spectrum for the ergosterol peak was C-18, 0.62 (s); C-26 and 27, 0.82 and 0.85 (d, J = 6 Hz); C-21, 1.03 (d, J = 6 Hz); C-19, 0.94 (s). Similarly the sterol composition of those larvae B (5.1 g) fed ergosterol and reaching the late sixth instar in 40 days was 13% cholesterol, 60% 7-dehydrocholesterol, and 27% ergosterol (Table 4). DISCUSSION H. zed is similar to other insects  in that it cannot complete its development using 4,4,14-trimethylsterols as its sole dietary sterols. However, the present study indicates that the larva can develop as far as the third instar not only on "lanosterol" but also on pure 24-dihydrolanosterol. When cholesterol was used to spare the "lanosterol" (in a 1:3 ratio, respectively) the larvae then reached the sixth instar. When the fate of the dietary "lanosterol" spared with cholesterol was examined in the metabolic studies, the ratio of cholesterol to the other sterols in the tissues increased to 1:l.Therefore, H. zea may be able to convert some of the 4,4,14-trimethylsterols to cholesterol because 1)the relative percentage of cholesterol in the tissues was at least double that in the diet; 2) there was little change in the relative percentages of sterols in the frass, which indicated that cholesterol was not selectively absorbed from the diet; 3) the relative percentage of lanosterol in the tissues was reduced by more than twofold, in contrast to the relative percentage of 24-dihydrolanosterol (which was only slightly below that in the diet), indicating that lanosterol can preferentially be converted to cholesterol or that it can first be converted to 24-dihydrolanosterol which may then be converted to cholesterol. The ratio of agnosterol to 24-dihydroagnosterol in the tissues also decreased from that of the diet. Despite this evidence, proof that 4,4,14trimethylsterol can be converted to cholesterol must await future studies using radioactive sterol. Since larvae did not develop at all on dietary 4,4-dimethylcholestero1, apparently the presence of the gem-dimethyl group at C-4 prevents this sterol from being a suitable component of membranes (andlor hormones) or a substrate that can be metabolized to other sterols such as cholesterol. There was some difference in the ability of the sterols to support the development of the two populations (A and B) of H. zed. Although some individuals in both groups reached the third instar with "lanosterol" as their dietary sterol, population B completed more of its total possible molts in 14 days (39% vs 13%). Population B was also able to use A5r7-sterolsmore efficiently than population A, because, unlike populations tested previously [3,10], some larvae molted when fed 7-dehydrocholesterol (24% of their total possible molts were completed in 14 days vs 0% for population A). Because a few individuals in population B actually reached the sixth instar when fed 7-dehydrocholesterol, unlike those in population A, it was possible to examine the sterol content of these individuals to ascertain the metabolic fate of this sterol. Since 7-dehydrocholesterol was the principal sterol recovered from their tissues, after the sterol was absorbed by the intestine much of it was probably used directly in the membranes. Therefore, the uptake and direct utilization of A5,7-desmethylstero1in tissues was similar to 360 Ritter that of the A0-,A5-, and A7-desmethylsterols, which also became the principal tissue sterols of larvae despite the differences in their B rings . However, since the rate of growth of the larva varied when it was fed these different sterols (A5 > A' > A7 > A5,7, with the rate being exceptionally slow on the A5,7-desmethylsterol, this indicated that the position and degree of unsaturation of the B ring affects the rate of absorption of the dietary sterol andlor synthesis of membranes using the dietary sterol andlor conversion of the molecule to other essential molecules (eg, ecdysteroids). The inability of 7-dehydrocholesterol to support normal growth was not limited to the first instar; development of second- third-, and fourth-instar larvae was also slowed when they were transferred from diet that contained cholesterol to diet that contained 7-dehydrocholesterol. Similar results were obtained when they were transferred to diet that contained no sterol (Table 5). When larvae reared on cholesterol were transferred to diet containing 7dehydrocholesterol, prior to the onset of feeding in the sixth instar, and the sterol composition of the tissues was determined at the end of the sixth instar, the ratio of cholesterol to 7-dehydrocholesterol in the larva (54:46) was similar to the percent of weight gained by the larva during the time spent on the two different sterols (60:40, respectively). This confirmed the evidence presented above that most of the dietary 7-dehydrocholesterol was incorporated directly into the tissues of the larvae. Although 7-dehydrocholesterol inhibited growth of larvae, the negative effect of 7-dehydrocholesterol on growth was reversible up to at least 9 days after incubation on this sterol; larvae that had remained in the first instar for this period of time finally completed their development and pupated after they were transferred to diet containing cholesterol. Other first-instar larvae also completed their development and pupated after they were transferred from diet containing no sterol to diet containing cholesterol. Therefore, the poor growth or absence of growth of larvae on diets containing 7-dehydrocholesterol, as well as no sterol, is reversible during at least the first 9 days of incubation and is probably due to lack of a suitable sterol substrate rather than a toxic effect of 7-dehydrocholesterol. As we have reported earlier, although ergosterol supports more rapid development of the larvae than 7-dehydrocholesterol, this A5t7f22-24/3-methylsterol supports slower development than the other A'-, A5-, and A7-sterols of the 24-ethyl, 24-methyl, and 24-desmethyl series that we tested . In the present study, when the sterol composition of the larvae fed ergosterol was examined, the ergosterol was metabolized to the same major end product in both populations (ie, 7-dehydrocholesterol). Also, when the sterol composition of members of population B that reached the sixth instar in 20 days was compared to that of larvae that required 40 days, their relative sterol compositions were similar. Therefore, the amount of time required to reach the sixth instar did not seem to affect (or was not the result of) the degree of metabolism of ergosterol. This is the first report of the conversion of ergosterol to 7-dehydrocholesterol in insects. In contrast, Blotella gemanica converts dietary ergosterol to 22-dehydrocholesterol [ll]. 7-Dehydrocholesterol is the principal tissue sterol of those insects, which consume this sterol directly, as well as those consuming ergosterol; this Utilization of Sterols by H. zed 361 suggests that the difference in the rates of growth of larvae on these two sterols involves differences in their rates of absorption, andlor ergosterol mi ht spare 7-dehydrocholesterol. It is not clear why the accumulation of A5/ -sterols in the tissues of H.zeu inhibits the development of this organism; other species of insects (eg, Tribolium confusum  and Attu cephulotes isthrnicola ) develop normally using A5i7-sterolsas tissue sterols. It is not known if the cholesterol, which was found in larvae fed ergosterol or 7-dehydrocholesterol, was a metabolic product of these dietary sterols. Minor amounts of cholesterol have been found consistently in those larvae fed A'-, A7-sterols and analyzed in the sixth instar . Ergosterol was the only sterol recovered from the ergosterol diet as well as from the frass, and 7-dehydrocholesterol was the only sterol isolated from the 7-dehydrocholesterol diet; this indicates that there was no contamination of the diet with other sterols and that no microorganisms in the digestive tract metabolize the dietary ergosterol to other sterols. Ergosterol, lanosterol, and 24-dihydrolanosterol, as well as other dietary 24-alkylsterols , can be recovered from the tissues of H. zeu; this indicates that these alkylated sterols enter the hemocoel of the larva prior to dealkylation. Therefore, differences in the position or number of double bonds (ie, A'-, A5-, A7-, As-, and A5r7-sterols)andlor the addition of alkyl groups (at C4/14! and C-24) do not prevent the uptake of certain dietary sterols. However, the slow rate of growth of H. zeu on A5,7- and As-sterols, relative to A5-, A'-, and A7-sterols, might be due to differences in the rates of uptake of these diverse sterols andlor the inability of A5r7- and As-sterols to fulfill all the essential metabolic roles necessary to support maximal growth of larvae of H. zed. 9 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, Nes WR: The effects of cholesterol on the development of Heliothis zea. J Insect Physiol, 27, 175 (1981). 3. Ritter KS, Nes WR: The effects of the structure of sterols on the development of Heliothis zea. J Insect Physiol, 27, 419 (1981). 4. Goodnight KC, Kircher HW: Metabolism of lathosterol by Drosophila puchea. Lipids, 6, 166 (1971). 5. Ritter KS: Metabolism of A'-, A5-, and A7-sterols by larvae of Heliothis zea. Arch Insect Biochem Physiol, 1, 281, (1984). 6. Nes WR, McKean ML: Biochemistry of Steroids and Other Isopentenoids. University Park Press, Baltimore, p 443 (1977). 7. Sekula BC, Nes WR: The identification of cholesterol and other steroids in Euphorbia pulcherimmu. Phytochemistry, 19, 1509 (1980). 8. Nes WR, Krevitz K, Joseph J, Nes WD, Harris B, Gibbons GF, Patterson GW: The phylogenetic distribution of sterols in tracheophytes. Lipids, 12, 511 (1977). 9. Kircher HW: Sterols and insects. In: Cholesterol Systems in Insects and Animals. Dupont J, ed. CRC Press, Inc. Boca Raton, pp 1-50 (1982). 10. Ritter KS: Some unusual aspects of the sterol biochemistry of insects. In: Isopentenoids in Plants, Biochemistry and Function. Nes WD, Fuller G, Tsai L, eds. Marcel Dekker, Inc. New York, pp 389-400 (1984). 362 Ritter 11. Clark AJ, Bloch K: Conversion of ergosterol to 22-dehydrocholesterol in Blattella germanica. J Biol Chem 234, 2589 (1959). 12. Svoboda JA, Robbins WE, Cohen CF, Shortino, TJ: Phytosterol utilization and metabolism in insects: recent studies with Tribolium confusurn. In: Insect and Mite Nutrition. Rodriguez JG, ed. North-Holland, Amsterdam, pp 505-516 (1972). 13. Ritter KS, Weiss BA, Norrbom AL, Nes WR: Identification of A5r7-24-methylene-and methylsterols in the brain and whole body of Atta cepkalotes istkmicola. Comp Biochem Physiol 7 2 B , 345 (1982).