Archives of Insect Biochemistry and Physiology 4:l-15 (1987)

Metabo I ism of 2 63' 4C] H y d rox y ecd y sone 26-Phosphate in the Tobacco Hornworm, Manduca sexta L., to a New Ecdysteroid Conjugate: 26-['4C]Hydroxyecdysone 22-Glucoside Malcolm J. Thompson, Mark F. Feldlaufer, Ruben Lozano, Huw H. Rees, William R. Lusby, James A. Svoboda, and Kenneth R. Wilzer, Jr. Insect and Nematode Hormone Laborato y, Agricultural Research Sem'ce, USDA, Beltsville, Ma yland (M.J. T., M.F.F., R. L., W.R. L., J.A.S., K.R. W.) and Department of Biochemistry, University of Liverpool, Liverpool, United Kingdom (H.H. R.)

Following injection into female Manduca sexta pupae, ['4C]cholesterol is converted to a radiolabeled C2, nonecdysteroid conjugate as well as ecdysteroid conjugates, which in ovaries and newly-laid eggs consist mainly of labeled 26-hydroxyecdysone 26-phosphate. During embryogenesis, as the level of 26-hydroxyecdysone 26-phosphate decreases there is a concurrent increase in the amount of a new, labeled ecdysteroid conjugate. This conjugate, which is the major ecdysteroid conjugate (9.4 pg/g) in 0- to l-hour- old larvae was identified as 26-hydroxyecdysone 22-glucoside by nuclear magnetic resonance and chemical ionization mass spectrometry. This is the first ecdysteroid glucoside to be identified from an insect. The disappearance of 26-hydroxyecdysone 26-phosphate in 0- to I-hour-old larvae indicates that the 26hydroxyecdysone 22-glucoside is derived from 26-hydroxyecdysone 26-phosphate. 3-Epi-26-hydroxyecdysone was the major free ecdysteroid isolated from these larvae and 3-epi-20,26-dihydroxyecdysone was the next most abundant ecdysteroid isolated. Interestingly, the 0- to I-hour-old larvae contained the highest levels of 3a-ecdysteroids per gram of insect tissue (8.7 &g) to be isolated from an insect, yet there was a complete absence of the corresponding free 3fi-epimers. The ecdysteroid conjugate profiles of ovaries and 0- to I-hour-old larvae are discussed. Methodology is presented that permits the efficient separation of free and conjugated ecdysteroids and nonecdysteroid conjugates (C2,-steroid conjugates).

Acknowledgments: We thank Dr. B.E. Mann, University of Sheffield, for the PMR spectra. The technical assistance of D. Harrison, M.D. Hollenbeck, and L.J. Liska is greatly acknowledged.

Received January 27,1986; accepted May 5,1986.

Address reprint requests to Malcolm J. Thompson, insect and Nematode Hormone Labora- tory, Bldg. 467, BARC-East, Beltsville, MD 20705.

2 Thompson et a1

Key words: ['4C]cholesterol, radiolabeled ecdysteroid conjugates, 26-hydroxyecdysone 22- glucosidel 26-hydroxyecdysone 26-phosphate, 3-epi-26-hydroxyecdysonel 3-epi- 20,26-dihydroxyecdysone, enzymatic hydrolysis

INTRODUCTION

The ovaries and newly-laid eggs of various insect species contain compar- atively large quantities of ecdysteroid conjugates and only minute amounts of free ecdysteroids [l-31. In ovaries and newly-laid eggs (0- to 1-h-old) of the tobacco hornworm, Munducu sexta, conjugates also account for more than 95% of the total ecdysteroids present, and we have identified 26-hydroxyec- dysone 26-phosphate as the major ecdysteroid conjugate [4]. In an effort to isolate and identify precursors of this ecdysteroid conjugate and to determine its metabolic fate, we examined the metabolism of [14C]cholesterol, a putative ecdysteroid precursor in M. sextu 151. Although a substantial amount of [14C]cholesterol was converted to 26-[14C]hydroxyecdysone 26-phosphate, the major metabolite of [14C]cholesterol in both ovaries and eggs was an unexpected C21 nonecdysteroid conjugate, namely 5-[14C]pregnen-3P,20P- diol glucoside containing three glucose units [6].

Quantitation of the ecdysteroid conjugate fraction, however, did show that the levels of radiolabeled 26-hydroxyecdysone 26-phosphate declined from 31 p g l g in ovaries of 4-day-old females, to 17 pglg in 72- to 88-h-old eggs [5]. As the levels of 26-hydroxyecdysone 26-phosphate in eggs de- creased, there was a concurrent increase in the amount of a new labeled ecdysteroid conjugate [5]. In this paper we provide evidence for the subse- quent metabolism of 26-[14C]hydroxyecdysone 26-phosphate in M. sextu to 26-hydroxyecdysone 22-glucoside and 3-epi-26-hydroxyecdysonc3, the major ecdysteroid conjugate and free ecdysteroid, respectively, in 0- to 1-h-old larvae of the tobacco hornworm. Methodology is presented that permits the separation of free and conjugated ecdysteroids and nonecdysteroid conju- gates (C21-steroid conjugates), which are major radiolabeled metabolites of labeled cholesterol in M. sextu ovaries and eggs.

MATERIALS AND METHODS Sterol

[4-14C]Cholesterol was purchased from Amersham Corp. * (Arlington Heights, IL). After purification by column chromatography the radiochemical purity was >99% by TLC?, specific activity 53.7 mCilmmo1.

+Abbreviations: ethanol = EtOH; high-performance liquid chromatography = HPLC; high- performance thin-layer chromatography = HP-TLC; methanol = MeOH; nuclear magnetic resonance = NMR; proton magnetic resonance = PMR; thin layer chromatography = TLC.

*Mention of a company name or proprietary product does not constitute an endorsement by the U.S. Department of Agriculture.

Free and Conjugated Ecdysteroids in Manduca larvae 3

Biological Material Tobacco hornworms were reared as described previously [7J. One pCi of

[14C]cholesterol in 25 p1 of a saline solution [8] containing 3% Tween 80 was injected into each female pupa (day 16) through the ventral intersegmental membrane between the fifth and sixth abdominal segments with a microsy- ringe [9]. As the moths emerged the females were kept in a flight cage with equal numbers of uninjected adult males and allowed to oviposit on tobacco plants. Eggs were removed from the tobacco plant within 18 h of laying and kept in Petri dishes at 28°C until hatch. The larvae (0- to 1-h-old) were collected every hour, counted as they were transfened into screw cap glass bottles, and stored in methanol at -20°C until workup. A total of 5,382 larvae (6.46 g) were collected. The larvae were extracted, and the free and conjugated ecdysteroids were isolated according to the schemes shown in Figures 1 and 2. In the scheme (Fig. 1) the partitionings were conducted as previously described [lo].

Purification of Ecdysteroid Conjugates from Aqueous Phase The aqueous phase from the butanol-water partition (Fig. 1) was reduced

to dryness under vacuum. The residue (245 mg) was redissolved in 4 ml of water and the solution was adjusted to pH 4 with 2N acetic acid. It was then

TDBACCO HORNMORH URVAE ( 6 . 5 g )

Homgenized i n MeOH, then i n 70% leOH

AQUEOUS &OH EXTRACTS

D r i e d under vacuum

RESIOUE (406 mg)

P a r t i t i o n e d a g a i n s t hexane and 70% MeOH

APDUR STEROLS IN H E M E 4 4.58 x lo6 dpm (94.4 mg)

ECDYSTEROIDS A)(D CONJUGATES IN 70Z &OH

Reduced t o dryness under vacuum

RESIDUE (291 mg; 0.41 x lo6 dpm) P a r t i t i o n e d between BuOH and H,O

FREE ECDYSTEROIDS. ECOYSTEROIO AND NOH- &CONJUGATED ECDYSTEROIDS I N H P c ECDYSTEROID CONJUGATES IN BuDH 4 Reduced t o dryness under v cuum RESIDUE (33.7 mg; 0.2 x 10% dpm)

Reduced t o dryness und r vacuum

Redissolved i n 4 ml o f H20 and a d j u s t e d t o pH 4 w i t h 2N AcOH

RESIDUE (245.2 mg; 0.12 x 105 dpm)

XM-2 C O l U M

2) EtOH f 150 ml D r i e d under vacuum

RESIDUE (19.5 mg; 0.12 x 106 dpm)

Dissolved i n 2.5 ml 10% MeOH

c1* YP-PAK 7

11 5 ml 10% MeOH 2) 5 ml 10% @OH Discarded

3) 5 m l 30% MeOH (3.2 mg. 5.28 x lo4 4) 10 ml 30% MeOH (1.5 mi; 5.42 x lo4 5) 5 ml 40% MeOH (10.7 mg; 0.46 x lo4

L Fig. 1. Procedure used for the isolation and purification of ecdysteroid conjugates from the aqueous phase of a butanol-water partition system.

4 Thompson et al

placed on a 1.5 x 16-cm column (bed volume 31 ml) of Amberlite XAD-2 beads (Rohm and Haas, Philadelphia, PA). The column was eluted with 150 ml of water followed by 150 ml of ethanol. The ethanol, which contained the ecdysteroid conjugates, was removed under vacuum, and the residue (19.5 mg) was further fractionated on a SEP-PAK cartridge (Millipore, Waters Chromatography Division, Milford, MA) as shown in Figure 1.

Purification of Ecdysteroid Conjugates from Butanol Phase The butanol phase from the butanol-water partition (Fig. 1) was reduced

to dryness under vacuum. The residue (33.7 mg) was further fractionated on a Florisil SEP-PAK cartridge (Waters) and then by SEP-PAK as shown in Figure 2.

High-Performance Liquid Chromatography and Radioassay of Ecdysteroid Conjugates

The ecdysteroid conjugates from the aqueous phase (combined c18 SEP- PAK fractions 3 and 4, Fig. 1) and from the butanol phase (combined SEP-PAK fractions 2 and 3, Fig. 2) were analyzed by ion suppression re- versed-phase HPLC with a Spectra-Physics 8700 solvent delivery system (Santa Clara, CA) on an IBM cf3 column (4.6 mm x 15 cm; 5-pm particle size, Danbury, CT) by isocratic elution with 26% methanol in 0.03 M aqueous NaHZP04 solution (pH 5) at 1.0 mllmin flow rate. Absorbance of the effluent at 254 nm was monitored with a Model 441 absorbance detector (Waters) and automatically recorded with a Shimadzu Model C-R3A integrator (Columbia, MD). Samples collected from HPLC were combined and concentrated under reduced pressure, desalted on SEP-PAK [4], and reanalyzed by HPLC in the above solvent system. When fractions (0.5 ml) were collected for moni- toring radioactivity, scintillation fluid (4 ml Hydrofluor, National Diagnostics, Somerville, NJ) was added directly to the solvent, and the fractions were counted in a Packard Tri Carb 46OCD scintillation counter or a Beckman LS 5801 liquid scintillation system.

High-Performance Liquid Chromatography and Radioassay of Free Ecdysteroids

The free ecdysteroids, which were only present in the butanol phase, were subsequently eluted in Florisil SEP-PAK fractions 3 and 4 (Fig. 2) with only a trace quantity present in fraction 5. The two fractions, both separately and combined, were analyzed by reversed-phase HPLC, as above, for the ecdy- steroid conjugates, except a Shandon (Sewickley, PA) ODs-Hypersil c18 column (4.6 mm X 25 cm, 5-pm particles) was used, and the solvent system was 38% methanol in water with a flow rate of 1.0 mllmin. Samples collected from HPLC were combined, concentrated under reduced pressure, and rean- alyzed by HPLC in the above solvent system. They were sufficiently pure for other physical and chemical analyses.

When fractions (0.5 ml) were collected for monitoring radioactivity, scintil- lation fluid (4 ml Hydrofluor) was added directly to the solvent, and the fractions were counted.

Free and Conjugated Ecdysteroids in Manduca Larvae 5

Hydrolysis of Ecdysteroid Conjugate by Enzyme Mixture An aliquot of the HPLC purified conjugate (40 pg), dissolved in 1 ml of 0.2

M sodium acetatelacetic acid buffer solution (pH 5), was added to a freshly prepared solution of 1 mg of P-glucuronidase (H-1; Sigma, St. Louis, MO), 1 mg of 0-glucuronidase (L-11; Sigma), and 1 mg P-glucosidase (Sigma) in 1 ml of 0.2 M sodium chloride. The mixture was incubated at 30°C for 48 h, and the released ecdysteroid was extracted into butanol and partially purified via C18 SEP-PAK prior to analysis by HPLC. The aqueous phase was reduced in volume with a stream of nitrogen and then placed on a 1 x 2-cm column of mixed bed resin (Bio-Rad AG 501-X8D, Richmond, CA). The released sugar was eluted with 100 ml of water.

High Performance Thin-Layer Chromatography Samples of the ecdysteroid fractions from SEP-PAK chromatography were

monitored by HJ.'-TLC (pre-coated plates for Nan0 TLC, Silica Gel 60F 254, E. Merck, Darmstadt, F.R.G.). For free ecdysteroid separations, the TLC plates were developed twice in the solvent system chloroformlethanol(65:35). For ecdysteroid conjugate separations, the TLC plates were developed once or twice in the solvent systems chloroformlmethanolllON ammonium hy- droxide (15:35:3.5). The spots were detected by UV, and the plates were sprayed with 50% H2S04 solution and charred in an oven at 105°C. The areas where spots developed were scraped from the plate, added to 4 ml of Hydrofluor, and counted.

Mass Spectrometry

4500 spectrometer equipped with an Incos data system. Mass spectral data were obtained via direct probe with a Finnigan Model

NMR Spectroscopy Fourier transform PMR spectra were recorded on a JEOL FX-60-Q or a

Bruker 400 MHz Fourier transform instrument. Samples were analyzed in C5D5N or D20, and PMR spectra were referenced to tetramethylsilane and 3-trimethylsilyl-2,2,3,3,-tetradeuteropropionic acid for spectra taken in C5D5N and D20, respectively.

RESULTS Isolation of Major Ecdysteroid Conjugate

The nature of ecdysteroid conjugates in 0- to 1-h-old larvae prevented the complete partitioning of the conjugated ecdysteroids into the aqueous phase by the butanol-water partitim system, which was successfully accomplished in the analysis of ecdysteroids and ecdysteroid conjugates of ovaries and eggs of the tobacco hornworm [4,10]. In the present case, whereas the aqueous phase contained only ecdysteroid conjugates, the butanol phase contained both free and conjugated ecdysteroids as well as nonecdysteroid conjugates (Fig. 1). After applying the conjugates in aqueous phase (adjusted

6 Thompson et a1

to pH 4 with 2N acetic acid) to an XAD-2 column (Fig. l), 93% of the total mass was removed from the column with water, and the partially purified conjugate was eluted with ethanol. The C18 SEP-PAK fractionation removed most of the remaining impurities (fractions 1, 2, and 5, Fig. 1). Although fraction 5 contained 4% of the total radioactivity of the conjugate fraction, it was not included in further analyses because of the large quantity of impuri- ties. The ion suppression reversed-phase HPLC and radioassay of 2% of the conjugates (combined fractions 3 and 4) gave the chromatogram shown in Figure 3B. The isolation of the ecdysteroid conjugates from the butanol phase was successfully achieved via Florisil SEP-PAK and CIS SEP-PAK fractiona- tions (Fig. 2). The ion suppression reversed-phase HPLC and radioassay of 1% of the conjugates (combined C18 SEP-PAK fractions 2 and 3) gave the chromatogram shown in Figure 3A. The CIS SEP-PAK fractions 5 and 6 gave the nonecdysteroid conjugate, 5-pregnen-3@,20P-diol triglucoside [6]. The combined ecdysteroid conjugate fractions from processing the aqueous phase (Fig. 1) and the butanol phase (Fig. 2) gave the ecdysteroid conjugate profile of 0- to 1-h-old larvae as shown in Figure 4B. The major peaks eluting at 7.97 and 7.31 min and the minor peaks eluting at 4.45, 9.77, and 16.76 min were radioactive. The profile of ecdysteroid conjugates from day-4 adult ovaries (Fig. 4A), previously determined [5], has been included for comparison.

For the identification of the major ecdysteroid conjugate, the isolation process in Figures 1 and 2 was conducted with 0- to 1-h-old larvae (21.4 g) derived from uninjected pupae. However, a Florisil column [lo g, (1.5 x 15 cm) 60-100 mesh, Fisher Sci., Fairlawn, NJ] rather than a Florisil SEP-PAK was used in the fractionation and purification of the butanol phase, and 50- ml fractions of 5, 15, 25, 25, 30, 30% ethanol in chloroform and two 50-ml

FRACTIWTION AND PURIFICATION OF B U N PHASE

Reduced to dryness under vacuum

RESIDUE 1 3 3 . 7 mg; 20.0 x lo4 dpml

FLORISIL SEP-PAY E t O H i n CHC13

t

1) 5 ml 5% E t O H 2) 5 ml 15% E t O H 3) 5 ml 25% E t O H 4) 5 ml 25% E t O H 5 1 5 ml 30% E t O H 6) 5 ml 30% E t O H

11.6 mg; (2.3 mg; 12.0 ma: (1.c m i ; (1.3 mg; (1.1 mg;

o 54 104 0'83 104 6:52 x 104 4.41 104 1.44 104 0.96 x lC4 -i

71 5 ml 40% EtOH 11.4 mg; 2.50 x lo4 dpm) E l 5 ml 60% E t O H (1.7 mg. 3.13 x lo4 dpm) 91 5 nl 100% E t O H (2 .5 mg1 0.53 x 104 dpml

Combined fract ions 7 through 9 and reduced to dryness under vacuum

ECDYSTEROID AND NONECOYSTEROID CONJUGATES

Dissolved i n 2 .5 ml 10% MeCH

Cia SEP-PAK 11 10 ml 10% MeOH 13.0 mg; 0 dpm) 21 5 mt 30% MeOH (0.4 mg. 2 16 x lo4 dpm) ECDYSTEROID 31 10 ml 30% &OH ( 0 . 2 mgI 0:84 x 104 dpmJcoNJUGATES 4) 5 m l 40% MeOH (0.1 mg; 0 dpml

51 5 ml 60% MeOH (0.5 mg' 3.23 x 104 6) 5 ml 100% MeOH 10.8 mg; 0.15 x lo4

Fig. 2. Procedure used for the isolation and purification of free ecdysteroids, ecdysteroid, and nonecdysteroid conjugates from the butanol phase of a butanol-water partition system.

Free and Conjugated Ecdysteroids in Manduca Larvae 7

n - , ~ , 1.p"'AU

E Q -0 4 8 12 16 20 U

>- k

300 l- 0 < 0

a a

200

100 AU

4 8 12 16 20

T IME (minl

Fig. 3. Ion suppression reversed-phase HPLC trace (U.V.) and radioassay analysis of partially purified ecdysteroid conjugates from 0- to I-h-old M. sexta larvae, partitioned into the butanol phase (A) or into the aqueous phase (B). Column conditions: IBM C8 column (4.6 mm x 15 cm) eluted isocratically with 26% methanol in 30 mM aqueous NaH2P04 solution (pH 5) at a flow rate 1.0 ml/min. Shaded areas indicate radioactivity.

fractions of 100% ethanol were collected. Fractionation of the residue from the ethanol effluent from the XAD-2 column via C18 SEP-PAK followed by HPLC analysis also gave a chromatogram similar to that of the chromatogram shown in Figure 3B. Final purification of the ecdysteroid conjugates from the SEP-PAK fractions of both the aqueous and butanol phases was achieved by collection from ion suppression HPLC followed by desalting on a C18 SEP- PAK cartridge [4]. The material eluting at 7.97 min exhibited an absorbance maximum at 240 nm (in methanol), which is characteristic of the a,fl-unsatu- rated keto group of ecdysteroids. Based on an average extinction coefficient of 12,000 for ecdysteroids, and a molecular weight of 642, approximately 200 p g of a chromatographically pure conjugate was isolated from 21.4 g of 0- to 1-h-old larvae according to the procedures outlined in Figures 1 and 2. The major ecdysteroid conjugate of 0- to 1-h-old larvae also exhibited a retention time (7.97 min) by ion suppression HPLC identical to one of the minor conjugates (4.9 min in 30% methanol in 0.03 M aqueous NaH2P04 solution) in 48- to 64-h-old and 72- to 88-h-old eggs [5].

Isolation of the Free Ecdysteroids The extraction of 0- to 1-h-old tobacco homworm larvae, followed by

partition and fractionation of the extracts according to the schemes in Figures 1 and 2, effectively separated the radiolabeled free and conjugated ecdyster- oids. The free ecdysteroids could now be readily detected by reversed-phase

8 Thompson et a1

600 n

n E

U

200 t > t- o _.

A

4 a 1

I .01 AU

L _7_;=i= 16 20

4 8 12 16 20

TIME ( m i n )

Fig. 4. ion suppression reversed-phase HPLC trace (U.V.) and radioassay analysis of partially purified ecdysteroid conjugates (A) from ovaries of day4 adult M. sexta, reanalyzed from [5] for comparison; (8) from 0- to I-h-old larvae. Column conditions are identical to Figure 3. Shaded areas indicate radioactivity.

HPLC when Florisil SEP-PAK fractions 3 and 4 (Fig. 2) were analyzed and radioassayed. Both fractions showed only two radioactive free ecdysteroid peaks. The reversed-phase HPLC and radioassay analysis of 3% of fraction 3 showed a minor radioactive peak which, eluting at 8.66 min, contained 12.9% of the total radioactivity of this fraction. The other peak, eluting at 17.64 min, contained 87.1% of the total radioactivity of fraction 3. Similarly, HPLC and radioassay analysis of 3% of fraction 4 showed that the peak eluting at 8.66 min contained 40% of the total radioactivity of this fraction; the peak eluting at 17.64 min contained 60% of the total activity of fraction 4. TLC analyses of Florisil SEP-PAK fractions 5 and 6 indicated that the bulk of the radioactive material was the nonecdysteroid conjugate, cholesterol sulfate. This conju- gate migrated slightly slower than 26-hydroxyecdysone in the free ecdyster- oid solvent system. Fractions 3 and 4 also contained some cholesterol sulfate.

Free and Conjugated Ecdysteroids in Manduca larvae 9

For the identification of the free ecdysteroids the isolation procedure in Figures 1 and 2 was repeated with 0- to l-h-old larvae (21.4 g) derived from uninjected pupae as indicated above for the conjugates. The fractions were monitored by TLC, and the combined fractions eluted from the Florisil column with 30% ethanol in chloroform gave a chromatogram identical to the combined Florisil SEP-PAK fractions 3 and 4 (Fig. 2). Final purification was achieved by collection from reversed-phase HPLC. Both ecdysteroids showed an absorbance maximum at 240 nm (in methanol), which is typical of the a$-unsaturated keto group of ecdysteroids. Based on an average extinction coefficient of 12,000 for ecdysteroids and a molecular weight of 496 and 480 for the ecdysteroids eluting at 8.66 and 17.64 min, respectively, approximately 24 and 158 ,ug of the chromatographically pure ecdysteroids were isolated from 21.4 g of larvae.

Analysis of the Major Ecdysteroid Conjugate Hydrolysis of the purified major ecdysteroid conjugate (HPLC retention

time 7.97 min) with the mixture of enzymes gave only 26-hydroxyecdysone (determined by HP-TLC and HPLC analyses).

Qualitative analysis of the sugar moiety via preparation of the TMS deriv- ative and capillary GLC analysis indicated the two major anomers of glucose Ell] eluting at 8.52 and 9.53 min. Authentic glucose under similar treatment gave the two anomers with identical retention time. These retention times differed from the TMS derivatives of the hexose sugars mannose, galactose, or fructose.

Mass Spectrometry (Ecdysteroid Conjugate) Ammonia chemical ionization of the major ecdysteroid conjugate showed

major peaks in the high mass region at mlz 660 (13'30, M + NH4)+, 642 (84Y0,

480 (91%, M - C6HI0O5), 464 (65%) and 446 (56%). The peaks at mlz 642 and 480 are coincidentally equivalent to the molecular ions of the ecdysteroid conjugate and ecdysteroid moiety, respectively. These results together with the results of enzymatic hydrolysis further indicate that 26-hydroxyecdysone glucoside is the major ecdysteroid conjugate in 0- to l-h-old larvae of the tobacco hornworm.

M + NH4 - HzO), 625 (loo%, M + H - H20), 607 (23'30, M + H - 2H20),

Mass Spectrometry (Free Ecdysteroids) Ammonia chemical ionization of the ecdysteroid eluting at 8.66 min

showed major peaks in the high mass region at mlz 514 (4%, M + NH4)+, 496 (21%, M + NH4 - H20) and 478 (12'30, M + NH4 - 2H20).

Similarly, ammonia chemical ionization of the ecdysteroid eluting at 17.64 min exhibited major peaks at mlz 498 (49%, M + NH4)+, 480 (loo%, M + N& - H20), and 462 (12'30, M + NH4 - 2H20).

The peaks at mlz 496 and 480 in the spectra of the compounds eluting at 8.66 and 17.64 min, respectively, are also coincidentally equivalent to the molecular ions of the ecdysteroids. These results indicate that the more polar

10 Thompson et al

ecdysteroid (elution time 8.66 min) contains one more hydroxy group than the major free ecdysteroid.

Fourier Transform PMR Spectroscopy (Identity of Major Ecdysteroid Conjugate)

The PMR spectra of the major ecdysteroid conjugate and 26-hydroxyec- dysone recorded at 400 MHz in D20 were compared (Fig. 5 ) . The major differences between the spectra of the conjugate and free 26-hydroxyecdy- sone were in the region of methyl resonances and the region for -CH-OH signals. The C-21 methyl, which occurred as a doublet at 0.953 and 0.938 ppm in the spectrum of 26-hydroxyecdysone1 occurred further downfield at 0.962 and 0.947 ppm. On the other hand, the C-26 proton signal of CHzOH, which occurred at 3.465 ppm in the spectrum of 26-hydroxyecdysone, ap- peared only slightly further upfield at 3.460 ppm in the spectrum of the ecdysteroid conjugate. The C-22 proton signal, which occurred as a doublet at 3.713 and 3.685 ppm in the spectrum of 26-hydroxyecdysone, could be in the spectrum of the conjugate (Fig. 5 ) as the two doublets that are centered further downfield at 3.905 and 3.872 ppm. The latter doublets could also be one of the protons of the sugar molecule. Additional NMR spectral analyses would be required to clanfy this situation. The other signals present in the spectrum of the conjugate in the region 3.24.2 ppm were the remaining -CH-OH protons of the ecdysteroid and glucose moieties. Comparison of the spectra of the conjugate and 26-hydroxyecdysone indicated that the reso- nances for the C-2 and C-3 protons were not shifted in the conjugate. The PMR spectrum nevertheless indicates that the glucose moiety of the ecdys-

Fig. 5. Partial PMR spectra of 26-hydroxyecdysone and 26-hydroxyecdysone 22-glucoside taken in D20 with a Bruker 400 MHz instrument.

Free and Conjugated Ecdysteroids in Manduca Larvae 11

teroid conjugate is at C-22 and the major ecdysteroid conjugate of 0- to l-h- old larvae of the tobacco hornworm is 26-hydroxyecdysone 22-glucoside.

Fourier Transform PMR Spectroscopy (Identity of Free Ecdysteroid) The PMR spectrum of the major free ecdysteroid (HPLC retention time

17.64 min; retention time for 26-hydroxyecdysone = 14.77 min) recorded at 60 MHz in CD5N showed methyl resonances at 0.74 (%Me), 1.08 (19-Me), 1.23, 1.30 (21-Me), and 1.47 (27-Me) ppm that were identical with authentic 26-hydroxyecdysone [l2]. However, the WLC and TLC, which both showed the compound (Rf 0.23) to be more apolar than 26-hydroxyecdysone (Rf 0.20), as well as PMR and mass spectral analyses, indicate that the major free ecdysteroid in 0- to l-h-old larvae of the tobacco hornworm is 3-epi-26- hydroxyecdysone. Similarly, the HPLC, TLC, and mass spectral analyses indicate that the ecdysteroid with the HPLC retention time of 8.66 min is 3- epi-20,26-dihydroxyecdysone (Rf 0.17; 20,26-dihydroxyecdysone Rf = 0.16, HPLC retention time = 7.42 min).

DISCUSSION

The partitioning systems and fractionation procedures employed in Fig- ures 1 and 2 effectively separate free ecdysteroids and the ecdysteroid and nonecdysteroid conjugates found in larvae of the tobacco hornworm. Final purification of the free ecdysteroids can then be achieved simply by reversed- phase HPLC, whereas ion suppression reversed-phase HPLC, followed by desalting on C18 SEP-PAK cartridge, affords the conjugates. This methodol- ogy should be applicable to the isolation of other free ecdysteroids and ecdysteroid glycosides and phosphates. It should be mentioned, however, that apolar ecdysteroid phosphates such as ecdysone monophosphates also partly partition into the butanol phase of a butanol-water partition system [2]. Since the phosphates are not readily eluted or recovered from a Florisil SEP-PAK or column, the ecdysteroid phosphates and certain other ecdyster- oid conjugates contained in the butanol phase (Figures 1 and 2) could be separated from the free ecdysteroids by use of the partition system of cyclo- hexane-butanol-water (4:6:10). Five transfers of upper phase over three tubes of the lower phase remove the apolar and most of the polar free ecdysteroids, which then can be further purified by use of Horisil SEP-PAK or column. The very polar ecdysteroids (for example 20,26-dihydroxyecdysones) and ecdysteroid conjugates contained in the combined lower phase can be further purified via silica SEP-PAK eluting with 5 ml of 5% MeOH in CHC13, 5 ml of 15% EtOH in CHC13, 10 ml of 30% EtOH in CHC13, followed by 5 ml each of 60% EtOH in CHCl3, MeOH, and 30% MeOH in water. The free ecdysteroids are eluted’in the 30 and 60% EtOH in CHCl, fractions and conjugates in MeOH and 30% MeOH in water fractions.

The reported PMR and mass spectral data, as well as the results of enzy- matic hydrolysis, clearly indicate that 26-hydroxyecdysone 22-glucoside is the major ecdysteroid conjugate in 0- to l-h-old larvae of the tobacco horn- worm. At this stage of development the level of the conjugate is nearly 10 pglg of tissue. The first insect ecdysteroid conjugates to be isolated and

12 Thompson et al

identified contained a phosphate moiety at C-22. These 22-phosphates of ecdysone and 2-deoxyecdysone were identified as the major conjugates in newly-laid eggs of Schistocercu greguriu [13]. 20-Hydroxyecdysone 22-phos- phate and 2-deoxy-20-hydroxyecdysone 22-phosphate have subsequently been identified from eggs of this species [14]. In extracts from newly laid eggs of Locustu rnigrutoria, ecdysteroid mononucleotides, namely the 22-aden- osine monophosphoric ester of 2-deoxyecdysone and 22-N6-(isopenteny1)- adenosine monophosphoric ester of ecdysone, have been identified as the conjugates [15,16]. Thus, it appears that the conjugation of the C-22 position of ecdysteroids plays an important role in metabolism of ecdysteroids.

In the first report of an in vitro conjugation of ecdysteroids by insect tissue, it was reported that 20-hydroxyecdysone and ponasterone A were converted into a-glucosides by transglucosylase in the body of Culliphoru erythrocephalu [lq. Similarly, following injection of Culliphoru larvae with tritium-labeled ponasterone A, the ecdysteroid is very quickly metabolized and appears as the ecdysteroid "glucoside" in the hemolymph, fat body, and epidermis. These results were the first evidence for the in vitro and in vivo formation of an ecdysteroid glucoside by an insect. Since then, ecdysteroid conjugates have been detected as metabolites of labeled ecdysteroids in larvae and pupae of various species, and based merely on enzymatic hydrolysis, have been designated as ecdysteroid glucosides, glucuronides, and sulfates [18]. The enzyme name under which a product is marketed, however, is not necessarily an indication of the major activity contained therein [19]. The present report of 26-hydroxyecdysone 22-glucoside is the first unequivocal identification of an ecdysteroid glucoside from an insect.

26-Hydroxyecdysone 26-phosphate is the major ecdysteroid in adult Mun- ducu ovaries (Fig. 4A) and early embryonated eggs [5]. Finding only trace amounts of 26-hydroxyecdysone 26-phosphate in 0- to 1-h-old larvae indi- cates that the 26-hydroxyecdysone 22-glucoside in these larvae (Fig. 4B) is derived from the 26-hydroxyecdysone 26-phosphate. This conversion occurs during the final hours prior to larval emergence (about 102 h), since 72- to 88-h-old eggs show as much as 17 pglg of 26-hydroxyecdysone 26-phosphate [5]. It remains to be determined whether the radioactive peak at about 8 min in the chromatogram of the ovarian ecdysteroid conjugate (Fig. 4A) is the 26- hydroxyecdysone 22-glucoside.

Although we have obtained approximately 50 pg by HPLC of a nearly pure sample of the material eluting in the peak at 7.31 min (Fig. 4B), the PMR, mass spectral data, and other results have not permitted us to deter- mine its complete structure. This compound was not detected in the HPLC chromatograms of ecdysteroid conjugates of 72- to 88-h-old eggs. We expect an increase in the level of this unknown conjugate in older larvae to be accompanied by a subsequent decrease in the level of 26-hydroxyecdysone 22-glucoside .

Both 3-epi-26-hydroxyecdysone and 3-epi-20t26-dihydroxyecdysone have been isolated from the tobacco hornworm at different developmental stages 120,211. Their occurrence as the free ecdysteroids of larvae was not surprising in view of the fact that the biological activity of the 3-epiecdysteroids strongly indicates that epimerization is a means of inactivation of the 36-hydroxy-

Free and Conjugated Ecdysteroids in Manduca Larvae 13

form [21]. It was surprising, however, that the corresponding 3P-epimers were undetectable. Furthermore, this is the largest quantity of 3a-ecdyster- oids per gram of insect tissue (8.7 pglg) to be isolated from an insect. It should be mentioned that no radiolabeled free ecdysteroids were detected in 48- to 64- or 72- to 88-h-old tobacco hornworm eggs stored in methanol [5]. Although we reported the first isolation and identification of 26-hydroxyec- dysone from 48- to 64-h-old tobacco hornworm eggs, our recent studies with this age group of eggs stored in methanol at -20°C indicate no free ecdyster- oids [5]. Previously, older eggs stored in glass bottles without solvent at -20" until work-up yielded a considerable quantity of free ecdysteroids [4,10,12]. We concluded that certain phosphatases of these eggs were acti- vated by lowering the temperature and subsequent hydrolysis of the ecdys- teroid conjugates caused an accumulation of free ecdysteroid [5].

The theoretical quantity of 26-hydroxyecdysone 26-phosphate available to be metabolized can be as high as 31 pg/g (ovaries) or as low as 25 pglg (48- to 64-h-old eggs) [5] . Thus, throughout embryonic development to the stage of 0- to 1-h-old larvae (21.4 g) the eggs could contain between 535-633 pg of 26- hydroxyecdysone 26-phosphate for metabolic purposes. Since the total quan- tity of free ecdysteroids (182 pg) plus ecdysteroid conjugates (250 pg) ac- counted for is below this theoretical minimum, all could have been formed from 26-hydroxyecdysone 26-phosphate.

The pathway from 26-hydroxyecdysone 26-phosphate to 26-hydroxyecdy- sone 22-glucoside probably proceeds via the ephemeral intermediate 26- hydroxyecdysone, which is then conjugated at C-22 with glucose to form the glucoside. Although a relatively high concentration of 3-epi-26-hydroxy- ecdysone (158 pg) was present in 21.4 g of 0- to 1-h-old larvae, no glucoside conjugate of this compound was detected. An alternate pathway could be that the 26-hydroxyecdysone 26-phosphate reacts at C-22 with glucose to form the phosphate-glucose conjugate followed by hydrolysis of the phos- phate moiety to give the 26-hydroxyecdysone 22-glucoside. Certainly, the reaction is rather specific and complete since presently there appears to be no intermediates or metabolites that suggest involvement in the pathway from 26-hydroxyecdysone 26-phosphate to 26-hydroxyecdysone 22-gluco- side. Our present knowledge, however, of the incorporation of [14C]~ho- lesterol into the ovarian ecdysteroids in M. sexta and subsequent metabolism during embryogenesis to larval emergence can best be expressed as shown in Figure 6.

In general, the profiles of ecdysteroids in insects indicate a complex mix- ture of different ecdysteroids occurring in each species [B], even at different developmental stages within a single species [22]. While the profile of ecdys- teroids in M. sexta ovaries is rather simple, the picture in developing em- bryos, which may only be capable of carrying out certain metabolic modifi- cations of ecdysteroids, such as hydrolysis, hydroxylation, epimerization, and conjugation [23], is more complex. The profiles of the free and conju- gated ecdysteroids in 0- to 1-h-old larvae encompass all of the reactions enumerated above. We believe that before we can fully understand the role and function of ecdysteroids in insects, we need to identdy the ecdysteroids resulting from various metabolic transformations occurring in insects at dif-

14 Thompson et al

3-EPI-26-[*'C]HYDROXYECDYSONE -t 3-EPI-20,26-[*4C]D1HYDROXYECDYSONE

[14C]CHOLESTEROL ..c- .-c --* 26-[*4C]HYDROXVECDYSONE 26-PHOSPHATE

\ \

26-['4C]HYDROXYECDYSONE 22-GLUCOSIDE

Fig. 6. Metabolic scheme of ecdysteroids in M. sexta following incorporation of ['4C]cholesterol into ovarian ecdysteroids through embryogenesis and up to 0- to I-hour-old larvae (+, known conversions; --+, presumed conversions; + +, assumed two-step reactions).

ferent developmental stages. However, some of the changes are so abrupt, as noted in this study, that the specific stages of insect development selected for ecdysteroid analysis are exceedingly critical. Rapid progress in the tech- niques for isolating and identdymg free ecdysteroids and their conjugates has made it possible to investigate the ecdysteroid profiles of M. sexta at any increment in time in the life cycle. Certainly, the conversion of [14C]cholesterol in M. sexta to labeled free and conjugated ecdysteroids greatly enhances our ability to identify them, and this knowledge will make it easier to gain a more thorough understanding of the metabolism and physiological roles of ecdys- teroids in M. sextu, and insects in general.

Warren et al. [J. Liquid Chromatography, 9, 1759 (1986)l also report the isolation of a glucoside conjugate of 26-hydroxyecdysone from M. sextu larvae.

LITERATURE CITED

1. Hsiao TH, Hsiao C: Ecdysteroids in the ovary and the egg of the greater wax moth. J Insect Physiol2.5, 45 (1979).

2. Dinan LN, Rees HH: The identification and titres of conjugated and free ecdysteroids in developing ovaries and newly-laid eggs of Schistocerca gregaria. J Insect Physiol 27, 51 (1981).

3. Lagueux M, Sall C, Hoffmann JA: Ecdysteroids during embryogenesis in Locusta migrato- ria. Am Zoo1 22, 715 (1981).

4. Thompson MJ, Weirich GF, Rees HH, Svoboda JA, Feldlaufer MF, Wilzer KR: New ecdysteroid conjugate: Isolation and identification of 26-hydroxyecdysone 26-phosphate from eggs of the tobacco hornworm, Manduca sextu (L.). Arch Insect Biochem Physiol 2, 227 (1985).

5. Thompson MJ, Svoboda JA, Feldlaufer MF, Lozano R: The fate of radiolabeled steroids in ovaries and eggs of the tobacco hornworm, Manduca sexta. Lipids 21, 76 (1986).

6. Thompson MJ, Svoboda JA, Lusby WR, Rees HH, Oliver JE, Weirich GF, Wilzer KR: Biosynthesis of a CZ1 steroid conjugate in an insect: the conversion of [14C]cholesterol to 5-[**C]pregnen-3P,20P-diol glucoside in the tobacco hornworm, Manduca sexta. J Biol Chem 260, 15410 (1985).

7. Kaplanis JN, Thompson MJ, Yamamoto RT, Robbins WE, Louloudes SJ: Ecdysones from the pupa of the tobacco hornworm, Manduca sexta (Johannson). Steroids 8, 605 (1966).

8. Telfer WH, Anderson LM: Functional transformations accompanying the initiation of a terminal growth phase in the cecropia moth oocyte. Dev Biol27, 512 (1968).

9. Kaplanis JN, Robbins WE, Thompson MJ, Baumhover AH: Ecdysone analog: Conversion to alpha ecdysone and 20-hydroxyecdysone by an insect. Science 166, 1540 (1969).

Free and Conjugated Ecdysteroids in Manduca larvae 15

10. Thompson MJ, Svoboda JA, Weirich GF: Ecdysteroids in developing ovaries and eggs of the tobacco hornworm. Steroids 43, 333 (1984).

11. Sweeley CC, Bentley R, Makita M, Wells WW: Gas-liquid chromatography of trimethyl- silyl derivatives of sugars and related substances. J Am Chem SOC 85, 2497 (1963).

12. Kaplanis JN, Robbins WE, Thompson MJ, Dutky SR: 26-Hydroxyecdysone: New insect molting hormone from the egg of the tobacco hornworm. Science 180, 307 (1973).

13. Isaac RE, Rose ME, Rees HH, Goodwin TW: Identification of ecdysone-22-phosphate and 2-deoxyecdysone-22-phosphate in eggs of the desert locust, Sckistocerca gregaria, by fast atom bombardment mass spectrometry and N.M.R. spectroscopy. J Chem SOC Chem Commun 249 (1982).

14. Isaac RE, Rose ME, Rees HH, Goodwin TW: Identification of the 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone from newly laid eggs of the desert locust, Schistocerca gregaria. Biochem J 213, 533 (1983).

W. Tsoupras G, Hetru C, Luu B, Lagueux M, Constantin E, Hoffmann JA: The major conjugates of ecdysteroids in young eggs and in embryos of Locusta migratoria. Tetrahedron Lett 23, 2045 (1982).

16. Tsoupras G, Luu B, Hoffmann JA: A cytokinin (isopentenyl-adenosyl-mononucleotide) linked to ecdysone in newly laid eggs of Locusta migratoria. Science 220, 507 (1983).

17. Heinrich G, Hoffmeister H: Bildung von Hormonglykosiden als Inaktivierungsmechanis- mus bei CaZliphora erythrocephala. Z Naturforsch 256, 358 (1970).

18. Koolman J: Ecdysone metabolism. Insect Biochem 12, 225 (1982). 19. Weirich GF, Thompson MJ, Svoboda JA: In vitro ecdysteroid conjugation by enzymes of

Manduca sexfa midgut cytosol. Arch Insect Biochem Physiol3, 109 (1986). 20. Kaplanis, JN, Thompson MJ, Dutky SR, Robbins WE: The ecdysteroids from young

embryonated eggs of the tobacco hornworm. Steroids 36, 321 (1980). 21. Kaplanis JN, Thompson MJ, Dutky SR, Robbins WE: The ecdysteroids from the tobacco

hornworm during pupal-adult development five days after peak titer of molting hormone activity. Steroids 34, 333 (1979).

22. Thompson MJ, Kaplanis JN, Weirich GF, Svoboda JA, Robbins WE: Moulting hormones of the tobacco hornworm. Sci Papers of the Inst of Org and Phys Chem of Wroclaw Technical University, No 212, Cod 7 (1981).

23. Sail C, Tsoupras G, Kappler C, Lagueux M, Zachary D, Luu B, Hoffmann JA: Fate of maternal conjugated ecdysteroids during embryonic development in Locusta rnigratoria. J Insect Physiol29, 491 (1983).