Biosynthesis of ecdysones: Metabolism of 7-dehydrocholesterol in Schistocerca gregaria

J. Insect Physiol..

1977. Vol. 23. pp. 1387 to 1392. Pergamon Press. Printed in Great Britain

BIOSYNTHESIS OF ECDYSONES: METABOLISM OF 7-DEHYDROCHOLESTEROL IN SCHlSTOCERCA GREGARIA P. JOHNSON and H. H. REES Department

of Biochemistry,

University

of Liverpool,

P.O. Box 147. Liverpool,

L69

3BX.

U.K.

(Received 25 May 1977) Abstract--The chemical synthesis of [3H]-7-dehydrocholesterol from [3H]-cholesterol is described and the incorporation of [“‘Cl-cholesterol, [3H]-7-dehydrocholesterol and [3H]-50c-cholest-7-en-3fi-ol into fi-ecdysone in the locust. Schistocerca gregaria, have been compared. The ratio of incorporation into j?-ecdysone of [3H]-7-dehydrocholesterol to that of [‘4C]-cholesterol administered simultaneously was 0.97:1. When these substrates were administered in the presence of non-radioactive 7-dehydrocholester01 this ratio was increased to 1.80:1. The incorporation of [‘HI-Sr-cholest-7-en-3j?-ol into P-ecdysone was low (0.019/,) compared with that of cholesterol (0.129;). suggesting that the former sterol is not a normal intermediate in ecdysone biosynthesis. The results are discussed m relation to the r61e of Y-dehydrocholesterol in ecdysone biosynthesis. Analysis of the radioactive sterols indicated that cholesterol, 7-dehydrocholesterol, Sr-cholest-7-en-3/S-ol and Sz-cholestan-3P-ol are interconvertlble. and that there is a large conversion of 7-dehydrocholesterol into cholesterol.

INTRODUCTION synthesise sterols de nouo from small molecules and rely upon a dietary source of these compounds for normal growth and development (CLAYTON, 1964). Cholesterol (I) will support normal growth and development of all species of insects with INSECTS cannot

two reported exceptions (HEED and KIRCHER. 1965; CHU et al.. 1970). It is now clear that during ecdysone biosynthesis from cholesterol at least some modification of the nucleus occurs before side-chain hydroxylation (THOMPSON et al., 1973 ; GALBRAI~ et al.. 1975). 7-Dehydrocholesterol (II) is a possible early inter-

,,& .,& l 1

(VI (VllRl

(U)

R,=H

(VIl)R2=

=Ac

1387

ii

(IV)

R4 =

(VlMR2=OAc

. R,=

IIX) R2=OAc

; R,=OH

i X) R,/R, Fig. 1.

R,=

iI

(IiT)

=

I;%

R,=OH R4=

R5=

OH

; R_,/R5 = ; R,/R5

+>c =

I:>(

1388

P. JOHNFON AND H. H.

mediate in the transformation of cholesterol into ecdysones (THOMPSONet al., 1973). The conversion of cholesterol (I) into 7-dehydrocholesterol (II) has been reported in several species of insects, the transformation being independent of associated microorganisms. In fact. the incorporation of [3HJ-7dehydrocholesterol (II) into fi-ecdysone (VII) in Calliphora stygia has been demonstrated, the incorporation (0.025%) being of the same order of magnitude as that of [3H]-cholesterol (I; 0.02%) (HORN et al., 1974). Because simultaneous administration of nonradioactive 7-dehydrocholesterol (II) with [‘HI-cholesterol (I) only slightly decreased the incorporation (0.012%) of the latter sterol, it was suggested that the biosynthesis of p-ecdysone (VII) may not proceed exclusively via 7-dehydrocholesterol (II), while not excluding other possible explanations. Many species of insects can be maintained for long periods with 7-dehydrocholesterol (II) as the sole dietary sterol (CLAYTON. 1964). The two reported species of insects in which cholesterol (I) will not support growth and development require a dietary source of a sterol which contains a A’-bond. e.g. a A7- or As*7-sterol. These species have presumably lost the capacity to introduce a A7 bond, which is also present in ecdysones. It is particularly significant that of a number of individual tissues from the tobacco homworm, Manduca sexta. and the American cockroach, Periplaneta americana, only the prothoracic glands possess a comparatively large percentage of 7-dehydrocholesterol (II). In both species the percentage of this sterol in the glands undergoes marked changes with development within an instar and only in this tissue can 7-dehydrocholesterol (II) account for as much as 25% and 60% of the total sterols in the cockroach and homworm. respectively. This implicates the prothoracic glands as a site of synthesis or storage of ‘I-dehydrocholesterol (II) (THOMPSONet al., 1973). The stereochemistry of introduction of the A7 bond during formation of 7-dehydrocholesterol (II) (JOHNSON et al.. 1975) and ecdysones (COOK et al.. 1973) is the same and this is consistent with the possible intermediacy of 7-dehydrocholesterol (II) in ecdysone biosynthesis. The present paper reports investigations on the relative conversions of 7-dehydrocholesterol (II). cholesterol (I) and the A7 sterol, 5a-cholest-7-en-3/?-o] (III), into fi-ecdysone (VII) in the desert locust, Schistocerca gregaria. Wide variations have been reported in the incorporation of labelled cholesterol (I) into z-ecdysone (V) 00.018%) and b-ecdysone (VII] (0.01 S-0.035%) in species of Calliphora (KARLSONand HOFFMEISTER, 1963; GALBRAITHet al., 1970; WILLIG et al., 1971); this can probably be attributed to differences in the developmental stages of the different groups of experimental animals. To circumvent such difficulties the present work has utilized a double label technique, which allows simultaneous determination of the relative conversion of two labelled sterols in one group of experimental animals.

REES

MATERIALS

AND METHODS

Chemicals Cholesterol (I) and 5r-cholestan-38-01 (IV) were bought from Koch-Light Laboratories Ltd., Colnbrook, Bucks, U.K., and 7-dehydrocholesterol (II) was purchased from Sigma (London) Chemical Co.. Kingston-upon-Thames, Surrey, U.K. Sa-Cholest-7-en3p-01 (III) was prepared by hydrogenation of 7-dehydrocholesterol (II) (JOHNSONet al., 1975). All nonradioactive sterols were recrystallized before use. [4-‘4C]-cholesterol (56 mCi/m mol) and [~cz, 2cc(nt3H]-cholesterol (43 Ci/m mol) were purchased from The Radiochemical Centre, Amersham. Bucks, U.K. 5a-[24-3H]-Cholest-7-en-3P-ol (21.2 mCi/m mol) was synthesized by Dr. R. BOID (unpublished). The purity of all radioactive substrates was checked before use by radio-GLC and in the case of 14C-labelled compounds by radioautography as well. r-Ecdysone (V) and /?-ecdysone (VII) were kindly provided by Simes S.p.A., Milan, Italy. Thin-layer chromatography Preparative TLC of sterols as a group was carried out routinely on silica gel (0.5 mm-thick Kieselgel G; E. Merck A.-G., Darmstadt, Germany) with chloroform for development. Constituent sterols were separated as acetates on 20% (w/w) Ag NO,-impregnated silica gel (0.5 mm-thick Kieselgel H). Development was without a wick in benzene-hexane (1: 1, v/v) to separate cholesteryl acetate (R,. 0.61) and 7-dehydrocholesteryl acetate (R,O.12) and in chloroform (ethanol-freethexane (1: 1, v/v) for separation of Sac-cholestan-38-y] acetate (Rf 0.57). Sr-cholest-7-en-3/?-yl acetate (R,. 0.34), cholesteryl acetate (R, 0.26) and 7-dehydrocholesteryl acetate (R, 0.05) (JOHNSONet al., 1975). Compounds were detected under U.V. light after TLC by spraying with a solution of Rhodamine 6G in acetone and sterols were eluted using dry redistilled diethyl ether. Preparative TLC separation of ecdysones was carried out on 0.5 mm thick Kieselgel GF,,, plates developing twice in chloroform-methanol (4: 1; v/w). The ecdysones were visualized under U.V. light and eluted well with chloroform-methanol (1: 1, v/v). In the case of ecdysone derivatives. chloroform-ethanol (9: 1, v/v) was used for development. Gas-liquid chromatography A Pye 104 gas chromatograph was employed fitted with a flame ionization detector and 4 mm i.d. glass columns. For collection of effluent material a metal splitter was fitted between the end of the column and the detector, so that approximately one part in ten passed to the detector. whereas the remainder was collected in glass capillary tubes at ambient temperature. Radiochemical methods Radioactivity

was measured

on a liquid

scintilla-

Biosynthesis of ecdysones in Schistocerca tion spectrometer. Samples were dissolved in 10ml of a dioxan scintillation solution containing 15g of 5-(4-biphenylyl~2~~(4-t-butylphenyl~l-oxa-3,4-diazole (butyl PBD) and 1OOg of naphthalene per litre of dioxan. 3H and 14C radioactivities are given after corrections for background, counting efficiency and quenching had been applied. Animals Schistocerca grcgaria were reared on a diet of wheat seedlings and rolled oats and were kept under a 12 hr “day” period when the temperature was maintained at 35°C and a corresponding 12 hr “night” period maintained at 25°C. Eggs were laid by mature female locusts into aluminium tubes containing a moist mixture of peat and sifted. washed sand. Tubes containing eggs were incubated at 30°C for approximately 17 days after which the young locusts hatched. Fifth instar larvae were csed in the present study, the instar lasting for 8 days under the rearing conditions employed. Preparation of [ 1g.2a(n)-3H]-7-dehydrocholestero[ 1 mCi of [ lr,20:(n)-3H]-cholesterol (43 Ci/m mole) and carrier non-radioactive cholesterol (8 mg) were dissolved in pyridine (1 ml) and acetic anhydride (0.5 ml) was added. The reaction mixture was left overnight. Ice was then added and after 30min the mixture was evaporated to a small volume under vacuum. Water (10ml) and ethanol (1Oml) were added, and the mixture was evaporated to dryness under vacuum yielding cholesteryl acetate. I,3-Dibromo-5,5-dimethyl hydantoin (8 mg) was added to a boiling solution of the cholesteryl acetate in redistilled light petroleum (b.p. 4&6O”C) (0.4 ml) and cyclohexane f3.16 ml), and the boiling was continued under reflux for 35 min. The reaction mixture was filtered and the residue washed with redistilled petrol (5 ml). Collidine (0.2 ml) was added to the combined filtrate and petrol washings and the volume of the resulting mixture was reduced to 0.2 ml under a stream of nitrogen. The mixture was heated under reflux for 20min at 140°C in an atmosphere of nitrogen, cooled to room temperature, filtered and the residue washed WIth ether (5 ml) (HALKES and VAN VLI~T, 1969). The filtrate and ether washings were combined and washed first with 2 M sulphuric acid (5 ml) and then three times with water (5 ml), and the solution was evaporated to dryness under vacuum. The reaction product was purified on AgNO,-impregnated silica gel developed in benzene and the band co-chromatographing with authentic 7-dehydrochoIesteryl acetate was eluted. This was saponified overnight at room temperature in 6% ethanolic KOH plus 1% pyrogallol under an atmosphere of nitrogen. 7-Dehydrocholesterol was isolated from the reaction mixture in the usual manner and purified by TLC on silica gel developed in chloroform (yield, 1.3 mg. 151.8 &i).

1389

The purity of the prepared [ 1~,2u(n~3H]-7-dehydrocholesterol was established by gas-liquid chromatography. Carrier cholesterol (1 pg) and 7-dehydrocholesterol (2 pg) were mixed with an aliquot of the [ lcc.2~(n)-3H]-7-dehydrocholesterol (0.05 &i) in cyclohexane (5 ~1) and the mixture subjected to micropreparative GLC on a 7 ft OV-17 column (temp. 250°C; carrier gas flow, 60 ml/min). Fractions were collected every 2 min and radioassayed. Only one radioactive peak, corresponding to 7-dehydrocholesterol, was observed. The radioactive 7-dehydrocholesterol was characterized additionally by U.V. spectrometry. In a trial reaction using non-radioactive cholesterol the product 7-dehydrocholesterol was also characterized by mass spectrometry. Transformation products of c(- and P-ecdysones Final purification of radioactive ecdysones was carried out by formation of derivatives. The 2-acetate derivatives of r-ecdysone and of /$ecdysone and 2.3-20.22-diacetonid-/?-ecdysone (X) were prepared by the methods of GALBRAITH and HORN (1969). 28Acetoxy-20,22-acetonid-P-ecdysone (IX) was prepared as described by LLOYD-JONES et al. (1973). The derivatives were purified by TLC using chloroformethanol (9:1, v/v) for development and recrystallized to constant specific radioactivity from various solvents (2-acetate derivatives of Y- and P-ecdysones. methanol-water; 2p-acetoxy-20.22-acetonid-/?-ecdysone (IX). chloroform
1390

P. JOHNSON AND

(200 ml). The aqueous phase was re-extracted with butanol (200ml) and then discarded. The combined butanol extracts were washed once with water (200ml) and evaporated to dryness under vacuum. The residue (4.1 g; 2.41 x 10’ dis/min) was partitioned between hexane (200ml) and 707; aqueous methanol (200 ml). The hexane phase was re-extracted once with 70% aqueous methanol and the 70% aqueous methanol phase was re-extracted once with hexane. Sterots and ecdysones were purified from the combined hexane (2.1 g; 1.87 x lO’dis/min) and the combined 70% aqueous methanol (567 mg; 1.67 x lo6 dis/min) fractions, respectively. The hexane extract was saponified in 6% ethanolic KOH and sterols (36.8 mg; 1.76 x 10’ dis/min) were isolated from the non-saponifiable lipid (207 mg; 1.87 x lO’dis/min by TLC on silica gel developed in chloroform. The sterols were acetylated and the steryl acetates (32.1 mg; 1.61 x lO’dis/min) were separated by TLC on AgNO,-impregnated silica gel chloroform (ethanol-freethexane developed in (1: 1, v/v). The individual steryl acetates were diluted with non-radioactive carrier material, recrystallized from chloroform-methanol to constant specific radioactivity and the total radioactivity associated with the original sterols calculated (Table 1). A small aliquot (l/lOth) of the 709/, aqueous methanol fraction was removed for analysis by high-performance liquid chromatography. Carrier cr-ecdysone (V, 20 mg) and p-ecdysone (VII, 20 mg) were added to the remaining material, which was then fractionated by TLC on Kieselgel GF,,, developed twice in chloroform-methanol (4: 1,v/v). Bands co-chromatographing with z-ecdysone (V) and b-ecdysone (VII) markers were scraped and eluted (r-ecdysone. 20.5 mg. 3.51 x 103dis/min; fi-ecdysone, 21.9mg. 1.71 x lo3 dis/min). The ecdysones were then purified further by formation of derivatives. When [4-‘4C]cholesterol was used as substrate, r*-ecdysone was transformed into the 2-acetate derivative (VI; 8.2 mg; 1.28 x 1O3 dis/min), whereas the 2,3-20,22-diacetonide derivative of p-ecdysone (X; 16.1 mg; 9.00 x lo3 dis/ min) was prepared (Table 1). In all other experiments, P-ecdysone was purified as the 28-acetoxy-20.22-acetonide derivative (IX).

Table

1. Transformation

of [4-‘4C]-cholesterol

H. H. REES RESULTS Biosynthesis of LY-and p-ecdysones from cholesterol [4-14C]-~olesterol (30 &i) was administered to twelve larvae of Schistocerca gregaria, the total lipid extracted, and the sterols and ecdysones were purified as derivatives (Table 1). All results for radioactive incorporations into ecdysones are corrected for the increase in molecular weight during derivative formation and are expressed as percentages of the radioactivity associated with the total lipid extract. thus allowing for losses of radioactivity occurring during injection of the substrates. Since the incorporation of [4-r4C]-cholesterol into r-ecdysone (0.0030,d) was much lower than into P-ecdysone (0.05’?), in subsequent experiments only the conversions of sterols into /3-ecdysone were considered. High-performance liquid chromatography of an aliquot of the 70% aqueous methanol fraction on a 4ft reverse-phase C,,/Corasil column eluted with 30% aqueous methanol, with collection of fractions every 5 min for radioassay showed that the majority of the radioactivity (69%) was associated with p-ecdysone (VII). with much less in the I-ecdysone (V) region. Relative incorporations of cholesterol (I). 7-dehydrocholesterol (II) and 5cc-cholest-7-en-3fl-ol(III) into /I-ecdysane

Groups of fifth instar larvae of S. gregaria were each injected with one of three different sterol substrate mixtures: (i) [4-14CJcholesterol (15 &i) and f1~,2c((nt3H1-7-dehydrocholesterol (30 PCi), (ii) [4-r4C]-cholesterol (25 PCi) and [24-3H]-5a-cholest-7-en-3B-ol (75 &i), and (iii) [4-‘4C]-cholesterol (25 &i). [ 1cc,2a(n)-3H]-7-dehydrocholesterol (50 &i) plus non-radioactive 7-dehydrocholesterol (II; 1 mg). The /J-ecdysone was isolated and purified via derivatives (Table 2). In the case of the experiments with the first two substrate mixtures, the constituent sterols were isolated from the hexane fraction and separated, as acetates, on AgNO,+ilica gel TLC before radioassay (Table 3). The radiochemical purity of the various steryl acetates ex TLC were checked by micropreparative GLC on a 7 ft 3% OV-17 column (temp. 255°C; carrier gas flow, 60ml/min). Radioactivity (3H and

into various

sterols

and ecdysones

in Schistocercn

grrgaria

Specific radioactinty a&r

Total

final

KCIyStalllZ?tlOll Substance

Total

(dls/mm/mg)

(dis/min)

hpld

Cholesteryl

PFXCntage lllCOrpOL&3fl

of cholesterol*

2.54 x IO’ acetate

7-Dehydrocholesteryl

19.884 acetate

2,444

51.Cholestan-3B-yl-acetate

75

5z-Cholest-7.en-3B-yl-acetate Z&Acetoxy-z-ecdysone

Based on radioactivity

911

x 106

34

x IO’

1.88 x IO’

-t

(VI)

2.3.20.22.Dtacetonid-8.ecdysone *

radmactlvity

171

31 (,I’)

in total lipid. t Purified

by GLC

x IO’ 693

476

I22

only.

x IO’

0.003”,, 0 OS”,,

Biosynthesis of ecdysones in Schistocerca Table 2. Relative incorporations

1391

of sterols into P-ecdysone in fifth instar S. gregaria specific

Wqht Compound

I mg)

Substrates: [C”C’j-cholesterol (15 ~CI) + rI1.2zln~‘H1-7-dehvdrocholesterol (30 UCI) L Total lipId cxt&t &Ecdysone (ex. TLC) 2fl-Acetoxy-/kcdysone t VIII1 (recrystallizedl ZB-Aceloxy-20.27-8ceionld-i3-ecdysone (IX) lrecrystalhzed) Radnactlwtv calculated ,n B-ecdwne Percentage lllcOrpOrat10”* Substrates: [&‘*C]-cholesterol (25 ~CI) + [24-‘H]-Sx-cholest-7-en-3~‘-ol (75 &i) Total hpid extract 0.Ecdysone (ex. TLC) ?a-Acetoxy-P-ecdysone ~recrystalbzed) ?I)-Acctox~-20.22-ace1oro,d-~-ecdysonc treccrystakedl Radmactwlty calculated ,n fkcdysone Percentage mcorporatlon* Substrates: [4-“~-cholesterol I25 ~CII + [I z.?l(nt’H]-7-dehydroholesterol (50 ~CI) + non-radmactnve 7-dehydrocholesterol (I mg) Total bpld extract P-Ecdysone (ex. TLC) 2B-Acetoxy-B-ecdysone (recrystalhzed) 2/J-Acetox?-20.22.acetor Id-/-ecdysone (recrystalhzedl Radmactlwty calculated m i(-ecdysone Percentage Incorpoiauon* *

radmactiwty alter final recrystallization (dls/mm/mg) ‘4C

ZOO0

Radmactlwy td@mn) ‘H

407

I I45

392

1033

‘4C

‘H

78 x IO” 900 x IO’

2.2 r 10’ 295 x 10’

9.01 x IO’ 0.1 l6”,

2.45 x IO’ 0.1 12” 0

1.1 x IO’

2.87 x IO’

2012 627 547

188 174

I33 x 104

21.41 377 338

I x IO’

0121” II

4.1 0014~”

342 I49

Y ItJ’ x IO’

6.94 x IO 74x * IO’

8.63 x IO’ 0.025”,

3.lh x 104 0 046”,

1415 I205

Based on radioactivity in total lipid extract.

Table 3. Distribution

Substrates

of ‘H and “‘C radioactivity amongst individual sterols (isolated as acetates) from fifth instar S. gregnria after administration of various substrates [C’“C]-Cholesterol (I5 ~CI) + [ 11.22(n~‘H]-7.dehydrocholesterol (30~0 ‘*C (drs/mm) ‘H (dwmin)

admmstercd

Steryl acetates w&ted Cholesteryl acetate 7.Dehydrocholesteryl aceti,te

2.16 583 514 4.67

51.Cholestan-38-yl-acetate 5z-Cholest-7-en-3~-yI-acetilte

x x x x

IOn IO’ IO’ IO’

Steryl acetates wtre separated by TLC on AgNO,-silica by m&o-preparative GLC. 14C) was apparenfly associated with cholesteryl acetate. Sa-cholestan-3p-yl acetate. 5a-cholest-7-en-3/?-yl acetate and 7-dehydrocholesteryl acetate in each experiment, indicating several nuclear double bond transformations. DISCUSSION The incorporation (0.05-O. 12%) of [4-l 4CJ-cholesterol into P-ecdysone in Schistocerca gregaria is of the same order of’ magnitude as that (0.015XJ.O35%) reported in species of Calliphora. Although care was taken to ensure that insects were at the same developmental stage, great variation was observed in the absolute conversions of cholesterol into P-ecdysone (0.05-0.12%) in diRerent groups of experimental animals. This does not affect the correlation of data from different experiments since a dual label technique was employed. The low incorporation of [4-“CJ-chole-

44S 3.46 407 628

x x x x

100 IO’ IO’ IO’

[C”C--Cholesterol (25 ~CI) + [?4-‘HI-k cholesi-7-m.3$oI (75 ~0) “C Idlsimm) % (dismm)

533 973

x 100 x IO”

571

x IO’

I20 x IO’

I65 106 I.21 726

x x x r

IOD 100 IO‘ 10’

gel. The radiochemical purity of the fractions was checked

sterol into r-ecdysone probably reflects the high C-20 hydroxylase activity in the insects at the developmental stage used in the present work (JOHNSON and REES. 1977). When [3H]-7-dehydrocholesterol and [‘4C]-cholesterol were simultaneously administered to S. gregaria. the conversion of the A5.7-sterol (II) into P-ecdysone (0.11%) was similar to that of cholesterol (Table 2). However. in the experiment where the two labelled compounds were administered simultaneously with non-radioactive 7-dehydrocholesterol. the ratio (1.8: 1) of 7-dehydrocholesterol incorporated (0.046%) to cholesterol incorporated (0.02X/,) was higher than in the previous experiment when nonradioactive 7-dehydrocholesterol was not adrninistered (0.97: 1). Hence. the simultaneous administration of non-radioactive 7-dehydrocholesterol reduced the conversion of cholesterol more than that of 7-dehydrocholesterol into fl-ecdysone. which is consistent

1392

P. JOHNSONAND H. H REES

with the intermediacy of the latter sterol in ecdysone biosynthesis: cholesterol G+ 7-dehydrocholesterol--+ ecdysones. If 7-dehydrocholesterol were not an intermediate in the biosynthesis of /I-ecdysone from cholesterol (e.g. 7-dehydrocholesterol $ cholesterol --• ecdysones). simultaneous administration of nonradioactive 7-dehydrocholesterol would reduce incorporation of the A5*’-sterol more than the incorporation of cholesterol. Interpretation of these expcriments is complicated by the large transformation of 7-dehydrocholesterol into cholesterol. The incorporation of the A’-sterol, 5cc-cholest-7-en-3fi-ol (III), was low (0.0140,/,) compared with that of cholesterol (0.12%) in the same experiment. and is probably not a normal intermediate in ecdysone biosynthesis. The distribution of radioactivity amongst the constituent sterols of S. gregaria after the administration of various labelled substrates (Table 3) suggests that cholesterol (A5), 7-dehydrocholesterol (A5,‘), Sa-cholestan-3/I-ol (A”) and See-cholest-7-en-3P-ol (A’) are apparently completely interconvertible. The sterol nuclear double bond transformations which have been demonstrated in various insect species are (THOMPSONrt al., 1973; SVOSODA et al., 1975; JOHNSON et d., 1975):

C. CAR~UTTfor maintenance of insects, and Professor T. W. GOODWIN,C.B.E.. F.R.S.. for his interest and encour-

agement, We are most grateful to SIMESS.p.A., Milan for generous gifts of z- and p-ecdysones. REFERENCES CHU H. M., NORRISD. M., and KOK L. T. (1970) Pupation requirement of the beetle. Xyleborus ferrugineus: sterols other than cholesterol. J. Insect Physiol. 16. 1379-1387. CLAYTONR. B. (1964) The utilization of sterols by insects. J. Lipid Res. 5, 3319. Coon I. F., LLOYD-JONES J. G.. REESH. H.. and GOODWIN, T. W. (1973) The stereochemistry of hydrogen elimination from C-7 during biosynthesis of ecdysones in insects and plants. Biochem. J. 136, 135-145. GALBRAITH M. N. and HORN D. H. S. (1969) Insect moulting hormones: crustecdysone (20-hydroxyecdysone) from Podocarpus elatus. Aust. J. C/tern. 22, 1045-1057.

GALBRAITHM. N., HORN D. H. S., MIDDLET~NE. J.. and THOMSON J. A. (1970) The biosynthesis of crustecdysone in the blowfly 179-I 80.

Calliphora stygia. Chrm. Commun. 1970,

GALBRAITHM. N., HORN D. H. S.. MIDDLETONE. J.. THOMWNJ. A.. and WILKIE J. S. (1975) Metabolism of 3jI,14a-dihydroxy-5~-(3a-3H)-cholest-7-en-6-one. J. Insect Phvsiol. 21, 23-32.

HALKESS. J. and VAN VLIET N. P. (1969) Investigations on sterols XXXV. Svnthesis of 25-hvdroxvcholecalciferol. Recueil Trar. Chim: des Pays-Bas k,

1&3@1083.

HEED W. B. and KIRCHERH. W. (1965) Unique sterol in the ecology and nutrition of Drosophila pachea. Science. Wash. 149, 758-761.

Because the S. greguria larvae were reared nonaxenically, many of the sterol transformations may be attributed to symbiotic micro-organisms. Several of the transformations, however, are of particular interest. The substantial conversion of 5a-cholest-7en-38-01 into cholesterol and 7-dehydrocholesterol explains the incorporation, albeit low, of the A’-sterol into /$ecdysone. although the latter sterol is probably not a normal precursor of moulting hormone. The Iarge conversion of 7-dehydrocholesterol into cholesterol indicates that an equilibrium probably exists between the two sterols, which suggests that the conversion of cholesterol into 7-dehydrocholesterol is not the rate-limiting step in ecdysone biosynthesis. It has been reported that 6-oxo-5r-cholestan-3/I-01 is formed from cholesterol and is further transformed into cc-ecdysone by organ cultures of prothoracic glands from Bomby nwri (SAKURAI et al., 1976). However, it is difficult to reconcile the intermediacy of both 6-oxo-5a-cholestan-3j?-ol and 7-dehydrocholesterol in ecdysone formation from cholesterol. Acknowledgements-We are indebted to the Medical Research Council for a research studentship to (P.J.), Mrs.

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