Metabolism of ecdysone and 20-hydroxyecdysone in adult drosophila melanogaster

Insect Biochem. Molec. Biol. Vol. 24, No. 1, pp. 49-58, 1994

0965-1748/94 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

Metabolism of Ecdysone and 20-Hydroxyecdysone in Adult Drosophila

melanogaster VERONIKA GRAU,* REN]~ LAFONT*t Received 4 January 1993; revised and accepted 15 April 1993 The metabolism of [3Hlecdysone injected into adult female and male Drosophila melanogaster was investigated. The metabolites present in flies and faeces were analysed separately after incubation times of 1, 2 or 4 h. In female flies ecdysone-22-fatty acid acyl esters were the major metabolites followed by 3-dehydroecdysone, 26-hydroxyecdysone, ecdysonoic acid, 20-hydroxyecdysone and a negatively charged conjugate of eedysone. In male flies the same compounds were formed, but their relative concentrations were somewhat different from those in female flies. All metabolites formed can be excreted. [3Hl20-hydroxyecdysone was metabolized in much the same way: 20-hydroxyecdysone22-acyl esters, 3-dehydro-20-hydroxyecdysone, 20-hydroxy-ecdysonoic acid and a negatively charged conjugate of 20-hydroxyecdysone were formed. However, 20,26-dihydroxyecdysone could not be detected after injection of [3H]20-hydroxyecdysone.

Ecdysteroids Metabolism Drosophila melanogaster

INTRODUCTION

Imago

though ecdysteroids can be detected in embryos of D. melanogaster (Mar6y et al., 1988). Embryonic ecdyDrosophila melanogaster is a model system of outstandsteroids are probably at least in part of maternal origin. ing importance for the study of the action of insect On the other hand, owing to its small size, D. moulting hormones ecdysteroids. The ecdysteroid recepmelanogaster has never been a favourable subject for tor (Koelle et al., 1991) and several ecdysone responsive biochemical studies. Therefore, the present knowledge genes from this insect have been characterized at the on ecdysteroid metabolism in adult flies is quite limited. molecular level (for a review see Andres and Thummel, After injection of unlabelled 20-hydroxyecdysone into 1992). adult flies metabolites of greater polarity were detected In adult insects of both sexes ecdysteroids play by radioimmunoassay (Smith and Bownes, 1985). important roles in reproduction (for a review see [3H]Ecdysone injected into adult females was Hagedorn, 1989). In adult Drosophila they are involved metabolized to [3H]20-hydroxyecdysone, high polarity among other factors in the regulation of vitellogenesis products, low polarity products and highly apolar com(for a review see Bownes, 1989). There are hints that the pounds (Diibendorfer and Mar6y, 1986). A more deformation of the egg shell is also directed by ecdysteroids tailed study was performed by Nohava (1987). She also because the expression of the s l5-chorion gene is reguinjected [3H]E into adult female flies and analysed the lated by CF1, a member of the thyroid/steroid receptor distribution of the metabolites in different organs and in superfamily (Shea et al., 1990). Ecdysteroids are the faeces. However, apart from 20-hydroxyecdysone detectable in adult Drosophila by radioimmunoassay none of the metabolites was identified. (Hodgetts et al., 1977; Garen et al., 1977; Bownes et al., Incubations in vitro of different organs from adult 1984). Drosophila with radiolabelled ecdysone and 20-hydroxySeveral zygotic proteins which are members of the ecdysone revealed the conversion to the same products thyroid/steroid receptor superfamily are necessary for in a tissue-specific manner. Only the highly apolar correct embryonic pattern formation (Nauber et al., compounds were not synthesized in vitro (Bownes et al., 1988; Oro et al., 1988, 1990; Pignoni et al., 1990; 1984; Diibendorfer and Mar6y, 1986). Apart from 20Lavorgna et al., 1991). However, up to now no ligands hydroxyecdysone the exact chemical nature of the for these receptor-like proteins have been identified metabolites was not determined. The metabolism of [3H]E and [3H]20E in Drosophila *ENS, Dtpartementde Biologic,CNRS URA 686, 75230Paris Cedex larvae has been analysed more closely (Sommt05, France. Martin et al., 1988): 3-dehydrocompounds, 3-epimers, ?Author for correspondence. 49

50

VERONIKA GRAU and RENI~LAFONT

ecdysonoic acids and conjugates were the metabolites identified. In this paper we describe more precisely the metabolism of ecdysone and 20-hydroxyecdysone as well as the excretion of these compounds and their metabolites in adult females and males of D. melanogaster. The chemical identity of the most abundant metabolites was determined. MATERIAL AND M E T H O D S

Chemicals Radioactive precursors. (23,23,24,24,-3H4)-2-deoxy ecdysone (original specific activity ca 100 Ci/mmol) was prepared according to H&ru et al. (1983) and was a generous gift from Dr Charles H&ru (Strasbourg). [3H]Ecdysone and [3H]20-hydroxyecdysone were prepared by incubating radiolabelled 2-deoxyecdysone with Locusta migrator& Malpighian tubules (Modde et al., 1984). All these compounds were regularly analysed by HPLC for radiochemical purity. Reference ecdysteroids. Ecdysone (E) and 20-hydroxyecdysone (20E) were from Simes (Milano, Italy) 2deoxyecdysone (2dE) and 2-deoxy-20-hydroxyecdysone (2d-20E) were generous gifts from Dr Maria Bathori (Szeged, Hungary) and Dr Denis Horn (Acheron, Australia). 3-Dehydroecdysone (3DE) and 3-dehydro20-hydroxyecdysone (3D20E) were prepared as described by Girault et al. (1989), and from them 3-epimers were obtained by chemical reduction (Dinan and Rees, 1978). 26-Hydroxyecdysone (26E) (25R and 25S isomers) was isolated from Manduca sexta eggs (a gift of Dr Bernard Mauchamp, Versailles, France); 20,26-dihydroxyecdysone (2026E) was isolated from Silene nutans (Girault et al., 1990). Ecdysonoic acid (Eoic) and 20hydroxyecdysonoic acid (20Eoic) were from Pieris brassicae pupae (Lafont et al., 1983). Various monoacetates of E and 20E were prepared according to Horn (1971). Various 22-acyl esters of E were generous gifts from Dr Laurence Dinah (Exeter, U.K.) (Dinah, 1988). The 22-oxo-20-hydroxyecdysone was isolated from Serratula tinctoria (Rudel et al., 1992). Synthesis of ecdysone 22-palmitoleate. The lack of commercially available palmitoleoyl anhydride led us to develop a synthesis procedure using palmitoleoyl chloride (Sigma). E (20 mg) was dissolved in 0.5 ml dry pyridine containing 2mg dimethylaminopyridine. The mixture was cooled to 0°C and 100 mg of palmitoleoyl chloride were added progressively. The mixture was allowed to stand at room temperature for 18 h, then 5 mi methanol were added and the whole was evaporated to dryness. The residue was dissolved with 5ml methanol:water 9:1 containing 0.6% K2CO3; after 20 min, 40 ml ethyl acetate and 30 ml water were added; the organic phase was collected and rinsed with 10 ml water, then evaporated and separated using NP-HPLC on a semi-preparative column (Zorbax-Sil, 250mm long, 9.4 mm i.d., solvent system dichloromethane:isopropanol:water 125:40:3, flow-rate 4 ml/min. The

materials of the major peaks were collected, then identified using mass spectrometry and NMR (Girault and Lafont, 1988):peak 1 (Ret. 4.0 min) = ecdysone 2-palmitoleate, peak 2 (Ret. 6.6min)=ecdysone 22-palmitoleate and peak 3 (Ret. 10.6 min)= ecdysone. Experimental animals The D. melanogasterwild type strain Oregon R was raised at room temperature on standard food with fresh baker's yeast. Adult flies were collected from the pupae during the first day after eclosion. Female and male flies were injected into the abdomen on the third day after eclosion with about 240,000 dpm [3H]E or [3H]20E in ca 0.3/~1 0.9% NaC1. Injected flies were kept (together with a small quantity of fresh baker's yeast mixed with some water) in micro test tubes with perforated lids. After 1, 2 or 4 h of incubation at room temperature flies and faeces were transferred separately into methanol and stored at -20°C. The eggs laid by the females were processed together with the faeces. Batches of 4-15 flies were used. All experiments were performed at least twice. Extraction method Flies were homogenized in a glass/glass homogenizer in methanol, centrifuged and the pellets were reextracted twice. Faeces were sonicated in methanol, but otherwise extracted in the same way. The eggs deposited by females during the time of incubation were normally extracted together with the faeces. Hydrolysis of conjugates Apolar ecdysteroid conjugates were hydrolysed with 13 U pig liver esterase (E-3128, Sigma) in 100 pl sodium borate buffer pH 8.0, overnight at 37°C in the presence of up to 20% methanol. For the digestion of polar conjugates 12-14,1 ml borate buffer with 130 U enzyme was used. The activity of the esterase in 10 or 20% methanol was checked by digesting reference products. //-glucuronidase (G-0751, Sigma; 400U) in 100pl of 50 mM sodium acetate buffer pH 5.3 was used to hydrolyse polar conjugates overnight at 37°C. Chromatographic analysis Five different high-performance liquid chromatography (HPLC) systems were used to analyse the metabolites. The flow-rate in all systems was 1 ml/min. All solvents used were of HPLC grade and were purchased from local suppliers. (1) A reversed-phase (RP) column Spherisorb 5ODS2 (25 cm long, 4.6 mm i.d.) eluted with a linear gradient from 20 to 1 0 0 % acetonitrile/isopropanol 50/20in 0.1% trifluoroacetic acid (TFA) over 30 rain followed by 20 min 100% acetonitrile/isopropanol. This system was suitable to separate the entire range from polar to apolar ecdysteroids.

METABOLISM OF [3H]E AND [3H]20E IN D. MELANOGASTER

(2) The same system as system 1 with 2 0 m M Tris/HCIO4 pH 7.5 instead of 0.1% TFA. (3) The same Spherisorb 5ODS-2 column eluted with a linear gradient from 8% acetonitrile to 40% in 2 0 m M Tris/HCIO4 pH7.5 over 40 rain. (4) An isocratic normal-phase (NP) system with isooctane/isopropanol/water 100/50/5 (v/v/v/) on a Z o r b a x ® - T M S column (15cm long, 4.6mm i.d.) to separate free and polar ecdysteroids. The column used was a quite old one with a long history of different applications in our laboratory. This caused a partial desorbtion of TMSOH, so this column behaved as a slightly deactivated silica, and proved to be especially suitable for this kind of separation. It was not possible to reproduce the retention times we observed on a new column of the same type except by using less polar solvent mixtures. (5) A linear gradient from isooctane/isopropanol/ water 660/70/2 to 100/40/30 in 40min for the

identification of apolar ecdysteroids on the same column as for system 4. To characterize and identify the metabolites of E and 20E the extracts of flies after 2 h of incubation and faeces after 4 h were separated in chromatographic system 1 and fractions of 0.5 ml were collected. The material from each peak was analysed individually using the appropriate chromatographic systems and compared to reference products. The identity of each product was ascertained on at least two independent chromatographic systems.

RESULTS

Metabolites o f [3HIE For the identification of the metabolites of radiol a b e l l e d E, s a m p l e s o f f e m a l e flies 2 h a f t e r i n j e c t i o n w i t h the precursor and the faeces after 4 h of incubation were s e p a r a t e d in c h r o m a t o g r a p h i c s y s t e m 1 a n d t h e m a t e r i a l from the major peaks was collected. The material from each peak was compared individually to reference sub-

TABLE 1. Behaviour of the metabolites of [H]E (a) and [H]20E (b) in different chromatographic systems 1-5 (see Material and Methods). The retention times are indicated in minutes. In the columns "Helix enzymes' and 'Esterase' the products liberated after digestion are indicated. Products which are not digested (not dig.) did not alter their chromatographic behaviour after enzyme treatment. (a) Retention time

Helix Peak 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Metabolite

l

2

HPP 20E 26E2 Eoic E 3DE APs E-22-Laurate 9 E-22Myristate E-22Oleate E-22Palmitate ? ? ?

7.3* 10.5 11.3 12.3 13.7 14.5 16-24 36.0 36.7 37.7

3.7 11.7 3.8

3

4

5

enzyme

Esterase

E 12.2 16.7

Not dig. Not dig.

17.0 9.7 7.8

14.6

Not dig. 25.8 24.1 27.0

38.5

26.0

39.7

25.7

41.0 42.5 44.3~,4.9

7.6 7.0 6.5

(b) Retention time

Helix Peak I 2 3 4 5 6

Metabolite

I

HPP 20Eoic 20E 3D20E AP HAPs

7.6 7.6 10.5 11.5 14.0 33.5~41.4

2

3

4

5.3 11.7

14.5

51

5

enzymes

Esterase

20E Not dig. 12.0 10.2 I1.I

Not dig.

Not dig. mostly 20E

*The HPP was a labile metabolite; when rechromatographed in chromatographic system 1, the retention time was 12.5 min.

VERONIKA

52

GRAU

stances using the appropriate chromatographic systems and digestion methods. The results are summarized in Table 1(a). Peak 1 was an unstable product; when rechromatographed in system 1 the retention time is 12.5 min instead of the 7.3 observed min during the first separation. As it eluted much earlier in 20 mM Tris/HClO, pH 7.5 (chromatographic system 2) than under acidic conditions (system 1) and its digestion with Helix enzymes liberated free E, this metabolite was probably a negatively charged double conjugate of E, A group of 2-4 apolar compounds (APs-peak group 7) with retention times from 16 to 24 min in chromatographic system 1 were not analysed because the individual peaks represented only minor metabolites of E. Among the highly apolar products (HAPS, peaks S-15) with retention times from 36 to 45 min some metabolites could not be fully identified. The metabolite 9 did not comigrate with any of the reference 22-acyl esters of E from L. Dinan. Therefore, we decided to synthesize ecdysone 22palmitoleate. Peak 9 however did not comigrate exactly with this product in chromatographic system 1. On NP system 5 the products 13, 14 and 15 eluted much earlier than the 22-acyl esters of E and during digestion with pig liver esterase E was liberated. They could be conjugates of E with fatty acids at another position, compounds which have not been described so far. As they were minor compounds it was not possible to fully characterize them. The relative amounts of the different HAPS formed by the flies showed considerable variations from experiment to experiment. This is possibly reflecting natural variations of the fatty acid composition of different batches of the food. Examples of chromatograms of the metabolites of female and male flies present in the bodies and in the faeces after 2 h of incubation separated in RP system 1 are shown in Fig. 1. Metabolites

of [‘H]2OE

[3H]20E was metabolized in much the same way as [“H]E [Table I(b); Fig. 21. Peaks I and 2 comigrated in chromatographic system 1, but were clearly separated in system 3. Peak 1 was digested by Helix enzymes to 20E, whereas peak 2 was stable. By comparison with the authentic standard, peak 2 was identified as 20Eoic. Peak 1 is probably a negatively charged conjugate of 20E. 20,26E did not accumulate although 2OEoic was detected. 20E was liberated after digestion with esterase from the HAPS, indicating that they are probably 20E fatty acid acyl esters. The biochemical nature of peak 5 could not be elucidated, It comigrated with 20E22Ac both in RP chromatographic system 1 and NP system 4. However, neither Helix enzymes nor esterase liberated free 20E as seen with authentic 20E22Ac reference substance. Peak 5 was not identical with E nor with 22-0x0-20E.

and RENfi LAFONT

Excretion The radiolabelled E and 20E injected into male and female flies and their metabolites were rapidly eliminated from the bodies in both sexes (Fig. 3a). The excretion of radioactivity after injection of t3H]20E was comparable (data not shown). All metabolites present in the female body were also found in the sample containing faeces and eggs (Figs 4 a+). Despite the presence of HAPS in the male flies, the faeces of male flies contained almost no HAPS. This sex-specific difference was not due to the fact that the HAPS would be sequestered preferentially into the eggs which were extracted together with the female feces: eggs separated from the faeces after 4 h of incubation only contained 0.003% of the total radioactivity; the HAPS in the faeces however represent 33% of the total radioactivity. 3DE was more abundant in the faeces of both males and females than in the bodies. The relative concentration of 20E compared to other metabolites within the flies respectively in the faeces was higher within the flies. Differences

between females

and males

Qualitatively the metabolites of [3H]E detected in males and in females were the same (Fig. 4). However, some quantitative differences existed. During the first hour after injection of the radiolabelled precursors the major radioactive compound in males was E, in females the HAPS. To compensate for this, the relative amounts of 26E, E and 3DE were higher in the faeces of males after 4 h of incubation. In females the concentrations of 26E and Eoic were about the same (Fig. 4 a<:); in males there was always less Eoic than 26E (Fig. 4 d-f). Dynamics

qf the

metabolism

of [-‘H]E

Owing to rather large changes in the rates of ecdysteroid metabolism and excretion during the first days of adult life (data not shown), the quantitative aspects of the data presented here cannot be considered as fully representative because they only cover a small period of the life-span. Nevertheless the nature and the relative abundance of the metabolites does not change to the same extent and some general trends can be pointed out (Fig. 4): Metabolites of higher polarity than E are preferentially formed during the first 2 h after injection of radiolabelled E. In females, the main products are the HAPS which are excreted quite efficiently (Fig. 4 c). They were accumulating in both sexes during the time of incubation. In males about the same quantity of HAPS as in females could be detected in the bodies after 4 h of incubation, but as mentioned above this group of metabolites was not excreted (Fig. 4 f). The total amount of HAPS in males made about one-third of the production in females. 3DE was the most important metabolite and the main excretion product in males. It was produced continuously during the time of incubation in both sexes.

METABOLISM

OF [3H]E AND

53

[3H]20E IN D. MELANOGASTER

8

I;

20

30

40

I min -

20

30

40

6 min -

C

10

FIGURE 1. RP-HPLC analysis of the metabolites of [ 3H]E produced by adult female (A, B) and male (C, D) D. melanogasrer. Flies (A, C) and faeces with eggs (B, D) were analysed separately after 2 h of incubation. The chromatographic system 1 was used. The arrowheads indicate the peaks listed in Table l(a).

DISCUSSION

Metabolites of [‘Hjkcdysone and [‘H]20hydroxyecdysone Injected [3H]E and [3H]20E are metabolized in adult female D. melanogaster following different pathways: (1) the most important is the esterification at position IB24,1--D

C-22 with different long-chain fatty acids; (2) oxidation in position C-3 leads to the formation of the 3dehydroecdysteroids 3DE and 3D20E; (3) 26E is a hydroxylation product at position C-26 which is then partially oxidized to Eoic (20,26E is not accumulated, however 20Eoic can be detected after injection of

54

VERONIKA GRAU and RENI~ LAFONT

[3H]20E); (4) the C-20-hydroxylation of E to 20E is only a minor pathway in adult flies; (5) anionic highly polar conjugates of E resp. 20E (HPPs); and (6) a group of non-identified apolar compounds (APs) are only formed in small amounts. Dfibendorfer and Mar6y (1986) performed a similar in vivo conversion experiment of [3HIE in adult female flies followed by thin-layer chromatography (TLC) analysis of the metabolites. They also detected highly apolar material probably corresponding to our HAPs. Their group of low polarity products might be composed of 3DE and the group of APs. The large amount of 20E found in their study conflicts with our results. The high concentration of high polarity products (D~bendorfer and Mar6y, 1986) is in contrast to the minute amounts of HPPs detected in our study and may be due to an artefact typical for TLC arising from non-specific adsorption of materials at the deposition site caused by polar impurities. After injection of huge concentrations of unlabelled 20E into adult D. melanogaster, mainly polar metabolites were detected after separation on RP-HPLC using a radioimmunoassay (Smith and Bownes, 1985). However, these results cannot be directly compared to ours because (1) the unphysiologically high concentrations of 20E injected may alter the metabolic pathways and (2) the radioimmunoassay used may fail to detect all metabolites of 20E, as only antibodies raised

against a 6-carboxymethoxime derivative of 20E were used in their study. Although comparable concentrations of radiolabelled ecdysteroids were used, the metabolism of 20E and E in larvae of D. melanogaster is quite different (Somm6Martin et al., 1988). Larvae do not produce any HAPs, the most significant metabolite in adult females. In larvae, E is mainly transformed to 20E, a minor product in adult flies. This quantitative difference could be stage-dependent, but it could also be due to the prolonged incubation of the larvae after injection (24h). Under these conditions excreted E may be ingested several times by the larvae and thus metabolized more efficiently. After oxidation at position C-3 leading to 3DE and 3D20E larvae form 3-epi-ecdysone (E') and 3-epi-20-hydroxyecdysone (20E') and metabolize them further on. 26-E is not accumulated in larvae, whereas it is detected in adults. The chemical nature of peak 5 material present in flies as well as in faeces of adult females 2 h after injection of [3H]20E could not be determined. Mar6y et al. (1988) described a metabolite after in vitro incubation of carcasses of adult females with similar chromatographic properties: comigration with 20E22Ac both in RP- and NP-chromatography. However, the authors did not try to digest the compound with esterase or Helix enzymes. The identification of this product as 20E22Ac should be reinvestigated.

13

A

d

10

20

30

40

min

5~0

d

1()

B

2~3

30

40

rain

50

FIGURE 2. RP-HPLC analysis of the metabolites of [3H]20E by adult female D. melanogaster. Flies (A) and faeces with eggs (B) were analysed separately after 2 h of incubation. The chromatographic system 1 was used. The arrowheads indicate the peaks listed in Table l(b).

METABOLISM OF [3HIE AND [3H]20EIN D. MELANOGASTER a) excretion

%

females

100 806040

Flies /eggs

~

2O Oh

lh

2h

4h

b) excretion males

Flies

e c e s

%O l O2o4o6o8Oo

Oh

lh

2h

4h

FIGURE 3. Excretion of radioactivity after injection of [3HIE into adult D. melanogaster and incubation times of 1, 2 or 4 h. The percentage of the radioactivity within the flies and in the faeces was calculated upon the total radioactivity (flies+ faeces) recovered: (a) excretion in females; (b) excretion in males•

Possible functions of the metabolites Highly apolar products• Ecdysteroids esterified at the C-22 position with long-chain fatty acids have been detected in the eggs of ticks and several insects (Diehl et al., 1985; Slinger et al., 1986; Slinger and Isaac, 1988a; Whiting and Dinan, 1989). An interaction of such compounds with vitellins of Drosophila has been proposed (Bownes et al., 1988). Up to now their function has not been unequivocally elucidated. They may represent storage forms of ecdysteroids for embryogenesis (Slinger and Isaac, 1988b) or excretion forms as proposed for ticks (Connat and Dotson, 1987; Connat et al., 1988). E-22-fatty acid acyl esters are only produced by adult flies, which would fit with their possible function in reproduction. In females they are the most important metabolite of E, however they are efficiently excreted into the faeces. Further investigations will reveal their localization within the body and the organs which synthesize them. 3-Dehydroecdysteroids. 3DE is the major metabolite when E is injected into males, and is one of the most important metabolites in females (Fig. 4). 3D20E is produced after injection of 20E. This also holds true for third instar larvae of Drosophila (Somm6-Martin et al., 1988). 3-Dehydrocompounds have been shown to be efficient inducers of the puffing of salivary gland chromosomes and of the expression of the P1 gene in

55

larval fat body (Richards, 1978; Spindler et al., 1977; Somm6-Martin et al., 1990). However a function as an active hormone in adult flies seems to be unlikely because of the fast excretion of 3DE we have shown (Fig. 4). In other insects 3DE is considered to be an intermediate of the inactivation pathway to 3-epiecdysone (E'). E' and 20E' are detected in third instar larvae as metabolites of E or 20E, respectively (Somm6-Martin et al., 1988), but, as mentioned above, there was no epimerization in adults. 26-Hydroxyecdysone. 26E is considered to be an intermediate product of the inactivation pathway to Eoic (Rees, 1989). A hormonal function of 26E has been proposed during Manduca sexta embryogenesis (Dorn et al., 1987). It is striking that 26E is detectable as a metabolite of E only in adult flies and not in third instar larvae (Somm6-Martin et al., 1988). 26E and Eoic are detected in female flies and in their faeces in about equal amounts. Males however produce more 26E than Eoic. After injection of 20E, no 20,26E accumulated although 20Eoic was present both in adult females and in faeces. The possible hormonal function of 26E in Drosophila adults and embryos needs further investigation. 20-Hydroxyecdysone. 20E is generally considered to be the active moulting hormone in insects. E is rapidly transformed to 20E in most insects (for a review see Lafont and Connat, 1989). However, 20-hydroxylation is only a minor pathway in adult flies whereas 20E is produced in large quantities in larvae (Somm6-Martin et al., 1988). Similar stage dependent differences of C-20-hydroxylation have been described for several insects (e.g. Bulenda et al., 1986) including dipteran flies (Koolman, 1978). As discussed above, 20E seems to be preferentially retained within the body of adult flies supporting the idea that 20E is still functioning as a hormone.

Storage and excretion In agreement with earlier publications on adult D. melanogaster (Smith and Bownes, 1985; Nohava, 1987) and Sarcophaga bullata (Briers et al., 1983) injected ecdysteroids were effectively eliminated from the body by excretion (Fig. 3). As in Sarcophaga practically all types of metabolites were excreted. However, some quantitative differences of the occurrence of 20E and 3DE after injection of [3HIE, and 3D20E after injection of [3H]20E, in bodies and faeces were observed. After conversion of E to 20E, 20E seemed to be preferentially retained within the body, whereas 3DE or 3D20E were almost restricted to the faeces. Incubations of larval organs in vitro in culture medium containing [3HIE (Somm6-Martin et al., 1988) showed that E is mainly converted into 20E in the fat body and into 3DE in the hindgut. If the sites of ecdysteroid metabolism are the same in adult flies a rapid excretion of the hindgut metabolites could eliminate 3DE from the flies. However, this does not explain why 20E compared for instance to 26E or Eoic was retained

56

V E R O N I K A G R A U and REN]~ L A F O N T

within the body. A specific interaction of 20E with proteins like 20E-binding proteins or the ecdysteroid receptor could diminish the excretion specifically. A 20E-binding protein of the haemolymph of Locusta migratoria with equilibrium constants of association for 20E which are 2.6 times higher for 20E than for 3DE was partially purified (Feyereisen et al., 1975; Cao et al., 1983; Feyereisen, 1985). However, related proteins with similar qualities have not been found in other insects where the binding proteins described seem to be less specific (for a review see Koolman, 1985). If 20E would be specifically retained within tissues like the fat body or the ovaries, it may be also protected from excretion. The sex-related difference of the excretion of the HAPs was striking. Almost all the HAPs formed in males were retained within the body, whereas in females after 4 h of incubation two-thirds were excreted. This result is not compatible with the idea that fatty acid conjugates of

ecdysone were synthesized and deposited in the oocytes which was described for instance for crickets and ticks (Diehl et aL, 1985; Whiting and Dinan, 1988) and proposed for D. melanogaster (Bownes et al., 1988). The interpretation of the sex specific differences of the metabolism of [ 3H]E and the excretion of the metabolites is not easy: preliminary experiments showed that the rate of metabolism of [3H]E in females and the relative amounts of the different metabolites formed fluctuate considerably during the first 3 days after eclosion (data not shown). In our experiments we only investigated flies on the third day after eclosion and it is possible that the sex specific differences can only be seen during a certain period of time after eclosion. Further investigations will be necessary to decide if there is a real sex-specific difference. The observed difference of the excretion of the HAPs may be due to the fact that their production is higher in females than in males (Fig. 4). It is possible

a) females 1 h

d) males 1 h

40

4°l • Flies ~ ]DFaeces

• Flies [] Faeces/eggs >

30'

O ~O

20'

J I

10.

_

I

HPPs

20E

26E

Eoic

E

3DE

APs

HAPs

HPPs

20E

26E

Eoic

E

3DE

APs

HAPs

3DE

APs

HAPs

APs

HAPs

e) males 2 h

b) females 2 h 30

30 1 •

• Flies [] Faeces/eggs

Flies

20

"~ 20. 0

10'

o~ 0

HPPs

20E

26E

Eoic

E

3DE

APs

HPPs

HAPs

20E

26E

c) females 4 h

Eoic

E

f) males 4 h

40

40

• Flies F'] Faeces/eggs

Flies ~>" .g 30

30' ¢t3 O "O 2 0 -

Faeces

o

"6 10-

0 HPPs

20E

26E

Eoic

E

3DE

APs

HAPs

o

,----~. HPPs

20E

~ 26E

~'. Eoic

E

3DE

•

F I G U R E 4. Comparison of the percentage of the different metabolites recovered from flies and faeces with the eggs of adult female (a-c) or male (d, f) D. melanogaster injected with [3H]E. The percentage was calculated upon the total radioactivity recovered. RP-HPLC analysis was performed using chromatographic system 1. The incubation times were I (a, d), 2 (b, e) or 4 (c, f)h.

METABOLISM OF [3H]E AND [3H]20E IN D. MELANOGASTER that the excretion o f those c o m p o u n d s starts when the storage capacity for the H A P s is exceeded. T h e a m o u n t o f H A P s r e m a i n i n g in the b o d y after 4 h was a b o u t the same in males a n d females (Fig. 4 c a n d f). A m e a n s to confirm this i n t e r p r e t a t i o n would be to inject [3H]E together with a bigger a m o u n t of unlabelled E to exceed the storage capacity for the metabolites. I n this case the H A P s should be also excreted by males. It m u s t however be kept in m i n d that the b e h a v i o u r of exogenous E does n o t necessarily perfectly reflect the fate o f the e n d o g e n o u s l y synthesized one.

CONCLUSIONS We have identified the m o s t i m p o r t a n t metabolites of E a n d 20E in a d u l t flies o f D. m e l a n o g a s t e r . C o m p a r e d with the m e t a b o l i s m o f larvae there were some c o m m o n p a t h w a y s like the o x i d a t i o n at position C-3, b u t also striking differences like the p r o d u c t i o n of the H A P s a n d the a c c u m u l a t i o n of 26E in a d u l t flies, b u t n o t in larvae. However, with the present data it was n o t possible to predict a n y f u n c t i o n s of the metabolites we f o u n d for the a d u l t flies a n d embryos: the c o m p a r i s o n between the m e t a b o l i s m of E a n d 20E in males a n d females did n o t reveal a n y female-specific metabolites which might be transferred into the developing eggs. The most likely candidates to be sequestered into the eggs, the H A P s , are eliminated from the b o d y o f female flies very efficiently by excretion. E x p e r i m e n t s are in progress to analyse the d i s t r i b u t i o n o f the metabolites within the b o d y of the flies, the c o n t r i b u t i o n of the different organs to the m e t a b o l i s m a n d to identify the p r o d u c t s which are t r a n s p o r t e d into the eggs.

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Acknowledgements--We wish to thank Dr M. Larchev~que for his contribution to the synthesis of ecdysone 22-palmitoleate, Dr L. Dinan for providing us with various 22-acyl esters of E, for critically reading the manuscript and for correcting our English. The authors are grateful to Dr C. Blais for helpful discussions, to Professor Dr A. Diibendorfer for communicating unpublished results and Professor Dr H. O. Gutzeit for constant support. We acknowledge the Deutsche Forschungsgemeinschaft for financial support (grant to V. G.).