Ecdysteroids during embryogenesis of the house cricket, Acheta domesticus: Occurrence of novel ecdysteroid conjugates in developing eggs

Insect Biochem. Molec. Biol. Vol.23, No. 3, pp. 319-329, 1993 Printedin GreatBritain.Allrightsreserved

0965-1748/93 $6.00+ 0.00 Copyright© 1993PergamonPressLtd

Ecdysteroids During Embryogenesis of the House Cricket, Acheta domesticus: Occurrence of Novel Ecdysteroid Conjugates in Developing Eggs PENSRI WHITING,* STANLEY SPARKS,* LAURENCE DINAN*'t Received 21 July 1992; revised and accepted 16 October 1992

The identity and titres of ecdysteroids during embryogenesis of the house cricket, Acheta domesticus, have been investigated by radioimmunoassay coupled to high-performance liquid chromatography (HPLC]RIA). It has previously been shown [Whiting and Dinan, J. Insect Physiol. 34, 625-631 (1988); Insect Biochem. 19, 759-765 (1989)l that the ecdysteroids present in newly-laid eggs are ecdysone (80 ng/g ft. wt) and a mixture of ecdysone 22-long-chain fatty acyl esters (732 ng ecdysone/g fr. wt). During the first 7 days of embryogenesis at 26°C, there is little change in the level or identity of the apolar conjugated ecdysteroids, but these increase 3-fold by day 10 and remain at this high level until the end of embryogenesis (13.5 days). Free ecdysteroid levels fluctuate during embryogenesis and there is a transition from ecdysone at the beginning of the embryological period to 20-hydroxyecdysone as the major free ecdysteroid at the end of embryogenesis. Novel ecdysteroid conjugates are increasingly formed as the embryo develops. These conjugates have unusual chromatographic properties, appearing polar on silicie acid, but apolar on reversed-phase. Evidence is presented, based on sensitivity to porcine liver esterase and Helix enzyme hydrolysis coupled with HPLC/RIA, that these conjugates are double conjugates consisting of ecdysone or 20-hydroxyecdysone esterified to a fatty acyi group at C-22 and to a neutral polar or negatively-charged group at another of the hydroxyls (possibly C-25). Steroid hormones chromatography

Ecoysone 20-Hydroxyecoysone

INTRODUCTION The roles of ecdysteroids in regulating reproduction in insects are the subject of considerable research interest (see Hagedorn, 1989; Lanot et al., 1989, for reviews). A number of functions have been postulated, depending on the insect species being investigated. It is apparent that different species use ecdysteroids to control a variety of aspects of reproduction and embryogenesis. In the house cricket, A c h e t a domesticus, ecdysteroids are present in both mature adult male and mature adult female insects, but the levels found in females are far higher (Whiting and Dinan, 1990a). In adult females, increasing levels of ecdysteroids correlate with ovarian development (Whiting et al., in prep.). Ecdysteroids are present in all *Departmentof BiologicalSciences,Universityof Exeter,Washington Singer Laboratories, Perry Road, Exeter, Devon EX44QG, England. tAuthor for correspondence. Abbreviations used: E: ecdysone; 20E: 20-hydroxyecdysone;HPLC: high-performance liquid chromatography; RIA: radioimmunoassay.

Radioimmunoassay

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tissues of mature adult females, with the ovaries, haemolymph and carcass as the major locations (Whiting and Dinan, 1990a), but the ovaries would appear to be the site of synthesis (Renucci and Strambi, 1981; Renucci et al., 1987). Ecdysone is the major ecdysteroid present, either in the free form or in a conjugated form; no evidence for the presence of 20-hydroxyecdysone has yet been obtained for this stage of development. The conjugates are mainly less polar than ecdysone (fatty acyl esters and possibly acetate esters too), but lower amounts of polar conjugates (identity unknown) are found in some tissues (ovaries, fat-body, gut; Whiting and Dinan, 1990a). The major ecdysteroids in the ovary are a mixture of ecdysone 22 long-chain fatty acyl esters (Whiting and Dinan, 1990a), and these, together with much smaller amounts of free ecdysone, are recoverable in newly-laid eggs (Whiting and Dinan, 1988a, b, 1989). The aim of this present study has been to investigate the fate of these ecdysteroids in developing eggs. Throughout embryogenesis, the majority of the ecdysteroids remains in a conjugated form and levels of total ecdysteroids increase

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dramatically during the second half of embryogenesis after catatrepsis. As embryogenesis proceeds, there is a transition from ecdysone to 20-hydroxyecdysone as the major free ecdysteroid. As the embryo develops, a new class of ecdysteroid conjugate is increasingly produced, and by the end of embryogenesis the level of these has become very significant. These new conjugates have very unusual chromatographic properties, appearing very polar on silica, but very apolar on reversed-phase. Evidence is presented that this unusual behaviour is a consequence of the new compounds being multiple conjugates, with an apolar fatty acyl group at C-22 of the ecdysteroids (ecdysone or 20-hydroxyecdysone) and a polar negatively-charged group esterified at one of the other hydroxyls (possibly at C-25). This work was presented in a preliminary form at the IXth Ecdysone Workshop, Paris (Whiting and Dinan, 1990b).

MATERIALS AND METHODS

Insects A. domesticus was cultured as described previously (Whiting and Dinan, 1988a). Females deposited eggs in beakers of moist sand, which were changed every day. The beakers containing eggs were covered-with cling-film and incubated for the required length of time at 26°C. At this temperature, hatching occurred between 13.5 and 14 days. Eggs of the appropriate chronological age were collected by flotation on a sugar solution (Whiting and Dinan, 1988a) and then stored at -20°C until use. Extraction of eggs Eggs were extracted with ethanol and the extract separated on silicic acid columns as described before (Whiting and Dinan, 1988a).

Radioimmunoassay The radioimmunoassay procedure was as described previously (Whiting and Dinan, 1988a), except that Packard Emulsifier-safe scintillation fluid was used in place of the more toxic Scintillator 299. Counting efficiency was only slightly reduced by this change. The major antiserum used in this work was DBL-1, generously provided by Professor J. Koolman (Universit/it Marburg, Germany). For differential RIA, H-22 and H-2 antisera were additionally used. These were kind gifts of Professor L. I. Gilbert and Professor J. D. O'Connor (University of North Carolina, Chapel Hill, U.S.A.).

High-performance liquid chromatography The HPLC equipment and general procedures have been described previously (Dinan, 1988). Details of columns and separation conditions were as follows: System NP1. Spherisorb SW-5 column (25cm x 4.6 mm i.d., 5 #m particle size) eluted isocratically with dichloromethane/propan-2-ol/water (125:25:2 v/v/v) at 1 ml/min.

System RPIA. Spherisorb ODS-2 column (25 cm x 4.6mm i.d., 5/~m) eluted at a constant flow rate of 1 ml/min with a linear gradient over 30 rain from 30% methanol in 20 mM citrate buffer (pH 4.5) to methanol, with which elution was continued for a further 50 min. System RP1B. As system RP1A, except that the citrate buffer was replaced with water. System RP2. Spherisorb ODS-2 column (25cm x 4.6ram i.d., 5/~m) eluted at 1 ml/min with a linear gradient over 40min from 20 to 50% methanol in 20 mM citrate buffer (pH 4.5). System RP3A. Spherisorb ODS-2 column (25 cm x 4.6mm i.d., 5/~m) eluted at 1 ml/min with a linear gradient over 30min from 20 to 100% methanol in 20 mM citrate buffer (pH 4.5), after which the column was eluted with methanol for a further 50 min. System RP3B. As system RP3A, except that citrate buffer was replaced by water. System RP4. Spherisorb ODS-2 column (25 cm x 4.6mm i.d., 5#m) eluted at 1 ml/min with a linear gradient over 20min from 80% methanol in citrate buffer (20mM; pH 4.5) to methanol, at which it was maintained for a further 20 min. Enzymic hydrolyses Generally, ecdysteroid conjugates were "completely" hydrolysed with a crude mixture of Helix pomatia gut juice hydrolases (Type H1 "Arylsulphatase"; Sigma, Poole, U.K.) as previously described [10mg/ml 0.1 M acetate buffer, pH 5.4, in the presence of 5% (v/v) ethanol for 5 days at 37°C; Whiting and Dinan, 1988a]. In order to investigate the nature of the conjugating moieties, partial hydrolyses with two enzyme preparations were used; (i) incubations with porcine liver esterases (20 ~1 [20 IU], Boehringer-Mannheim, Lewes, U.K.) were conducted in 0.1 M borate buffer (pH 8.4; 180/H) at 37°C (Diehl et al., 1985) for 24h and (ii) incubations with Helix enzymes (2 mg/ml) under the general conditions for 48 h. Hydrolyses were stopped by the addition of ethanol. RESULTS

Initial analyses by RIA In our colony of A. domesticus, embryogenesis lasts 13.5 days at 26°C. Consequently, eggs were incubated for 3, 7, 10 or 13 days ( + 8 h ) and these were analysed for their ecdysteroid content to provide an overview of how ecdysteroid titres change during embryological development. Results are compared to those previously obtained for 0-1 day eggs (Whiting and Dinan, 1988, 1989). The extraction and partial purification procedure results in three fractions from open column chromatography on silicic acid; chloroform (containing apolar lipids), chloroform:methanol (7:3 v/v; containing free ecdysteroids and apolar ecdysteroid conjugates) and methanol (containing polar ecdysteroids and polar ecdysteroid conjugates). Each fraction was analysed for ecdysteroid content by RIA (DBL-1 antiserum) both before and after hydrolysis with Helix enzymes. The

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FIGURE 1. Quantification of RIA-positive free and conjugated ecdysteroids in extracts of eggs of A. domeslicus. Eggs of known age were extracted with ethanol and separated by silicic acid chromatogaphy to yield 0, 30 and 100% methanol in chloroform fractions. Aliquots of each of these fractions were assessed by RIA (DBL-1 antiserum). Solid columns represent the RIA-positive material in the column fractions and the hatched areas the extra RIA-positive material released by Helix enzyme hydrolysis.

results, presented in Fig. 1, are expressed in pg ecdysone equivalents per egg, as the weight of the egg approximately doubles between day 3 and day 7, owing to the uptake of water from the external environment (Fig. 1; Schneider, 1973). The small amount of RIA-positive material in the chloroform fractions is probably a result of interference by the large amount of triglyceride present in these fractions. The levels of "free" ecdysteroids in the chloroform: methanol (7:3 v/v) fraction fluctuates during embryogenesis, being maximal in the day 13 extract at c 100pg ecdysone equivalents/egg. The level of apolar conjugates in the chloroform: methanol (7: 3 v/v) fraction remains initially constant at c 260 pg ecdysone equivalents/egg and then increases 3-fold to 825 pg ecdysone equivalents/egg in 10 and 13 day eggs. Hydrolysable ecdysteroid conjugates and RIA-positive polar ecdysteroids are essentially absent from the methanol fraction deriving from the newly-laid egg extract, but the level of RIA-positive material in these fractions, both before and after hydrolysis with Helix enzymes, increases steadily during embryogenesis, reaching a level of 325 pg ecdysone equivalents/egg (after hydrolysis) in 13 day eggs.

Analysis of chloroform :methanol (7:3 v/v) fractions These fractions were analysed by RIA after separation on HPLC [silica (system NP1) or C18 reversed-phase

(system RP1B)]. This revealed that in eggs of all ages the majority of directly RIA-positive material co-chromatographed with ecdysone or 20-hydroxyecdysone, with the contribution from 20-hydroxyecdysone fluctuating with developmental age (molar ratio 20E:E=4.0, 2.3, 4.1, and 7.5 at 3, 7, 10 and 13 days, respectively). After enzymic hydrolysis of the HPLC fractions, RIA revealed that the apolar conjugates present in all these fractions co-chromatographed with ecdysone 22-fatty acyl reference compounds, which elute together on the normal-phase system (Dinan, 1988). Further evidence for this identification was provided by co-chromatography of the RIA-positive material released from the apolar conjugates with ecdysone on both normal-phase and reversed-phase HPLC (data not shown). Insignificant amounts of 20hydroxyecdysone 22-fatty acyl esters were present, even in older eggs. The results from RP-HPLC/RIA (system RP1B) substantiated all the above findings. In addition, since the various ecdysone 22-fatty acyl esters separate in this system (Dinan, 1988; Whiting and Dinan, 1988b), it is possible to state that the fatty acid composition of the endogenous ecdysone 22fatty acyl esters does not significantly change during embryogenesis, even after the large increase in apolar conjugates between days 7 and 10 (data not shown).

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are broad and, when using the ion-suppression system at least, there is partial resolution into two broad peaks [fractions 24-31 (18% of the RIA-positive material released) and fractions 34-39 (72%), in the ion-suppression system]. This indicates that the conjugates are in fact a mixture of compounds. The conjugates in fractions 24-31 (Fig. 2) do not appear to elute earlier in the absence of citrate and these may not therefore be charged molecules. The small peaks detected in fractions 8-10, 12 13 and 15-16 (together 10% of the RIApositive material released) correspond to the elution region of ecdysteroid phosphates, acetate phosphates and ecdysteroid acid reference compounds.

Analysis of methanol fractions Initially, the polarity on silicic acid of the conjugates in the methanol fractions deriving from extracts of older eggs suggested that they might be similar to the ecdysteroid phosphates identified in eggs of Schistocerca gregaria (Isaac et al., 1983). Therefore, a portion of the methanol fraction deriving from 13 day eggs was separated on an ion-suppression RP-HPLC system (RP2) suitable for the separation and identification of ecdysteroid phosphates and ecdysteroid acids (reference ecdysteroid phosphates and acids were very kindly provided by Dr M. Kabbouh and Professor H. H. Rees, University of Liverpool, Dr R. E. Isaac, University of Leeds, U.K. and Professor R. Lafont, Ecole Normale Sup6rieure, Paris, France). Surprisingly, only very low levels of conjugates eluted under these conditions. Consequently, this fraction was separated on an ionsuppression system of greater eluting power (system RP1A; Fig. 2), and this clearly demonstrated that the bulk of the ecdysteroid conjugates (fractions 24-39) elute after reference ecdysone, but slightly before reference ecdysone 22-fatty acyl esters. Omission of the ion suppression buffer (system RP1B) caused the conjugates to elute earlier in fractions 24-34. This indicates that a significant proportion of the conjugates present in the methanol fractions can carry a negative charge. Also the shape of the conjugate peak is informative. The peaks

Identity of ecdysteroids released from the new conjugates The RIA-positive material released after hydrolysis of fractions 24-31 and 34-39 (Fig. 2; ion-suppression separation) were identified by reversed-phase HPLC/RIA (system RP1B). Two peaks of RIA-positive material were obtained in each case, co-chromatographing with ecdysone and 20-hydroxyecdysone. Once the cross-reactivity of the DBL-1 antiserum for these two ecdysteroids is taken into account, the ratio of 20-hydroxyecdysone to ecdysone is c 2.5:1 for both broad conjugate peaks. Differential RIA of new conjugates The methanol fraction deriving from the separation on silica of the 13 day egg extract was assessed with three

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CRICKET EGG ECDYSTEROIDS different antisera after various periods of incubation with Helix enzymes at either 1 mg/ml (sufficient to hydrolyse ecdysteroid 22-phosphates; Dinan and Rees, 1981) or 10mg/ml (required to hydrolyse ecdysteroid 22-fatty acyl esters effectively; Whiting and Dinan, 1988a). In addition to the DBL-1 antiserum, which is sensitive to changes in both the side-chain and A-ring of antigenic molecules, the H-2 and H-22 antisera were used (Warren and Gilbert, 1986). The unhydrolysed conjugates are very poorly recognized by the DBL-I antiserum (Fig. 3), and the low concentration of Helix enzymes only causes a 6-fold increase in RIA-positive material even after 5 days' incubation. The high concentration of Helix enzymes causes a 16-fold increase over the same period. The H-22 antiserum, which is mainly sensitive to changes in the A-ring, recognizes the unhydrolysed conjugates to a significant extent and therefore it is likely that the conjugating moieties are located in the side-chain. Incubations with Helix enzymes at ! or 10mg/ml both raise the RIA-positive response almost equivalently, by 2-3-fold only. The H-2 antiserum, which is sensitive to changes in the side-chain, does not recognize the unhydrolysed conjugates. Further, hydrolysis with Helix enzymes even at 10mg/ml does not provide the large increase in RIA-positive material seen with the two other antisera. These observations can all be accounted for if there are two sites of conjugation, both on the side-chain, and if the major ecdysteroid involved is (as already shown) 20-hydroxyecdysone rather than ecdysone. Partial purification of ecdysteroids in 13 day eggs The ethanol extract of 13 day eggs was separated on silica to give the following fractions: (i) 0%, (ii) 10%,

323

TABLE 1. Distribution of free and conjugated ecdysteroids in fractions from the separation of an ethano|ic extract of 13 day-old eggs (1.45 g) by silicic acid (2 g) chromatography. The column was eluted sequentially with 20ml each of 0, 10, 15, 20, 30, 50, 80 and 100% methanol in chloroform. Each fraction was assessed for RIA-positive (DBL-1 antiserum) ecdysteroid content before and after enzymic hydrolysis (Helix enzymes at 10 mg/ml for 5 days) ng Ecdysone Equivalents/g eggs Before hydrolysis

Methanol (%) 0 10 15 20 30 50 80 100

0 20.5 119.2 30.5 0 20.5 53.0 47.7

After hydrolysis 17.9 821.0 218.5 36.4 26.5 152.3 199.0 92.7

(iii) 15%, (iv) 20%, (v) 30%, (vi) 50%, (vii) 80% and (viii) 100% methanol in chloroform. Each was assessed for RIA-positive material both before and after hydrolysis with Helix enzymes (Table 1). Portions of each fraction containing significant amounts of ecdysteroids (10, 15, 50 and 80% methanol in chloroform fractions) were also separated by reversed-phase H P L C (with water methanol and citrate-methanol gradients). Also, hydrolysed material was separated by reversedphase H P L C to identify the released ecdysteroid. The 10% methanol in chloroform fraction contained only ecdysone 22-fatty acyl esters. The 15% m e t h a n o l chloroform fraction contained a small amount of ecdysone 22-fatty acyl esters and both free ecdysone and 20-hydroxyecdysone (1:9, after correction for crossreactivities). The 50% methanol in chloroform fraction contained most of the novel p o l a r - a p o l a r conjugates,

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DAYS FIGURE 3. Helix hydrolysis of the embryonic conjugates from 13 day-old eggs of A. domesticus monitored by differential RIA. Portions of the conjugates were hydrolysed with Helix enyzmes at 1 mg/ml (solid symbols) or 10 mg/ml (open symbols) and the amount of RIA-positive material was separately determined using three antisera (DBL-I: - - , H-2:... and H-22: - - -).

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whereas the 80% fraction contained lower levels of these plus the low levels of polar conjugates.

T A B L E 2. Ratios of 20-hydroxyecdysone to ecdysone found in conjugate peaks designated in Fig. 4. The RIA-positive material released by enzymic hydrolysis corresponding to each of the peaks was pooled and separated by R P - H P L C (system RP2) and monitored by RIA (DBL-1 antiserum). Allowance has been made for the different cross-reactivities of ecdysone and 20-hydroxyecdysone. N D = not determined

pH optimum for Helix hydrolysis of the novel conjugates The ability of the Helix enzymes (10 mg/ml at 3°C for 2 days) to hydrolyse the novel conjugates was assessed by their ability to release RIA-positive material from aliquots of the 30-50% methanol in chloroform fraction in a range of 50 mM acetate buffers from pH 3 to 6. A broad optimum for hydrolysis was obtained between pH 4.5 and 5.5. Ion-suppression reversed-phase HPLC of novel conjugates When the novel conjugates are eluted from C~8 reversed-phase with a citrate buffer-methanol gradient beginning at 30% methanol, they elute as minor and major broad peaks (Fig. 2), suggesting that each peak represents a complex mixture. In order to improve the resolution of the component compounds, the conjugates were separated on a gradient beginning at 80% methanol. Enzymic hydrolysis followed by RIA revealed two main areas of RIA-positive material (Fig. 4). The first (33% of the RIA-positive material released) coincided with the void volume, whereas the second (67%) consisted of a series of peaks, reminiscent of the separation obtained with the endogenous ecdysone 22-fatty acyl esters from newly-laid eggs (Whiting and Dinan, 1988b, 1989), except that these conjugates elute significantly earlier than those. The identities of the ecdysteroids released from the major peaks were determined by reversed-phase HPLC/RIA (Table 2), showing that the early peaks were predominantly conjugates of 20hydroxyecdysone, while the later ones are conjugates of ecdysone. The peak at the void volume is composed of a mixture conjugates of 20-hydroxyecdysone and 400

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Partial enzymic hydrolysis and (ion-suppression) reversed-phase HPLC of novel conjugates An aliquot of the partially purified novel conjugates was treated with Helix enzymes for 2 days and the ecdysteroid mixture was separated by (ion-suppression) reversed-phase HPLC, followed by complete hydrolysis of fractions with Helix enzymes and RIA [Fig. 5(A)]. This demonstrates significant peaks of released ecdysone and 20-hydroxyecdysone and, in addition to the peak of 100

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FIGURE 5. Partial hydrolysis of embryonic conjugates from l 3 day-old eggs of A. domesticus with (A) Helix enzymes (2 mg/ml for 2 days) or (B) porcine liver esterase (20 IU for 24 h). Ethanol extracts of the hydrolysis mixtures were separated by R P - HPLC [system RPIB for (A) and system RP3B for (B)]. Fraction of 1 min duration were collected and subjected to "complete" enzymic hydrolysis before RIA.

remaining novel conjugates, there is a small, but definite, series of peaks eluting in the same region as the ecdysone 22-fatty acyl esters. Thus, it would appear that one of the conjugating moieties is a fatty acyl group, probably located at C-22. Partial hydrolysis of the novel conjugates with porcine liver esterases followed by ion-suppression reversedphase HPLC, complete hydrolysis of fractions with Helix enzymes and RIA [Fig. 5(B)] demonstrates that several new peaks are formed, all eluting at positions more polar than the novel conjugates. Most of these peaks are shifted to earlier elution times when citrate is omitted from the mobile phase. DISCUSSION Early studies on the identification of ecdysteroids in newly-laid eggs of Bombyx mori, Locusta migratoria and

S. gregaria revealed the presence of maternally-derived polar conjugated ecdysteroids (reviewed in Hoffmann and Lagueux, 1985). These were identified as ecdysteroid phosphates in B. mori (Ohnishi et al., 1989), S. gregaria (Isaac et al., 1983) and Manduca sexta (Thompson et al., 1988). In the course of embryogenesis, the maternal ecdysteroids in eggs of L. migratoria are hydrolysed to produce a series of peaks of free ecdysteroids, the occurrence of which correlates with cuticulogenic events (Lagueux et al., 1979). Ecdysteroids are then further converted to new (embryonic) conjugates and inactivation metabolites. These inactivation products have been identified as a mixture of ecdysteroid acids and acetyl ecdysteroid phosphates in S. gregaria and L. migratoria. Correlation of embryonic free ecdysteroid titres with cuticular events in the egg has also been suggested for some other species (Bulli~re et al., 1979; Imboden and Lanzrein, 1982; Bordes-All6aume and

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PENSRI WHITING et al.

Sami, 1987), but is not so apparent for other species (Warren et al., 1986). The complex nature of the relationship between egg ecdysteroids and embryonic events is further underlined by the existence of some species, the newly-laid eggs of which do not apparently contain any maternally-derived conjugates (e.g. phasmids: Fournier and Radallah, 1988; Dinan, unpubl, observ.). Even within an order, a constant pattern for the identity and titres of ecdysteroids during embryogenesis is not observed, as demonstrated by several studies on lepidopteran species (Hsiao and Hsiao, 1979; Ohnishi et al., 1981; Smith and Bollenbacher, 1985; Warren et al., 1986; Thompson et al., 1988) and, which is pertinent to this study, also for the Orthoptera; newly-laid eggs of the desert or migratory locusts contain very high levels of polar conjugates, whereas house cricket eggs contain only moderate levels of apolar conjugates (Whiting and Dinan, 1988a), which have been conclusively identified as a mixture of ecdysone 22-fatty acyl esters (Whiting and Dinah, 1989). Similar apolar ecdysteroid esters are present in the eggs of other insect species (Slinger et al., 1986; Slinger and Isaac, 1988) and are probably of widespread occurrence in many arthropod species at different developmental stages (Connat and Diehl, 1986). They may also serve a role in the detoxification of dietary ecdysteroids (Kubo et al., 1987; Robinson et al., 1987; Connat et al., 1988a). Significant levels of polar or apolar ecdysteroid esters are not present in newly-laid eggs of the Mediterranean field cricket, Gryllus bimaculatus (Espig et al., 1989). Differences between Orthopteran species also extend to the occurrence and distribution of ecdysteroids in adult insects. They are present in male crickets (Hoffmann and Behrens, 1982; Whiting and Dinan, 1990a), but absent from male L. migratoria (Hoffmann et al., 1975; Lagueux et al., 1977). In adult female locusts, the ecdysteroids are confined essentially (98%) to the ovaries, whereas in A. domesticus only one third of the total ecdysteroid is associated with the ovaries (Renucci and Strambi, 1981; Whiting and Dinan, 1990a). In order to provide a firm basis for the identification of the functions of ecdysteroids during reproduction and embryogenesis in house crickets, we have expanded our initial studies on the identification of the ecdysteroids in newly-laid eggs. On the one hand, we are investigating the identity, distribution and developmental profiles of ecdysteroids in adult female insects (Whiting and Dinan, 1990a; Whiting et al., in prep.). This report represents a complementary approach; analysis of the fate of the maternally-derived fatty acyl esters during embryogenesis. We have examined the ecdysteroid content of developing eggs at five timepoints evenly spread throughout embryogenesis in order to provide an overview of the changes before determining complete titre curves for each of the identified ecdysteroids. It is clear that changes in ecdysteroid content occur throughout embryogenesis, and that the rate and extent

of these changes increases as the embryo develops. Initially (day 3 and day 7 samples), the changes are confined to the appearance of free 20-hydroxyecdysone and the formation of low levels of new (embryonic) conjugates. 20-Hydroxyecdysone could derive from the hydroxylation of the pre-existing pool of free ecdysone or from the hydrolysis and hydroxylation of a small portion of the ecdysone 22-fatty acyl esters. De novo biosynthesis is also a possibility, but seems unlikely in view of the large amount of potential precursors present. The level of ecdysone 22-fatty acyl esters (and consequently of the total ecdysteroids) increases dramatically between days 7 and 10, indicating that the embryo is capable of biosynthesizing ecdysteroid (in the form of ecdysone 22-fatty acyl esters). Hydrolysis and hydroxylation produce 20-hydroxyecdysone, which might be expected to be the active molecule. The continued increase in the levels of novel conjugates during embryogenesis implies that the formation of these are the means by which the free 20-hydroxyecdysone and ecdysone are inactivated once they have had their action. The hypothesized ecdysteroid interconversions are summarized in Scheme 1.

Cholesterol

E-22FA hydrolysis E ~ydroxyla

lion

20E c o n j u g a lion 1

JL V

P-E

P-20E conjugation

P-E-22FA

2

,l

P-20E-22FA

SCHEME 1. Putative composite scheme for ecdysteroidmetabolism during embryogenesis of A. domesticus. E = ecdysone; 20E = 20hydroxyecdysone;22FA= 22-fattyacylester and P = unknownpolar conjugate moiety.

CRICKET EGG ECDYSTEROIDS

The absence of significant levels of 20-hydroxyecdysone 22-fatty acyl esters indicates that the ecdysone 22-fatty acyl esters are not directly hydroxylated and also that, in the process of inactivation, the free ecdysteroids (20-hydroxyecdysone and ecdysone) are conjugated with the polar moiety before they are re-acylated. The occurrence of low levels of (purely) polar ecdysteroid conjugates in developing eggs is in accord with this hypothesis. The validity of the suggested conversions needs to be proved by the direct demonstration of each step in Scheme 1. In this context, we have recently shown that homogenates of developing eggs show an increasing ability to hydrolyse [3H]ecdysone fatty acyl esters (West and Dinan, unpubl, observ.). The identity of the novel conjugates produced in large amounts towards the end of embryogenesis is intriguing. A number of lines of evidence indicate that they are doubly-conjugated ecdysteroids. Their unusual chromatographic behaviour can be explained if one conjugating group is a fatty acyl moiety, which would be responsible for the strong retention on C~8 reversedphase columns, while the presence of an additional polar conjugate group would provide the strong interaction with silica. The novel conjugates are hydrolysed only slowly by H e l i x enzymes (a heterogeneous hydrolase preparation), just like ecdysone 22-fatty acyl esters (Whiting and Dinan, 1988a), whereas polar ecdysteroid conjugates (phosphates or glucosides) are generally rapidly hydrolysed (Sannasi and Karlson, 1974). Partial hydrolysis with H e l i x enzymes releases, in addition to larger amounts of ecdysone and 20-hydroxyecdysone, a small amount of material which co-chromatographs with ecdysteroid 22-fatty acyl esters. Treatment of the new conjugates with porcine liver esterases releases no free ecdysteroids, but polar conjugates instead. Thus, the existence of multiply-conjugated ecdysteroid seems certain. Double conjugates seem most likely, with a fatty acyl group at C-22 and the polar moiety on one of the other hydroxyls. The differential radioimmunoassay data suggest that the site of polar conjugation is also on the side-chain (C-257), because the novel conjugates are recognized by the H-22 antiserum, but not by the H-2 or DBL-1 antisera. The absence of significant amounts of polar conjugates released from the novel conjugates on hydrolysis with H e l i x enzymes (i.e. after removal of the fatty acyl moiety) can be explained if the polar-apolar conjugates are a poor substrate for, for example the phosphatase activity in the H e l i x hydrolases, so that the rate limiting step is the removal of the fatty acyl group; the subsequent removal of the polar conjugating moiety being very rapid by comparison. Ecdysteroid fatty acyl esters were first identified as metabolites of exogenous (ingested or injected) ecdysteroid in ticks (Connat et al., 1984; Wigglesworth et al., 1985; Diehl et al., 1985; Crosby et al., 1986b). The biological significance of these compounds in the argasid tick Ornithodoros m o u b a t a has recently been questioned,

327

as the apolar metabolites of exogenous ecdysteroid do not appear to have constant levels of endogenous counterparts in different batches of eggs and are not metabolized during embryogenesis (Connat et al., 1988b). It has been suggested that the formation of apolar metabolites in ticks serves to inactivate dietary ecdysteroids (Connat et al., 1986; Connat and Dotson, 1988; Connat et al., 1988b). This is obviously not the case in house cricket eggs as we have shown that ecdysone fatty acyl esters are constant endogenous components of newly-laid eggs (Whiting and Dinan, 1988a, 1989) and undergo changes in titre and conversions (this report). The studies on ticks also revealed the presence of a second class of esterase-susceptible apolar ecdysteroid conjugate derived from exogenous ecdysteroids, via the ecdysteroid 22-fatty acyl esters. These were called AP1 by Connat et al. (1984) and were characterized by eluting slightly earlier from reversed-phase than the 20-hydroxyecdysone 22-fatty acyl esters (AP2), but no further indication as to their identity has been published. Their behaviour on reversed-phase is strikingly similar to that of the novel conjugates found in developing eggs of A. domesticus. The AP1 conjugates of ticks also appear polar on silicic acid (J.-L. Connat, pets. commun.). Thus, circumstantial evidence exists that the novel conjugates in house cricket eggs and the AP1 conjugates from ticks belong to the same class. Similar chromatographic behaviour is observed with a class of metabolites of [3H]ecdysone found in developing eggs of the hard tick Boophilus microplus (Crosby et al., 1986a), but preliminary evidence suggests that these are fatty acyl ecdysonoic acids. The identification of the novel conjugates as doublyconjugated ecdysteroids needs to be confirmed by isolation of the compounds and unambiguous characterization by spectroscopic methods. Although the peculiar chromatographic properties will no doubt assist their purification, their heterogeneity will not facilitate the obtaining of sufficient amounts of each conjugate. It is apparent that two ecdysteroids (20-hydroxyecdysone and ecdysone) are involved, each of which can be esterified at C-22 to any one of about nine major long-chain fatty acids and at one of the other hydroxyl groups to any one of two polar moieties. Thus, the novel conjugates are a complex mixture of >36 different components. REFERENCES

Bordes-All6aumeN. and Sami L. (1987) Ecdysteroidtitre and cuticle depositions in embryosof the dipteran Calliphora erythrocephala. Int. J. invert, reprod. Dev. 11, 109 122. Bulli6~'e D., Bulli6re F. and de Reggi M. (1979) Ecdysteroidtitres during ovarian and embryonic development in Blaberus craniifer. Roux's archs dev. Biol. 186, 103-114. Connat J.-L. and Diehl P. A. (1986) Probable occurrence of ecdysteroid fatty acid esters in different classes of arthropods. Insect Biochem. 16, 91-97. Connat J.-L. and Dotson E. M. (1988) Comparativeinvestigationof the egg ecdysteroidsof ticksusingradioimmunoassayand metabolic studies. J. Insect Physiol. 34, 639-645.

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Connat J.-L., Diehl P. A. and Morici M. (1984) Metabolism of ecdysteroids during the vitellogenesis of the tick Ornithodoros moubata (Ixodoidea, Argasidae): accumulation of apolar metabolites in the eggs. Gen. comp. Endocr. 56, 100-110. Connat J.-L., Diehl P. A. and Thompson M. J. (1986) Possible inactivation of ingested ecdysteroids by conjugation with long-chain fatty acids in the female tick Ornithodoros moubata (Acarina:Argasidae). ,4rchs Insect Biochem. Physiol. 3, 235-252. Connat J.-L., Dotson E. M. and Diehl P. A. (1988a) Apolar conjuates of ecdysteroids are not used as a storage form of molting hormone in the argasid tick Ornithodoros moubata. Archs Insect Biochem. Physiol. 9, 221-236. Connat J.-L., Ffirst P.-A. and Zweilin M. (1988b) Detoxification of injected and ingested ecdysteroids in spiders. Comp. Biochem. PhysioL 91B, 257265. Crosby T., Wigglesworth K. P., Lewis D. and Rees H. H. (1986a) Moulting hormones in the development of the cattle tick, Boohilus microlus. In Proceedings o f the Vero Beach Symposium; Host Regulated Developmental Mechanisms in Vector Arthropods (Edited by Borovsky D. and Spielman A.), pp. 37-45. University of Florida, Fla. Crosby T., Evershed R. P., Lewis D., Wigglesworth K. P. and Rees H. H. (1986b) Identification of ecdysone 22-long-chain fatty acyl esters in newly laid eggs of the cattle tick Boophilus microplus. Biochem. J. 24, 131-138. Diehl P. A., Connat J.-L., Girault J. P. and Lafont R. (1985) A new class of apolar ecdysteroid conjugates: esters of 20-hydroxyecdysone with long-chain fatty acids in ticks. Int. J. invert, reprod. Dev. 8, 1-13. Dinan L. (1988) Separation of ecdysone acyl esters and their 2,3acetonide derivatives by high-performance liquid chromatography. J. Chromatogr. 436, 279-288. Dinan L. N. and Rees H. H. (1981) The identification and titres of conjugated and free ecdysteroids in developing ovaries and newlylaid eggs of Schistocerca gregaria. J. Insect Physiol. 27, 51-58. Espig W., Thiry E. and Hoffmann K. H. (1989) Ecdysteroids during ovarian development and embryogenesis in the cricket, Gryllus bimaculatus de Geer. Invert. reprod. Dev. 15, 143-154. Fournier B. and Radallah D. (1988) Ecdysteroids in Carausius eggs during embryonic development. Archs Insect Biochem. Physiol. 7, 211-224. Hagedorn H. H. (1989) Physiological roles of hemolymph ecdysteroids in the adult insect. In Ecdysone; From Chemistry to Mode of Action (Edited by Koolman J.), pp. 279289. Thieme, Stuttgart. Hoffmann K. H. and Behrens W. (1982) Free ecdysteroids in adult male crickets, Gryllus bimaculatus. Physiol. Ent. 7, 269-279. Hoffmann J. A. and Lagueux M. (1985) Endocrine aspects of embryonic development in insects. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. I, pp. 435-460. Pergamon Press, Oxford. Hoffmann J. A., Lagueux M., Him M., De Reggi M., Goltzen~ F. and Feyereisen R. (1975) Evolution du taux des ecdyst~roides chez les imagos m~les et femelles de Locusta migratoria L. Colloques internationaux CNRS No. 251; Actualitbs sur les Hormones d'Invertbbrbs (Edited by Durchon M. M.), pp. 359-365 CNRS, Paris. Hsiao T. H. and Hsiao C. (1979) Ecdysteroids in the ovary and the egg of the greater wax moth. J. Insect Physiol. 25, 45-52. Imboden H. and Lanzrein B. (1982) Investigations on ecdysteroids and juvenile hormones and on morphological aspects during early embryogenesis in the ovoviviparous cockroach Nauphoeta cinerea. J. lnsect Physiol. 28, 37-46. Isaac R. E., Rose M. E., Rees H. H. and Goodwin T. W. (1983) 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, 533-541. Kubo I., Komatsu S., Asaka Y. and De Boer G. (1987) Isolation and identification of apolar metabolites of ingested 20-hydroxyecdysone in frass of Heliothis virescens larvae. J, chem. Ecol. 13, 785 794.

Lagueux M., Hirn M. and Hoffmann J. A. (1977) Ecdysone during ovarian development in Locusta migratoria. J. Insect Physiol. 23, 109-119. Lagueux M., Hetru C., Goltzen6 F., Kappler C. and Hoffmann J. A. (1979) Ecdysone titre and metabolism in relation to cuticulogenesis in embryos of Locusta migratoria. J. Insect Physiol. 25, 709 724. Lanot R., Dorn A., Giinster B., Thiebold J., Lagueux M. and Hoffmann J. A. (1989) Functions of ecdysteroids in oocyte maturation and embryonic development of insects. In Ecdysone; From Chemistry to Mode of Action (Edited by Koolman J.), pp. 262-270. Thieme, Stuttgart. Ohnishi E., Mizuno T. Ikekawa N. and Ikeda T. (1981) Accumulation of 2-deoxy ecdysteroids in ovaries of the silkworm, Bombyx mori. Insect Biochem. 11, 155 159. Ohnishi E., Hiramoto M., Fujimoto Y., Kakinuma K. and Ikekawa N. (1989) Isolation and identification of major ecdysteroid conjugates from the ovaries of Bombyx mori. Insect Biochem. 19, 95-101. Renucci M. and Strambi A. (1981) Evolution des ecdystrroides ovariens et hrmolymphatiqes au cours de la maturation ovarienne chez Acheta domesticus L. (Orthopt~re). C. R. Acad. Sci. Paris, S~rie III, 293, 825-830. Renucci M., Strambi A. and Strambi C. (1987) Ovarian development and endocrine control of vitellogenesis in the house cricket Acheta domesticus. Life Sci. Adv. 6, 83-92. Robinson P. D., Morgan E. D., Wilson I. D. and Lafont R. (1987) The metabolism of ingested and injected [3H]ecdysone by final instar larvae of Heliothis armigera. Physiol. Ent. 12, 321-330. Sannasi A. and Karlson P. (1974) Metabolism of ecdysone: phosphate and sulphate esters as conjugates of ecdysone in Calliphora vicina. Zool. Jb. Physiol. 78, 378-386. Schneider R. (1973) Protein-Analysen an einzelnen Eiern und Eiteilen w/ihrend der Embryonalentwicklung von Acheta domesticus L. Ph. D. thesis, University of Marburg, Germany. Slinger A. J. and Isaac R. E. (1988) Synthesis of apolar ecdysone esters by ovaries of the cockroach Periplaneta americana. Gen. comp. Endocr. 70, 74-82. Slinger A. J., Dinan L. N. and Isaac R. E. (1986) Isolation of apolar ecdysteroid conjugates from newly-laid oothecae of Periplaneta americana. Insect Biochem. 16, 115-119. Smith S. L. and Bollenbacher W. E. (1985) Ovarian ecdysteroids and their secretion in late-pharate adults of Galleria mellonella. J. Insect Physiol. 31, 419-424. Thompson M. J., Svoboda J. A., Lozano R. and Wilzer K. R. (1988) Profile of free and conjugated ecdysteroids and ecdysteroid acids during embryonic development of Manduca sexta (L.) following maternal incorporation of [t4C]cholesterol. Archs Insect Biochem. Physiol. 7, 157-172. Warren J. T. and Gilbert L. I. (1986) Ecdysone metabolism and distribution during the pupal-adult development of Manduca sexta. Insect Biochem. 16, 65-82. Warren J. T., Steiner B., Dorn A., Pak M. and Gilbert L. I. (1986) Metabolism of ecdysteroids during embryogenesis of Manduca sexta. J. Liq. Chromatogr. 9, 1759-1782. Whiting P. and Dinan L. (1988a) The occurrence of apolar ecdysteroid conjugates in newly-laid eggs of the house cricket, Acheta domesticus. J. Insect Physiol. 34, 625q531. Whiting P. and Dinan L. (1988b) The formation of apolar ecdysteroid conjugates by ovaries of the house cricket, Acheta domesticus, in vitro. Biochem. J. 252, 95-103. Whiting P. and Dinan L. (1989) Identification of the endogenous apolar ecdysteroid conjugates present in newly-laid eggs of the house cricket (Acheta dornesticus) as 22-long-chain fatty acyl esters of ecdysone, lnsect Biochem. 19, 759-767. Whiting P, and Dinan L. (1990a) Chromatographic separations of ecdysone acyl esters and their application to the distribution and identification of ecdysteroids in adult house crickets, Acheta domesticus. In Chromatography and Isolation of Insect Hormones

CRICKET EGG ECDYSTEROIDS and Pheromones (Edited by McCaffery A. R. and Wilson I. D.), pp. 53~7. Plenum Press, New York. Whiting P. and Dinan L. (1990b) Ecdysteroids in adults and eggs of the house cricket, Acheta domesticus. Int. J. invert, reprod. Dev. 18, 132-133. Wigglesworth K. P., Lewis D. and Rees H. H. (1985) Ecdysteroid titre and metabolism to novel apolar derivatives in adult female Boophilus microplus (Ixodidae). Archs Insect Biochem. Physiol. 2, 39-54.

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Acknowledgements--Antisera were very kindly provided by Professors Jan Koolman, Larry Gilbert and Denny O'Connor. Professors Ren6 Lafont and Huw Rees and Drs Mohammed Kabbouh and Elwyn Isaac generously provided authentic ecdysteroid phosphate and acid reference compounds. We thank Chris C16ment for his help with the preparation of the figures and his comments on the manuscript.