A re-investigation on the ecdysteroids during embryogenesis in the desert locust, Schistocerca gregaria

J. /nsrct PI~y.sio/. Vol. 28, No. 7, pp. 647-654. Printed in Great Brifain.

0022-1910/82/070647-08803.00/O 0 1982 Pergamnn Prrss Lid

1982

A RE-INVESTIGATION ON THE ECDYSTEROIDS DURING EMBRYOGENESIS IN THE DESERT LOCUST, SCHISTOCERCA GREGARIA E. D. MORGAN*

S. SCALIA and

Department of Chemistry. University of Keele. Keele. Staffordshire. ST5 SBG, U.K.

(Rewired 30 Octohrr 1981; rerisrd

4 Jan~ur,v 1982)

Abstract-Comparison of gas chromatography and high-pressure liquid chromatography methods for determination of ecdysteroids in insect eggs revealed that published values for Schistocerca gregariu were very low. The error has been traced to incomplete hydrolysis of conjugates. Revised values for the levels of ecdysone, 20-hydroxy-ecdysone and 2-deoxy-ecdysone are given here. The same general pattern of falling ecdysteroid titre during early stages of embryo development followed by an increase and a final decrease is observed. Experiments have been made on the effect of phosphate, ionic strength and a specific /?-glucuronidase competitive inhibitor on the hydrolysis of the polar ecdysteroid conjugates. in an attempt to provide more evidence on the chemical nature of these compounds. Key Words. Schistocercu grrgariu. ecdysteroids. ecdysteroid conjugates. embryogenesis

INTRODUCTION THE PRESENCEof ecdysteroids

in the eggs of insects

has been recognized for some time (OHNISHI et al., 1971) and identified in several species. Detailed investigations have been carried out on eggs of Manduca xxtu (KAPLANIS et al., 1973). Oncopeltus fasciatus (KAPLANIS er al., 1975), Blabera craniifer (BULLIERE rt al.. 1976), Bombyx mori (OHNISHI et al., 1971; MIZUNO and OHNISHI. 1975), Leucophaea maderae (MATZ and HOFFMANN (1975) Galleria mellonella (HSIAO and HSIAO, 1979), Nauphoeta cinerea (IMBODEN et al.. 1978), Locusta migratoria (LAGUEUX et al., 1977) and Schistocerca gregaria (GANDE and MORGAN, 1979; DINAN and REES, 198la). In spite of the existence of all these reports the function of the moulting hormones in embryonic development is not yet well understood, being possibly related to the embryo cuticulogenesis (KURODA, 1971; CHIHARA et al., 1972; BULLIERE and BULLIERE, 1973: DORN and ROMER, 1976; LAGUEUX et al., 1979;). How-

ever the hypothesis of a neuroendocrine control of the embryonic apolysis has been dismissed by other authors on the evidence that under in vitro conditions, embryonic abdomens are capable of producing several cuticles long after having been severed from head and thorax (MUELLER, 1963; TAKAMI, 1963: SBRENNA-MICCIARELLI and SBRENNA, 1972; HAGET, 1977). We have approached this problem by determinat-

ing the hormone titre in the developing eggs in order to discover any correlation between the varying amount of ecdysteroids and distinct stages of embryogenesis. A previous investigation has been carried out, in our group. on the variation in ecdysteroids level in the eggs of S. greguriu from the time of oviposition to hatching (GANDE and MORGAN, 1979). Determi* Author

nations were by gas chromatography with electroncapture detection after silylation of the ecdysteroids. Ecdysone and 20-hydroxy-ecdysone, present both as free and, mainly, as polar conjugates, were identified. However, later, in the course of comparison of gas chromatography (GC) and high-pressure liquid chromatography (HPLC) methods we found large differences in the amounts of conjugated ecdysteroid isolated from eggs with the two different procedures. Checking GC determination against HPLC on a single sample showed complete agreement for the two methods of detection. Re-investigation showed that incomplete hydrolysis of ecdysteroid conjugates had caused the low values in our earlier work. Recently, work from another group has also shown that the amount of conjugates detected in newly laid eggs of S. gregaria, by HPLC was ten times greater than that reported in Gande’s work (DINAN and REES. 198la). We report here the revised values for the levels of the three major ecdysteroids found in the eggs; ecdysone, 20-hydroxy-ecdysone and 2-deoxy-ecdysone. throughout the period of development from oviposition to hatching. The exact nature of the conjugating group in the polar ecdysteroids remains unknown. A phosphate, sulphate or glucuronide has been suggested but it is still uncertain whether one or several such groups are present in the egg conjugates. In the course of this work we have looked at factors affecting the hydrolysis of the conjugates including the nature of the buffer, ionic strength, and a specific glucuronidase inhibitor, which eliminate the possibility of the conjugates being glucuronides. MATERIALS

AND

METHODS

Eggs

The eggs of Schistocerca gregaria Forskal were obtained from a colony of locusts reared in crowded

for correspondence. 647

648

S. SCALIAand E. D. MORGAN

conditions in a 12: 12 hr light/dark photoperiod regime. The ambient temperature during the light phase was 32” falling to 28°C in the dark phase. The locusts were fed on fresh grass and dry bran. Mature female adult S. gregaria laid their eggs in aluminium tubes containing moist sand. The egg pods were removed from the sand and allowed to develop for different lengths of time at a constant temperature of 30°C in moist cotton-wool. The eggs were dated by the number of days elapsed from oviposition to the extraction, and the embryos staged according to the scheme of SHULOVand PENER (1963). Extraction procedure

The eggs were ground in methanol in a glazed mortar and the resulting slurry filtered through a sintered filter (porosity 3). The filtrate was evaporated down and partitioned between hexane and methanol/water (8:2 V/V) to remove apolar impurities. The residue from the aqueous methanol phase was then partitioned between countersaturated n-butanol and water. Each fraction was backwashed with a small volume of the appropriate counter phase which was added to the main fraction. During this partition the free ecdysteroids are found in the butanol phase, whereas the polar conjugates were in the water phase. PuriJication of the conjugated ecdysteroids

The aqueous phase residue from the butanol-water partition was dissolved in 5 ml of IOOmM acetate buffer (pH 5.2) and subjected to overnight enzymatic hydrolysis at 37°C in the presence of sufficient amount of the digestive juice of the snail Helix pomatia (5 $/ml) (Koch-Light Laboratories Ltd. Colnbrook) to give 4000 Roy units/ml of aryl sulphatase (measured by the method of PASQUALINI,1967), 500 Fishman units/ml of /?-glucuronidase (measured by the method of FISHMANet al., 1967) and 100 Sigma units/ml of acid phosphatase (measured by the method of SOMMER, 1954). In order to purify the ecdysteroids, freed by the enzymatic hydrolysis, from the impurities present in the incubation medium, the latter was injected into a SEP-PAK C-18 cartridge (Waters Associates, Milford) and eluted successively with 5 ml of 20% (V/V) methanol in water and 7 ml of 80% (V/v) methanol in water. The latter fraction contains all the different ecdysteroids present in the egg extract and can be directly analysed by reverse-phase high pressure liquid chromatography (RP-HPLC) with ultra-violet detection of the eluate, as described below. PuriJication of the free ecdysteroids

The residue from the butanol phase of the butanolwater partition was purified by reverse-phase medium pressure liquid chromatography (RP-MPLC) which was performed with a MPL series II Micro-pump (Metering Pump Ltd., London), a Tefzel slider valve (Magnus Scientific Instrumentation Ltd., Sandbach) injection system and a IEC column (Whatman Ltd., Maidstone) 10mm i.d. x 45cm dry packed with 3@40-pm particles of C,,-Magnusil H (Magnus Scientific Instrumentation Ltd., Sandbach). The sample, dissolved in 50% (V/V) methanol-water (4ml), was injected into the column and eluted successively with 40% (V/V) methanol-water (80 ml) and

80% (V/V) methanol-water (70 ml) collecting in 10 ml fractions. The ecdysteroids were eluted in fractions 11 to 15, which were then analysed by HPLC on a silica column (NP-HPLC). HPLC analysis of ecdysteroids

The HPLC analysis was performed with a Pye Unicam LC3XP Pump Liquid Chromatograph (Pye Unicam Ltd., Cambridge) equipped with a Pye Unicam gradient elution system, a sample injection valve (Rheodyne Inc., Cotati) and a model 220 Mixed wavelength Absorbance Detector (Chromatronix Inc., Berkeley) set at 254 nm. Two types of columns were used: (i) a reverse phase column 5 mm i.d. x 25 cm, packed with 5 pm particles of Hypersil ODS (Shandon Southern, Runcorn) was eluted under isocratic conditions (60% V/V methanol in water) at a flow rate of 0.8 ml/min. (ii) a normal phase column 5 mm i.d. x 25 cm packed with 5 pm particles of Hypersil (Shandon Southern, Runcorn) was run under isocratic conditions (methylene chloride-isopropanol-water 125:25:2) at a flow rate of 1 ml/min. The quantities of the ecdysteroids present in the biological extracts were calculated from standard curves. obtained by plotting peak area against ng of pure ecdysone (Simes, Milano), 20-hydroxy-ecdysone (Simes, Milano) and 2-deoxy-ecdysone (D. H. S. Horn, Melbourne). ldentijkation of ecdysteroids

Identification of the hormones was based on the observation that the biological sample co-chromatographed with authentic compound on RP-HPLC, normal phase HPLC and silica gel (Merck, Darmstadt) thin-layer chromatography. Furthermore the trimethylsilyl ethers derivative of the biological sample co-chromatographed with authentic compound derivative on gas chromatography with a 63Ni electron-capture detector. Glucuronidase inhibition experiment

A portion of the aqueous layer containing conjugated ecdysteroids (0.3 pmol) was evaporated to dryness, redissolved in 0.1 M acetate buffer (10 ml, pH 5.2) and divided into two equal parts; to one of them digestive juice of Helix pomatia was added (5 PI/ml, to give 4000 Roy units/ml of aryl sulphatase, 500 Fishman units/ml of /I-glucuronidase and 100 Sigma units of acid phosphatase), to the other one D-saccharic acid-lP-lactone (Sigma, London) was added (10 -3 M after dilution in the buffer). The pH of the buffered inhibitor solution was checked (glass electrode) and adjusted to pH 5.2, after which the enzyme (5pl/ml) was added as in the other sample. Both samples were incubated for 1 hr at 37°C and then separately purified and analysed by RP-HPLC as described above. Phosphate-inhibition experiment

The aqueous phase containing polar ecdysteroid conjugates from the butanol-water partition was divided in two equal parts. The two portions were evaporated to dryness and redissolved, respectively, in 0.1 M acetate buffer (5 ml, pH 5.2) and 0.1 M phosphate buffer (5 ml, pH 5.2). Both fractions were incubated overnight in presence of the digestive juice of Helix pomatia (5 PI/ml) after which they were separately purified and analysed as previously.

Ecdysteroids

and embryogenesis

649

in the desert locust

Hydrolysis of conjugates at difSerent ionic strength

Titre of,free hormone

The aqueous phase from the butanol-water partition was divided in two equal portions which were evaporated to dryness and redissolved, one in 0.1 M acetate buffer (5 ml, pH 5.2) and the other in 1.8 M acetate buffer (5 ml, pH 5.2). Both samples were incubated for 1 hr in the presence of the same amount of enzyme and analysed for ecdysteroids as in the previous section.

The butanol phase, from the butanol/water partition containing the free ecdysteroids had to be subjected to a further purification step by reverse-phase medium-pressure chromatography (RP-MPLC) in order to remove impurities which interfere in the NP-HPLC system. During the investigation, the levels of free ecdysone have also been measured in each of the 14 days of development (Table 1). A decrease of amount of hormone during the first 8 days is followed by a peak on days 11-12 (55 rig/egg). Thereafter the level falls to the minimal values reported (IO ng/egg) (Fig. 2). The total weight of each egg increases by 2009, during the first 8 days due to the absorption of water therefore the difference between the variation of the levels of the hormone expressed in ng/egg and pg/g (Table 1). Free 20-hydroxy-ecdysone and free 2-deoxy-ecdysone were also detected. Because of the small amount (l-5 ng/egg) found, the quantitation of these two ecdysteroids. in the free state, during embryonic development has not been undertaken. The values for 20-hydroxy-ecdysone are given in GANDE and MORGAN (1979).

Comparison qf HPLC and CC methods The aqueous layer containing the conjugated ecdysteroids was divided in two equal parts, which were then evaporated to dryness. One of them was redissolved in 0.1 M acetate buffer (5 ml, pH 5.2) to which 5 &ml of enzyme was added, the other one was redissolved in 0.1 M phosphate buffer (5 ml, pH 5.2) to which 25 $/ml of enzyme was added. After the overnight incubation of 37’C, the acetate buffered fraction was analysed by HPLC as previously described, whereas the phosphate buffered fraction by GC as reported in GANDE and MORGAN (1979).

Titre oj hydroxysable hormones

RESULTS Under the conditions chosen for the incubation of the eggs. it required 14 days from oviposition to the hatching of the first-instar Schistocerca larvae. Each batch of egg pods was dated, extracted and purified as described in Materials and Methods. The purified extracts were analysed for the ecdysteroids present by reverse phase HPLC (RP-HPLC) and normal phase or absorption HPLC (NP-HPLC) and quantified by recording the UV absorbance of the effluent from the column. A typical HPLC trace of the purified extract is shown in Fig. 1. C

I

0

A

1

I

I

I

In this paper the term conjugated hormones refers to the highly polar ecdysteroids which are hydrolysed by the digestive juice of the snail Helix pomatia to release the free compounds. DINAN and REES (1981b) have shown, using labelled conjugates. the presence in the eggs of S. americana gregaria of highly polar ecdysteroids which are not enzymatically hydrolysable. Consequently this class of compound cannot be detected by the method developed in our investigation The polar conjugated ecdysteroids are partitioned quantitatively into the water phase of the butanolwater partition provided that careful attention is paid to phase separation and back-washing of fractions to be discarded (MIZUNO and OHNISHI, 1975; WILSON Table

Day

[min]

1;

lb

5

0

Fig. 1. Typical RP-HPLC trace of a purified extract of hydrolysed ecdysteroid conjugates. Retention time of compounds are indicated by (A) 20-hydroxy-ecdysone; (B) ecdysone; (C) 2-deoxy-ecdysone. (X) represents a U.V. absorbing impurity from the enzymes. (1) indicates the injection point.

1 2 3 4 5 6 7 8 9 10 II 12 13 I4

1. Mean levels of free ecdysone developing eggs of S. grrqariu Embryonic stage

VI XI XIV-XV xv XVI XVIIXVIll XVIII XIX xx

XXI XXII XXIII

m the

Free ecdysone pgig

ng/egg 37.7 28.2 32.5 17.5 15.3 11.4 9.5 I I.1 24.9 26.8 44 55.1 10.2 17.8

3.0 2.2 2.6 1.4 I.’ 0.9 0.7 0.4 0.9 I.0 1.7 2.2 0.4 0.7

Each determination is the mean of 2 separate extractions. Levels of free 20-hydroxy-ecdysone and ?-deoxy-ecdysone were below 5 ng/egg throughOUI.

650

S. SCALIA

Free

and E. D. MORGAN

Ecdysone

-

1 n

L_

10 11

123456789

Fig. 2. Level of ecdysone cerccl gregaria

12 13 14

days

in the developing eggs of Schistofrom oviposition to hatching.

and MORGAN, 1978). We have also compared the partition method of separating the free and conjugated ecdysteroids with a chromatographic method. In the latter case the extract of eggs was dissolved in 20”/0 (v/v) methanol in water (5 ml) injected into a Sep-pak C-18 cartridge and eluted successively with 40% (v/v) methanol in water (4ml) and 80% (v/v) methanol in water (7 ml). The 20 and 40% methanol in water fractions were collected, combined, evaporated to dryness, dissolved in 0.1 M acetate buffer (5 ml, pH 5.2) and hydrolysed as previously described. The partition and chromatographic methods have been shown to give consistent results in the quantitation of the conjugated ecdysteroids contained in the same sample of eggs. Because of their high polarity the conjugates form a class of compound which is difficult to analyse directly by chromatography. For this reason they are first hydrolysed in order to release the free hormones from the polar conjugated compounds and these are chromatographed.

Table 2. Mean levels of conjugated

Embryonic

Day

stage

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

VI

XI XIV-XV xv XVI XVI-XVIII XVIII XIX xx XXI XXII XXIII

Each determination

The hydrolysis was accomplished by the mixture of enzymes contained in the digestive juice of the snail Helix pomatia (HOLDEN and TRACEY. 1950). The amount of enzyme used was shown to give complete hydrolysis of the conjugates. increasing enzyme caused no incremental change in ecdysteroids released. A blank determination made with buffer and enzyme was checked for the presence of ecdysteroids. With our detection method no ecdysteroids were found in the batches of enzyme used in this study. Furthermore no solvolysis of conjugates was observed in the absence of enzyme. In the developing eggs of 5. yrrgaria considerable amounts of conjugates were found. Conjugated ecdysone. 20-hydroxy-ecdysone and 2-deoxy-ecdysone were detected. and the amount of each ecdysteroid at each of the 14 days of the embryonic development is shown in Table 2. Note that the mass of the eggs increases during development due to absorption of water, so results are expressed both in terms of ng per egg, which indicates changes in the total amount of ecdysteroids and in terms of pg per g which shows changes in concentration of ecdysteroids. The total amount of conjugates in newly laid eggs is 755.7 ng/egg, it decreases to a value of 513.4 ng/egg at day 6 and then it rises again to a maximum on day 11-12 (870ng/egg). Ecdysone is the main hormone present (60% of the total amount of conjugates detected) followed by 2-deoxy-ecdysone (30%) and 20-hydroxy-ecdysone (IO”/,). Inhibition qf’ enzymatic hydrolysis of conjugates To determine whether phosphate ions affected the rate of hydrolysis of conjugates, experiments were carried out comparing the yield of ecdysteroids obtained from the hydrolysis in 0.1 M acetate buffer (pH 5.2) and 0.1 M phosphate buffer (pH 5.2) respectively. The same amount of substrate (0.3 x 1O-6 moles) and enzyme (5~1/ml) were found to give. in phosphate buffer 52.5 ng/egg for ecdysone. 23.8 ng/egg for 2-deoxy-ecdysone, 5 ng/egg for 20-hydroxy-ecdysone whereas in acetate buffer the values were: 436 ng/egg for ecdysone, 217 ng/egg for 2-deoxy-ecdysone,

ecdysteroids

in the developing

Conjugated

Conjugated

ecdysone /G/P n8/3 gg

20-l.ydroxy-ecdysone n&88 I&?

465 391 304 218 285 187 236 326 338 372 411 477 314 282

37.2 31.2 24.3 17.4 22.8 15.0 9.4 13.0 13.5 14.8 16.4 19.0 12.5 11.2

48.4 44.6 21.5 27.0 25.0 27.3 32.3 40.6 51.7 88.3 84.9 61.0 57.7 54.2

is the mean of at least 3 different extractions.

eggs of S. grrgurio Conjugated

3.8 3.5 1.7 2.1 2.0 2.1 1.25 1.6 2.0 3.5 3.4 2.4 2.3 2.1

2-deoxy-ecdysone n&88 /%I8 242 222 138 127 144 100 162 198 243 342 374 315 234 298

19.3 17.7

11.0 10.2 Il.5 8.0 6.5 7.9 9.7 13.7 14.9 12.6 9.3 11.9

Ecdysteroids and embryogenesis in the desert locust 44.6 ng/egg for 20-hydroxy-ecdysone. Thus phosphate ions caused a 9O;b decrease in the amount of ecdysteroids released when the enzymic hydrolysis is carried out in acetate buffer. The effect of D-saccharic acid-1,4-lactone, a known competitive inhibitor of j3-glucuronidase (LEVVY, 1952; ROY, 1970) on the hydrolysis of ecdysteroid conjugates of the eggs of S. gregaria was determined to see if any inhibition was observed. Experiments were made on newly laid eggs and nine-day-old eggs (i.e. after blastokinesis) to compare the hydrolysis of the same amount of conjugates in the absence and presence of 1 mM inhibitor. We observed no diminution of the amount of ecdysteroids released from the conjugated form by the Helix pomatia digestive juice when the incubation medium contains D-saccharic acid-1,4-lactone 1 mM, a concentration which caused, under the same experimental conditions, complete inhibition of the hydrolysis of 10m3 M phenolphthalein glucuronide, the substrate with the highest known affinity for P-glucuronidase (SPENCER and WILLIAMS, 1951). The influence of the ionic strength on the rate of the hydrolysis of conjugates has also been studied. Different ionic strength buffered solution (i.e. acetate buffer 0.1. 1 and 1.8 M) were tested on the standard assay for the measurement of the P-glucuronidase, aryl sulphatase and acid phosphatase activity of the digestive juice of Helix pomutia. Acetate buffer 1.8 M was found to inhibit completely the fi-glucuronidase activity, to produce 89% inhibition of the arylsulphatase activity whereas it did not affect the acid phosphatase activity. The effect of 1.8 M acetate buffer was thus studied on the hydrolysis of the conjugates by comparing the amount of ecdysteroids released by the Helir pomatia enzymes in presence of 0.1 M acetate buffer (pH 5.2) and 1.8 M acetate buffer (pH 5.2). The results obtained showed that the efficiency of the hydrolysis is little affected (109,) by the higher ionic strength of the 1.8 M acetate buffer. The values for the 0.1 M buffer are: ecdysone = 124 ng/egg, 2-deoxyecdysone = 48.1 ng/egg, 20-hydroxy-ecdysone = 13.7 ngiegg whereas 1.8 M buffer gives 112 ng/egg for ecdysone, 43.6 ng/egg for 2-deoxy-ecdysone. 12 ng/egg for 20-hydroxy-ecdysone.

DISCUSSION When the large difference in the amount of conjugated ecdysteroids in S. gregaria eggs, between our GC and HPLC methods was discovered, it was important to find the reason for this difference and to provide corrected values. It was easily demonstrated that GC and HPLC determinations gave consistent results. and the error was eventually traced to incomplete hydrolysis of the ecdysteroid conjugates caused by enzyme inhibition by the phosphate buffer. Our first studies in this area had been on the conjugated 20-hydroxy-ecdysone excreted in the faeces of S. gwyctriu (WILSON and MORGAN, 1978). We found the acetate buffer, normally used with Helix pomatia juice for the hydrolysis of conjugates, caused problems with the conversion of 20-hydroxy-ecdysone to its trimethylsilyl ether (WILSON, 1979) (the acetate has a catalytic effect and causes the formation of more than one de-

651

rivative). Phosphate buffer caused some inhibition of the H. pomatia enzymes (JARRIGE and HENRY. 1952) but satisfactory conditions were found using a fivefold increase in the amount of enzyme, and these conditions were used in later work with the conjugates in eggs. However, either because of the different nature of the egg conjugates or because they are present in much greater quantity, the phosphate buffer method proved inadequate and caused an error. HOFFMAN et al. (1980) have shown in Locusta migratorin and DINAN and REES (1981a) in S. gregaria that 2-deoxy-ecdysone in an important ecdysteroid in the conjugated fraction in eggs. In GC, 2-deoxy-ecdysone elutes much earlier than the other ecdysteroids. and was not determined in our earlier work (GANDE and MORGAN, 1979). In the HPLC method, the only compounds found representing more than 19, of the total conjugates were ecdysone, 2-deoxy-ecdysone and 20-hydroxy-ecdysone. All three have been determined here. Free ecdysone and 20-hydroxy-ecdysone were determined in the original study, here ecdysone alone has been quantified, the amount of 20-hydroxy-ecdysone is on the limit of detection by HPLC with the quantity of eggs used, and the results did not differ greatly from those published earlier. A difference in the level of free ecdysone is also seen for the first five days between this and the previous investigation. Although we are not able to give any certain explanation, this discrepancy is probably attributable to differences of methodology. The amount of free 2-deoxyecdysone was below the limit of accurate detection (less than 5 ng/g). As much as 477 ng/egg of conjugated ecdysone was found in this study by HPLC in 12 day old eggs (the maximal titre) in contrast to 28.4 ng/egg the maximal value for the same compound in GANDE and MORGAN (1979). Although this is so widely different. the pattern of the titre (Fig. 3) is the same in both investigations. with the levels falling from oviposition to day 5 or 6 and then rising steadily to a maximum three days before hatching and then falling again. The levels we now record (Tables 1 and 2) are in good agreement with those reported for newly laid eggs of the same species by DINAN and REES(1981 a), for ecdysone, 2-deoxy-ecdysone and 20-hydroxy-ecdysone. However. these authors suggested that their values for free 2-deoxy-ecdysone and free 20-hydroxy-ecdysone were grossly underestimated, we did not find this to be so. The occurrence of polar conjugated ecdysteroids in eggs has been reported in several species of insects. Nevertheless the positive chemical identification of these compounds has not been established yet. They have been tentatively identified in larvae as esters of sulphuric and phosphoric acid or ethers of glucose or glucuronic acid on the basis of their electrophoretic behaviour and by enzymatic hydrolysis (KARLSON and SHAAYA, 1964; HEINRICH and HOFFMEISTER, 1970; WILLIG et uI., 1971; K~~LMAN rt a!.. 1973). More recently (HETRU et (I/., 1981) the first physico-chemical identification of ecdysteroid conjugates in newly laid eggs of Locusta migratoria has been reported. In this study we have tried to shed some light on the chemical nature of the conjugated ecdysteroids present in the eggs of S. gregariu by an indirect approach: namely by studying the effect. on the rate of the hydrolysis. of a selective inhibition of one of the

652

S. SCALIA and

E. D. MORGAN

and TRACEY. 1950). However the results obtained indicate the absence from the eggs of S. gregaria of ecdysteroids conjugated with glucuronic acid and suggested that the aryl sulphatase enzyme (measured in the assay) is not involved in the hydrolysis, but a sulphate or phosphate conjugate is possible. Since the first study on ecdysteroids titre in the eggs of insects a number of investigations have been carried out, chiefly by determining the identity and amount of hormones by bioassay. radioimmunoassay, gas chromatography, gas chromatography with mass spectrometry, or HPLC. However, the mass of data available seems to point to divergences more than to common patterns. Our results, showing that the majority (90%) of ecdysteroids exist in the embryo in the conjugated form, are in agreement with previous reports on the same species (GANDE and MORGAN. 1979; DINAN and REES, 1981a) and on other insect embryos (LAGUEUXef al.. 1977; HSAIO and HSAIO, 1979) whereas other authors have reported the presence mainly of free hormones (BULLIEREer al., 1975; DARN and ROMER,1976; IMBODEN et a/., 1978). Remarkable divergences can also be elicited on the Key physico-chemical nature and ratio of the different q Coqugated 20- hydroxyecdysone hormones in the embryo of insects. Ecdysone, 20-hyq Conjugated 2 - deoxyecdysone droxy-ecdysone and 2-deoxy-ecdysone both in the 0 Conjugated ecdyaone free and conjugated form have been detected in this investigation, ecdysone being the main hormone. Not Fig. 3. Levels of conjugated ecdysteroids in the developing only the presence of others ecdysteroids has been eggs of Schistocerca gregariu from oviposition to hatching. evinced (KAPLANISet al., 1973, 1975; HSIAOand HSIAO, Vertical bars indicate the range of results for conjugated ecdysone. 1979; LAGUEUXet al., 1979: DINAN and REES,1981a) but also the role of ecdysone as main hormone (OHNISHI et al., 1977; GANDE and MORGAN, 1979; enzymes contained in the H. pomatia juice: i.e. /?-glu- LAGUEUXet a/., 1979; DINAN and REES, 1981a) does not accord with the observations reported in other curonidase. studies (KAPLANISet al.. 1973, 1975: BULLIEREet al.. As also reported in a previous study (JARRIGEand 1976; IMBODEN er al., 1978; HSIAOand HSIAO.1979). HENRY, 1952) a high ionic strength (produced by The fluctuation of ecdysteroid titre during embryo1.8 M acetate buffer) has been shown here, using the genesis is very similar for the free (Fig. 2) and conjustandard assay, to inhibit completely the p-glucuronidase activity of the H. pomatia digestive juice. On the gated forms (Fig. 3) of the hormones and it is characterized by a decrease in the level of the ecdysteroids in other hand 1.8 M acetate buffer caused only a small the early stages of the embryonic development foldecrease of the efficiency of the hydrolysis of the conlowed by an increase after blastokinesis when the jugated ecdysteroids compared with 0.1 M acetate prothoracic glands have differentiated. Blastokinesis buffer, normally used as incubation medium. occurs between stages XVI and XVIII. according to D-Saccharic acid-1,4-lactone is known to be a competitive inhibitor of the /%glucuronidase (LEVVY,1952; the scheme of SHULOVand PENER(1963), that is, on ROY, 1970). Its high specificity has been confirmed by day 8 of development in our experiments. The single peak found here compares favourably with previously showing that the P-glucuronidase of the Helix pomapublished results (KAPLANISet al., 1973. 1975; BULtia digestive juice is powerfully inactivated. It lost LIEREef al., 1976; DORN and ROMER,1976; GANDE 95% of its activity, in the standard assay, with 1 mM and MORGAN,1979) although there is other evidence D-saccharic acid-1,4-lactone, whereas a concentration of 20mM did not affect the aryl sulphatase and acid either of a more complicated pattern with several peaks (IMBODEN ef al., 1978; LANGUEUXet al., 1979) or phosphate activity present in the digestive juice. Bearing in mind that the /?-glucuronidase is not specific of no remarkable variation of the hormone level during embryogenesis (DINAN and REES,1981b). It is not with respect to the aglycone (ROY, 1970) we have then possible to formulate any conclusive explanations of studied the effect of D-saccharic acid-1,Clactone on the role of ecdysteroids in embryonic development the hydrolysis of the conjugated ecdysteroids. The from the data so far available. However, the determiresults reported show that the p-glucuronidase comnations that have been made suggest that the rising petitive inhibitor did not influence the yield of the ecdysteroid titre in the later phase of embryonic enzymatic hydrolysis of the conjugates. growth is due to the synthetic activity of embryonic At this stage no conclusive proof can be formulated neuroendocrine glands. Also, the correlation between from the study of the influence of high ionic strength of the buffer and /%glucuronidase competitive inhibithe time of ecdysone peak (days 11-12 in this study) tor, on the efficiency of the enzymatic hydrolysis of and the time of apolysis of the second embryonic conjugates, because the H. pomatia juice contains cuticle and the deposition of the third cuticle (stage 28 such a large number of non-specific enzymes (HOLDEN in the study of SBRENNA-MICCIABELLI and SBRENNA

Ecdysteroids

and embryogenesis

which correspond to stage XXI of Shulov and and to day 12 in our development period), supports the hypothesis of embryonic moults being controlled by hormones in a manner similar to the control of larvae moults.

(1972). Pener,

Arknowledgemen~s-We thank Dr. VECCHIETTI (Simes. Milan) and Dr. D. H. S. HORN (CSIRO, Melbourne) for gifts of ecdysteroids and C. R. BIELBY for carrying out gas chromatography determinations. S. S. wishes to thank Stiftelsen Blanceflor Boncompagni-Ludovisi for a grant and the University of Keele for a Departmental Studentship.

REFERENCES BULLIERE F. and BULLIERE D. M. (1973) Action de l‘hormone de mue SLITla diffkrenciation des cellules tpidermiques d’un appendice d’embryon de Blatte isolt in culture in vitro. C. r. hehd. Shanc. Acad. Sci., Paris (D), 276, 2533-2539.

BULLIERE D.. BULLIERE F.. MAUCHAMP 9.. LAFONT R., DE REGGI M. and HIRN M. (1976) Etude des variations du taux d’hormone de mue au tours du dCroulement du deuxitme cycle de la vie embryonnaire chez une Blatte. C. r. hebd. Seanc. Acad. Sci. Paris (D) 283, 647-650. CHIHARA’C. J., PETRIW. H.. FRISTROM J. W. and KING D. S. (1972) The assay of ecdysones and juvenile hormone on Drosophila imaginal disks in vitro. J. Insect Physiol. 18, I 115-l 123. DINAN L. N. and REES H. H. (1981a) The identification and titres of conjugated and free ecdysteroids in developing ovaries and newly-laid eggs of Schisrocerca gregaria. 1. Insect

Physiol.

27, 51-58.

DINAN L. N. and REES H. H. (1981b) Incorporation in oivo of [4-‘4C]-ch10resterol into the conjugated ecdysteroids in ovaries and eggs of Schistocerca americana gregaria. Insect Biochem. 11, 255-265. DORN A. and ROMER F. (1976) Structure and function of prothoracic glands and oenocytes in embryos and last larval instars of Oncopeltus fasciatus Dallas (Insecta, Heteroptera). Cell Tissue. Res. 171, 331-350. FISHMAN W. H., KATO K.. ANSTISS C., and GREEN S. (1967) Human serum /l-glucuronidase: its measurement and some of its properties. Clinica Chim. Acta. IS, 435447. GANDE A. R. and MORGAN E. D. (1979) Ecdysteroids in the developing eggs of the desert locust. Schistocerca grrgaria. J. Insect Ph~~slol. 25, 289-293. HAC~ETA. (1977) In Traiti de Zoologie, Tome 8 (Ed. by GRASSE P.). pp. 2-387. Masson Paris. HEINRICH G. and HOFFMEISTERH. (1970) Bildung von Hormonglykosiden als Inaktivierungsmechanismus bei Cnlliphora erythrocephala. Z. Naturforsch. 25b, 358-361. HETRU C., KAPPLER C., Luu 9.. TSOUPRAS G., HORND. H. S. and HOFFMANN J. A. (1981) Biosynthetic pathway of ecdysone in Locusta migratoria ovarioles: in vitro studies with labelled precursors. Psysico-chemical studies on ecdysteroids conjugates in newly-laid eggs. Vth Ecdysone symposium. Gurten-Berne (unpublished). HOFFMANN. J. A., LAGUEUX M., HETRU C.. CHARLET M. and GOLTZENE G. (1980) Ecdysone in reproductively competent female adults and in embryos of insects. In Progress in Ecdysone Research (Ed. by HOFFMANN J. A.), pp. 431-165. Elsevier North Holland, Amsterdam. HOLDEN M. and TRACEY M. V. (1950) A study of enzymes that can break down Tobacco-leaf components. Biochum. J. 47, 407.-414.

HSIAO T. H. and HSIAO C. (1979) Ecdysteroids in the ovary and the eggs of the Greater Wax Moth. J. Insect Physiol. 25. 45-52.

653

in the desert locust

IMBODEN H., LANZREIN B., DELBECQUE J. P. and L~~SCHER M. (1978) Ecdysteroids and juvenile hormone during embryogenesis in the ovoviviparous cockroach Nauphoeta

cinerea.

Gen. camp. Endocr.

36, 638-635.

JARRIGE P. and HENRY R. (1952). Etude de I’activitt glucuronidasique du sue digestif d’Helix pomatia (L.) et de sa phenolsulfatase. Bull. Sot. Chim. biol. E34, 872-885. KAPLANIS J. N.. ROBBINS W. E., THOMPSON M. J. and DUTKY S. R. (1973) 26-Hydroxyecdysone: new insect moulting hormone from the egg of the tobacco hornworm. Science, Wash. 180, 307-308. KAPLANIS J. N.. DUTKY S. R., ROBBINS W. E.. THOMPSON M. J.. LIND~UIST E. L., HORN D. H. S. and GALBRAITH M. N. (1975) Makisterone A: a 28-carbon hexahydroxy moulting hormone from the egg of the tobacco hornworm. Science, Wash. 190. 681-682. KARL~ON P. and SHAAYA E. (1964) Der ecdysontiter Wahrend der Insectenentwicklung. I. Eine methode zur bestimmung des ecdysongehalts. J. Insect Physiol. 10, 797-804.

K~~LMAN J.. HOFFMANN J. A. and KARL~ON P. (1973) Sulphate esters as inactivation products of ecdysone in Locusta migratoria. Hoppe-Seyler’s 2. Physiol. Chem. 354, 1043-1048. KURODA Y. (1971) Effects of substances with ecdysone and juvenile hormone activity on the growth of embryonic tissues from D. melanogaster. Drosoph. inf: Serc. 46, 104. LANGUEUX M., HIRN M. and HOFFMANN J. A. (1977) Ecdysone during ovarian development in Locusta migratoria. J. Insect

Physiol.

23, 109-l

19.

LANGUEUX M., HETRU C.. GOLTZENE 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, 709-723.

LEVVY G. A. (1952) The preparation and properties of p-glucuronidase. Biochem. J. 52, 464-472. MATZ G. and HOFFMANN J. A. (1975) Titre d’hormone de mue et metabolisme de I’a-ecdysone radioactive au tours du diveloppement embryonnaire de Leucophaeu maderae. 3rd Co/l. Physiol. Insecles, Strasbourg. France. MIZUNO T. and OHNISHI E. (1975) Conjugated ecdysone in the eggs of the silkworm. Bombyu mori. Deuel. Growth Differ.

17. 219-225.

MUELLER N. S. (1963) An experimental analysis of moulting in embryos of Melanopus difirmrialis. Deal Biol. 8. 2X-240.

OHNISHI E.. OHTAKI T. and FUKUDA S. (1971) Ecdysone in the eggs of Bombvx silkworm. Proc. Japan Acad. 47. 413-415. OHNISHI E.. MIZUNO T., IKEKAWA N.. Aw \TA N. and SAKURAI S. (19771 Occurrence of r-ecdysone in the developing embryos of the silkworm. Bombyu mori. J. Insecr Physiol.

23, 317-319.

PASQUALINI J. R. (1967) Analysis and identification of steroid conjugates. In Steroid Hormone analysis, Vol. I (Ed. by CARSTENSEN H.). pp. 436-444. Marcel Dekker. New York. ROY A. 9. (1970) Enzymological aspects of Steroid Conjugation. In Chemical and Biological aspects of Steroid Conjugation (Ed. by BERNSTEIN S. and SALOMON S.), pp. 9&95. Springer-Verlag. New York. SBRENNA-MICCIARELLI A. and SBRENNA G. (1972). The embryonic apolyses of Schistocerca greguria (Orthoptera). J. Insect Physiol. 18, 1027-1037. SHULOV A. and PENER M. P. (1963) Studies on the development of eggs of the desert locust (Schistocerca gregarin Forskal) and its interruption under particular conditions of humidity. Anti-Locust Bulletin No. 41. Anti-Locust Research Centre, London. SOMMER A. J. (1954) The determination of acid and alkalme phosphatase using p-nitrophenyl phosphate as substrate. .4rn. J. med. Tec,hno/. 20, 244.

654

S. SCALIA and E. D. MORGAN

SPENCER B. and WILLIAMS R. T. (1951) Studies in detoxication. Spectrophotometric determination of /Cglucuronidase using p-chlorophenylglucuronide as substrate. Biothem.

J. 48. 537-546.

TAKAMI T. (1963) In vitro culture of embryos in the silkworm Bombyx mcri L. III. Moult of brainlesss embryos in culture. J. exp. Bid. 40. 735-739. WILLIG A., REES H. H. and GCODWIN T. W. (1971) Biosynthesis of insect moulting hormones in isolated ring

glands and whole larvae of Culliphoru. J. Insecf Phyvrol. 17, 2317-2326. WILSON I. D. (1979)Gas chromatographic analysis of ecdysteroids in the desert locust Schistocercu greguria. Ph.D. Thesis. Keele University. WILSON I. D. and MORGAN E. D. (1978) Variations in ecdysteroid levels in 5th instar larvae of Schistocrrcu grrguriu in gregarious and solitary phases. J. Insrcr Physiol. 24, 75 I-756.