Archives of Insect Biochemistry and Physiology 41:107–116 (1999)

© 1999 Wiley-Liss, Inc.

3-Dehydroecdysone Secretion by the Molting Gland of theCockroach, Periplaneta americana (L.)

Klaus Richter1*, Gustav-Adolf Böhm1, and Frank Leubert2

1Sächsische Akademie der Wissenschaften zu Leipzig, Forschungsgruppe Prof.Dr.H.Penzlin, Jena, Germany2Institut für Allgemeine Zoologie und Tierphysiologie der Friedrich-Schiller-Universität Jena, Jena, Germany

Prothoracic glands of Periplaneta americana were tested un-der in vitro conditions for their ability to release 3-dehydro-ecdysone (3DE) besides ecdysone. 3DE was identified by RIAafter conversion to ecdysone by a ketoreductase-containinghemolymph preparation as well as in HPLC fractions of incu-bation medium by an antiserum with affinity to 3DE and ecdys-one. The ratio of 3DE to ecdysone secreted into the culturemedium was found to vary between 2:1 and 7:1, depending onthe period in the last nymphal instar. Enzyme activity of 3b-ketoreductase showed fluctuations that coincided with thehemolymph ecdysteroid titre. Homogenates of prothoracicglands do not contain ecdysone but contain 3DE. Neverthelessthe glands release 3DE as well as ecdysone under in vitro con-ditions. Different sites where conversion of 3DE to ecdysonetakes place are discussed. Arch. Insect Biochem. Physiol.41:107–116, 1999. © 1999 Wiley-Liss, Inc.

Key words: 3-dehydroecdysone; ecdysone; ecdysteroids; 3b-ketoreductase;prothoracic gland; Periplaneta americana

Contract grant sponsor: Deutsche Forschungsgemeinschaft;Contract grant number: Ri-641/1-3.

*Correspondence to: Klaus Richter, Sächsische Akademie derWissenschaften zu Leipzig Forschungsgruppe Jena, P.O.Box100322, D-07703 Jena, Germany. E-mail: b6rikl@cnve.rz.uni-jena.de

Received 15 January 1999; accepted 29 January 1999

INTRODUCTION

Ecdysteroids are the main secretory productsof the molting glands in arthropods. Their titerin the hemolymph in coordination with that ofjuvenile hormone is essential in the regulation ofpostembryonic development and molting. Biosyn-thesis and release of ecdysteroids by the moltingglands of insects are regulated by neuropeptides(ecdysiotropins and ecdysiostatins) (Bollenbacher,1988; Gilbert, 1989, Hua et al., 1997) and, in someinsect species, for example in cockroaches, addi-tionally by innervation (Richter, 1993).

Originally, it was assumed that the moltinghormone ecdysone is the primary secretory prod-uct of the molting glands (Rees, 1985). Studies indifferent lepidopteran species and in other insects(Sakurai et al., 1989; Kiriishi et al., 1990; Smith,

1995), as well as in crustacea (Rudolph et al.,1992; Böcking et al., 1995), have provided evi-dence that, at least in the majority of these spe-cies, 3-dehydroecdysone (3DE) is the primaryecdysteroid released into the hemolymph by themolting gland. 3DE may function as a prohor-mone during the development of some arthropodspecies (Böcking et al., 1995; Oeh et al., 1998).

It has been documented that 3DE is rapidly

108 Richter et al.

converted to ecdysone in the hemolymph by a ke-toreductase (3-oxoecdysteroid-3β-reductase) (Blaisand Lafont, 1984) present in the hemolymph(Warren et al., 1988; Roussel, 1992; Chen et al.,1996; Nomura et al., 1996) and in other tissuessuch as the midgut (Webb et al., 1995) and eggs(Kelly et al., 1990). The ratio of 3DE to ecdysonesecreted by the prothoracic glands varies greatlyamong the investigated lepidopteran species be-tween 2.5:1 (Galleria mellonella) and 36:1 (Papilioxuthus) (Kiriishi et al., 1990). As an exception,the prothoracic glands in Bombyx mori larvae,produce predominantly ecdysone (Nomura et al.,1996). Surprisingly, no 3DE could be detected inthe prothoracic glands of larvae of the beetleTenebrio molitor and in the ring glands of the flySarcophaga peregrina (Kiriishi et al., 1990) andthe blowfly Calliphora erythrocephala (Budd etal., 1993). In the beetle Zophobas atratus (Aribiet al., 1997), 3DE was not detected neither in vivonor in vitro. In this insect, however, 2-deoxyec-dysone is secreted by the prothoracic glands andseems to serve as a prohormone.

Recently, Webb et al. (1995) and Chen et al.(1996) provided evidence that in last instar lar-vae of the cotton leafworm, Spodoptera littoralis,3β-ketoreductase exhibits peak activity late in theinstar. The significance of the 3β-ketoreductaseactivity late in the instar may be related to theproduction peak of the molting hormone. In theprothoracic glands of the last instar nymphs ofthe cockroach, Periplaneta americana, a 1:1 ratioof 3DE and ecdysone was reported by Kiriishi etal. (1990). In addition, they found very low 3β-ketoreductase activity in the hemolymph of P.americana nymphs.

In comparison with lepidopteran larvae, thelast nymphal instar of P. americana is quite long(30 days on average; Richter, 1992). Nothing isknown about the profile of the ratio of 3DE toecdysone during the last nymphal stage of thecockroach, nor of the profile of activity of 3β-ke-toreductase in the hemolymph. This study wasdesigned to consider these aspects.

MATERIALS AND METHODSAnimals

Prothoracic glands from last instar nymphsof laboratory-bred P. americana were used in all

the experiments. To make the determination oftheir developmental stage as precise as possible,the animals were selected within some hours af-ter molting into the last instar from mass cul-ture and kept in groups in piacryl containers withperforated lids (15 × 25 × 10 cm) under constantconditions (28°C, 50–60% relative humidity, 12:12light-dark cycle, water and food [standard rat pel-lets] ad libitum, with folded paper to providecover). The average duration of the last instarnymphs under these conditions was 30.5 ± 0.6days (n = 366).

Gland Preparation and Incubation

The prothoracic glands were dissected fromnon-anaesthetized nymphs under Ringer’s solution(Yamasaki and Narahashi, 1959) immediately af-ter decapitation. Unless otherwise described, indi-vidual glands were washed twice for 10 s each, oncein Ringer’s solution and once in the incubation me-dium to remove the hemolymph adhering to theglands. The glands were then divided into twohalves by cutting transversely with iridectomy scis-sors. This operation must be performed very care-fully to avoid compressing or stretching theglands. The two halves of the gland were trans-ferred separately into two Eppendorf tubes (1 ml)with 25 µl culture medium (Medium 199/Parker,plus 0.1% BSA). One half served as the controland the other as the experimental. When incu-bated for 2 h at 30°C, ecdysteroid production byboth the halves was nearly the same and with-out any significant differences (unpublished data).

Determination of 3-Dehydroecdysone andRadioimmunoassay

3DE was determined in the experimentalpreparation after conversion of the secreted 3DEto ecdysone by incubation with a 3β-ketoreduc-tase preparation and, subsequently determinationby RIA the ecdysone equivalents. After enzymaticconversion, the ecdysone content of the mediumincreased by an amount equivalent to the amountof 3DE. The amount of ecdysone in the controlpreparation served as the base line.

Ketoreductase solution was prepared by col-lecting hemolymph from 18–20-day-old last in-star nymphs on ice. In this period of the lastnymphal stage, the ratio of 3DE to ecdysone inthe hemolmph is relatively low, indicating a high

3-Dehydroecdysone in Periplaneta americana 109

level of enzymatic activity in the hemolymph.Hemolymph was diluted 2:1 with Medium 199containing 0.2 mM NADPH, centrifuged (15.000g,10 min, 4°C) and was added to the incubationmedium (final concentration 44 µM NADPH).

To convert 3DE to ecdysone in the incuba-tion medium, after an initial 2 h incubation pe-riod the prothoracic glands were removed fromthe medium, a cocktail of 20 µl ketoreductasepreparation was added to 10 µl of the incubationmedium and was incubated for 1 h at 30°C(Gelman et al., 1991). The medium of the controlhalf gland (10 µl) was incubated with 20µl Me-dium 199 containing 0.2 mM NADPH but with-out hemolymph for 1 h. Ecdysteroid RIA datafrom this preparation showed the amount ofecdysone secreted by the gland.

The difference between the RIA-detectableecdysone content in the medium after incubationwith the hemolymph preparation and the ecdysonecontent in the corresponding control half of thegland, indicated the 3DE content of the medium.

The ecdysteroid RIA data in the experimentalpreparations was corrected for the amount of ecdys-teroids present in the ketoreductase preparation.

To determine ecdysteroids in the prothoracicglands, the glands were washed immediately af-ter isolation twice, once in Ringer’s solution andonce in incubation medium, and the ecdysteroidswere extracted after sonication with methanol/0.1% TFA.

Every experimental series consisted of atleast 10 samples. The ratio of 3DE to ecdysonewas calculated as the mean (± SEM) of all theratios of samples in a series. The statistical sig-nificance of the differences was determined byvariance analysis (Mann-Whitney’s t-test) usingPrism Software (GraphPad, San Diego, CA).

Ecdysteroids in the culture medium or ingland homogenates were assayed quantitativelyby radioimmunoassay on a methanolic extract (ex-tracted twice with tenfold volume of 70% metha-nol/water) using two antisera with differentaffinities to different ecdysteroids. Serum 1(Eibisch et al., 1980) recognized ecdysone and 20-OH-ecdysone equally and does not show any af-finity to 3DE. Serum 2, a generous gift from Dr.Sho Sakurai, Kanazawa, Japan, recognized ecdy-sone and 3DE equally (Table 1). In experimentswhere 3DE was converted to ecdysone, serum 1

was used in RIA assays to determine the amountof ecdysone equivalents. Differential determina-tion of ecdysone and 3DE, respectively, in HPLCfractions was performed by the use of serum 2.Ecdysone was used as the standard; thus resultsin all RIA determinations are expressed as ecdys-one equivalents.

For determination of ecdysone titers in thehemolymph, 20 µl hemolymph was obtained byinserting a micropipette between tergits of the2nd and 3rd abdominal segments. Ecdysone wasassayed quantitatively by radioimmunoassay ofa methanolic extract (extracted twice with ten-fold volume of 70% methanol/water) using anti-serum 1.

Tritiated ecdysone (89 Ci/mM; New EnglandNuclear Corp., Boston, MA) was used as theradioligand.

RP-HPLC

To identify ecdysone and 3DE by RP-HPLCin the medium after incubation of the prothoracicglands (2 h), and in the glands, medium or glandhomogenates were extracted twice with 10 vol-umes of 70% methanol/water. After centrifugation,the supernatant was dried in a vacuum rotatoryevaporator, redissolved in 60 µl of 16% acetoni-trile/water, and subjected to RP-HPLC fraction-ation (System Knauer, Berlin, Germany). Theanalysis was carried out on a 250 × 4 mm I.D.RP-column packed with 10-µm particles of Spheri-sorb ODS-2, eluted at 1 ml/min with a lineargradient from 5 to 30% acetonitrile in water, con-taining 0.1% trifluoroacetic acid (TFA). The elu-ate was monitored by UV detection at 254 nm.Reference ecdysteroids (ecdysone and 3DE) werechromatographed before and after sample analy-sis. Fractions of 15 s duration were collected over40 min at room temperature. The ecdysteroid con-tent in the fractions was determined by RIA withserum 2.

Reference solutions of 3DE in methanol and

TABLE 1. Cross-Reactivity of Ecdysteroids to theSerum 1 and Serum 2 Antisera in Relation toEcdysone (ED-50 Ecdysteroid/Ecdysone)

Ecdysteroids Serum 1 Serum 2

Ecdysone 1 13-Dehydroecdysone 11.2 1.320-Hydroxyecdysone 1.1 n.d.

110 Richter et al.

methanolic extractions for the determination of3DE were stabilized by the addition of 0.1%TFA.3DE as a reference compound was a gift fromProf. René Lafont, Paris. Ecdysone was purchasedfrom Sigma, Deisenhofen, Germany. All solventswere HPLC grade.

RESULTSSecretion of Ecdysone and 3-DehydroecdysoneDuring the Last Nymphal Stage

During a 2-h incubation period 0.64 ± 0.07ng ecdysone (n = 60) was released by one half ofthe prothoracic gland from 23-day-old last instarnymphs. Exposure of the incubation medium af-ter removing the half-gland to a hemolymph 3β-ketoreductase preparation for 1 h resulted in anet increase of antiecdysteroid serum 1-detectableecdysteroid content to 3.0 ± 0.89 ng per half-gland(n = 14). In the medium of the half-glands actingas control, without addition of ketoreductasepreparation, the RIA detectable ecdysteroids didnot significantly change during the 1-h incuba-tion interval (0.69 ± 0.13 ng ecdysone per half-gland) (n = 15; P = 0.1753). After subtraction ofthe amount of ecdysone in incubation mediumwithout 3β-ketoreductase treatment from the

amount of ecdysone detected after treatment with3β-ketoreductase, the ratio of 3DE to ecdysonesecreted by the gland halves from a 23-day-oldlast instar nymph during 2 h was 3.2:1.

Exposure of the incubation medium from onehalf of a prothoracic gland for 1 h to a hemolymphpreparation, previously boiled for 2.5 min to in-activate the ketoreductase, resulted in an antiec-dysteroid serum 1-detectable ecdysteroid contentof 0.78 ± 0.17 ng ecdysone equivalents (n = 10).This is not significantly different from sampleswithout ketoreductase treatment (P = 0.8204).

The ratio of 3DE to ecdysone produced bythe prothoracic gland was not constant during thelast nymphal instar. This is due to variations inthe level of ecdysone as well as of 3DE secretedinto the culture medium by the prothoracic glandsfrom different periods of the nymphal stadium(Fig. 1). As was shown by Richter and Baumann(1997), ecdysone production by the prothoracicgland is low in the first half of the last nymphalstage. From day one to day 17 in this stage, theecdysone production did not exceed 1 ng per half-gland/2 h. Nevertheless, glands from the 7th daynymphs secreted twice the amount of 3DE asecdysone (1 ng:0.5 ng per half-gland/2h). Whereasthe ecdysone content of the secreted ecdysteroids

Fig. 1. Level of ecdysone and 3-dehydroecdysone (3DE af-ter conversion to ecdysone, RIA with serum 1, in ecdysoneequivalents), secreted by prothoracic glands from last in-

star nymphs of different ages during a 2-h incubation inter-val (M ± SEM, n = 8–10). Days without columns are notdetermined.

3-Dehydroecdysone in Periplaneta americana 111

remained fairly constant up to the 17th day, the3DE level increased on day 10 to 500% of theecdysone level. The 3DE level decreased betweenday 10 to 17 and the ratio of 3DE to ecdysone dur-ing this period ranged between 6:1 and 7.5:1. Onabout the 20th day, there is a small peak of ecdys-one production in last instar nymphs (Richter andBaumann, 1997). In the samples from day 20nymphal glands, ecdysone levels in the mediumwere higher (about 2 ng per half-gland) and the 3DElevel in the incubation medium exceeded the levelof ecdysone by about 200%. In the last one third ofthe nymphal stage, before the rise of the main peakof ecdysteroid production, the 3DE level increasedto 400– 500% of the level of ecdysone (Fig. 1). Im-mediately before the imaginal molt on day 30, theecdysone synthesis reached a maximum (about 6ng per half-gland), and the ratio of 3DE to ecdys-one decreased to 1.5:1 (Figs. 1 and 2).

Identification of 3DE and Ecdysone by HPLC

Ecdysone and 3DE as reference standardswere eluted from the column in an acetonitrile/water gradient with a difference in the retentiontimes of 3 min (Fig. 3). In HPLC separation ofmethanolic extracts of the medium in which pro-thoracic glands were incubated for 2 h, the ecdy-sone and 3DE peaks eluted at the same retention

times as the corresponding reference compounds(Fig. 3A).

In the experiments demonstrating the ratioof ecdysone to 3DE in ecdysteroid secretion byprothoracic glands directly, medium was nottreated with hemolymph. However, 3β-ketoreduc-tase is present in hemolymph as well as inhemocytes (Nomura et al., 1996). In order to re-move hemocytes adherent on the outer surface ofthe prothoracic glands to a larger extent, someexperimental glands were washed more inten-sively than usual. These glands were washed 6times for 10 s each, once in Ringer’s solution and5 times in incubation medium. After in vitro in-cubation (2 h) of intensively washed glands, ecdys-teroids secreted into the incubation mediumshowed the same ratio of ecdysone to 3DE as wasfound in incubations of less intensively washedglands (Fig. 3A).

In homogenates of the prothoracic glands, noecdysone was detected by radioimmunological as-says on the HPLC-eluated fractions, but 3DE andsome unidentified compounds of lower polaritywere detected (Fig. 3B).

Activity of 3 b-Ketoreductase

The enzymatic activity of 3β-ketoreductase inthe hemolymph preparation was tested on the re-ference compound 3DE as a substrate and the pro-duct was monitored on HPLC. 3DE (75 ng) wasincubated for 30 and 60 min, respectively, with 300µl of hemolymph preparation containing ketoreduc-tase. After incubation for these periods, the decreasein 3DE due to its conversion to ecdysone was de-termined in the corresponding fractions of HPLCby radioimmunological assay.

During the 30- and 60-min incubations, theRIA detectable amount of 3DE continuously de-creased while the level of ecdysone increased (Fig.4). This result shows that the enzymatic activityin the hemolymph preparation is really a 3β-ke-toreductase, which is active in the conversion of3DE to ecdysone.

The variations in the amount of 3DE pro-duced in vitro by prothoracic glands from last in-star nymphs of diverse ages suggest changes inthe hemolymph 3β-ketoreductase activity at dif-ferent periods of the nymphal instar.

In order to determine the changes in 3β-ke-toreductase level, hemolymph preparations from

Fig. 2. Ratio 3-dehydroecdysone/ecdysone (3DE:E), secretedby prothoracic glands in vitro at different periods in the lastnymphal stage (M±SEM, n = 10–20).

112 Richter et al.

Fig. 3. Identification of ecdysone (E) and 3-dehydroecdysone(3DE) by RP-HPLC in an acetonitrile/water gradient (5–30%). Radioimmunological determination in the fractionsevery 15 s, as compared by the marker compound. A: Blackcolumns, methanolic extraction of medium after 3-h incuba-

Fig. 4. Ecdysone (E) and 3-dehydroecdysone (3DE) (blackcolumns), resulting after 30- and 60-min incubation inter-vals of 75 ng 3DE with ketoreductase preparation from

hemolymph, identified by the marker compounds. Determi-nation of ecdysone and 3DE with serum 2 in ecdysoneequivalents.

tion of 30 prothoracic glands from the 23rd day of the lastnymphal instar. B: Black columns, methanolic extraction ofa homogenate of 25 prothoracic glands from the 23rd day ofthe last nymphal instar.

3-Dehydroecdysone in Periplaneta americana 113

different periods of the last nymphal instar (iden-tical volumes 20 µl), were examined for their en-zyme activity in converting a standard amountof 3DE (0.75 ng) to ecdysone. The enzyme activ-ity was quantified by RIA determination of theecdysone, resulting from the conversion of 3DE.

Up to the 10th day of the stadium, the en-zyme activity was extremely low (basic level). Af-ter the 10th day, it increased steadily up to the20th day. The main increase in enzyme activityoccurred during the last few days of the nymphalinstar. The variation in the hemolymph ketore-ductase activity is in accordance with the changesin ecdysone titer in the hemolymph during thelast nymphal instar (Fig. 5).

DISCUSSION

Arthropod molting glands are distinguishedby the major types of their secretory ecdysteroids.In some insect species, like Locusta migratoriaand C. erythrocephala, ecdysone is the majorsecretory product, in others (e.g., Manduca sexta),3DE, and in Crustacea, ecdysone and 25-deoxyec-dysone (Lafont, 1997).

As shown in the experiments reported here,

using the indirect method of enzymatic conversionas well as HPLC, the ecdysteroid release from theprothoracic gland in P. americana is a mixture of3DE and ecdysone. The ability of the prothoracicgland of P. americana to produce significantly more3DE than ecdysone is shared with several lepi-dopteran species like Heliothis zea, M. sexta, Papilioxuthus, Pieris rapae, and Trichoplusia ni (Kiriishiet al., 1990; Lafont, 1997). At all time points exam-ined in the last instar of the cockroach, the amountof 3DE released by the prothoracic gland exceededthat of ecdysone.

The largest differences in 3DE and ecdys-one content (6–8:1) produced by the prothoracicgland are in the middle of the last nymphalstage (14th to 17th day, near the head criticalperiod; Richter et al., 1995), and after the 25thday (before the main peak of ecdysteroid pro-duction; Richter and Böhm, 1997). In the lastinstar larvae of S. littoralis, the ratio of 3DEto ecdysone at the end of the larval stage is 4:1(Chen et al., 1996). In a xanthid crab, Menippemercenaria, secretion of 3DE always exceedsthat of another secretory ecdysteroid, 25-deoxy-ecdyone with a ratio of 14.4:1.9 in de-eyestalkedcrabs (Rudolph et al., 1992).

Fig. 5. Activity of 3β-ketoreductase and ecdysone titer in the hemolymph (determinationwith serum 1 in ecdysone equivalents) during the last nymphal instar (M ± SEM; n = 10–15).

114 Richter et al.

In the only previous investigation on P.americana (Kiriishi et al., 1990), a ratio of 3DEto ecdysone of 1:1 was found in incubations ofprothoracic glands. Although the authors em-ployed the same principle of conversion and de-termination, their results are at variance withours. The reason for this difference may be thedifferent antisera. Furthermore, the age of thenymphs in the stadium was not specified andhence the results may not be comparable.

3DE is an intermediate compound in themetabolism of ecdysteroids. A cytosolic oxygen-dependent oxidase catalyses the formation of3DE from ecdysone in different insect tissues(Koolman and Karlson, 1978; Weirich, 1989).3DE can be reduced to 3-epi-ecdysone by 3α-reductase as has been reported based on inves-tigation on cytosolic enzyme preparations fromthe midgut (Milner and Rees, 1985). The for-mation of this endproduct is irreversible. Theconversion from 3DE to ecdysone is catalyzedby 3β-ketoreductase (Webb et al., 1995).

The enzyme requires NADPH as a cofactor.Besides hemolymph, 3β-ketoreductase activityhas also been observed in the brain, midgut, andproctodaeum in Ostrinia nubilalis, M. sexta, andCancer antennarius (Warren et al., 1988; Gelmanet al., 1990; Spaziani et al., 1989). In prothoracicgland cells, the titer of ecdysone oxidase was de-scribed as being very low (Gilbert, 1989). Othertissues or fragments were not present in our in-cubations. Therefore, the ratio of 3DE to ecdys-one observed in our studies reflects the secretionby the prothoracic glands and is not affected byproducts from other tissues.

However, the reason for the secretion ofecdysone as well as of 3DE by the prothoracicgland, at least under in vitro conditions, remainsunclear. In the prothoracic gland cells, ecdysonecould not be detected. The secretion of ecdysoneas well as of 3DE was also not changed after re-moving hemocytes, possibly adherent to the outersurface of the gland cell membrane to a large ex-tent, by washing of the glands more intensively.The main ecdysteroid in the prothoracic glandsof P. americana nymphs is 3DE. It is conceivablethat 3β-ketoreductase is bound to the outer sur-face of the gland cell membrane, so that 3DE ispartially converted to ecdysone while passingthrough the membrane. Nevertheless, the high-

est enzyme activity is in the hemolymph. In dia-lyzed midgut cytosol of S. littoralis, 3α- as wellas 3β-reductase activity was detected throughoutthe last larval instar (Webb et al., 1995). No ke-toreductase could be seen in the prothoracicglands of O. nubilalis (Gelman et al., 1989). Fur-thermore in S. littoralis no conversion of ecdys-one and 3DE by the prothoracic gland could bedetected (Chen et al., 1996).

The profile of 3β-ketoreductase activity inthe hemolymph preparation from P. americananymphs suggests that this enzyme activity is de-tectable throughout the last nymphal instar, butit shows maxima at different times. Peaks of en-zyme activity coincide with periods of higher rateof secretion by the gland and higher ecdysonelevel in the hemolymph (Richter and Baumann,1997). This correlation holds true for the profileof 3β-ketoreductase in the midgut cytosol of thelast larval instar of S. littoralis (Webb et al., 1995;Chen et al., 1996). Thus, 3DE secretion by theprothoracic gland as well as 3β-ketoreductase ac-tivity in the hemolymph of P. americana nymphsare related to the periodicity of production of themolting hormone. The prothoracic gland releasesmainly 3DE as a prohormone. The required levelof molting hormone is produced in peripheral tis-sues by the enzymatic conversion of 3DE to ecdys-one and ecdysone to 20-hydroxyecdysone.

Obviously, at least in P. americana nymphs,there are two different sites where conversion of3DE to ecdysone takes place: the outer surface ofthe prothoracic gland cells and, predominantly, inthe hemolymph. The supposition is that 3β-ketore-ductase activities in the prothoracic gland and inthe hemolymph are regulated independently. Infavour of this assumption are the various levels ofecdysone and 3DE released by the gland under invitro conditions on the one hand, and the level ofecdysone and 3β-ketoreductase activity in thehemolymph on the other hand, at different periodsof the nymphal stadium. The low level of ecdysonein the hemolymph, as well as in the incubation me-dium around the 10th day of the instar coincideswith the low 3β-ketoreductase enzyme activity inthe hemolymph, though 3DE secretion is quite highin this period. The function of 3DE in this period ofthe larval life is unclear.

On about the 16th day, the ratio of ecdysone

3-Dehydroecdysone in Periplaneta americana 115

and 3DE secreted in vitro remains unchanged fromday 10, but the hemolymph titer of ecdysone in-creases parallel with the enzyme activity. On the20th day, under in vitro conditions, i.e., withouthemolymph, the gland releases more ecdysone thanthe glands from 10- to 17-day nymphs. The ratio of3DE to ecdysone in vitro and the enzyme activityin the hemolymph do not appear to be correlatedduring the last period of the nymphal stage. Themechanisms responsible for the regulation of theenzymatic conversion of 3DE to ecdysone at the twosites remain unknown.

ACKNOWLEDGMENTS

We greatly appreciate the generous gift of3-dehydroecdysone from Prof. René Lafont (ÉcoleNormale Supérieure, Départment de Biologie,Paris) and of an ecdysteroid antiserum from Dr.Sho Sakurai (Faculty of Science, Department ofBiology, Kanazawa). We thank Mrs. Gisela Radtkefor her valuable technical assistence.

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