Investigations on ecdysteroids and juvenile hormones and on morphological aspects during early embryogenesis in the ovoviviparous cockroach Nauphoeta cinerea

INVESTIGATIONS ON ECDYSTEROIDS AND JUVENILE HORMONES AND ON MORPHOLOGICAL ASPECTS DURING EARLY EMBRYOGENESIS IN THE OVOVIVIPAROUS COCKROACH NA UPHOETA CINEREA H. Division

of Zoophysiology.

IMBODEN

and B. LANZKEIN*

Zoological Institute. University CH-3012 Bern, Switzerland

of Bern. Engehaldenstrasse

6.

early embryogenesis. which is from ovulation (day 0) until dorsal closure (day 19). the quantity of free and conjugated ecdysteroids in the egg cases. as measured bq radioimmunoassay (RIA). increases. Thin-layer chromatography (TLC) and high-performance-hquid-chromatography (HPLC) analyses combined with RIA suggest that 20-hqdrouy-ecdysone is the predominant ecdlsteroid. Hydrolysis of the highly polar products of day-0 and day- 17 eg_ecases by Helix ~mmrrri enzymes indicates the presence of some conjugates of 20-hydroxy-ecdysone. hydrolyzable under these conditions. However. important quantities of RIA-reactive highly polar products are not hydrolyzed particularly in da)-17 egg cases. These results demorstrate that the highly polar products of day-0 egg cases are qualitatively as well as quantitatively different from the highly polar products of day-17 egg cases. Morphological investigations show that the peak of 20-hydroxy-ecdysone at the time of the dorsal closure coincides with the synthesis of an embryonic cuticle. LJsing the Gulleriu wax test only traces, or no juvenile hormone activity could be detected in embryos during the entire period of early embryonic development. Morphological investigations of the brood sac suggest that this organ 1s very important to facilitate the initial uptake of water into the eggs. Thereafter the embryos can develop independently of the female when kept in a humid environment. &J>, UOx/ frtci~,~: Embryonic development. ecdystcroids. cuticle synthesis. brood sac Abstract-During

endocrinology ovoviviparous

INTRODUCTION THE PKESENCE of ecdysteroids in adult females has been reported for several insects (for a review see HOFFMANN et a/., 1980). In Locusta migratoria it has

been shown that ecdysteroids are synthesised at the end of the oscyte-maturation cycle in the follicle cells of the terminal obcytes and then pass into the oijplasm (LAGUEUX et al., 1977). Later the majority of these ecdysteroids can bc found in newly laid eggs as conjugates (HOFFMANN et al.. 1980). Reports on the presence of ecdyster,sids in newly laid eggs have been published also for B#w$~.Y nzori ( OHNISHI et al., 197 1. 1977a. 1977b; MRUNO and OHNISHI, 1975). for Marduca se.ytu (KAPLANIS et al.. 1973. 1975). for Otlcopeltus jhsciatu;, (Dam and ROMEK, 1976). for Galleriu nwllonella (HSIAO and HSIAO, 1979) and for

Schistocrrca grrgaritr (GANIE et cd., 1979; DINAN and REES, 1981). In Locusta it has been postulated by LAGUEUXer al. (1977) that the ‘maternal ecdysteroids’ produced in the ovaries are destined for the embryo to be used during early phase:. of embryogenesis and that in the older embryo Ihe prothoracic glands produce ecdysone. As concerns the function of these ecdysteroids in Locmta, it has been shown that. during embryogenesis, each peak in ecdysteroid concentration is correlated with the onset of the deposition of a cuticle ( LAGUEUX et al., 1979). In

our

laboratory.

*To whom correspondence

we

have

investigated

should

bc addressed.

the

of embryonic development in the Nauphoeta cinvreu cockroach (IMBODEN rt al., 1978). In this insect the embryonic period of 37-40 days occurs within the brood the female, and we have found in embryos

sac of

stagespecific quantities of free ecdysone and 20-hydroxyecdysone as well as juvenile hormone. Ecdysteroids have been observed in small amounts at the time of the dorsal closure (day 18) and in higher amounts around day 28 and 32 when also large quantities of juvenile hormone-active material have been measured (days 27 and 33). Gas-liquid chromatography-mass spectrometry (glc-ms) analysis of the ecdysteroids at day 32 have indicated the presence of ecdysone, 20-hydroxyecdcsone (which is the predominant ecdlsteroid). 26-hydroxyecdysone, and probably 20,26-dihydroxyecdysone. We still know very little of the function and the site of origin of these hormones. As concerns the late ecdysteroid peaks which are paralleled by juvenile hormone peaks we suppose that they are correlated with the deposition of the first larval cuticle (IMBODEN. unpublished). We have found that embryos isolated from the mother at the time of dorsal closure, (this is after the first uptake ofwater has occurred) are able to develop and show similar ecdysteroid titres as embryos developing within the brood sac of the female (IMBODEN~~al., 1978). This indicates that embryoscan produce their own hormones after dorsal closure has occurred. Histological investigations have revealed that the corpora allata and prothoracic glands are differentiated after dorsal closure. However, the first ecdysteroid peak (day 18) is observed before the

differentiation of the prothoracic glands, indicating another site of origin for these ecdysteroids. When egg cases are removed from the mother directly after ovulation the embryos never develop, which suggests that the female is very important for the completion of early embryonic development. In the present work, we report upon investigations of the early phase of embryogenesis from ovulation until dorsal closure. We measured and characterised the free ecdysteroids as well as the highly polar products. which is the fraction containing the conjugates. We have also made careful morphological studies in order to find out whether the ecdysteroid peak at dorsal closure is correlated with the deposition of cuticle. In addition. we investigated the role of the female and report some morphological examinations of the egg case surface and the brood sac.

MATERIALS

AND METHODS

Nauphorta cinerea were kept at 26’ C and 60”,,, r.h. on flakes of dog food and water at a photoperiod of I2 hr. The juvenile hormone-dependent oiicyte maturation lasts 12 days. Between 36 and 40 odcytes are then packed (after ovulation) into an egg case, which is first extruded and afterwards retracted into the brood sac. Here the eggs are incubated until the larvae hatch 3740 days later. The dorsal closure occurs around day I9 after ovulation. Eutructiorl procedures Egg cases were homogenized directly in 65”, methanol/water for extraction of ecdqsteroids and in ethyl acetatejethanol 5:1 (v/v) for extraction of juvenile hormones. Haemolymph of pregnant females was collected after cutting off a leg. after cooling the insects in ice water. The extraction procedure was the same as for the egg cases. Brood sacs were pressed out and cut away from the pregnant females, washed in distilled water and extracted in 65”, methanol/water. Pur$cation

ur7d

quant$ication

oj’ecdysteroids

The 65”,, msthanol!water extracts of whole egg cases. haemolymph of pregnant females and brood sacs were dryed on a rotatory evaporator and purified by thin layer chromatography (TLC) using 1 x diisopropyl-ether and 2 x chloroform/ethanol In some experiments 4 96” <,. SO/20 as solvents. different zones were scraped off for ecdysteroid determinations by radioimmunoassay (RIA): Zl. the highly polar products from the origin up to the 20hydroxy-ecdysone zone: 22, the 20-hydroxy-ecdysone zone; 23. the ‘ecdysone’ zone: 24. the low polaritS products (after the ecdysone zone up to the front). In other experiments the TLC-plates were subdivided into smaller bands and eluted in methanol. Every time, ecdysone and 20-hydroxy-ecdysone were used as references. High performance liquid chromatography (HPLC): TLC-purified extracts were injected into 2 different high pressure liquid chromatographs: (a) System I:

Model Du Pont 848, equipped with a variablewavelength U.V. detector (Perkin-Elmer) using a reverse phase column Zorbax ODS (4.6 mm in diameter and 24 cm long; DuPont) and methanol; water 50:50 (viv) as solvent. (b) System II: Model Waters Associates M 6000 A pump, equipped with Waters Associates M 440 spectrometer (254 nm) using a reverse-phase C I8 column (Merck RP 18) with a solvent system methanol/water 50:50 (v/v). As references we used ecdysone and 20-hydrosy-ecdysone from Simes (Milan. Italy). 2-deoxyecdqsone and 2-deoxy-20-hydroxy-ecdysone from Dr. D. H. S. HOKN fMelhourne) and 20,26-dihydro\-ecd~sone from Dr. J. N. KAPLASIS (Beltsville). RlA: After TLC-purification or after HPLC separation the different zones or fractions were assayed in a RIA which was carried out according to the method ot BORSTand O’CONNOR (1974) and HORX er (I/., (1976). Ecdysone and 20-hydroxy-ecdysone used as standards for the RIA were purchased from Simes (Milan. Italy). All results are expressed as ng of ecdysone equivalent with the only exception for the 20-hqdroxq-ecdysone zone and 20-hydroxy-ecdysone fraction, respectivel). For these the results are expressed as nanograms of 20-hydroxy-ecdysone equivalent. Antibodies H I-2 or H II-I from Dr. J. D. O’CONNOR were used with a twofold and five-fold higher sensitivity respective11 for ecdysone than for 20-hydroxy-ecdysone. Pur$cation

arld quant$cutiorl

c!f .jurer~ik

hotnronr

After extraction in ethyl acetatel’ethanol. 5.1 (v/v) extracts were purified by TLC (6”,, ethyl acetate, benzene) as described b> LA’V~REIUc/ tr/. (lY75). For determination of juvenile hormone activity the Galleria wax test was used (DE WILDE et al.. 1968). Under our conditions I Galleria unit corresponds to 8 pg juvenile hormone 111 (Calbiochem). Erlzymutic IrJdrolysis

of

polar products

After TLC purification the origin-zone (Zl) was extracted with methanol. These highly polar products were then incubated in a 50 mM acetate buffer at pH 5.0 for I8 hr at 37’C with [1-l)-glucuronide glucuronhydrolase and Arylsulphatase (Sigma, U.S.A.: Partially purified powder from Helispomutiu. Type H-L) whilst shaking. This product showed no immunoreactive material in our RIA. After incubation the samples were purified by TLC or HPLC. Fixation

o/ embryos

md histologic

Egg cases were tixed in alcoholic Bouin’s solution for 24 hr and afterwards transferred into 70”, ethanol. Using a dissecting microscope we prepared the embryos in 50”,, glycerol and put them on a slide. There the embryos were stained with haematoxylin according to the method of DELAFIELI) (see ROMEIS. 1968). After washing with water, dehydration was performed through a graded ethanol series. Then the embryos were incubated in xylene and embedded in a transparent glue (Eukitt). The brood sacs were fixed in alcoholic Bouin’s solution for 24 hr and afterwards transferred into 70”,, ethanol. Dehydration was performed through a graded ethanol series and the fixed tissues were embedded in paraplast. Sections were cut at 5 {trn and were stamed m haemytosylm according to the method of DELAFIELI) (see ROM~IS, 1968).

39

5d

6d

7d

8d

10d

9d

haad th

abd

I d

mm lld

12d

13d

14d

15d

16d

lfd

18d

Fig. 1. Early embryonic development. (abd) abdomen, (ant) antenna. (md) mandible. (mx,J maxillae. pz, pA) legs. (pleur) pleuropodium. (proct) proctodaeum. (stem) stomodaeum, (th) thorax.

(p,,

Fig. 7. Inner surface Fig. 8. Egg-case

surface,

of a 14-day-old showing

the imprints

Fig. 9. Top of a brood-sac Fig. 10. Openings Fig.

brood

1I. Opening structures

of the brood

papilla

at the top of a papilla

sac with papillae

with canals

of a 14-day-old

on the top of a papilla

(scale bar: 50 pm).

sac papillae

(scale bar: 50 pm).

(scale bar: 7 pm). brood-sac

of a O&-old

brood

(scale bar: 4 bm). sac (scale bar: 4 /Irn).

Earl> embryogenesis

in the ovoviviparous

RESULTS Embryonic development The embryonic development of Nauphoeta cineren lasts about 37-40 days and dorsal closure takes place around day 19. Em bryogenesis can thus be subdivided into early embryogenesis (from ovulation until dorsal closure) and late embryogenesis (from dorsal closure until hatching). The different developmental stages of early embryogenesis are shown in Fig. 1. At day 5 after ovulation. outgrowths at the cephalic and caudal ends are recognizable. At day 7. the segmentation of the germ band becomes clearly visible, and a day later rudiments of appendages appear in the cephalic and the thoracic regions. At day 10, all segments become distinguishable and the pleuropodia begin to grow from the first abdominal segment. At day 11, the caudal end of the embryo begins to fold ventrally leading to dorsal c.osure at day 19. The pleuropodia still exist after dorsal closure, but with our preparation method the long filaments broke away. Analysis cases

qf juvenik

hormone arzd ecdysteroids

in egg

Egg cases of difi‘erent age were collected between ovulation and dorsal closure and investigated for the

cockroach

Nucc$~wta

c~b~r~~/

presence of juvenile hormone and ecdysteroids. Only traces or no juvenile hormone activity could be detected throughout early embryogenesis using the Galleria wax-test. For ecdysteroid analysis, the TLC plate was divided here into 4 different zones which were analyzed separately (Fig. 3). In zone 1 with the highly polar products, small amounts of RIA-active material were detected from ovulation until about day 8 or 9. On day 10 more RIAactive material was detectable and was found to increase continuously until dorsal closure. In zone 2 into which 20-hydroxy-ecdysone would migrate, an increase in RIA-active material could be found after day 11 and a sharp peak was observed on day 18, immediately before dorsal closure. In O-dayold egg cases about 54 pmol 20-hydroxy-ecdysone equivalent/g egg case were measured while in 1g-dayold egg cases about 1.4 nmol 20-hydroxy-ecdysone equivalent/g egg cases were found. In zone 3 corresponding to ecdysone. smaller quantities of RIA-active material compared to zone 2 were measured and again the amount of RIA-active material was found to increase after day 11 and to show a peak on day 18. In O-day-old egg cases we found about 4 pmol ecdysone equivalent/g egg case and in 18-day-old egg cases about 330 pmol ecdysone


HPP

z2x----x I3 zzo .._...... 0 o(

OVUhtiifl

41

clOE.al

ClOSUre

Fig. 2. RI.4 activity in egg cases in zones 1. 2 and 3 from ovulation until dorsal closure. The results are expressed for zone 1 and 3 in ng ecdqsone equivalents/egg case and for zone _7 in ng ?O-hydroxy-ecdysone equivalents/egg case. Each symbol represents one determination (4 egg cases were pooled).

H. thlBOl)I:\ ANI) B.

41 Table

I. Comparison

of free ecdyateroids

LAhZKLli\

in newI)-laid

egg

of different

insects

free X-hgdroxy-

ecd.eq./egg “9

case

TLC

100

30

20

10

0

Fig. 3. EcdJsterod pattern in O-day-old (black bars. aupernnposed) and In I7-Jay-old (white bars) eg cases after TLC separation (Zone 5 expressed in 20-hydroxy-ecdysone

equivalents. all other zones in ecdysone equivalents). equivalent/g egg case. In zone 4 with the low polarity products only traces of ecdysteroids were detected from ovulation until dorsal closure (data not presented in Fig. 2). If we compare quantitatively the zones 1,2 and 3 we can see that before day 8 only small amounts of RIA activity were detected. Simultaneously with the beginning of the uptake of water into the egg cases on day IO (IMBODEN et al., 1978) the quantity of RIAactive material was found to increase in all 3 zones. We then concentrated on 2 stages during early embryogenesis, namely O-day-old egg cases and l7day-old egg cases. Several times the data given in Fig. 2 could be confirmed. In order to investigate more precisely the identity of the ecdysteroids we prepared large pools of the 2 stages for more detailed TLC analysis (Fig. 3) and HPLC analysis (Fig. 4) as well as for further characterization of the highly polar products (Figs. 5 and 6). For the Figs, 3,5 and 6 results from the same egg case pool are presented. In Fig. 3 we can see the results where the TLC plates were divided into 13 zones. In O-day-old egg cases, only small quantities of RIA-active material were found whereas

free rcdysone (pm01 cq.,gl

ecdysone (pm01 eq.:gl

6500 5657

92 IO4

X50

60

600

5

6

56

in 17-day-old egg cases high amounts of RIA activity were found in the zones corresponding to highly polar products. 20-hydroxy-ecdysone and ecdysone. The results of an HPLC analysis performed according to System II is shown in Fig. 4. A comparison with the retention times of 5 different ecdysteroids strongly indicates the presence of particularly highly polar products together with 20hydroxy.-ecdysone and ecdysone. In the following, RIA-reactive material eluting with the retention time of 20-hydroxy-ecdysone in HPLC or comigrating with the same in TLC will be called 20-hydroxy-ecdysone. The same experiment was repeated several times using HPLC System I, and the data given in Fig. 4 could always be confirmed. In addition, Figs. 3 and 4 also suggest the presence of immunoreactive products less polar than ecdysone. The highly polar products were then further analyzed and compared between day-0 and day-l 7 egg cases. First we incubated the quantity of highly polar products (RIA-active material) from the zones I corresponding to one O-day-old and to one 17-day-old egg case (see Fig. 3) with buffer only or with Hrlis enzymes, and measured the ecdysteroid contents after TLC separation into 8 zones (Fig. 5). We can see that the highly polar products of day 0 as well as day 17 consist to a large extent of 20-hydroxy-ecdysone conjugates (confirmed by HPLC) which can be hydrolyzed by the Helix enzymes. However, remarkable quantities of highly polar products especially in day-17 egg cases remain in zone 1 after Hdis treatment. Additional incubation of the zones 1 with He/i.u enzymes did not result in a further increase of free ecdysteroids demonstrating that the RIA-active material of zone 1 consists of different types of compounds and is different in day-0 and day-17 egg cases, This result was confirmed in several independent experiments. The highly polar products were further investigated in the following experiment. Here we incubated the same quantity of RIA-reactive highly polar products from day 0 (about 2 egg-case equivalents) and day 17 (about 0.04 egg-case equivalent) with the same amount of hydrolases from Helix ponu~tia (Fig. 6). The RIA results after TLC separation demonstrate that the highly polar products in day-0 egg cases consist to a comparatively much larger extent of Helix hydrolyzable 20-hydroxy-ecdysone conjugates than day-l 7 egg cases. As before, additional incubation of the remaining highly polar products did not result in a

Early embryogenesis ecd.eq.legg

cas.e

“9

in the ovoviviparous

cockroach

Nauphorta cinrrea

43

HPLC

60

a:

ZO-26-dihydroxy-ecdysone

b:

ZO-hydroxy-ecdysone

c: d:

ecdysone 2-dewy-2V-hydrnxy-ecdysone

e:

2-dewy-ecdysone

/ I

in,

Fig. 4. Ecdysteroid pattern in O-day-old (black bars, superimposed) and in 17-day-old (white bars) eggcases after HPLC separation (System II). (Fraction 10 expressed in 20-hydroxy-ecdysone equivalents, all other fractions in ecdysone equivalents.) ecd

eq /egg

case

Y 90 40

30

20

10

0

B

1

front

Fig. 5. TLC s,eparation of highly polar products corresponding to DW O-day-old and to DW 17.day-old egg case incubated without (left) and with (right) Helix enzymes. O-day-old egg cases: black bars (superimposed). 17.day-old egg cases: white bars. (Zone 4 expressed in 20-hydroxy-ecdysone equivalents. all other zones in ecdysone equivalents.)

further increase of free ecdysteroids in both cases. These results show that the highly polar products from day-0 and day-l 7 egg cases are both qualitatively and quantitatively different. This finding could be confirmed several times. In the following we calculated whether the free 20-hydroxy-ecdysone present in day- I8 eggcasescould arise from He/is hydrolyzed conjugates present in day-0 egg cases. The quantity of free hormone present in day-18 egg cases is about 120 ng (Fig. 2) while the quantity of Helix hydrolyzable 20-hydroxy-ecdysone from conjugates present in day-0 egg cases is about 18 ng (Fig. 5). Also a comparison of the quantity of Helix hydrolyzable 20-hydroxy-ecdysone conjugates between day-0 (about 18 ng hormone equivalent) and

day-17 (about 32 ng hormone equivalent) egg cases where the titre of free 20-hydroxy-ecdysone has reached about 30 ng (Fig. 3) shows that the free 20hydroxy-ecdysone found in 1&day,-old egg cases cannot arise from Helix hydrolyzable conjugates deposited in the eggs at ovulation. Function of the mother in embryonic development In a series of experiments we tried to elucidate whether egg cases can develop outside the mother or not. Egg cases isolated immediately after ovulation were never observed to develop (IMBODEN et al., 1978). However, egg cases isolated after the first important uptake of water (IMBODEN et al., 1978) at day 13-15 after ovulation did develop, when kept in sterile and

ecd eq “9 60

1 4

3-

2-

1

O-

I 1

2

Fig. 6. TLC separation of the .s(,,ac’~/[/a/r/i/~, of RIA-reactive hrghl! polar products from da!-0 and Jay-l 7 egg cases incubated without (left) and with (right) HL’II’Ienq mes. O-day-old egg cw~s: white bars. 17-&t! -old egg cases: blad

bars (superimposed).

(Note: data presented

different11

from Fig. 4.) (Zone 4 expressed equivalents.)

in

20-h!clru\)-ec~i~sone equivalents. all other zones in ccdywne humid environment. When developing egg cases which were isolated at day 13 were analyzed for their ecdysteroid titres at day 17 we found in zone 1 with the highly polar products approx. 100 ng ecdysone equivalentsiegg case, in zone 2 (20-hvdroxv-ecdysone zone) appros. 100 ng 20-h> &ox),-eddy sone equivalents/egg case and in zone 3 (ecdysone zone) approx. 10 ng ecdysone equivalents/egg case. These quantities compare very well with those detected in normal egg cases at day 17 (see Fig. 2). Also morphological development is very similar in embryos isolated from the mother at day 13 and in embryos developing in the female (see Fig. 1). Dipping of whole egg cases into distilled water before day 8 after ovulation showed that the eggs are unable to take up water. This result suggests that the mother plays an important role in the uptake of water into the egg case. Since the egg case is in very close contact with the brood sac we investigated the morphology and histology of this tissue. A brood sac at day 14, the time when the first important uptake of water takes place into the eggs, was photographed by scanning electron microscopy. Figure 7 shows that the inner surface of the brood sac is covered with papillae which are in close contact with the egg case as shown by the imprints of the papillae on the surface of the egg case (Fig. 8). Such imprints could never be observed on day-O-old egg cases. Histological investigations show that there are thin canals which penetrate the surface of the papillae (Fig. 9). The openings of these canals are shown in Fig. 10. A comparison with the papillae structure at day 0 (Fig. 11) reveals that these openings are not yet differentiated at day 0. These photographs show a very tight morphological contact between brood sac and egg case particularly at day 14 and suggest that the brood sac plays a role in the uptake of water into the egg case. Due to the intimate connection between egg case and brood sac we also investigated this tissue for the

presence ofecdysteroids. Brood sacs at different stages between ovulation and dorsal closure (day 3. 6, 9. 10. 13, 17, 18, 20) were extracted and assayed for RIA activity. Always small quantities of highly polar products, 20-hydroxy-ecdysone, ecdysone and low polarity products were found which make it unlikely that ecdysteroids are stored in the brood sac and then transported into the egg case. Since water passes from the mother to the egg case the possible transport of ecdysteroids was also investigated. Only little RIA activity was found in all TLC zones in the haemolymph of the female (lo-20 pg ecd. eq.:nl) compared to the quantities in egg cases (Fig. 1) from ovulation until dorsal closure. This makes the transport of ecdy steroids from the haemolymph of the mother to the eggs highly improbable. DISCUSSION Our results show that only very small quantities of free ecdysteroids are present in O-day-old egg cases (Fig. 2 and Fig. 3) and that after day 8 the quantity of ecdysteroids increases (Fig. 21. At the time of dorsal closure we detected a peak of 20-hydroxy-ecdysone coinciding with the synthesis of an ‘embryonic cuticle’. It seems likely that one function of the free 20-hydroxy-ecdysone at day 18 is the control of the synthesis of the ‘embryonic cuticle’. The synthesis of this ‘embryonic cuticle’ is probably not dependent on the presence of juvenile hormone since we could find only traces or no juvenile hormone activity during early embryonic development using the Galkria wax test. This ‘embryonic cuticle’ is thinner (about 400 nm) than the ‘larval cuticle’ (about 7 Ltm), which is synthesised in the presence of juvenile hormone (IMBODEN et a/., 1978) at the end of the embryonic development. It was observed also in Oncopeltus (DORN, 1976). Locustu (LACWEUX rt al.. 1979) and in Blabcrus

(BULLIEKErt al., 1979) that moulting-hormone peaks during embryonic development are correlated with the onset of the deposition of a cuticle. As in Nauphoeta, also in Leucophaeu the deposition of an ‘embryonic cuticle’ begins just as dorsal closure is being completed, and the beginning qf the ‘larval cuticle’ secretion occurs after apolysis of the ‘embryonic cuticle’ (RINTEKK~ECHT, 1977). Also in Schistocerca (SBRENNA-MICCIAKELLI and SBRENNA, 1972) and in Blaberus (BULLIERE. 1973) two cuticles are deposited successively during! embryonic life. The latter author could show that the addition of inokosterone to the medium resulted in the early onset of cuticle deposition in legs of embryos previously cut at the base of the tarsus. From this result she concluded that embryonic moulting cycles are controlled by the same hormonal mechanism as larval cycles. A comparison of the titres of free ecdysone and free 20-hydroxy-ecdysone in O-day-old eggs from Nauphoeta (Figs. .! and 3) with other insects shows remarkable differences. In Table 1 the results of measurements of free ecdysone and 20-hydroxyecdysone in O-day-old eggs in different insects are compared. These results are rather confusing. The values for 20-hydroxy-ecdysone are similar while the ecdysone values are extremely different in the insects represented. We do not know how to explain these differences, however, some discrepancies could be due to the different techniques used for extraction, purification and quantification. Since we were particularly puzzled b> the data of Leucophaea. a cockroach closely related to Na~pho~~t~~. in a preliminary experiment we analyzed. with our methods (TLC and HPLC combined with RIA). day-0 egg cases of Lrucophaeu. We observed quantities comparable to those given for Nauphoeta and could thus not confirm the data given bq MATZ (1980). According to our results, 20-hydroxy-ecdysone (TLC and HPLC) is the predominant free ecdysteroid in both cockroach species and free ecdysone is present only in small quantities. As concerns the highly polar products, results have been published for newly laid eggs of Schistocerca gre~~uriu (GA~IX ef trl.. 1979: DIYAI\ and REPS. 1981 ). of Lomstu migraforiu (LAGUEUX et al.. 1979) and of G‘alleria n~ellonellu ( HSIA~and HSIAO,1979) indicating high amounts of ecdysone conjugates hydrolyzable bj Helix enzymes. In Nuuphoeta we found, in O-day-old egg cases. large c uantities of 2O-hydrosJ,-ecdysone conjugates hydrolyzable by Helix enzymes (Figs. 5 and 6). In addition, our results show. that the quantity of RIA-reactive highly polar products increases from day 0 towards dorsal closure (Figs. 2 and 3). Similar observations are reported for Locustu ( LAGUKX ef al., 1979). Our data indicate (Fig. 6) that the highly polar products consist of different RIA-reactive compounds and that they ar: qualitativeI], and quantitatively different in day-0 and day-17 egg cases. Since the titre of free 20-hydroxy-ecdysone measured in day-l 8 egg cases (Fig. 2) is much higher than the quantities of free and conjugated 20-h) droxb -ecclysone (h>,drolyzable by Hc,/i.\-enzymes) present in O-day-old egg cases (Fig. 4) this hormone could arise from conjugates which cannot be hydrolyzed by Heli.\- enzymes. or from a new synthesis in the egg case or from the mother. The latter

possibility seems unlikely because we observed only small quantities of ecdysteroids in the very haemolymph of pregnant females. The site of origin of the small quantities of ecdysteroids in the haemolymph of pregnant females is not yet known. However, it is obvious that they are not produced by the ovary because we found the same quantities of ecdysteroids in females ovariectomized immediately after ovulation as in normal pregnant females. Ovariectomy did also not affect embryonic development nor did decapitation or ligation of the abdomens of the pregnant females just after ovulation (IMBODEN, unpublished). However, when egg cases were isolated at ovulation they were never observed to develop. Development occurred only when egg cases were isolated from the female after day 13-l 5, i.e. when the first important uptake of water into the egg case has taken place (IMBODENet al., 1978). This indicates an important role of the mother for the uptake of water. It is known that in the cockroach Diplopteru pu~~ctutu. the dry weight of the egg case increases during embryomc development (STAY and COOP, 1973). This increase in dry weight does not begin until shortly after dorsal closure but thereafter parallels the increase in fresh weight. The milk secreted by the walls of the brood sac. in which the embryos develop, provides for this increase (STAY and COOP, 1974). In contrast. in Nut~phoeru. the dry weight of the egg case decreases gradually throughout embryonic development and it seems probable that the observed increases in fresh weight are due to the uptake of water only (IMBOD~N (11 al., 1978). Incubation of egg cases at different stages in distilled water shows that they are not able to take up water before day 8 (at about 20” () gestation time) after ovulation. The same phenomenon has been observed in Diplopteru where the ability to take up water develops after about 13”, of the gestation period (STAY and COOP, 1973). Careful morphological and histological investigations of the brood sac reveal the presence of papillae. The same kind of brood-sac papillae have been found in Diplopteru (HAGAN, 1951) and in Leucophaea (ENGELMANN. 1957). A photograph of the egg-case surface and a comparison of the papillae structure between day 0 (Fig. I I ) and day 14 (Fig. 10) show a very tight connection between the brood sac and the egg case which develops towards the time of uptake of water. This suggests that, in Nuuphoeta. the brood sac is very important in the process of water uptake and explains the inability of egg cases to develop if isolated before that time. Since ecdysteroids in the egg cases increase simultaneously with the uptake of water we have included ecdysteroid measurements of brood sacs of different stages. We found only small quantities which suggests that this tissue does not contribute significantly to the rise of ecdysteroids in egg cases. Experiments under way with labelled cholesterol applied to females during o&q te maturation, leading to the formation of labelled, free and conjugated ecdysteroids promise to help elucidate the question of the origin of ecdysteroids during early embryonic development. ,4[,Xllo~~/~,(/~~[,~~l~,/fr,s~Thanks are due to Dr. J. D. O’COUNOK (Los Angeles) for the ecd>sone antiserum. to Dr. J. KOOLMAN (Marburg) for 23,14-“Hz-rcdysone. We wish to

H. IMBODENAND B. I.ANZKEII\;

46

thank also Mrs. CHRISTINE KAPPLEK and Dr. J. A. HOFFMANN (Strasbourg) for accepting us to perform some HPLCanalyses and for providing us with ecdysteroid references. Thanks are also due to Mrs. GUNILLA KL’HNI and Mrs. ANNA TSCHAN for technical assistance and to Mrs. Dr. MI(.H~LE CAKON (University of Fribourg, Switzerland) for doing scanning electron microscopy. Financial support by the Swiss National Science Foundation (Grant 3.188.77) is gratefully acknowledged.

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IMBODENH., LANZKEIN B.. DELBE~QUEJ. P. and LOsct~t~ M. (1978) Ecdysteorids and juvemle hormone during embryogenesis in the ovoviviparous cockroach Narrphorta cinereu. Gm. camp. Endow. 36, 628435. KAPLANISJ. N., ROBBINSW. E., THOMPSON M. J. and DUTKY S. R. (1973) 26-hqdroxy-ecdysone: New insect molting hormone from the egg of the tobacco hornworm. Scirr~t,, Wash., 180, 307-308. KAPLANIS J. N.. DUTKY S. R.. ROBBINS W. E.. THOMPSOX M. J.. LINDQUISTE. L.. HORX D. H. S. and GALBRAITH M. N. (1975) Makisterone A: A 2%carbon hexahydroxq moulting hormone from the embryo of the milhweed bug. Science, wush. 190, 68 l-682. LAUXLIX M., HIRN M. and HOFFMANNJ. A. (1977) Ecdqsone during ovarian development in Lorusru migratoriu. J. Insect PI7ysiol. 23, 109-l 19. LA(;UEIJX M.. HETRU C.. GOLTZENE F.. KAPPLEK C. and HOFFMANN J. A. (1979) Ecdysone titre and metabolism m relation to cuticulogenesis in embryos of Lo~c.vtu migrutorla. J. Insect Pllysivl. 25, 709-724. LANZKE~NB.. HASHIMOTO M.. PAKMAKOVIC.CI V.. NAKANISHI K., WILHELM R. and L~~SCHERM. (1975) Identification and quantification of juvenile hormones from different developmental stages of the cockroach Nuuphorra cinereu. Life SC;. 16, 1271-1284. MATZ G. (1980) Variations du taux des ecdystt-roides au tours de l’embryoge&se de Lrucophueu mudwue. C. R. iwhd. Sknc. .4cutl. %i. Puris. 291. 501-504. MIZUY~ T. and OHNISHI E. (1975) Conjugated ecdysone in the eggs of the silkworm. Devel. Growth Dqjer. 17, 219-225. OHNISH~ E., OHTAU T. and FUKUVA S. (197 I) Ecdysone in the eggs of BombJ.v silkworm. Proc. Jupun. Acad. 47, 413415. OHNISHI E., MIZLIX~ T., IKEKAWA N. and SAKL~RAIS. (1977a) 2-deoxy-r-ecdysone from ovaries and eggs of the silkworm Bomb?\- mori. Sciencr,Wush 197, 66-61. OHNISHI E.. MIZUNO T.. IKE~AWA N.. AWATA N. and SAKURAI S. (1977b) Occurrence of z-ecdysone in the developing embryos of the silkworm Bon?hy.~ mori. J. Imrct Physiol. 23, 3 17-3 19. RINTERK;N~C.~IT E. et MATZ G. (1977) Etude ultra-structurale des dephts cuticulaires cher I’embrlon de Leucophura ntuderue. C. R. hebd. .Sk~nc~. .4cud. Sci. Paris. 284, 23Y5-23YX. ROMEIS B. (1968) hIllirc.rkopis~he Technik. R. Oldenbourg. Miinchen. SBKENNA-MICCIAKELLI A. and SBKFNNA G. (1972) The embryonic apolysis of Schistocercu greguriu (Orthoptera). J. Insect PI?Jsio/. 18. 1027-1037. STA’I. B. and COOP A. (1973) Developmental stages and chemical composltion in embryos of the cockroach. Diplopterupunc~tutu, with observations on the effect ofdiet. J. Insect Physiol. 19, 147-171. STAY B. and COOP A. (1974) ‘Milk’ secretion for embryogenesis in a viviparous cockroach. Tiss~r and Cr,// 6(4), 669-693.