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Ecdysteroids and juvenile hormone during embryogenesis in the ovoviviparous cockroach Nauphoeta cinerea
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
36, 628-635 (1978)
Ecdysteroids and Juvenile Hormone during Embryogenesis in the Ovoviviparous Cockroach Nauphoeta cinereal H. IMBODEN,* B. LANZREIN,*
J. P. DELBECQUE,~ AND M. L~SCHER*
*Division of Zoophysiology, Zoological Institute of the University of Bern, Engehaldenstr. 6, CH-3012 Bern, Switzerland, and tVniversitC de Dijon, Laboratoire de Zooiogie, Boulevard Gabriel, F-21000 Dijon, France Accepted October 17, 1978 Embryos of the ovoviviparous cockroach Nauphoetn cinerea are shown to contain ecdysteroids and juvenile hormone (JH) dependent on their stage of development. Ecdysteroid determinations were made by radioimmunoassay and Musca bioassay and for JH determinations the Galleria wax test was used. Mass fragmentography allowed the identification of ecdysone and ecdysterone and indicates the presence of still other ecdysteroids. After the dorsal closure these hormones are produced in the embryo, but the site of synthesis is not yet known.
Eggs of Manduca sexta (Kaplanis et al., 1973, 1976), Bombyx mori (Ohnishi et al., 1977a,b), Locusta migratoria (Lagueux et al., 1977; Lagueux and Hoffmann submitted), Macrotermes bellicosus and Macrotermes subhyalinus (Delbecque et al., 1978), as well as embryos of Blabera (Bulhere et al., 1976), Leucophaea (Matz and Hoffman, 197% Oncopeltus (Kaplanis et al., 1975; Dorn and Romer, 1976), and other insects have been found to contain ecdysteroids. JH-active material has been detected in eggs and embryos of Oncopeltus (Dorn, 1975) as well as of Hyalophora cecropia (Gilbert and Schneiderman, 1961). However, to our knowledge, ecdysteroid and JH determinations have never been made simultaneously and the hemolymph of the embryos has never been investigated. Here we report on the presence, isolation, and quantification of ecdysteroids and JH during the course of embryonic development in Nauphoeta cinerea. This is an ovoembryoviviparous cockroach where ’ Dedicated to Professor Peter Karlson on the occasion of his 60’” birthday.
genesis occurs in an egg case within the uterus of the female. Ecdysteroid and JH determinations were performed using whole egg cases or hemolymph of embryos and pregnant females. MATERIALS
Nauphoeta cinerea were kept at 26” and 60% relative humidity on dog flakes and water, at a photoperiod of 12 hr. The JH-dependent oocyte maturation lasts 12 days. The oocytes are then packed into an egg case, which is extruded and then retracted into a brood sac (uterus). There the eggs are incubated until the larvae hatch 37-40 days later.
Extracti&
Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Procedure
Whole egg cases were homogenized in 65% methanol/water for extraction of ecdysteroids and in ethyl acetate/ethanol 5/l (v/v) for extraction of JHs. For collection of egg content, eggs between ovulation and dorsal closure were punctured and the outflowing liquid was sucked into a microcapillary and dropped into 65% methanol/water for extraction of ecdysteroids and into ethyl acetate/ethanol 5/l (v/v) for extraction of JHs. For collection of embryo hemolymph, the head of embryos after dorsal closure was punctured and the outflowing hemolymph was collected in a microcapillary and extracted in the same way as the egg content. 628
0016-6480/78/0364-0628$01.00/O
AND METHODS
Insects
ECDYSTEROIDS
AND JH IN COCKROACH
Hemolymph of pregnant females was collected after cooling the whole insects in ice water by cutting away a leg. The extraction procedure was the same as for egg content and embryo hemolymph.
Pmrijication and QuantiJication Ecdysteroids
of
Whole egg cases. After homogenization in 65% methanol/water, precipitation in the cold ensued. Extracts were then dried and purified by chromatography on a column of silicic acid and by thin-layer chromatography (tic) (1 X diisopropyl ether, 1 x chloroform/ethanol 96%, 80/20) (Delbecque et al., 1975. 1978). Egg content and hernoiymph. After extraction in 65% methanol/water extracts were dried and purified directly by tic using the same solvents as for whole egg cases. Radioimmunoassay (RIA). After tic purification the ecdysone and ecdysterone zone were assayed separately in a RIA which was carried out according to the method’of Borst and O’Connor (1974) and Horn et al. (1976). This RIA has a five- to seven-fold higher sensitivity for ecdysone than for ecdysterone. Ecdysone and ecdysterone used as standards for the RIA were purchased from Simes (Milan, Italy). Results are expressed as nanograms of ecdysone and ecdysterone equivalent. Musca bioassay. The Musca bioassay was carried out according to the method of Kaplanis et al. (1966) but by a ligature of the larvae in rubber bands (Staal, 1967). Gas-liquid chromatography--mass spectrometry (glc-ms). The glc-ms was performed on a LKB 9000 instrument using a 2-m x 2-mm silanized coiled glass column with 1% OV-1 on 100-120 mesh silanized Gas Chrom P. Gas liquid chromatography conditions were the following: column temperature, 28.5”, flow rate of carrier gas (helium) 30 mllmin. Fragmentograms and mass spectra were recorded at 28 eV. Silylniion. Ecdysteroids were silylated with trimethylsilyhmidazole from Koch-Light. Silylation was performed at 6S0 overnight (Delbecque et al., 1975, 1978). Ecdysone (ol-ecdysone), makisterone A, and 26-hydroxyecdysone used as standards for silylation were generous gifts from Hoffmann-La Roche (Basle, Switzerland), Simes (Milan, Italy), and Dr. Kaplanis, respectively. Ecdysterone (fi-ecdysone, 20-hydroxy-ecdysone) and inokosterone were purchased from Schwarz/Mann.
Purification
and Quantification
of JH
After extraction in ethyl acetate/ethanol, S/l (v/v) extracts were purified by tic (6% ethyl acetate/ benzene) as described by Lanzrein et a/. (1975). For determination of JH activity the Galleria wax test was
EMBRYOS
629
used (De Wilde et al., 1968). Under our conditions 1 Galleriu unit (GU) corresponds to 8 pg IH III (Calbiochem). For characterization of JH, tic-purified extracts were injected into a high pressure liquid chromatograph (Du Pont 848) equipped with a variablewavelength uv detector (Perkin-Elmer) using a column Zorbax SIL (Du Pont) and 6% ether hexane as solvent.
RESULTS Changes in Weight of Egg Cases during Embryagenesis Egg cases throughout embryonic development were observed and their fresh weight and dry weight were measured (Fig. 1). From Day 0 to 13 the egg content appears to be very viscous. On Day X3 the fresh weight begins to increase rapidly and at that time lipid droplets appear in the egg. The fresh weight increases until the 19th day when dorsal closure occurs. Another minor increase in’fresh weight is observed between Days 26’and 30. Three days later the egg case and the chorion break on the side of the embryos’ heads which from now on are only covered by the embryonal cuticle. Since the dry weight of the egg case decreases gradually throughout embryonic development it seems likely that increases in fresh weight are due to uptake of water only. A similar uptake of wat,~r-although less drastic-was observed by Engelmann (19.57) in Leucophaea. Ecdysteroid Cases
and JH Activity in Whole Egg
Egg cases were divided into halfs: One half was extracted and purified for erdysteroid determination with the Musca bioassay and the other half for JH determination with the Galleria wax test (Fig. 2). Ecdysteroid activity was detected in small amounts at the time of the dorsal closure and in higher amounts around Days’29 and 32. JH-active material could be detected only after the dorsal closure had occurred. Two peaks were observed, namely one on Day 27 and one on Day 33 at the time of the
630
IMBODEN mgkgg
ET AL.
case
250
--f-f-------*
dry weight f
-*--I--------.5-* 50 0
0
Nletton
lb
2b dorsaltclos”re
so hatching
FOG. 1. Fresh weight and dry weight of egg cases from ovulation to hatching of the larvae. Mean values k SE; 30-70 determinations per point.
G.Ulg .I04
ecd.eq./g
n.urg
n9 a30
50
ecdysone ecdysterone JH
A-....... A x-x
I F
,’:?b /Ii I 400
00
2oQ
a
FIG. 2. Ecdysone, ecdysterone, and JH activity in whole egg cases during embryogenesis. Ecdysone and ecdysterone were determined by Mtiscu bioassay after separate elution from thinlayerplates and the results are expressed in ecdysone or ecdysterone equivalents and in Musca units (MU). JH activity was determined by Galieria bioassay and is expressed in Galkria units (CU). Each symboi represents one determination.
ECDYSTEROIDS
AND
JH
rupture of the chorion. Thus, the Galleria and Musca bioassay results demonstrate, dependent on the stage of embryogenesis, the presence of JH-active and ecdysteroidactive material in egg cases. In order to further identify the ecdysteroids, tic-purified extracts of 32 dayold-egg cases were analyzed by glc-ms. A mass fragmentogram of the combined tic zones (except the origin) is given in Fig. 3. The mass of m/e 561 is a characteristic fragment of fully silylated ecdysterone and other 20-hydroxy-ecdysteroids which only differ from ecdysterone by the side chain (e.g., inokosterone, makisterone); the mass of m/e 567 is characteristic for fully silylated ecdysone and other ecdysteroids having no hydroxyl at C-20 and differing from 21
IN
COCKROACH
, 5
,
* 10 ’
’
’
I xia *
’
’
I 20I r
I min
FIG. 3. Mass chromatogram of ecdysteroid derivatives in silic acid-purified egg case (32 days otd). Penta-trimethylsilyl (TMS) derivative of ecdysone (A) is detected at m/e 567 (retention time 9 min). HexaTMS derivative of ecdysterone (B) is detected at m/e 561 (retention time 11.5 min). (C) Corresponds to penta-TMS derivative of ecdysterone and/or traces of makisterone A (retention time 1.5 min). TMS derivative of 26-OH-ecdysone (D) is detected at m/t 567 (retention time 14.5 min). (E) Possibly represents 20,26 dihydroxyecdysone. (*) Indicates unknown compounds (possibly not ecdysteroids). (y/) indicate unidentified isomers of ecdysterone. Taking into account the electronic amplification and the relative response of ions, track at m/e 567 is 4.3-fold more sensitive.
431
ecdysone by the side chain, as 26hydroxyecdysone (Delbecque ei; nl., in preparation). According to mass fragments and retention times, Fig. 3 shows the presence of ecdysone, ecdysterone, which is the predominant ecdysteroid, and 26 hydroxyecdysone, However, the presence of other 20-hydroxy-ecdysteroids, having fragments at pnle 561, is indicated: One of them shouldering ecdysterone peak could be an isomer, possibly inokosterone or an epimer: another molecule is detected, with a greater retention time than makisterone, thus a heavier side chain, suggesting it could be 20,2~hy~oxyecdyso~e. Thinlayer chromatography zones corresponding to ecdysone and to ecdysterone were analyzed separately and again demonstrated the presence of ecdysone and ecdysterone. The results indicate a, good separation of ecdysone, ecdysterone, and other ecdysteroids in the tic system used. In order to characterize the Jo-active material, tic-purified extracts were injected into a high pressure liquid chromat~g~a~h~ Fractions were collected and tested in the Galieria wax test. It was found th’at most of the JH activity elutes with the retention time of JH III, thus suggesting the presence of JN III in egg cases. Ecdysteroids
,
EMBRYOS
and JH in Egg Canrenr arzd
After having demonstrated the presence of JH, ecdysone, and ecdysterone in whole egg cases, we investigated whether these hormones are accumulated in the egg content and later in the h~olym~h of cmbryos. The analysis is given in Fig. 4 and reveals close similarities to the resuhs with whole egg cases (Fig. 2). ~~dyster~~ds are detectable already before dorsal closure and shortly after each JH peak,, Again JH appears in two peaks after dorsal closure has occurred. A comparison of Fig. 4 with Fig. 2 reveals that ecdysteroids as well as JH are accumulated in the l~~rnolyrn~~ of the embryo.
632
IMBODEN
ET
AL.
FIG. 4. Ecdysone, ecdysterone, and JH activity in egg content and hemolymph of embryo during embryogenesis. Ecdysone and ecdysterone were determined by RIA after separate elution from thin-iayer~plates and the results are expressed in ecdysone or ecdysterone equivalents, respectively. JH activity was determined by G~~Ileria bioassay and is expressed in Gallerid units (GU). Each symbol represents one determinarion.
Ecdysteroids and JH in the Hemolymph Pregnant Females
of
stage of dorsal closure were isolated and put into a humid environment. Thirteen days later, at the time corresponding to Day 32 of normal development when JH and ecdysteroid titers are high, parts of the egg cases were extracted and tested for the presence of JH and ecdysteroids. We measured 120,000 GU JH, 300 ng ecdysone, and 2,300 ng ecdysterone equivalents/ml hemolymph. These values are similar to those of Day 32 in normal development (Fig. 4). The other parts of the egg cases were left in the humid environment and the embryos hatched at the expected time. These findings demonstrate that embryos after dorsal closure produce JH and ecdysteroids themselves and are able to develop without interference of the endocrine system of the mother.
Nothing is known on the origin of these hormones in the embryos and since embryogenesis occurs within the female we also included ecdysteroid and JH determinations in the hemolymph of the pregnant females (Fig. 5). Small amounts of ecdysone and ecdysterone .could be detected in the hemoIym~h of the pregnant female from ovulation until dorsal closure of the embryo. Thereafter only traces of ecdysteroids were measurabIe. No JH could be detected before the rupture of the chorion. Thereafter the titer of JH increases continuously. Examination of the ovaries revealed that simultaneously with the increase of JH titer the growth of the next generation of oocytes is initiated DISCUSSION (Imboden, unpublished). We also investigated the development of Our results with the ovoviviparous cockeggs in isolated egg cases. Egg cases at the roach Nauphoeta cinerea demonstrate the
ECDYSTEROIDS
AND JH IN COCKROACH
EMBRYOS
633
ecd.eq./mi
FIG. 5. Ecdysone, ecdysterone (RIA activity), and JH activity in the hemolyl~ph of females during pregnancy. Each symbol represents one determination. Note that the scale for ecdysteroids is IOO-fold enlarged as compared to Fig. 4.
presence of ecdysteroids and JH in egg cases and hemolymph of embryos. The use of glc-ms allowed the identi~cation in egg cases of ecdysone, ecdysterone, and 26hydroxyecdysone. In addition the presence of other ecdysteroids (as possibly 20,26dihydroxyecdysone~ is suggested. Analysis of the JH-active material by hplc and Gallevia bioassay suggests the presence of mostly JH III. Since little information is available on changes of ecdysteroid and JH titers as a function of embryonic development we first made JH and ecdysteroid determinations in egg cases and hemolymph of embryos at different developmental stages. If one compares the curves obtained with egg cases (Fig. 2) with those obtained with egg content and hemol~m~h of embryos (Fig. 4) strong similarities are obvious and indicate an accumulation of ecdysteroids and JH in the hem~lymph of the embryo. The role of these hormones in embryo-
genesis is not clear yet, but in Eocldsru a role of ecdysteroids in controlling the secretion of serosal cuticle is suggested (Lagueux and Hoffmann, submitted), A small peak of ecdysteroids is observed before dorsal closure and two high peaks, ranging up to 1-3 pgiml, appear adored Days 25 and 33, respectively. Ecdysterone is always by far more abundant than ecdysone. At about the same time, i&e., around Days 25 and 33, we found two peaks of JH, 10 x lO$ and 15 x lo4 GLJlml, resflectively. These quantities are higher than ithe highest values observed in reproducing females which reach only 1.5 x 1P Gulml (Lanzrein et nl., in pre~a~~io~}. Since it is generally believed that the coincidence of a high molting hormone titer with a high JH titer Causes rno~~j~g, to a larva ~Sc~~eid’~rrnan and Gilbert, 1964: Gilbert and King, 1973)’ the peaks of eedysteroidsand JH are perhaps ~~n~~~ted ix/ith one or two embryonic molts. jeweler, we
634
IMBODEN
do not know yet if and at what time embryonic molts occur in Nauphoeta. A simultaneous peak of JH activity (Tenebrio bioassay) and ecdysteroid activity (Calliphora bioassay) has also been observed during embryogenesis in Oncopeltus by Dorn (1975, 1976) who suggested a correlation with an embryonic molt. Little is known about the site of origin of ecdysteroids and JH in embryos. The ecdysteroids present in toccata in early embryonic development (i.e., before the endocrine glands of the embryo are differentiated) are synthesized in the ovaries of the mother and then accumulated in the cells of the serosa (Lagueux et al., 1977; Lagueux and Hoffmann, submitted). In ~~alo~hora cecropia (Gilbert and Schneiderman, 1961) eggs of allatectomized females do not contain JH-active material whereas eggs from normal females do contain JH, thus suggesting that JH of the hemolymph .of the mother is transmitted to the eggs. We do not know yet where JH and ecdysteroids are synthesized during embryogenesis in ~u~p~oet~. Before dorsal closure eggs contain only small amounts of ecdysteroids and no detectable JH. lt is noteworthy that also the hemolymph of young pregnant females, whose prothoracic glands are degenerated (Lanzrein, 1975), contains small amounts of ecdysteroids. It is not yet known where the ecdysteroids of the embryo and the mother are produced and what their function is. After dorsal closure embryos produce JH and ecdysteroids themselves since it was found that eggs in cases which were removed from the mother at the time of dorsal closure were able to develop and have similar titers of JH and ecdysteroids as eggs developing in the brood sac of the mother. Histological investigations revealed that after dorsal closure the CA and prothoracic glands of the embryos are well differentiated and that their cells have voluminous nuclei. This may indicate that ecdy-
ET AZ,.
steroids and JH are synthesized by the embryos’ endocrine glands. Electron microscopic investigations of the embryonic CA of Oncopeltus (Darn, 1975) also led to the conclusion that the glands might be functional. Sites of molting hormone synthesis other than the prothoracic glands, like oenocytes, have been repeatedly suggested in the literature (Dorn and Romer, 1976; Locke, 1969; Romer et al., 1974; Studinger and Willig, 1975; Hsiao et crl., 1975) and cannot be excluded for the embryo of Nauphoeta. ACKNOWLEDGMENTS Thanks are due to Professor J. D. O’Connor (Department of Biology, University of Cahfornia, Los Angeles) for the ecdysone antiserum and to Dr. P. Masner (Biological Laboratory of Dr. R. Maag, Dielsdorf) for his help with the Musca bioassay. We also thank Professors Maume and Padieu (Departement de Bioehimie, Universite de Dijon) for the use of the LKB 9000 glc-ms apparatus. Financial suppoft by Swiss National Foundation Grant 3.188.77 and by a grant for international collaboration of the CNRS (France) is gratefully acknowledged.
REFERENCES Borst, D. W., and O’Connor, J. D. (1974). Trace analysis of ecdysones by gas-liquid chromatography, radioimmunoassay and bioassay. Steroids 24, 637-656. Bull&e, D., Bulliere, F., Mauchamp, B., Lafont, R., De Reggi, M., et Hirn, M. (1976). Etude des variations du taux d’hormone de mue au tours du d~roulement du deuxieme cycle de la vie embryonnaire chez une blatte. C.R. Acad. Sci. Ser. D 283, 647-650. Delbecque, J. P., Lanzrein, B., Bordereau, C., Imboden, H., Hirn, M., O’Connor, J. D., Noirot, C., and Lfischer, M. (1978). Ecdysone and ecdysterone in physogastric termite queens and eggs of Macrotermes bellicosus and Macrotermes subhyaknus. Gen. Camp. Endocrino(., 36, 40-47. Delbecque, J. P,, Prost, M., Maume, B. F., Deiachambre, J., Lafont, R., et Mauchamp, B. (1975). Dosage de la Pecdysone par fragmentographie de masse au tours du dernier stade larvaire de TeRebrio molitor L. (Insecte, Coliopt&e). C. R. Acad. Sci. Ser. D 281, 309-312. De Wilde, J., Staal, G. B., De Kort, C. A. D., De Loof, A., and Baard, G. (1968). Juvenile hormone
ECDYSTEROIDS
AND
JH
titre. in the haemolymph as a function of photoperiodic treatment in the adult Colorado beetle (Leptinotursa decemlineata). Proc. Ken, Ned. Akad. Wetensch. Ser. C 71, 321-326. Darn, A. (1975). Struktur und Funktion des embryonalen Corpus allatum von Oncopeltus fasciatus Dallas. Verh. Deut. Zoof. Ges. 85-89. Darn, A., and Romer, F. (1976). Structure and Rmction of prothoracic glands and oenocytes in embryos and last larval instars of Oncopeltus fasciatus Dallas. Cell Tissue Res. 171, 331-3.50. Engelmann, F. (1957). Bau und Funktion des weibIichen Geschlechtsapparates bei der ovoviviparen Schabe Lelacophaea maderae (Orthoptera) und einige Beobaehtungen iiber die Ent~cklung. Biol. Zentralbl. 76(6), X2-740. Gilbert, L. I., and King, D. S. (1973). Physiology of growth and deveIopment: Endocrine aspects. In “The PhysioIogy of Insecta” (Rockstein, ed.) Vol. 1, pp. 249-370. Academic Press, New York. Gilbert, L. I., and S~hneiderman, II. A. (1961). The content of juvenile hormone and lipid in Lepidoptera: Sexual differences and developmental changes. Gen. Comp. Endocrinol. 1,453-472. Horn, D. H. S., Sage, B., and O’Connor, J. D. (1976). A high affinity antiserum specific for the ecdysone nucleus. 1. Insect Physiol. 22, 901-905. Hsiao, T. H., Hsiao, C., and De Wilde, J. (1975). Moulting hormone production in the isolated abdomen of the Colorado beetle. Nature (London) 255, 727-728. Kaplanis, J. N., Dutky, S. R., Robbins, W. E., Thompson, M. J., Lindquist, E. L., Horn, D. H. S., and Galbraith, M. N. (1975). Makisterone A: A Z&carbon hexahydroxy moul~ng hormone from the embryo of the milkweed bug. Science 190, 681-682. Kaplanis, J. N., Robbins, W. E., Thompson, M. J., and Dutky, S. R. (1973). 26-Hydroxyecdysone: New insect molting hormone from the egg of the tobacco harnworm. Science 180, 307-308. Kapianis,J. N., Robbins, W. E.~Thompson,M.3., and Dutky, S. R. (1976). The molting hormones from the embryonated egg of the tobacco hornworm, ~u~d~c~ ‘sexta (L.). steroids 27(.5), 675-679. Kaplanis, .I. N., Tabor, M. J., Thompson, W. E., Robbins, W. E., and Shortino, T. J. (1966). Assay for ecdysone (moit~ng hormone) activity using the
IN
COCKROACH
EMBRYOS
635
housefly, ~uscff dor~est~ca L. steroids 8, 625631. Lagueux, M., Hirn, M., and Hoffmann, 9. A. (I977). Ecdysone during ovarian deve~opment,~n i;acusta migratoria. J. Ivrsect Physiol. 23, 109,119. Lagueux, M., and Hoffmann, J. A. Maternal ecdysteroids and early embryonic development in Locusfu migrator~u L., submitted. Lanzrein, B. (1975). Programming, induction, or prevention of the breakdown of the prothoracic gland in the cockroach, ~aup~oeta cinerea. J. Insect Physiol. 21, 367-389. Lanzrein, B., Hashimoto, M., Parmakovich, V., Nakanishi, K,, Wilhelm, R., and Liischer, M. (1975). Identification and quanti~cat~on of juvenile hormones from different deveiopmentaI stages of the cockroach Nauphoeta cinerea. Life Sci. 16, 1271-1284. Locke, M. (1969). The ultrastructure of oenocytes in molt/intermolt cycle of an insect. Tisscla Cell 1, 103-154. Matz, G., and Hoffmann, J. A. (1975). “Tine d’hormone de mue et metabolisme d’a-ecdysone radioactive au cours du dhetoppement embrysnnaire de Leucophaea maderae.” Reported at 3” Gall. Physioi. Insectes, Strasbourg, France. Ohnishi, E., Mizuno, T., Chatani, F., Ikekawa, N.i and Sakurai, S. (1977). Z-Deoxy-tw-ecdysoae from ovaries and eggs of the silkworm, Bomhyx mari, Science 197, 66-67. Ohnishi, E., Mizuno, T., Ikekawa, N., Awata, N., and Sakurai, S. (1977). Occurrence of a-ecdysone in the developing embryos of the silkworm, Bombyx mori. J. Insect Physiol. 23, 337-319. Romer, R., Emmerich, H., and Nowock, .I-(1974)). Biosynthesis of ecdysones in isolated protboradc glands and oenocytes in Tenebrio molitor in vitro. J. IrLsrct. Pizysjol.20, 1975-1987. Schneiderman, H. A., and Gilbert, L. I. (1964). Control of growth and development in insects. Srience 143, 325-333. Staal, G. B. (1967). Plants as a source of insect hormones. Proc. Ken. Ned. Aked. Wetensch. Ser. C 70(4), 409-418. Studinger. G., and Wihig, A. (1975). Bjasynthesi~ of o- and @-ecdysone in isolated abdomens of larvae of Musca domestica. 1. Insect Physiol. 21, 17931798.
AND
COMPARATIVE
ENDOCRINOLOGY
36, 628-635 (1978)
Ecdysteroids and Juvenile Hormone during Embryogenesis in the Ovoviviparous Cockroach Nauphoeta cinereal H. IMBODEN,* B. LANZREIN,*
J. P. DELBECQUE,~ AND M. L~SCHER*
*Division of Zoophysiology, Zoological Institute of the University of Bern, Engehaldenstr. 6, CH-3012 Bern, Switzerland, and tVniversitC de Dijon, Laboratoire de Zooiogie, Boulevard Gabriel, F-21000 Dijon, France Accepted October 17, 1978 Embryos of the ovoviviparous cockroach Nauphoetn cinerea are shown to contain ecdysteroids and juvenile hormone (JH) dependent on their stage of development. Ecdysteroid determinations were made by radioimmunoassay and Musca bioassay and for JH determinations the Galleria wax test was used. Mass fragmentography allowed the identification of ecdysone and ecdysterone and indicates the presence of still other ecdysteroids. After the dorsal closure these hormones are produced in the embryo, but the site of synthesis is not yet known.
Eggs of Manduca sexta (Kaplanis et al., 1973, 1976), Bombyx mori (Ohnishi et al., 1977a,b), Locusta migratoria (Lagueux et al., 1977; Lagueux and Hoffmann submitted), Macrotermes bellicosus and Macrotermes subhyalinus (Delbecque et al., 1978), as well as embryos of Blabera (Bulhere et al., 1976), Leucophaea (Matz and Hoffman, 197% Oncopeltus (Kaplanis et al., 1975; Dorn and Romer, 1976), and other insects have been found to contain ecdysteroids. JH-active material has been detected in eggs and embryos of Oncopeltus (Dorn, 1975) as well as of Hyalophora cecropia (Gilbert and Schneiderman, 1961). However, to our knowledge, ecdysteroid and JH determinations have never been made simultaneously and the hemolymph of the embryos has never been investigated. Here we report on the presence, isolation, and quantification of ecdysteroids and JH during the course of embryonic development in Nauphoeta cinerea. This is an ovoembryoviviparous cockroach where ’ Dedicated to Professor Peter Karlson on the occasion of his 60’” birthday.
genesis occurs in an egg case within the uterus of the female. Ecdysteroid and JH determinations were performed using whole egg cases or hemolymph of embryos and pregnant females. MATERIALS
Nauphoeta cinerea were kept at 26” and 60% relative humidity on dog flakes and water, at a photoperiod of 12 hr. The JH-dependent oocyte maturation lasts 12 days. The oocytes are then packed into an egg case, which is extruded and then retracted into a brood sac (uterus). There the eggs are incubated until the larvae hatch 37-40 days later.
Extracti&
Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Procedure
Whole egg cases were homogenized in 65% methanol/water for extraction of ecdysteroids and in ethyl acetate/ethanol 5/l (v/v) for extraction of JHs. For collection of egg content, eggs between ovulation and dorsal closure were punctured and the outflowing liquid was sucked into a microcapillary and dropped into 65% methanol/water for extraction of ecdysteroids and into ethyl acetate/ethanol 5/l (v/v) for extraction of JHs. For collection of embryo hemolymph, the head of embryos after dorsal closure was punctured and the outflowing hemolymph was collected in a microcapillary and extracted in the same way as the egg content. 628
0016-6480/78/0364-0628$01.00/O
AND METHODS
Insects
ECDYSTEROIDS
AND JH IN COCKROACH
Hemolymph of pregnant females was collected after cooling the whole insects in ice water by cutting away a leg. The extraction procedure was the same as for egg content and embryo hemolymph.
Pmrijication and QuantiJication Ecdysteroids
of
Whole egg cases. After homogenization in 65% methanol/water, precipitation in the cold ensued. Extracts were then dried and purified by chromatography on a column of silicic acid and by thin-layer chromatography (tic) (1 X diisopropyl ether, 1 x chloroform/ethanol 96%, 80/20) (Delbecque et al., 1975. 1978). Egg content and hernoiymph. After extraction in 65% methanol/water extracts were dried and purified directly by tic using the same solvents as for whole egg cases. Radioimmunoassay (RIA). After tic purification the ecdysone and ecdysterone zone were assayed separately in a RIA which was carried out according to the method’of Borst and O’Connor (1974) and Horn et al. (1976). This RIA has a five- to seven-fold higher sensitivity for ecdysone than for ecdysterone. Ecdysone and ecdysterone used as standards for the RIA were purchased from Simes (Milan, Italy). Results are expressed as nanograms of ecdysone and ecdysterone equivalent. Musca bioassay. The Musca bioassay was carried out according to the method of Kaplanis et al. (1966) but by a ligature of the larvae in rubber bands (Staal, 1967). Gas-liquid chromatography--mass spectrometry (glc-ms). The glc-ms was performed on a LKB 9000 instrument using a 2-m x 2-mm silanized coiled glass column with 1% OV-1 on 100-120 mesh silanized Gas Chrom P. Gas liquid chromatography conditions were the following: column temperature, 28.5”, flow rate of carrier gas (helium) 30 mllmin. Fragmentograms and mass spectra were recorded at 28 eV. Silylniion. Ecdysteroids were silylated with trimethylsilyhmidazole from Koch-Light. Silylation was performed at 6S0 overnight (Delbecque et al., 1975, 1978). Ecdysone (ol-ecdysone), makisterone A, and 26-hydroxyecdysone used as standards for silylation were generous gifts from Hoffmann-La Roche (Basle, Switzerland), Simes (Milan, Italy), and Dr. Kaplanis, respectively. Ecdysterone (fi-ecdysone, 20-hydroxy-ecdysone) and inokosterone were purchased from Schwarz/Mann.
Purification
and Quantification
of JH
After extraction in ethyl acetate/ethanol, S/l (v/v) extracts were purified by tic (6% ethyl acetate/ benzene) as described by Lanzrein et a/. (1975). For determination of JH activity the Galleria wax test was
EMBRYOS
629
used (De Wilde et al., 1968). Under our conditions 1 Galleriu unit (GU) corresponds to 8 pg IH III (Calbiochem). For characterization of JH, tic-purified extracts were injected into a high pressure liquid chromatograph (Du Pont 848) equipped with a variablewavelength uv detector (Perkin-Elmer) using a column Zorbax SIL (Du Pont) and 6% ether hexane as solvent.
RESULTS Changes in Weight of Egg Cases during Embryagenesis Egg cases throughout embryonic development were observed and their fresh weight and dry weight were measured (Fig. 1). From Day 0 to 13 the egg content appears to be very viscous. On Day X3 the fresh weight begins to increase rapidly and at that time lipid droplets appear in the egg. The fresh weight increases until the 19th day when dorsal closure occurs. Another minor increase in’fresh weight is observed between Days 26’and 30. Three days later the egg case and the chorion break on the side of the embryos’ heads which from now on are only covered by the embryonal cuticle. Since the dry weight of the egg case decreases gradually throughout embryonic development it seems likely that increases in fresh weight are due to uptake of water only. A similar uptake of wat,~r-although less drastic-was observed by Engelmann (19.57) in Leucophaea. Ecdysteroid Cases
and JH Activity in Whole Egg
Egg cases were divided into halfs: One half was extracted and purified for erdysteroid determination with the Musca bioassay and the other half for JH determination with the Galleria wax test (Fig. 2). Ecdysteroid activity was detected in small amounts at the time of the dorsal closure and in higher amounts around Days’29 and 32. JH-active material could be detected only after the dorsal closure had occurred. Two peaks were observed, namely one on Day 27 and one on Day 33 at the time of the
630
IMBODEN mgkgg
ET AL.
case
250
--f-f-------*
dry weight f
-*--I--------.5-* 50 0
0
Nletton
lb
2b dorsaltclos”re
so hatching
FOG. 1. Fresh weight and dry weight of egg cases from ovulation to hatching of the larvae. Mean values k SE; 30-70 determinations per point.
G.Ulg .I04
ecd.eq./g
n.urg
n9 a30
50
ecdysone ecdysterone JH
A-....... A x-x
I F
,’:?b /Ii I 400
00
2oQ
a
FIG. 2. Ecdysone, ecdysterone, and JH activity in whole egg cases during embryogenesis. Ecdysone and ecdysterone were determined by Mtiscu bioassay after separate elution from thinlayerplates and the results are expressed in ecdysone or ecdysterone equivalents and in Musca units (MU). JH activity was determined by Galieria bioassay and is expressed in Galkria units (CU). Each symboi represents one determination.
ECDYSTEROIDS
AND
JH
rupture of the chorion. Thus, the Galleria and Musca bioassay results demonstrate, dependent on the stage of embryogenesis, the presence of JH-active and ecdysteroidactive material in egg cases. In order to further identify the ecdysteroids, tic-purified extracts of 32 dayold-egg cases were analyzed by glc-ms. A mass fragmentogram of the combined tic zones (except the origin) is given in Fig. 3. The mass of m/e 561 is a characteristic fragment of fully silylated ecdysterone and other 20-hydroxy-ecdysteroids which only differ from ecdysterone by the side chain (e.g., inokosterone, makisterone); the mass of m/e 567 is characteristic for fully silylated ecdysone and other ecdysteroids having no hydroxyl at C-20 and differing from 21
IN
COCKROACH
, 5
,
* 10 ’
’
’
I xia *
’
’
I 20I r
I min
FIG. 3. Mass chromatogram of ecdysteroid derivatives in silic acid-purified egg case (32 days otd). Penta-trimethylsilyl (TMS) derivative of ecdysone (A) is detected at m/e 567 (retention time 9 min). HexaTMS derivative of ecdysterone (B) is detected at m/e 561 (retention time 11.5 min). (C) Corresponds to penta-TMS derivative of ecdysterone and/or traces of makisterone A (retention time 1.5 min). TMS derivative of 26-OH-ecdysone (D) is detected at m/t 567 (retention time 14.5 min). (E) Possibly represents 20,26 dihydroxyecdysone. (*) Indicates unknown compounds (possibly not ecdysteroids). (y/) indicate unidentified isomers of ecdysterone. Taking into account the electronic amplification and the relative response of ions, track at m/e 567 is 4.3-fold more sensitive.
431
ecdysone by the side chain, as 26hydroxyecdysone (Delbecque ei; nl., in preparation). According to mass fragments and retention times, Fig. 3 shows the presence of ecdysone, ecdysterone, which is the predominant ecdysteroid, and 26 hydroxyecdysone, However, the presence of other 20-hydroxy-ecdysteroids, having fragments at pnle 561, is indicated: One of them shouldering ecdysterone peak could be an isomer, possibly inokosterone or an epimer: another molecule is detected, with a greater retention time than makisterone, thus a heavier side chain, suggesting it could be 20,2~hy~oxyecdyso~e. Thinlayer chromatography zones corresponding to ecdysone and to ecdysterone were analyzed separately and again demonstrated the presence of ecdysone and ecdysterone. The results indicate a, good separation of ecdysone, ecdysterone, and other ecdysteroids in the tic system used. In order to characterize the Jo-active material, tic-purified extracts were injected into a high pressure liquid chromat~g~a~h~ Fractions were collected and tested in the Galieria wax test. It was found th’at most of the JH activity elutes with the retention time of JH III, thus suggesting the presence of JN III in egg cases. Ecdysteroids
,
EMBRYOS
and JH in Egg Canrenr arzd
After having demonstrated the presence of JH, ecdysone, and ecdysterone in whole egg cases, we investigated whether these hormones are accumulated in the egg content and later in the h~olym~h of cmbryos. The analysis is given in Fig. 4 and reveals close similarities to the resuhs with whole egg cases (Fig. 2). ~~dyster~~ds are detectable already before dorsal closure and shortly after each JH peak,, Again JH appears in two peaks after dorsal closure has occurred. A comparison of Fig. 4 with Fig. 2 reveals that ecdysteroids as well as JH are accumulated in the l~~rnolyrn~~ of the embryo.
632
IMBODEN
ET
AL.
FIG. 4. Ecdysone, ecdysterone, and JH activity in egg content and hemolymph of embryo during embryogenesis. Ecdysone and ecdysterone were determined by RIA after separate elution from thin-iayer~plates and the results are expressed in ecdysone or ecdysterone equivalents, respectively. JH activity was determined by G~~Ileria bioassay and is expressed in Gallerid units (GU). Each symbol represents one determinarion.
Ecdysteroids and JH in the Hemolymph Pregnant Females
of
stage of dorsal closure were isolated and put into a humid environment. Thirteen days later, at the time corresponding to Day 32 of normal development when JH and ecdysteroid titers are high, parts of the egg cases were extracted and tested for the presence of JH and ecdysteroids. We measured 120,000 GU JH, 300 ng ecdysone, and 2,300 ng ecdysterone equivalents/ml hemolymph. These values are similar to those of Day 32 in normal development (Fig. 4). The other parts of the egg cases were left in the humid environment and the embryos hatched at the expected time. These findings demonstrate that embryos after dorsal closure produce JH and ecdysteroids themselves and are able to develop without interference of the endocrine system of the mother.
Nothing is known on the origin of these hormones in the embryos and since embryogenesis occurs within the female we also included ecdysteroid and JH determinations in the hemolymph of the pregnant females (Fig. 5). Small amounts of ecdysone and ecdysterone .could be detected in the hemoIym~h of the pregnant female from ovulation until dorsal closure of the embryo. Thereafter only traces of ecdysteroids were measurabIe. No JH could be detected before the rupture of the chorion. Thereafter the titer of JH increases continuously. Examination of the ovaries revealed that simultaneously with the increase of JH titer the growth of the next generation of oocytes is initiated DISCUSSION (Imboden, unpublished). We also investigated the development of Our results with the ovoviviparous cockeggs in isolated egg cases. Egg cases at the roach Nauphoeta cinerea demonstrate the
ECDYSTEROIDS
AND JH IN COCKROACH
EMBRYOS
633
ecd.eq./mi
FIG. 5. Ecdysone, ecdysterone (RIA activity), and JH activity in the hemolyl~ph of females during pregnancy. Each symbol represents one determination. Note that the scale for ecdysteroids is IOO-fold enlarged as compared to Fig. 4.
presence of ecdysteroids and JH in egg cases and hemolymph of embryos. The use of glc-ms allowed the identi~cation in egg cases of ecdysone, ecdysterone, and 26hydroxyecdysone. In addition the presence of other ecdysteroids (as possibly 20,26dihydroxyecdysone~ is suggested. Analysis of the JH-active material by hplc and Gallevia bioassay suggests the presence of mostly JH III. Since little information is available on changes of ecdysteroid and JH titers as a function of embryonic development we first made JH and ecdysteroid determinations in egg cases and hemolymph of embryos at different developmental stages. If one compares the curves obtained with egg cases (Fig. 2) with those obtained with egg content and hemol~m~h of embryos (Fig. 4) strong similarities are obvious and indicate an accumulation of ecdysteroids and JH in the hem~lymph of the embryo. The role of these hormones in embryo-
genesis is not clear yet, but in Eocldsru a role of ecdysteroids in controlling the secretion of serosal cuticle is suggested (Lagueux and Hoffmann, submitted), A small peak of ecdysteroids is observed before dorsal closure and two high peaks, ranging up to 1-3 pgiml, appear adored Days 25 and 33, respectively. Ecdysterone is always by far more abundant than ecdysone. At about the same time, i&e., around Days 25 and 33, we found two peaks of JH, 10 x lO$ and 15 x lo4 GLJlml, resflectively. These quantities are higher than ithe highest values observed in reproducing females which reach only 1.5 x 1P Gulml (Lanzrein et nl., in pre~a~~io~}. Since it is generally believed that the coincidence of a high molting hormone titer with a high JH titer Causes rno~~j~g, to a larva ~Sc~~eid’~rrnan and Gilbert, 1964: Gilbert and King, 1973)’ the peaks of eedysteroidsand JH are perhaps ~~n~~~ted ix/ith one or two embryonic molts. jeweler, we
634
IMBODEN
do not know yet if and at what time embryonic molts occur in Nauphoeta. A simultaneous peak of JH activity (Tenebrio bioassay) and ecdysteroid activity (Calliphora bioassay) has also been observed during embryogenesis in Oncopeltus by Dorn (1975, 1976) who suggested a correlation with an embryonic molt. Little is known about the site of origin of ecdysteroids and JH in embryos. The ecdysteroids present in toccata in early embryonic development (i.e., before the endocrine glands of the embryo are differentiated) are synthesized in the ovaries of the mother and then accumulated in the cells of the serosa (Lagueux et al., 1977; Lagueux and Hoffmann, submitted). In ~~alo~hora cecropia (Gilbert and Schneiderman, 1961) eggs of allatectomized females do not contain JH-active material whereas eggs from normal females do contain JH, thus suggesting that JH of the hemolymph .of the mother is transmitted to the eggs. We do not know yet where JH and ecdysteroids are synthesized during embryogenesis in ~u~p~oet~. Before dorsal closure eggs contain only small amounts of ecdysteroids and no detectable JH. lt is noteworthy that also the hemolymph of young pregnant females, whose prothoracic glands are degenerated (Lanzrein, 1975), contains small amounts of ecdysteroids. It is not yet known where the ecdysteroids of the embryo and the mother are produced and what their function is. After dorsal closure embryos produce JH and ecdysteroids themselves since it was found that eggs in cases which were removed from the mother at the time of dorsal closure were able to develop and have similar titers of JH and ecdysteroids as eggs developing in the brood sac of the mother. Histological investigations revealed that after dorsal closure the CA and prothoracic glands of the embryos are well differentiated and that their cells have voluminous nuclei. This may indicate that ecdy-
ET AZ,.
steroids and JH are synthesized by the embryos’ endocrine glands. Electron microscopic investigations of the embryonic CA of Oncopeltus (Darn, 1975) also led to the conclusion that the glands might be functional. Sites of molting hormone synthesis other than the prothoracic glands, like oenocytes, have been repeatedly suggested in the literature (Dorn and Romer, 1976; Locke, 1969; Romer et al., 1974; Studinger and Willig, 1975; Hsiao et crl., 1975) and cannot be excluded for the embryo of Nauphoeta. ACKNOWLEDGMENTS Thanks are due to Professor J. D. O’Connor (Department of Biology, University of Cahfornia, Los Angeles) for the ecdysone antiserum and to Dr. P. Masner (Biological Laboratory of Dr. R. Maag, Dielsdorf) for his help with the Musca bioassay. We also thank Professors Maume and Padieu (Departement de Bioehimie, Universite de Dijon) for the use of the LKB 9000 glc-ms apparatus. Financial suppoft by Swiss National Foundation Grant 3.188.77 and by a grant for international collaboration of the CNRS (France) is gratefully acknowledged.
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ECDYSTEROIDS
AND
JH
titre. in the haemolymph as a function of photoperiodic treatment in the adult Colorado beetle (Leptinotursa decemlineata). Proc. Ken, Ned. Akad. Wetensch. Ser. C 71, 321-326. Darn, A. (1975). Struktur und Funktion des embryonalen Corpus allatum von Oncopeltus fasciatus Dallas. Verh. Deut. Zoof. Ges. 85-89. Darn, A., and Romer, F. (1976). Structure and Rmction of prothoracic glands and oenocytes in embryos and last larval instars of Oncopeltus fasciatus Dallas. Cell Tissue Res. 171, 331-3.50. Engelmann, F. (1957). Bau und Funktion des weibIichen Geschlechtsapparates bei der ovoviviparen Schabe Lelacophaea maderae (Orthoptera) und einige Beobaehtungen iiber die Ent~cklung. Biol. Zentralbl. 76(6), X2-740. Gilbert, L. I., and King, D. S. (1973). Physiology of growth and deveIopment: Endocrine aspects. In “The PhysioIogy of Insecta” (Rockstein, ed.) Vol. 1, pp. 249-370. Academic Press, New York. Gilbert, L. I., and S~hneiderman, II. A. (1961). The content of juvenile hormone and lipid in Lepidoptera: Sexual differences and developmental changes. Gen. Comp. Endocrinol. 1,453-472. Horn, D. H. S., Sage, B., and O’Connor, J. D. (1976). A high affinity antiserum specific for the ecdysone nucleus. 1. Insect Physiol. 22, 901-905. Hsiao, T. H., Hsiao, C., and De Wilde, J. (1975). Moulting hormone production in the isolated abdomen of the Colorado beetle. Nature (London) 255, 727-728. Kaplanis, J. N., Dutky, S. R., Robbins, W. E., Thompson, M. J., Lindquist, E. L., Horn, D. H. S., and Galbraith, M. N. (1975). Makisterone A: A Z&carbon hexahydroxy moul~ng hormone from the embryo of the milkweed bug. Science 190, 681-682. Kaplanis, J. N., Robbins, W. E., Thompson, M. J., and Dutky, S. R. (1973). 26-Hydroxyecdysone: New insect molting hormone from the egg of the tobacco harnworm. Science 180, 307-308. Kapianis,J. N., Robbins, W. E.~Thompson,M.3., and Dutky, S. R. (1976). The molting hormones from the embryonated egg of the tobacco hornworm, ~u~d~c~ ‘sexta (L.). steroids 27(.5), 675-679. Kaplanis, .I. N., Tabor, M. J., Thompson, W. E., Robbins, W. E., and Shortino, T. J. (1966). Assay for ecdysone (moit~ng hormone) activity using the
IN
COCKROACH
EMBRYOS
635
housefly, ~uscff dor~est~ca L. steroids 8, 625631. Lagueux, M., Hirn, M., and Hoffmann, 9. A. (I977). Ecdysone during ovarian deve~opment,~n i;acusta migratoria. J. Ivrsect Physiol. 23, 109,119. Lagueux, M., and Hoffmann, J. A. Maternal ecdysteroids and early embryonic development in Locusfu migrator~u L., submitted. Lanzrein, B. (1975). Programming, induction, or prevention of the breakdown of the prothoracic gland in the cockroach, ~aup~oeta cinerea. J. Insect Physiol. 21, 367-389. Lanzrein, B., Hashimoto, M., Parmakovich, V., Nakanishi, K,, Wilhelm, R., and Liischer, M. (1975). Identification and quanti~cat~on of juvenile hormones from different deveiopmentaI stages of the cockroach Nauphoeta cinerea. Life Sci. 16, 1271-1284. Locke, M. (1969). The ultrastructure of oenocytes in molt/intermolt cycle of an insect. Tisscla Cell 1, 103-154. Matz, G., and Hoffmann, J. A. (1975). “Tine d’hormone de mue et metabolisme d’a-ecdysone radioactive au cours du dhetoppement embrysnnaire de Leucophaea maderae.” Reported at 3” Gall. Physioi. Insectes, Strasbourg, France. Ohnishi, E., Mizuno, T., Chatani, F., Ikekawa, N.i and Sakurai, S. (1977). Z-Deoxy-tw-ecdysoae from ovaries and eggs of the silkworm, Bomhyx mari, Science 197, 66-67. Ohnishi, E., Mizuno, T., Ikekawa, N., Awata, N., and Sakurai, S. (1977). Occurrence of a-ecdysone in the developing embryos of the silkworm, Bombyx mori. J. Insect Physiol. 23, 337-319. Romer, R., Emmerich, H., and Nowock, .I-(1974)). Biosynthesis of ecdysones in isolated protboradc glands and oenocytes in Tenebrio molitor in vitro. J. IrLsrct. Pizysjol.20, 1975-1987. Schneiderman, H. A., and Gilbert, L. I. (1964). Control of growth and development in insects. Srience 143, 325-333. Staal, G. B. (1967). Plants as a source of insect hormones. Proc. Ken. Ned. Aked. Wetensch. Ser. C 70(4), 409-418. Studinger. G., and Wihig, A. (1975). Bjasynthesi~ of o- and @-ecdysone in isolated abdomens of larvae of Musca domestica. 1. Insect Physiol. 21, 17931798.