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The effects of juvenile hormone, 20-hydroxyecdysone, precocene II, and ovariectomy on the activity of the corpora allata (in vitro) in adult female Gryllus bimaculatus
J. Insect Physiol. Vol. 35, No. 4, pp. 299-304, Printed in Great Britain. All rights reserved
1989
Copyright 0
OO22-1910/89 $3.00 + 0.00 1989 Pergamon Press plc
THE EFFECTS OF JUVENILE HORMONE, 20-HYDROXYECDYSONE, PRECOCENE II, AND OVARIECTOMY ON THE ACTIVITY OF THE CORPORA ALLATA (hV ?K!‘-‘RO) IN ADULT FEMALE GRYLLUS R. Allgemeine
Zoologie
WENNAUER,
(Biologic
BIMACULATUS
L. KASSEL
I), Universitlt
and K. H.
Ulm. Oberer
HOFFMANN*
Es&berg,
D-7900
Ulm/Donau,
F.R.G.
(Received 20 June 1988: rerised 19 September 1988) Abstract-Corpora allata from adult female Gry//us bimacularus exhibit rates of juvenile hormone III synthesis in vitro which are closely correlated with oocyte maturation. Juvenile hormone III treatments in uino (injection of 0.5 gg juvenile hormone III) of females reared at 20°C (animals with spontaneously inactive corpora allata) stimulated juvenile hormone synthesis within 12 h. The results could be verified as a de noun biosynthesis, by following the incorporation of radioactivity from [2-14C]acetate and L-[methyl-‘Hlmethionine into juvenile hormone III. Addition of juvenile hormone III in vitro (10-8-10-“ M) did not stimulate de now synthesis of juvenile hormone III. In females from a rearing temperature of 20°C a stimulating effect of 20-hydroxyecdysone injection (0.2555 pg) on juvenile hormone III synthesis became apparent in riro within 24 h. No stimulation was observed when isolated corpora allata were maintained for 34 h in medium containing 5. 1O-5 M ecdysone or 20-hydroxyecdysone. Female adult crickets quickly recover from treatment in tk~o with precocenes. After incubation of isolated corpora allata in the presence of precocenes, juvenile hormone III synthesis was diminished in a dosedependent manner. In ovariectomized females juvenile hormone III synthesis was drastically suppressed. Key Word Index:
Corpora allata, juvenile hormone III, precocene, 20-hydroxyecdysone, ovariectomy, reproduction, Gryllus bimaculatus
INTRODUCTION
Females of many insect species exhibit cyclical patterns of reproductive activity. Associated with the gonotrophic cycles are changes in activity of the corpora allata. Moreover, perception of social, chemical or physical factors by the insects may result in altered corpora allata activity (Feyereisen, 1985). The various patterns of corpora allata activity during reproduction and their modulation by external factors imply the existence of control mechanisms for corpora allata activity. Extensive experimental evidence shows that the corpora allata are regulated by stimulatory and inhibitory signals which reach the glands either via the haemolymph (humorally) or via nervous connections (for reviews see Tobe, 1980; de Kort and Granger, 1981; Feyereisen, 1985). A convenient direct method for estimating the spontaneous activity of the corpora allata is the short-term in rho radiochemical assay, as introduced by Pratt and Tobe (1974) and Tobe and Pratt (1974, 1975). This method relies on the incorporation of the methyl moiety of r_-[methyl-‘4C]methionine into juvenile hormone. The assay, adapted for Gryllus bimaculutus (Koch and Hoffmann, 1985), has been employed recently to show that corpora allata activity in adult female crickets is regu-
*To whom
correspondence
should
be addressed.
lated in a temperature-dependent manner (Espig and Hoffmann, 1985). In corpora allata of females reared at 27°C or at a daily alternating temperature regime of 24: 12°C (16:8 h) (in both temperature regimes reproduction rate is high), a first peak in juvenile hormone III synthesis is reached 48 days earlier than in females from 20°C (where reproduction rate is low). Furthermore, in animals from 20°C corpora allata activity is low during the entire oviposition period, whereas at 24: 12°C and 27°C respectively, high corpora allata activity is found during this period of adult life. Application of juvenile hormone III to females with a low corpora allata activity induced a dose-dependent stimulation of egg production and oviposition (Koch and Hoffmann, 1985). Further experiments had shown that in female adults of G. bimaculatus the concentrations of free ecdysteroids in haemolymph and tissues are also temperature-dependent (Hoffmann et al., 1981) and that exogenous ecdysteroids stimulate egg deposition rates in females with a low endogenous ecdysteroid titre (Behrens and Hoffmann, 1983). The chronological sequence of increasing corpora allata activity, vitellogenin synthesis, ovarian weight gain, ecdysteroid production, and oviposition in G. bimaculatus (Hoffmann, 1986) suggests that the onset of vitellogenin synthesis and reproduction is related to the first increase in juvenile hormone III synthesis. The results from the ecdysteroid injection experiments, however, indicate that in G. bimaculatus, as well as in the
R.
300
WENNAUER et
house cricket, Acheta domesh.ts (Chudakova et al., 1982), both of the hormones, juvenile hormone III and moulting hormone, are involved in the control of reproduction . In the present paper we investigated regulatory interactions between 20-hydroxyecdysone and juvenile hormone III synthesis in G. bimaculatus. using a combination of in vivo and in vitro techniques. The injection of juvenile hormone III or 20-hydroxyecdysone into females or the presence of the hormones in the incubation medium were assayed for effects on juvenile hormone III biosynthesis by the corpora allata in vitro. The effect of precocene II. which has been reported to influence the corpora allata of hemimetabolous insect species (Soderlund et al., 1981), is included in this study. Finally, we studied the effects of ovariectomy on juvenile hormone III biosynthesis.
MATERIALS
AND METHODS
Animals and surgery
Mediterranean field crickets, G. bimaculatus de Geer, were reared either at a constant 20 and 27°C respectively, or at a daily alternating temperature regime of 24 : 12°C (16 : 8 h) and a 16 h light-8 h dark cycle as described previously (Behrens et al., 1983). Freshly ecdysed females (l- 12 h) were collected from the stock and maintained separately. Injections and operations were performed with carbon dioxideanaesthetized animals. Corpora cardiaca-corpora allata complexes were dissected as described in Koch and Hoffmann (1985). Special care was taken to remove adjacent tissue from the glands. (In G. bimaculatus salivary glands are tightly attached to the corpora allata.) Ovariectomy was performed in lastinstar females from 27°C as described in KempaTomm (1987). In vivo treatment of females
Female crickets were injected 2 days after the imaginal ecdysis through the intersegmental membrane between the hind leg coxa and the abdomen by means of a 10~1 Hamilton syringe. Females from a rearing temperature of 20°C were injected either with 20-hydroxyecdysone (Simes, Milan; 0.25-5 pg of hormone) in insect Ringer (85 mM NaCl, 6 mM KCl, and 3 mM CaCl,; 5~1) (Behrens and Hoffmann, 1983) or with juvenile hormone III (Fluka, Neu-Ulm; 0.5 fig) in insect Ringer (5 ~1) containing 0.05% (v/v) Tween 80 (Merck, Darmstadt). Approximate haemolymph volume of 2-day-old females from 20°C is 200~1 (Ehler et al., 1986). Precocene II (6,7-dimethoxy-2,2-dimethyl-3-chromen; Sigma, Munich; 100 pg) was dissolved in acetone (5 ~1) and topically applied onto the less sclerotized pleural region of the abdomen of 2-day-old females reared at 27°C. Control animals were treated with insect Ringer and acetone, respectively. 3-48 h after treatment the corpora cardiacs-corpora allata complexes were dissected for the radiochemical assay. In vitro treatment of corpora allata Corpora cardiaca-corpora allata complexes were removed from 2- to I-day-old females. Individual
al.
pairs of gland complexes were thoroughly rinsed in insect Ringer and transferred into 100 ~1 of incubation medium 199 (Flow Laboratories, England; with Hanks’ salts, without L-glutamine and Na,CO,. buffered with 20 mM Hepes), fortified with 1% (w/v) Ficoll 400 (Sigma, Munich). The pH of the final medium was 6.5 (Koch and Hoffmann, 1985). Test compounds (juvenile hormone III dissolved in 0.55% (v/v) Tween 80, 20-hydroxyecdysone in distilled water, and precocene II in 0.55% (v/v) Tween 80; 10 ~1 each) were added to the medium (for final concentrations see Results). Control groups consisted of gland complexes incubated in medium containing the solvents for the test compounds only. If not otherwise stated incubations were performed at 20°C in darkness for 180-240 min. Juvenile hormone biosynthesis by the corpora allata
Biosynthesis of juvenile hormone III by individual pairs of corpora allata was determined using the radiochemical in vitro assay, as introduced by Pratt and Tobe (1974) and Tobe and Pratt (1974, 1975). The incorporation of the methyl moiety of L-[methyl“C]methionine (Amersham, England; final sp. act. 34-40 mCi/mmol; final concentration of L-methionine 0.26mM; Koch and Hoffmann, 1985) into juvenile hormone III was followed for 3-4 h at 20°C. To determine de novo juvenile hormone III biosynthesis incorporation of radiolabel was measured after 4 h incubation of gland complexes at 27°C in medium supplemented with L-[methyl-3H]methionine (Amersham, England; initial sp. act. 15 Ci/mmol) and [2-14C]acetate (Amersham, England; initial sp. act. 56 mCi/mmol) and appropriate amounts of unlabelled methionine and acetate to give final concentrations of 0.26mM methionine (final sp. act. about 55 mCi/ mmol) and 2 mM acetate (final sp. act. about 10 mCi/ mmol). Juvenile hormone III and methyl famesoate were measured following separation by thin-layer chromatography (silicagel 60 F,, plates; toluene:ethyl acetate: acetic acid, 70: 30: 1 (v/v) as solvent system). 14C and ‘H radioactivity were determined by liquid scintillation counting in Rotiscint 2211 (Roth, Karlsruhe) scintillation fluid. The results expressed as “JH III synthesis” indicate that gland complexes plus the incubation medium were extracted (Tobe and Stay, 1977). Values are means + SEM for the number of individual measurements indicated on each figure. A t-test was used in statistical analyses. RESULTS
Synthesis of radiolabelled products by the corpora alfata in vitro
The corpora allata of G. bimaculatus produce juvenile hormone III (Koch and Hoffmann, 1985). There is a linear relationship between the rate of juvenile hormone III synthesis and its release. A further incubation product, which is not released into the incubation medium, was identified as the 2E, 6E-isomer of the juvenile hormone III precursor, methyl farnesoate by co-chromatography with the reference substance in thin-layer chromatography (silicagel 60 F2%) and HPLC (Waters PBondapak
Regulation of corpora allata activity in crickets
301
- 61 20
==6
2: j3
c 3
12
24 t?e
(h)
Fig. 1. The effect of juvenile hormone III injections after various periods of time (348 h) on the rate of juvenile hormone III (JH III) synthesis (in oirro) of 2-day-old females reared at 20°C. Experimental animals (crosshatched columns) were injected once with 0.5 pg juvenile hormone III. Controls (open columns, C) received 5 ~1 of insect Ringer with 0.05% (v/v) Tween 80.
Cl8 reversed phase column; 1985).
Koch and Hoffmann,
Effect of juvenile hormone III injection
After a single injection of 0.5 pg juvenile hormone III into females reared at 20°C at day 2 after ecdysis activity of corpora allata in vitro increased about 7-fold (Fig. 1). The increase in juvenile hormone III synthesis became apparent 12 h after injection, and the activity remained high, even 48 h after treatment (P < 0.057 at 12 h; P < 0.019 at 48 h). The incorporation of radioactivity from [2-‘4C]acetate into juvenile hormone III and its immediate precursor, methyl farnesoate (Fig. 2) indicates that this increase is due to a de novo synthesis of juvenile hormone III from acetate. Figure 2 also demonstrates that already active glands (juvenile hormone III synthesis of 7.4 pmol h-’ per pair vs 0.4 pmol h-’ per pair of glands in Fig. 1) were further stimulated by juvenile hormone III treatment in vivo. Effect in vitro of exogenous juvenile hormone
In order
to test whether
the hormone
influ-
e2 ,” 1 E ~Id-IL 4
1
l3
20
control
0.25
0.5
L 5 w.X-WE
Fig. 3. Juvenile hormone III (JH III) synthesis 24 h after injection of 0.25-5 pg 20-hydroxyecdysone (20-OH-E) or control solution (5 ~1 insect Ringer) into 2-day-old females reared at 20°C.
ences glands directly, 10-s-10-4 M juvenile hormone III were included in the incubation medium of corpora cardiaca-corpora allata complexes from 2-day-old females reared at 20°C and rates of juvenile hormone biosynthesis were followed by the incorporation of radioactivity from [2-14C]acetate and L-[methyl-3H]methionine into juvenile hormone III. Addition of juvenile hormone III in vitro did not stimulate de novo synthesis of juvenile hormone III (all values lower than OSpmol h-’ per pair; not shown in figures). In vivo effect of 20-hydroxyecdysone
Figure 3 shows that injection of the hormone into females with a low endogenous moulting hormone titre (20°C; 2 days after ecdysis) causes a stimulation of juvenile hormone III biosynthesis within 24 h. The synthesis of juvenile hormone III increased from a basal level of 1.2 + 1.3 pmol h-l per pair up to 8.1 + 2.2 pmol h-’ per pair in response to 5 pg 20-hydroxyecdysone (P < 0.030 at 2 pg; P < 0.014 at 5 fig). Effect in vitro of free ecdysteroids
3500
I 3000
2500
2000
.j P g 2 p
Glands were incubated over a 4 h period in medium containing 5. 10m5M ecdysone or 20-hydroxyecdysone. No differences were observed in the rate of juvenile hormone III synthesis between the corpora allata incubated in the presence (3.9 + 1.5 pmol h-’ per pair for ecdysone) or absence (3.7 + 1.0 pmol h-’ per pair) of the ecdysteroids.
.c
rsoop 1000
h 0
600
0.5 po .Jn III
Fig. 2. Juvenile hormone III (JH III) synthesis (in oitro) (b) and the amount of methyl famesoate (MF) (a) in corpora allata of 3-day-old 20°C females 24 h after injection of 0.5pg juvenile hormone III. Incorporation of the methyl moiety of [‘Hlmethionine was used as mass-marker (open columns). Right ordinates and cross-hatched columns: incorporation of [Wlacetate (dpm) into MF (a) and JH III (b) after 4 h incubation at 27°C. Controls (C) received 5 ~1 of insect Ringer with 0.05% (v/v) Tween 80. Mean values of 5 determinations k SEM.
Eflect of precocene II
Previous experiments had shown that repeated topical application or injections of 150 pg precocene II did not affect ovarian development and oiicyte growth in maturing females (W. Espig, unpublished results). These results agree well with the present observations on the in vivo effect of precocene II on juvenile hormone synthesis. When 100 pg precocene II were applied onto 2-day-old females from a rearing temperature of 27°C (animals with spontaneously active glands), their corpora allata activity was reduced 12 h later, but restored to control values within 24 h (Fig. 4). After an incubation of isolated corpora allata (from 8-day-old females reared at 24: 12°C; 16: 8 h)
302
R. WENNAUER et al
40. 2 k
35. 30
L g
25.
-
20.
l
15.
P
IO-
i 5
c 12
C24
5-
Fig. 6. Juvenile hormone III (JH III) synthesis (in vitro) by corpora allata of adult females ovariectomized (Ovv) or sham-operated (Co) as last instars. Rearing temperature 27°C. Mean values of 10 determinations.
C48 time (h)
Fig. 4. The effect of topical application of 1OOng of precocene II dissolved in 5 ~1 acetone on the rate of juvenile hormone III (JH III) biosynthesis of spontaneously active corpora allata (from females reared at 27°C). Open columns, controls (C) which had been treated with 5 ~1 acet-
one; cross-hatched columns, experimental animals 1248 h after treatment.
for 3 h in various concentrations of either precocene I (not shown in figure) or precocene II (Fig. 5) a dose-dependent inactivation of the glands was found. Spontaneously active corpora allata were inhibited completely by exposure in vitro to lOa3 M precocenes. The amount of methyl farnesoate in the glands which were treated with precocenes was also reduced. Ovariectomy
In this experiment we investigated the interdependence between corpora allata and the ovary.
r 13
12 11 10 SP s* 7k 6. 5 Ii 4% 3 2 I control
lo-'
10-0
lo-. 10-a M precocene II
Fig. 5. Effects of exposure of corpora allata to precocene 11 in vitro(10-7-10-3 M in the incubation medium) on juvenile hormone III (JH III) biosynthesis (open columns) and on the amount of methyl farnesoate (MF) within the glands (cross-hatched columns). Controls were treated with 10 ~1 of 0.55% (v/v) Tween 80.
Ovaries were excised from female last larval instars and the corpora allata activity (in vitro) was determined between days 0 and 22 of adult life (Fig. 6). In sham-operated controls we measured corpora allata activities which were similar to those of untreated females (Koch and Hoffmann, 1985). In ovariectomized females juvenile hormone III synthesis was high shortly after ecdysis, but suppressed during the subsequent period of adult life. DISCUSSION
The combination of in uivo application of effecters and in vitro determination of corpora allata activity allows the study of potential factors which may be involved in the regulation of corpora allata activity. Evidence is available which demonstrates that the rate of juvenile hormone synthesis in vitro shortly after extirpation of the glands is a good indicator of the rate of juvenile hormone synthesis of those glands in situ (for reviews see Tobe and Feyereisen, 1983; Feyereisen, 1985). A linear relationship between the rate of juvenile hormone III synthesis and its release shows that corpora allata of G. bimaculatus do not store juvenile hormone III. Similar observations were done by Tobe and Stay (1977) for corpora allata of Diploptera punctata, but also for many other insect species. The existence of humoral factors directly controlling corpora allata activity has been postulated for a long time, but none has been characterized to date (Feyereisen, 1985). Mechanisms for the control of copora allata activity by experimentally modified titres of juvenile hormone have been described, however, and demonstrate the existence of complex feedback loops (indirect control). Evidence for a negative feedback effect of exogenous juvenile hormone (or juvenile hormone analogues) on juvenile hormone biosynthesis has been presented, e.g. for Leptinotarsa decemlineata by Schooneveld et al. (1979) and Khan et al. (1982), and for Diploptera punctata by Tobe and Stay (1979, 1980). In larvae of Manduca sexta juvenile hormone levels were suppressed in a dose-dependent fashion following topical treatment with the juvenoids fenoxycarb or hydroprene (Baker et al., 1986). The
Regulation of corpora allata activity in crickets
of a positive feedback loop, which would stimulate juvenile hormone synthesis in response to an elevated juvenile hormone titre, was suggested by Tobe and Stay (1979). Topical application of 2.5 pg of hydroprene to newly emerged female D. punctuta stimulated juvenile hormone synthesis on day 4 after moulting. The feedback stimulation is suggested to occur by way of the ovary (Stay and Rankin, 1986). Further indication of a positive feedback mechanism has been given by Kozhanova and Chudakova (1985) who suggested that the juvenile hormone analog methoprene activates corpora allata of the house cricket, Acheta domesticus, to secrete endogenous juvenile hormone. Our results (Figs 1 and 2) also indicate that during an experimental elevation of the juvenile hormone titre in oioo (injection of 0.5 pg juvenile hormone III into adult females from 2O”C, which have a low endogenous juvenile hormone level) positive feedback regulation occurs. The increase in juvenile hormone III synthesis is due to a de nova synthesis from acetate. Our experimental data, however, do not show that juvenile hormone influences corpora allata directly. Addition of juvenile hormone III to the incubation medium did not stimulate de novo synthesis of juvenile hormone III from acetate within the incubation time of 4 h. Several studies have been conducted on juvenile hormone-ecdysteroid interactions in adult insects, and most of them suggest that ecdysteroids have an allatostatic effect (Friedel et al., 1980; Stay et al., 1980; Lanzrein et al., 1981; Rankin and Stay, 1985). In former experiments we have shown that exogenous ecdysteroids stimulate ovarian growth and egg deposition rates in females with a low endogenous ecdysteroid titre (Behrens and Hoffmann, 1983). Repeated injections of 2-5 pg ecdysone or 20-hydroxyecdysone, respectively, caused an increase in the number of eggs per female more than twice. The results of this paper demonstrate that 20-hydroxyecdysone may be a positive regulator of juvenile hormone synthesis in adult females of G. bimacufotus. A dose-dependent stimulatory effect of injected 20-hydroxyecdysone on juvenile hormone synthesis has also been demonstrated by Whisenton et al. (1985) for last instar larvae of Manduca sexta. Also in this study 20-hydroxyecdysone applied in vitro did not activate the corpora allata. It is possible that the incubation time of 4 h is too short to detect longer lasting effects, but it is also possible that disconnected glands are not able to respond directly to stimulation by ecdysteroids (Couillaud and Girardie, 1985). Watson et a/. (1986) recently demonstrated that 20-hydroxyecdysone stimulates corpora allata activity of Manduca sexta during larval-pupal development indirectly via the brain-corpora cardiaca. Only long-term in vitro experiments could resolve the question as to whether the slow in vivo responses, as observed in G. bimaculutus (Figs 1 and 3), occur via direct effect of the compounds on the corpora allata or whether the effects are mediated by the brain and nerves. Surgical removal of the ovaries resulted in lower than normal corpora allata activity, suggesting that the presence of developing ovaries is necessary for the attainment of high juvenile hormone synthetic existence
303
activity. The observed effect of ovariectomy can be interpreted in at least 2 ways: (1) Ovariectomy results in the appearance of large quantities of haemolymph vitellogenins (Kempa-Tomm, 1987) and the high levels of haemolymph proteins could be part of a negative feedback signal. (2) Reduced levels of juvenile hormone synthesis in ovariectomized animals could result from the absence of a stimulatory (humoral) factor from the ovary. Ovariectomy suppresses juvenile hormone biosynthesis also in other insect species (Stay and Tobe, 1978; Riiseler et al., 1980; Weaver, 1981; Lanzrein et al., 1981; Strambi, 1981). In the cockroach, Diploptera puntncta, the ovary provides two different signals on corpora allata activity: a stimulatory one early in the gonotrophic cycle, which stimulates juvenile hormone synthesis and oiicyte growth, and an inhibitory one, which results in a decline in juvenile hormone biosynthesis at the end of a gonotrophic cycle (Stay et al., 1980). The nature of the stimulating factor is unknown, whereas 20-hydroxyecdysone from the mature ovary seems to inhibit juvenile hormone synthesis at the end of the gonotrophic cycle. Similar results have been reported for another cockroach, Nauphoeta cinerea (Lanzrein et al., 1981). Our data clearly show that the “insensitivity” of adult crickets in viuo to precocenes does not result from the lack of intrinsic sensitivity of their corpora allata to these allatotoxins. Similar results have been reported by Soderlund et al. (1981) for various insect species. The reduced level of methyl farnesoate within the glands of crickets which had been treated with precocene II in vitro might suggest that the site of action is earlier than the final epoxidative step in the juvenile hormone biosynthesis (Pratt and Bowers, 1977). Detoxification of the compound by nontarget tissues, along with binding by peripheral tissues may help to explain the ability to recover quickly from treatment with precocenes in uiuo (Bowers and Feldlaufer, 1982). On the other hand, there are several reports in the literature that mention reversible effects of precocenes on corpora allata also in oitro (Staal, 1986). Acknowledgements-The investigations were supported by the Deutsche Forschungsgemeinschaft (Ho 631/l l-1 and 1l-2). We are grateful to Professor H. Rembold, MPI Martinsried, for performing juvenile hormone titre determinations by means of combined gas chromatography-selected ion monitoring mass spectrometry. Methyl famesoate was kindly provided by Dr A. Schooley, Zoecon Research InstituteSandoz Croo Protection. We would also like to thank Dr F. C. Baker,-Zcecon Research Institute for his critical reading of the manuscript.
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Lanzrein B.. Wilhelm R. and Buschor J. (1981) On the regulation of the copora allata activity in’ adult females of-the ovoviviparous cockroach Nauphoeta cinerea. In Juvenile Hormone Biochemistry (Ed. by Pratt G. E. and Brooks G. T.), pp. 147-160: Elsevidr/North-Holland. Amsterdam. Pratt G. E. and Bowers W. S. (1977) Precocene II inhibits iuvenile hormone biosynthesis by cockroach corpora allata in vitro. Nature 2&S, 548-550. Pratt G. E. and Tobe S. S. (1974) Juvenile hormones radiobiosynthesized by corpora allata of adult female locusts in vitro. Life Sri. 14, 575-586. Rankin S. M. and Stay B. (1985) Ovarian inhibition of
Stay B. and Rankin S. (1986) Regulation of corpus allatum activity during reproduction: the role of ovaries and nerves. In Advances in Invertebrate Reproduction 4 (Ed. by Porchet M., Andries J.-C. and Dhainaut A.), pp. __ 43-50. Elsevier Science, Amsterdam. Stay B., Friedel T., Tobe S. S. and Mundall E. C. (1980) Feedback control of juvenile hormone synthesis in cockroaches: possible role for ecdysterone. Science 207, 898-900.
Stay B. and Tobe S. S. (1978) Control of juvenile hormone biosynthesis during the reproductive cycle of a viviparous cockroach. II. Effects of unilateral allatectomy, implantation of supernumerary corpora allata and ovariectomy. Gen. camp. Endocr. 34, 276286. Strambi C. (1981) Some data obtained by radioimmunoassay of juvenile hormones. In Juvenile Hormone Biochemistry (Ed. by Pratt G. E. and Brooks G. T.), pp. 59-63. Elsevier/North-Holland, Amsterdam. Tobe S. S. (1980) Regulation of the corpora allata in adult female insects. In Insect Biology in the Future (Ed. by Locke M. and Smith D. S.), pp. 345-367. Academic Press, New York. Tobe S. S. and Feyereisen R. (1983) Juvenile hormone biosynthesis: regulation and assay. In Endocrinology of Insects (Ed. by Downer R. H. G. and Laufer H.), pp. 161-178. Liss, New York. Tobe S. S. and Pratt G. E. (1974) The influence of substrate concentrations on the rate of insect juvenile hormone biosynthesis by corpora allata of the dessert locust in virro. Biochem. J. 144, 107-113.
Tobe S. S. and Pratt G. E. (1975) Corpus allatum activity in uirro during maturation in the desert locust, Schistocerca gregaria. J. exp. Biol. 62, 611627.
Tobe S. S. and Stay B. (1977) Corpus allatum activity in vitro during the reproductive cycle of the viviparous cockroach, Diploptera punctata (Eschscholtz). Gen. camp. Endocr. 31, 1388147.
Tobe S. S. and Stay B. (1979) Modulation of juvenile hormone biosynthesis by an analogue in the cockroach. Nature 281, 481482. Tobe S. S. and Stay B. (1980) Control of juvenile hormone biosynthesis during the reproductive cycle of a viviparous cockroach. III. Effects of denervation and age on compensation with unilateral allatectomy and supernumerary corpora allata. Gen. camp. Endocr. 40, 89-98. Watson R. D., Whisenton L. R., Bollenbacher W. E. and Granger N. A. (1986) Interendocrine regulation of the corpora allata and prothoracic glands of Manduca sexta. Insect Biochem. 16, 149-155.
Weaver R. J. (1981) Radiochemical assays of corpus allatum activity in adult female cockroaches following ovariectomy in the last nymphal instar. Experientia 37, 435436.
Whisenton L. R., Bowen M. F., Granger N. A., Gilbert L. J. and Bollenbacher W. E. (1985) Brain-mediated 20-hydroxyecdysone regulation of juvenile hormone synthesis by the corpora allata of the tobacco homworm, Manduca sexta. Gen. camp. Endocr. 58, 31 l-318.
1989
Copyright 0
OO22-1910/89 $3.00 + 0.00 1989 Pergamon Press plc
THE EFFECTS OF JUVENILE HORMONE, 20-HYDROXYECDYSONE, PRECOCENE II, AND OVARIECTOMY ON THE ACTIVITY OF THE CORPORA ALLATA (hV ?K!‘-‘RO) IN ADULT FEMALE GRYLLUS R. Allgemeine
Zoologie
WENNAUER,
(Biologic
BIMACULATUS
L. KASSEL
I), Universitlt
and K. H.
Ulm. Oberer
HOFFMANN*
Es&berg,
D-7900
Ulm/Donau,
F.R.G.
(Received 20 June 1988: rerised 19 September 1988) Abstract-Corpora allata from adult female Gry//us bimacularus exhibit rates of juvenile hormone III synthesis in vitro which are closely correlated with oocyte maturation. Juvenile hormone III treatments in uino (injection of 0.5 gg juvenile hormone III) of females reared at 20°C (animals with spontaneously inactive corpora allata) stimulated juvenile hormone synthesis within 12 h. The results could be verified as a de noun biosynthesis, by following the incorporation of radioactivity from [2-14C]acetate and L-[methyl-‘Hlmethionine into juvenile hormone III. Addition of juvenile hormone III in vitro (10-8-10-“ M) did not stimulate de now synthesis of juvenile hormone III. In females from a rearing temperature of 20°C a stimulating effect of 20-hydroxyecdysone injection (0.2555 pg) on juvenile hormone III synthesis became apparent in riro within 24 h. No stimulation was observed when isolated corpora allata were maintained for 34 h in medium containing 5. 1O-5 M ecdysone or 20-hydroxyecdysone. Female adult crickets quickly recover from treatment in tk~o with precocenes. After incubation of isolated corpora allata in the presence of precocenes, juvenile hormone III synthesis was diminished in a dosedependent manner. In ovariectomized females juvenile hormone III synthesis was drastically suppressed. Key Word Index:
Corpora allata, juvenile hormone III, precocene, 20-hydroxyecdysone, ovariectomy, reproduction, Gryllus bimaculatus
INTRODUCTION
Females of many insect species exhibit cyclical patterns of reproductive activity. Associated with the gonotrophic cycles are changes in activity of the corpora allata. Moreover, perception of social, chemical or physical factors by the insects may result in altered corpora allata activity (Feyereisen, 1985). The various patterns of corpora allata activity during reproduction and their modulation by external factors imply the existence of control mechanisms for corpora allata activity. Extensive experimental evidence shows that the corpora allata are regulated by stimulatory and inhibitory signals which reach the glands either via the haemolymph (humorally) or via nervous connections (for reviews see Tobe, 1980; de Kort and Granger, 1981; Feyereisen, 1985). A convenient direct method for estimating the spontaneous activity of the corpora allata is the short-term in rho radiochemical assay, as introduced by Pratt and Tobe (1974) and Tobe and Pratt (1974, 1975). This method relies on the incorporation of the methyl moiety of r_-[methyl-‘4C]methionine into juvenile hormone. The assay, adapted for Gryllus bimaculutus (Koch and Hoffmann, 1985), has been employed recently to show that corpora allata activity in adult female crickets is regu-
*To whom
correspondence
should
be addressed.
lated in a temperature-dependent manner (Espig and Hoffmann, 1985). In corpora allata of females reared at 27°C or at a daily alternating temperature regime of 24: 12°C (16:8 h) (in both temperature regimes reproduction rate is high), a first peak in juvenile hormone III synthesis is reached 48 days earlier than in females from 20°C (where reproduction rate is low). Furthermore, in animals from 20°C corpora allata activity is low during the entire oviposition period, whereas at 24: 12°C and 27°C respectively, high corpora allata activity is found during this period of adult life. Application of juvenile hormone III to females with a low corpora allata activity induced a dose-dependent stimulation of egg production and oviposition (Koch and Hoffmann, 1985). Further experiments had shown that in female adults of G. bimaculatus the concentrations of free ecdysteroids in haemolymph and tissues are also temperature-dependent (Hoffmann et al., 1981) and that exogenous ecdysteroids stimulate egg deposition rates in females with a low endogenous ecdysteroid titre (Behrens and Hoffmann, 1983). The chronological sequence of increasing corpora allata activity, vitellogenin synthesis, ovarian weight gain, ecdysteroid production, and oviposition in G. bimaculatus (Hoffmann, 1986) suggests that the onset of vitellogenin synthesis and reproduction is related to the first increase in juvenile hormone III synthesis. The results from the ecdysteroid injection experiments, however, indicate that in G. bimaculatus, as well as in the
R.
300
WENNAUER et
house cricket, Acheta domesh.ts (Chudakova et al., 1982), both of the hormones, juvenile hormone III and moulting hormone, are involved in the control of reproduction . In the present paper we investigated regulatory interactions between 20-hydroxyecdysone and juvenile hormone III synthesis in G. bimaculatus. using a combination of in vivo and in vitro techniques. The injection of juvenile hormone III or 20-hydroxyecdysone into females or the presence of the hormones in the incubation medium were assayed for effects on juvenile hormone III biosynthesis by the corpora allata in vitro. The effect of precocene II. which has been reported to influence the corpora allata of hemimetabolous insect species (Soderlund et al., 1981), is included in this study. Finally, we studied the effects of ovariectomy on juvenile hormone III biosynthesis.
MATERIALS
AND METHODS
Animals and surgery
Mediterranean field crickets, G. bimaculatus de Geer, were reared either at a constant 20 and 27°C respectively, or at a daily alternating temperature regime of 24 : 12°C (16 : 8 h) and a 16 h light-8 h dark cycle as described previously (Behrens et al., 1983). Freshly ecdysed females (l- 12 h) were collected from the stock and maintained separately. Injections and operations were performed with carbon dioxideanaesthetized animals. Corpora cardiaca-corpora allata complexes were dissected as described in Koch and Hoffmann (1985). Special care was taken to remove adjacent tissue from the glands. (In G. bimaculatus salivary glands are tightly attached to the corpora allata.) Ovariectomy was performed in lastinstar females from 27°C as described in KempaTomm (1987). In vivo treatment of females
Female crickets were injected 2 days after the imaginal ecdysis through the intersegmental membrane between the hind leg coxa and the abdomen by means of a 10~1 Hamilton syringe. Females from a rearing temperature of 20°C were injected either with 20-hydroxyecdysone (Simes, Milan; 0.25-5 pg of hormone) in insect Ringer (85 mM NaCl, 6 mM KCl, and 3 mM CaCl,; 5~1) (Behrens and Hoffmann, 1983) or with juvenile hormone III (Fluka, Neu-Ulm; 0.5 fig) in insect Ringer (5 ~1) containing 0.05% (v/v) Tween 80 (Merck, Darmstadt). Approximate haemolymph volume of 2-day-old females from 20°C is 200~1 (Ehler et al., 1986). Precocene II (6,7-dimethoxy-2,2-dimethyl-3-chromen; Sigma, Munich; 100 pg) was dissolved in acetone (5 ~1) and topically applied onto the less sclerotized pleural region of the abdomen of 2-day-old females reared at 27°C. Control animals were treated with insect Ringer and acetone, respectively. 3-48 h after treatment the corpora cardiacs-corpora allata complexes were dissected for the radiochemical assay. In vitro treatment of corpora allata Corpora cardiaca-corpora allata complexes were removed from 2- to I-day-old females. Individual
al.
pairs of gland complexes were thoroughly rinsed in insect Ringer and transferred into 100 ~1 of incubation medium 199 (Flow Laboratories, England; with Hanks’ salts, without L-glutamine and Na,CO,. buffered with 20 mM Hepes), fortified with 1% (w/v) Ficoll 400 (Sigma, Munich). The pH of the final medium was 6.5 (Koch and Hoffmann, 1985). Test compounds (juvenile hormone III dissolved in 0.55% (v/v) Tween 80, 20-hydroxyecdysone in distilled water, and precocene II in 0.55% (v/v) Tween 80; 10 ~1 each) were added to the medium (for final concentrations see Results). Control groups consisted of gland complexes incubated in medium containing the solvents for the test compounds only. If not otherwise stated incubations were performed at 20°C in darkness for 180-240 min. Juvenile hormone biosynthesis by the corpora allata
Biosynthesis of juvenile hormone III by individual pairs of corpora allata was determined using the radiochemical in vitro assay, as introduced by Pratt and Tobe (1974) and Tobe and Pratt (1974, 1975). The incorporation of the methyl moiety of L-[methyl“C]methionine (Amersham, England; final sp. act. 34-40 mCi/mmol; final concentration of L-methionine 0.26mM; Koch and Hoffmann, 1985) into juvenile hormone III was followed for 3-4 h at 20°C. To determine de novo juvenile hormone III biosynthesis incorporation of radiolabel was measured after 4 h incubation of gland complexes at 27°C in medium supplemented with L-[methyl-3H]methionine (Amersham, England; initial sp. act. 15 Ci/mmol) and [2-14C]acetate (Amersham, England; initial sp. act. 56 mCi/mmol) and appropriate amounts of unlabelled methionine and acetate to give final concentrations of 0.26mM methionine (final sp. act. about 55 mCi/ mmol) and 2 mM acetate (final sp. act. about 10 mCi/ mmol). Juvenile hormone III and methyl famesoate were measured following separation by thin-layer chromatography (silicagel 60 F,, plates; toluene:ethyl acetate: acetic acid, 70: 30: 1 (v/v) as solvent system). 14C and ‘H radioactivity were determined by liquid scintillation counting in Rotiscint 2211 (Roth, Karlsruhe) scintillation fluid. The results expressed as “JH III synthesis” indicate that gland complexes plus the incubation medium were extracted (Tobe and Stay, 1977). Values are means + SEM for the number of individual measurements indicated on each figure. A t-test was used in statistical analyses. RESULTS
Synthesis of radiolabelled products by the corpora alfata in vitro
The corpora allata of G. bimaculatus produce juvenile hormone III (Koch and Hoffmann, 1985). There is a linear relationship between the rate of juvenile hormone III synthesis and its release. A further incubation product, which is not released into the incubation medium, was identified as the 2E, 6E-isomer of the juvenile hormone III precursor, methyl farnesoate by co-chromatography with the reference substance in thin-layer chromatography (silicagel 60 F2%) and HPLC (Waters PBondapak
Regulation of corpora allata activity in crickets
301
- 61 20
==6
2: j3
c 3
12
24 t?e
(h)
Fig. 1. The effect of juvenile hormone III injections after various periods of time (348 h) on the rate of juvenile hormone III (JH III) synthesis (in oirro) of 2-day-old females reared at 20°C. Experimental animals (crosshatched columns) were injected once with 0.5 pg juvenile hormone III. Controls (open columns, C) received 5 ~1 of insect Ringer with 0.05% (v/v) Tween 80.
Cl8 reversed phase column; 1985).
Koch and Hoffmann,
Effect of juvenile hormone III injection
After a single injection of 0.5 pg juvenile hormone III into females reared at 20°C at day 2 after ecdysis activity of corpora allata in vitro increased about 7-fold (Fig. 1). The increase in juvenile hormone III synthesis became apparent 12 h after injection, and the activity remained high, even 48 h after treatment (P < 0.057 at 12 h; P < 0.019 at 48 h). The incorporation of radioactivity from [2-‘4C]acetate into juvenile hormone III and its immediate precursor, methyl farnesoate (Fig. 2) indicates that this increase is due to a de novo synthesis of juvenile hormone III from acetate. Figure 2 also demonstrates that already active glands (juvenile hormone III synthesis of 7.4 pmol h-’ per pair vs 0.4 pmol h-’ per pair of glands in Fig. 1) were further stimulated by juvenile hormone III treatment in vivo. Effect in vitro of exogenous juvenile hormone
In order
to test whether
the hormone
influ-
e2 ,” 1 E ~Id-IL 4
1
l3
20
control
0.25
0.5
L 5 w.X-WE
Fig. 3. Juvenile hormone III (JH III) synthesis 24 h after injection of 0.25-5 pg 20-hydroxyecdysone (20-OH-E) or control solution (5 ~1 insect Ringer) into 2-day-old females reared at 20°C.
ences glands directly, 10-s-10-4 M juvenile hormone III were included in the incubation medium of corpora cardiaca-corpora allata complexes from 2-day-old females reared at 20°C and rates of juvenile hormone biosynthesis were followed by the incorporation of radioactivity from [2-14C]acetate and L-[methyl-3H]methionine into juvenile hormone III. Addition of juvenile hormone III in vitro did not stimulate de novo synthesis of juvenile hormone III (all values lower than OSpmol h-’ per pair; not shown in figures). In vivo effect of 20-hydroxyecdysone
Figure 3 shows that injection of the hormone into females with a low endogenous moulting hormone titre (20°C; 2 days after ecdysis) causes a stimulation of juvenile hormone III biosynthesis within 24 h. The synthesis of juvenile hormone III increased from a basal level of 1.2 + 1.3 pmol h-l per pair up to 8.1 + 2.2 pmol h-’ per pair in response to 5 pg 20-hydroxyecdysone (P < 0.030 at 2 pg; P < 0.014 at 5 fig). Effect in vitro of free ecdysteroids
3500
I 3000
2500
2000
.j P g 2 p
Glands were incubated over a 4 h period in medium containing 5. 10m5M ecdysone or 20-hydroxyecdysone. No differences were observed in the rate of juvenile hormone III synthesis between the corpora allata incubated in the presence (3.9 + 1.5 pmol h-’ per pair for ecdysone) or absence (3.7 + 1.0 pmol h-’ per pair) of the ecdysteroids.
.c
rsoop 1000
h 0
600
0.5 po .Jn III
Fig. 2. Juvenile hormone III (JH III) synthesis (in oitro) (b) and the amount of methyl famesoate (MF) (a) in corpora allata of 3-day-old 20°C females 24 h after injection of 0.5pg juvenile hormone III. Incorporation of the methyl moiety of [‘Hlmethionine was used as mass-marker (open columns). Right ordinates and cross-hatched columns: incorporation of [Wlacetate (dpm) into MF (a) and JH III (b) after 4 h incubation at 27°C. Controls (C) received 5 ~1 of insect Ringer with 0.05% (v/v) Tween 80. Mean values of 5 determinations k SEM.
Eflect of precocene II
Previous experiments had shown that repeated topical application or injections of 150 pg precocene II did not affect ovarian development and oiicyte growth in maturing females (W. Espig, unpublished results). These results agree well with the present observations on the in vivo effect of precocene II on juvenile hormone synthesis. When 100 pg precocene II were applied onto 2-day-old females from a rearing temperature of 27°C (animals with spontaneously active glands), their corpora allata activity was reduced 12 h later, but restored to control values within 24 h (Fig. 4). After an incubation of isolated corpora allata (from 8-day-old females reared at 24: 12°C; 16: 8 h)
302
R. WENNAUER et al
40. 2 k
35. 30
L g
25.
-
20.
l
15.
P
IO-
i 5
c 12
C24
5-
Fig. 6. Juvenile hormone III (JH III) synthesis (in vitro) by corpora allata of adult females ovariectomized (Ovv) or sham-operated (Co) as last instars. Rearing temperature 27°C. Mean values of 10 determinations.
C48 time (h)
Fig. 4. The effect of topical application of 1OOng of precocene II dissolved in 5 ~1 acetone on the rate of juvenile hormone III (JH III) biosynthesis of spontaneously active corpora allata (from females reared at 27°C). Open columns, controls (C) which had been treated with 5 ~1 acet-
one; cross-hatched columns, experimental animals 1248 h after treatment.
for 3 h in various concentrations of either precocene I (not shown in figure) or precocene II (Fig. 5) a dose-dependent inactivation of the glands was found. Spontaneously active corpora allata were inhibited completely by exposure in vitro to lOa3 M precocenes. The amount of methyl farnesoate in the glands which were treated with precocenes was also reduced. Ovariectomy
In this experiment we investigated the interdependence between corpora allata and the ovary.
r 13
12 11 10 SP s* 7k 6. 5 Ii 4% 3 2 I control
lo-'
10-0
lo-. 10-a M precocene II
Fig. 5. Effects of exposure of corpora allata to precocene 11 in vitro(10-7-10-3 M in the incubation medium) on juvenile hormone III (JH III) biosynthesis (open columns) and on the amount of methyl farnesoate (MF) within the glands (cross-hatched columns). Controls were treated with 10 ~1 of 0.55% (v/v) Tween 80.
Ovaries were excised from female last larval instars and the corpora allata activity (in vitro) was determined between days 0 and 22 of adult life (Fig. 6). In sham-operated controls we measured corpora allata activities which were similar to those of untreated females (Koch and Hoffmann, 1985). In ovariectomized females juvenile hormone III synthesis was high shortly after ecdysis, but suppressed during the subsequent period of adult life. DISCUSSION
The combination of in uivo application of effecters and in vitro determination of corpora allata activity allows the study of potential factors which may be involved in the regulation of corpora allata activity. Evidence is available which demonstrates that the rate of juvenile hormone synthesis in vitro shortly after extirpation of the glands is a good indicator of the rate of juvenile hormone synthesis of those glands in situ (for reviews see Tobe and Feyereisen, 1983; Feyereisen, 1985). A linear relationship between the rate of juvenile hormone III synthesis and its release shows that corpora allata of G. bimaculatus do not store juvenile hormone III. Similar observations were done by Tobe and Stay (1977) for corpora allata of Diploptera punctata, but also for many other insect species. The existence of humoral factors directly controlling corpora allata activity has been postulated for a long time, but none has been characterized to date (Feyereisen, 1985). Mechanisms for the control of copora allata activity by experimentally modified titres of juvenile hormone have been described, however, and demonstrate the existence of complex feedback loops (indirect control). Evidence for a negative feedback effect of exogenous juvenile hormone (or juvenile hormone analogues) on juvenile hormone biosynthesis has been presented, e.g. for Leptinotarsa decemlineata by Schooneveld et al. (1979) and Khan et al. (1982), and for Diploptera punctata by Tobe and Stay (1979, 1980). In larvae of Manduca sexta juvenile hormone levels were suppressed in a dose-dependent fashion following topical treatment with the juvenoids fenoxycarb or hydroprene (Baker et al., 1986). The
Regulation of corpora allata activity in crickets
of a positive feedback loop, which would stimulate juvenile hormone synthesis in response to an elevated juvenile hormone titre, was suggested by Tobe and Stay (1979). Topical application of 2.5 pg of hydroprene to newly emerged female D. punctuta stimulated juvenile hormone synthesis on day 4 after moulting. The feedback stimulation is suggested to occur by way of the ovary (Stay and Rankin, 1986). Further indication of a positive feedback mechanism has been given by Kozhanova and Chudakova (1985) who suggested that the juvenile hormone analog methoprene activates corpora allata of the house cricket, Acheta domesticus, to secrete endogenous juvenile hormone. Our results (Figs 1 and 2) also indicate that during an experimental elevation of the juvenile hormone titre in oioo (injection of 0.5 pg juvenile hormone III into adult females from 2O”C, which have a low endogenous juvenile hormone level) positive feedback regulation occurs. The increase in juvenile hormone III synthesis is due to a de nova synthesis from acetate. Our experimental data, however, do not show that juvenile hormone influences corpora allata directly. Addition of juvenile hormone III to the incubation medium did not stimulate de novo synthesis of juvenile hormone III from acetate within the incubation time of 4 h. Several studies have been conducted on juvenile hormone-ecdysteroid interactions in adult insects, and most of them suggest that ecdysteroids have an allatostatic effect (Friedel et al., 1980; Stay et al., 1980; Lanzrein et al., 1981; Rankin and Stay, 1985). In former experiments we have shown that exogenous ecdysteroids stimulate ovarian growth and egg deposition rates in females with a low endogenous ecdysteroid titre (Behrens and Hoffmann, 1983). Repeated injections of 2-5 pg ecdysone or 20-hydroxyecdysone, respectively, caused an increase in the number of eggs per female more than twice. The results of this paper demonstrate that 20-hydroxyecdysone may be a positive regulator of juvenile hormone synthesis in adult females of G. bimacufotus. A dose-dependent stimulatory effect of injected 20-hydroxyecdysone on juvenile hormone synthesis has also been demonstrated by Whisenton et al. (1985) for last instar larvae of Manduca sexta. Also in this study 20-hydroxyecdysone applied in vitro did not activate the corpora allata. It is possible that the incubation time of 4 h is too short to detect longer lasting effects, but it is also possible that disconnected glands are not able to respond directly to stimulation by ecdysteroids (Couillaud and Girardie, 1985). Watson et a/. (1986) recently demonstrated that 20-hydroxyecdysone stimulates corpora allata activity of Manduca sexta during larval-pupal development indirectly via the brain-corpora cardiaca. Only long-term in vitro experiments could resolve the question as to whether the slow in vivo responses, as observed in G. bimaculutus (Figs 1 and 3), occur via direct effect of the compounds on the corpora allata or whether the effects are mediated by the brain and nerves. Surgical removal of the ovaries resulted in lower than normal corpora allata activity, suggesting that the presence of developing ovaries is necessary for the attainment of high juvenile hormone synthetic existence
303
activity. The observed effect of ovariectomy can be interpreted in at least 2 ways: (1) Ovariectomy results in the appearance of large quantities of haemolymph vitellogenins (Kempa-Tomm, 1987) and the high levels of haemolymph proteins could be part of a negative feedback signal. (2) Reduced levels of juvenile hormone synthesis in ovariectomized animals could result from the absence of a stimulatory (humoral) factor from the ovary. Ovariectomy suppresses juvenile hormone biosynthesis also in other insect species (Stay and Tobe, 1978; Riiseler et al., 1980; Weaver, 1981; Lanzrein et al., 1981; Strambi, 1981). In the cockroach, Diploptera puntncta, the ovary provides two different signals on corpora allata activity: a stimulatory one early in the gonotrophic cycle, which stimulates juvenile hormone synthesis and oiicyte growth, and an inhibitory one, which results in a decline in juvenile hormone biosynthesis at the end of a gonotrophic cycle (Stay et al., 1980). The nature of the stimulating factor is unknown, whereas 20-hydroxyecdysone from the mature ovary seems to inhibit juvenile hormone synthesis at the end of the gonotrophic cycle. Similar results have been reported for another cockroach, Nauphoeta cinerea (Lanzrein et al., 1981). Our data clearly show that the “insensitivity” of adult crickets in viuo to precocenes does not result from the lack of intrinsic sensitivity of their corpora allata to these allatotoxins. Similar results have been reported by Soderlund et al. (1981) for various insect species. The reduced level of methyl farnesoate within the glands of crickets which had been treated with precocene II in vitro might suggest that the site of action is earlier than the final epoxidative step in the juvenile hormone biosynthesis (Pratt and Bowers, 1977). Detoxification of the compound by nontarget tissues, along with binding by peripheral tissues may help to explain the ability to recover quickly from treatment with precocenes in uiuo (Bowers and Feldlaufer, 1982). On the other hand, there are several reports in the literature that mention reversible effects of precocenes on corpora allata also in oitro (Staal, 1986). Acknowledgements-The investigations were supported by the Deutsche Forschungsgemeinschaft (Ho 631/l l-1 and 1l-2). We are grateful to Professor H. Rembold, MPI Martinsried, for performing juvenile hormone titre determinations by means of combined gas chromatography-selected ion monitoring mass spectrometry. Methyl famesoate was kindly provided by Dr A. Schooley, Zoecon Research InstituteSandoz Croo Protection. We would also like to thank Dr F. C. Baker,-Zcecon Research Institute for his critical reading of the manuscript.
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