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Effects of anti-juvenile hormone “ETB” on the development and metamorphosis of the silkworm, Bombyx mori
J. hrsect Ph,vs;o~.Vol. 30. No. 6, pp. 499-506. 1984 Printed in Great Britain. All rights reserved
0022-1910184 53.00 + 0.00 Copyright (‘1 1984 Pergamon Press Ltd
EFFECTS OF ANTI-JUVENILE HORMONE “ETB” ON THE DEVELOPMENT AND METAMORPHOSIS OF THE SILKWORM, BOMBYX MORI KENJI
The Sericultural
Experiment
KIGUCHI,
TADASU
MORI*
HIROMU AKAI
and
Station, Yatabe, Ibaraki 305, Japan and *Kinjo Gakuin Moriyama-ku. Nagoya-shi, Aichi 463. Japan
University,
Ohmori.
(Received IO October 1983; revised 5 Junuary 1984)
Abstract-As in the tobacco homworm Munduca sexta, the synthetic juvenile hormone analogue ETB (ethyl 4-[2-(tert-buthylcarbonyloxy)butoxy]benzoate) showed both juvenile hormone-like and anti-juvenile hormone activities in the silkworm, Bombyx mori. When ETB was topically applied to allatectomized 4th-instar larvae, the compound counteracted the effects of allatectomy, such as induction of precocious metamorphosis and black pigmentation in the larval markings. Therefore, ETB had juvenile hormone activity, but it could neither induce brown pigmentation in the markings nor induce an extra-larval moult as can juvenile hormone. When intact 3rd-instar larvae were treated with the compound, the majority underwent precocious metamorphosis in the 4th-instar, and later formed fertile miniature adults. Some moulted into larval-pupal intermediates or Sth-instar larvae with darkened larval markings and/or with abnormality of specific regions of the silk-gland. The optimal dose for such anti-juvenile effects was about I-lOpg/larva, and higher doses showed less activity. Such anti-juvenile hormone effects of ETB were counteracted by administration of the juvenile hormone analogue, methoprene, before a certain critical time in the 4th-instar. The corpora allata of treated larvae appeared cytologically normal, and the corpora allata from ETB-induced miniature moths secreted juvenile hormone when implanted into allatectomized 4th-instar larvae.
Kqv Word &de-x: ETB, anti-juvenile
Bombyx mori, precocious
hormone,
MATERIALS
INTRODUCTION
The discovery of compounds with anti-juvenile hormone activity gave a new perspective to the development of potent insect control agents. Moreover, studies on anti-juvenile hormones were expected to lead to the better understanding of juvenile hormone action and endocrine regulation of the corpora allata. Nevertheless, a relatively limited number of such compounds is known, such as the ageratochromenes (precocenes) (Bowers, 1976), ETB, piperonyl butoxide (Staal, 1977), compactin (Monger et al., 1982; Hiruma et al., 1983). fluoromevalonate (Quistad et al., 1981), piron and piridon derivatives (Murakoshi and Ichimoto, 1972), abietic acid derivatives (Murakoshi et al., 1975) and terpenoid imidazoles (Kuwano et al., 1983). Among them, ETB is one of the most intriguing chemicals because of its peculiar and complex actions (Staal, 1977; Staal et al., 1981). The compound was first synthesized as a juvenile hormone mimic (Kondo et al.. 1973). Subsequently its anti-juvenile hormone activity was discovered in the tobacco hornworm, Manduca sextu, although other lepidopteran insects so far tested seem to be unaffected (Staal, 1977; Staal et al., 1981). We examined the effects of ETB administration on the silkworm, Bombyx mori, and found that it had both typical anti-juvenile hormone and potent juvenile hormone-like activities, just as seen in Munduca sexta. 1.P 30 h
E
metamorphosis
499
Experimental
AND METHODS
animals
The F, hybrid strain of Bombyx mori resulting from the cross between N. 124 and C.124 was used as experimental animals. Larvae were reared routinely at 25°C on fresh mulberry leaves. Developmental stages were determined by the Spiracle Index described by Kiguchi and Agui (198 1). Application
of ETB
and juvenile
hormone
unulogue
ETB, ethyl 4-[2-(tert-butylcarbonyloxy)butoxy] benzoate, was a gift from Dr G. B. Staal, Zoecon Corporation, U.S.A. Various doses (0.001-500 pg) of ETB dissolved in ethanol were applied topically onto the whole dorsal surface of the larvae anaesthetized with ethyl ether. Third-instar larvae were treated with 0.5-2 ~1 of solution, 4th-instar larvae with 2-6 ~1 and Sth-instar larvae with l&20 ~1, respectively. Control animals received the corresponding volume of ethanol. resulting in negligible mortality. For the counteraction experiments, a juvenile hormone analogue, methoprene (ZR 515, Zoecon). dissolved in peanut oil was used. Surgical
methods
Allatectomy was as described by Kiguchi and Riddiford (1978). Corpora allata were implanted through a lateral incision in the intersegmental membrane between the 7th and 8th larva1 segments. The
500
KENJI KIGUCHI
CI (I/.
B
A
C
D
Fig. I. Normal silk gland (A) and various degree of abnormal silk glands (B. C, D) induced by juvenile hormone injection 12-24 h after allatectomy in the silkworm, Bomhyx mori. Abnormal degeneration occurs only at the middle division of the middle silk gland. a. anterior silk gland; b, anterior division of the middle silk gland; c, middle division of the middle silk gland: d, posterior division of the middle silk gland: e, posterior silk gland.
tectomy, and score I to brown markings which could be caused by the injection of a high dose of juvenile hormone. Scores 2 and 4 indicated intermediate pigmentation.
wound sites were not sealed, but little blood leakage resulted. Evaluation
qf t$%cts
ETB-treated larvae were reared at 25 C. At an appropriate time, they were scored carefully under a dissecting microscope. Special attention was paid to the appearance of precocious pupal characters. pigmentation of larval markings and a morphological abnormality of a specific region of the silk-glands (Fig. I), because these are all related to juvenile hormone action or function of corpora allata (Kiguchi, 1976) (see Discussion section). The peculiar degeneration of only the mid-region of the middle silkgland (c in Fig. 1A) was first noted by Fukuda (1956) when he reimplanted corpora allata into allatectomized 4th-instar larvae within 24 h of allatectomy, and thus prevented precocious metamorphosis except for this specific degeneration of the silkgland. Effects on marking pigmentation was scored as described previously (Kiguchi, 1972): the silkworm larva has two pairs of larval markings on the ventral side of the abdomen. The outer side of each marking is black and the inner side brown in colour. Normally there is little variation in this marking colouration in this strain. Score 3 was given to non-treated control larvae, score 5 to completely black markings which could be induced by alla-
Table I. Effect of ETB on mduction
Histology For electron microscopical study, corpora allata were dissected from the ETB-induced precocious wandering larvae. The corpora allata were fixed with 2.5”/:, glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 M sucrose. They were post-fixed in l9/, O,O, in the same buffer and embedded in Epon 812. Sections were stained with uranyf acetate and lead citrate, and examined in a JEOL JEM-100CX II electron microscope.
RESULTS
The juvenile hormone-mimicking activity of ETB was tested according to the method described by Kiguchi et al. (1974). Larvae were allatectomized 24 h after the 3rd ecdysis, and different doses of ETB dissolved in 5 ~1 of ethanol were applied topically within 1Omin after the allatectomy. Control larvae which had been allatectomized and then treated with 5 ~1 of ethanol underwent precocious metamor-
of larval moulting larvae
m allatectomized
No. of larvae D
IO0 I I)
I 0. I II.0 I Allatectomirrd control
activity qf’ ETB
Juvenile hormone-mimicking
4th.mstar
transformed
Bomhw
into
No. ot lawaL: tested
Precocmus PuPa
5 IO IO IO ICI IO
0 0 (I 0 7 IO
0 0 0 0 2 0
5 IO IO IO I 0
I0
IO
0
0
Larval-pupal intermediate
5th~Instar IXW
Effect of anti-juvenile
hormone “ETB” on Bombyx
Table 2. Effect of ETB and synthetic juvenile hormone on the marking pigmentation of the allatectomized Born& larvae Score (Mean + Standard deviation)
---
Dose @g/larva) 500 100 50 10 5
I 0.1 0.01 0.001 0.0001 Allatectomized control
ETB*
Synthetic juvenile hormonet
3.0 f 0 (3) 2.9 + 0.3 (10) Not tested 3.0 f 0 (10) Not tested 3.0 + 0 (10) 3.0 f 0 (10) 3.2 f 0.4 (10) 4.8 f 0.4 (10) 5.0 &O (10)
1.0&O (8) 1.050 (8) 2.1 + 0.4 (8) 4.0 5 0 (8)
5.0? 0 (30)
5.0 * 0 (IO)
*Larvae were allatectomized just before apolysis in the 4th~instar, and the given amount of ETB was topically applied within IOmin. Scoring was done after the 4th ecdysis. tData from Kigucbi (1972) (Juvenile hormone used was a stereoisometric mixture of juvenile hormone 1). A number of larvae tested was shown in the parentheses in each column.
phosis. Administration of 1-5001.18 of ETB prevented this precocious metamorphosis so that all treated animals underwent a larval moult. No acute lethal effect was observed. Lower doses (0.001-0.1 pg) showed no or little effect. The dose response curve gave a 5004 effective dose of 0.25 pg/larva. Next, the juvenile hormone activity was assayed based on the colour change of the larval markings as previously reported (Kiguchi, 1974). Allatectomy at the time of general apolysis in the 4th instar causes black pigmentation of the larval markings in the 5th instar. This darkening can be prevented by an injection of an appropriate dose of juvenile hormone just after allatectomy. Moreover, a high dose of the hormone induces brown pigmentation of the larval markings in the dose-response manner in both intact and allatectomized 4th instar larvae (Table 2). To determine whether ETB has the same effect as juvenile hormone, the compound was applied topically to allatectomized test animals. They ecdysed into the 5th instar about 30 h after the ETB treatment, and their marking pigmentation was scored carefully under the dissecting microscope. As shown in Table 2, at a low dose of 0.01 pgg/larva, ETB inhibited the black pigmentation of the markings caused by allatectomy. Yet large doses of l-500 pg/larva did not
501
induce the brown pigmentation of the larval markings (score 1 or 2). Thus ETB differs from juvenile hormone and many juvenile hormone derivatives. Another peculiar phenomenon was observed when ETB was applied to the final Sth-instar larvae. A supernumerary larval moult can be induced by the application of high doses of juvenile hormone and many of its analogues at the middle stage of the 5th instar (Akai et al., 1973). Such supernumerary moulting was not induced by ETB, even at an extremely high dose (100-500 pg/g of larval body weight). The only effect of ETB at this time and dosage was the induction of dauer (permanent) larvae. Thus, there are some essential differences between the actions of juvenile hormone and of ETB. Anti-juvenile hormone activity of ETB To check the anti-juvenile hormone effects of ETB, 10 pg of the chemical was applied topically to 3rd or 4th-instar Bombyx larvae. The results were shown in Table 3. When ETB was applied to the 3rd-instar larvae, morphologically normal 4th~instar larvae were formed. As shown in Table 3, many of these larvae then moulted into precocious miniature pupae and larval-pupal intermediates. Those treated earliest in the 3rd-instar were most affected. Some of the test animals formed Sth-instar larvae, but most of these had abnormal silk glands (Fig. 1) and dark colouration of the larval marking spots. When ETB was applied to clth-instar larvae, none of the treated animals showed any sign of precocious metamorphosis. The only recognizable effect was the darkening of the larval markings in the 5th instar. To determine the optimal amount of ETB needed to induce anti-juvenile hormone effects, various doses of the chemical (0.1-200 pg/larva) were administered topically to freshly ecdysed 3rd-instar larvae. As shown in Table 4, induction of precocious metamorphosis occurred in a comparatively narrow dose range of 1-1Opg per larva. Higher doses (50, 100, 200pgg/larva) showed little or no activity in this respect. By contrast, both darkening of larval markings and the abnormal region-specific degeneration of silkglands in the 5th instar were induced over a wider dose range of l-2OOpg. The anti-juvenile hormone effects of ETB could be counteracted by administration of a juvenile hormone analogue, methoprene. When 1 pg of methoprene was injected into 0 and 24-h-old 4th~instar
Table 3. Effects of ETB (10 pg/larva) on 3rd and 4th-instar Eombyx larvae No. of larvae transformed into Time ETBadministered (h after ecdysis)
___
No. of larvae tested
Precocious pupae
Larval-pupal intermediate
33 32 24
23 7 0
4 13 II
12 12 12
0 0 0
0 0 0
~
..__~
Marking pigmentation (mean score)
O0Of larvae with silk gland abnormality
6 12 13
4.3 3.8 3.3
IO0 x.7 x5
12 12 12
3.8 3.6 3.0
0 0 0
Sth-lnstar larvae
3rd instar
I 24 48 4th mstar
I 24 48
502
KENJI KIGU(‘HI Table 4. Dose -response
for ET6 admmibtered
PI ul. on r~ewly moulted
3rd.mstar
No. of larvae transformed
Marking plgmentalion (mean score)
Dose (~g;larval
4.0
200 100 50 I0 5 I 0.5 0.1 COlltrOl
41 43 4 (I 4.u 73 ? (I 30 30
larvae which have previously received 10 pg of ETB just after ecdysis into the 3rd instar. all of the experimental animals moulted into Sth-instar larvae with no silk gland abnormality. Importantly. their larval marking colour was brown (score I), possibly due to the action of methoprene. When methoprene was given at 48 or more hours after ecdysis to the 4th stage, it only partially or no longer prevented the effects of ETB. Efect
of ETB on the c’orporu ulIutu
Precocious pupae caused by ETB became fertile miniature moths. To determine whether ETB affected the functioning of the corpora allata, one pair of corpora allata were dissected from the ETB-treated host animal on the day after adult eclosion and implanted into an allatectomized young 4th-instar larva. All recipients moulted into normal Sth-instar larvae (N = 15), indicating that ETB-affected corpora allata were able to secrete juvenile hormone. The ultrastructure of the corpora allata from ETBtreated precocious wandering larvae was also observed. As in the normal Sth-instar wandering larvae (Fig. 2A). the corpus allatum cells of ETB-treated insects were found to be characterized by well developed large nuclei, many mitochondria and often conspicuous Golgi complexes (Fig. 2. B and C). Neurosecretory axons derived from the brain were also observed in the peripheral regions of the organs (Fig. 2D). In the axons there were two types ot granules: electron-dense and transparent granules (Fig. 2D), just as in the normal insects. In the three corpora allata observed. no significant degradation was recognizable.
Allatectomized 4th-instar Bonzbw larvae become precocious pupae and then fertile moths (Bounhiol. 1938). As might be expected. both male and female miniature moths caused by ETB mated normally, and produced fertilized diapausing eggs. The eggs showed good hatchability (>954;) after chilling at 5°C for about 4 months. These results indicate that juvenile hormone is not involved in the sexual maturation (sex pheromone production, spermatogenesis and egg formation etc.) in the silkworm, Bomb~~r wzori. DISCUSSION
Anti-juvenile
larvae
into
hormone USSUJ h_v Bombyx
Compounds with anti-juvenile hormone activity are the chemicals which can induce the effects of
(‘,, Of larvae with silk gland abnormality 70 x5 x3 IO0 100 13 (1 0 0
surgical removal of the corpora allata, the source of juvenile hormone. The effects of allatectomy in Bombys are summarized in Fig. 3. As in other lepidopteran insects, allatectomy during the early and middle stage of the penultimate instar causes precocious pupation. When allatectomized just at the beginning of spiracular apolysis. 1arvalLpupal intermediates are induced (Kiguchi and Agui, 1981). Thereafter, removal of the corpora allata can no longer prevent the larval moult, but it still induces black pigmentation of the larval markings (Kiguchi,1972). In addition, degeneration of a specific region of the silk glands is caused by the temporary decrease in haemolymph juvenile hormone after allatectomy. More pronounced degeneration is prevented by the subsequent implantation of corpora allata or injection of synthetic juvenile hormone within 12-24 h of allatectomy (Fukuda, 1956; Kiguchi. 1976). Such silk gland degeneration occurs during the moult to the 5th instar in these juvenile hormone-treated larvae. Apparently, the cells of the region (the middle division of the middle silk gland) are committed in the pupa by the sudden decrease (or absence) of juvenile hormone and a low but significant level of ecdysteroids in the haemolymph (Kiguchi and Agui. 1981). so that they then degenerate later when exposed to the high level of ecdysteroids during the 4th-larval moult (unpublished). Since the effects of allatectomy in Body.\- mori are well understood it is a good bioassay for anti-juvenile hormones. Charucteristics
gf’ actions qf’ ETB
ETB shows both juvenile hormone-like and antijuvenile hormone action against the silkworm, Bornb~*s tori. Just as natural juvenile hormone and many derivatives. ETB effectively inhibits precocious metamorphosis and black pigmentation of the larval markings seen in allatectomized 4th-instar larvae. Importantly, ETB could not mimic all of the effects of juvenile hormone; namely it neither induced brown pigmentation in the larval markings nor induced extra-larval moult even when large doses were applied at the appropriate developmental stage. These results may suggest that ETB has little intrinsic juvenile hormone activity, but acts to synergise endogenous juvenile hormone. In other insects it also can mimic juvenile hormone at high doses (Staal, 1977: Staal et al., 1981). and in Man&u sextu it acts as a juvenile hormone directly on the epidermis in ci/ro (Riddiford er al., 1983). By contrast, ETB produces almost all the same
Fig. 2. The corpora allata from the normal 5th~instar larva (A) and the precocious wandering larva ted by ETB-administration (B, C, D). N: Nucleus, M: Mitochondria. G: Golgi complex, DG: Electron-dense granules, TG: Transparent granules.
503
Effect of anti-juvenile
hormone “ETB”
PreCOClOUS
Larval
-pupa1
Black
intermediate
PuPa
505
on Bon1h~a.u
of
plqmentatlon
larval
morklnqs
3rd Larval ecdysis
1 ä*_ .-. __~
24
-
I
72h
Splroculor apolysls 12 -24
h
‘-
I - CA
General opolysls +JH
~
Fig. 3. Summary
+JH
I
End of period
JH
pigmentation
secretIon
JH for
sensitive marklnq
by CA
t Sllkqland
Larval
abnormol~ty
internedlate
of effects of allatectomy
End of critical period for
- pupal
(-CA)
effects as surgical removal of the corpora allata in the penultimate-instar larvae when applied to young 3rd-instar larvae. The treated morphologically normal 4th~instar larvae became precocious pupae. larval--pupal intermediates or Sth-instar larvae with darkened larval markings and/or silk gland abnormality. Thus, ETB shows anti-juvenile hormone activity in the silkworm, B0mby.x mori, as well as in the tobacco hornworm. Munduca se.xtu (Staal. 1977; Riddiford et ul., 1983). How ETB exerts its anti-juvenile hormone action is unknown. It effectively reduces juvenile hormone output by Manducu corpora allata in vitro (Kramer and Staal. 1981). Our cytological observations show that ETB had no noticeable cytotoxic effect on BombJ).u corpora allata. Moreover, corpora allata from miniature moths produced by ETB treatment secreted juvenile hormone as assayed by implantation into young larvae. Obviously, ETB is different type of anti-juvenile hormone from the ageratochromenes (precocenes) (Bowers, 1976). Staal rt al., (1981) suggested that ETB has complex activity, including competition at juvenile hormone receptor level, inhibition of early steps in the hormone synthesis and depression of the biosynthesis through negative feedback. Edwards ef al. (1983) also suggested the possible mode of action of ETB as an inhibitor of feedback system responsible for switching the corpora allata off and on. Since ETB effects can be counteracted by methoprene applied 3-4 days after the ETB treatment. there is little likelihood that the compound competes for juvenile hormone receptors at the target tissues to inhibit the action of endogenous juvenile hormone. Rather, ETB seems to be able to depress the juvenile hormone-secreting activity of the corpora allata (therefore. the biosynthesis) through a feedback mechanism. Immediately after application, ETB might act synergistically. Then, as the compound is metabolized, its anti-juvenile hormone activity may take over and prevent either the release or action of juvenile hormone. Possibly the persistent low concentration during the 3rd to 4th-larval moult acts as a juvenile hormone on the
in the silkworm. Bombyu mori. See text for delail corpora allata and prevents the corpora allata from turning back on after ecdysis. Thus, there is insufficient juvenile hormone in the 4th-instar and precocious metamorphosis results. More experiments are necessary to test this hypothesis, but the data thus far indicate that ETB may be a useful compound for the analysis of the feedback mechanisms controlling juvenile hormone biosynthesis. Aclino~~ledgemenfs-We would like to thank Dr G. B. Staal of Zoeconcorporation for gift of ETB and Professor Lynn M. Riddiford. Universitv of Washington. U.S.A.. for critical reading and improvement of- this manuscript. We also would like to thank Professor S. Morohoshi, the president of Tokyo University of Agriculture and Technology, Japan for his helpful suggestion and encouragement. This work was partly supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture. Japan (A-436030). REFERENCES Akai H.. Kiguchi K. and Mori K. (1973) The influence of juvenile hormone on the growth and metamorphosis of Bombyx larvae. Bull. seric. Exp. Stn Japair 25, 287-305. Bounhiol J. J. (1938) Recherches exp&imentales sur le determinisme de la mCtamorphose chez les Ltpidoptcres. Suppl. Bull. Biol., France et Belgique. Bowers W. S., Ohta T.. Cleere J. S. and Marsella P. A. (1976) Discovery of insect anti-juvenile hormones in plants. Science, 193, 542-547. Edwards. J. P.. Bergot. B. J. and Staal, G. B. ( 1983) Effects of three compounds with anti-juvenile hormone activity and a juvenile hormone analogue on endogenous juvenile hormone levels in the tobacco hornworm. Manduca sesta. J. Insect Physiol.. 29, 83-89. Fukuda S. (1956) Hormone balance in the metamorphosis of insects, with special reference to the effect of hormone unbalance on the silkgland in Bombvx mori. Jap. J. rsp. Morph., 10, 14-26. Hiruma K.. Yagi S. and Endo A. (1983) ML-236B (compactin) as an inhibitor of juvenile hormone biosynthesis. Appl. Ent Zool.. 18, 111~115. Kieuchi K. (1972) Hormonal control of the colouration of l&al bod‘y and the pigmentation of larval markings in Bombrs mori. (1) Endocrine organs affecting the colouration of larval body and the pigmentation of markings. J. seric. Sri.. Japan 41, 407-412.
506
KENJIKIGIJCHI
Kiguchi K. (1974) A simple method to evaluate the juvenile hormone activity based on the color change of the larval marking in the silkworm, B0mh.r.v mori. J. seric. Sci.. Japan 43, 340-343. Kiguchi K. (1976)Physiological action of juvenile hormone in Bomb.~u mori with special reference to the larval colouration. (In Japanese). Doctoral thesis. University of Tokyo. Kiguchi K. and Riddiford L. M. (1978) A role of juvenile hormone in pupal development of the tobacco homworm, Manduca sexta. J. Insect Physiol. 24, 673-680. Kiguchi K. and Agui N. (1981) Ecdysteroid levels and developmental events during larval moulting in the silkworm, Bombvx mori. J. Insect Phvsioi. 27. 805-812. Kiguchi K.. Ohtaki T.. Akai H. and Mori K. (‘1974) Juvenile hormone activity of terpenoid esters and phenyl-geranylethers with different alkyl substituents against the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). Appl. Em. 2001. 9, 29-33. Kondo K. (1973) /j-Phenoxy-oder substituierte Phenoxyethanol-Verbindungen Offenlegungschrift 24 56 082, Bundesrepublik Deutschland, May 28, 1973. Kramer S. J. and Staal G. B. (1981) In vitro studies on the mechanism of action of anti-juvenile hormone agents in larvae of Manduca se.xta. In Juvenile Hormone Biochemistry: Action, Agonism and Antagonism (Ed. by Pratt G. E. and Brooks G. T.), pp. 425437. Elsevier/North-Holland Biomedical Press, Amsterdam. Kuwano E., Takeya R. and Eto M. (1983) Terpenoid imidazoles: New anti-juvenile hormones. Agric. Biol. Chem. 47, 921-923.
et
uf.
Monger D. J.. Lim W. A., Kezdy F. J. and Law J. H. (1982) Compactin inhibits insect HMG-CoA reductase and juvenile hormone biosynthesis. Biochem. biophys. Res. Commun. 105, 13741380. Murakoshi S. and Ichimoto I. (1972) On the appearance of three-moulters from the F, hybrid of four-moulters in the silkworm, Bombyx mori L. due to oral administration of a 2-hydroxymethyi-5-hydroxy-4-pyridone. Jap. J. appl. Em. Zool. 16, 159-161. Murakoshi S., Nakata T., Ohtsuka Y., Akita H., Tahara A. and Tamura S. (1975) Appearance of three-moulters from larvae of the silkworm, Bomby.u mori L., by oral administration of abietic acid derivatives. Jap. J. appl. Em. Zooi. 19, 267-272. Quistad G. B., Cerf D. C., Schooley D. A. and Staal G. B. (1981) Fluoromevalonate acts as an inhibitor of insect juvenile hormone biosynthesis. Mature 289, I76 177. Riddiford L. M., Roseland C. R.. Thalberg S. and Curtis A. T. (1983) Action of two juvenile hormone antagonists on lepidopteran epidermis. J. Insect Phvsiol. 29, 281-286. Staal G. B. (1977) Insect control with insect growth regulators based on insect hormones. Ser. varia pont. Acad. Sci. 41, 353-383. Staal G. B., Henrick C. A., Bergot B. J., Cerf D. C.. Edwards J. P. and Kramer S. J. (1971) Relationships and interactions between JH and anti JH analogues in Lepidoptera. In Regulation of Insect Development and Behaviour (Ed. by Sehnal F.. Zabza A., Menn J. J. and Cymborowski B.). pp. 323-340. Wroclaw Technical University Press. Wroclaw.
0022-1910184 53.00 + 0.00 Copyright (‘1 1984 Pergamon Press Ltd
EFFECTS OF ANTI-JUVENILE HORMONE “ETB” ON THE DEVELOPMENT AND METAMORPHOSIS OF THE SILKWORM, BOMBYX MORI KENJI
The Sericultural
Experiment
KIGUCHI,
TADASU
MORI*
HIROMU AKAI
and
Station, Yatabe, Ibaraki 305, Japan and *Kinjo Gakuin Moriyama-ku. Nagoya-shi, Aichi 463. Japan
University,
Ohmori.
(Received IO October 1983; revised 5 Junuary 1984)
Abstract-As in the tobacco homworm Munduca sexta, the synthetic juvenile hormone analogue ETB (ethyl 4-[2-(tert-buthylcarbonyloxy)butoxy]benzoate) showed both juvenile hormone-like and anti-juvenile hormone activities in the silkworm, Bombyx mori. When ETB was topically applied to allatectomized 4th-instar larvae, the compound counteracted the effects of allatectomy, such as induction of precocious metamorphosis and black pigmentation in the larval markings. Therefore, ETB had juvenile hormone activity, but it could neither induce brown pigmentation in the markings nor induce an extra-larval moult as can juvenile hormone. When intact 3rd-instar larvae were treated with the compound, the majority underwent precocious metamorphosis in the 4th-instar, and later formed fertile miniature adults. Some moulted into larval-pupal intermediates or Sth-instar larvae with darkened larval markings and/or with abnormality of specific regions of the silk-gland. The optimal dose for such anti-juvenile effects was about I-lOpg/larva, and higher doses showed less activity. Such anti-juvenile hormone effects of ETB were counteracted by administration of the juvenile hormone analogue, methoprene, before a certain critical time in the 4th-instar. The corpora allata of treated larvae appeared cytologically normal, and the corpora allata from ETB-induced miniature moths secreted juvenile hormone when implanted into allatectomized 4th-instar larvae.
Kqv Word &de-x: ETB, anti-juvenile
Bombyx mori, precocious
hormone,
MATERIALS
INTRODUCTION
The discovery of compounds with anti-juvenile hormone activity gave a new perspective to the development of potent insect control agents. Moreover, studies on anti-juvenile hormones were expected to lead to the better understanding of juvenile hormone action and endocrine regulation of the corpora allata. Nevertheless, a relatively limited number of such compounds is known, such as the ageratochromenes (precocenes) (Bowers, 1976), ETB, piperonyl butoxide (Staal, 1977), compactin (Monger et al., 1982; Hiruma et al., 1983). fluoromevalonate (Quistad et al., 1981), piron and piridon derivatives (Murakoshi and Ichimoto, 1972), abietic acid derivatives (Murakoshi et al., 1975) and terpenoid imidazoles (Kuwano et al., 1983). Among them, ETB is one of the most intriguing chemicals because of its peculiar and complex actions (Staal, 1977; Staal et al., 1981). The compound was first synthesized as a juvenile hormone mimic (Kondo et al.. 1973). Subsequently its anti-juvenile hormone activity was discovered in the tobacco hornworm, Manduca sextu, although other lepidopteran insects so far tested seem to be unaffected (Staal, 1977; Staal et al., 1981). We examined the effects of ETB administration on the silkworm, Bombyx mori, and found that it had both typical anti-juvenile hormone and potent juvenile hormone-like activities, just as seen in Munduca sexta. 1.P 30 h
E
metamorphosis
499
Experimental
AND METHODS
animals
The F, hybrid strain of Bombyx mori resulting from the cross between N. 124 and C.124 was used as experimental animals. Larvae were reared routinely at 25°C on fresh mulberry leaves. Developmental stages were determined by the Spiracle Index described by Kiguchi and Agui (198 1). Application
of ETB
and juvenile
hormone
unulogue
ETB, ethyl 4-[2-(tert-butylcarbonyloxy)butoxy] benzoate, was a gift from Dr G. B. Staal, Zoecon Corporation, U.S.A. Various doses (0.001-500 pg) of ETB dissolved in ethanol were applied topically onto the whole dorsal surface of the larvae anaesthetized with ethyl ether. Third-instar larvae were treated with 0.5-2 ~1 of solution, 4th-instar larvae with 2-6 ~1 and Sth-instar larvae with l&20 ~1, respectively. Control animals received the corresponding volume of ethanol. resulting in negligible mortality. For the counteraction experiments, a juvenile hormone analogue, methoprene (ZR 515, Zoecon). dissolved in peanut oil was used. Surgical
methods
Allatectomy was as described by Kiguchi and Riddiford (1978). Corpora allata were implanted through a lateral incision in the intersegmental membrane between the 7th and 8th larva1 segments. The
500
KENJI KIGUCHI
CI (I/.
B
A
C
D
Fig. I. Normal silk gland (A) and various degree of abnormal silk glands (B. C, D) induced by juvenile hormone injection 12-24 h after allatectomy in the silkworm, Bomhyx mori. Abnormal degeneration occurs only at the middle division of the middle silk gland. a. anterior silk gland; b, anterior division of the middle silk gland; c, middle division of the middle silk gland: d, posterior division of the middle silk gland: e, posterior silk gland.
tectomy, and score I to brown markings which could be caused by the injection of a high dose of juvenile hormone. Scores 2 and 4 indicated intermediate pigmentation.
wound sites were not sealed, but little blood leakage resulted. Evaluation
qf t$%cts
ETB-treated larvae were reared at 25 C. At an appropriate time, they were scored carefully under a dissecting microscope. Special attention was paid to the appearance of precocious pupal characters. pigmentation of larval markings and a morphological abnormality of a specific region of the silk-glands (Fig. I), because these are all related to juvenile hormone action or function of corpora allata (Kiguchi, 1976) (see Discussion section). The peculiar degeneration of only the mid-region of the middle silkgland (c in Fig. 1A) was first noted by Fukuda (1956) when he reimplanted corpora allata into allatectomized 4th-instar larvae within 24 h of allatectomy, and thus prevented precocious metamorphosis except for this specific degeneration of the silkgland. Effects on marking pigmentation was scored as described previously (Kiguchi, 1972): the silkworm larva has two pairs of larval markings on the ventral side of the abdomen. The outer side of each marking is black and the inner side brown in colour. Normally there is little variation in this marking colouration in this strain. Score 3 was given to non-treated control larvae, score 5 to completely black markings which could be induced by alla-
Table I. Effect of ETB on mduction
Histology For electron microscopical study, corpora allata were dissected from the ETB-induced precocious wandering larvae. The corpora allata were fixed with 2.5”/:, glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 M sucrose. They were post-fixed in l9/, O,O, in the same buffer and embedded in Epon 812. Sections were stained with uranyf acetate and lead citrate, and examined in a JEOL JEM-100CX II electron microscope.
RESULTS
The juvenile hormone-mimicking activity of ETB was tested according to the method described by Kiguchi et al. (1974). Larvae were allatectomized 24 h after the 3rd ecdysis, and different doses of ETB dissolved in 5 ~1 of ethanol were applied topically within 1Omin after the allatectomy. Control larvae which had been allatectomized and then treated with 5 ~1 of ethanol underwent precocious metamor-
of larval moulting larvae
m allatectomized
No. of larvae D
IO0 I I)
I 0. I II.0 I Allatectomirrd control
activity qf’ ETB
Juvenile hormone-mimicking
4th.mstar
transformed
Bomhw
into
No. ot lawaL: tested
Precocmus PuPa
5 IO IO IO ICI IO
0 0 (I 0 7 IO
0 0 0 0 2 0
5 IO IO IO I 0
I0
IO
0
0
Larval-pupal intermediate
5th~Instar IXW
Effect of anti-juvenile
hormone “ETB” on Bombyx
Table 2. Effect of ETB and synthetic juvenile hormone on the marking pigmentation of the allatectomized Born& larvae Score (Mean + Standard deviation)
---
Dose @g/larva) 500 100 50 10 5
I 0.1 0.01 0.001 0.0001 Allatectomized control
ETB*
Synthetic juvenile hormonet
3.0 f 0 (3) 2.9 + 0.3 (10) Not tested 3.0 f 0 (10) Not tested 3.0 + 0 (10) 3.0 f 0 (10) 3.2 f 0.4 (10) 4.8 f 0.4 (10) 5.0 &O (10)
1.0&O (8) 1.050 (8) 2.1 + 0.4 (8) 4.0 5 0 (8)
5.0? 0 (30)
5.0 * 0 (IO)
*Larvae were allatectomized just before apolysis in the 4th~instar, and the given amount of ETB was topically applied within IOmin. Scoring was done after the 4th ecdysis. tData from Kigucbi (1972) (Juvenile hormone used was a stereoisometric mixture of juvenile hormone 1). A number of larvae tested was shown in the parentheses in each column.
phosis. Administration of 1-5001.18 of ETB prevented this precocious metamorphosis so that all treated animals underwent a larval moult. No acute lethal effect was observed. Lower doses (0.001-0.1 pg) showed no or little effect. The dose response curve gave a 5004 effective dose of 0.25 pg/larva. Next, the juvenile hormone activity was assayed based on the colour change of the larval markings as previously reported (Kiguchi, 1974). Allatectomy at the time of general apolysis in the 4th instar causes black pigmentation of the larval markings in the 5th instar. This darkening can be prevented by an injection of an appropriate dose of juvenile hormone just after allatectomy. Moreover, a high dose of the hormone induces brown pigmentation of the larval markings in the dose-response manner in both intact and allatectomized 4th instar larvae (Table 2). To determine whether ETB has the same effect as juvenile hormone, the compound was applied topically to allatectomized test animals. They ecdysed into the 5th instar about 30 h after the ETB treatment, and their marking pigmentation was scored carefully under the dissecting microscope. As shown in Table 2, at a low dose of 0.01 pgg/larva, ETB inhibited the black pigmentation of the markings caused by allatectomy. Yet large doses of l-500 pg/larva did not
501
induce the brown pigmentation of the larval markings (score 1 or 2). Thus ETB differs from juvenile hormone and many juvenile hormone derivatives. Another peculiar phenomenon was observed when ETB was applied to the final Sth-instar larvae. A supernumerary larval moult can be induced by the application of high doses of juvenile hormone and many of its analogues at the middle stage of the 5th instar (Akai et al., 1973). Such supernumerary moulting was not induced by ETB, even at an extremely high dose (100-500 pg/g of larval body weight). The only effect of ETB at this time and dosage was the induction of dauer (permanent) larvae. Thus, there are some essential differences between the actions of juvenile hormone and of ETB. Anti-juvenile hormone activity of ETB To check the anti-juvenile hormone effects of ETB, 10 pg of the chemical was applied topically to 3rd or 4th-instar Bombyx larvae. The results were shown in Table 3. When ETB was applied to the 3rd-instar larvae, morphologically normal 4th~instar larvae were formed. As shown in Table 3, many of these larvae then moulted into precocious miniature pupae and larval-pupal intermediates. Those treated earliest in the 3rd-instar were most affected. Some of the test animals formed Sth-instar larvae, but most of these had abnormal silk glands (Fig. 1) and dark colouration of the larval marking spots. When ETB was applied to clth-instar larvae, none of the treated animals showed any sign of precocious metamorphosis. The only recognizable effect was the darkening of the larval markings in the 5th instar. To determine the optimal amount of ETB needed to induce anti-juvenile hormone effects, various doses of the chemical (0.1-200 pg/larva) were administered topically to freshly ecdysed 3rd-instar larvae. As shown in Table 4, induction of precocious metamorphosis occurred in a comparatively narrow dose range of 1-1Opg per larva. Higher doses (50, 100, 200pgg/larva) showed little or no activity in this respect. By contrast, both darkening of larval markings and the abnormal region-specific degeneration of silkglands in the 5th instar were induced over a wider dose range of l-2OOpg. The anti-juvenile hormone effects of ETB could be counteracted by administration of a juvenile hormone analogue, methoprene. When 1 pg of methoprene was injected into 0 and 24-h-old 4th~instar
Table 3. Effects of ETB (10 pg/larva) on 3rd and 4th-instar Eombyx larvae No. of larvae transformed into Time ETBadministered (h after ecdysis)
___
No. of larvae tested
Precocious pupae
Larval-pupal intermediate
33 32 24
23 7 0
4 13 II
12 12 12
0 0 0
0 0 0
~
..__~
Marking pigmentation (mean score)
O0Of larvae with silk gland abnormality
6 12 13
4.3 3.8 3.3
IO0 x.7 x5
12 12 12
3.8 3.6 3.0
0 0 0
Sth-lnstar larvae
3rd instar
I 24 48 4th mstar
I 24 48
502
KENJI KIGU(‘HI Table 4. Dose -response
for ET6 admmibtered
PI ul. on r~ewly moulted
3rd.mstar
No. of larvae transformed
Marking plgmentalion (mean score)
Dose (~g;larval
4.0
200 100 50 I0 5 I 0.5 0.1 COlltrOl
41 43 4 (I 4.u 73 ? (I 30 30
larvae which have previously received 10 pg of ETB just after ecdysis into the 3rd instar. all of the experimental animals moulted into Sth-instar larvae with no silk gland abnormality. Importantly. their larval marking colour was brown (score I), possibly due to the action of methoprene. When methoprene was given at 48 or more hours after ecdysis to the 4th stage, it only partially or no longer prevented the effects of ETB. Efect
of ETB on the c’orporu ulIutu
Precocious pupae caused by ETB became fertile miniature moths. To determine whether ETB affected the functioning of the corpora allata, one pair of corpora allata were dissected from the ETB-treated host animal on the day after adult eclosion and implanted into an allatectomized young 4th-instar larva. All recipients moulted into normal Sth-instar larvae (N = 15), indicating that ETB-affected corpora allata were able to secrete juvenile hormone. The ultrastructure of the corpora allata from ETBtreated precocious wandering larvae was also observed. As in the normal Sth-instar wandering larvae (Fig. 2A). the corpus allatum cells of ETB-treated insects were found to be characterized by well developed large nuclei, many mitochondria and often conspicuous Golgi complexes (Fig. 2. B and C). Neurosecretory axons derived from the brain were also observed in the peripheral regions of the organs (Fig. 2D). In the axons there were two types ot granules: electron-dense and transparent granules (Fig. 2D), just as in the normal insects. In the three corpora allata observed. no significant degradation was recognizable.
Allatectomized 4th-instar Bonzbw larvae become precocious pupae and then fertile moths (Bounhiol. 1938). As might be expected. both male and female miniature moths caused by ETB mated normally, and produced fertilized diapausing eggs. The eggs showed good hatchability (>954;) after chilling at 5°C for about 4 months. These results indicate that juvenile hormone is not involved in the sexual maturation (sex pheromone production, spermatogenesis and egg formation etc.) in the silkworm, Bomb~~r wzori. DISCUSSION
Anti-juvenile
larvae
into
hormone USSUJ h_v Bombyx
Compounds with anti-juvenile hormone activity are the chemicals which can induce the effects of
(‘,, Of larvae with silk gland abnormality 70 x5 x3 IO0 100 13 (1 0 0
surgical removal of the corpora allata, the source of juvenile hormone. The effects of allatectomy in Bombys are summarized in Fig. 3. As in other lepidopteran insects, allatectomy during the early and middle stage of the penultimate instar causes precocious pupation. When allatectomized just at the beginning of spiracular apolysis. 1arvalLpupal intermediates are induced (Kiguchi and Agui, 1981). Thereafter, removal of the corpora allata can no longer prevent the larval moult, but it still induces black pigmentation of the larval markings (Kiguchi,1972). In addition, degeneration of a specific region of the silk glands is caused by the temporary decrease in haemolymph juvenile hormone after allatectomy. More pronounced degeneration is prevented by the subsequent implantation of corpora allata or injection of synthetic juvenile hormone within 12-24 h of allatectomy (Fukuda, 1956; Kiguchi. 1976). Such silk gland degeneration occurs during the moult to the 5th instar in these juvenile hormone-treated larvae. Apparently, the cells of the region (the middle division of the middle silk gland) are committed in the pupa by the sudden decrease (or absence) of juvenile hormone and a low but significant level of ecdysteroids in the haemolymph (Kiguchi and Agui. 1981). so that they then degenerate later when exposed to the high level of ecdysteroids during the 4th-larval moult (unpublished). Since the effects of allatectomy in Body.\- mori are well understood it is a good bioassay for anti-juvenile hormones. Charucteristics
gf’ actions qf’ ETB
ETB shows both juvenile hormone-like and antijuvenile hormone action against the silkworm, Bornb~*s tori. Just as natural juvenile hormone and many derivatives. ETB effectively inhibits precocious metamorphosis and black pigmentation of the larval markings seen in allatectomized 4th-instar larvae. Importantly, ETB could not mimic all of the effects of juvenile hormone; namely it neither induced brown pigmentation in the larval markings nor induced extra-larval moult even when large doses were applied at the appropriate developmental stage. These results may suggest that ETB has little intrinsic juvenile hormone activity, but acts to synergise endogenous juvenile hormone. In other insects it also can mimic juvenile hormone at high doses (Staal, 1977: Staal et al., 1981). and in Man&u sextu it acts as a juvenile hormone directly on the epidermis in ci/ro (Riddiford er al., 1983). By contrast, ETB produces almost all the same
Fig. 2. The corpora allata from the normal 5th~instar larva (A) and the precocious wandering larva ted by ETB-administration (B, C, D). N: Nucleus, M: Mitochondria. G: Golgi complex, DG: Electron-dense granules, TG: Transparent granules.
503
Effect of anti-juvenile
hormone “ETB”
PreCOClOUS
Larval
-pupa1
Black
intermediate
PuPa
505
on Bon1h~a.u
of
plqmentatlon
larval
morklnqs
3rd Larval ecdysis
1 ä*_ .-. __~
24
-
I
72h
Splroculor apolysls 12 -24
h
‘-
I - CA
General opolysls +JH
~
Fig. 3. Summary
+JH
I
End of period
JH
pigmentation
secretIon
JH for
sensitive marklnq
by CA
t Sllkqland
Larval
abnormol~ty
internedlate
of effects of allatectomy
End of critical period for
- pupal
(-CA)
effects as surgical removal of the corpora allata in the penultimate-instar larvae when applied to young 3rd-instar larvae. The treated morphologically normal 4th~instar larvae became precocious pupae. larval--pupal intermediates or Sth-instar larvae with darkened larval markings and/or silk gland abnormality. Thus, ETB shows anti-juvenile hormone activity in the silkworm, B0mby.x mori, as well as in the tobacco hornworm. Munduca se.xtu (Staal. 1977; Riddiford et ul., 1983). How ETB exerts its anti-juvenile hormone action is unknown. It effectively reduces juvenile hormone output by Manducu corpora allata in vitro (Kramer and Staal. 1981). Our cytological observations show that ETB had no noticeable cytotoxic effect on BombJ).u corpora allata. Moreover, corpora allata from miniature moths produced by ETB treatment secreted juvenile hormone as assayed by implantation into young larvae. Obviously, ETB is different type of anti-juvenile hormone from the ageratochromenes (precocenes) (Bowers, 1976). Staal rt al., (1981) suggested that ETB has complex activity, including competition at juvenile hormone receptor level, inhibition of early steps in the hormone synthesis and depression of the biosynthesis through negative feedback. Edwards ef al. (1983) also suggested the possible mode of action of ETB as an inhibitor of feedback system responsible for switching the corpora allata off and on. Since ETB effects can be counteracted by methoprene applied 3-4 days after the ETB treatment. there is little likelihood that the compound competes for juvenile hormone receptors at the target tissues to inhibit the action of endogenous juvenile hormone. Rather, ETB seems to be able to depress the juvenile hormone-secreting activity of the corpora allata (therefore. the biosynthesis) through a feedback mechanism. Immediately after application, ETB might act synergistically. Then, as the compound is metabolized, its anti-juvenile hormone activity may take over and prevent either the release or action of juvenile hormone. Possibly the persistent low concentration during the 3rd to 4th-larval moult acts as a juvenile hormone on the
in the silkworm. Bombyu mori. See text for delail corpora allata and prevents the corpora allata from turning back on after ecdysis. Thus, there is insufficient juvenile hormone in the 4th-instar and precocious metamorphosis results. More experiments are necessary to test this hypothesis, but the data thus far indicate that ETB may be a useful compound for the analysis of the feedback mechanisms controlling juvenile hormone biosynthesis. Aclino~~ledgemenfs-We would like to thank Dr G. B. Staal of Zoeconcorporation for gift of ETB and Professor Lynn M. Riddiford. Universitv of Washington. U.S.A.. for critical reading and improvement of- this manuscript. We also would like to thank Professor S. Morohoshi, the president of Tokyo University of Agriculture and Technology, Japan for his helpful suggestion and encouragement. This work was partly supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture. Japan (A-436030). REFERENCES Akai H.. Kiguchi K. and Mori K. (1973) The influence of juvenile hormone on the growth and metamorphosis of Bombyx larvae. Bull. seric. Exp. Stn Japair 25, 287-305. Bounhiol J. J. (1938) Recherches exp&imentales sur le determinisme de la mCtamorphose chez les Ltpidoptcres. Suppl. Bull. Biol., France et Belgique. Bowers W. S., Ohta T.. Cleere J. S. and Marsella P. A. (1976) Discovery of insect anti-juvenile hormones in plants. Science, 193, 542-547. Edwards. J. P.. Bergot. B. J. and Staal, G. B. ( 1983) Effects of three compounds with anti-juvenile hormone activity and a juvenile hormone analogue on endogenous juvenile hormone levels in the tobacco hornworm. Manduca sesta. J. Insect Physiol.. 29, 83-89. Fukuda S. (1956) Hormone balance in the metamorphosis of insects, with special reference to the effect of hormone unbalance on the silkgland in Bombvx mori. Jap. J. rsp. Morph., 10, 14-26. Hiruma K.. Yagi S. and Endo A. (1983) ML-236B (compactin) as an inhibitor of juvenile hormone biosynthesis. Appl. Ent Zool.. 18, 111~115. Kieuchi K. (1972) Hormonal control of the colouration of l&al bod‘y and the pigmentation of larval markings in Bombrs mori. (1) Endocrine organs affecting the colouration of larval body and the pigmentation of markings. J. seric. Sri.. Japan 41, 407-412.
506
KENJIKIGIJCHI
Kiguchi K. (1974) A simple method to evaluate the juvenile hormone activity based on the color change of the larval marking in the silkworm, B0mh.r.v mori. J. seric. Sci.. Japan 43, 340-343. Kiguchi K. (1976)Physiological action of juvenile hormone in Bomb.~u mori with special reference to the larval colouration. (In Japanese). Doctoral thesis. University of Tokyo. Kiguchi K. and Riddiford L. M. (1978) A role of juvenile hormone in pupal development of the tobacco homworm, Manduca sexta. J. Insect Physiol. 24, 673-680. Kiguchi K. and Agui N. (1981) Ecdysteroid levels and developmental events during larval moulting in the silkworm, Bombvx mori. J. Insect Phvsioi. 27. 805-812. Kiguchi K.. Ohtaki T.. Akai H. and Mori K. (‘1974) Juvenile hormone activity of terpenoid esters and phenyl-geranylethers with different alkyl substituents against the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). Appl. Em. 2001. 9, 29-33. Kondo K. (1973) /j-Phenoxy-oder substituierte Phenoxyethanol-Verbindungen Offenlegungschrift 24 56 082, Bundesrepublik Deutschland, May 28, 1973. Kramer S. J. and Staal G. B. (1981) In vitro studies on the mechanism of action of anti-juvenile hormone agents in larvae of Manduca se.xta. In Juvenile Hormone Biochemistry: Action, Agonism and Antagonism (Ed. by Pratt G. E. and Brooks G. T.), pp. 425437. Elsevier/North-Holland Biomedical Press, Amsterdam. Kuwano E., Takeya R. and Eto M. (1983) Terpenoid imidazoles: New anti-juvenile hormones. Agric. Biol. Chem. 47, 921-923.
et
uf.
Monger D. J.. Lim W. A., Kezdy F. J. and Law J. H. (1982) Compactin inhibits insect HMG-CoA reductase and juvenile hormone biosynthesis. Biochem. biophys. Res. Commun. 105, 13741380. Murakoshi S. and Ichimoto I. (1972) On the appearance of three-moulters from the F, hybrid of four-moulters in the silkworm, Bombyx mori L. due to oral administration of a 2-hydroxymethyi-5-hydroxy-4-pyridone. Jap. J. appl. Em. Zool. 16, 159-161. Murakoshi S., Nakata T., Ohtsuka Y., Akita H., Tahara A. and Tamura S. (1975) Appearance of three-moulters from larvae of the silkworm, Bomby.u mori L., by oral administration of abietic acid derivatives. Jap. J. appl. Em. Zooi. 19, 267-272. Quistad G. B., Cerf D. C., Schooley D. A. and Staal G. B. (1981) Fluoromevalonate acts as an inhibitor of insect juvenile hormone biosynthesis. Mature 289, I76 177. Riddiford L. M., Roseland C. R.. Thalberg S. and Curtis A. T. (1983) Action of two juvenile hormone antagonists on lepidopteran epidermis. J. Insect Phvsiol. 29, 281-286. Staal G. B. (1977) Insect control with insect growth regulators based on insect hormones. Ser. varia pont. Acad. Sci. 41, 353-383. Staal G. B., Henrick C. A., Bergot B. J., Cerf D. C.. Edwards J. P. and Kramer S. J. (1971) Relationships and interactions between JH and anti JH analogues in Lepidoptera. In Regulation of Insect Development and Behaviour (Ed. by Sehnal F.. Zabza A., Menn J. J. and Cymborowski B.). pp. 323-340. Wroclaw Technical University Press. Wroclaw.