Role of juvenile hormone esterase in Diptera (Drosophila virilis) metamorphosis

J. Insect Physiol.Vol.37, No. 7, pp. 541-548,1991 Printedin Great Britain. All rights reserved

0022-1910/91 $3.00+ 0.00 Copyright0 1991PergamonPressplc

ROLE OF JUVENILE HORMONE ESTERASE IN DIPTERA (DROSOPHILA W&X9) METAMORPHOSIS I. Yu. RAUSCHENBACH,’ N. S. LUKASHINA,’T. M. KHLEBODAROVA’ and L. I. KOROCHKIN* ‘Institute of Cytology and Genetics, Siberian Department, U.S.S.R. Academy of Sciences, Novosibirsk, 630090 and *Institute of Developmental Biology, U.S.S.R. Academy of Sciences, Vavilova 26, Moskow, 117808,U.S.S.R. (Received 5 October 1990; revised 26 February 1991)

Abstract-Juvenile hormone metabolism and its esterase activity during Drosophila uirilis development and the effects of juvenile hormone esterase inhibition on metamorphosis have been investigated. The mid 3rd-instar larvae have juvenile hormone degradation by IUPAC. Juvenile hormone esterase activity appears at the beginning of the wandering stage and sharply rises by the end. After puparium formation, the esterase activity continues to rise attaining peak values in 48 h pupae; thereafter the enzyme activity starts to decline. Esterase inhibition by paraoxon inhibits D. uirilis metamorphosis. Data obtained are compared to the results of juvenile hormone and ecdysone titres in other Drosophila species. The key role of juvenile hormone esterase for genome reprogramming in Diptera is discussed. Key Word Index: Drosophila virilis; juvenile hormone; juvenile hormone esterase; metamor-

phosis

INTRODWTION The reprogramming required to switch from larval to pupal development is induced by 20-hydroxyecdysone in the absence of juvenile hormone. With the latter present, 20-hydroxyecdysone causes larva moulting but the programme of development remains unchanged and a new larval instar takes place (Riddiford, 1978, 1980, 1981, 1984; Hwang-Hsu et al., 1979). The major pathways for juvenile hormone degradation in insects is hydrolysis of the ester moiety and hydration of the epoxide ring. The primary products of these reactions are juvenile hormone acid and juvenile hormone diol (Hammock, 1985). In insect haemolymph, juvenile hormone is degraded only through hydrolysis of the ester by a specific group of esterases. According to Hammock (1985) these esterases are “capable of rapidly hydrolyzing juvenile hormone in the presence or absenceof carrier protein”. The other tissues contain, besides esterases (juvenile hormone esterases and general esterases), IUPACs and mixed function oxidases capable of degrading unbound juvenile hormone (De Kort and Granger, 1981). The view is held that juvenile hormone esterase plays the key role in the metamorphosis of insects of the lepidopteran order, since it inactivates juvenile hormone in the haemolymph before the reprogramting from larval to pupal development and the switchover to a new programme (Riddiford, 1980,

1981; Hammock, 1985; Newitt and Hammock, 1986). Studies of esterase activity in the haemolymph of the species of the dipteran order, Drosophila melunogaster (Wilson and Gilbert, 1978), Surcophuga bullutu (Weirich and Wren, 1976), D. hydei (Klages and Emmerich, 1979) raised the question whether these esterases play a key role in metamorphosis of this order. The reason was that esterase activity was undetectable in the haemolymph of the last-instar larvae in the dipteran order which was contrary to the observations made in the lepidopteran order. Activity of the esterase in the haemolymph appeared at the pre-pupal stage (Klages and Emmerich, 1979). However, the absence of juvenile hormone esterase activity from the haemolymph of dipteran larvae does not mean that the esterase has no part in the key role in the metamorphosis. The possibility exists that the degradation of juvenile hormone occurs directly in the larval tissues and not in the haemolymph. To see whether this is the case, measurements of esterase activity in the whole body of larvae are needed. The functional importance of the enzyme in switching the developmental programme to metamorphosis may also be examined by inhibiting the esterase activity in larvae at the wandering and prepupal stages. The present work has accumulated data on juvenile hormone metabolism in whole body homogenates of D. virilis during development and on the effect of the juvenile hormone esterase inhibition on the course of D. virilis metamorphosis. 541

I. Yu.

542 MATERIALS

RAUSWENBACH et

AND METHODS

The study was performed on D. virilis wild-type stock 101. D. virilis were raised at 25°C with 20 larvae on 7 ml nutrient medium; they were synchronized three times-when hatched, at the second moult and at puparium formation. The larval instar before puparium formation was determined by morphological criteria. As shown earlier (Rauschenbach et al., 1984) 12-13 h before puparium formation starts, the bases of anterior spiracles of larvae of D. virilis turn yellowish, and 2 h before puparium formation the larvae stop moving. Radioactive juvenile hormone metabolism and its esterase activity were analysed in larvae of these two groups, in the third-instar larvae 48 h after the second moult, in white prepupae (1 h after puparium formation), and, finally, in pupae 24, 48, 77 and 90 h after puparium formation. All the homogenate of 12 individuals prepared in 120~1 distilled water was 5 min centrifuged at 8000 rpm. The supernatant obtained was diluted with 0.1 M buffer phosphate (pH 7.4) in the 1: 4 ratio, and used to evaluate the juvenile hormone degradation using the partition method of Hammock and Sparks (1977) modified by Shapiro et al. (1986). The ‘H chainlabelled juvenile hormone III (11.9 Ci/mmol at C-10, NEN Research Products) and the unlabelled juvenile hormone III (95% E, E, Sigma) were used as well. Unlabelled (0.1 pg) and radioactive (12,500 dpm) juvenile hormone III were mixed and added to 100 pl buffer-diluted supematant. The reaction was performed in siliconized test tubes at 27°C for 120 min. The reaction time was specified earlier experimentally (Rauschenbach et al., 1989). The hydrolysis products were analysed by means of thinlayer chromatography (TLC) according to Renucci (1986). A silicagel plate with samples applied thereupon was placed into a chamber with a chloroform/ethylacetate mixture (2 : 1, v/v). Zones corresponding to juvenile hormone III, its acid, acid-dial and diol were identified by means of cochromatography with radioactive standards which we obtained using the techniques of Slade and Zibbit (1972). Chromatograms of these standards correlated well with those of the work by Renucci (1986). In experiments studying the effect of juvenile hormone esterase inhibitor-paraoxon (Sigma)--on D. virilis metamorphosis, the reagent was diluted first with a small amount of ethanol and then water. The solution was either applied to white prepupae (controls were applied with water containing a corresponding concentration of ethanol) or sprayed on to food onto which wandering larvae were placed. For controls the food was sprayed with water containing the corresponding amount of ethanol. The juvenile hormone, diluted with acetone, was sprayed on to food onto which wandering larvae were placed (for controls the food was sprayed with acetone). The esterase activity was estimated by the intensity of the

al.

staining of a corresponding fraction in the D. virilis esterase spectrum (using /I-naphthyl acetate as a substrate). Earlier we performed some experiments to identify juvenile hormone esterase in this spectrum (Rauschenbach et al., 1987). It was identified on the basis of the following data: (1) the corresponding fraction is the only esterase in D. virilis which is not inactivated by diisopropylphosphofluoridate; (2) it is the only esterase which has activated by juvenile hormone in vivo; (3) juvenile hormone is the substrate for this fraction; (4) the increase in its activity at the wandering stage, in prepupae and early pupae correlates well with the decrease of juvenile hormone titre; (5) this fraction K, value for juvenile hormone is close to the K,,, value for /I-naphthyl acetate and is lower by a factor of lo2 than the K,,, value for a-naphthyl acetate. The details of the procedure has been described (Rauschenbach et al., 1987). RESULTS

Juvenile hormone metabolism in D. virilis development

Figure 1 presents the result of a typical TLC separation of samples of radiolabelled juvenile hormone degradation by the supematants of larval (48, 60 and 70 h after the second moult) and pupal (48 h after pupariation) homogenates. The results from TLC for the mid third-instar larvae (48 h after the second moult) are clear. Juvenile hormone is degraded by IUPAC through hydration of the epoxide ring, since only its diol is chromatographically separated. Our results are in agreement with those obtained on larvae of two other dipteran species, S. bullata (Slade and Zibbit, 1972) and D. melanoguster (Wilson and Gilbert, 1978). They are at variance, however, with those of Klages and Emmerich (1979) who have demonstrated the presence of juvenile hormone acid in the peripheral tissues of third-instar larvae of D. hydei. This may be explained by differences in the methods used to measure the degradation of ‘H-juvenile hormone, such as the different substrate used, juvenile hormone III, in our case, and juvenile hormone I in that of Klages and Emmerich (1979). It is worth noting that our results are also in agreement with the recent investigations of Richard et al. (1989) who found the juvenile hormone III bisepoxide to be naturally occurring juvenile hormone of D. melanogaster larvae. Twelve hours before pupariation, 60 h after the second moult, juvenile hormone is degraded mainly by IUPAC. Juvenile hormone esterase activity, however, appears, since TLC recovered some trace of the acid (Fig. 1). By the end of the wandering stage (70 h after the second moult) juvenile hormone degradation is effected solely by the esterase, as it is evident by the appearance of only the acid on the chromatograms (Fig. 1). In pupae, 48 h after pupariation, the activity of the esterase is high (the major metabolite of juvenile

Role of juvenile hormone esterase in Diptera

cl EZI

Ill

N

1

2

543

L 46

L 60

L 70

P 40

4

3

5

Fraction

6

7

6

numbers

Fig. 1. Analysis of the degradation products of )H-juvenile hormone III by thin-layer chromatography. L 48, 60, 70, P 48-juvenile hormone degradation after incubation with larval extracts 48, 60 and 70 h after the second moult and with pupal extracts 48 h after pupariation, respectively. I-10 designate the fraction numbers on the thin-layer chromatograms. JH-Juvenile hormone non-degradated; JH-A-JHacid; JH-D-JH-diol; JH-D-A-JH-diol-acid. degradation is the acid) but IUPAC functions, also, as judged from the slight amounts of the metabolite juvenile hormone diol-acid separated by TLC (Fig. 1). hormone

Juvenile hormone development

esterase

activity in D.

virilis

The estimates for esterase activity in D. virifis during development are given in Table 1. In larvae of the mid third instar, esterase activity is undetectable. It appears weakly at the beginning of the wandering stage (12 h before pupariation) and rises sharply by the end of the stage. After puparia-

tion, esterase activity continues to rise attaining peak values in 48-h pupae; thereafter it starts to decline. On the basis of our observations, it is concluded that juvenile hormone degradation at the time of wandering appears to be effected by juvenile hormone esterase directly in the larval tissues and not in the haemolymph in Drosophila, unlike the insects of the lepidopteran order. Juvenile hormone esterase inhibition in wandering larvae and in prepupae of D. virilis

Data on the effect of the application of paraoxon on white prepupae on their ability to metamorphose

Table I. Activity of juvenile hormone esterase during development of

D. virilis Esterase activity fmol/min/animal*

Groups Larvae White prepupae Pupae

0 0.07 + 0.01 0.41 f 0.05 0.62 k 0.1 1.15 * 0.15 1.80 + 0.2 1.18kO.2 1.15 f 0.2

48 h after second moult 12 h before pupariation 2 h before pupariation 24 h 48 h 77 h 90 h

after after after after

pupariation pupariation pupariation pupariation

*Means f SE (5-7 independent extracts, determinations

in triplicate).

Table 2. Capacity of Drosophilato metamorphose after paraoxon application to white prepupae Experiment experment

I 2 3 4

(pm$Zimal) 7 0.7 0.1 0.1 x 10-r

Control

Number of prepupae

Metamorphosed W)

Number of prepupae

17 22 31 23

0 0 90 100

16 17 18 19

Metamorphosed (“/) 100 100 100 100

I.

544

Yu.RAU~CI~ENBACHer al.

Table 3. Capacity of Drosophilato metamorphose from paraoxon-treated

wandering larvae Died (%)

N experiment 1

2 3

Dose (nmol/animal)

Number of larvae

Metamorphosed (%)

2.8 x lo-) control 0.18 control 2.5 control

23 18 58 63 12 9

87 88 34 92 0 100

are presented in Table 2. Paraoxon at small doses affects the metamorphosis little (experiment 3) or not at all (experiment 4). The 7 pmol/animal and 0.7 pmol/animal doses (experiments 1 and 2, respectively) prevent matamorphosis and inhibit esterase activity (Fig. 2). The higher dose (7 pmol/animal) is problematical since all esterases are inhibited (sample 5, Fig. 2); at the 0.7 pmol/animal dose only juvenile hormone esterases are inhibited (samples 1 and 2, Fig. 2). The latter dose is unlikely to be toxic to prepupae since wandering larvae treated with the 2.8 pmol/animal dose display a normal course of metamorphosis and their juvenile hormone esterase is not inhibited. (The reason why the 2.8 pmol/animal dose does not inhibit this esterase in wandering larvae is still to be determined.) When dose is increased to 0.18 nmol/animal the number of metamorphosed larvae sharply declines and there are many intermediates which simultaneously display features of larvae and pupae: larvae with a pupal cuticle [Fig. 3(B)], pupae with a larvae cuticle and intermediates with the cuticular segmentation, variation in size of pupae, and with the fore part of the body unpigmented [Fig. 3 (A)]. The presence of such forms may be evidence of increased levels of juvenile hormone during pupation which is not surprising since its esterase is inhibited. Further increases in the paraoxon dose for larval treatment (Table 3, experiment 3) blocks metamorphosis in all individuals. Intermediates are produced and, of greater importance, a fraction of the larvae live for 7 days (controls pupate in 1S-20 h) and becoming enormous in size. Again, this is interpreted as evidence of the considerably increased juvenile hormone levels in these larvae. Such interpretation is supported by the results with juvenile hormone-treated larvae (Table 4). DISCUSSION

The contention that juvenile hormone esterase is the key metamorphic enzyme in lepidopteran insects has been derived from comparisons of the dynamics

Intermediates (%) 0 0 24 0 17 0

Pupae

Larvae

0 0 6 0 42 0

13 12 36 8 41 0

of the esterase activity, juvenile hormone level and ecdysteroid titre: an increase in esterase activity and a decrease in juvenile hormone titre precede the titre peaks of the ecdysteroids reprogramming the genome (Riddiford, 1978; Hwang-Hsu ef af., 1979; Jones ef al., 1982). In the context of the data supporting this contention for Diptera, it would be appropriate to compare with ecdysteroid titre, the decrease in juvenile hormone level and increase in juvenile hormone esterase activity in Drosophila. In the studies carried out on D. melanogaster through the years, either an ecdysteroid titre peak occurs during puparium formation (Hodgetts et al., 1977; Garen et al., 1977; Klose et al., 1980; Bainbridge and Bownes, 1988) or there is an increase in titre 5-6-h before pupariation with highest values 34 h after puparium formation (Borts et al., 1974; Berreur et al., 1979). The observation common to all these studies was the basal level of ecdysteroids 7-8 h before pupariation. Thus in D. melunoguster the metamorphosis-related increase in ecdysteroid titre starts at the wandering stage which lasts for 6-10 h in this particular species (Richards, 1981) and the major peak in the ecdysteroid titre which occurs during puparium formation (the first peak). Our data indicate that in Drosophila it is precisely at the time of wandering that juvenile hormone esterase activity rises sharply, six-fold. This rise correlates well with the decrease in the juvenile hormone titre reported for another species of the uiriIis group, D. hydei (Biihrlen et al., 1984). It should be emphasized that the events involved in the reprogramming from a larval to a pupal development are not limited to pupariation. In Drosophila, a sequence of inductive interactions unfolds at the prepupal and early pupal stages, as evidence by the appearance of a second ecydysteroid titre peak in prepupae (Klose et al., 1980; Handler, 1982) and a third peak in early pupae (Handler, 1982). It is pertinent to note that in Lepidopteru the

Table 4. Capacity of Drosophilato metamorphose from juvenile hormone III-treated wandering larvae Died (%) N experiment

Dose (@g/animal)

Number of larvae

Metamorphosed (%)

Intermediates 1%)

Pupae

I

4 20 control

10 13 14

0 0 86

50 69.3 0

40 23 0

2 3

Larvae 10 7.7 14

1

2

3

4

5

Fig. 2. Esterase spectrum in 18-h pupae when paraoxon is applied at the white prepupal stage. 1-4- -Esterase fractions; JH-JH-esterase. Samples 1, 2-paraoxon application at a 0.7pmo1, dose. Samples 3, 4-control. Sample 5-paraoxon application at a 7 pmol/animal dose.

545

(A)

(B 1

I

I 1 mm

Fig. 3. Effect of paraoxon on metamorphosis in D. virilislarvae. (A) On the left: two larvae with pupal cuticles; on the right: normal pupa. (B) On the left: intermediate with pigmented cuticles of the hind part of the body; on the right: normal pupa.

546

Role of juvenile hormone esterase in Diptera

reprogramming-related second peak of ecdysteroids also occurs at the prepupal stage. Juvenile hormone titre has to be extremely low for ecdysteroids to exert their prepupal inductive effects in Diptera. This is obvious from a body of evidence. Richards (1978) has established that the presence of juvenile hormone at an ecdysone-free period (the one at the prepupal stage between the first and second peaks in ecdysterone titres is a case in point) suppressed the late puffs in polytene chromosomes of the salivary gland cells of D. melunoguster; furthermore, Lezzi and Wyss (1977) have shown that the presence of juvenile hormone blocks the appearance of the prepupal ecdysone puff C 1-18 in the salivary glands of Chironomus. Cherbas et al. (1989) have also found that the juvenile hormone blocks the ecdysone response of D. melanogaster K, cells. Further support is provided by the data of Biihrlen et al. (1984) which demonstrate an extremely low juvenile hormone content during the prepupal stage of D. hydei and ours according to which juvenile hormone esterase activity is high during this stage in D. uirilis. Klages and Emmerich (1979), too, reported high esterase activity in prepupae and early pupae of D. hydei.

The high esterase activity through these developmental stages seems to be related to the need for a rapid juvenile hormone metabolism. In his extensive review concerned with the mechanisms of juvenile hormone degradation, Hammock (1985) entertains the possibility that juvenile hormone may be necessary at the prepupal stage to synchronize development by slowing down the maturation of tissues that are particularly sensitive to ecdysone. In fact, a prepupal juvenile hormone pulse occurs in many lepidopteran species (Hammock, 1985). Should levels of juvenile hormone be measured in prepupal Drosophila at time intervals shorter than Biihrlen et al. (1984) used? Perhaps, these titre data would reveal juvenile hormone pulses also in Drosophila? However, as noted above, the presence of juvenile hormone at the prepupal stage blocks the switchover of the developmental programme in the target tissues, and accordingly, one would anticipate a high juvenile hormone metabolic rate in prepupae and, possibly, early pupae: rapid synthesis and degradation in the haemolymph. What is especially noteworthy is that juvenile hormone activates its esterase directly, by passing the brain, at the prepupal stage (Jones and Hammock, 1983). The major role of this esterase in metamorphosis of Lepidoptera was advocated in work showing that enzyme inhibition blocks metamorphosis in Trichoplusia ni prepupae (Sparks and Hammock, 1980) and in studies reporting disturbances in the dynamics of the titre of ecdysteroids in prepupae of the same species with inhibited juvenile hormone esterase: titre did not decrease to the normal level by the end of the stage (Jones

et al., 1986).

547

As shown in our experiments, inhibition of juvenile hormone esterase in D. virilis wandering larvae and prepupae results in blocked metamorphosis and argues for the important role this enzyme plays in switching over the developmental programme for a larval to a pupal one in Diptera. Summarizing, the role of this esterase in the switchover of the developmental programme in the course of the metamorphosis of insects of the dipteran order, as exemplified by Drosophila, may be envisioned as follows. The decrease in juvenile hormone titre necessary for the larval-to-pupal reprogramming of the tissue cells, which are the first to be involved in the events of metamorphosis, occurs during the wandering stage. The decrease is effected through the esterase which hydrolyses juvenile hormone directly in the larval tissues. Of importance is that the reprogramming may not proceed simultaneously, but as in Hyalophora cecropiu where the epidermal cells are reprogrammed first, with the cells of the gonads, muscles, intestine following (Riddiford, 1972, 1975). The further decrease in the titre of juvenile hormone (or its maintenance at low levels) requisite for the development program switchover, is provided by juvenile hormone esterase hydrolysis in the haemolymph. Significantly high activity of the esterase throughout the pupal development is likely to remain to degrade juvenile hormone to completion (see Biihrlen et al., 1984; Bownes and Rembold, 1987) and that seems to be a necessary condition for the normal course of metamorphosis. Acknowledgement-We are grateful to MS N. Anufrieva for

translating the manuscript into English. REFERENCES

Bainbridge S. P. and Bownes M. (1988) Ecdysteroid titres during Drosophila metamorphosis. Insect Biochem. 18, 185-197. Berreur P., Porcheron P., Berreur-Bonnenfant J. and Simpson P. (1979) Ecdysteroid levels and pupariation in Drosophilamelanogaster.J. exp. Zool. 210, 347-352. Borts D. W., Bollenbacher W. E., O’Connor J. D., King D. S. and Fristrom J. W. (1974) Ecdysone level during metamorphosis of Drosophilamelanogaster.Devl Biol. 39, 308-316.

Bownes M. and Rembold H. (1987) The titre of juvenile hormone during the pupal and adult stages of the life cycle of Drosophila melanogaster. Eur. J. Biochem. 164, 709-712. Biihrlen U., Emmerich H. and Rembold H. (1984) Titer of juvenile hormone III in Drosophilahydeiduring metamorphosis determined by GC-MS-MIS. Z. Naturf. 39, 115c&ll54. Cherbas L., Koehler M. M. D. and Cherbas P. (1989) Effects of iuvenile hormone on the ecdysone response of Drosophila KC cells. Devl Genet. 10, 177-188. _ De Kort C. A. D. and Granter N. A. (1981) Regulation of the juvenile hormone titer: A. Rev. bt. i6, 1-28. Garen A., Kauver L. and Lepesant J.-A. (1977) Roles of ecdysone in Drosophila development. Proc. natn. Acad. Sk, U.S.A. 74, 5099-5113. Hammock B. D. (1985) Regulation of juvenile hormone: degradation. In

548

I. Yv. RAUSC~~ENBACI+ et al.

Pharmacology (Eds Kerkut G. A. and Gilbert L. I.), Vol. 7, pp. 431472., Pergamon Press, Oxford. Hammock B. D. and Sparks T. C. (1977) A rapid assay for insect juvenile hormone esterase activity. Analyr. Biothem. 82, 573-579.

Handler A. M. (1982) Ecdysteroid titers during pupal and adult development in D. melanogaster. Devl Biol. 93, 73-82.

Hodgetts R. B., Sage B. and O’Connor J. D. (1977) Ecdysone titers during postembryonic development of D. melanogaster. Devl Biol. 60, 3 lCL317.

Hwang-Hsu K., Reddy G., Krishna Kumaran A. (1979) Correlations between juvenile hormone esterase activity, ecdysone titer and cellular reprogramming in Galleria mellonella. J. Insect Physiol. 25, 105-l 11. Jones G. and Hammock B. D. (1983) Prepupal regulation of juvenile hormone esterase through direct induction by juvenile hormone. J. Insect. Physiol. 29, 471475. Jones D., Jones G., Wing K. D., Rudnicka M. and Hammock B. D. (1982) Juvenile hormone esterase of Lepidoprera-I. Activity in the haemolymph during the last larval instar of 11 species. J. camp. Physiol. MB, I-10. Jones D., Jones G., Rudnica M., Click A. J. and Sreekrishna S. S. (1986) High resolution isoelectric focusing ofjuvenile hormone esterase activity from hemolymph of Trichoplusia ni. Experienria 42, 4547.

Klages G. and Emmerich H. (1979) Juvenile hormone metabolism and juvenile hormone esterase titer in hemolymph and peripheral tissues of Drosophila hydei. J. camp. Physiol. 132, 319-326.

Klose W., Gateff E., Emmerich H. and Beikirch H. (1980) Developmental studies on two ecdysone deficient mutants of Drosophila melanogaster. Wilhelm Roux Arch. Devl Biol. 189, 5167.

Lezzi M. and Wyss C. (1976) The antagonism between juvenile hormone and ecdysone. In The Juvenile Hormone (Ed. Gilbert L. I.), pp. 252-269. Plenum Press, New York. Newitt R. A. and Hammock B. D. (1986) Relationship between juvenile hormone and ecdysteroid in larval-pupal development of Trichoplusia ni (Lepidoptera: Noctuiidae). J. Insect Physiol. 32, 835-844. Rauschenbach I. Y., Lukashina N. S. and Philippova M. S. (1984) Genetico-endocrine regulation of Drosophila development under extreme environmental conditions-V. The-existence of gene-regulator controlling the activity of the JH-esterase in D. virilis. Genetica 20. 239-247 (in Russian). Rauschenbach I. Y., Lukashina N. S., Budker V. G. and Korochkin L. I. (1987) Genetics of esterases in Drosophila-IX. Characterization of the JH-esterase in D. virilis. Biochem. Genet. 25, 687-705.

Rauschenbach I. Y., Lukashina N. S. and Budker V. G. (1989) Juvenile hormone metabolism during ontogenesis

in Drosophila virilis. Dokl. Akad. Nauk SSSR 306, 1262-1265 (in Russian). Renucci M. (1986) Juvenile hormone degradation in nerve tissues and fat body of female Acheta domesticus (Insecta, Orthoptera). Camp. Biochem. Physiol. 84A, 101-106. Richard D. S., Applebaum S. W., Sliter T. J., Baker F. C., Schooley D. A., Reuter C. C., Henrich V. C. and Gilbert L. I. (1989) Juvenile hormone bisepoxide biosynthesis in vitro by the ring gland of Drosophila melanogaster: a putative juvenile hormone in the higher Diptera. Proc. nafn. Acad. Sci., U.S.A. 86, 1421-1425. Richards G. (1978) Sequential gene activation by ecdysone in politene chromosomes of D. melanogaster-VI. Inhibition by juvenile hormone. Devl Biol. 66, 32-42. Richards G. (1981) Insect hormones in development. Biol. Rev. 56, 501-509.

Riddiford L. M. (1972) Juvenile hormone in relation to the larval-pupal transformation of the cecropia silkworm. Biol. Bull. mar. biol. Woods Hole. 142, 310-325. Riddiford L. M. (1975) Juvenile hormone-induced delay of metamorphose of the viscera of the cecropia silkworm. Biol. Bull. mar. biol. Woods Hole 148, 429-439.

Riddiford L. M. (1978) Ecdysone-induced change in cellular commitment of the epidermis of the tobacco homworm. Manduca sexta, at the initiation of metamorphosis. Gen. romp. Endocr. 34, 438-446.

Riddiford L. M. (1980) Insect endocrinology: actions of hormones at the cellular level. A. Rev. Physiol. 42, 51 l-528.

Riddiford L. M. (198 1) Hormonal control of epidermal cell development. Am. Zool. 21, 751-762. Riddiford L. M. (1984) Hormonal control of sequential gene expression in insect epidermis. In Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormone,

pp. 265-272. Springer, Berlin. Shapiro A. B., Wheelock G. D., Hagedorn H. H., Baker F. C., Tsai L. W. and Schooley D. A. (1986) Juvenile hormone and juvenile hormone esterase in adult females of the mosquito Aea’es aegypti. J. Insect Physiol. 32, 867-875.

Slade M. and Zibitt C. H. (1972) Metabolism of cecropia juvenile hormone in insects and mammals. In Insecr Juvenile Hormone (Eds Menn J. J. and Beroza M.), pp. 155-176. Academic Press, New York. Sparks T. C. and Hammock B. D. (1980) Comparative inhibition of the juvenile hormone esterase from Trichoplusia ni, Tenebrio molitor and Musca domestica. Pest. Biochem. Phvsiol. 14. 290-121.

Weirich G. and Wren J: (1976) Juvenile hormone esterase in insect development: a comparative study. Zool. 49, 341-350.

Wilson T. G., Gilbert L. I. (1978) Metabolism of juvenile hormone I in D. melanogaster. Camp. Biochem. Physiol. 6OA, 85-89.