Biological, radiochemical and physicochemical evidence for the low activity of disconnected corpora allata in locust

J. InsectPhysiol.Vol. Printed

in Great

33, NC’. 4, pp. 223-228, Britain. All rights reserved

1987 Copyright

0022-1910/87 $3.00 + 0.00 Q 1987 Pergamon Journals Ltd

BIOLOGICAL, RADIOCHEMICAL AND PHYSICOCHEMICAL EVIDENCE FOR THE LOW ACTIVITY OF DISCONNECTED CORPORA ALLATA IN LOCUST FRANCK

COUILLAUD*,

*Laboratoire de Neuroendocrinologie, 33405 Talence Ccdex and tLaboratoire

BERNARD MAUCHAMPt

and

UA CNRS 1683, Universitt de Phytopharmacie, INRA,

ADRIEN GIRARDIE*

de Bordeaux I Avenue des Facultks, Route de Saint Cyr. 78000 Versailles,

France (Received 21 March 1986; revised20 June 1986)

Abstract-The rate of juvenile hormone biosynthesis by locust corpora allata after transection of the nervi corporis allati 1, was measured in vifro using both radiochemical assay and gas chromatography-mass spectroscopy analyses. Incubations in different culture media or in pure haemolymph result in a low rate of juvenile hormone biosynthesis by disconnected glands. In vivo studies using juvenile, chromatotropic and gonadotrop.lc effects of the corpora Furthermore, animals with disconnected implantation than control hosts.

Key Word Indw: Insect, disconnected bioassays

allata confirm the low activity of the disconnected glands. corpora allata appear to be more sensitive to corpora allata

corpora

allata, juvenile

INTRODUCTION In vitro techniques are currently used to study juvenile hormone biosynthesis by insect corpora allata (see Schooley and Baker, 1985). Evidence suggests that in vitro measurements are a faithful representation of in vivo biosynthesis, but no definitive proof can be produced until it is possible to measure juvenile homtone biosynthesis in vivo (De Kort and Granger, 1981; Tobe and Feyereisen, 1983). A successful application of the in vitro approach

(radiochemical assay) was developed by Tobe and Pratt (1974), Pratt and Tobe (1974), and Pratt et al. (1975). Furthermore, gas chromatography-mass spectroscopy (GC-MS) analysis corroborates data obtained by radiochemical assay for in vitro juvenile hormone measuremem. (Mauchamp et al., 1985). In Locusta migratoria an.d Diploptera punctata the in vitro rates of juvenile hormone biosynthesis by corpora allata determined by radiochemical assay reflect both juvenile hormone titre (determined by GC-MS) and reproductive events known to be influenced by juvenile hormone (Couillaud et al., 1985; Tobe et al., 1985). Surprisingly in Locustn, the disconnection of the corpora allata by severance of the nervi corporis allati 1 (NCA-I) results in $ery low activity of the glands (evaluated by radiochemical assay) whereas juvenile hormone-mediated physiological events appear to occur normally and juvenile hormone titre is maintained or even slowly increases (evaluated by GCMS; Couillaud et al., 1984, 1985). Several hypotheses can be proposed to explain this situation. Data previously obtained by our group and our studies in

hormone

biosynthesis,

juvenile

hormone

progress allow us to summarize the situation as follows: (1) Allatectomy leads to the complete disappearance of juvenile hormone from the haemolymph and the absence of juvenile hormone-mediated physiological events (Couillaud et al., 1985), suggesting that no tissue except the corpora allata can synthesize juvenile hormone in locust. (2) Half-life of racemic exogenous juvenile hormone is similar in sham operated and NCA-I-transectioned females (Couillaud et al., 1985). (3) Recovery of labelled racemic juvenile hormone in the haemolymph after injection into the haemocoel is higher in NCA-I-transectioned animals than in controls (Couillaud et al., 1985) but this difference alone does not account for the high juvenile hormone titre in operated animals. (4) Disconnected corpora allata do not produce either juvenile hormone precursors of juvenile hormone acid as terminal product of biosynthesis (Couillaud and De Kort, unpublished). The present paper attempts to disprove the possibility that the low rate of juvenile hormone biosynthesis by disconnected corpora allata results from an artefact of the radiochemical assay. A new incubation medium and pure haemolymph have been tested and the results confirmed the low rate of juvenile hormone biosynthesis by disconnected corpora allata in vitro. GC-MS analysis of incubation medium also corroborated the large difference in the rate of juvenile hormone release between normal and disconnected corpora allata. Finally, the low rate of juvenile hormone release of disconnected corpora allata was exhibited by in z&o biological tests using the chromatotropic and gonadotropic and juvenile effects of the corpora allata. 223

224

FRANCK COUILLAUD MATERIAL

AND METHODS

Animals

The African locust (Locusta migratoria migratorioydes) was reared under crowded conditions essentially as described previously (Girardie, 1967). In adults and larvae, transection of the NCA-I and allatectomy were performed on the first day after ecdysis on carbon dioxide-anaesthetized locusts using Girardie’s method (1967). Sham-operated animals underwent the same surgery except the transection of the NCA. Corpora allata incubation

Glands were quickly removed from the animals and carefully dissected to remove fat body or other contaminating tissues. Incubations were performed in TC 199 or minimal essential medium (Flow lab) fortified by ficoll. [‘4C]methyl methionine was added and the specific radioactivity of the incubation medium (approx. 5 GBq/mmol) was determined by direct assay of an aliquot. Pure haemolymph was also used as incubation medium. Eight-to nine 9-day-old adult females with intact or day-l transectioned NCA-I, were bled through a slit in the neck membrane. After addition of a small amount of glutathione, haemocytes were removed by centrifugation (10,OOOg for 2 min at 4°C) and the supernatant was stored at -20°C until use. Corpora allata were incubated in 100 ~1 of the haemolymph after addition of 0.6 mM of 2.2 GBq/mmol [‘4C]methyl methionine. Lack of data concerning the size of the endogenous methyl methionine pool did not permit quantitative results and gave only comparative values.

et al.

levels of activity were defined in relation to the pigmentation of the Sth-instar larvae. Host animals were 4th-instar larvae intact or with corpora allata disconnected on day 1 of the stadium. Implanted glands came from 9-day-old adult females intact or NCA-I transectioned on the first day after emergence. All the donor females had vitellogenic oiicytes. Gonadotropic biological test

In locusts, allatectomy prevents oijcyte growth and corpora allata implantation restores odcyte development (Joly, 1960). The ability of the corpora allata to restore oiicyte development in allatectomized females was used as indicator of implanted gland activity. Implanted glands came from 9-day-old adult females intact or with the NCA-I transectioned on ,the first day of the adult life. On day 8, 10 oiicytes were measured in each host female to calculate the mean length of the oiicytes. Juvenile biological test

In 4th-instar larval locusts, allatectomy performed before critical period results in anticipated metamorphosis (Joly, 1960). Corpora allata implantation on day 1 restores the normal larval moult (Joly, 1960). Furthermore, high activity of implanted glands also induces green colour formation as previously described for the chromatotropic test. So, 4th-instar larvae, allatectomized on day 1 were implanted with corpora allata. Implanted glands came either from 9-day-old intact adult females or with the NCA-I transectioned. The comparative activity of the implanted glands was estimated according to morphological aspects of the host.

Radiochemical assay

Statistical tests

Protocol for the radiochemical assay was previously described in Locusta (Girardie et al., 1981) according to Tobe and Pratt’s method (1974). Organic extracts of the incubation medium was submitted to thin-layer chromatography separation for determination of the rate of juvenile hormone biosynthesis.

The comparison of oiicyte lengths and rates of juvenile hormone biosynthesis were analysed by Student’s t-test. Ovarian maturation states and green colour formation were compared by x2 test.

Gas chromatography-mass

spectroscopy analysis

Juvenile hormone from culture medium was extracted with hexane and directly analysed by GC-MS using the positive chemical ionization mode with ammonia as reagent gas (Mauchamp et al., 1985). C-16 juvenile hormone (juvenile hormone-III) was known to be the exclusive juvenile hormone produced by locust corpora allata in vitro (Tobe and Stay, 1977; Pratt and Weaver. 1978: Mauchamo et al.. 1985). So. the three following ions were mon;tored for selected ion monitoring analysis (SIM): 267 (MH+), 252 (MH+-CH,OH + NH,) and 235 (MH+ + CH,OH). Retention time was determined with synthetic juvenile hormone III (Calbiochem-Behring). Chromatotropic biological test

Corpora allata activity in vivo was determined by their ability to induce green colour in Sth-instar larvae following implantation into day-3 4th-instar larvae. The induced green colour was shown to be proportional to implanted corpora allata activity (Joly, 1967). According to Joly’s scale, six different

RESULTS

Juvenile hormone biosynthesis by intact or disconnected corpora allata in various incubation media

Differences in rates of juvenile hormone biosynthesis in vitro between intact and disconnected corpora allata were investigated in various incubation media (Fig. 1). Standard procedures for radiochemical assay in locusts have been described using TC 199 (see Tobe and Feyereisen, 1983). Figure 1A corroborates the large differences in rate of juvenile hormone biosynthesis on day 8 between normal and corpora allata disconnected on day 1. To investigate any TC 199 involvement in the low rate of juvenile hormone biosynthesis by the denervated glands, corpora allata activity was assayed with minimal essential medium. Figure 1B shows a large difference in rate of juvenile hormone biosynthesis between disconnected and intact corpora allata and results similar to those found with TC 199. Although tissue culture media are routinely used for in vitro assays of corpus allatum activity, we employed haemolymph directly as a culture medium to assess corpus allatum activity under more physio-

Low activity of disconnected corpora allata (B)

CC

CA

‘j

(D)

(C)

c

CC

CA

S

CC CA

s

Fig. 1. Rate of juvenile hormone biosynthesis by corpora allata in oitro in different incubation media. (A) in TC 199; (B) in minimal essential medium; (C) in pure haemolymph from 9-day-old adult females; (D) in pure haemolymph from 9-day-old adult females with the NCA-I transectioned on day 1. Means & SEM, n = 6. logical conditions. Because we were unable to determine either the endogenous methyl pool in the haemolymph or juvenile hormone esterase activity, results are only comparative and dpm attributed to juvenile hormone was only the net difference between juvenile hormone biosynthesis and its degradation. Furthermore, to investigate any possibility of qualitative changed in the haemolymph after NCA transection (i.e. hormonal composition) we used haemolymph from sham operated females or from females with corpora allata disconnected on day 1. Again control corpora allata exhibited a high rate of juvenile hormone biosynthesis whereas disconnected glands had very low activity in both haemolymph from controls (Fig. 1C) and from day - 1, NCAtransectioned females (Fig. 1D). In conclusion, the low rate of juvenile hormone biosynthesis by disconnected corpora allata is independent of the incubation medium. GC-MS determination of juvenile hormone production by corpora allata in vitro A radiochemical assay for corpus allatum activity was developed using the rate of methyl group incorporation of L-methyl,-[i4C]methionine into juvenile hormone (Tobe and Pratt, 1974). In locusts,

225

incubation of corpora allata with L-methyl[‘4C]methionine leads to biosynthesis of [r4C]methyl farnesoate which is converted into C-16 juvenile hormone (Judy et al., 1973; Hammock, 1975). Hypothetical modifications of the biosynthetic pathway in disconnected corpora allata could result in suppression of the methyl group incorporation into juvenile hormone and hence in a breakdown of the radiochemical assay. In this respect, nothing is known about the biosynthetic pathway in disconnected corpora allata. In an attempt to elucidate this question, we determined juvenile hormone biosynthesis by disconnected corpora allata in vitro using GC-MS. This method allowed analysis of total biosynthetized juvenile hormone without any consideration of the biosynthetic pathway. Figure 2 shows that data found by GC-MS corroborate the results found with radiochemical assay. Rate of juvenile hormone biosynthesis by ten pairs of disconnected corpora allata was very low (Fig. 2B) when compared to synthesis by only two pairs of sham-operated glands (Fig. 2A). Ratio of juvenile hormone biosynthesis, as determined by the number of captured ions, of individual pair of disconnected to intact corpora allata was around 20 and similar to that found with radiochemical assay. This result proves radiochemical assay measurements and discards the possibility of another pathway for juvenile hormone biosynthesis by disconnected corpora allata. In vivo bioassays of corpora allata activity In vivo biological tests for corpus allatum activity, in spite in their relative low sensitivity, provide interesting information on juvenile hormone synthesis in physiological conditions. In an attempt to corroborate differences in juvenile hormone biosynthesis between disconnected and control corpora allata in vivo, we determined corpora allata activity using chromatotropic, gonadotropic and juvenile biological tests. Table 1 shows the results of implanting one pair of corpora allata into 4th-instar larvae on day 3 of the stadium. Donors were 9-day-old adult females with intact NCA-I (line 1) or the NCA-1 transectioned on day 1 (line 2). The results clearly show that disconnected corpora allata were less competent than control corpora allata to induce changes in host pigmentation from brown to green by the subsequent stadium. Thus, according to this test, disconnected corpora allata appeared to be less active in vivo than the control glands. We also screened the implantation experiment using host larvae with corpora allata which were disconnected on the first day of the stadium. These operated host larvae received extra corpora allata implantations from 9-day-old females. Again, control implanted corpora allata (line 3) were more potent in inducing the green colour than the disconnected glands (line 4). Furthermore, comparison of the two types of host larvae (control or with transsectioned NCA-I) revealed that the implantation of corpora allata was more efficient in hosts with disconnected corpora allata than in intact hosts. Allatectomy performed on day 1 of the 4th-larval stadium resulted in prothetelic adults (Joly, 1960). AS shown in Table 2, muscle implantation had no effect

226 (A)

4 17

TIC

?;,,

(B)

417

100%

: 54240

100%

: 17800

RIG = 252

cl

2

1

4

3

5

Fig. 2. Total thin-layer chromatography and selected-ion chromatograms (RIC = 235 and RIC = 252) from GC-MS analysis of the hexane extract of culture medium containing (A) 2 pairs of corpora allata from 9-day-old adult females and (B) 10 pairs of corpora allata from 9-day-old adult females with the NCA-I transectioned on day 1.

on restoration of a normal larval moult. Only one animal moulted into a normal Sth-instar larvae certainly as a result of one incomplete allatectomy. Implantation of corpora allata restores the larval Table

1. Chromatotrooic

effect of coroora

allata

imolanted

the pigmentation of the moult. Furthermore, Sth-instar larvae resulted from implanted corpora allata activity as described previously for the chromatotrophic test. Again, using the juvenile biological

into 4th-instar

larvae. determined

Sth-instar Hosts Sth-instar

Implanted larvae

Sth-instar larvae with disconnected

CA

CA

Gregarious

Reduced gregarious

bv 5th-instar

larval

aiementation

larval pigmentation

Few green spots

Discreet green

Green with dark spots

Very green

I Control CA 2 Disconnected

CA

3 23

3 5

6 4

6 0

12 0

I 0

3 Control CA 4 Disconnected

I CA

14

2 6

2 5

IO 3

8 0

3 0

Donors were 9-day-old adult females with vitellogenic x2 test. CA = corpora allata.

o&ytes.

Highly significant

differences between lines (1,2) (3,4) (1.3) and (2,4) using

Low activity of disconnected corpora allata Table 2. Juvenile

and chromatotropic effects of corpora allata implanted into 34-day-old 4th-instar larvae allatectomized of the stadium, determined by morphology and pigmentation of the Sth-instar larvae. Sth-instar

Imolanted

Donor

Prothetelic adults

tissues

Muscle Control CA Disconnected animals

Greearious

45 2 9

CA were 9-day-old

Table 3. Gonadotrophic

effecl: of corpora

Discrete green

Green with dark soots

Very green

0 I3 4

0 IO 2

0 7 0

0 II 0

0 0 0

oacytes.

Highly significant

Muscle Control CA Disconnected

CA

1 2 3

differences (x’ test). CA = corpora

allata implanted into day-l allatectomized adult females. determined and oijcyte maturation states on day I2

OGcyte length mean f SEM

tissues

larval pigmentation

Few green sLwts

I 10 43

adult females with vitellogenic

Previtellogenesis

0.96 + 0.01 4.30 * 0.31 2.89 f 0.27

Donor animals were 9-day-old adult females with vitellogenic (Student’s and x2 tests). CA = corpora allata.

on the first day

Reduced ereearious

06cyte Implanted

221

0 24 27

I 2 oBcytes. Highly

DISCUSSION

The present data Iusing different experimental methods including physicochemical techniques and in vivo approaches provided strong evidence for the low activity of the disconnected corpora allata. The nature of the incubation medium for in vitro radiochemical assay was not involved in the low rate of juvenile hormone biosynthesis by disconnected corpora allata. Neither minimal essential medium nor haemolymph were able to suppress the large difference between activities of control and disconnected glands. Cross incubations suggested that haemolymph from disconnected and control females was similar or that corpora allata were not sensitive to the haemolymph factors during the 3-h incubation. Determinations of labelled juvenile hormone in haemolymph after a 3-h incubation period were only the result of difference between labelled juvenile hormone biosynthesis and its degradation. Under our experimental conditions, juvenile hormone biosynthesis exceeded deg:radation. These results are in agreement with previous observations that degradation of juvenile hormone in Locusta haemolymph is rather low (De Kort et al., 1979), particularly for the natural enantiomer (10R) juvenile hormone III synthesized by the corpora allata (Schooley et al., 1978) which is protected by a preferential association with the juvenile hormone-binding protein (Peter et al., 1979; De Kort et al., 1983). Cross incubations

significant

by mean of &cyte

lengths

states on day 12

Vitellogenesis

21

test, day-l disconnected corpora allata from 9-day-old adult females were less active than control corpora allata in restoring the normal larval moult and inducing green pigmentation. To evaluate the activity of implanted corpora allata, we followed their ability to restore oiicyte growth by observing ovarian maturation states and by measuring terminal oiicyte length (Table 3). Day-l disconnected corpora allata from 9-day-old adult females were able to promote normal oiicyte maturation, but again they appeared less potent than the control glands.

maturation

allata

Chorioaenesis 0 5 2

differences

Ege. laying 0 6 2

between lines (1.2); (1,3) and (2.3)

revealed similar juvenile hormone production by corpora allata in haemolymph from normal and NCA-I-transectioned females. This observation suggested no difference in rate of juvenile hormone degradation between haemolymph from normal and operated females. GC-MS analysis of incubation media corroborates the low juvenile hormone production by disconnected corpora allata which is around 20 times lower than in control glands. Radiochemical assay determination is only the quantification of the incorporation of the labelled methyl group into juvenile hormone. The present data show that in control glands, the rate of methylation of farnesoi’c acid by disconnected corpora allata is a faithful representation of the in vitro juvenile hormone biosynthesis. No alternative pathway of juvenile hormone biosynthesis was used by disconnected glands to produce juvenile hormone. To investigate if the low rate of juvenile hormone biosynthesis in vitro by disconnected corpora allata reflected a low activity in vivo, we explored gland activity using different biological tests. All the results indicate a lower in vivo activity by disconnected corpora allata than by controls. Also control glands probably lose their high rate of juvenile hormone biosynthesis after implantation. According to our previous findings (Couillaud and Girardie, 1985), corpora allata activity breaks down 30 h after transection of the NCA-1. Physiological responses of host animals probably resulted from the difference in activity during the first 30 h after implantation. In an attempt to explore further the regulation of juvenile hormone titre in disconnected animals, we compared chromatographic effect of implanted corpora allata both in intact and in NCA-Itransectioned larval hosts. Our results clearly show that implanted glands were more potent in inducing chromatotropic effects in disconnected corpora allata larvae than in control larvae. This result is in agreement with our previous data (Couilland et al., 1985) which suggested some process in disconnected corpora allata animals to “save” juvenile hormone.

FRANCK COUIILLAUD et ai.

228

Involvement of juvenile hormone retention by tissues had been suggested (Nowock et al., 1976; Couillaud et al., 1985; Tobe et al., 1985) and studies on the role of the juvenile hormone binding protein and juvenile hormone catabolism (De Kort et al., 1979; 1983) are still in progress (Couillaud and De Kort, unpublished). To summarize, bioassays and physicochemical methods corroborate data obtained by radiochemical assay and provide strong evidence for the low rate of juvenile hormone biosynthesis by the corpora allata after transection of the NCA-I. Acknowledgements-We thank F. Rossignol and Malosse for their technical assistance and B. Proux illustration of this paper.

C. for

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