gruetzmacher1984- the effect of juvenile hormone on

8/16/2019 Gruetzmacher1984- The Effect of Juvenile Hormone On

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J. Insect Physi ol.

Vol. 30, No. 4, pp. 331-340, 1984

Printed in Great Britain. All rights reserved

0022-1910/84 3.00+ 0.00

Copyright 0 1984Pergamon Press Ltd

THE EFFECT OF JUVENILE HORMONE ON

PROTHORACIC GLAND FUNCTION DURING THE

LARVAL-PUPAL DEVELOPMENT OF

MANDUCA SEXTA :

AN IN SITU AND IN VITRO ANALYSIS

M. C. GRUETZMACHER*, L. I. GILBERT?~, N. A. GRANGER~. W. GOODMANQ: nd

W. E. BOLLENBACHER~

*Committee on Virology, The University of Chicago, Chicago, Illinois 60637. TDepartment of Biology,

The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514, fDepartment of

Anatomy, Th’e University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514 and

§Department of Entomology. University of Wisconsin, Madison, Wisconsin 53706. U.S.A.

Receioed 21 September 1983)

Abstract-The application of juvenile hormone I or ZR 512 to neck-ligated. day-5 fifth instar (V,) larvae

reduced the time to pupation in a dose-dependent manner

when compared to neck-ligated controls treated

with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA)

reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the

ecdysteroid tilres were similar to those of normally developing larvae although the ecdysteroid peak

elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged

the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V, larvae that were untreated ultimately

pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 pg/ml

haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated

larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine

whether this stimulation was direct or indirect, an in

vitro

approach was taken. Prothoracic glands from

V,, V, and V, larvae were incubated in vitro under conditions in which they could be stimulated by

prothoracicotropic hormone, and were

exposed to concentration of free juvenile hormones I, Il. III or

ZR 512 ranging from 10m5 M to IO-“‘M. In no case were the prothoracic glands stimulated in a

dose-dependent manner that

would be indicative

of hormone activation. Similar results were obtained

when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with

the acids of the three juvenile hormone homologues revealed them to be ineffective in activating

prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone

by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from

these data tha? juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so

indirectly.

Key Word Index: Juvenile hormone. Manduca sexta, prothoracicotropic effects, ecdysteroid titre

INTRODUCTION

Although the prothoracicotropic hormone (PTTH) is

accepted as the primary effector of prothoracic gland

activity, studies as early as 1959 suggested a direct

interaction between the corpora allata and pro-

thoracic glands. Ichikawa and Nishiitsutsuji-Uwo

(1959) were able to elicit adult development in brain-

less

Philosamia Cynthia ricini

pupae by implanting

corpora allata and concluded that PTTH released

from the implanted corpora allata elicited pro-

thoracic gland activation and subsequent devel-

opment. Williams (1959) conducted similar studies on

brainless, diapausing

Hyalophora cecropia

pupae and

concluded that juvenile hormone from the corpora

allata stimulated the prothoracic glands. At the same

time, Gilbert and Schneiderman (1959) succeeded in

eliciting development in brainless saturniid pupae

with crude juvenile hormone extracts and postulated

qcorrespondence to: Professor L. I. Gilbert, Department of

Biology, Wilson Hall 046A, The University of North

Carolina at Chaoel Hill. Chaoel Hill, North Carolina

27514, U.S.A. _

that the stimulatory effect of these extracts was due

to the presence of juvenile hormone. These early

studies were subsequently interpreted to indicate that

juvenile hormone stimulates the prothoracic glands

directly, although the possibility remains that PTTH

was released from the implanted corpora allata since

in some species this gland is the neurohaemal organ

(see Agui et al., 1980). Further, if juvenile hormone

was the active factor, it could have elicited its effect

indirectly by stimulating the synthesis and/or release

of a prothoracicotropic factor from other tissues, or

one or more juvenile hormone metabolites could have

been responsible for the observed effects (e.g. Bhaska-

ran

et al.,

1980).

In recent years, several in vitro studies utilizing a

variety of Lepidoptera have supported the idea that

juvenile hormone affects prothoracic gland activity

(e.g. Hiruma et al., 1978; Cymborowski and Stolarz,

1979; Hiruma, 1980; Safranek

et al.,

1980; Hiruma

and Agui, 1982). There are indications that juvenile

hormone can either inhibit or activate the pro-

thoracic glands depending on developmental state

(e.g. Cymborowski and Stolarz, 1979; Hiruma and

331

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33

M. C.

GRUETZMACHER e6 al.

Agui, 1982); that is, juvenile hormone inhibits the

prothoracic glands of young iast-instar larvae but

stimulates the glands late in the last instar.

Although more than 2 decades have passed since

the first studies suggested that juvenile hormone acts

on the prothoracic glands to stimulate the synthesis

of ecdysone, there is still no evidence that the effect

is direct. The recent availability of techniques to

assess prothoracic gland activity

in

v tro has now

provided the means to study the putative effects of

juvenile hormone upon prothoracic gland activity.

The present study addresses the question of juvenile

hormone control of prothoracic gland activity using

the

in vitro

techniques utilized so successfully in

studies of prothoracic gland activation by PTTH

(Bollenbacher

et al.,

1979; see Gilbert et al., 1981).

M TERI LS ND METHODS

Animals

Munduca sexta

larvae were reared on an artificial

diet under a long-day photoperiod (16L:8D) at 25°C

(Vince and Gilbert, 1977) and approximately 70%

relative humidity. Fifth-instar larvae were separated

into gates and only gate-2 animals were used (Tru-

man, 1972; Granger et

al.,

1979).

Juvenile ~zormoneb~nd~ngrotein

Juvenile hormone binding protein was partially

purified by affinity chromatography (Goodman and

Goodman, 1981). Haemolymph was collected from

late fourth-instar larvae and concentrated by ultra-

filtration (Amicon UMIO). The binding protein in

the concentrated haemolymph was labelled by add-

ing [3Hljuvenile hormone I (New England Nuclear

Corp., 10 Ci/mmol) and the labelled material was

separated by gel filtration on an S-200 (Pharmacia)

column (2.5 x 135cm) (Goodman

et al.,

1978). The

[”HIjuvenile hormone binding protein fractions were

pooled and dialyzed overnight against a borate-

glycerol buffer (0.1 M sodium borate, 20% glycerol,

pH 7.8). Juvenile hormone was removed from the

dialyzed binding protein by addition of an equal

amount of heptane-toluene (9: 1, v/v), and then

gently shaking the mixture at 25°C for 3 h. The

delipidated protein (aqueous fraction) was removed

and centrifuged at 12,OOOg for 30min (4*C) to

remove denatured protein. The protein solution was

then incubated for 30min with phenylmethyl-

sulphonylfluoride ( 10m3M), mixed and passed

through the affinity column which would be the

column bed. The resin mixture was incubated for 2 h

at 4°C with occasional stirring, brought to room

temperature and the unbound protein eluted with

glycerol borate buffer. The column was washed with

sufficient borate buffer to reduce the U.V. (280nm)

absorbance of the effluent to zero. Another 50 ml of

buffer was then passed through the column before

eluting the juvenile hormone-associated protein. The

latter was accomplished by incubation of the column

at room temperature with a hormone-saturated

buffer (2 mg juvenile hormone I, 0.25 m1 of ethanol

and 49.75ml of borate buffer) for 2 h. During the

incubation period the column bed was occasionally

stirred to ensure complete distribution of the hor-

mone.

The hormone-containing buffer was then col-

lected and the column washed with an additional

50 ml of hormone-free buffer. The combined eluates

were pooled and concentrated.

The partially purified binding protein was stored at

-20°C until use and the amount of protein recovered

was quantified by U.V. s~ctrophotometry at 230 nm.

For

in vitro

incubations with binding protein, juvenile

hormone-saturated binding protein was added di-

rectly to the incubation medium in concentrations of

juvenile hormone ranging from 1O--6 o lo-“M.

Juvenile hormone and juvenile hormone acids

Juvenile hormones I, II and III were purchased

from Calbiochem. The hormone analogue ZR 512

was a gift from Zoecon Corp. and methyl epoxy

stearate was purchased from Allied Science Labora-

tories.

Juvenile hormone acid(s) was prepared by incu-

bating 10 mg of the appropriate hormone homologue

with 5 ml methanol- 1 N NaOH (1:

1, v/v)

in the dark

in a shaking water bath at 40°C for 4 h. The reaction

mixture was then carefully titrated to pH 5.0 with

I-ICI and extracted 5 times with chloroform-toluene

(9: 1, v/v). The aqueous methanol phase containing

the juvenile hormone acid was then dried under NZ,

taken up in chloroform and the acid purified by high

performance liquid chromatography (HPLC). The

purity of the generated juvenile hormone acid was

established by NMR and i.r. spectroscopy. The juve-

nile hormone acids were stored dry at -20°C until

use and were quantifi~ spectrophotometrically in

spectra-grade methanol (A,,,, 213-217 nm; ex-

tinction coefficient, 14,770).

Uptake of ffifjuvenile hormone I

To determine the degree to which topically applied

juvenile hormone homologues were taken up by

treated larvae, 100 pg of [-‘HIjuvenile hormone I (sp.

act. 0.015 pCi/mmol) was applied to neck-ligated

day-5 fifth instar (V,) larvae. To assess uptake and

metabolism of the hormone, haemolymph samples

were taken periodically over 48 h from different

animals at each point to minimize the potentially

adverse effects of repeated bleeding from a single

animal. Samples were immediateIy extracted for juve-

nile hormone I with hexane-methanol (1:2, v/v>

(Granger et al., 1979). The efficiency of extraction of

[‘HIjuvenile hormone by this partition was 8004. To

separate juvenile hormone I from its metabolites, an

aliquot of the hexane epiphase containing the

[3H]juvenile hormone I was mixed with unlabelled

juvenile hormone I to serve as a carrier, and the

sample was subjected to thin layer chromatography

(TLC) in hexane-ethyl acetate (3

: 1, v/v).

The devel-

oped plates were divided into 1 cm fractions, the

fractions scraped individually into scintillation vials

with Aquasol (NEN) and the samples radioassayed

by liquid scintillation counting. The efficiency of

recovery from the TLC plate was 80%. The aqueous

hypophase from the organic solvent partition con-

tained polar metabolites of juvenile hormone, e.g.

juvenile hormone acid, and the amount of this mate-

rial present in the haemoiymph was determined by

counting an aliquot of the hypophase added to

Aquasol. The total amount of [‘HIjuvenile hormone

I metabolites in the haemolymph was determined by


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334

M. C. GRUETZMACHER et al.

123456789

Days after llgatlon

Fig. 2. Effects of treatment on the haemolymph ecdysteroid

titre of V, neck-ligated larvae. normally developing

larvae; 0 neck-ligated larvae treated with 100 pg ZR 512;

A neck-ligated larvae treated with 100 pg juvenile hormone

I;

n

eck-ligated larvae treated with solvent only (20~1

cyclohexane); 0 neck-ligated larvae treated with IOOpg

methyl epoxy stearate. W denotes wandering and P denotes

pupation.

Haemolymph ecdysteroid titre

If topically applied juvenile hormone were affecting

the prothoracic glands this effect should be reflected

by characteristic differences in the haemolymph ecdy-

steroid titre of the various experimental populations.

These differences should more likely be temporal

than quantitative, since pupal development occurs

even in ligated, untreated animals. Therefore, as

another means of assessing the putative tropic effect

of juvenile hormone on the prothoracic glands, the

haemolymph ecdysteroid titres were determined in

normal,

ligated and treated larvae during

larval-pupal development.

Haemolymph ecdysteroid titres of neck-ligated lar-

vae treated with ZR 512 and juvenile hormone I were

compared to the ecdysteroid titre of normally devel-

oping V, animals and neck-ligated controls treated

with cyclohexane (Fig. 2). For unmanipulated, wan-

dering larvae, the titre was essentially that reported

previously (Bollenbacher

et al.,

1981): the titre was

low (0.25 pg/ml haemolymph) on day 0 (equivalent

to wandering V, larvae), peaked on day 1 (equivalent

to V, larvae) at 3.5 pg/ml haemolymph, and then

declined to approximately 1.2 pg/ml haemolymph on

day 3 (equivalent to V, larvae). These animals pu-

pated 5 days after wandering. The haemolymph

ecdysteroid titre of ZR 512-treated animals was

about 0.25 pg/ml haemolymph on day 0, and in-

creased gradually to 0.75 pg/ml haemolymph on day

2. The titres on days 1 and 2 in these larvae were

significantly lower than the titres on comparable days

in normal, developing animals (P < 0.01). Three days

after treatment the titre in the ZR 512-treated larvae

peaked at 4.5 pg/ml haemolymph, a level similar to

the peak in the titre of untreated day-2 animals

(P > 0.05), even though this peak occurred 1 day

later. By day 4 the titre fell to about 0.9 pg/ml

haemolymph and pupation occurred at the normal

time, 5 days after ligation. Thus, it appeared that ZR

512 restored a normal developmental time frame by

evoking an increase in the ecdysteroid titre, although

the peak in the titre occurred 1 day later than in the

untreated larvae. In juvenile hormone I-treated,

ligated animals, which also pupated 5 days after

treatment, the peak ecdysteroid titre reached a level

comparable to that in the untreated and ZR

512-treated larvae. However, this peak lagged that in

the normal titre by 2 days and that of ZR 512-treated,

neck-ligated animals by 1 day.

The slow development of the ligated and

cyclohexane-treated larvae was characterized by a

haemolymph ecdysteroid titre that increased gradu-

ally from the time of ligation to approx 5 days after

ligation, 0.5 pg/ml-0.9 pg/ml, respectively. After this

gradual rise, the titre increased rapidly, peaking at the

time of pupation on day 9 at 7 pg/ml haemolymph.

This peak is approx double those determined for

normal animals and ZR 512- and juvenile hormone

I-treated, ligated larvae. The significance of this

relatively enormous ecdysteroid titre is conjectural at

present.

Lastly, to confirm the analogue specificity of the

response obtained with ZR 5 12 and juvenile hormone

I treatment of ligated larvae, methyl epoxy stearate

was applied to V, neck-ligated larvae and the ecdy-

steroid titre was assessed. The haemolymph ecdy-

steroid titre was similar to that determined for the

cyclohexane-treated larvae, as suggested by the time

to pupation (10 days). From days O-9 after treat-

ment, the titre in the methyl epoxy stearate-treated

larvae was essentially the same as that of the ligated

controls, with a peak of approx 4.5 pg/ml hae-

molymph on the day of pupation. This ecdysteroid

level, as well as the level on day 12, was not

significantly different than those of the cyclohexane-

treated controls at the same times (P > 0.05). This

indicated that methyl epoxy stearate had no effect on

the ecdysteroid titre either quantitatively or tem-

porally, which is consistent with data on its lack of

an effect on the time required for the pupation of

ligated, control larvae.

The above results revealed that ZR 512 and juve-

nile hormone I appear to affect prothoracic gland

function so that a near-normal haemolymph ecdy-

steroid titre results from their application to ligated

larvae. The only apparent difference between the

titres of normal animals and those of ZR 512 - and

juvenile hormone I-treated ligated larvae is the tem-

poral lag in the rise of the haemolymph ecdysteroid

titre by one and two days in ZR 512 - and juvenile

hormone I-treated, ligated larvae, respectively. Thus,

it appears that the prothoracic glands could have

been activated by juvenile hormone to increase their

rate of ecdysone synthesis, but to study the changes

in the ecdysteroid titre in response to juvenile hor-

mone is merely to examine a covert effect for which

the overt expression is the acceleration of devel-

opment. The results do not demonstrate that the

effect on the prothoracic glands is direct.

[‘H]Juvenile hormone I penetration of the cuticle

To demonstrate a possible direct effect of juvenile

hormone on the prothoracic glands an

in vitro

ap-

proach was required. This necessitated an initial

determination of the effective concentration of juv-

enile hormone to be used in the in vitro studies; this

concentration could be established by determining

the in situ haemolymph titre of juvenile hormone

resulting from the dose of topically applied hormone

(100 /*g) needed to elicit normal pharate pupal devel-


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Effect of juvenile hormone on prothoracic gland

335

present in the haemolymph was apparently due to a

relatively constant rate of catabolism of the

[3H]juvenile hormone I together with a similar rate of

sequestration and/or excretion of metabolites. Given

the high levels of these metabolites in the hae-

molymph, of which a large portion appeared to be

juvenile hormone acid, the possibility existed that

these metabolites could affect the larval-pupal devel-

opment of the ligated larvae; such a possibility has

been suggested previously (Bhaskaran

et al.,

1980;

Granger et al., 1982).

Once the effective in

situ

concentration of juvenile

hormone I had been determined, it was possible to

initiate the

in v i tro

studies probing the function of

juvenile hormone in the regulation of the prothoracic

glands during the larval-pupal development of Man-

duca.

24

40

Time h)

Fig. 3. Haemolymph titre of juvenile hormone and metab-

elites after topical application of [‘Hljuvenile hormone I to

V, neck-ligated larvae. 0 juvenile hormone I; 0 juvenile

hormone I metabolites. 1 pig juvenile hormone I

(0.015 ~Ci/mmol) was applied in cyclohexane at zero time.

In vitro

conditions or prothoracic gland incubations

w it h uvenil e hormone

opment in Vj wandering, ligated larvae. (It is as-

sumed here that the endogenous juvenile hormone

titre is negligible in V, larvae -J. Bergot et al., pers.

comm.-and especially in neck-ligated larvae.) In

addition, determination of the haemolymph juvenile

hormone titre resulting from the topical application

of juvenile hormone would provide information re-

garding the temporal relationship between the in-

creased juvenile hormone titre and the increased

ecdysteroid titre. This information would aid in the

design and interpretation of

in v i tro

studies, since a

direct effect of increased juvenile hormone titre on

the prothoracic glands should be followed by an

increase in the ecdysteroid titre. Finally, an analysis

of [3H]juvenile hormone uptake would also provide

information on the rate and nature of degradation of

juvenile hormone to metabolites that could possibly

influence prothoracic gland activity.

To test for a possible direct stimulatory effect of

juvenile hormone and ZR 512 on the prothoracic

glands in v i tro glands from V,, V, and V, larvae were

used. The selection of these days was based on the

observation that these stages of larval-pupal devel-

opment of either normal or juvenile hormone-treated,

ligated animals occurred before or during the increase

in the haemolymph ecdysteroid titre (Bollenbacher et

al.,

1981), and thus, represented times at which the

prothoracic glands were presumably responding to

juvenile hormone or ZR 512

in sit u.

A time course analysis of [3H]juvenlle hormone I

levels in larval haemolymph after topical application

revealed that the hormone was present at a level in

excess of lo-’ M within 10 min (Fig. 3). At 0.5, 1 and

2 h, the concentration of [3H]juvenile hormone I

fluctuated around lo-’ M, falling by 6 h to IO-‘M.

At 24 h it had dropped to 7 x 10m9M, and finally

reached approx 1 x 10m9M at 48 h. Thus, these re-

sults indicated that the topical application of 100 yg

of juvenile hormone I needed to elicit deveiopment in

ligated larvae did not result in pharmacological hae-

molymph titres of juvenile hormone, at least after

24 h. The titre had fallen to about low9 M 1S-2 days

before the increase in the ecdysteroid titre occurred,

an observation which suggested that the effect of

juvenile hormone on the prothoracic glands may not

be direct.

Time courses of ecdysone synthesis by these pro-

thoracic glands were generated to determine the

length of time the glands could be incubated

in v i tro

during which potential JH effects might be observed.

Previous studies on prothoracic gland activation in

v i t ro indicated that an incubation time of 8 h was the

maximal time within which activation could be dis-

cerned, since beyond this time rates of synthesis by

activated glands were no longer linear relative to the

rate of synthesis by unactivated glands. This effect on

synthesis rates was presumably a result of substrate

depletion of the activated glands (Bollenbacher

et al .,

1979). Thus, although incubation times longer than

8 h could be used, the resulting data might be difficult

to interpret since any activation of synthesis would be

occurring under non-linear conditions for both con-

trol and experimental glands.

This study also revealed that a substantial degree

of degradation of the exogenous juvenile hormone I

occurred. The concentration of rH] polar metabolites

was well in excess of 10e6M, and remained at this

level for the 48 h of the time course study (Fig. 3).

This relatively constant amount of metabolites

To verify that the basal rates of ecdysone synthesis

by day 5, 6 and 7 larval prothoracic glands were

linear during an incubation period of 8 h, time

courses of ecdysone synthesis by these glands were

determined (Fig. 4). For day-5 and -6 prothoracic

glands, the rates of ecdysone synthesis were both

linear and comparable (_ 5 ng h-l gland-‘)

during

the incubation period, while the mean rate of ecdy-

sone synthesis by day-7 prothoracic glands was con-

siderably less, approx 1.4 ng h-’ gland-‘. The

significance of the different rates is not clear, but for

the purpose of this study it was only important to

know that the glands were capable of linear synthesis

in v i tro,

and that they possessed sufficient substrate to

demonstrate an increased rate of ecdysone synthesis

if activated within the incubation period. Thus, these

glands were suitable for probing the putative direct

tropic effect of juvenile hormone and ZR 512 on the


6/10

336 M. C. GRUETZMACHER et al.

10

2 4

6 6

Time h)

Fig. 4. Basal rates of

ecdysone synthesis by prothoracic

glands in vitro. lands from V, larvae; 0 glands from V,

larvae: a glands from V, larvae. Bars signify + SEM.

prothoracic glands. It is important to note that under

these in vitro conditions, prothoracic glands from

days 5-7 can be activated by PTTH (Gruetzmacher,

Gilbert and Bollenbacher, unpublished observations)

in a manner similar to that reported for day-0 pupal

prothoracic glands (Bollenbacher

et al.,

1983).

Short-term efJects in vitro of juvenile hormone and

ZR 512 on prothoracic glands

To conduct these

in vitro

activation studies both

critically and thoroughly, a time course protocol was

used with a broad range of concentrations of the test

compounds. Prothoracic glands from V,. V, and V,

larvae were incubated

in vitro

with either juvenile

hormone or ZR 512 in concentrations ranging from

10m6 to lO_“M, and a time course of ecdysone

synthesis by experimental (+ test compound) and

control glands (- test compound) was then gener-

ated at each concentration to monitor potential

effects on the rate of ecdysone synthesis. A dose-

dependent stimulation of ecdysone synthesis would

be indicative of direct activation of the glands by the

test compound; i.e. the latter would be functioning as

a prothoracicotropin.

Since the corpora allata of Munduca synthesize

2

4’ 1

juvenile hormones II and III as well as juvenile hor-

mone I during larval-pupal development (Granger

et al.,

1982), these homologues were assayed

in vitro

with the same protocol used for juvenile hormone I.

Since juvenile hormone I in the haemolymph of

Munduca

is normally associated with its binding

protein (Goodman and Gilbert, 1978) the in vitro

studies testing the effect of juvenile hormone I were

conducted not only with the hormone free in solution

(unbound), but also complexed to the binding pro-

tein. Each assay consisted of 3 replicates and was

conducted at least 2 times. More than 1100 pairs of

prothoracic glands were utilized during the course of

this study.

Time courses of ecdysone synthesis by V, pro-

thoracic glands in response to various concentrations

of unbound juvenile hormone I (Fig. 5) failed to

reveal prothoracic gland stimulation, i.e. the activa-

tion ratio was approx one for all time points. Some

variability was noted at 10m7M and lo-’ M juvenile

hormone I at 1 and 2 h but the increased activation

ratios were not significantly different and were not

dose-dependent. Since the activation ratios were less

than 2 and were not significantly different between

juvenile hormone I concentrations at a given incu-

bation time, nor between different incubation times at

a given hormone concentration, it was concluded that

this hormone was not exerting a tropic effect on V,

prothoracic glands. This same protocol was used to

test the effect of juvenile hormone I on V, and V,

glands and of juvenile hormone II and III on V,, V,

and V, glands. Overall, the time courses of ecdysone

synthesis by these various prothoracic glands with

differing concentrations of the juvenile hormone ho-

mologues yielded results almost identical to those

noted above for juvenile hormone I on V, prothoracic

glands, i.e. a tropic effect was not observed. These

studies are summarized in Table 1 with mean activa-

tion ratios obtained for all the concentrations of

unbound juvenile hormone used at a given incubation

time. The range of the activation ratios obtained at

each time point is also presented to illustrate the

general reproducibility and small variation occurring

over the entire concentration range.

Careful analysis of these dose-response time

course data revealed that there were isolated cases,

e.g. juvenile hormone I with V, prothoracic glands at

1 h and for hormone II with V, prothoracic glands at

1h, when the range of activation ratios obtained with

different concentrations

of hormone varied

significantly. However, these were isolated occur-

rences and the response was never observed for more

4

I I I I

2

4

6

6

Time h)

Fig. 5. Effects of uvenile hormone I on the in vitro activity of prothoracic glands from V, larvae.

10m6 M; 0 IO-‘M; n o-‘M; 0 10m9M; A IO-“‘M; A control prothoracic glands. A, denotes

activation ratio.


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Effect of juvenile hormone on prothoracic gland

Table 1. The effects of unbound juvenile hormones on prothoracic gland activity in uizro

337

Treatment

Developmental

Stage

v,

Juvenile

hormone I

V,

V,

Juvenile

hormone II

Juvenile

hormone III

ZR 512

“S

“6

V,

VS

“6

“7

VS

V6

V,

lh

Mean A, +

SEM*

(Range)

2h 4h

1.27JrO.18

(0.81-1.77)

-

0.81 2 0.07

(0.69-1.10)

0.86*o.li

(0.62- 1.36)

-

1.40&0.16

(0.98-2.01)

1.15+0.06

(0.95-1.34)

1.06 + 0.09

I 02 rfr0.05

(0.79.-1.32) (0.93-1.21)

0.8

1

0.02

1.02 0.05

(0.75-0.88) (0.90- 1.21)

0.88 i 0.04

0.98 f 0.02

(0.79-1.02)

(0.93-1.02)

0.98 f: 0.08

1.03 t 0.08

(0.82-I .32) (0.84.--1.24)

0.93 +0.16

0.91 10.15

(0.36- 1.46)

(0.33-1.35)

1.31 kO.12

1.2s+o.t5

(0.96-l .63) (0.91--1.89)

1.10 & 0.05

(0.98-1.31)

0.88

* 0.09

0.89 & 0.60

-

(0.56-1.19) (0.75-1.08)

1.03

*0.14

(0.62-I .46)

1.19 & 0.15

-

(0.76-1.77)

1.08

t 0.05

(0.96-1.21)

1.14&0.07

-

(1.04 1.38)

1.~21_0.13

(0.56-1.42)

1.2550.21

-

(0.65-1.82)

0.90

& 0.04

(0.76-0.97)

0.96 f 0.08

-

(0.70-1.16)

8h

1.06 rt 0.07

(0.94-1.35)

I .02 t 0.06

(0.85-1.19)

1.01 rt 0.01

(0.98--l .06)

1.08 i 0.07

(0.90.. 1.33)

0.94 rt 0.06

(0.82-1.84)

0.95 i 0.1 I

(0.65-I .40)

1.08 0.09

(0.9% 1.08)

0.97 + 0.05

(0.78-1.08)

1.01 rtO.08

(0.66-1.15)

1.00 + 0.07

(0.84-I .23)

1.20f0.10

(0.89-l .43)

0.90 f 0.06

(0.72-1.01)

*The mean A, (activation ratio) values represent the mean for all con~ntrations of juvenile hormones or ZR 512 assayed. concentrations

assayed were lOme, lo-‘, 10w8, 10s9 and IO-“M. h signifies hours of incubation.

than one time point during the incubation period.

Furthermore, a dose-dependent response was never

observed.

Except with V, prothoracic glands at 2 h, no effects

on the glands were observed with ZR 512. The lack

of an effect is exemplified by the response of V,

prothoracic glands to ZR 512 (Fig. 6) where the

activation ratio varied nominally over the 8-hr incu-

bation period. That ZR 512 did not stimulate the

glands was a notable observation since it was the

most potent material in evoking a biological response

in

situ

and is not subject to catabolism by esterases

(Kramer and DeKort, 1976). Therefore, it appears

that in the unbound state, neither juvenile hormone

nor ZR 512 stimulate the prothoracic glands

in vitro

Since juvenile hormone I is bound to the binding

protein in sit u, it was possible that the hormone/bind-

ing protein complex could activate the prothoracic

glands since this would be a more “physiological”

condition than the use of unbound hormone. The

same experimental design was employed as in the

previous in vitro studies and similar results were

obtained: juvenile hormone/binding protein did not

elicit a significant dose-dependent effect on gland

activity over time (Table 2). Therefore, the presence

of binding protein does not appear to be adequate for

demonstrating a tropic effect of juvenile hormone I

on the prothoracic glands during 8 h in

vitro

In summary, these in

vitro

studies reveal that

during 8 h

in vitro

under defined incubation condi-

tions, juvenile hormone does not have a tropic effect

on the prothoracic glands. Therefore, its effect on the

larval-pupal development of neck-ligated V, larvae is

probably a result of the indirect activation of the

2

t

d ,

.

t :

1

i

I

I

2

4

6

8

Time h)

Fig. 6. Effects

of ZR

512 on the in vitro

activity of prothoracic glands from

V,

larvae. 0 10F6M; 0

10m7M; 1Om8M; 0 10d9M; A 1O-‘oM; A control prothoracic glands.

A, denotes

activation ratio.


8/10

338

M. C.

GRUETZMACHER efal.

Table 2. The effects of juvenile hormone I-juvenile hormone

binding protein complex on prothoracic gland activity in trilro

Mean A,kSEM

Developmental

(Range)

stage

2h 4h

8h

_.-. . .._-- ----- --

V,

0.98 i 0.09 1.07 i 0.09 1.25 f 0.11

(0.80-1.31) (0.84-1.31) (0.98-1.66)

“*

1.17+0.16 1A4 * 0.30 1.35 0.07

(0.72-1.74) (3.11-2.80) (1.21.-1.61)

V,

1.16iO.04 1.32 k 0.09 1.20 i: 0.08

(1.00-1.28) (1.01-1.58) (0.92-1.48)

*The mean A, (activation ratio) values represent the mean for all

con~nt~dtions ofjuvenile hormone I bound to juvenile hormone

binding protein assayed. Concentrations of juvenile hormone I

bound to the binding protein assayed were IO-‘, IO-‘, IO-‘, IO+’

and IO-” M. h signifies hours of incubation.

prothoracic glands. To ensure that our inability to

demonstrate a direct effect was not due to lack of

exposure time between the juvenile hormone and

prothoracic glands, longer incubation periods were

also examined.

Long t erm t ects of ZR 512 on prothor acic gland

act iv i ty

in vitro

It has been suggested that juvenile hormone and

ZR 512 may affect prothoracic gland activity during

very narrowly defined developmental periods in the

fifth instar of Manduca, and that these effects might

be observed

in v i tro

if the glands were maintained in

culture for at least 48 h in the presence of the

hormone (Sho Sakurai, personal communication). To

test this possibility, prothoracic glands were exposed

to ZR 5 12 for 48 h in v i t ro under sterile conditions in

two separate, preliminary studies. For V, glands the

activation ratio values at 48 h varied more than those

for the 8-hr incubations, but still fluctuated around

one. Because of these fluctuations, only one concen-

tration of ZR 512 (10m9M) yielding an activation

ratio of

1.5

appeared to be significantly different. This

result is probably not physiologically significant for

the same reasons detailed for the 8-hr incubation

results, i.e. lack of a dose-response.

In summary, the results indicate that juvenile hor-

mone does not affect prothoracic gland activity

directly under either the long-term or short-term

conditions utilized here.

Ot her efect s ?~j at i e~i I e ormone and t s metab~l ~t es on

prothoracic glands in vitro

In addition to the possible tropic effect of juvenile

hormone on the prothoracic glands, an inhibitory

effect has also been proposed (see Introduction). A

preliminary in vitro analysis of juvenile hormone I on

V, larval prothoracic gland activity indicated a lack

of effect, although topical application of juvenile hor-

mone I to V, larvae resulted in an arrest of develop-

ment. However, preliminary analysis of the effects of

juvenile hormone acids on day-0 pupal prothoracic

glands in v i t ro suggests that the acid of juvenile

hormone III, but not of hormones I or II, inhibits

ecdysone synthesis by the prothoracic glands in a

dose-dependent manner (lo-* M juvenile hormone

III acid to lO-‘M). Neither methyl epoxy stearate

nor linoleic acid were effective and no change in the

pH of the culture medium was noted after the

addition of juvenile hormone III acid. There was no

effect of any of the juvenile hormone acids on pro-

thoracic glands from larvae (V,, V,, V,, V,). The

physiological significance of this preliminary obser-

vation on day-0 pupal prothoracic glands is not clear:

further research is necessary to ascertain if this effect

is physiological or pharmacological. Nevertheiess,

this is the only result from all of the in

v i t ro

studies

on juvenile hormone indicating a direct effect on the

prothoracic glands in v i tro.

This investigation of the potential direct tropic

effect of juvenile hormone on the prothoracic glands

of

M anduca sext a

has revealed by whole-animal

experimentation, i.e. by indirect means, that juvenile

hormone exerts a positive effect on the glands of

neck-ligated, V, larvae as reported previously (e.g.

Hiruma

et al.,

1978; Cymborowski and Stolarz, 1979;

Safranek et al., 1980). That is, topical application of

the hormone or ZR 512 results in a decrease in the

time to pupation and this is a result of an increased

haemolymph ecdysteroid titre. Further analysis of

this phenomenon assessed the putative activation of

the prothoracic glands by juvenile hormone directly

with an in v i tro protocol and revealed that neither

unbound juvenile hormone I, II, III or ZR 512, nor

the hormone bound to binding protein affected pro-

thoracic gland activity

in v i tro

in a dose-dependent

manner at concentrations ranging from 10d6 to

lo-“M. However, prothoracic glands of the stages

tested can be activated by PTTH extract. At least in

the case of M anduca, juvenile hormone does not

appear to activate the glands directly, but probably

acts indirectly through another factor which, in turn,

stimulates ecdysone synthesis by the prothoracic

glands.

In the past 5 years, there have been several

in s i tu

studies demonstrating that topically applied juvenile

hormone affects prothoracic gland activity in Lepi-

dopteran larvae. In

M amest ra br assi cae,

the time

required for neck-ligated, wandering-stage larvae to

pupate was reduced in a dose-dependent manner by

topically applied ZR 512 (Hiruma et al ., 1978). When

the prothoracic glands of these ZR 5l2-treated larvae

were transplanted into untreated recipients, the time

to pupation was also reduced. Similar studies on

unmanipulated larvae of

Spodopkra li tt oral&

gave

essentially the same results, in that larvae treated with

a juvenile hormone analogue after dorsal vessel ex-

posure underwent pupal development more quickly

than untreated animals (Cymborowski and Stolarz,

1979). As in the

M amestra

study, it was concluded

that juvenile hormone acted directly on the pro-

thoracic glands and had a prothoracicotropic role at

the time of the second head-critical period (pharate

pupal development). When wandering V,

Manduca

larvae were neck-ligated the length of time to the

pupal moult doubled, similar to the effect observed in

M amestra.

Topical application of ZR 512 reduced

this delay which was progressively eliminated by

the analogue in a dose-dependent manner (see also

Safranek et al., 1980).

While the

in sit u

investigations conducted thus far

on the putative prothoracicotropic action of juvenile


9/10

E&et

of juvenile hormone on prothoracic

gland

339

hormone have unqu~~sti nably demonstrated such an

effect, the indirect nature ofthe approaches employed

prevented a determination of the level at which the

effect was exerted. However, our attempt to establish

a direct causal relationship between this hormone and

increased prothoracic gland activity using an in

v i t ro

approach was unsucc~essful. Stimulation of the glands

was not observed in the presence of concentrations of

juvenile hormone sp.anning the normal physiological

range of the hormone, nor with concentrations well

above and below this range. These same results were

obtained irrespective of the homologue used and the

manner in which it was presented to the glands (i.e.

rt: binding protein). AIthough it is possible that this

failure to stimulate the prothoracic glands was a

function of unfavourabie in vitro incubation condi-

tions, this seems unlikely since these same conditions

support PTTH activation of the prothoracic glands.

Thus, it appears that juvenile hormone does not

activate the prothoracic glands directly but does so

indirectly. Results suggesting such a possibility have

been reported previously (Sehnal et al., 1981). Fur-

ther analysis of this phenomenon in

~f f~d~ u

using

the in u&o approach has revealed the existence of an

as yet uniden~~ed tropic factor whose synthesis

and/or release is controlled by juvenile hormone

(~ollenbacher ef a&, ~~etzmacher et al., un-

pubi~sh~ obs~~ations~~ It is apparently this factor

that accelerates development. The chemical proper-

ties of the factor are ~igni~~ntly different from those

of PTTH, and its nature and function ipr situ will be

the subject of future reports from our laboratories.

A physiological role for juvenile hormone as a

protharacicotropin driving the larval-pupal devel-

opment of ~u~~~~ff is temporally consistent with the

haemolymph titres of juvenile hormone I and II

@ergot

et

al., personal communication), and with the

in

v i t ro

biosynthetic activity of the corpora allata

(Cranger et ul., f982j during this period. Why FTTH

and juvenile hormone are both necessary to regulate

the prothoracic glands is not clear, but one expla-

nation may be the need to sustain an elevated level

of gland activity over a long period of time. This

possibility is supported by the extended increase in

the ecdysteroid titre during larval-pupal devel-

opment in M anduca,

an

increase which begins on day

5.5 and peaks on day 7.5 (BolIenbach~~

et al.,

1981).

Conceivably, juvenile hormone could first exert its

indirect effect after commitment (first head-critical

period for PTTH release), elevating the basal activity

of the glands. The increase in corpora allata activity

found on day 5 supports this possibility @ranger ~1

ai., 1982). PTTH would then exert a direct and far

more dramatic effect on ecdysone synthesis at the

second head-critical period of the instar, which would

affect the sharp rise in the ecdysteroid titre on day

7”-8. The experimental evidence tits this hypothesis,

since ligated, wandering larvae which lack a source of

PTTH are nevertheless capable of undergoing meta-

morphosis to the pupa in a normal period of time if

treated with exogenous juvenile hormone, thus indi-

cating the possibility of multiple levels of control of

the prothoracic glands.

Under experimental conditions, either juvenile hor-

mone or PTTH may act alone, the latter directly on

the prothoracic glands and the former indirectly, but

to achieve a normal developmental sequence in a

~p~ation of insects, both must act. This, of course,

does not explain why larvae whose entire

brain-retrocerebral complex have been removed ulti-

mately develop (Judy, 1972). In the neck-ligated

M unduca

larvae in the present study not treated with

juvenile hormone, the haemolymph ecdysteroid titre

increased gradually until it was N 175% greater than

that of normal larvae when pupation occurred

-4 days after ligation. This corroborates the studies

conducted with cialleria (Sehnal et ai., 1981 f and

C+JQ&S (Dean and Steel, 1982). Therefore, the

prothoracic glands which are never completely qui-

escent in the last-larval instar of

~~~u~a

(Bollen-

bather et al ., 1981) may also become active spontane-

ously.

In contrast to the tropic effect of juvenile hormone

on figated, wandering Vs larvae is the inhibitory effect

of the hormone on the development of last-instar

larvae treated before the first head-critical period

(pre-commitment). This phenomenon has been dem-

onstrated with several species of Lepidoptera (Cym-

borowski and Stolarz, 1979; Safranek et al., 1980;

Hiruma and Agui, 1982) and has been corroborated

by this study. As with the tropic effect of juvenile

hormone, the inhibition of prothoracic gland activity

by

juvenile

hormone appears to occur only at specific

developmental stages. Although our attempts to

demonstrate this effect in vitro with V, (pre-

commitments larval prothoracic glands were un-

successful, a metabolite of juvenile hormone XII,

juvenile hormone III acid, did inhibit day-0 pupal

prothoracic gland activity

in v i tro

in a dosc-

dependent manner. The physiological significance of

the inhibition of this stage of development is not

known, but is currently being investigated and may

be a fail-safe mechanism for preventing precocious

pupal-adult development.

As can be seen from the above, control of pro-

thoracic gland activity during

a single instar is more

complex than thought previously. New hypo~eses

in~rporatin~ these data derived from in &ro studies

must be formulate and tested before we can hope to

elucidate the mechanisms of prothoracic gland regu-

lation. An understanding of these regulating pro-

cesses is requisite for an understanding of moulting

and metamorphosis.

Acknow l edgement s-Thi s

work was supported by grants

from NIH (AM-30118) to L.G., (M-18791) to W.B. and

from

NSF (PCM-8116931) to L.G.

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