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Juvenile hormone modifies larvae and silk gland development in Bombyx mori
BIOCHIMIE, 1979, 61, 275-281.
Juvenile hormone modifies larvae and silk gland
development in Bombyx mort. Jacques DAILLIE.
D~partement de Bioloyie G~ndrale et AppliquEe, Universit~ Claude Bernard Lyon-l, Laboratoire associ~ au C.N.B.S. n ° 92, 69621 Villeurbanne, France.
R6sum6.
Summary.
L'application locale d ' u n a n a l o g u e d e l'horm o n e juv6nile (ZR 515) p e n d a n t la p r e m i e r e moiti6 du dernier (cinqui~me) s t a d e l a r v a i r e chez B o m b y x m o t / d 6 t e r m i n e un a l l o n q e m e n t de ce s t a d e et u n a c c r o i s s e m e n t du p o i d s d e s l a r v e s et de la s6cr6tion de sole. Un t r a i t e m e n t plus tardif p e u t p r o v o q u e r u n e m u e l a r v a i r e surnum6raire. En outre, l ' h o r m o n e p r o v o q u e l'arr~t du d 6 v e l o p p e m e n t ou de l'activit6 s6cr6toire d e l a q l a n d e s6riciq~ne selon le m o m e n t d u traitement. Lorsque l ' a n a l o q u e est a p p l i q u 6 a u d6but du cinqui6me s t a d e i a r v a i r e , la c r o i s s a n c e de la g l a n d e ainsi q u e l a s y n t h b s e du RNA et d e s prot6ines sont d ' a b o r d inhib6es. Plus lard, elles r e p r e n n e n t et m ~ m e se t r o u v e n | amplifi6es. Finalement, la production de sole atteint IS0 p o u r cent de celle d e s t6moins. Un d e s effets p r / m a i r e s du traitement s e m b l e se situer a u n i v e a u de l a s y n t h b s e d e s RNA. Ainsi, le trult e m e n t p a r l ' h o r m o n e juv6nile p o u r r a i t d e v e n i r un n o u v e a u m o y e n d'6tude de la transcription e t / o u l a m a t u r a t i o n d e s RNA.
Topical application of a Juvenile Hormone Analogue (ZR 515) during the first half of the last (fifth) larval instar of Bombyx mori induces a prolongation of this instar and increases larv a e weight a n d silk secretion. Later treatment can lead to an extra larval molt. Moreover, the hormone a n a l o g u e stops either the development or the secretory activity of the silk gland, depending on the period of treatments. W h e n Juvenile Hormone is administered at the beginning of the fifth instar, silk gland growth as well as RNA and protein syntheses are first inhibited and later resumed and amplified. Finaly silk production increases up to 150 per cent of controls. One of the primarly effects of treatments seems to be RNA synthesis. Thus Juvenile Hormone analogue application could be a new tool for studying RNA transcription a n d / o r processing.
Introduction. Treatments with natural or synthetic juvenile hormones (J. H.) induce in B o m b y x mort fifth instar larvae a prolongation of the larval life and, depending on the administered doses and on the application times, a delay in spinning cocoon, the occurrence of an additional (fifth) larval molt or the formation of so-called permanent larvae [14]. When the J. H. treatment is done during the first half of the fifth instar, the silkworms are generally able of spinning cocoon and, in this case, the silk shell is alnvays heavier than in controls [2-5J.
Key words : Bombyx, silk gland, juvenile hormone.
On the basis of such preliminary data 'were hypothesized J'. H. should act not only at the organism level but also, directly or not, on growth and physiological activity of the silk gland itself. With the aim of verifying our hypothesis we confirm both the above-mentioned works and bring new data concerning the J. H. effects on silk gland development and activity. Particularly, it becomes clear that J.H. acts on DNA, RNA and protein syntheses. Detailed data are reported elsewhere [6]. We just recall here the most significanl and present same additional results in viev¢ to discuss their
J. Daillie.
276
b i o l o g i c a l m e a n i n g a n d the p o s s i b i l i t i e s o f f e r e d by hormonal experimentation.
I.
EFFECTS DURATION
OF
J'.H.
AND
ANALOGUE
LARVAE
ON
THE
FIFTH
INSTAR
GROWTH.
Results (table I, fig. 2). B
In all cases the feeding period was increased, the m a x i m u m being observed in the type B treatment. days
,
,
i
,
t
=
,
i
x
,
t ~
x
t
,
,=.
i
of ,
L
5thinstar ~
i
•
c
FIG. I. - - Procedures of treatmenl. Juvenile hormone analogue (ZR. 515.) was administered by topical appl,ication. One dose of 1 ~g par g of body weight was applied every day at 3 periods (A, B, C) of 3 days each, at times indicated by arrows.
I n t h e s e e x p e r i m e n t s , w e u s e d t h e J.H. a n a l o g u e Z R 515 (Gift of Z o e c o n Co) d i s s o l v e d in a c e t o n and topically administered upon dorsal tegument, to J 124 × C 124 F 1 h y h r i d s . T h e e f f e c t i v e dose, c h o s e n o n t h e basis of v a r i o u s assays, w a s 1 ~g p e r g of f r e s h b o d y w e i g h t . W e first l o o k e d at the g l o b a l effects of t r e a t m e n t p e r f o r m e d at t h r e e diff e r e n t p e r i o d s of t h e fifth i n s t a r , as i n d i c a t e d in f i g u r e 1. T h e n ~re f o c a l i z e d e x p e r i m e n t s on t h e earlier type treatment.
Larvae .treated according to procedures A and B, exhibited a normal spinning behaviour ; however, some B-treated individuals were not able to achieve spinning and died. O n the contrary, silkw o r m s submitted to the type C treatment did not spin ; they entered in a fifth larval type molting period but died because being unable to come out of the old cuticle. The larvae weight reached at the end of the feeding phase was increased by 20 to 30 per cent after type A and B treatments. No change was observed with C-treated silkworms. W h e n J.H. was administered early in the fifth instar (Type A), the growth curve did not change in its initial phase
(fig. 2). Discussion. T h e late ( t y p e C) t r e a t m e n t m o d i f i e s t h e h o r m o nal c o n d i t i o n s l e a d i n g to p u p a l e c d y s i s a n d m e t a m o r p h o s i s as a l r e a d y o b s e r v e d b y A k a i et al. [1, 4]. A l t h o u g h an a d d i t i o n a l l a r v a l m o l t in I n s e c t s is q u i t e d i f f i c u l t to o b t a i n b y an i n t e r v e n t i o n d u -
TABLE I.
T r e a t m e n t effects on the feeding phase duration and the w e i g h t of larvae, silk shell and posterior silk gland. A f t e r data reported by Kurata and Daillie [6]. Type oi treatment (see fig. t)
Length o[ feeding phase(t){days}
Weight (per cent) ol control of larvae
of silk shell
of P.(2)S. G.
9
14.5
C~
13.5
122 121
132 140
124 131
B
Q C~
17.0 17.0
131 130
121 (3) 85 (3)
117 98.5
C
Q cf
14.5 13.5
107 91
A
(1) (2) (3) (4)
Sex
ALM (4) ALM (4)
The duration for control larvae was 8 days. P.S.G. : posterior silk gland. A few larvae did not spin then died. No spinn,ing but ooeurrenee of an additional larva/ molt ALM.
BIOCHIMIE, 1979, 61, n ° 2.
71.5 49
Effect of juvenile hormone. r i n g the last instar t7J, it should be c o n c l u d e d that J.H. i n t e r a c t s w i t h e c d y s o n e w h i c h is i n c r e a sing at that time [8] and leading to f a v o r a b l e h o r m o n a l c o n d i t i o n s to p r o m o t e an a d d i t i o n a l l a r v a l molt.
277
strongly i n c r e a s e d but n o r m a l m e t a m o r p h o s i s is yet possible. It should be o b s e r v e d that the p e r i o d of ,treatment is i n c l u d e d , in the f o r m e r case, but not e n t i r e l y in the later one, in t h e so-called <> [9]. F u r t h e r m o r e , in
c~
cn
-30 ~.
? ~ o
2 ¸
i.
FI6. 2.
o 2
3
4
5
-
-
10
ays of 1
20
6
7
8
9
10
Circles for body weight, squares for silk glands weight and triangles for silk glands/ body weight ratio. O.pen signs for controls. Closed s.igns for t~eated silkworms.
5 th i n s t a r
11
12
On the c o n t r a r y , the early (type A) t r e a t m e n t seems only to result in a delay in the d e v e l o p m e n t p r o g r a m since s p i n n i n g then p u p a t i o n o c c u r norm a l y but later t h a n in c o n t r o l animals. W h e n s y n t h e t i c J.H. is given a c c o r d i n g to type B treatment, t h e d u r a t i o n of the fifth instar is m o r e
Effects of type a treatment on larvae and silk gland growth.
1'3
both e x p e r i m e n t a l situations, the la.rval life extension is a c c o m p a n i e d by a d d i t i o n a l g r o w t h . So, J.H. c o u l d act by i n c r e a s i n g the o b l i g a t o r y f e e d i n g phase, i n v o l v i n g ei,ther h y p e r m e t a b o l i s m as desc r i b e d in Dermestes [10] or s w e l l i n g as seen in Pieris [11] like in B o m b y x . Our results seem fur.
TABLE II.
W e i g h t (1) of posterior and m i d d l e silk glands after various types of treatment. Time ol treatment (2) Development aiter the |st meal
-- 2 hrs (i applicatiou)
--2 hrs and + 24 hrs +[,8 hrs and+72 hrs (2 applications) (2 applications)
Posterior
3 days
65
52
75
Silk Gland
5 days
100
81
53
3 days
42
34
72
5 days
55
34
31
Middle Silk Gland
(1} Per cent of controls. (2) Time zero corresponds to the first meal, so that the applications of - - 2 , + 24 et + 48 hours were done 2 hrs before and one or two days after the first meal. J. H. dose applied was 1 ~g/g body weight.
BIOCHIM1E, 19'/9, 61, n ° 2.
19
J. Daillie.
27'8
ther i n agreement w i t h the hypothesis w h e r e b y a high level of J'.H. w o u l d i n h i b i t the secretion of the b r a i n ( p r o t h o r a c o t r o p i c ) h o r m o n e as expected by Nijhout a n d W i l l i a m s [12]. II. EFFECTS OF J.H. APPLICATION ON SILK GLAND GROWTH AND SILK PRODUCTION.
Results (tables I, II, III and IV ; figure 2). The silk gland growth kinetics was only studied i n the case of p r o c e d u r e A (fig. 1). D u r i n g the t r e a t m e n t p e r i o d and the following two days, the silk glands were very slighty g r o w i n g up, t h e i r development b e i n g therefore c l e a r l y different from the total larvae gro~vth (fig. 2). However, this d i s c r e p a n c y quickly recovered t h e n the glands grew bigger t h a n i n controls. F i n a l y treated w o r m s p r o d u c e d more silk as s h o w n in table I : the A-treated larvae p r o d u c e d cocoon shells heavier by 30 to 50 per cent t h a n those of n o r m a l silkworms. C o n c e r n i n g the other conditions, only the B-treated females capable of spinn i n g made shells heavier (20 per cent more) than u n t r e a t e d i n d i v i d u a l s . The weight of the posterior silk glands (table I) at the end of the feeding period w a s b r o a d l y keeping w i t h the changes observed for larvae a n d shell 'weight, d e p e n d i n g on the types of treatment. A d d i t i o n a l data s h o w n in table II f u r t h e r indicate that a single dose applied just before the first meal was yet effective three days later. It was only on day 5 that posterior siLkgland r e s u m e d its delay of growth. An a d d i t i o n a l applica,tion after one d a y of development p r o l o n g e d the growth in-
h i b i t i o n . Moreover, t r e a t m e n t a d m i n i s t e r e d in tile course of growth phase (on days 2 and 3) resulted clearly i n s i m i l a r effects. It should also be noted that in all cases we observed an accentuated relative decrease of weight i n the m i d d l e p o r t i o n of the gland. This r e i n f o r c e d effect was p r o b a b l y due to the i n h i b i t i o n of siLk p r o t e i n synthesis w h i c h failed of course to accumulate in the reservoir. Since the p e r i o d of earlier t r e a t m e n t s c o i n c i d e with the phase of DNA synthesis in controls and with the b e g i n n i n g of RNA a n d p r o t e i n a c c u m u lation, it w a s i n t e r e s t i n g to look at the effects of J.H. analogue on such m a c r o m o l e c u l a r events. Using pulse labelling e x p e r i m e n t s after a type A-treatment [6], we o b t a i n e d the following results (table III) : m a x i m u m i n c o r p o r a t i o n of glycine, a p p e a r i n g mostly i n siLk fibroin [13], w h i c h occ u r r e d by days 4-5 i n controls, was delayed by two days and r e m a i n e d at a high rate for six more days i n posterior silk glands of treated silkworms. C o n c e r n i n g n u c l e i c a,cids syntheses, m a x i m u m inc o r p o r a t i o n arised on day 3 i n c o n t r o l larvae for t h y m i d i n e as well as for u r i d i n e w h i l e in treated silkworms, we noted that the labelling p a t t e r n was modified : a high level of i n c o r p o r a t i o n lasted from day 3 up to day 7. I n the case of t h y m i d i n e , even two peaks were clearly seen (the n o r m a l one on day 3, a n o t h e r on day 7) to w h i c h c o r r e s p o n d ,changes of t h y i n i dine k i n a s e a n d DNA p o l y m e r a s e activities [6, 14]. However, DNA a c c u m u l a t i o n (table III) measured i n the course of the experiment, i n d i c a t e d that the final a m o u n t of DNA was the same i n silk
TABLE III.
DNA content and incorporation of [SHl thffnIidine, [SHl uridine and [l~Cl glgcine in posterior silk gland of treated and control silkworms. [3H] thymidine [aH] uridine dpm(X t0~}/PSG dpm (× t03)/PSG Days of ~st instar 1 3 5 7 9
C 360 790 147 100 87
T 380 590 513 866 141
C 320 852 255 170 115
T 400 675 670 705 245
[taG] glyeine dpm (X t0a)/PSG C
T
2 110 230 192 133
2 20 75 196 210
DNA content p.g/PSG C
T
28 85 95 104 110
26 61 81 92 102
11
--
112
--
200
--
231
--
112
13
--
140
--
213
--
200
--
105
Silk glands of silkworms treated according to procedure A (fig. 1) were excised at indicated days and incubated as previously described [20, 21] with [3H] thymidirre or [3H] uridirm (1 IxGi/m~) pins [14C] glyoine (0.2 ixGi/ml). Aeido-insoluble ma~eri,al was then counted. DNA was also quantified in the course of experiment.
BIOCHIMIE, 1979, 61, n ° 2.
Effect of juvenile hormone.
279
glands of both treated a n d u n t r e a t e d i n d i v i d u a l s . Therefore, J.H. applica,tion seems to d e t e r m i n e a n extension of the d u r a t i o n of the two r e p l i c a t i o n cycles w h i c h take place at the b e g i n n i n g of the fifth i n s t a r [15].
a p p e a r e d very slowly : in the case of experim e n t II, for instance, I~NA synthesis (or accumulation) d u r i n g the 48-72 h p e r i o d was r e d u c e d b y 70 per cent of the control but only 50 per cent d u r i n g the previous 24 hours.
C o n c e r n i n g RNA a c c u m u l a t i o n , a c c o r d i n g to the a b o v e - m e n t i o n e d results of l a b e l l i n g expriments, we observed a r e t a r d a t i o n (three days) of the o c c u r r e n c e of the m a x i m u m RNA a m o u n t i n treated silk glands. I n fact, the t r e a t m e n t i n d u c e d a rather p a r a d o x i c a l effect ~ ' h i c h has to be noticed. At first, d u r i n g at least the time of ~reatment, a m a r k e d i n h i b i t i o n of RNA synthesis was occurring w h i c h then r e s u m e d a n d finaly the m a x i m u m a m o u n t f o u n d was about 50 per cent h i g h e r t h a n i n the controls. T y p i c a l curves of RNA accumulation in posterior silk glands of both treated a n d u n t r e a t e d a n i m a l s are s h o w n in PavCs p a p e r in this issue [16].
I n another e x p e r i m e n t (results not shown) we observed that in g r o w i n g silk g l a n d (day 4) the effect of a single J.H. a p p l i c a t i o n on RNA synthesis was detectable by pulse labelling on six h o u r s after the time of treatment.
Discussion. As we have already p o i n t e d out, the J.H. analogue, a,pplied in the first t h i r d of the fifth i n s t a r provokes an increase of both the larval life duration a n d the weight of larvae. The last p h e n o m e -
TABLE IV.
Inhibition of RNA accumulation in posterior silk gland after earlier treatments. Inhibition (per cent ol controls) at indicated times lollowing the first meal 2t, hrs I 1 application at indicated time
48 hrs
72 hrs
33
46 41
60 60
30
48
- - 1 hr 0 -~- 2 hrs
30 28 17
- - 1 hr -~- 6 hrs
45
It
1 application at indicated time III 3 applications
( t
- - 6 hrs -~- 4 hrs
+ 18 hrs
Results expressed in per cent of controls values. The dose of J.H. used for each applications was 1 ~tg/g body weight. We verified, in c o m p l e m e n t a r y e x p e r i m e n t s (table IV) that the previous i n h i b i t o r y effect ~¢as effective w h e n the analogue was applied at the time of the firs,t meal or even six hours later. In respect w i t h RNA a c c u m u l a t i o n rates in the controls, i n h i b i t i o n at 24, 48 a n d 72 hours after the first meal, was of 25-45, 4.1-48 a n d 60 per cent, respectively. Reiteration of the ~reatment (3 times) in the range of - - 6 to + 1 8 hours did not m o d i f y the results. Such data i n d i c a t e that, as for the silk gland growth, tile i n h i b i t o r y effect las.ted a long time after the J. H. analogue was applied a n d dis-
BIOCHIMIE, 1979, 61, n ° 2.
n o n is p r o b a b l y a consequence of the former, if we c o n s i d e r that the obligatory feeding phase is itself increased, Short t e r m and long term effects of J.H. t r e a t e m e n t on the silk gland development appear quite different since weight as well as RNA a n d p r o t e i n syntheses ( i n c l u d i n g silk proteins) are p r i m a r i l y i n h i b i t e d a n d later resume and amplify. Figure 2 clearly shows such diffe. rences but also indicates that the p r o p o r t i o n of th~ silkgland weight ( i n c l u d i n g silk secretion) in the total body weight is finaly the same in treated and u n t r e a t e d i n d i v i d u a l s . This fact suggests that
280
J. Dailtie.
all the p a r a m e t e r s except time, w h i c h d e t e r m i n e the relative g r o w t h of organs and tissues do not change u n d e r such h o r m o n a l c o n d i t i o n s . P a r t i c u larly, it seems that J.H. acts in a m p l i f y i n g a disc o r d a n c e in g r o w t h w h i c h n o r m a l y exists b e t w e e n silk glands a n d the other tissues of the larvae. W h e n J.H. is a p p l i e d along the s e c o n d t h i r d of the instar, the silk g l a n d d e v e l o p m e n t is stoped a n d delayed. It can be m o r e or less c o m p l e t e l y r e s t o r e d c o n s e q u e n t l y to an e n h a n c e d d u r a t i o n of the l a r v a l life. Even in the best cases, the silk p r o d u c t i o n is l o w e r than after an e a r l i e r t r e a t m e n t . C o n c e r n i n g s i l k w o r m s t r e a t e d a c c o r d i n g to the p r o c e d u r e C., too little i n f o r m a t i o n is k n o w n aboul silk gland. In such animals, w h i c h are unable to s p i n a n d to e n t e r t h e p u p a l stage, the w e i g h t of t h e p o s t e r i o r silk gland becomes l o w e r than in controls. This r e s u l t leads us to t h i n k that the gland stops fibroin synthesis. Two DNA replica.tion cycles o c c u r in t r e a t e d p o s t e r i o r silk gland as ~,ell as in n o r m a l one. In the former, J.H. a p p l i c a t i o n affects DNA s y n t h e s i s o n l y b y i n c r e a s i n g t h e time n e e d e d to c o m p l e t e the c o r r e s p o n d i n g DNA content and the onset of repli,cation does not seem d e l a y e d . T h e r e f o r e the two DNA s y n t h e s i s cycles w h i c h are o v e r l a p p e d in c o n t r o l l a r v a e [15] b e c o m e m o r e d i s t i n c t follow i n g an e a r l y a p p l i c a t i o n of the J.H. analogue. Such a result r e i n f o r c e s the feeling that the degree of p o l y p l o i d y in silk g l a n d cell nuclei is m a i n l y d e p e n d i n g on genetic c h a r a c t e r i s t i c s and c a n n o t be easily m o d i f y b y any k i n d of factors [17, 187. It has been c l a i m e d , in some cases, that J.H. acts first b y i n c r e a s i n g DNA s y n t h e s i s ; in fact, such effects w e r e only c l e a r l y o b s e r v e d in fat b o d y cells of Locusta [19]. As DNA a m o u n t r e m a i n s b r o a d l y u n c h a n g e d a n d as t r a n s c r i p t i o n rate a p p e a r s r a t h e r constant in e u k a r y o t e s , t h e question a r i s e s h o w a J.H. treatmen.t m a y i n d u c e an i n c r e a s e of the m a x i m u m RNA amount a c c u m u l a t e d in silk gland. In fact, w h e n t r e a t m e n t starts on the onset of the fifth instar, RNA a c c u m u l a t i o n is first d r a s t i c a l l y inhibited. The length of the i n h i b i t i o n p h a s e d e p e n d s on the n u m b e r of a p p l i c a t i o n s , but a single one is sufficient to o c c u r a severe d e l a y in RNA synthesis. Even a t r e a t m e n t a d m i n i s t e r e d after the b e g i n n i n g of s y n t h e s i s has been s h o w n effective. One can t h e r e f o r e c o n c l u d e that one of the p r i m a r i l y effects of t h e J.H. t r e a t m e n t is to i m p r o v e the i n i t i a t i o n of RNA t r a n s c r i p t i o n at a n o r m a l rate. T h e r e is m o r e o v e r a n ~ r r o w c o r r e l a t i o n b e t w e e n the RNA s y n t h e s i s i n h i b i t i o n and the d e l a y observed in silk gland g r o w t h and silk p r o t e i n secretion.
BlfOCHIMIE, 1979, 61, n ° 2.
After b e i n g i n h i b i t e d and delayed, RNA c o n t e n t i n c r e a s e s a n d r e a c h e s a h i g h e r level in treated t h a n in c o n t r o l larvae, as for silk g l a n d growth. Using a m a t h e m a t i c a l m o d e l to s t u d y the quantitative changes of RNA, Pav6, in this issue [16], finds that the d e g r a d a t i v e p a r a m e t e r of the d y n a mic a c c u m u l a t i o n p r o c e s s is m o s t l y m o d i f i e d in t r e a t e d silk glands w i t h r e s p e c t to the c o n t r o l ~nes. He thus h y p o t h e t i z e s that the e n h a n c e m e n t ~f final a m o u n t of RNA f o l l o w i n g the J.H. application to be d u e to a d e c r e a s e of t u r n o v e r r a t h e r of s y n t h e s i s processes. Such a h y p o t h e s i s h a s of course to be v e r i f i e d but seems a l r e a d y interesting to c o n s i d e r : since silk gland d e v e l o p m e n t was a little m o r e d e l a y e d that RNA synthesis, it seems p o s s i b l e that the d e l a y in g r o w t h involves a c o r r e s p o n d i n g late in t h e a p p e a r e n c e of nuclease activities. An e a r l y J.H. t r e a t m e n t i n d u c e s a r e t a r d a t i o n then an i n c r e a s e of the silk p r o t e i n s synthesis. This o b s e r v a t i o n is quite in a g r e e m e n t w i t h the p r e v i o u s p o i n t s of the discussion, since o t h e r studies c l e a r l y i n d i c a t e that the fibroin p r o d u c t i o n is e n t i r e l y d e p e n d e n t of RNA a c c u m u l a t i o n [17, 181.
Conclusion.
The J'.H. analogue ZR 515 i n d u c e s v a r i o u s changes in Bombyx mort development, d e p e n d i n g on the p e r i o d s of t r e a t m e n t . Moreover, it acts d i f f e r e n t l y u p o n silk gland and other tissues. The most i n t e r e s t i n g effects a p p e a r w h e n the analogue is a p p l i e d at the b e g i n n i n g of the fifth l a r v a l instar. D u r i n g this p e r i o d , a decrease of n a t u r a l h o r m o n e o c c u r s in n o r m a l larvae. Thus our results seem to be a c o n s e q u e n c e of a p e r s i s t e n t h i g h level of h o r m o n e o w i n g to analogue a p p l i c a tions. The i n h i b i t i o n of RNA s y n t h e s i s in silk gland as a s h o r t t e r m effect of t r e a t m e n t s m a y a l l o w us of u n d e r s t a n d i n g the m e c h a n i s m s of changes in transcrip.tion a c t i v i t y d u r i n g the molting p e r i o d and the e a r l y feeding phase. Acknowledgements.
I would like to thank G. Deeaussin for her technical assista.nce ; G. B,osquet, B. Gal~ez, P. Coalble and J.-C. Prudhomme for their critical advising. REFERENCES. l. Akai, H. & Kobayashi, M. (1971) Appl. Ent. Zool. (Japan), 6, 138-139. ~. Akai, H., Kiguehi, K. & Mort, K. (1971) Appl. Ent, Zool. (Japan), 6, 218-220,
Effect of juvenile hormone. 3. Murakoshi, S., Chang, C. F. a Tamura, S. (1972) Agr. Biol. Chem. (Japan), 36, 692-694. 4. Akai, H., Kiguchi, K. ~ Mori, K. (1973) Bull. Sericul. Exp. Sta. (Tokyo)., 25, 287-305. 5. Nihmura, M., Aomori, S., Mori, K. • Matsui, M. (1972) Agr. Biol. Chem. (Japan), 36, 889-8,92. 6. Kurata, K. & Daillie, J. (19,78) Bull. Sericul. Exp. Sta., 27, u n d e r press. 7. Slama, K., ~ o m a n u k , M. a Sorm, F. (19'74) ¢ Insect h o r m o n e s and Bioanalogues >>, Springer Verlag, Wien, New-York, 447 pp. 8. Calvez, B., I-Iirn, M. a De Reggi, M. (1976)FEBS Letters, 71, 5%61. 9. Legay, J.-M. (1966) La prise de nourriture chez le V e t ~ sole. Thesis, Paris. 10. Sblama, M. ~ H~odkov,a, M. (1'975) Biol. Bull., 148, 320332. 11. Srihari, T. ~ G~ahukar, R. T. (197'5) Bull. Soc. Zool. Fr., 100, 327-3~,3.
BIOCHIMIE, 197@, 61, n ° 2.
281
I2. Nijhout, H. F. ~ Williams, G. M. (1974) J. Exp. Biol., 61, 493-501. 13. P r u d h o m m e , J.-C., Sridhara, S. & Daillie, J. (1973) Arch. Biochem. Biophys., 159, 97-104. 14. De Turenne, M. (19q:8) La synth$se du DNA dans les glandes sdricig~nes de Bombyx morl au dernier stade larvaire. Thesis, Lyon. 15. Perdrix, S. (1979) Biochimie, 61, this iss,ue. 16. Pave, A.. (1979) Biochimie, 61, this issue. 17. Shigematsu, H., Kurata, K. & Takeshita, H. (1978) Comp. Bioehem. Physiol., 61 B, 237-242. 18. P r u d h o m m e , J.-C. & Couble, P. (1979) Biochimie, 61, this issue. 19. Chen, T., Couble, P., Ahuhakima, R. & Wyatt, G. R. (1979') Deo. Biol. (under press). 20. De Turenne, M. & Daillie, J. (1969) Biochim. Biophys. Acta, 186, 267-2'79. 21. Couble, P., P r u d h o m m e , J.-C. & Dail.lie, J. (1977) Exp. Cell Res., 109, 139-150.
Juvenile hormone modifies larvae and silk gland
development in Bombyx mort. Jacques DAILLIE.
D~partement de Bioloyie G~ndrale et AppliquEe, Universit~ Claude Bernard Lyon-l, Laboratoire associ~ au C.N.B.S. n ° 92, 69621 Villeurbanne, France.
R6sum6.
Summary.
L'application locale d ' u n a n a l o g u e d e l'horm o n e juv6nile (ZR 515) p e n d a n t la p r e m i e r e moiti6 du dernier (cinqui~me) s t a d e l a r v a i r e chez B o m b y x m o t / d 6 t e r m i n e un a l l o n q e m e n t de ce s t a d e et u n a c c r o i s s e m e n t du p o i d s d e s l a r v e s et de la s6cr6tion de sole. Un t r a i t e m e n t plus tardif p e u t p r o v o q u e r u n e m u e l a r v a i r e surnum6raire. En outre, l ' h o r m o n e p r o v o q u e l'arr~t du d 6 v e l o p p e m e n t ou de l'activit6 s6cr6toire d e l a q l a n d e s6riciq~ne selon le m o m e n t d u traitement. Lorsque l ' a n a l o q u e est a p p l i q u 6 a u d6but du cinqui6me s t a d e i a r v a i r e , la c r o i s s a n c e de la g l a n d e ainsi q u e l a s y n t h b s e du RNA et d e s prot6ines sont d ' a b o r d inhib6es. Plus lard, elles r e p r e n n e n t et m ~ m e se t r o u v e n | amplifi6es. Finalement, la production de sole atteint IS0 p o u r cent de celle d e s t6moins. Un d e s effets p r / m a i r e s du traitement s e m b l e se situer a u n i v e a u de l a s y n t h b s e d e s RNA. Ainsi, le trult e m e n t p a r l ' h o r m o n e juv6nile p o u r r a i t d e v e n i r un n o u v e a u m o y e n d'6tude de la transcription e t / o u l a m a t u r a t i o n d e s RNA.
Topical application of a Juvenile Hormone Analogue (ZR 515) during the first half of the last (fifth) larval instar of Bombyx mori induces a prolongation of this instar and increases larv a e weight a n d silk secretion. Later treatment can lead to an extra larval molt. Moreover, the hormone a n a l o g u e stops either the development or the secretory activity of the silk gland, depending on the period of treatments. W h e n Juvenile Hormone is administered at the beginning of the fifth instar, silk gland growth as well as RNA and protein syntheses are first inhibited and later resumed and amplified. Finaly silk production increases up to 150 per cent of controls. One of the primarly effects of treatments seems to be RNA synthesis. Thus Juvenile Hormone analogue application could be a new tool for studying RNA transcription a n d / o r processing.
Introduction. Treatments with natural or synthetic juvenile hormones (J. H.) induce in B o m b y x mort fifth instar larvae a prolongation of the larval life and, depending on the administered doses and on the application times, a delay in spinning cocoon, the occurrence of an additional (fifth) larval molt or the formation of so-called permanent larvae [14]. When the J. H. treatment is done during the first half of the fifth instar, the silkworms are generally able of spinning cocoon and, in this case, the silk shell is alnvays heavier than in controls [2-5J.
Key words : Bombyx, silk gland, juvenile hormone.
On the basis of such preliminary data 'were hypothesized J'. H. should act not only at the organism level but also, directly or not, on growth and physiological activity of the silk gland itself. With the aim of verifying our hypothesis we confirm both the above-mentioned works and bring new data concerning the J. H. effects on silk gland development and activity. Particularly, it becomes clear that J.H. acts on DNA, RNA and protein syntheses. Detailed data are reported elsewhere [6]. We just recall here the most significanl and present same additional results in viev¢ to discuss their
J. Daillie.
276
b i o l o g i c a l m e a n i n g a n d the p o s s i b i l i t i e s o f f e r e d by hormonal experimentation.
I.
EFFECTS DURATION
OF
J'.H.
AND
ANALOGUE
LARVAE
ON
THE
FIFTH
INSTAR
GROWTH.
Results (table I, fig. 2). B
In all cases the feeding period was increased, the m a x i m u m being observed in the type B treatment. days
,
,
i
,
t
=
,
i
x
,
t ~
x
t
,
,=.
i
of ,
L
5thinstar ~
i
•
c
FIG. I. - - Procedures of treatmenl. Juvenile hormone analogue (ZR. 515.) was administered by topical appl,ication. One dose of 1 ~g par g of body weight was applied every day at 3 periods (A, B, C) of 3 days each, at times indicated by arrows.
I n t h e s e e x p e r i m e n t s , w e u s e d t h e J.H. a n a l o g u e Z R 515 (Gift of Z o e c o n Co) d i s s o l v e d in a c e t o n and topically administered upon dorsal tegument, to J 124 × C 124 F 1 h y h r i d s . T h e e f f e c t i v e dose, c h o s e n o n t h e basis of v a r i o u s assays, w a s 1 ~g p e r g of f r e s h b o d y w e i g h t . W e first l o o k e d at the g l o b a l effects of t r e a t m e n t p e r f o r m e d at t h r e e diff e r e n t p e r i o d s of t h e fifth i n s t a r , as i n d i c a t e d in f i g u r e 1. T h e n ~re f o c a l i z e d e x p e r i m e n t s on t h e earlier type treatment.
Larvae .treated according to procedures A and B, exhibited a normal spinning behaviour ; however, some B-treated individuals were not able to achieve spinning and died. O n the contrary, silkw o r m s submitted to the type C treatment did not spin ; they entered in a fifth larval type molting period but died because being unable to come out of the old cuticle. The larvae weight reached at the end of the feeding phase was increased by 20 to 30 per cent after type A and B treatments. No change was observed with C-treated silkworms. W h e n J.H. was administered early in the fifth instar (Type A), the growth curve did not change in its initial phase
(fig. 2). Discussion. T h e late ( t y p e C) t r e a t m e n t m o d i f i e s t h e h o r m o nal c o n d i t i o n s l e a d i n g to p u p a l e c d y s i s a n d m e t a m o r p h o s i s as a l r e a d y o b s e r v e d b y A k a i et al. [1, 4]. A l t h o u g h an a d d i t i o n a l l a r v a l m o l t in I n s e c t s is q u i t e d i f f i c u l t to o b t a i n b y an i n t e r v e n t i o n d u -
TABLE I.
T r e a t m e n t effects on the feeding phase duration and the w e i g h t of larvae, silk shell and posterior silk gland. A f t e r data reported by Kurata and Daillie [6]. Type oi treatment (see fig. t)
Length o[ feeding phase(t){days}
Weight (per cent) ol control of larvae
of silk shell
of P.(2)S. G.
9
14.5
C~
13.5
122 121
132 140
124 131
B
Q C~
17.0 17.0
131 130
121 (3) 85 (3)
117 98.5
C
Q cf
14.5 13.5
107 91
A
(1) (2) (3) (4)
Sex
ALM (4) ALM (4)
The duration for control larvae was 8 days. P.S.G. : posterior silk gland. A few larvae did not spin then died. No spinn,ing but ooeurrenee of an additional larva/ molt ALM.
BIOCHIMIE, 1979, 61, n ° 2.
71.5 49
Effect of juvenile hormone. r i n g the last instar t7J, it should be c o n c l u d e d that J.H. i n t e r a c t s w i t h e c d y s o n e w h i c h is i n c r e a sing at that time [8] and leading to f a v o r a b l e h o r m o n a l c o n d i t i o n s to p r o m o t e an a d d i t i o n a l l a r v a l molt.
277
strongly i n c r e a s e d but n o r m a l m e t a m o r p h o s i s is yet possible. It should be o b s e r v e d that the p e r i o d of ,treatment is i n c l u d e d , in the f o r m e r case, but not e n t i r e l y in the later one, in t h e so-called <
c~
cn
-30 ~.
? ~ o
2 ¸
i.
FI6. 2.
o 2
3
4
5
-
-
10
ays of 1
20
6
7
8
9
10
Circles for body weight, squares for silk glands weight and triangles for silk glands/ body weight ratio. O.pen signs for controls. Closed s.igns for t~eated silkworms.
5 th i n s t a r
11
12
On the c o n t r a r y , the early (type A) t r e a t m e n t seems only to result in a delay in the d e v e l o p m e n t p r o g r a m since s p i n n i n g then p u p a t i o n o c c u r norm a l y but later t h a n in c o n t r o l animals. W h e n s y n t h e t i c J.H. is given a c c o r d i n g to type B treatment, t h e d u r a t i o n of the fifth instar is m o r e
Effects of type a treatment on larvae and silk gland growth.
1'3
both e x p e r i m e n t a l situations, the la.rval life extension is a c c o m p a n i e d by a d d i t i o n a l g r o w t h . So, J.H. c o u l d act by i n c r e a s i n g the o b l i g a t o r y f e e d i n g phase, i n v o l v i n g ei,ther h y p e r m e t a b o l i s m as desc r i b e d in Dermestes [10] or s w e l l i n g as seen in Pieris [11] like in B o m b y x . Our results seem fur.
TABLE II.
W e i g h t (1) of posterior and m i d d l e silk glands after various types of treatment. Time ol treatment (2) Development aiter the |st meal
-- 2 hrs (i applicatiou)
--2 hrs and + 24 hrs +[,8 hrs and+72 hrs (2 applications) (2 applications)
Posterior
3 days
65
52
75
Silk Gland
5 days
100
81
53
3 days
42
34
72
5 days
55
34
31
Middle Silk Gland
(1} Per cent of controls. (2) Time zero corresponds to the first meal, so that the applications of - - 2 , + 24 et + 48 hours were done 2 hrs before and one or two days after the first meal. J. H. dose applied was 1 ~g/g body weight.
BIOCHIM1E, 19'/9, 61, n ° 2.
19
J. Daillie.
27'8
ther i n agreement w i t h the hypothesis w h e r e b y a high level of J'.H. w o u l d i n h i b i t the secretion of the b r a i n ( p r o t h o r a c o t r o p i c ) h o r m o n e as expected by Nijhout a n d W i l l i a m s [12]. II. EFFECTS OF J.H. APPLICATION ON SILK GLAND GROWTH AND SILK PRODUCTION.
Results (tables I, II, III and IV ; figure 2). The silk gland growth kinetics was only studied i n the case of p r o c e d u r e A (fig. 1). D u r i n g the t r e a t m e n t p e r i o d and the following two days, the silk glands were very slighty g r o w i n g up, t h e i r development b e i n g therefore c l e a r l y different from the total larvae gro~vth (fig. 2). However, this d i s c r e p a n c y quickly recovered t h e n the glands grew bigger t h a n i n controls. F i n a l y treated w o r m s p r o d u c e d more silk as s h o w n in table I : the A-treated larvae p r o d u c e d cocoon shells heavier by 30 to 50 per cent t h a n those of n o r m a l silkworms. C o n c e r n i n g the other conditions, only the B-treated females capable of spinn i n g made shells heavier (20 per cent more) than u n t r e a t e d i n d i v i d u a l s . The weight of the posterior silk glands (table I) at the end of the feeding period w a s b r o a d l y keeping w i t h the changes observed for larvae a n d shell 'weight, d e p e n d i n g on the types of treatment. A d d i t i o n a l data s h o w n in table II f u r t h e r indicate that a single dose applied just before the first meal was yet effective three days later. It was only on day 5 that posterior siLkgland r e s u m e d its delay of growth. An a d d i t i o n a l applica,tion after one d a y of development p r o l o n g e d the growth in-
h i b i t i o n . Moreover, t r e a t m e n t a d m i n i s t e r e d in tile course of growth phase (on days 2 and 3) resulted clearly i n s i m i l a r effects. It should also be noted that in all cases we observed an accentuated relative decrease of weight i n the m i d d l e p o r t i o n of the gland. This r e i n f o r c e d effect was p r o b a b l y due to the i n h i b i t i o n of siLk p r o t e i n synthesis w h i c h failed of course to accumulate in the reservoir. Since the p e r i o d of earlier t r e a t m e n t s c o i n c i d e with the phase of DNA synthesis in controls and with the b e g i n n i n g of RNA a n d p r o t e i n a c c u m u lation, it w a s i n t e r e s t i n g to look at the effects of J.H. analogue on such m a c r o m o l e c u l a r events. Using pulse labelling e x p e r i m e n t s after a type A-treatment [6], we o b t a i n e d the following results (table III) : m a x i m u m i n c o r p o r a t i o n of glycine, a p p e a r i n g mostly i n siLk fibroin [13], w h i c h occ u r r e d by days 4-5 i n controls, was delayed by two days and r e m a i n e d at a high rate for six more days i n posterior silk glands of treated silkworms. C o n c e r n i n g n u c l e i c a,cids syntheses, m a x i m u m inc o r p o r a t i o n arised on day 3 i n c o n t r o l larvae for t h y m i d i n e as well as for u r i d i n e w h i l e in treated silkworms, we noted that the labelling p a t t e r n was modified : a high level of i n c o r p o r a t i o n lasted from day 3 up to day 7. I n the case of t h y m i d i n e , even two peaks were clearly seen (the n o r m a l one on day 3, a n o t h e r on day 7) to w h i c h c o r r e s p o n d ,changes of t h y i n i dine k i n a s e a n d DNA p o l y m e r a s e activities [6, 14]. However, DNA a c c u m u l a t i o n (table III) measured i n the course of the experiment, i n d i c a t e d that the final a m o u n t of DNA was the same i n silk
TABLE III.
DNA content and incorporation of [SHl thffnIidine, [SHl uridine and [l~Cl glgcine in posterior silk gland of treated and control silkworms. [3H] thymidine [aH] uridine dpm(X t0~}/PSG dpm (× t03)/PSG Days of ~st instar 1 3 5 7 9
C 360 790 147 100 87
T 380 590 513 866 141
C 320 852 255 170 115
T 400 675 670 705 245
[taG] glyeine dpm (X t0a)/PSG C
T
2 110 230 192 133
2 20 75 196 210
DNA content p.g/PSG C
T
28 85 95 104 110
26 61 81 92 102
11
--
112
--
200
--
231
--
112
13
--
140
--
213
--
200
--
105
Silk glands of silkworms treated according to procedure A (fig. 1) were excised at indicated days and incubated as previously described [20, 21] with [3H] thymidirre or [3H] uridirm (1 IxGi/m~) pins [14C] glyoine (0.2 ixGi/ml). Aeido-insoluble ma~eri,al was then counted. DNA was also quantified in the course of experiment.
BIOCHIMIE, 1979, 61, n ° 2.
Effect of juvenile hormone.
279
glands of both treated a n d u n t r e a t e d i n d i v i d u a l s . Therefore, J.H. applica,tion seems to d e t e r m i n e a n extension of the d u r a t i o n of the two r e p l i c a t i o n cycles w h i c h take place at the b e g i n n i n g of the fifth i n s t a r [15].
a p p e a r e d very slowly : in the case of experim e n t II, for instance, I~NA synthesis (or accumulation) d u r i n g the 48-72 h p e r i o d was r e d u c e d b y 70 per cent of the control but only 50 per cent d u r i n g the previous 24 hours.
C o n c e r n i n g RNA a c c u m u l a t i o n , a c c o r d i n g to the a b o v e - m e n t i o n e d results of l a b e l l i n g expriments, we observed a r e t a r d a t i o n (three days) of the o c c u r r e n c e of the m a x i m u m RNA a m o u n t i n treated silk glands. I n fact, the t r e a t m e n t i n d u c e d a rather p a r a d o x i c a l effect ~ ' h i c h has to be noticed. At first, d u r i n g at least the time of ~reatment, a m a r k e d i n h i b i t i o n of RNA synthesis was occurring w h i c h then r e s u m e d a n d finaly the m a x i m u m a m o u n t f o u n d was about 50 per cent h i g h e r t h a n i n the controls. T y p i c a l curves of RNA accumulation in posterior silk glands of both treated a n d u n t r e a t e d a n i m a l s are s h o w n in PavCs p a p e r in this issue [16].
I n another e x p e r i m e n t (results not shown) we observed that in g r o w i n g silk g l a n d (day 4) the effect of a single J.H. a p p l i c a t i o n on RNA synthesis was detectable by pulse labelling on six h o u r s after the time of treatment.
Discussion. As we have already p o i n t e d out, the J.H. analogue, a,pplied in the first t h i r d of the fifth i n s t a r provokes an increase of both the larval life duration a n d the weight of larvae. The last p h e n o m e -
TABLE IV.
Inhibition of RNA accumulation in posterior silk gland after earlier treatments. Inhibition (per cent ol controls) at indicated times lollowing the first meal 2t, hrs I 1 application at indicated time
48 hrs
72 hrs
33
46 41
60 60
30
48
- - 1 hr 0 -~- 2 hrs
30 28 17
- - 1 hr -~- 6 hrs
45
It
1 application at indicated time III 3 applications
( t
- - 6 hrs -~- 4 hrs
+ 18 hrs
Results expressed in per cent of controls values. The dose of J.H. used for each applications was 1 ~tg/g body weight. We verified, in c o m p l e m e n t a r y e x p e r i m e n t s (table IV) that the previous i n h i b i t o r y effect ~¢as effective w h e n the analogue was applied at the time of the firs,t meal or even six hours later. In respect w i t h RNA a c c u m u l a t i o n rates in the controls, i n h i b i t i o n at 24, 48 a n d 72 hours after the first meal, was of 25-45, 4.1-48 a n d 60 per cent, respectively. Reiteration of the ~reatment (3 times) in the range of - - 6 to + 1 8 hours did not m o d i f y the results. Such data i n d i c a t e that, as for the silk gland growth, tile i n h i b i t o r y effect las.ted a long time after the J. H. analogue was applied a n d dis-
BIOCHIMIE, 1979, 61, n ° 2.
n o n is p r o b a b l y a consequence of the former, if we c o n s i d e r that the obligatory feeding phase is itself increased, Short t e r m and long term effects of J.H. t r e a t e m e n t on the silk gland development appear quite different since weight as well as RNA a n d p r o t e i n syntheses ( i n c l u d i n g silk proteins) are p r i m a r i l y i n h i b i t e d a n d later resume and amplify. Figure 2 clearly shows such diffe. rences but also indicates that the p r o p o r t i o n of th~ silkgland weight ( i n c l u d i n g silk secretion) in the total body weight is finaly the same in treated and u n t r e a t e d i n d i v i d u a l s . This fact suggests that
280
J. Dailtie.
all the p a r a m e t e r s except time, w h i c h d e t e r m i n e the relative g r o w t h of organs and tissues do not change u n d e r such h o r m o n a l c o n d i t i o n s . P a r t i c u larly, it seems that J.H. acts in a m p l i f y i n g a disc o r d a n c e in g r o w t h w h i c h n o r m a l y exists b e t w e e n silk glands a n d the other tissues of the larvae. W h e n J.H. is a p p l i e d along the s e c o n d t h i r d of the instar, the silk g l a n d d e v e l o p m e n t is stoped a n d delayed. It can be m o r e or less c o m p l e t e l y r e s t o r e d c o n s e q u e n t l y to an e n h a n c e d d u r a t i o n of the l a r v a l life. Even in the best cases, the silk p r o d u c t i o n is l o w e r than after an e a r l i e r t r e a t m e n t . C o n c e r n i n g s i l k w o r m s t r e a t e d a c c o r d i n g to the p r o c e d u r e C., too little i n f o r m a t i o n is k n o w n aboul silk gland. In such animals, w h i c h are unable to s p i n a n d to e n t e r t h e p u p a l stage, the w e i g h t of t h e p o s t e r i o r silk gland becomes l o w e r than in controls. This r e s u l t leads us to t h i n k that the gland stops fibroin synthesis. Two DNA replica.tion cycles o c c u r in t r e a t e d p o s t e r i o r silk gland as ~,ell as in n o r m a l one. In the former, J.H. a p p l i c a t i o n affects DNA s y n t h e s i s o n l y b y i n c r e a s i n g t h e time n e e d e d to c o m p l e t e the c o r r e s p o n d i n g DNA content and the onset of repli,cation does not seem d e l a y e d . T h e r e f o r e the two DNA s y n t h e s i s cycles w h i c h are o v e r l a p p e d in c o n t r o l l a r v a e [15] b e c o m e m o r e d i s t i n c t follow i n g an e a r l y a p p l i c a t i o n of the J.H. analogue. Such a result r e i n f o r c e s the feeling that the degree of p o l y p l o i d y in silk g l a n d cell nuclei is m a i n l y d e p e n d i n g on genetic c h a r a c t e r i s t i c s and c a n n o t be easily m o d i f y b y any k i n d of factors [17, 187. It has been c l a i m e d , in some cases, that J.H. acts first b y i n c r e a s i n g DNA s y n t h e s i s ; in fact, such effects w e r e only c l e a r l y o b s e r v e d in fat b o d y cells of Locusta [19]. As DNA a m o u n t r e m a i n s b r o a d l y u n c h a n g e d a n d as t r a n s c r i p t i o n rate a p p e a r s r a t h e r constant in e u k a r y o t e s , t h e question a r i s e s h o w a J.H. treatmen.t m a y i n d u c e an i n c r e a s e of the m a x i m u m RNA amount a c c u m u l a t e d in silk gland. In fact, w h e n t r e a t m e n t starts on the onset of the fifth instar, RNA a c c u m u l a t i o n is first d r a s t i c a l l y inhibited. The length of the i n h i b i t i o n p h a s e d e p e n d s on the n u m b e r of a p p l i c a t i o n s , but a single one is sufficient to o c c u r a severe d e l a y in RNA synthesis. Even a t r e a t m e n t a d m i n i s t e r e d after the b e g i n n i n g of s y n t h e s i s has been s h o w n effective. One can t h e r e f o r e c o n c l u d e that one of the p r i m a r i l y effects of t h e J.H. t r e a t m e n t is to i m p r o v e the i n i t i a t i o n of RNA t r a n s c r i p t i o n at a n o r m a l rate. T h e r e is m o r e o v e r a n ~ r r o w c o r r e l a t i o n b e t w e e n the RNA s y n t h e s i s i n h i b i t i o n and the d e l a y observed in silk gland g r o w t h and silk p r o t e i n secretion.
BlfOCHIMIE, 1979, 61, n ° 2.
After b e i n g i n h i b i t e d and delayed, RNA c o n t e n t i n c r e a s e s a n d r e a c h e s a h i g h e r level in treated t h a n in c o n t r o l larvae, as for silk g l a n d growth. Using a m a t h e m a t i c a l m o d e l to s t u d y the quantitative changes of RNA, Pav6, in this issue [16], finds that the d e g r a d a t i v e p a r a m e t e r of the d y n a mic a c c u m u l a t i o n p r o c e s s is m o s t l y m o d i f i e d in t r e a t e d silk glands w i t h r e s p e c t to the c o n t r o l ~nes. He thus h y p o t h e t i z e s that the e n h a n c e m e n t ~f final a m o u n t of RNA f o l l o w i n g the J.H. application to be d u e to a d e c r e a s e of t u r n o v e r r a t h e r of s y n t h e s i s processes. Such a h y p o t h e s i s h a s of course to be v e r i f i e d but seems a l r e a d y interesting to c o n s i d e r : since silk gland d e v e l o p m e n t was a little m o r e d e l a y e d that RNA synthesis, it seems p o s s i b l e that the d e l a y in g r o w t h involves a c o r r e s p o n d i n g late in t h e a p p e a r e n c e of nuclease activities. An e a r l y J.H. t r e a t m e n t i n d u c e s a r e t a r d a t i o n then an i n c r e a s e of the silk p r o t e i n s synthesis. This o b s e r v a t i o n is quite in a g r e e m e n t w i t h the p r e v i o u s p o i n t s of the discussion, since o t h e r studies c l e a r l y i n d i c a t e that the fibroin p r o d u c t i o n is e n t i r e l y d e p e n d e n t of RNA a c c u m u l a t i o n [17, 181.
Conclusion.
The J'.H. analogue ZR 515 i n d u c e s v a r i o u s changes in Bombyx mort development, d e p e n d i n g on the p e r i o d s of t r e a t m e n t . Moreover, it acts d i f f e r e n t l y u p o n silk gland and other tissues. The most i n t e r e s t i n g effects a p p e a r w h e n the analogue is a p p l i e d at the b e g i n n i n g of the fifth l a r v a l instar. D u r i n g this p e r i o d , a decrease of n a t u r a l h o r m o n e o c c u r s in n o r m a l larvae. Thus our results seem to be a c o n s e q u e n c e of a p e r s i s t e n t h i g h level of h o r m o n e o w i n g to analogue a p p l i c a tions. The i n h i b i t i o n of RNA s y n t h e s i s in silk gland as a s h o r t t e r m effect of t r e a t m e n t s m a y a l l o w us of u n d e r s t a n d i n g the m e c h a n i s m s of changes in transcrip.tion a c t i v i t y d u r i n g the molting p e r i o d and the e a r l y feeding phase. Acknowledgements.
I would like to thank G. Deeaussin for her technical assista.nce ; G. B,osquet, B. Gal~ez, P. Coalble and J.-C. Prudhomme for their critical advising. REFERENCES. l. Akai, H. & Kobayashi, M. (1971) Appl. Ent. Zool. (Japan), 6, 138-139. ~. Akai, H., Kiguehi, K. & Mort, K. (1971) Appl. Ent, Zool. (Japan), 6, 218-220,
Effect of juvenile hormone. 3. Murakoshi, S., Chang, C. F. a Tamura, S. (1972) Agr. Biol. Chem. (Japan), 36, 692-694. 4. Akai, H., Kiguchi, K. ~ Mori, K. (1973) Bull. Sericul. Exp. Sta. (Tokyo)., 25, 287-305. 5. Nihmura, M., Aomori, S., Mori, K. • Matsui, M. (1972) Agr. Biol. Chem. (Japan), 36, 889-8,92. 6. Kurata, K. & Daillie, J. (19,78) Bull. Sericul. Exp. Sta., 27, u n d e r press. 7. Slama, K., ~ o m a n u k , M. a Sorm, F. (19'74) ¢ Insect h o r m o n e s and Bioanalogues >>, Springer Verlag, Wien, New-York, 447 pp. 8. Calvez, B., I-Iirn, M. a De Reggi, M. (1976)FEBS Letters, 71, 5%61. 9. Legay, J.-M. (1966) La prise de nourriture chez le V e t ~ sole. Thesis, Paris. 10. Sblama, M. ~ H~odkov,a, M. (1'975) Biol. Bull., 148, 320332. 11. Srihari, T. ~ G~ahukar, R. T. (197'5) Bull. Soc. Zool. Fr., 100, 327-3~,3.
BIOCHIMIE, 197@, 61, n ° 2.
281
I2. Nijhout, H. F. ~ Williams, G. M. (1974) J. Exp. Biol., 61, 493-501. 13. P r u d h o m m e , J.-C., Sridhara, S. & Daillie, J. (1973) Arch. Biochem. Biophys., 159, 97-104. 14. De Turenne, M. (19q:8) La synth$se du DNA dans les glandes sdricig~nes de Bombyx morl au dernier stade larvaire. Thesis, Lyon. 15. Perdrix, S. (1979) Biochimie, 61, this iss,ue. 16. Pave, A.. (1979) Biochimie, 61, this issue. 17. Shigematsu, H., Kurata, K. & Takeshita, H. (1978) Comp. Bioehem. Physiol., 61 B, 237-242. 18. P r u d h o m m e , J.-C. & Couble, P. (1979) Biochimie, 61, this issue. 19. Chen, T., Couble, P., Ahuhakima, R. & Wyatt, G. R. (1979') Deo. Biol. (under press). 20. De Turenne, M. & Daillie, J. (1969) Biochim. Biophys. Acta, 186, 267-2'79. 21. Couble, P., P r u d h o m m e , J.-C. & Dail.lie, J. (1977) Exp. Cell Res., 109, 139-150.