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Progress of developmental programme during the last larval instar of Bombyx mori: Relationships with food intake, ecdysteroids and juvenile hormone
J. lnsecf Physiol., Vol. 27, No. 4,pp. 233-239, 1981 Primed in Greaf Brifain.
0022-1910/81/040233-07$02.00/O 01981 Pergamon Press Ld
PROGRESS OF DEVELOPMENTAL PROGRAMME DURING THE LAST LARVAL INSTAR OF BUMBYX MORI: RELATIONSHIPS WITH FOOD INTAKE, ECDYSTEROIDS AND JUVENILE HORMONE BERNARD CALVEZ Liniversite Claude Bernard Lyon-l, Departement de Biologie Generale Associt au C.N.R.S. no. 243.43. Boulevard du 11 Novembre 1918.69622
et Appliquee. Laboratoire Villeurbanne Cedex, France
(Received14 July 1980:revised 27 Ocfoher 1980) Abstract---The progress of developmental programme in the epidermal cells of last instar larvae of Bomh~r mori was determined by ecdysteroid injections in normal and in JH-treated larvae. To clarify the importance of food intake in the control of development, starved animals were also used. The instar begins with a period during which the larval programme is expressed: this occurs in the presence of 20-hydroxyecdysone. Epidermal cells can thereafter secrete pupal cuticle after ecdysteroid injection although the larval programme is normally still present. During the last period only pupal characters can be expressed either in normal or in 20-hydroxyecdysone-injected larvae. These different developmental phases are not correlated with obligatory and facultative feeding periods. Transition from the first to the second phases is correlated with the absence of JH effects on pupal genes. JH applications during the second period, however, prevent the expression of pupal characters after 20hydroxyecdysone injection. Thus, during this period, the pupal programme is not stabilized. Cellular reprogramming itself occurs at the onset of the last developmental period and is probably under the control of ecdysteroids. Ker Word Index: Bomh~~\mori. development, juvenile hormone. ecdysones, feeding periods
INTRODUCTION
THE DEVELOPMENTof most animals is directly or indirectly controlled by hormonal mechanisms which can be partly regulated by exogenous factors like food intake. The effect of food intake is not limited only to the growth of the animal: it can act also as a developmental signal (WIGGLESWORTH, 1935; CLARKE and LANGLEY, 1963; COULON, 1977; BOSQUET, 1979). A larval instar in Lepidoptera can be divided into two periods related to food intake: the obligatory feeding period and the facultative feeding period dehned by BOUNHIOL(1938) and described in Bombvx mori by LEGAY (1955), in Galleria by WOOLEVERand PIPA (1970). If a larva is starved completely during the first period. growth is stopped and development terminates as the larva dies without moulting. On the other hand if the larva is starved completely during the second period, development continues and a moult occurs after a few days. Depending on the instar, the moult is larval or larvo-pupal. Transition from one feeding period to the next is accompanied by important metabolic changes: protein synthesis in the fat body (CALVEZ and FOURCHE. 1980). as well as haemolymph proteins (CALVEZ, 1977) are modified in Bombys mori. GULLET and FOURCHE (1980) have also observed mitochondrial modifications in Pieris. FOURCHE (1967a, b) concluded that, in Drosophila, imposed starvation during the obligatory feeding period prevented the production of ecdysone in the larva but he noticed also that food intake was
important for the larva to react in the presence of ecdysone. Furthermore, NIJHOG’T(1976) in Manduca, HWANG Hsu et al. (1979) in Galleria have demonstrated that an ecdysone injection during the first days of the last larval instar induced the synthesis of new larval cuticle. If the injection was made later, the new cuticle was pupal. According to these authors, these results show that the change in developmental programme in the epidermal cells probably occurs before circulating ecdysteroids normally appear at a high level in the haemolymph while the larva is still feeding. The purpose of this work was to determine whether correlations exist between the transition from the obligatory to the facultative feeding periods and the change in developmental programme. It was also hoped to clarify whether these two periods depend upon hormonal changes.
MATERIALS
AND METHODS
1. E.xperimental procedures Several experiments were carried out (Fig. I) to study the relationships between feeding periods and reprogramming, and between reprogramming and hormonal variations during the last larval instar of to precise developmental Bombyx mori. Due synchronisation and to the prolonged 8-day feeding period of the insect (Fig. la), the different developmental periods could be determined easily. (a) Determination qf the duration of the obligator? 233
BERNARDCALVEZ
234
-----_
la)
feeding period 1 Spinmng
1 Pupal ecdysls L$ath
(b) -------_-----j
/L__
t
(c)
Pupal moult?
day I, 2,3,4 or5)
Starvohon
(at
LOrVal or I Pupal moult?
-----t 20- hydroxyecdysane Cotday I, 2, 3, 4 or 5)
(d)
(e)
-tJH-
_---_-b 1Spinning
+
1Pupal ecdysk Dealt-
_
-___-___-_____,
/_____-_
or
PUpal m0Jlt? t
storvatlon
LIXMl
_-----
(f)
Cf I Pupal mxxlll?
2O-hydroxet.d! ysone
Death _--------
(g)
f
/-----
t Starvatbon
OP:pa1 -It?
LOWOI th)
____
ZO-hydroxecdyxxle t
Or
I Pupa1mcult 7
**JH.
(i)
._
--_-_ jSpinnin(l
___-----
(1)
-t Pup01 ecdysls
1’
t Starvation
_--_
(k) ZO- hydroxyecdyscnet
I 4th larval ecdysis
Fig. 1. Diagrammatic representation of experimental procedures. Criteria to be observed at the end of each procedure are given (death, larval moult or pupal moult). ~ feeding period; ----natural or imposed starvation period; period of JH, topical applications. and facultative feeding periods. Larvae were starved during the fifth instar after feeding periods of a variable length (Fig. lb). According to BOUNHIOL (1938) and LEGAY (1955) only two results were possible: the starvation period would lead either to death of the larva, or to a normal larval-pupal moult. Two parameters chosen for these experiments were the time elapsed between beginning of starvation and pupal ecdysis, and the percentage of larvae moulting into pupae compared with the duration of the previous feeding period. (b) Determination of a possible relationship between the level of ecdysteroids and the change in feeding
periods. During the last larval instar, measurements made every 24 hr showed that ecdysteroids appeared at a high level only after the animal had normally finished eating, at the onset of spinning (CALVEZ et al., 1976). However, the time between each measurement was too long to assess important and fast hormone variations during the feeding period, and individual measurements were made every 3 hr in groups of 4-5 larvae. (c) Determination of the time of expression of the pupalprogramme after an ecdysteroid injection. It was possible to induce a moult using 20-hydroxyecdysone injections. The cuticle morphology revealed the way in
Developmental programme of Bornb~.~ marl which the developmental programme could be expressed in the presence of ecdysteroid at the time of injection: it could be larval or pupal. (d) The effect of juvenile hormone on the duration of the obligatory andfacultativefeedingperiods and on the progress of the developmental programme. This objective was achieved by two complementary experiments. (i) In providing exogenous juvenile hormone from the time of the fourth ecdysis to the fifth day of the last larval instar, it was hoped to prolong the high endogenous JH phase which characterized the beginning of the instar (HsIAoand HSIAO, 1977; BAEHR et al., 1978; MAUCHAMP~~al., 1979; RIDDIFORD, 1980). Following this treatment, several experimental procedures were attempted including: continuation of normal feeding (Id); complete starvation (le); ecdysteroid injection f 1D: continuation of feeding until day 7.5 and starvation imposed (lg): or ecdysteroid injected (1 h). (ii) The second experiment consisted of artificially increasing the level of endogenous juvenile hormone at a period during which this level normally was very low. Thus, the treatment started on the fifth day after ecdysis and continued until day 7.5. At the end of the treatment. the larvae were fed normally (li), were starved (lj, or were injected with ecdysteroid (lk). In each case, the groups included 15-20 larvae of both sexes and experiments were repeated two to three times.
Bombyx mori larvae of the European strain 200 x 300 were reared according to procedures described earlier (CALVEZ et al., 1976). During the last larval instar, the larva feeds for 8 days, after which spinning begins. Larvae pupate about 5 days thereafter. Thus, the total duration from the last larval ecdysis to pupal ecdysis is 13 days (Fig. la). 3. Chemical.9 20-Hydroxyecdysone was purchased from RohtoPharmaceutical, Osaka, Japan. For ecdysteroid radioimmunoassay, antibodies and radioactive 20hydroxyecdysone were kindly supplied by Dr. DE REGGI, C.N.R.S. Marseille, France. JH, was a gift graciously obtained from Procida, Marseille, France. 4. General meth0d.s (al ZO-H~~dro.~.vecdy.rone injections. The ecdysteroid Table
35 -
\ \ \ \
$, E xtrapolakn
1 \ \ \.
25 r 8 B El
15 -
\
. \
Injections* (Time in hr) Age of larva < 3 days Age of larva 2 3 days
*The hormone concentration of
into last instar 1st (01
2nd (3)
3rd (6)
4th (12)
1.0
3.5
3.5
2.0
I
I
2.0
6.0
in Ringer’s
6.0
solution
5.0
to a
l\._ . I 8
I
4
2
6
Feedmg period,
days
Fig. 2. Influence of the duration ofprevious feeding period on the delay between beginning of starvation and pupal ecdysis.
peak which appears normally at the end of the last larval instar (CALVEZ et a/., 1976) was produced by 4 injections of 20-hydroxyecdysone at regular intervals into the prolegs as indicated in Table 1. The larvae were starved immediately after the first injection. Larvae apolysed and deposited a new cuticle within 60 hr of the last injection, but they did not ecdyse. The nature of the newly-deposited cuticle was examined by peeling away the old cuticle. (b) Juvenile hormone treatment. JH, was topically applied along the dorsal vessel of the abdomen. Preliminary experiments have shown that oil solutions are harmful to the insects: the hormone should be diluted in pure acetone. If this treatment is applied only once at a very high dose (30 y/larval, just after ecdysis, or if daily applications are made for 5 days (1 lj/g fresh weight), normal developmental duration is not changed. It is likely that JH,, one of the natural juvenile hormones in the insect, is recognized by specific esterases and metabolized more rapidly than mimetics used in other works (RI~DIFORD, 1972; BENSKINand VINSON, 1973: SRIHARIand GAHUKAR, 1975; GELBIC and NEMEC, 1978; CYMBOROWSKIand STOLARZ, 1979). It was thus necessary to repeat
Table 2. Influence of age of the larva on morphological features of the newly secreted cuticle after 20hydroxyecdysone injection Days
I
?
100
100
Larval-pupal
0
Pupal
0
Morphology of cuticle
Larval*
was dissolved
I y/fil.
larvae
l\
5-
I. Experimental procedures for 20-hydroxyecdysone injections
Dose y/larva
235
3
4
5
2s
0
(I
0
70
0
0
0
5
100
100
*Animals which present larval cuticle wing discs are scored in ‘larval’ moult. Data are expressed in percentage.
with evaginated
236
BERNARDCALVEZ of both results was obtained. The number of pupae increased with the duration of the feeding period. Thus, in Bombyx mori, the average duration of the obligatory feeding period was 4 days.
2.Dehy between beginning starvation andpupalecdysis
Age,
For those starved animals which could pupate, the duration of the instar was never shortened: the earlier the animals were starved, the longer was the last instar. Indeed, there was an important delay between the beginning of starvation and pupal ecdysis (Fig. 2). Moreover, among the groups fed for 3 and 4 days which resulted in death or a moult, the delay corresponded to a critical period in starvation resistance, since all starved larvae that survived this delay pupated.
days
Fig. 3. Ecdysteroid changes in the haemolymph of Bomb~x mori larvae during the few first days of last larval instar. The day ‘zero’ corresponds to larval ecdysis. Standard error of the mean is given for each point. topical applications (1 v/g fresh weight) every 8 hr, during the whole treatment. (c) E&steroid content. Ecdysteroid content was determined in haemolymph by the RIA method of DE REGGI et al. (1975).
RESULTS 1.Duration of the obligatory and facultative feeding periodr The duration of the obligatory and facultative feeding periods previously determined in Bombyx mori by LECAY (1955) was examined again under the conditions of this experiment. When larvae were starved after a 2-day feeding period, all died without pupating. On the other hand, when larvae were starved after at least a S-day feeding period, all moulted into pupae. In the case of intermediate feeding periods (3 and 4 days), a mixture
3. EC&steroid injection and age of larva After the larvae were injected with 20-hydroxyecdysone and immediately starved, they all moulted (Fig. lc). Within 60 hr following the last injection, the animals could be separated into three groups according to the morphology of cuticle: larvae, pupae and larval-pupal intermediates. A larval cuticle and the presence of prolegs were the criteria retained at larval moults. Animals which presented a larval cuticle with everted wing discs were also scored as a larval moult. The pupal moult was characterized by a tanned cuticle without prolegs. Larval-pupal intermediates presented a mosaic of the two types of cuticle. The proportions of larvae, pupae and intermediates varied with the previous feeding periods (Table 2). When 20-hydroxyecdysone was injected after a 1- or 2-day feeding period, all animals secreted larval cuticle. When the hormone was injected at day 3, larvae, pupae and larval-pupal intermediates were observed. When the hormone was injected after a 4day feeding period. all the animals secreted a pupal cuticle. The developmental programme could thus express pupal characters after only a 4-day feeding
Larval ecdysis
Characters
Pupal ecdysis
Larval
normally
expressed
Characters expressed after 20- hydroxyecdysone injection
Larvol
-------
Pu pal ----------
J
7-I Hormonal variations
Days
Fig. 4. Summary of progress of developmental programme in last instar larvae of Bomb_v.umot?.
Developmental programme of Bomhr.~ mori
237
feeding period to the next was not accompanied by a change in the basic level of ecdysteroids or by a short, sudden increase in their level of haemolymph. Thus, 4. Ecdysteroid content of the haemolymph offed larvae the existence of both feeding periods could not be related to the presence of circulating ecdysteroids. Results of ecdysteroid measurements (Fig. 3) Two results, however, suggested the involvement of indicate that, except for larvae at ecdysis, circulating juvenile hormone in the control of both feeding ecdysteroid levels were low. No peak was detected. periods. Two important phenomena occurred between 5. Ejfects qfjuvenile hormone treatment the fourth and fifth days of the last instar in untreated (a) Duration of last larval instar. Larvae treated with larvae: at day 4 all larvae injected with 20JH 1 from the ecdysis to the fifth day, fed for 13 days. hydroxyecdysone moulted into pupae and at day 5 all The duration of the feeding period increased by 60”/ went through the facultative feeding period. Thus, it compared with that of untreated larvae. seemed as if a larva could only go through the (b) Duration of the obligatory andfacultativefeeding facultative feeding period when the pupal programme periods. When larvae were treated with JH, from was expressed completely, i.e. when the endogenous ecdysis to the fifth day and starved immediately juvenile hormone had no effect on pupal genes. The afterwards (treatment le) SO-950/, died without duration of the obligatory feeding period could be moulting (100”; of the untreated larvae could pupate prolonged on average by topical applications of JH,, under these conditions-see paragraph 1). When However, in all day-5 larvae treated with juvenile larvae were starved 2.5 days after the end of JH hormone, the larval programme alone was expressed treatment (Ig), only 40-50:; could pupate. On in the presence of 20-hydroxyecdysone. Nevertheless, average, JH, increased the duration of obligatory a few larvae went through the facultative feeding feeding period. period. Though the juvenile hormone still had an Five to twenty per cent of the larvae treated with effect on the developmental programme. the larva had JH,, however, pupated about 17 days after being reached the threshold of the facultative feeding period. starved from the fifth day. The presence of hormone at Thus, juvenile hormone did not control the transition a high level did not prevent these larvae from going from the obligatory to the facultative feeding period. through the facultative feeding period. Since ecdysteroids did not control this transition When the JH, treatment began on the fifth day of either. progress of developmental programme could the feeding period (this day normally occurred during no longer be considered as a preliminary to the the facultative feeding period) and lasted 2.5 days transition from the first to the second feeding period. (Fig. 1i), the total duration of feeding was prolonged: The definition of feeding periods was based on the spinning started on day 11. observation of what happened to the starved larvae: When starved at the end of the JH, treatment death or moult. It was then obvious that the effects of (Fig. Ij) all larvae started spinning 6 days later and starvation were shown by a response of the all-or-none then pupated. Although the larvae were treated with principle which suggested that transition from one JH!, they still went through the facultative feeding feeding period to another corresponded to a period. developmental commitment. In reality, another (cl l@cts qf‘injection of 20-hydroxyecdysone. When important parameter had to be considered: the delay 20-hydroxyecdysone was injected at day 5 into larvae after which death or a moult occurred after starvation. treated with JH, from 0 to day 5, all larvae underwent The previous results showed that the earlier the a complete larval moult. When the injection was made larvae were starved during the last larval instar, the 2.5 days after the end of treatment (Fig. lh), the later they moulted. Moreover, in each group, larvae animals secreted larval cuticle with evaginated wing which resisted starvation the longest, were able to discs. LJnder these conditions, JH, prolonged the moult: the others died before moulting. Since there period in all larvae during which the larval programme was no developmental commitment at the origin of could be expressed. transition from one feeding period to another, this led When larvae treated with JH, from day 5 to day 7.5 to the conclusion that energy reserves of the larva at (Fig. 1k) were injected with 20-hydroxyecdysone, no the beginning of starvation alone was a critical factor. typical pupal moult occurred, only wing discs Besides, LEGAY (1955) in B0mby.u mori and NIJH~~JT evaginated. In these 7.5-day-old larvae treated with (1975) in Manduca sexta have demonstrated prevJH, the developmental programme expressed larval iously that the pupal moult was not possible if characters, although ecdysteroid injection revealed a larvae had not reached a critical size. Thus, if the pupal programme in 5-day-old larvae, i.e. just before energy reserves of a Bombyx mori larva previously fed JH treatment. Thus in 5-day-old larvae the pupal for 2 days during the last larval instar were sufficient to programme was not stabilized. resist a 33-day starvation period (Fig. 21 it could pupate. This hypothesis implied that. while starvation was imposed even during the obligatory feeding period, larval development as well as all physiological DISCUSSION functions (BOSQUET. 1979) continued slowly, but did I Feeding period.? not cease. Could the transition from the obligatory to the 2. Development of the developmentalprogramme during facultative feeding periods correspond to important the last larval instar changes in hormonal secretions such as variations in NIJHOLX ( 19751 in Manduca, KUMARAN ( 1978) and ecdysteroids and juvenile hormone? Data reported in HWANG Hsc et al. (1979) in Galleria demonstrated this study demonstrated that the transition from one period while these characters normally appeared later, at the end of the instar.
much
238
BERNARDCALVEZ
that, during the last larval instar, the epidermal cells could synthesize pupal cuticle in the presence of ecdysteroids. This ability to synthesize pupa1 cuticle was normally revealed only at the end of the last larval instar. Data from present work are consistent with these results. Also, they show that it is necessary to distinguish between 3 phases during the last stage (Fig. 41. The instar began with a period during which the larval programme was expressed. This programme was the only one to be expressed if ecdysone reached a high level in the haemolymph. The duration of this phase depended upon the organ considered. For wing discs, they evaginated 2448 hr following an injection of 20-hydroxyecdysone after only 2 days of feeding. For the rest of the body larval cuticle could be obtained after hormone injection during the first 3 days. This phase was followed by a period during which the pupal programme itself could be expressed but only in the presence of 20-hydroxyecdysone. In untreated larvae, the larval programme was still being expressed and the morphology did not change: the larval endocuticle was still secreted (BARBIER, 19711. Thus cellular reprogramming did not take place normally during this period: it could be induced only by the injection of 20-hydroxyecdysone. Some authors suggested that the first small peak of ecdysone which was noted previously in Manduca (BOLLENBACHERet al., 1975; WIELGUS et al.. 1979). in Pieris (LAFONT et al., 1977) at the end of feeding just before the very high ecdysteroid peak, could play an important part in the onset of the developmental programme and was responsible for the ability to secrete pupa1 characters in presence of ecdysone. In Galleria, however, HWANC Hsu et al. (1979) observed that some larvae were capable of secreting pupa1 cuticle at a stage before the appearance of the first ecdysone peak. In Bomby.u mori, the result was all the more significant that the pupa1 programme could be revealed 4 days before the onset of the ecdysone peak. Furthermore, measurements confirmed that the ability to express pupa1 characters did not appear in the presence of circulating ecdysteroids. However, it is clear that the first phase was closely related to the presence of juvenile hormone. The transition from the first to the second phase occurred while this hormone had no further effects on pupal genes. JH thus prolonged the period during which the larval programme alone could be expressed and it prevented the pupal programme from being expressed in the presence of 20-hydroxyecdysone. Moreover, HWANC Hsu et al. (1979) have pointed out that epidermal cell ability to express pupal characters occurs just after the increase in haemolymph JH esterase activity. These results showed the importance of the juvenile hormone .statusquo effects (RIDDIFORD, 19801 which prevent the transition from one developmental state to another. During the second phase, if both programmes can be expressed in the larva, the pupal programme is not stabilized since the presence of JH alone is enough to prevent its expression. RIDDIFORD (1972, 1980), NIJHOUT (1976), CYMBOROWSKIand STOLARZ (1979), HWANG Hsu et al. (1979) showed that at the end of the last instar the larvae became insensitive to exogenous JH. Thus another factor is necessary for the
stabilization of the pupal programme. In this case, the first ecdysone peak could play a part, as HWANG Hsu et al. (1979) suggest. This stage alone corresponds to true cellular reprogramming since only pupal characters can be expressed. During the second phase, modifications of synthesis are obtained only if 20hydroxyecdysone is injected into larvae. These data support the concept of MCCALEB and KUMARAN (1980): reprogramming of the insect larval genome occurs only on exposure to ecdysteroids in the absence of juvenile hormone. Acl;no&dgemcnt~I technical assistance.
thank JOCELYNE DEM~NT for her
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239
juvenile hormone esterase activity in Galleria me/lone/la larvae. J. Insect Physiol. 26, 171-177. NIIHOUT H. F. (1975) A threshold size for metamorphosis in the tobacco hornworm, MunducasextatL). Biol. Bull. mar. biol. Lab.. Woods Hole 149, 214-225. NIIHOUT H. F. (1976) The role of ecdysone in pupation of Munduca sexta. J. Insect Physiol. 22, 453463. RIDDIFOR5 L. M. (1972) Juvenile hormone in relation to the larval pupal transformation of the cecropia silkworm. Biol. Bull. mar. biol. Lab., Woods Hole 142. 310-325. RIDDIFORD L. M. (1980) Interaction of ecdysteroids and juvenile hormone in the regulation of larval growth and metamorphosis of the tobacco hornworm. In: Progress in Ecdysone Research (Ed. by HOFFMAN) ElsevieriNorthHolland, Amsterdam. SRIHARI T. and GAHUKAR R. T. (1975) The influence of juvenile hormone on food consumption and growth in the last instar larva of Pieris brassicue L. (Lepidoptera Pieridae). Bull. Sot. ~001. Fr. 100, 327-333. WIELGUS J. J., BOLLENBACHERW. E. and GILBERT L. I. (1979) Correlations between epidermal DNA synthesis and haemolymph ecdysteroid titre during the last larval instar of the tobacco hornworm Manducu selta. J. Insert Ph_v.riol. 25, 9-16. WIGGLESWORTH V. B. (1935) The physiology of ecdysis in Rhodniw prolixus (Hemiptera). II. Factors controlling moulting and ‘metamorphosis’. Q. Jl. microsc. Sci. 77, 191-222. W~C~LEVER P. and PIPA R. (1970) Spatial and feeding requirements for pupation of last instar larval Galleria mellonella (Lepidoptera). J. Imeet Physiol. 16. 25 l-262.
0022-1910/81/040233-07$02.00/O 01981 Pergamon Press Ld
PROGRESS OF DEVELOPMENTAL PROGRAMME DURING THE LAST LARVAL INSTAR OF BUMBYX MORI: RELATIONSHIPS WITH FOOD INTAKE, ECDYSTEROIDS AND JUVENILE HORMONE BERNARD CALVEZ Liniversite Claude Bernard Lyon-l, Departement de Biologie Generale Associt au C.N.R.S. no. 243.43. Boulevard du 11 Novembre 1918.69622
et Appliquee. Laboratoire Villeurbanne Cedex, France
(Received14 July 1980:revised 27 Ocfoher 1980) Abstract---The progress of developmental programme in the epidermal cells of last instar larvae of Bomh~r mori was determined by ecdysteroid injections in normal and in JH-treated larvae. To clarify the importance of food intake in the control of development, starved animals were also used. The instar begins with a period during which the larval programme is expressed: this occurs in the presence of 20-hydroxyecdysone. Epidermal cells can thereafter secrete pupal cuticle after ecdysteroid injection although the larval programme is normally still present. During the last period only pupal characters can be expressed either in normal or in 20-hydroxyecdysone-injected larvae. These different developmental phases are not correlated with obligatory and facultative feeding periods. Transition from the first to the second phases is correlated with the absence of JH effects on pupal genes. JH applications during the second period, however, prevent the expression of pupal characters after 20hydroxyecdysone injection. Thus, during this period, the pupal programme is not stabilized. Cellular reprogramming itself occurs at the onset of the last developmental period and is probably under the control of ecdysteroids. Ker Word Index: Bomh~~\mori. development, juvenile hormone. ecdysones, feeding periods
INTRODUCTION
THE DEVELOPMENTof most animals is directly or indirectly controlled by hormonal mechanisms which can be partly regulated by exogenous factors like food intake. The effect of food intake is not limited only to the growth of the animal: it can act also as a developmental signal (WIGGLESWORTH, 1935; CLARKE and LANGLEY, 1963; COULON, 1977; BOSQUET, 1979). A larval instar in Lepidoptera can be divided into two periods related to food intake: the obligatory feeding period and the facultative feeding period dehned by BOUNHIOL(1938) and described in Bombvx mori by LEGAY (1955), in Galleria by WOOLEVERand PIPA (1970). If a larva is starved completely during the first period. growth is stopped and development terminates as the larva dies without moulting. On the other hand if the larva is starved completely during the second period, development continues and a moult occurs after a few days. Depending on the instar, the moult is larval or larvo-pupal. Transition from one feeding period to the next is accompanied by important metabolic changes: protein synthesis in the fat body (CALVEZ and FOURCHE. 1980). as well as haemolymph proteins (CALVEZ, 1977) are modified in Bombys mori. GULLET and FOURCHE (1980) have also observed mitochondrial modifications in Pieris. FOURCHE (1967a, b) concluded that, in Drosophila, imposed starvation during the obligatory feeding period prevented the production of ecdysone in the larva but he noticed also that food intake was
important for the larva to react in the presence of ecdysone. Furthermore, NIJHOG’T(1976) in Manduca, HWANG Hsu et al. (1979) in Galleria have demonstrated that an ecdysone injection during the first days of the last larval instar induced the synthesis of new larval cuticle. If the injection was made later, the new cuticle was pupal. According to these authors, these results show that the change in developmental programme in the epidermal cells probably occurs before circulating ecdysteroids normally appear at a high level in the haemolymph while the larva is still feeding. The purpose of this work was to determine whether correlations exist between the transition from the obligatory to the facultative feeding periods and the change in developmental programme. It was also hoped to clarify whether these two periods depend upon hormonal changes.
MATERIALS
AND METHODS
1. E.xperimental procedures Several experiments were carried out (Fig. I) to study the relationships between feeding periods and reprogramming, and between reprogramming and hormonal variations during the last larval instar of to precise developmental Bombyx mori. Due synchronisation and to the prolonged 8-day feeding period of the insect (Fig. la), the different developmental periods could be determined easily. (a) Determination qf the duration of the obligator? 233
BERNARDCALVEZ
234
-----_
la)
feeding period 1 Spinmng
1 Pupal ecdysls L$ath
(b) -------_-----j
/L__
t
(c)
Pupal moult?
day I, 2,3,4 or5)
Starvohon
(at
LOrVal or I Pupal moult?
-----t 20- hydroxyecdysane Cotday I, 2, 3, 4 or 5)
(d)
(e)
-tJH-
_---_-b 1Spinning
+
1Pupal ecdysk Dealt-
_
-___-___-_____,
/_____-_
or
PUpal m0Jlt? t
storvatlon
LIXMl
_-----
(f)
Cf I Pupal mxxlll?
2O-hydroxet.d! ysone
Death _--------
(g)
f
/-----
t Starvatbon
OP:pa1 -It?
LOWOI th)
____
ZO-hydroxecdyxxle t
Or
I Pupa1mcult 7
**JH.
(i)
._
--_-_ jSpinnin(l
___-----
(1)
-t Pup01 ecdysls
1’
t Starvation
_--_
(k) ZO- hydroxyecdyscnet
I 4th larval ecdysis
Fig. 1. Diagrammatic representation of experimental procedures. Criteria to be observed at the end of each procedure are given (death, larval moult or pupal moult). ~ feeding period; ----natural or imposed starvation period; period of JH, topical applications. and facultative feeding periods. Larvae were starved during the fifth instar after feeding periods of a variable length (Fig. lb). According to BOUNHIOL (1938) and LEGAY (1955) only two results were possible: the starvation period would lead either to death of the larva, or to a normal larval-pupal moult. Two parameters chosen for these experiments were the time elapsed between beginning of starvation and pupal ecdysis, and the percentage of larvae moulting into pupae compared with the duration of the previous feeding period. (b) Determination of a possible relationship between the level of ecdysteroids and the change in feeding
periods. During the last larval instar, measurements made every 24 hr showed that ecdysteroids appeared at a high level only after the animal had normally finished eating, at the onset of spinning (CALVEZ et al., 1976). However, the time between each measurement was too long to assess important and fast hormone variations during the feeding period, and individual measurements were made every 3 hr in groups of 4-5 larvae. (c) Determination of the time of expression of the pupalprogramme after an ecdysteroid injection. It was possible to induce a moult using 20-hydroxyecdysone injections. The cuticle morphology revealed the way in
Developmental programme of Bornb~.~ marl which the developmental programme could be expressed in the presence of ecdysteroid at the time of injection: it could be larval or pupal. (d) The effect of juvenile hormone on the duration of the obligatory andfacultativefeedingperiods and on the progress of the developmental programme. This objective was achieved by two complementary experiments. (i) In providing exogenous juvenile hormone from the time of the fourth ecdysis to the fifth day of the last larval instar, it was hoped to prolong the high endogenous JH phase which characterized the beginning of the instar (HsIAoand HSIAO, 1977; BAEHR et al., 1978; MAUCHAMP~~al., 1979; RIDDIFORD, 1980). Following this treatment, several experimental procedures were attempted including: continuation of normal feeding (Id); complete starvation (le); ecdysteroid injection f 1D: continuation of feeding until day 7.5 and starvation imposed (lg): or ecdysteroid injected (1 h). (ii) The second experiment consisted of artificially increasing the level of endogenous juvenile hormone at a period during which this level normally was very low. Thus, the treatment started on the fifth day after ecdysis and continued until day 7.5. At the end of the treatment. the larvae were fed normally (li), were starved (lj, or were injected with ecdysteroid (lk). In each case, the groups included 15-20 larvae of both sexes and experiments were repeated two to three times.
Bombyx mori larvae of the European strain 200 x 300 were reared according to procedures described earlier (CALVEZ et al., 1976). During the last larval instar, the larva feeds for 8 days, after which spinning begins. Larvae pupate about 5 days thereafter. Thus, the total duration from the last larval ecdysis to pupal ecdysis is 13 days (Fig. la). 3. Chemical.9 20-Hydroxyecdysone was purchased from RohtoPharmaceutical, Osaka, Japan. For ecdysteroid radioimmunoassay, antibodies and radioactive 20hydroxyecdysone were kindly supplied by Dr. DE REGGI, C.N.R.S. Marseille, France. JH, was a gift graciously obtained from Procida, Marseille, France. 4. General meth0d.s (al ZO-H~~dro.~.vecdy.rone injections. The ecdysteroid Table
35 -
\ \ \ \
$, E xtrapolakn
1 \ \ \.
25 r 8 B El
15 -
\
. \
Injections* (Time in hr) Age of larva < 3 days Age of larva 2 3 days
*The hormone concentration of
into last instar 1st (01
2nd (3)
3rd (6)
4th (12)
1.0
3.5
3.5
2.0
I
I
2.0
6.0
in Ringer’s
6.0
solution
5.0
to a
l\._ . I 8
I
4
2
6
Feedmg period,
days
Fig. 2. Influence of the duration ofprevious feeding period on the delay between beginning of starvation and pupal ecdysis.
peak which appears normally at the end of the last larval instar (CALVEZ et a/., 1976) was produced by 4 injections of 20-hydroxyecdysone at regular intervals into the prolegs as indicated in Table 1. The larvae were starved immediately after the first injection. Larvae apolysed and deposited a new cuticle within 60 hr of the last injection, but they did not ecdyse. The nature of the newly-deposited cuticle was examined by peeling away the old cuticle. (b) Juvenile hormone treatment. JH, was topically applied along the dorsal vessel of the abdomen. Preliminary experiments have shown that oil solutions are harmful to the insects: the hormone should be diluted in pure acetone. If this treatment is applied only once at a very high dose (30 y/larval, just after ecdysis, or if daily applications are made for 5 days (1 lj/g fresh weight), normal developmental duration is not changed. It is likely that JH,, one of the natural juvenile hormones in the insect, is recognized by specific esterases and metabolized more rapidly than mimetics used in other works (RI~DIFORD, 1972; BENSKINand VINSON, 1973: SRIHARIand GAHUKAR, 1975; GELBIC and NEMEC, 1978; CYMBOROWSKIand STOLARZ, 1979). It was thus necessary to repeat
Table 2. Influence of age of the larva on morphological features of the newly secreted cuticle after 20hydroxyecdysone injection Days
I
?
100
100
Larval-pupal
0
Pupal
0
Morphology of cuticle
Larval*
was dissolved
I y/fil.
larvae
l\
5-
I. Experimental procedures for 20-hydroxyecdysone injections
Dose y/larva
235
3
4
5
2s
0
(I
0
70
0
0
0
5
100
100
*Animals which present larval cuticle wing discs are scored in ‘larval’ moult. Data are expressed in percentage.
with evaginated
236
BERNARDCALVEZ of both results was obtained. The number of pupae increased with the duration of the feeding period. Thus, in Bombyx mori, the average duration of the obligatory feeding period was 4 days.
2.Dehy between beginning starvation andpupalecdysis
Age,
For those starved animals which could pupate, the duration of the instar was never shortened: the earlier the animals were starved, the longer was the last instar. Indeed, there was an important delay between the beginning of starvation and pupal ecdysis (Fig. 2). Moreover, among the groups fed for 3 and 4 days which resulted in death or a moult, the delay corresponded to a critical period in starvation resistance, since all starved larvae that survived this delay pupated.
days
Fig. 3. Ecdysteroid changes in the haemolymph of Bomb~x mori larvae during the few first days of last larval instar. The day ‘zero’ corresponds to larval ecdysis. Standard error of the mean is given for each point. topical applications (1 v/g fresh weight) every 8 hr, during the whole treatment. (c) E&steroid content. Ecdysteroid content was determined in haemolymph by the RIA method of DE REGGI et al. (1975).
RESULTS 1.Duration of the obligatory and facultative feeding periodr The duration of the obligatory and facultative feeding periods previously determined in Bombyx mori by LECAY (1955) was examined again under the conditions of this experiment. When larvae were starved after a 2-day feeding period, all died without pupating. On the other hand, when larvae were starved after at least a S-day feeding period, all moulted into pupae. In the case of intermediate feeding periods (3 and 4 days), a mixture
3. EC&steroid injection and age of larva After the larvae were injected with 20-hydroxyecdysone and immediately starved, they all moulted (Fig. lc). Within 60 hr following the last injection, the animals could be separated into three groups according to the morphology of cuticle: larvae, pupae and larval-pupal intermediates. A larval cuticle and the presence of prolegs were the criteria retained at larval moults. Animals which presented a larval cuticle with everted wing discs were also scored as a larval moult. The pupal moult was characterized by a tanned cuticle without prolegs. Larval-pupal intermediates presented a mosaic of the two types of cuticle. The proportions of larvae, pupae and intermediates varied with the previous feeding periods (Table 2). When 20-hydroxyecdysone was injected after a 1- or 2-day feeding period, all animals secreted larval cuticle. When the hormone was injected at day 3, larvae, pupae and larval-pupal intermediates were observed. When the hormone was injected after a 4day feeding period. all the animals secreted a pupal cuticle. The developmental programme could thus express pupal characters after only a 4-day feeding
Larval ecdysis
Characters
Pupal ecdysis
Larval
normally
expressed
Characters expressed after 20- hydroxyecdysone injection
Larvol
-------
Pu pal ----------
J
7-I Hormonal variations
Days
Fig. 4. Summary of progress of developmental programme in last instar larvae of Bomb_v.umot?.
Developmental programme of Bomhr.~ mori
237
feeding period to the next was not accompanied by a change in the basic level of ecdysteroids or by a short, sudden increase in their level of haemolymph. Thus, 4. Ecdysteroid content of the haemolymph offed larvae the existence of both feeding periods could not be related to the presence of circulating ecdysteroids. Results of ecdysteroid measurements (Fig. 3) Two results, however, suggested the involvement of indicate that, except for larvae at ecdysis, circulating juvenile hormone in the control of both feeding ecdysteroid levels were low. No peak was detected. periods. Two important phenomena occurred between 5. Ejfects qfjuvenile hormone treatment the fourth and fifth days of the last instar in untreated (a) Duration of last larval instar. Larvae treated with larvae: at day 4 all larvae injected with 20JH 1 from the ecdysis to the fifth day, fed for 13 days. hydroxyecdysone moulted into pupae and at day 5 all The duration of the feeding period increased by 60”/ went through the facultative feeding period. Thus, it compared with that of untreated larvae. seemed as if a larva could only go through the (b) Duration of the obligatory andfacultativefeeding facultative feeding period when the pupal programme periods. When larvae were treated with JH, from was expressed completely, i.e. when the endogenous ecdysis to the fifth day and starved immediately juvenile hormone had no effect on pupal genes. The afterwards (treatment le) SO-950/, died without duration of the obligatory feeding period could be moulting (100”; of the untreated larvae could pupate prolonged on average by topical applications of JH,, under these conditions-see paragraph 1). When However, in all day-5 larvae treated with juvenile larvae were starved 2.5 days after the end of JH hormone, the larval programme alone was expressed treatment (Ig), only 40-50:; could pupate. On in the presence of 20-hydroxyecdysone. Nevertheless, average, JH, increased the duration of obligatory a few larvae went through the facultative feeding feeding period. period. Though the juvenile hormone still had an Five to twenty per cent of the larvae treated with effect on the developmental programme. the larva had JH,, however, pupated about 17 days after being reached the threshold of the facultative feeding period. starved from the fifth day. The presence of hormone at Thus, juvenile hormone did not control the transition a high level did not prevent these larvae from going from the obligatory to the facultative feeding period. through the facultative feeding period. Since ecdysteroids did not control this transition When the JH, treatment began on the fifth day of either. progress of developmental programme could the feeding period (this day normally occurred during no longer be considered as a preliminary to the the facultative feeding period) and lasted 2.5 days transition from the first to the second feeding period. (Fig. 1i), the total duration of feeding was prolonged: The definition of feeding periods was based on the spinning started on day 11. observation of what happened to the starved larvae: When starved at the end of the JH, treatment death or moult. It was then obvious that the effects of (Fig. Ij) all larvae started spinning 6 days later and starvation were shown by a response of the all-or-none then pupated. Although the larvae were treated with principle which suggested that transition from one JH!, they still went through the facultative feeding feeding period to another corresponded to a period. developmental commitment. In reality, another (cl l@cts qf‘injection of 20-hydroxyecdysone. When important parameter had to be considered: the delay 20-hydroxyecdysone was injected at day 5 into larvae after which death or a moult occurred after starvation. treated with JH, from 0 to day 5, all larvae underwent The previous results showed that the earlier the a complete larval moult. When the injection was made larvae were starved during the last larval instar, the 2.5 days after the end of treatment (Fig. lh), the later they moulted. Moreover, in each group, larvae animals secreted larval cuticle with evaginated wing which resisted starvation the longest, were able to discs. LJnder these conditions, JH, prolonged the moult: the others died before moulting. Since there period in all larvae during which the larval programme was no developmental commitment at the origin of could be expressed. transition from one feeding period to another, this led When larvae treated with JH, from day 5 to day 7.5 to the conclusion that energy reserves of the larva at (Fig. 1k) were injected with 20-hydroxyecdysone, no the beginning of starvation alone was a critical factor. typical pupal moult occurred, only wing discs Besides, LEGAY (1955) in B0mby.u mori and NIJH~~JT evaginated. In these 7.5-day-old larvae treated with (1975) in Manduca sexta have demonstrated prevJH, the developmental programme expressed larval iously that the pupal moult was not possible if characters, although ecdysteroid injection revealed a larvae had not reached a critical size. Thus, if the pupal programme in 5-day-old larvae, i.e. just before energy reserves of a Bombyx mori larva previously fed JH treatment. Thus in 5-day-old larvae the pupal for 2 days during the last larval instar were sufficient to programme was not stabilized. resist a 33-day starvation period (Fig. 21 it could pupate. This hypothesis implied that. while starvation was imposed even during the obligatory feeding period, larval development as well as all physiological DISCUSSION functions (BOSQUET. 1979) continued slowly, but did I Feeding period.? not cease. Could the transition from the obligatory to the 2. Development of the developmentalprogramme during facultative feeding periods correspond to important the last larval instar changes in hormonal secretions such as variations in NIJHOLX ( 19751 in Manduca, KUMARAN ( 1978) and ecdysteroids and juvenile hormone? Data reported in HWANG Hsc et al. (1979) in Galleria demonstrated this study demonstrated that the transition from one period while these characters normally appeared later, at the end of the instar.
much
238
BERNARDCALVEZ
that, during the last larval instar, the epidermal cells could synthesize pupal cuticle in the presence of ecdysteroids. This ability to synthesize pupa1 cuticle was normally revealed only at the end of the last larval instar. Data from present work are consistent with these results. Also, they show that it is necessary to distinguish between 3 phases during the last stage (Fig. 41. The instar began with a period during which the larval programme was expressed. This programme was the only one to be expressed if ecdysone reached a high level in the haemolymph. The duration of this phase depended upon the organ considered. For wing discs, they evaginated 2448 hr following an injection of 20-hydroxyecdysone after only 2 days of feeding. For the rest of the body larval cuticle could be obtained after hormone injection during the first 3 days. This phase was followed by a period during which the pupal programme itself could be expressed but only in the presence of 20-hydroxyecdysone. In untreated larvae, the larval programme was still being expressed and the morphology did not change: the larval endocuticle was still secreted (BARBIER, 19711. Thus cellular reprogramming did not take place normally during this period: it could be induced only by the injection of 20-hydroxyecdysone. Some authors suggested that the first small peak of ecdysone which was noted previously in Manduca (BOLLENBACHERet al., 1975; WIELGUS et al.. 1979). in Pieris (LAFONT et al., 1977) at the end of feeding just before the very high ecdysteroid peak, could play an important part in the onset of the developmental programme and was responsible for the ability to secrete pupa1 characters in presence of ecdysone. In Galleria, however, HWANC Hsu et al. (1979) observed that some larvae were capable of secreting pupa1 cuticle at a stage before the appearance of the first ecdysone peak. In Bomby.u mori, the result was all the more significant that the pupa1 programme could be revealed 4 days before the onset of the ecdysone peak. Furthermore, measurements confirmed that the ability to express pupa1 characters did not appear in the presence of circulating ecdysteroids. However, it is clear that the first phase was closely related to the presence of juvenile hormone. The transition from the first to the second phase occurred while this hormone had no further effects on pupal genes. JH thus prolonged the period during which the larval programme alone could be expressed and it prevented the pupal programme from being expressed in the presence of 20-hydroxyecdysone. Moreover, HWANC Hsu et al. (1979) have pointed out that epidermal cell ability to express pupal characters occurs just after the increase in haemolymph JH esterase activity. These results showed the importance of the juvenile hormone .statusquo effects (RIDDIFORD, 19801 which prevent the transition from one developmental state to another. During the second phase, if both programmes can be expressed in the larva, the pupal programme is not stabilized since the presence of JH alone is enough to prevent its expression. RIDDIFORD (1972, 1980), NIJHOUT (1976), CYMBOROWSKIand STOLARZ (1979), HWANG Hsu et al. (1979) showed that at the end of the last instar the larvae became insensitive to exogenous JH. Thus another factor is necessary for the
stabilization of the pupal programme. In this case, the first ecdysone peak could play a part, as HWANG Hsu et al. (1979) suggest. This stage alone corresponds to true cellular reprogramming since only pupal characters can be expressed. During the second phase, modifications of synthesis are obtained only if 20hydroxyecdysone is injected into larvae. These data support the concept of MCCALEB and KUMARAN (1980): reprogramming of the insect larval genome occurs only on exposure to ecdysteroids in the absence of juvenile hormone. Acl;no&dgemcnt~I technical assistance.
thank JOCELYNE DEM~NT for her
REFERENCES BAEHR J. C., PORCHERON P. and DRAY F. (1978) Dosages
radioimmunologiques des ecdysttroides et des hormones juvkniles au tours des derniers stades larvaires de Rhodnius prolixus (StU). Insecte Hemiptere Reduviidae. C.r.hebd. Skanc, Acad. Sri.. Paris 287 D, 523-526. BARBIER R. (1971) Recherches sur la morphogentse tigumentaire d’un insecte holomktabole: Galleria me//oneNa L. (Lepidopttre Pyralidae). Thise Doctorat, Rennes, France. BENSKINJ. and VINSONB. (I 973) Factors affecting juvenile hormone analogue activity in the tobacco budworm. J. econ. Em. 66, 15-20. BOLLENBACHERW. E., VEDECKIS W. V., GILBERT L. I. and
O’CONNORJ. D. (1975) Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca .serta. Devl. Biol. 44, 46-53. BOSQUET G. (1979) Jelne et rkgulation du m&abolisme prottosynth&ique chez les larves de Bombyx mori et Phi/o.ramia cvnrhia Walkeri (Lepidopttres). Thkse de Doctorat, Lyon, France.
BOUNHIOLJ. J. (1938) Recherches exptrimentales sur le dkterminisme de la mttamorphose chez les LBpidoptires. Bull. biol. Fr. Belg., suppl. 24, 199. CALVEZ B., HIRN M. and DE REGGI M. (1976) Ecdysone changes in the haemolymph of two silkworms (Bombyx mori and Philo.samia Cynthia) during larval and pupal development. FEBS Left. 71, 57-61. CALVEZ B. (1977) Modifications des protkines de l’h&molymphe en relation avec deux phases du 5tme stade larvaire: les pkriodes d’alimentation. C.r. hebd. SPanc. Acad. Sci.. Pari.r 2% D. 1127-l 130. CALVEZ B. and FOURCHE J. (1980) Protein synthesis in the fat body of the fifth larval instar Philosamiu Cynthia larvae. Relation to two feeding periods. Devl. Growth Diff: 22, 93-102. CLARKE K. U. and LANGLEY P. A. (1963) Studies on the initiation of growth and moulting in Locusta migratoria migratorioides R. and F. I. The time and nature of the iniiiating stimulus. J. Insect PI?ysiol. 9, 287-292. COULON M. (1977) Vers la dkfinition d’un programme de dkveloppement larvaire. Ann. 2001. Ecol. anim. 9,649-663. CYMBOROWSKIB. and STOLARZ G. (1979) The role ofjuvenile hormone during larval-pupal transformation of Spodoptera littorallr: switchover in the sensitivity of the prothoracic gland to juvenile hormone. J. Insect Phv.tio/. 25, 939-942. DE REGCI M., HIRN M. H. and DELAACE M. A. (1975) Radioimmunoassay of ecdysone: an application to Drosophila larvae and pupae. Biochem. biophys. Res. Commun. 66, 1307-I 3 15. FOURCHE J. (1967a) Le dtterminisme de la formation du puparium chez Dro.rophila melanoga.rter. Influence du jeDne et de la fourniture d’ecdysone. Archs. Anat. microsc. Morph. exp. 56, 141-152.
Developmental
programme
FOURCHE J. (1967b) Action de l’ecdysone sur les larves de Drosophila melanogaster soumises au jetme. Existence d’un doubleconditionementpourlaformationdupuparium. C.r. hebd. Seam Acad. Sri., Paris 264 D, 2398-2400. GELBIC I and NFMEC V. (1978) Effect of a juvenoid on the nutrient content and growth of larvae of Spodoptera littoralk Acta. ent. bohenmoslov. 15, 15-24. GULLET C. and FOURCHE J. (1980) Fonctionnement du systtme mitochondrial et respiration au tours du Stme age larvaire de Pieris. Reorod. Nut. Dev. 20. 961-973. HSIAO T. H. and ‘HSIAO C. (1977) Simultaneous determination of molting and juvenile hormone titers of the greater wax moth. J. Insect Physiol. 23, 89-93. HWANG Hsu K., REDDY G.. KRISHNA KUMARAN A., BOLLENBACHERW. E. and GILBERT L. I. (1979) Correlation between juvenile hormone esterase activity, ecdysone titre and cellular reprogramming in Galleriamellonella. J. Insect Physiol. 25, 105-l 1I. KUMARAN K. A. (1978) Reprogramming and DNA synthesis in Galleria mellonella larval epidermal cells. Dtfferenfiation 12, 121-125. LAFONT R., MAUCHAMP B., BLAIS C. and PENNETIER J. L. (1977) Ecdysones and imaginal disc development during the last larval instar of Pieris brassicae. J. Insect Physiol. 23, 277-283. LEGAY J. M. (1955) La prise de nourriture chez le Vera soie. These Paris. A 2757.3630, France. MAU~HAMP B.. LAFONT R. and JOURDAIN D. (1979) Mass fragmentographic analysis of juvenile hormone 1 levels during the last larval instar of Pieris brassicae. J. Insect Physiol. 25, 545-550. MCCALEB D. and KRISHNA KUMARAN A. (1980) Control of
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