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Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae
J. Insect Physiol., Vol. 21, No. 8, pp. 535-544, Printed in Great Britain.
1981
0022-1910/81/080535-10$02.00/O ‘c 1981 Pergamon Press Ltd.
REGULATION AND SIGNIFICANCE OF ECDYSTEROID TITRE FLUCTUATIONS IN LEPIDOPTEROUS LARVAE AND PUPAE FRANTISEK SEHNAL*, PETER MARbY? *$Institute of Entomology, Czechoslovakia and tInstitute
Czechoslovak of Genetics.
and
JAROSLAVAMALA$
Academy of Sciences, Na Folimance 5, 12000 Prague, Hugarian Academy of Sciences. H-6701 Szeged, Hungary
(Received 10 December 1980)
Abstract-The last larval moult of GaNeria mellonella is induced by an elevation of ecdysteroid titre to more than 200 rig/g.. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr. is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 rig/g)) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500-2500 rig/g)) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 rig/g)) in the middle of pupal instar. Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 3040 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing increase of the prothoracit:otropic neurohormone (PTTH). In either case, the pupation-inducing ecdysteroids is ? times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed. Neither in G’. mellonella larvae. nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid prosduction or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5-15 times lower than usual ecdvsteroid concentration when the last-instar larvae are exposed to JH.
INTRODUCTION THE PRIMARY sources of ecdysteroids in most immature insects are the prothoracic glands (CHINO et al., 1974; KING er al.. 1974). They produce a prohormone, ecdysone, which is converted to the actual hormone, 20- hydroxyecdysone in peripheral tissues (KING, 1972). 20Both ecdysone. hydroxyecdysone and their hormonally-active metabolites are inactivated biochemically and/or depleted by excretion I:KOOLMAN,1978). Their content in the body obviously depends on the balance between the rate of ecdysone secretion and the rate of ecdysteroid metabolism. Available information demonstrates that the level of secretion plays the crucial role (AGUI and YAGI, 1973; BOLLENBACHERet al., 1975; HIRN et al., 1979). Isolated larval abde3mens of some insects produce enough ecdysteroids tc3 support metamorphosis to the adult stage (HSIAO et LI/., 1976; DELBEQUEet al., 1978). With the exception of some hybrids of Bombay mori (cf. NISHIITSUTSUJI-Uwo and NISHIMURA, 1972), no *To whom correspondence I,= 27.8
-c
should
be sent.
such development occurs in Lepidoptera. The low titre of ecdysteroids in the isolated abdomens of caterpillars demonstrates that their prothoracic glands are indispensable for development (LAFONT et al., 1977). This is further proven by the induction of development in isolated larval (FUKUDA, 1944) and pupal (WILLIAMS, 1947) abdomens of Lepidoptera which are supplied by implants of prothoracic glands. Prothoracic glands of both larvae (GIBBS AND RIDDIFORD, 1977; MALA et al., 1977) and pupae by pro1947) are stimulated (WILLIAMS, thoracicotropic neurohormone (PTTH) from the brain (AGUI et al., 1980). The release of PTTH depends both on the environmental and intrinsic factors which are integrated in the central nervous system (MALL and SEHNAL, 1978; RIDDIFORD and TRUMAN, 1978). The intrinsic factors seem to include the juvenile hormone (JH). which in the last-instar larvae of Manduca sexta reportedly inhibits the PTTH release (NIJHOUT and WILLIAMS. 1974). Delay of the larval-pupal transformation, which is induced by JH and juvenoids in many endopterygote insects (SEHNAL, 1976) and in some species occurs naturally as larval diapause (YIN and CHIPPENDALE. 1973; YAGI and 535
536
FRANTI~EK SEHNAL.PETER MARTY
FUKAYA, 1974), has been explained as a result of such an inhibition of PTTH production (CHIPPENDALEand YIN, 1976: TAKEDA, 1978). An alternate explanation is that JH directly suppresses the function of larval prothoracic glands (CIEMIOR er a/., 1979), although it seems to activate the prothoracic glands of decerebrate or diapausing pupae (GILBERT and SCHNEIDERMAN, 1959?. It is obvious that the control of ecdysteroid production is complex and involves PTTH action on prothoracic glands. JH action on the brain and prothoracic glands, and apparently also influence of the thoracic ganglia on the glands (MALA et al., 1977; GERSCH, 1978). The larvae of Galleria mellonella and some other species moult when the brain, corpora allata and the thoracic ganglia are removed (MALL er al.. 1977). Their prothoracic glands clearly secrete enough ecdysone spontaneously. It is not known. however, if low quantities of ecdysteroids are continuously present in the body and cause a sequence of ‘covert effects’ (WILLIAMS, 1968) until an ‘overt effect.’ the moult, is accomplished, or if there is a delayed spontaneous surge ofecdysone release, similar to that which occurs in intact insects upon stimulation of prothoracic glands by PTTH. In the present study we examine regulation of ecdysteroid production by measuring ecdysteroid titre in G. mellonella subjected to various experimental manipulations. The effects of JH are also investigated in diapausing pupae of Hyalophora cecropia and Celerio euphorbiae.
MATERIALS
AND METHODS
Insects and surgery The wax moth, Galleria mellonella L. (Pyralidae) was reared under standard conditions (SEHNAL, 1966). Larvae and pupae of precisely-known age in respect to the preceding ecdysis were used for the experiments. Behaviouristic (wandering, spinning and mobility of the larvae) and morphological markers (progress of apolysis and eye-pigment migration) were employed as additional criteria for assessing the physiological age of the last-instar larvae. Decapitated larvae and isolated larval abdomens were obtained by means of ligation applied behind the head and behind mesothorax respectively; the body part anterior to the ligation was cut off. One day after ligation some insects received implants of brains and/or endocrine glands. All the surgical procedures performed with Galleria mellonella have been described earlier (MALT et al., 1977). The diapausing pupae of Hvalophora cecropia (Saturniidae) were kindly provided by Dr. W. H. TELFER of the University of Pennsylvania, Philadelphia, and Dr. J. H. WILLIS of the University of Illinois, Urbana-Champaign. No difference between these two groups of pupae was found. The diapausing pupae of Celerio euphorbiae L. (Sphingidae) were obtained through the courtesy of Dr. K. GRZELAK of the Institute of Biophysics and Biochemistry, Polish Academy of Sciences, Warsaw. These pupae came from caterpillars, which were collected in central Poland on Euphorbia cyparisias and raised afterwards under a short photoperiod indoors.
AND
JAROSLAVA MALA
Operations were performed on pupae anaesthetized in a stream of carbon dioxide and surface-sterilized with 70‘!/, ethanol. The head and thorax were cut off and the abdomen was supplied, according to the protocol, with implants taken from the removed front parts. The cut was then covered with a round piece of plastic coverslip and sealed along the margin with beeswax. Hormones and their application Synthetic JH-II (methyl [2E,6E,lOZ]-3,7,1 ltrimethyl-10,l l-epoxy- 2,6_tridecadienoate) and the juvenoid SJ-42-F (ethyl [2E]-3,7,11-trimethyl-2dodecenoate) were kindly given to use by Dr. V. JAROL~Mof the Institute of Organic Chemistry and Biochemistry, CSAV, Prague. In the experiments with G. mellonella the compounds were dissolved in acetone and adminstered topically. The diapausing pupae and isolated pupal abdomens of H. cecropia and C. euphorbiae were injected with undiluted juvenoid. Monitoring the MH titre In all experimental insects we recorded initiation and progress of the moulting process as indications of in ecdysteroid titre. For direct an increase measurements of ecdysteroids, 2-25 specimens were grouped to provide a sample weight of 100-600 mg. Each sample was homogenized in a motor-driven Potter-Elvehjem glass homogenizer in 4 ml 60% methanol, which was supplemented with 10e4 M phenylthiourea. The homogenates were stored for 2-8 weeks and then used for MH determination by radioimmunoassay described previously (MAROY et al., 1977; MAR~)Y and TARN~)Y, 1978). No separation of individual ecdysteroids was attempted; our data do not distinguish between ecdysone, 20-hydroxyecdysone and their metabolites. The antiserum we used was equally sensitive to ecdysone and 20hydroxyecdysone. A few complementary estimations of ecdysteroid titre in the penultimate-instar larvae were done by Dr. J. P. DELBECQLJE of the University of Dijon. The background level was about 5 times lower but the titre changes during the instar were indentical with those found by our assay.
RESULTS Changes of ecdysteroid titre during development of Galleria mellonella During the examined period, the titre of ecdysteroids rose to a peak in the second third of the penultimate-instar and then dropped to levels below 20 rig/g at the beginning of the last larval instar (Fig. 1). A slight but significant (P
Regulation
of ecdysteroid
537
titre
AE II 2ooc 600 -
0 I
24
VI
0
40
ARREST
96
144
VII
I
192
0
I
I
48
96
144
192
P
I
A
Fig. I. The ecd ysteroid titre in Galleriu me/lonellu during the penultimate and last larval instars (VI, VII), the pupal instar (P), and in freshly-emerged adults (A). Note that two different scales of ecdysteroid concentration are used and that the data for female pupae and adults (f) are presented separately from those concerning the males (m). The length of each instar is divided into 24 hr intervals (development of the lastinstar larvae w,as arrested for 8 days by preventing the larvae to leave their food). Arrows indicate apolyses (A), ecdyses (E), wandering (W), and spinning (S), respectively. End of the spinning was accompanied by a drop in the titre at 152 hr of the instar. In apolysing larvae the
ecdysteroid content reached a maximum of 780 rig/g (over 1000 rig/g in a few individuals), followed by a decrease to about 2013 rig/g in pharate pupae. Female pupae consistently possessed a higher ecdysteroid titre than the maIes, although the relative fluctuations during most of the instar were similar in both sexes (Fig. 1). Ecdysteroid concentration reached a high peak at the beginning of the instar and a smaller peak in the middle of the instar. This second peak was confined to a brief time interval: the amount of ecdysteroids determined at 96 hr was significantly higher (P
Stage Last-instar larvae 120 hr after ecdysis Female and male pupae (1:1) 24 hr after ecdysis 120 hr larvae 2 days after the removal of head and thorax by ligation
1. Distribution
Head
of ecdysteroids
+ prothorax
751 + 124
The source of ecdysteroids In wandering last-instar larvae and in pupae 24 hr after ecdysis we determined ecdysteroids separately in the head and prothorax, and in the remaining body part. Summation of the two values compared well with the content established in intact insects of the same age (Table 1). In both larvae and pupae, however, we found that the concentration expressed per gram of fresh weight was one order of magnitude higher in the front parts than in the hind parts. When the prothoracic glands contained in the front parts were removed from the wandering larvae by means of a ecdysteroid ligation behind prothorax, the concentration in isolated abdomens decreased within 2 days to 40 rig/g.. We interpreted these findings as a confirmation that the prothoracic glands are the source of ecdysteroids in the larvae and early pupae of G. mellonella. A drop of ecdysteroid titre and cessation of development was observed in all larval abdomens isolated prior to 152 hr of the last-instar. The in larvae and pupae
of G. meNoneNu
Meso- and metathorax + abdomen
Total
Intact control
69 + 1
95 * 2
100 + 13
(8.3 mg)
(184 mg)
(194 mg)
(208 mg)
3290 + 1205
411 & 141
650 + 230
901 + 153
(12 mg)
(122 mg)
(137 mg)
(153 mg)
40 f
19
(170 mg)
The ecdysteroid content is given in rig/g fresh weight (mean + SE.): the fresh weight of examined parentheses. Two groups of 10 insects were taken for each determination.
body parts is indicated
in
FKANTISEK SEHNAL. PETERMARRY AND JAROSLAVA MALA
538
abdomens isolated at 152 hr occasionally produced patches of pupal cuticle. These individuals contained an elevated titre of ecdysteroids (198 rig/g)) and we assume that they were ligated after the final ecdysteroid increase in the last-larval instar had begun. Stimulation of ecdysteroid content The ecdysteroid titre of larvae also decreased when only the head had been removed. For example, decapitation of penultimate-instar larvae caused within 2 days a decrease from 205 + 32 rig/g to 51 + 18 rig/g (averages from 30 animals analyzed in 3 samples each time). In larvae decapitated 120 hr into the last-instar, the titre was below 50 rig/g on days 3 and 10 but increased to 200 rig/g by day 20 after decapitation (Fig. 2A). This level was also found in about 60% of the samples taken for the determination between 30 and 40 days. The remaining samples contained well over 1000 rig/g;; no intermediate figures were found. Since the percentage of samples with high ecdysteroid content corresponded to the number of insects pupating between 30 and 40 days (MALA et al., 1977), we assume that the individuals with a high ecdysteroid content are those which had initiated the larval-pupal moult. The decapitated larvae, which underwent apolysis as revealed by their body shape and reduced mobility, contained invariably over 2000 rig/g ecdysteroids. This concentration was much higher than that found in intact larvae (Fig. 1) but, as
in intact larvae, the titre dropped abruptly and concurrent with the secretion of the pupal cuticle (Fig. 2A). In pupae freshly formed from the decapitated larvae the content of ecdysteroids was only 200 rig/g.. Both the increase in ecdysteroid titre and the secretion of pupal cuticle were accelerated following implantation of 3 larval brains into each decapitated larva. The titre was elevated substantially already on day 10 after ligation (9 days after implantation) and most insects apolysed within the next 2 days. In these insects the ecdysteroid content reached values over 2500 rig/g,, as were found in decapitated larvae without brain implants only 3040 days after ligation (cf. Figs. 2A and B). The titre dropped in freshly-formed pupae; it was higher than in spontaneously-pupating insects but the difference was probably due to variations in the age of analyzed specimens. The increase of ecdysteroids was also accelerated in decapitated larvae which received implants of 3 braincorpora cardiaca-corpora allata complexes (Fig. 3A) and in those which were implanted with 3 brains and simultaneously treated with 0.5 pg of JH-II (Fig. 3B). These insects apolysed already 6-10 days after the implantation and produced either pupae with patches of larval cuticle or larval-pupal intermediates (cf. SEHNAL and SCHNEIDERMAN.1973). The general course of ecdysteroid titre was similar as in the previouslydescribed groups of decapitated insects (Fig. 2A and B), but the maximal concentration was only 1000 rig/g
No sign of moult Apolyzed Freshly
u
insects ecdysed
No sign of moult Apolyzed
insects
Freshly
g
insects ecdysed
insects
B
140(
I $
120(
Q 2
lOO(
E f
60(
B 8 60( n” s 40(
No Treatment Days After
I Indicated
Brain
l--dYf+ 6
Implants k-CC-CA
implants
7
1 JH d Brain
E-10
Implants
Treatments Days
Fig. 2. Changes in the ecdysteroid content after decapitation (A) and in the decapitated larvae which were supplied with the implants of 3 brains (B). The larvae were decapitated 120 hr of the last-instar and the brains provided by donors the same age.
at of
After
Indicated
Treatments
Fig. 3. Changes in the ecdysteroid content in the decapitated larvae which received either implants of 3 complexes braincorpora cardiaca+orpora allata (A) or implants of 3 brains supplemented with topical application of 0.5 pg JH II (B).
Regulation
of ecdysteroid
in insects with implanted complexes (Fig. 3A) and 600 rig/g in those supplied with brains and treated with JH-II (Fig. 3B). In decapitated larvae, which developed into larva-pupal intermediates following an implantation of 3 brains and a treatment with 10 pg SJ-42-F (not shown in Fig. 3). the ecdysteroid titre also reached a maximum of only 608 rig/g of body weight (average value from the analysis of 3 specimens at the time of apolysis).
S $ $
The increase in ecdysteroid content followed by secretion of pupal cuticle occurred in more than 80’; of the decapitated larvae. A similar proportion of decapitated larvae moulted when implanted with 3 brains either with or without corpora cardiaca+orpora allata. or when implanted with 3 brains and treated with SJ-42-F (Table 2, upper part). On the other hand, implantation of corpora cardiaca+orpora allata without the brain reduced the proportion of moulting insects to 159, and the treatment with SJ-42-F to Y0 (Table 2). The average ecdysteroid content in the last 2 groups remained very low for 20 days after treatment. The slight increase which was found 20-30 days after implantation of corpora cardiaca+orpora allata apparently indicated that a few of the insects initiated the moulting process (Fig. 4A). A single specimen. which actually did produce a new larval cuticle, contained 192 rig/g.. The majority of those decapitated larvae, which were treated with 0.5 pg JH-II on days 1, 16 and 25 after ligation, maintained a low ecdysteroid titre throughout 40 days of the experiment. Only in a few of them, which apolysed between days 8 and 10, the titre increased to 624 rig/g (Fig. 4B). It is obvious from these results that corpora cardiaca
Table 2. The incidence,
I
Nosign
EB
Apolyzed Insects
m
Freshly ecdysed Insects
of moult
600
ij 400
D
e F L
ZCKJ
3
Days after lndlcoted treatments
Fig. 4. Changes in the ecdysteroid content of decapitated larvae which were either implanted with 3 pairs of copora cardiacs-corpora allata (A) or treated with 0.5 pg JH II on days 1. 16 and 26 after decapitation (B).
of ecdysteroids and consequently reduced the number of spontaneously-moulting decapitated larvae. The bottom part of Table 2 demonstrates that the moults of isolated abdomens, which had been supplied with prothoracic glands, were also hampered by corpora allata implants and the juvenoid. Clearly, intact inervation and localization of prothoracic glands are irrelevant for this JH effect. A decrease in ecdysteroids was observed also in the
timing and type of moult in decapitated larvae and isolated larval abdomens subjected to various treatments
0
Treatment Decapitated
Moulted 0
Isolated implanted + 3 br implanted + 3 cc-ca implanted implanted + 10 pg SJ-42-F
Days
Type of moult
larvae 83 88 71 67 15 5
None 3 br implanted 3 br-cc-ca implanted 3 br implanted + IO pg SJ-42F 3 ccsa implanted 10 /q SJ-42-F
None 2 PC 2 PC 2 PC 2 PC
1
e
Suppression of ecdvs teroid titre
539
titre
41.8 14.0 12.6 14.8 30.0
f 9.6 + 3.6 +_ 4.1 f 2.1 + 13.4 24
Pupal Pupal Intermediate Intermediate Intermediate Intermediate
larval abdomens 0 32 35 12 0
29.8 + 3.1 15.8 f 1.9 36.0 + 6.1
No moult Pupal Pupal Intermediate No moult
Twenty insects were used in each experiment. The figures under Days represents means + S.D. The accomplished moults were perfect in every respect but the insects could not escape from the old exuvia. The new cuticle was either exclusively pupal of both larval and pupal cuticles were produced (intermediate moult). The non-moulting insects usually died more l.han 40 days after ligation. Abbreviations: (br) brains, (cc) corpora cardiaca, (ca) corpora allata, (PG) prothoracic glands.
FRANTISEK SEHNAL. PETER MARTY
540 Table
3. Ecdysteroid
AND
JAROSLAVA MALL
titre (mean + SE.) at comparable physiological treated and control larvae of G. melioneilu Treated
Stage Crawling Apolysis Apolysis Ecdysed
168* 168.f 180: 192:
14 41 135 72
* + + +
Titre
Age 4 7 12 6
120 168 168 184
112 756 756 278
* + f +
The age of analyzed insects is given in hr after the last larval ecdysis. Untreated ecdysed into pupae whereas the treated insects developed into superlarvae. * 1 pg SJ-42F applied at 84 hr of the last-instar. t 100 pg SJ-42-F at 84 hr. $10 pg JH II at 120 hr.
Table 4. Induction
of development
of JH-
Controls Titre
Age
stages
in isolated abdomens euphoribiae
of diapausing
21 96 96 54 controls
pupae of Celerio
Ecdysed Dead
Treatment (number of insects) 500 pg 3 PTG 3 PTG 3 PTG 3 br +
Number
SJ (8) + 500 pg oil (6) f 500 pg SJ (6) + 3 br + 500 pg SJ (5) 500 pg SJ (5)
(day)
Day
0 0 0 4 0
65 + 12”
89 ) 36 69 + 28 104 +_ 43 180 60 + 11
The implants were taken from the same pupae which provided the isolated abdomens. Olive oil and the juvenoid SJ-42-F were injected 2 weeks after the abdomens had been isolated and supplied with the implants. Abdomens which completed secretion of new cuticle and resorbed the moulting fluid were classified as ‘ecdysed.’ Implants: (PTG) prothoracic glands; (br) brains. * Represents mean + S.D.
Table
5. Induction
of development in the isolated abdomens Hyalophoro cecropia
of diapausing
pupae
of
Ecdysed Treatment (number of insects)
Number
500 pg oil (7) 500 pg SJ or 100 pg JH (7) 3 PTG + 500 ~1 oil (13) 3 PTG + 500 ~1 SJ or 100 ~1 JH (16) 3 PIG + 2 or 3 br (8) 2 or 3 br + 500 ~1 oil (10) 2 or 3 br + 500 ~1 SJ or 100 ~1 JH (10)
0 1 1 2 8 10 10
Dead Day
204 240 177 f 79 + 90 * 82 f
(day)
61 34 30 19
442 372 520 434
i 213 I 153 i 141 i 169
Only abdomens which survived for 40 days were evaluated. Most experiments were performed with males; female abdomens received 3 brains, whereas male abdomens 2 brains. Other procedures were identical to those described for C. euphorbia (Table 4). No differences were found between insects injected with SJ-42-F and those injected with JH (JH-I and JH-II 8:l). The figures represent means + S.D. non-ligated
last-instar
larvae
which
were treated
with
JH or the juvenoid (Table 3). Application of 1 pg SJ-42-F at 84 hr of the instar caused a great delay in the pupa1 moult, whereas treatment with 100 pg SJ-42-F induced an extra larval moult (cf. CIEMIOR et al., 1979). The ecdysteroid concentration decreased in both cases. The insects treated with 100 pg SJ-42-F underwent larval apolysis at an extremely low concentration of less than 50 rig/g.. A similar extra larva1 moult was also induced with 10 pg JH-II adminstered at 120 hr of the last-instar. The highest ecdysteroid titre found in these insects was 165 rig/g,,
which is one fifth of the level found in the untreated controls undergoing the pupal moult (Table 3). The stimulation
of development
in diapausing pupae
Isolated abdomens of diapausing pupae of Celerio euphorbiae did not develop although some of them survived for half of a year (Table 4). Implantation into each abdomen of 3 prothoracic glands, which were taken from the discarded front parts of the same pupae, was insufficient to induce development. Simultaneous application of 500 pg/specimen of SJ-42-F, a treatment which induced development in 8
Regulation of ecdysteroid titre
intact diapausing pupae, (not in the table) failed to stimulate the implanted glands. By contrast, when the glands were implanted simultaneously with 3 brains (taken also from diapausing pupae), 80% of the abdomens developed and produced new cuticle with adult scales. In some regions the new cuticle possessed pupal features, obviously due to the juvenilizing action of SJ-42-F. Implanalion of brains without prothoracic glands had no effect (Table 4). Experiments with diapausing pupae of Hyalophora cecropia yielded similar results in that all preparations receiving prothoracb: glands and 2-3 brains developed into adults. In addition, in H. cecropia the moult was also induced in isolated abdomens without the gland implants (Table 5). When these abdomens were supplied with 2-3 ‘brains, all of those which have survived for 50 days moulted within the next 60 days. For this effect it was irrelevant if the abdomens received injection of olive oil or of JH; in the latter cuticle was case, however, the newly-secreted predominantly pupal. Only exceptionally a late moult (134-240 days after the isolation) occurred in abdomens supplied with prothoracic glands and treated with olive oil or with JH without the brain implants.
Significance of chanpes in ecdysteroid content during development
The ecdysteroimd amounts established in G. mellonella in the TIresent study are comparable with the figures published by HSIAOand HSIAO(1977) and BOLLENBACHER et al. (1978). Our results indicate that larval moults, including apolysis, epidermal proliferation and cuticle secretion, are caused by single ecdysteroid peaks (Fig. 1). The larval-pupal and pupal-adult transformations are controlled by ecdysteroids in a complex way. The larval-pupal transformation, including epidermal reprogramming, begins at about 60 hr of the last larval instar, as indicated by the loss of tissue sensitivity to JH (SEHNAL, 1968; SEHNAL and SCHNEIDERMAN, 1973). The transformation is probably released by, and its progress depends on, the increase in ecdysteroid titre in the absence of JH. Coincidently, secretion of JH from the corpora allata is prevented via nerves at 48-60 hr (SEHNAL and GRANGER,1975). The reprogramming of the epidermis extends over most of the last larval instar (HWANGHSUet al., 1979). The responsiveness of epidermal cells to JH decays rapidly during the ecdysteroid peak between 120 and 144 hr, concurrently with increased epidermal mitotic activity (PIEPHO,1939; SEHNALand NOVAK, 1969). The epidermis retains its sensitivity to high JH doses until 152 hr when the major ecdysteroid peak begins. This pe,ak controls the completion of the larval-pupal moul ting process and associated metamorphic events. For example, evertion of the wing discs and degeneration of specialized larval organs such as the silk glands and proventriculus occur at this time (S~HNAL,1968); ecdysteroid control over these processes was documented in experiments with isolated larval abdomens (SEHNAL.1972). It would seem that completion of pupal
541
development can be controlled by a single broad ecdysteroid peak at the end of the last larval instar. The occurrence of 2 distinct peaks in Lepidoptera (BOLLENBACHER et al., 1975; LAFONT et al., 1977; MARTYand TARN~Y,1978; DEANet al., 1980). may be advantageous, however, because it provides a mechanism for adjusting the development to environmental conditions. The ecdysteroid increase leading to the second peak may be delayed and the insects, such as caterpillars of G. mellonella exposed to unfavourable conditions (SEHNAL, 1966) enter diapause. Fluctuations of ecdysteroids also affect the behaviour of larvae. Several observations indicate that in G. mellonella the ecdysteroid increase at 120 hr during the last-instar terminates feeding and releases wandering followed by cocoon spinning. For example, caterpillars which maintain a permanently low ecdysteroid titre after JH treatment (Table 3), continue to feed over extended periods of time and do not spin (CIEMIORet al., 1979). When in normal larvae the ecdysteroid increase is interrupted after it has begun by depriving the larvae of sufficient space, the spinning is also prevented (Fig. 1). On the other hand, injection of 20-hydroxyecdysone into feeding larvae causes termination of feeding and at least in some cocoon cases induces spinning (unpublished observation). A high ecdysteroid titre in the freshly-ecdysed pupae of G. mellonella apparently supports the early onset of the pupal-adult transformation. Imaginal apolysis and extensive epidermal differentiation, which take place between 30 and 40 hr after pupal ecdysis (MARCUS, 1962), are probably caused by the high ecdysteroid peak at 36 hr (Fig. 1). The small ecdysteroid increase at 96 hr is associated with the secretion of imaginal cuticle. The following rise of ecdysteroids in females has been elucidated by BOLLENBACHER et al. (1978) and HSIAO and HSIAO (1977). Our results provide ample support for observations that moults may be induced with much lower concentrations of ecdysteroids than those found in normally-developing insects (CLARET et al., 1977; LAFONT et al., 1977). The excessive peaks of ecdysteroids apparently serve as pace-makers which synchronize development of various body parts. SEHNAL(1972) observed that when isolated larval abdomens were induced to develop by repeated exposures to a steady concentration of 20-hydroxyecdysone, the progress of metamorphosis was very uneven. Induction of secretion in the prothoracic glands
The prothoracic glands appear to be the sole primary source of ecdysteroids in G. mellonella larvae. The decrease in ecdysteroid titre in decapitated larvae shows that without the brain the activity of prothoracic glands decreases and in some specimens is never restored. In most decapitated larvae, however, the glands are inactive for 30-40 days and then burst into activity, as indicated by the sudden increase of ecdysteroid titre (Fig. 2A). Initiation of development in decerebrate larvae (MALA et al., 1977) and pupae (MCDANIEL, 1979) and also in isolated abdomens supplied with prothoracic gland implants (FUKUDA,
542
FRANTISEK SEHNAL.PETERMAR~)YAND JAROSLAVA MALA
1944; WILLIAMS, 1947) is obviously caused by this ecdysteroid surge. The ecdysteroid concentration drops when the decapitated insects accomplish secretion of the new cuticle (Fig. 2A), hence, the prothoracic glands return to a state of relative inactivity. The induction of such a surge is apparently a characteristic attribute of prothoracic glands, which is independent of the nature of their stimulation. It was consistently observed in all our experiments. The decline in activity at the end of secretion may be regulated through a feed-back mechanism by the blood concentration of ecdysteroids and/or by their effects, because it always occurs after the insects had produced new cuticle. Ecdysteroid production from unkown organs in isolated abdomens of Leptinotarsa decemlineata also shows spontaneous fluctuations (HSIAO et al., 1976). Brain implants accelerate the ecdysteroid increase and pupation of decapitated larvae (Fig. 2B). Except for this acceleration, the magnitude of the surge of ecdysteroid titre is similar as in insects whose prothoracic glands were activated spontaneously (cf. Figs. 2A and B). It is obvious that the brain implants influence neither the rate of ecdysone secretion, nor the velocity of ecdysteroid degradation and excretion. The prothoracicotropic neurohormone (PTTH), which is apparently released from the implanted brains, acts as a trigger inducing secretion in the prothoracic glands. The regulation of ecdysteroid production
by JH
Our data show that the moult-inducing spontaneous surge of ecdysteroids is prevented with JH (Table 2). The spontaneous moults are also rare in decerebrate larvae, whose corpora allata remain in situ and apparently produce some JH (MALL et al., 1977). When these insects moult in spite of the presence of JH, their ecdysteroid titre never reaches the values found in insects of comparable age but lacking JH (Fig. 4). These data demonstrate clearly that JH suppresses the spontaneous activation of prothoracic glands and restrains the function of active glands. On the other hand, the activation of prothoracic glands by PTTH is not prevented by JH. As seen from Table 2, JH affects neither the number of moulting insects, nor the timing of moult in decapitated larvae which have been supplied with brain implants. The ecdysteroid titre in treated insects is always lower than in the non-treated controls (cf. Figs. 2 and 3) but nevertheless increases to a moult-inducing peak. In the presence of JH the prothoracic glands apparently respond to PTTH but secrete less ecdysone. The data Of CHIPPENDALEand YIN (1976) indicate existence of a similar mechanism in Diatraea grandiosellu. They noted that intact diapausing caterpillars moult in the presence of JH but the decapitated caterpillars do not moult. We suggest that the prothoracic glands of intact insects are activated by PTTH whereas the decapitated larvae not only lack PTTH but a spontaneous activation of their prothoracic glands is prevented by JH. Induction of a prompt moult by brain implants in JH-treated decapitated larvae of G. mellonella (Table 2) indicates that JH does not influence the PTTH liberation from the implanted brain. The regulation by JH of PTTH release from the brain in situ is more
complex. Thus, JH applied in intact larvae at the start of the last-instar induces a larval moult within 4-5 days whereas the controls undergo a pupal moult in 7-8 days (CIEMIORet al.. 1979). The prothoracic glands of treated larvae clearly become active earlier than those of the controls. In this case, JH apparently accelerates the release of PTTH, which in turn stimulates the prothoracic glands. By contrast, moderate doses of JH administered in the middle of the last-instar suppress the ecdysteroid production. The long period of inactivity of the prothoracic glands indicate that in this case JH prevents the PTTH release. When the jH-treated larvae receive PTTHproducing brain implants, the prothoracic glands become active and a mouit ensues (CIEMIOR ef al.. 1979). Inhibition of PTTH production was considered by some authors as the only way by which JH hinders the function of prothoracic glands (NIJHOUT and WILLIAMS, 1974; CHIPPENDALE, 1977; TAKEDA, 1978). We stress that it is just one of the mechanisms by which JH regulates the titre of ecdysteroids. In our opinion, the body concentration of JH is one of the factors which are integrated in the brain system of regulation of its prothoracicotropic activity. Depending on the other information the brain receives, JH can either accelerate or delay the liberation of PTTH. Independently of this action, JH acts directly on the prothoracic glands and inhibits their function to the extent that virtually no ecdysone is produced unless the glands are stimulated by PTTH. The rate of ecdysone production thus depends on a balance between PTTH and JH. This tentative model can explain the endocrine regulation of larval diapause and all experimental data on the effects of JH and juvenoids on the insect moult, e.g. the inhibition of moulting by JH in the larvae of Blatella gemanica (MASNER et al., 1975). Does JH ever stimulate
the prothoracic glands?
GILBERT and SCHNEIDERMANN (1959) and KRISHNAKUMARAN and SCHNEIDERMANN (1965) demonstrated that JH and juvenoids stimulate development of diapausing and decerebrate saturniid pupae. HIRUMA et al. (1978) made an identical observation on the decerebrate last-instar larvae and pupae of a noctuid moth, Mamestra brassicae. The stimulation of development in such insects was interpreted as indicating direct activation of prothoracic glands by JH. HIRUMA et al. (1978) noted, however, that the juvenoid treatment had no effect on larvae, which had been decapitated within first 4Oy; of the length of the last-instar. In our experiments with diapausing pupae of H. cecropia and C. euphorbiae (Tables 4 and S), JH never stimulated the prothoracic glands which were implanted into isolated abdomens. This contrasts with the reported action of JH on brainless pupae and reveals that JH stimulates only the prothoracic glands in situ. The hormone apparently affects the glands indirectly, via some organ(s) in the head or thorax. The glands in situ are stimulated by various agents (WILLIAMS, 1967; NISHIITSUTSUJI-UWO and NISHIMURA, 1972). Our present results provide an alternative explanation of the moult-inducing action of JH in the diapausing and decerebrate insects. We have
Regulation
of ecdysteroid
consistently observed that after a JH treatment the larvae of G. mellone!la undergo apolysis and secrete cuticle at very low levels of ecdysteroids (Table 2). Table 3 shows that the treated larvae accomplish a moult at a 5-15 times lower concentration of ecdysteroids than the untreated controls. This implies, consistently with the observations of MASNER et al. (1975), MITSUI and RIDDIFORD (1978) and other authors, that JH lowers the hormonal requirements of epidermis for cuticle secretion. Accordingly, JH should stimulate moulting in diapausing and brainless insects if their prothoracic glands produce some ecdysone. The ecdyxeroid titre in these insects would be too low to induce development in the absence of JH but could support a moult when JH is present. This kind of moult stimulation by JH very likely occurs in the diapsuing caterpillars which undergo stationary larval moults (YIN and CHIPPENDALE, 1973; YAGI and FUKAYA, 1974; TAKEDA, 1978). It has been shown that the activity of their prothoracic glands is curtailed by moderate concentrations of JH (BERGOT ef al., 1976). CHIPPENDALE(1977) and TAKEDA (1978) concluded that the diapausing larvae contain just enough ecdysteroids to undergo stationary larval moults.
titre
543
southwestern corn borer, Diarraea grandioselia. J. Insect
Physiol. 22, 989-995. CIEMIOR K. E., SEHNAL F. and SCHNIEDERMANH. A. (1979) Moulting, growth and survival of Galleria mellonella L. (Lep., Pyralidae) treated withjuvenoids. Z. angew. Ent. 88, 414-425. CLARET J., PORCHERONP. and DRAY F. (1977) Critical period and ecdysone titers in the pupae of Pieris brassicae. Experientia 33, 1389-1390. DEAN R., BOLLENBACHERW. E., LOCKE M., SMITH S. L. and GILBERT L. I. (1980) Haemolymph ecdysteroid levels and cellular events in the intermoult/moult sequence of Calpodes ethlius. J. Insect Physiol. 26, 267-280. DELBEQUEJ .-P., DELACHAMBREJ., HIRN M. and DE REGCI M. (1978) Abdominal production of ecdysterone and pupal-adult development in Tenebrio molitor (Insecta, Coleoptera). Gen. camp. Endocr. 35, 436-444. FUKUDA S. (1944) The hormonal mechanisms of larval molting and metamorphosis in the silkworm. J. Fat. Sci. Tokyo Univ. Section IV 6, 477-532. GERSCH M. (1978) Hormonale und humorale Steuerung durch neurosekretorische Peptide. In Probleme der Korrelation neuronaLP und endokriner Regulation bei Evertebraten (Ed. by PENZLIN H.) pp. 8-31. FriedrichSchiller-Universitit, Jena. GIBBS D. and RIDDIFOKD L. M. (1977) Prothoracicotropic hormone in Manduca sexta: Localization by a larval assay. J. exp. Biol. 66, 255-266. GILBERT L. I. and SCHNEIDERMANH. A. (1959) Prothoracic gland stimulation by juvenile hormone extracts of insects. Acknowledgements--We acknowledge the gift of Nature. Lond. 184, 171-173. hormones from Drs. V. JAROL~Mand H. EMMERICH HIRN M., HETRU C., LAGLIEUX M. and HOFFMANN J. A. (3H-ecdysone), and the supply of pupae from Drs. K. (1979) Prothoracic gland activity and blood titres of GRZELAK, W. H. TELFI:R, and .I. H. WILLIS. Skillful technical ecdysone and ecdysterone during the last larval instar of assistance was provided by A. VARADI, H. PECINOVA and Locusra migratoria L. J. Insect Physiol. 25, 255-261. L. SENOHR,&BKOV.&. We are grateful to Dr. J. P. DELBECQUE HIRUMA K., SHIMADAH. and YAGI S. (1978) Activation of the for checking some of our data in an independent assay. prothoracic gland by juvenile hormone and Critical reading of the manuscript by Dr. K. A. KUMARAN is prothoracicotropic hormone in Mamestra brassicae. also much appreciated. J. Insect Physiol. 24, 215-220. H~~Ao T. H. and HSAIO C. (1977) Simultaneous determination of molting and juvenile hormone titers of REFERENCES the greater wax moth. J. Insect Physiol. 23, 89-93. HSIAO T. H., HSIAO C. and DE WILDE J. (1976) Molting hormone titer changes and their significance during AGUI N., GRANGER N. A., GILBERT L. I. and BOLLENBACHER development of the Colorado beetle, Leptinotarsa W. E. (1980) Cellular localization of the insect decemlineata. J. Insect Physiol. 22, 1257-i 261. prothora&otropic hormone: In vitro assay of a single HWANC -Hsu K., REDDY G., KURMARAN A. K., neurosecretory cell. Proc. natn. Acad. Sci. U.S.A. 16, BOLLENBACHER W. E. and GILBERT L. I. (1979) 5694-5698. Correlations between juvenile hormone esterase activity. AGLJI N. and YAG~ S. (1973) Relationship between ecdysone titre and cellular reprogramming in Galleria endogenous ecdysone titer and in vitro activity of mellonella. J. Insect Physiol. 25, 105-l 11. prothoracic glands of Mamestra brassicae (Lepidoptera: KING D. S. (1972) Ecdysone metabolism in insects. Am. Zool. Noctuidae). Appl. Em. 2001. 8, 239-240. 12, 343-345. BERGOT B. J., SCHO~LEY D. A., CHIPPENDALEG. M. and YIN KING D. S., BOLLENBACHERW. E., BORST D. W., VEDECKIS C.-M. (1976) Juveniie hormone titer determinations in the W. V.. O’CONNOR J. D., ITTYCHERIAHP. I. and GILBERT southwestern corn borer, Diatraea grandosella, by electron L. I. (1974) The secretion of I-ecdysone by the prothoracic capture-gas chromatography. Life Sri. 18, 8 1 I-820. glands of Manduca sexta in vitro. Proc. natn. Acad. Sci. BOLLENBACHERW. E.. VEDECKIS W. V., GILBERT L. I. and U.S.A. 71, 793-796. O’CONNOR J. D. (1975) Ecdysone titers and prothoracic KOOLMAN J. (1978) Metabolism of ecdysteroids. In gland activity during the larval-pupal development of Comparative Endocrinology (Ed. by GAILLARD P. J. and Manduca sexta. Devl. Biol. 44, 46-53. BOER H. H.) pp. 495-498. Elsevier/North Holland, BOLLENBACHER W. E., ZVENKO H., KUMARAN A. K. and Amsterdam. GILBERT L. I. (1978 I Changes in ecdysone content during KRISHNAKUMARAN A. and SCHNEIDERMAN H. A. (1965) postembryonic development of the wax moth, Galleria Prothoracotrophic activity of compounds that mimic mellonella: The role of the ovary. Gen. camp. Endocri. 34, juvenile hormone. J. Insect Physiol. 11, 1517-l 532. 169-179. LAFONT R., MAUCHAMP B., BLAIS C. and PENNETIER J.-L. CHINO H., SAKURA~S., OHTAKI T., IKEKAWA N.. MIYAZAKI (1977) Ecdysones and imaginal disc development during H., ISHIBASHI M. and ABUKI H. (1974) Biosynthesis of the last larval instar of Pieris brassicae. J. Insect Physiol. ecdysone by protho,-acic glands in vitro. Science N. Y. 183, 23, 277-283. 529-530. MALL J.. GRANGER N. A. and SEHNAL F. (1977) Control of CHIPPENDALE G. M. (1977) Hormonal regulation of larval prothoracic glands in the larvae of Galleria mellonella. diapause. A. Rev. I%. 22, 121-138. J. Insect Physiol. 23, 309-3 16. CHIPPENDALE G. M. and YIN C.-M. (1976) Endocrine MALL J. and SEHNAL F. (1978) Role of the nerve cord in the interactions controlling the larval diapause of the
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control of prothoracic glands in Galieria mellonella L. Evperientia 34, 1233-I 235. MARCUS W. (1962) Untersuchungen iiber die Polarit%t der Rumpfhaut von Schmetterlingen. IWhelm Roux Arch. 154, 56-102. MAR& P. and TARNOY K. (1978) Moulting hormone level during the last instar of Galleria mellonella larva. J. Insect Phvsiol. 24, 325-327. MAR~)Y P., VARC~HA J. and HORVATH K. (1977) Rapid heterologous haptene radioimmunoassay for insect moulting hormone. FE&S Letters 81, 319-322. MASNERP.. HANGARTNEKW. and SUCHY M. (1975) Reduced titres ofecdysone followingjuvenile hormone treatment in the German cockroach. Blattella germanica. J. Insect Physiol. 21, 1755-l 762. MCDANIEL C. N. (1979) Haemolymph ecdysone concentratlons in Hyalophora cecropia pupae, dauer pupae and adults. J. Insect Pilysiol. 25, 143-145. MITSUI T. and RIDDIFORD L. M. (1978) Hormonal requirements for the larval-pupal transformation of the epidermis of Manducu serta in vitro. Dev. Biol. 62, 193-205. NI.IHOUT H. F. and WILLIAMS C. M. (1974) Control of moulting and metamorphosis in the tobacco hornworm. Manduca sezta (L.): Cessation of juvenile hormone secretion as a trigger for pupation. J. esp. &a/. 61, 493-501. NISHIITSL!TSUJI-~JWOJ. and NISHIMURA M. S. (1972) Adult development induced by the injection of non-hormonal agents into brainless pupae of silkworms. Appl. Ent. 2001. 7, 207-216. PIEPHO H. (1939) iiber den Determinationszustand der Vorpuppenhypodermis bei der Wachsmotte Galleria mellonella L. Biol. Zbl. 59, 314-326. RIDDIFORD L. M. and TRUMAN J. W. (1978) Biochemistry of insect hormones and insect growth regulators. In Biochemistry qf Insects (Ed. by ROC.KSTEIN M.) pp. 307-357. Academic Press, New York. SEH&AL F. (1966) Kritisches Studium der Bionomie und Biometrik der in verschiedenen Lebensbedingungen geziichteten Wachsmotte, Galieria mellonella L. (Lepidoptera). 2. wk. 2001. 174, 53-82.
AND
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SEHNAL F. (1968) Influence of the corpus allatum on the development of internal organs of Galleria mellonella L. (Lepidoptera). J. Insect Physiol. 14, 73-86. SEHNAL F. (1972) Action of ecdysone on ligated larvae of Galleria mellonella L.. (Lepidoptera): Induction of development. Acta ent. bohemoslav. 69, 143-155. SEHNAL F. (1976) Action of juvenoids on different groups of insects. In The Juvenile Hormones (Ed. by GILBERT L. I.) pp. 301-322.. Plenum Press. New York. SEHNAL F. and GRANGER N. A. (1975) Control of corpora allata function in larvae of Galleria mellonella. Bicl. Bull. 148, 106-I 16. SEHNAL F. and NOVAK V. J. A. (1969) Morphogenesis of the pupal integument in the waxmoth (Galleria mellonella) and its analysis by means of juvenile hormone. Acra ent. bohemoslov. 66, 137-145. SEHNAL F. and SCHNIEDERMANH. A. (1973) Action of the corpora allata and of juvenilizing substances on the larval-pupal transformation of Galleria mellonella L. (Lepidoptera). Acta enf. bohemoslov. 70, 289-302. TAKEDA N. (I 978) Hormonal control of prepupal diapause in Monemaflavescens (Lepidoptera). Gen. camp. Endocr. 34, 123-131. WILLIAMS C. M. (1947) Physiology of insect diapause. II. Interaction between the pupal brain and prothoracic glands in the metamorphosis of the giant silkworm, Platysamia cecropia. Biol. Bull. 93, 89-98. WILLIAMS C. M. (1967) The present status of the brain hormone. In Insect and Physiology (Ed. by BEAMENT J. W. L. and TREHERNE J. E.1 pp. 133-139. Oliver and Boyd, Edinburgh. WILLIAMS C. M. (1968) Ecdysone and ecdysone-analogues: their assay and action on diapausing pupae of the Cynthia silkworm. Biol. Bull. 134, 344-355. YAGI S. and FUKAYA M. (1974) Juvenile hormone as a key factor regulating larval diapause of the rice stem borer, Chilo suppressalis. Appl. Em. 2001. 9, 247-255. YIN C.-M. and CHIPPENDALEG. M. (1973) Juvenile hormone regulation of the larval diapause of the southwestern corn borer, Diatraea grandiosella. J. Insecr Physiol. 19, 2403-2420.
1981
0022-1910/81/080535-10$02.00/O ‘c 1981 Pergamon Press Ltd.
REGULATION AND SIGNIFICANCE OF ECDYSTEROID TITRE FLUCTUATIONS IN LEPIDOPTEROUS LARVAE AND PUPAE FRANTISEK SEHNAL*, PETER MARbY? *$Institute of Entomology, Czechoslovakia and tInstitute
Czechoslovak of Genetics.
and
JAROSLAVAMALA$
Academy of Sciences, Na Folimance 5, 12000 Prague, Hugarian Academy of Sciences. H-6701 Szeged, Hungary
(Received 10 December 1980)
Abstract-The last larval moult of GaNeria mellonella is induced by an elevation of ecdysteroid titre to more than 200 rig/g.. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr. is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 rig/g)) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500-2500 rig/g)) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 rig/g)) in the middle of pupal instar. Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 3040 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing increase of the prothoracit:otropic neurohormone (PTTH). In either case, the pupation-inducing ecdysteroids is ? times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed. Neither in G’. mellonella larvae. nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid prosduction or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5-15 times lower than usual ecdvsteroid concentration when the last-instar larvae are exposed to JH.
INTRODUCTION THE PRIMARY sources of ecdysteroids in most immature insects are the prothoracic glands (CHINO et al., 1974; KING er al.. 1974). They produce a prohormone, ecdysone, which is converted to the actual hormone, 20- hydroxyecdysone in peripheral tissues (KING, 1972). 20Both ecdysone. hydroxyecdysone and their hormonally-active metabolites are inactivated biochemically and/or depleted by excretion I:KOOLMAN,1978). Their content in the body obviously depends on the balance between the rate of ecdysone secretion and the rate of ecdysteroid metabolism. Available information demonstrates that the level of secretion plays the crucial role (AGUI and YAGI, 1973; BOLLENBACHERet al., 1975; HIRN et al., 1979). Isolated larval abde3mens of some insects produce enough ecdysteroids tc3 support metamorphosis to the adult stage (HSIAO et LI/., 1976; DELBEQUEet al., 1978). With the exception of some hybrids of Bombay mori (cf. NISHIITSUTSUJI-Uwo and NISHIMURA, 1972), no *To whom correspondence I,= 27.8
-c
should
be sent.
such development occurs in Lepidoptera. The low titre of ecdysteroids in the isolated abdomens of caterpillars demonstrates that their prothoracic glands are indispensable for development (LAFONT et al., 1977). This is further proven by the induction of development in isolated larval (FUKUDA, 1944) and pupal (WILLIAMS, 1947) abdomens of Lepidoptera which are supplied by implants of prothoracic glands. Prothoracic glands of both larvae (GIBBS AND RIDDIFORD, 1977; MALA et al., 1977) and pupae by pro1947) are stimulated (WILLIAMS, thoracicotropic neurohormone (PTTH) from the brain (AGUI et al., 1980). The release of PTTH depends both on the environmental and intrinsic factors which are integrated in the central nervous system (MALL and SEHNAL, 1978; RIDDIFORD and TRUMAN, 1978). The intrinsic factors seem to include the juvenile hormone (JH). which in the last-instar larvae of Manduca sexta reportedly inhibits the PTTH release (NIJHOUT and WILLIAMS. 1974). Delay of the larval-pupal transformation, which is induced by JH and juvenoids in many endopterygote insects (SEHNAL, 1976) and in some species occurs naturally as larval diapause (YIN and CHIPPENDALE. 1973; YAGI and 535
536
FRANTI~EK SEHNAL.PETER MARTY
FUKAYA, 1974), has been explained as a result of such an inhibition of PTTH production (CHIPPENDALEand YIN, 1976: TAKEDA, 1978). An alternate explanation is that JH directly suppresses the function of larval prothoracic glands (CIEMIOR er a/., 1979), although it seems to activate the prothoracic glands of decerebrate or diapausing pupae (GILBERT and SCHNEIDERMAN, 1959?. It is obvious that the control of ecdysteroid production is complex and involves PTTH action on prothoracic glands. JH action on the brain and prothoracic glands, and apparently also influence of the thoracic ganglia on the glands (MALA et al., 1977; GERSCH, 1978). The larvae of Galleria mellonella and some other species moult when the brain, corpora allata and the thoracic ganglia are removed (MALL er al.. 1977). Their prothoracic glands clearly secrete enough ecdysone spontaneously. It is not known. however, if low quantities of ecdysteroids are continuously present in the body and cause a sequence of ‘covert effects’ (WILLIAMS, 1968) until an ‘overt effect.’ the moult, is accomplished, or if there is a delayed spontaneous surge ofecdysone release, similar to that which occurs in intact insects upon stimulation of prothoracic glands by PTTH. In the present study we examine regulation of ecdysteroid production by measuring ecdysteroid titre in G. mellonella subjected to various experimental manipulations. The effects of JH are also investigated in diapausing pupae of Hyalophora cecropia and Celerio euphorbiae.
MATERIALS
AND METHODS
Insects and surgery The wax moth, Galleria mellonella L. (Pyralidae) was reared under standard conditions (SEHNAL, 1966). Larvae and pupae of precisely-known age in respect to the preceding ecdysis were used for the experiments. Behaviouristic (wandering, spinning and mobility of the larvae) and morphological markers (progress of apolysis and eye-pigment migration) were employed as additional criteria for assessing the physiological age of the last-instar larvae. Decapitated larvae and isolated larval abdomens were obtained by means of ligation applied behind the head and behind mesothorax respectively; the body part anterior to the ligation was cut off. One day after ligation some insects received implants of brains and/or endocrine glands. All the surgical procedures performed with Galleria mellonella have been described earlier (MALT et al., 1977). The diapausing pupae of Hvalophora cecropia (Saturniidae) were kindly provided by Dr. W. H. TELFER of the University of Pennsylvania, Philadelphia, and Dr. J. H. WILLIS of the University of Illinois, Urbana-Champaign. No difference between these two groups of pupae was found. The diapausing pupae of Celerio euphorbiae L. (Sphingidae) were obtained through the courtesy of Dr. K. GRZELAK of the Institute of Biophysics and Biochemistry, Polish Academy of Sciences, Warsaw. These pupae came from caterpillars, which were collected in central Poland on Euphorbia cyparisias and raised afterwards under a short photoperiod indoors.
AND
JAROSLAVA MALA
Operations were performed on pupae anaesthetized in a stream of carbon dioxide and surface-sterilized with 70‘!/, ethanol. The head and thorax were cut off and the abdomen was supplied, according to the protocol, with implants taken from the removed front parts. The cut was then covered with a round piece of plastic coverslip and sealed along the margin with beeswax. Hormones and their application Synthetic JH-II (methyl [2E,6E,lOZ]-3,7,1 ltrimethyl-10,l l-epoxy- 2,6_tridecadienoate) and the juvenoid SJ-42-F (ethyl [2E]-3,7,11-trimethyl-2dodecenoate) were kindly given to use by Dr. V. JAROL~Mof the Institute of Organic Chemistry and Biochemistry, CSAV, Prague. In the experiments with G. mellonella the compounds were dissolved in acetone and adminstered topically. The diapausing pupae and isolated pupal abdomens of H. cecropia and C. euphorbiae were injected with undiluted juvenoid. Monitoring the MH titre In all experimental insects we recorded initiation and progress of the moulting process as indications of in ecdysteroid titre. For direct an increase measurements of ecdysteroids, 2-25 specimens were grouped to provide a sample weight of 100-600 mg. Each sample was homogenized in a motor-driven Potter-Elvehjem glass homogenizer in 4 ml 60% methanol, which was supplemented with 10e4 M phenylthiourea. The homogenates were stored for 2-8 weeks and then used for MH determination by radioimmunoassay described previously (MAROY et al., 1977; MAR~)Y and TARN~)Y, 1978). No separation of individual ecdysteroids was attempted; our data do not distinguish between ecdysone, 20-hydroxyecdysone and their metabolites. The antiserum we used was equally sensitive to ecdysone and 20hydroxyecdysone. A few complementary estimations of ecdysteroid titre in the penultimate-instar larvae were done by Dr. J. P. DELBECQLJE of the University of Dijon. The background level was about 5 times lower but the titre changes during the instar were indentical with those found by our assay.
RESULTS Changes of ecdysteroid titre during development of Galleria mellonella During the examined period, the titre of ecdysteroids rose to a peak in the second third of the penultimate-instar and then dropped to levels below 20 rig/g at the beginning of the last larval instar (Fig. 1). A slight but significant (P
Regulation
of ecdysteroid
537
titre
AE II 2ooc 600 -
0 I
24
VI
0
40
ARREST
96
144
VII
I
192
0
I
I
48
96
144
192
P
I
A
Fig. I. The ecd ysteroid titre in Galleriu me/lonellu during the penultimate and last larval instars (VI, VII), the pupal instar (P), and in freshly-emerged adults (A). Note that two different scales of ecdysteroid concentration are used and that the data for female pupae and adults (f) are presented separately from those concerning the males (m). The length of each instar is divided into 24 hr intervals (development of the lastinstar larvae w,as arrested for 8 days by preventing the larvae to leave their food). Arrows indicate apolyses (A), ecdyses (E), wandering (W), and spinning (S), respectively. End of the spinning was accompanied by a drop in the titre at 152 hr of the instar. In apolysing larvae the
ecdysteroid content reached a maximum of 780 rig/g (over 1000 rig/g in a few individuals), followed by a decrease to about 2013 rig/g in pharate pupae. Female pupae consistently possessed a higher ecdysteroid titre than the maIes, although the relative fluctuations during most of the instar were similar in both sexes (Fig. 1). Ecdysteroid concentration reached a high peak at the beginning of the instar and a smaller peak in the middle of the instar. This second peak was confined to a brief time interval: the amount of ecdysteroids determined at 96 hr was significantly higher (P
Stage Last-instar larvae 120 hr after ecdysis Female and male pupae (1:1) 24 hr after ecdysis 120 hr larvae 2 days after the removal of head and thorax by ligation
1. Distribution
Head
of ecdysteroids
+ prothorax
751 + 124
The source of ecdysteroids In wandering last-instar larvae and in pupae 24 hr after ecdysis we determined ecdysteroids separately in the head and prothorax, and in the remaining body part. Summation of the two values compared well with the content established in intact insects of the same age (Table 1). In both larvae and pupae, however, we found that the concentration expressed per gram of fresh weight was one order of magnitude higher in the front parts than in the hind parts. When the prothoracic glands contained in the front parts were removed from the wandering larvae by means of a ecdysteroid ligation behind prothorax, the concentration in isolated abdomens decreased within 2 days to 40 rig/g.. We interpreted these findings as a confirmation that the prothoracic glands are the source of ecdysteroids in the larvae and early pupae of G. mellonella. A drop of ecdysteroid titre and cessation of development was observed in all larval abdomens isolated prior to 152 hr of the last-instar. The in larvae and pupae
of G. meNoneNu
Meso- and metathorax + abdomen
Total
Intact control
69 + 1
95 * 2
100 + 13
(8.3 mg)
(184 mg)
(194 mg)
(208 mg)
3290 + 1205
411 & 141
650 + 230
901 + 153
(12 mg)
(122 mg)
(137 mg)
(153 mg)
40 f
19
(170 mg)
The ecdysteroid content is given in rig/g fresh weight (mean + SE.): the fresh weight of examined parentheses. Two groups of 10 insects were taken for each determination.
body parts is indicated
in
FKANTISEK SEHNAL. PETERMARRY AND JAROSLAVA MALA
538
abdomens isolated at 152 hr occasionally produced patches of pupal cuticle. These individuals contained an elevated titre of ecdysteroids (198 rig/g)) and we assume that they were ligated after the final ecdysteroid increase in the last-larval instar had begun. Stimulation of ecdysteroid content The ecdysteroid titre of larvae also decreased when only the head had been removed. For example, decapitation of penultimate-instar larvae caused within 2 days a decrease from 205 + 32 rig/g to 51 + 18 rig/g (averages from 30 animals analyzed in 3 samples each time). In larvae decapitated 120 hr into the last-instar, the titre was below 50 rig/g on days 3 and 10 but increased to 200 rig/g by day 20 after decapitation (Fig. 2A). This level was also found in about 60% of the samples taken for the determination between 30 and 40 days. The remaining samples contained well over 1000 rig/g;; no intermediate figures were found. Since the percentage of samples with high ecdysteroid content corresponded to the number of insects pupating between 30 and 40 days (MALA et al., 1977), we assume that the individuals with a high ecdysteroid content are those which had initiated the larval-pupal moult. The decapitated larvae, which underwent apolysis as revealed by their body shape and reduced mobility, contained invariably over 2000 rig/g ecdysteroids. This concentration was much higher than that found in intact larvae (Fig. 1) but, as
in intact larvae, the titre dropped abruptly and concurrent with the secretion of the pupal cuticle (Fig. 2A). In pupae freshly formed from the decapitated larvae the content of ecdysteroids was only 200 rig/g.. Both the increase in ecdysteroid titre and the secretion of pupal cuticle were accelerated following implantation of 3 larval brains into each decapitated larva. The titre was elevated substantially already on day 10 after ligation (9 days after implantation) and most insects apolysed within the next 2 days. In these insects the ecdysteroid content reached values over 2500 rig/g,, as were found in decapitated larvae without brain implants only 3040 days after ligation (cf. Figs. 2A and B). The titre dropped in freshly-formed pupae; it was higher than in spontaneously-pupating insects but the difference was probably due to variations in the age of analyzed specimens. The increase of ecdysteroids was also accelerated in decapitated larvae which received implants of 3 braincorpora cardiaca-corpora allata complexes (Fig. 3A) and in those which were implanted with 3 brains and simultaneously treated with 0.5 pg of JH-II (Fig. 3B). These insects apolysed already 6-10 days after the implantation and produced either pupae with patches of larval cuticle or larval-pupal intermediates (cf. SEHNAL and SCHNEIDERMAN.1973). The general course of ecdysteroid titre was similar as in the previouslydescribed groups of decapitated insects (Fig. 2A and B), but the maximal concentration was only 1000 rig/g
No sign of moult Apolyzed Freshly
u
insects ecdysed
No sign of moult Apolyzed
insects
Freshly
g
insects ecdysed
insects
B
140(
I $
120(
Q 2
lOO(
E f
60(
B 8 60( n” s 40(
No Treatment Days After
I Indicated
Brain
l--dYf+ 6
Implants k-CC-CA
implants
7
1 JH d Brain
E-10
Implants
Treatments Days
Fig. 2. Changes in the ecdysteroid content after decapitation (A) and in the decapitated larvae which were supplied with the implants of 3 brains (B). The larvae were decapitated 120 hr of the last-instar and the brains provided by donors the same age.
at of
After
Indicated
Treatments
Fig. 3. Changes in the ecdysteroid content in the decapitated larvae which received either implants of 3 complexes braincorpora cardiaca+orpora allata (A) or implants of 3 brains supplemented with topical application of 0.5 pg JH II (B).
Regulation
of ecdysteroid
in insects with implanted complexes (Fig. 3A) and 600 rig/g in those supplied with brains and treated with JH-II (Fig. 3B). In decapitated larvae, which developed into larva-pupal intermediates following an implantation of 3 brains and a treatment with 10 pg SJ-42-F (not shown in Fig. 3). the ecdysteroid titre also reached a maximum of only 608 rig/g of body weight (average value from the analysis of 3 specimens at the time of apolysis).
S $ $
The increase in ecdysteroid content followed by secretion of pupal cuticle occurred in more than 80’; of the decapitated larvae. A similar proportion of decapitated larvae moulted when implanted with 3 brains either with or without corpora cardiaca+orpora allata. or when implanted with 3 brains and treated with SJ-42-F (Table 2, upper part). On the other hand, implantation of corpora cardiaca+orpora allata without the brain reduced the proportion of moulting insects to 159, and the treatment with SJ-42-F to Y0 (Table 2). The average ecdysteroid content in the last 2 groups remained very low for 20 days after treatment. The slight increase which was found 20-30 days after implantation of corpora cardiaca+orpora allata apparently indicated that a few of the insects initiated the moulting process (Fig. 4A). A single specimen. which actually did produce a new larval cuticle, contained 192 rig/g.. The majority of those decapitated larvae, which were treated with 0.5 pg JH-II on days 1, 16 and 25 after ligation, maintained a low ecdysteroid titre throughout 40 days of the experiment. Only in a few of them, which apolysed between days 8 and 10, the titre increased to 624 rig/g (Fig. 4B). It is obvious from these results that corpora cardiaca
Table 2. The incidence,
I
Nosign
EB
Apolyzed Insects
m
Freshly ecdysed Insects
of moult
600
ij 400
D
e F L
ZCKJ
3
Days after lndlcoted treatments
Fig. 4. Changes in the ecdysteroid content of decapitated larvae which were either implanted with 3 pairs of copora cardiacs-corpora allata (A) or treated with 0.5 pg JH II on days 1. 16 and 26 after decapitation (B).
of ecdysteroids and consequently reduced the number of spontaneously-moulting decapitated larvae. The bottom part of Table 2 demonstrates that the moults of isolated abdomens, which had been supplied with prothoracic glands, were also hampered by corpora allata implants and the juvenoid. Clearly, intact inervation and localization of prothoracic glands are irrelevant for this JH effect. A decrease in ecdysteroids was observed also in the
timing and type of moult in decapitated larvae and isolated larval abdomens subjected to various treatments
0
Treatment Decapitated
Moulted 0
Isolated implanted + 3 br implanted + 3 cc-ca implanted implanted + 10 pg SJ-42-F
Days
Type of moult
larvae 83 88 71 67 15 5
None 3 br implanted 3 br-cc-ca implanted 3 br implanted + IO pg SJ-42F 3 ccsa implanted 10 /q SJ-42-F
None 2 PC 2 PC 2 PC 2 PC
1
e
Suppression of ecdvs teroid titre
539
titre
41.8 14.0 12.6 14.8 30.0
f 9.6 + 3.6 +_ 4.1 f 2.1 + 13.4 24
Pupal Pupal Intermediate Intermediate Intermediate Intermediate
larval abdomens 0 32 35 12 0
29.8 + 3.1 15.8 f 1.9 36.0 + 6.1
No moult Pupal Pupal Intermediate No moult
Twenty insects were used in each experiment. The figures under Days represents means + S.D. The accomplished moults were perfect in every respect but the insects could not escape from the old exuvia. The new cuticle was either exclusively pupal of both larval and pupal cuticles were produced (intermediate moult). The non-moulting insects usually died more l.han 40 days after ligation. Abbreviations: (br) brains, (cc) corpora cardiaca, (ca) corpora allata, (PG) prothoracic glands.
FRANTISEK SEHNAL. PETER MARTY
540 Table
3. Ecdysteroid
AND
JAROSLAVA MALL
titre (mean + SE.) at comparable physiological treated and control larvae of G. melioneilu Treated
Stage Crawling Apolysis Apolysis Ecdysed
168* 168.f 180: 192:
14 41 135 72
* + + +
Titre
Age 4 7 12 6
120 168 168 184
112 756 756 278
* + f +
The age of analyzed insects is given in hr after the last larval ecdysis. Untreated ecdysed into pupae whereas the treated insects developed into superlarvae. * 1 pg SJ-42F applied at 84 hr of the last-instar. t 100 pg SJ-42-F at 84 hr. $10 pg JH II at 120 hr.
Table 4. Induction
of development
of JH-
Controls Titre
Age
stages
in isolated abdomens euphoribiae
of diapausing
21 96 96 54 controls
pupae of Celerio
Ecdysed Dead
Treatment (number of insects) 500 pg 3 PTG 3 PTG 3 PTG 3 br +
Number
SJ (8) + 500 pg oil (6) f 500 pg SJ (6) + 3 br + 500 pg SJ (5) 500 pg SJ (5)
(day)
Day
0 0 0 4 0
65 + 12”
89 ) 36 69 + 28 104 +_ 43 180 60 + 11
The implants were taken from the same pupae which provided the isolated abdomens. Olive oil and the juvenoid SJ-42-F were injected 2 weeks after the abdomens had been isolated and supplied with the implants. Abdomens which completed secretion of new cuticle and resorbed the moulting fluid were classified as ‘ecdysed.’ Implants: (PTG) prothoracic glands; (br) brains. * Represents mean + S.D.
Table
5. Induction
of development in the isolated abdomens Hyalophoro cecropia
of diapausing
pupae
of
Ecdysed Treatment (number of insects)
Number
500 pg oil (7) 500 pg SJ or 100 pg JH (7) 3 PTG + 500 ~1 oil (13) 3 PTG + 500 ~1 SJ or 100 ~1 JH (16) 3 PIG + 2 or 3 br (8) 2 or 3 br + 500 ~1 oil (10) 2 or 3 br + 500 ~1 SJ or 100 ~1 JH (10)
0 1 1 2 8 10 10
Dead Day
204 240 177 f 79 + 90 * 82 f
(day)
61 34 30 19
442 372 520 434
i 213 I 153 i 141 i 169
Only abdomens which survived for 40 days were evaluated. Most experiments were performed with males; female abdomens received 3 brains, whereas male abdomens 2 brains. Other procedures were identical to those described for C. euphorbia (Table 4). No differences were found between insects injected with SJ-42-F and those injected with JH (JH-I and JH-II 8:l). The figures represent means + S.D. non-ligated
last-instar
larvae
which
were treated
with
JH or the juvenoid (Table 3). Application of 1 pg SJ-42-F at 84 hr of the instar caused a great delay in the pupa1 moult, whereas treatment with 100 pg SJ-42-F induced an extra larval moult (cf. CIEMIOR et al., 1979). The ecdysteroid concentration decreased in both cases. The insects treated with 100 pg SJ-42-F underwent larval apolysis at an extremely low concentration of less than 50 rig/g.. A similar extra larva1 moult was also induced with 10 pg JH-II adminstered at 120 hr of the last-instar. The highest ecdysteroid titre found in these insects was 165 rig/g,,
which is one fifth of the level found in the untreated controls undergoing the pupal moult (Table 3). The stimulation
of development
in diapausing pupae
Isolated abdomens of diapausing pupae of Celerio euphorbiae did not develop although some of them survived for half of a year (Table 4). Implantation into each abdomen of 3 prothoracic glands, which were taken from the discarded front parts of the same pupae, was insufficient to induce development. Simultaneous application of 500 pg/specimen of SJ-42-F, a treatment which induced development in 8
Regulation of ecdysteroid titre
intact diapausing pupae, (not in the table) failed to stimulate the implanted glands. By contrast, when the glands were implanted simultaneously with 3 brains (taken also from diapausing pupae), 80% of the abdomens developed and produced new cuticle with adult scales. In some regions the new cuticle possessed pupal features, obviously due to the juvenilizing action of SJ-42-F. Implanalion of brains without prothoracic glands had no effect (Table 4). Experiments with diapausing pupae of Hyalophora cecropia yielded similar results in that all preparations receiving prothoracb: glands and 2-3 brains developed into adults. In addition, in H. cecropia the moult was also induced in isolated abdomens without the gland implants (Table 5). When these abdomens were supplied with 2-3 ‘brains, all of those which have survived for 50 days moulted within the next 60 days. For this effect it was irrelevant if the abdomens received injection of olive oil or of JH; in the latter cuticle was case, however, the newly-secreted predominantly pupal. Only exceptionally a late moult (134-240 days after the isolation) occurred in abdomens supplied with prothoracic glands and treated with olive oil or with JH without the brain implants.
Significance of chanpes in ecdysteroid content during development
The ecdysteroimd amounts established in G. mellonella in the TIresent study are comparable with the figures published by HSIAOand HSIAO(1977) and BOLLENBACHER et al. (1978). Our results indicate that larval moults, including apolysis, epidermal proliferation and cuticle secretion, are caused by single ecdysteroid peaks (Fig. 1). The larval-pupal and pupal-adult transformations are controlled by ecdysteroids in a complex way. The larval-pupal transformation, including epidermal reprogramming, begins at about 60 hr of the last larval instar, as indicated by the loss of tissue sensitivity to JH (SEHNAL, 1968; SEHNAL and SCHNEIDERMAN, 1973). The transformation is probably released by, and its progress depends on, the increase in ecdysteroid titre in the absence of JH. Coincidently, secretion of JH from the corpora allata is prevented via nerves at 48-60 hr (SEHNAL and GRANGER,1975). The reprogramming of the epidermis extends over most of the last larval instar (HWANGHSUet al., 1979). The responsiveness of epidermal cells to JH decays rapidly during the ecdysteroid peak between 120 and 144 hr, concurrently with increased epidermal mitotic activity (PIEPHO,1939; SEHNALand NOVAK, 1969). The epidermis retains its sensitivity to high JH doses until 152 hr when the major ecdysteroid peak begins. This pe,ak controls the completion of the larval-pupal moul ting process and associated metamorphic events. For example, evertion of the wing discs and degeneration of specialized larval organs such as the silk glands and proventriculus occur at this time (S~HNAL,1968); ecdysteroid control over these processes was documented in experiments with isolated larval abdomens (SEHNAL.1972). It would seem that completion of pupal
541
development can be controlled by a single broad ecdysteroid peak at the end of the last larval instar. The occurrence of 2 distinct peaks in Lepidoptera (BOLLENBACHER et al., 1975; LAFONT et al., 1977; MARTYand TARN~Y,1978; DEANet al., 1980). may be advantageous, however, because it provides a mechanism for adjusting the development to environmental conditions. The ecdysteroid increase leading to the second peak may be delayed and the insects, such as caterpillars of G. mellonella exposed to unfavourable conditions (SEHNAL, 1966) enter diapause. Fluctuations of ecdysteroids also affect the behaviour of larvae. Several observations indicate that in G. mellonella the ecdysteroid increase at 120 hr during the last-instar terminates feeding and releases wandering followed by cocoon spinning. For example, caterpillars which maintain a permanently low ecdysteroid titre after JH treatment (Table 3), continue to feed over extended periods of time and do not spin (CIEMIORet al., 1979). When in normal larvae the ecdysteroid increase is interrupted after it has begun by depriving the larvae of sufficient space, the spinning is also prevented (Fig. 1). On the other hand, injection of 20-hydroxyecdysone into feeding larvae causes termination of feeding and at least in some cocoon cases induces spinning (unpublished observation). A high ecdysteroid titre in the freshly-ecdysed pupae of G. mellonella apparently supports the early onset of the pupal-adult transformation. Imaginal apolysis and extensive epidermal differentiation, which take place between 30 and 40 hr after pupal ecdysis (MARCUS, 1962), are probably caused by the high ecdysteroid peak at 36 hr (Fig. 1). The small ecdysteroid increase at 96 hr is associated with the secretion of imaginal cuticle. The following rise of ecdysteroids in females has been elucidated by BOLLENBACHER et al. (1978) and HSIAO and HSIAO (1977). Our results provide ample support for observations that moults may be induced with much lower concentrations of ecdysteroids than those found in normally-developing insects (CLARET et al., 1977; LAFONT et al., 1977). The excessive peaks of ecdysteroids apparently serve as pace-makers which synchronize development of various body parts. SEHNAL(1972) observed that when isolated larval abdomens were induced to develop by repeated exposures to a steady concentration of 20-hydroxyecdysone, the progress of metamorphosis was very uneven. Induction of secretion in the prothoracic glands
The prothoracic glands appear to be the sole primary source of ecdysteroids in G. mellonella larvae. The decrease in ecdysteroid titre in decapitated larvae shows that without the brain the activity of prothoracic glands decreases and in some specimens is never restored. In most decapitated larvae, however, the glands are inactive for 30-40 days and then burst into activity, as indicated by the sudden increase of ecdysteroid titre (Fig. 2A). Initiation of development in decerebrate larvae (MALA et al., 1977) and pupae (MCDANIEL, 1979) and also in isolated abdomens supplied with prothoracic gland implants (FUKUDA,
542
FRANTISEK SEHNAL.PETERMAR~)YAND JAROSLAVA MALA
1944; WILLIAMS, 1947) is obviously caused by this ecdysteroid surge. The ecdysteroid concentration drops when the decapitated insects accomplish secretion of the new cuticle (Fig. 2A), hence, the prothoracic glands return to a state of relative inactivity. The induction of such a surge is apparently a characteristic attribute of prothoracic glands, which is independent of the nature of their stimulation. It was consistently observed in all our experiments. The decline in activity at the end of secretion may be regulated through a feed-back mechanism by the blood concentration of ecdysteroids and/or by their effects, because it always occurs after the insects had produced new cuticle. Ecdysteroid production from unkown organs in isolated abdomens of Leptinotarsa decemlineata also shows spontaneous fluctuations (HSIAO et al., 1976). Brain implants accelerate the ecdysteroid increase and pupation of decapitated larvae (Fig. 2B). Except for this acceleration, the magnitude of the surge of ecdysteroid titre is similar as in insects whose prothoracic glands were activated spontaneously (cf. Figs. 2A and B). It is obvious that the brain implants influence neither the rate of ecdysone secretion, nor the velocity of ecdysteroid degradation and excretion. The prothoracicotropic neurohormone (PTTH), which is apparently released from the implanted brains, acts as a trigger inducing secretion in the prothoracic glands. The regulation of ecdysteroid production
by JH
Our data show that the moult-inducing spontaneous surge of ecdysteroids is prevented with JH (Table 2). The spontaneous moults are also rare in decerebrate larvae, whose corpora allata remain in situ and apparently produce some JH (MALL et al., 1977). When these insects moult in spite of the presence of JH, their ecdysteroid titre never reaches the values found in insects of comparable age but lacking JH (Fig. 4). These data demonstrate clearly that JH suppresses the spontaneous activation of prothoracic glands and restrains the function of active glands. On the other hand, the activation of prothoracic glands by PTTH is not prevented by JH. As seen from Table 2, JH affects neither the number of moulting insects, nor the timing of moult in decapitated larvae which have been supplied with brain implants. The ecdysteroid titre in treated insects is always lower than in the non-treated controls (cf. Figs. 2 and 3) but nevertheless increases to a moult-inducing peak. In the presence of JH the prothoracic glands apparently respond to PTTH but secrete less ecdysone. The data Of CHIPPENDALEand YIN (1976) indicate existence of a similar mechanism in Diatraea grandiosellu. They noted that intact diapausing caterpillars moult in the presence of JH but the decapitated caterpillars do not moult. We suggest that the prothoracic glands of intact insects are activated by PTTH whereas the decapitated larvae not only lack PTTH but a spontaneous activation of their prothoracic glands is prevented by JH. Induction of a prompt moult by brain implants in JH-treated decapitated larvae of G. mellonella (Table 2) indicates that JH does not influence the PTTH liberation from the implanted brain. The regulation by JH of PTTH release from the brain in situ is more
complex. Thus, JH applied in intact larvae at the start of the last-instar induces a larval moult within 4-5 days whereas the controls undergo a pupal moult in 7-8 days (CIEMIORet al.. 1979). The prothoracic glands of treated larvae clearly become active earlier than those of the controls. In this case, JH apparently accelerates the release of PTTH, which in turn stimulates the prothoracic glands. By contrast, moderate doses of JH administered in the middle of the last-instar suppress the ecdysteroid production. The long period of inactivity of the prothoracic glands indicate that in this case JH prevents the PTTH release. When the jH-treated larvae receive PTTHproducing brain implants, the prothoracic glands become active and a mouit ensues (CIEMIOR ef al.. 1979). Inhibition of PTTH production was considered by some authors as the only way by which JH hinders the function of prothoracic glands (NIJHOUT and WILLIAMS, 1974; CHIPPENDALE, 1977; TAKEDA, 1978). We stress that it is just one of the mechanisms by which JH regulates the titre of ecdysteroids. In our opinion, the body concentration of JH is one of the factors which are integrated in the brain system of regulation of its prothoracicotropic activity. Depending on the other information the brain receives, JH can either accelerate or delay the liberation of PTTH. Independently of this action, JH acts directly on the prothoracic glands and inhibits their function to the extent that virtually no ecdysone is produced unless the glands are stimulated by PTTH. The rate of ecdysone production thus depends on a balance between PTTH and JH. This tentative model can explain the endocrine regulation of larval diapause and all experimental data on the effects of JH and juvenoids on the insect moult, e.g. the inhibition of moulting by JH in the larvae of Blatella gemanica (MASNER et al., 1975). Does JH ever stimulate
the prothoracic glands?
GILBERT and SCHNEIDERMANN (1959) and KRISHNAKUMARAN and SCHNEIDERMANN (1965) demonstrated that JH and juvenoids stimulate development of diapausing and decerebrate saturniid pupae. HIRUMA et al. (1978) made an identical observation on the decerebrate last-instar larvae and pupae of a noctuid moth, Mamestra brassicae. The stimulation of development in such insects was interpreted as indicating direct activation of prothoracic glands by JH. HIRUMA et al. (1978) noted, however, that the juvenoid treatment had no effect on larvae, which had been decapitated within first 4Oy; of the length of the last-instar. In our experiments with diapausing pupae of H. cecropia and C. euphorbiae (Tables 4 and S), JH never stimulated the prothoracic glands which were implanted into isolated abdomens. This contrasts with the reported action of JH on brainless pupae and reveals that JH stimulates only the prothoracic glands in situ. The hormone apparently affects the glands indirectly, via some organ(s) in the head or thorax. The glands in situ are stimulated by various agents (WILLIAMS, 1967; NISHIITSUTSUJI-UWO and NISHIMURA, 1972). Our present results provide an alternative explanation of the moult-inducing action of JH in the diapausing and decerebrate insects. We have
Regulation
of ecdysteroid
consistently observed that after a JH treatment the larvae of G. mellone!la undergo apolysis and secrete cuticle at very low levels of ecdysteroids (Table 2). Table 3 shows that the treated larvae accomplish a moult at a 5-15 times lower concentration of ecdysteroids than the untreated controls. This implies, consistently with the observations of MASNER et al. (1975), MITSUI and RIDDIFORD (1978) and other authors, that JH lowers the hormonal requirements of epidermis for cuticle secretion. Accordingly, JH should stimulate moulting in diapausing and brainless insects if their prothoracic glands produce some ecdysone. The ecdyxeroid titre in these insects would be too low to induce development in the absence of JH but could support a moult when JH is present. This kind of moult stimulation by JH very likely occurs in the diapsuing caterpillars which undergo stationary larval moults (YIN and CHIPPENDALE, 1973; YAGI and FUKAYA, 1974; TAKEDA, 1978). It has been shown that the activity of their prothoracic glands is curtailed by moderate concentrations of JH (BERGOT ef al., 1976). CHIPPENDALE(1977) and TAKEDA (1978) concluded that the diapausing larvae contain just enough ecdysteroids to undergo stationary larval moults.
titre
543
southwestern corn borer, Diarraea grandioselia. J. Insect
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