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Factors affecting change in sensitivity of prophoracic glands to juvenile hormone in Mamestra brassicae
J. Insecr Physiol., Vol. 3X. No. 2. pp. 193-199. Printed irr Grew Bri/uijl.
1982.
0022-1910/82/020193~7$03.00/0 (0 1982 Pergamon Pws.s Ltd.
FACTORS AFFECTING CHANGE IN SENSITIVITY OF PROTHORACIC GLANDS TO JUVENILE HORMONE IN MAMESTRA BRASSICAE KIYOSHI HIRUMA Institute
01‘
Agriculture and Forestry.
University of Tsukuba. Ibaraki-ken 305, Japan
Sakura-mura.
Niihari-gun.
Abstract--The prothoracic glands of the early last-instar larva of Mamestra brassicae (day O-3) were found previously to be insensitive to stimulation by juvenile hormone, whereas those later in the instar (from day 4 on) were activated by this hormone. When neck-ligatured young larvae (day-l, day-2 and day-3) were given
juvenile hormone 5-10 days after ligation, pupation was induced. Similarly, juvenile hormone induced pupation of isolated abdomens which contained prothoracic glands taken from neck-ligatured day-3 larvae 5 days after ligation. If the glands were exposed to prothoracicotropic hormone (PTTH) from implanted brains before they were transplanted to isolated abdomens, their sensitivity to juvenile hormone activation was enhanced. Ecdysone but not 20-hydroxyecdysone given every 3 hr for 12 hr also slightly enhanced sensitivity. These results suggest that prothoracic glands from either day-l. day-2 or day-3 larvae can slowly acquire a sensitivity to juvenile hormone activation by prolonged incubation in the absence of factors from the head. The acquisition of sensitivity occurs more rapidly in the presence of both a factor from the brain. presumably PTTH. and ecdysone released from the prothoracic glands themselves.
INTRODUCTION WILLIAMS(1946. 1947. 1952) found that implantation of the chilled brain of Hvulophora cecropia activated the prothoracic glands of diapausing or brainless pupae and thereby induced adult development. Recently, the brain of Manduca sexta has been shown to secrete prothoracicotropic hormone (PTTH) which activates the prothoracic glands (BOLLENBACHERet al., 1979; AGUI et al., 1979). Juvenile hormone has also been shown to activate the pupal prothoracic glands of some lepidopterans (GILBERT and SCHNEIDERMAN, 19.59; WILLIAMS, lY5Y; KRISHNAKUMARAN and SCHNEIDERMAN. 1965; R~~LLER and DAHM, 1968: R~~LLER~~al., 1969). but its prothoracicotropic role in the larva is less clear. In Mamrstra brassicae. HIRUMA et ul. (1978) and HIRUMA (unpublished) found that juvenile hormone I, II or the analogue (methoprene) could not activate the prothoracic glands during the early part of the last-larval instar, but could activate the glands beginning about the time of cessation of feeding and during the prepupal period. At all times during the last-larval instar. PTTH could activate the glands. In Spodopteru littoralis, juvenile hormone analogue stimulated the prothoracic glands shortly before pupation (CYMBOROWSKIand STOLARZ, 1979). Similar effects have been reported for Manduca sexta (SAFRANEK et ul.. lY80). It has not, however. been reported that physiological concentrations ofjuvenile hormone can stimulate the prothoracic glands in vitro. The precise roles ofjuvenile hormone and of PTTH in the activation of the glands during the normal larval-pupal transformation in Lepidoptera however remain unclear. In Manzestra the level of juvenile hormone decreases rapidly l-2 days after ecdysis to the final instar, then increases again after the cessation
of feeding (VARJAS et al., 1976; Y AGI. 1976). This latter increase has a dual function: activation of the prothoracic glands and suppression of adult development (HIRUMA, 1980). Since, during the feeding period, the glands seem to be insensitive to the prothoracicotropic effect of juvenile hormone, the experiments reported here were done to determine how the prothoracic glands gain competence to respond to juvenile hormone. MATERIALS Insect reuring
AND METHODS
conditions
of the cabbage armyworm, Marnestru were reared on an artificial diet ( AGUI et al.. 1975) under aseptic conditions until the 5th instar. and then semi-aseptically during the 6th (last) instar. The larvae emptied their guts and entered the wandering stage 5 days after the last-larva1 ecdysis and developed into non-diapausing pupae under a long-day photoperiod of 16 hr light and 8 hr dark at 25°C. Larvae
brassicae,
Treatment
of hormones
The juvenile hormone analogue. isopropyl-l I methoxy-3,7-l l-trimethyl-2,4_dodecadienoate, (methoprene, provided by Otsuka Pharmaceutical Co.). Cl8juvenilehormone(JHI)(SigmaChemicalCo..Lot, 98(3-0109-l), Cl7-juvenile hormone (JHII) (Sigma, Lot, 108-0057-l) and C16-juvenile hormone (JHIII) (Sigma, Lot, 87C-02232) wereused for these experiments. One pg of hormone in I ~1 acetone was applied topically by microsyringe (Terumo Co.) to the dorsal region of larvae once a day for 3 days. As a control, 1.0 ~1 of acetone was applied in the same manner. 20-Hydroxyecdysone(RohtoPharmaceuticalCo.)was
193
KIYOSHIHIRUMA
194
dissolved in 107, isopropanol, and the concentration was checked spectrophotometrically (Earn = 12670; MELTZER, 1971) as described by RIDDIFORD et al. (1979). Ecdysone (Eco-Chemical Intermediate) was prepared in the same way as the 20-hydroxyecdysone (a 241 = 12300; MELTZER, 1971). The final hormone concentrations were adjusted to 0.05-0.1 pg/pl. Larvae were anaesthetized with CO,. and 5-10 ~1 of ecdysteroid solution was injected through the 4th abdominal proleg. The proleg was tied off with a silk thread ligature. implantation of brains and prothoracic glands Donor larvae were sterilized in 70;; ethyl alcohol for 2 min. then washed in sterilized distilled water. Both brains and prothoracic glands were removed under sterile 0.9’!, NaCl solution and washed thoroughly. These organs were implanted into a larval host through the 4th proleg, which was subsequently ligatured with a silk thread. Both dissection and implantation were performed under ethyl ether anaesthesia. To remove the implanted brains, the host larva was anaesthetized with ethyl ether, the ligature loosened, and the implants removed using forceps. The proleg was tied off again with a ligature. Injection of homogenized brains Brains from the day-7 last-instar larvae were used as a PTTH source. PTTH was prepared as follows: the brains were removed and stored in a 0.9% NaCl solution at -30°C. Before use the sample was reconstituted in water and homogenized. The homogenate was heated to 90°C for 3 min (to sterilize) and 10 ~1 containing 10 brain equivalents was injected into each larval abdomen as described above for 20-hydroxyecdysone. Ligations Ligations were performed either behind the head or thorax of the larvae with a silk thread ligature. The part anterior to the ligature was cut off to ensure complete removal of the brain or both the brain and prothoracic glands. All experimental animals were observed for 25 days after treatment.
RESULTS Initiation of pupation of neck-ligatured exogenous juvenile hormone
larvae by
When day-2 or day-3 final-instar Mamestra larvae were neck-ligatured, few pupated. Since 0.5 pgjuvenile hormone analogue had been found to activate the prothoracic glands at the time of the gut purge (HIRUMA et al., 1978) it was of interest to determine if this analogue could play a similar role in the neckligatured larvae of various stages. Therefore, 1 pg analogue was applied daily for 3 days to the neckligatured larvae beginning at various times after ligation. As seen in Fig. 1, day-2 larvae were most sensitive to juvenile hormone analogue application beginning 10 days after neck-ligation, whereas day-3 larvae became sensitive 3-5 days after ligation.
01
3
IO
5 DAYS JHA
AFTER APPLICATION
I5
25
50
LIGATION BEGUN
Fig. 1. Effects of the timing of juvenile hormone analogue (JHA) application on the pupation of last-instar larvae neckligatured on either day 2 or day 3. One pg of analogue in I pl of acetone was applied topically once daily for 3 days. The pupation rates of the ligates to which acetone was applied were 4.O”,d (in 21 days) for day-2 and 2.296 (in 20 days) for day-3 larvae. When days required for pupation were calculated from the day of the end of analogue treatment. day-2 ligates to which juvenile hormone analogue was applied 3, 5, IO and 17 days after ligation pupated in an average of 9.0,9.0,9.7 and 14.0 days, respectively. In day-3 larvae, the ligates to which analogue was applied I, 3. 5, 10 and 25 days after ligation pupated in 9.0, 8.4, 7.9, 10.8 and 15.3 days, respectively. Twenty-one to 46 ligates were scored per point. The data should not be compared with the data of the Tables and Figs since larvae from different batches were used.
Furthermore, twice as many day-3 larvae pupated in response to analogue application at the time of peak sensitivity as did day-2 larvae. Day-O and day-l larvae were excluded, because when neck-ligatured they did not survive for more than 10 days. To show that this effect was due to juvenile hormone rather than some other action of the analogue, juvenile hormone I, II and III were applied to neck-ligatured day-3 larvae for 3 days beginning either immediately after neck-ligation or 5 days later. Table 1 shows that both juvenile hormone I and II promoted pupation and as with the analogue were most effective 5 days after neck-ligation. Importantly. these 2 natural hormones caused pupation in 44.5 days as compared to the 8 days required after analogue treatment. In further experiments, the analogue has been utilized, since its effectiveness on the activation of prothoracic glands closely paralleled that of juvenile hormone 1 and II. Action of juvenile prothoracic glands
hormone
analogue
on isolated
To determine if juvenile hormone acted directly on the prothoracic glands, day-3 larvae were neekligatured, then the glands were removed either immediately or 5 days later and implanted into abdomens isolated from day-5 larvae which had emptied their guts. The abdomens were then given juvenile hormone analogue daily for 3 days. Table 2 shows that when the glands were removed 5 days after neck-ligation, most were responsive to the applied analogue and subsequently caused pupation of the abdomens. By contrast, those taken immediately after
Activation Table
I. Effects of naturally
Treatment
occurring
juvenile
of prothoracic
hormones
0 7 0 0 0
24 26 19 25
0 92 84 4
I II III
Acetone Juvenile Juvenile Juvenile
hormone hormone hormone
1 II III
5 5 5 5
One ng of each hormone in 1 ,Aof acetone was applied *Calculated from the day of the end of treatment.
hormone
hormone
analogue
topically
18.0
4.5 * 1.7 4.0 * 0.9 6.0
once a day for 3 days.
No. of abdomens
0 0 5 5
day 3 larvae Average days required for pupation* (Mean A- S.D.)
(“,) pupated
analogue on the pupation of isolated abdomens neck-ligatured day-3 final instar larvae* Days after neck-ligation of prothoracic gland removal
Treatment of isolated abdomen? analogue
of neck-ligatured
21 15 15 18 20
hormone hormone hormone
hormone
on the pupation
Days after ligation
0 0 0 0
Juvenile Acetone Juvenile Acetone
application
195
No. of larvae used
None Acetone Juvenile Juvenile Juvenile
Table 2. Effects of juvenile
gland
25 18 21 15
containing
prothoracic
(“,) pupated g: 0 62$ 0
glands
from
Average days required for pupation (Mean + SD.1 9.5 * 0.7 x.5 * I.3
* Day-3 last-instar larvae were neck-ligatured. Then either immediately or 5 days later, their prothoracic glands were removed. One pair was implanted into an abdomen isolated by ligature behind the thorax of a day-5 final-instar larvae. Experimental animals were observed for 25 days. Isolated abdomens without implanted prothoracic glands never pupated. even after analogue application (N = 23). t One pg of juvenile hormone analogue and 1 nl of acetone were applied topically once a day for 3 days. 1 Significantly different at P > 0.001.
Table 3. Effects ofjuvenile
analogue
on the pupation of isolated abdomens glands*
Time of application Days after ( implantation
Treatmentt Juvenile Acetone Juvenile Acetone
hormone
hormone
analogue
hormone
analogue
0 0 5 5
)
No. of abdomens 25 18 24 19
5 days after implantation
(I’,,) pupated 8+ 0+ 29: 0
of prothoracic
Average days required for pupation (Mean k SD.) 4.5 + (1.7 7.3 * 2.1
* Prothoracic glands were taken from day-3 last-instar larvae. One pair of prothoracic glands was implanted into each abdomen isolated from day-5 last-instar larvae. t One gg of juvenile hormone analogue or I ~1 of acetone was applied topically once a day for 3 days. $ Significantly different at P > 0.05. Experimental animals were observed for 25 days. There was no pupation of isolated abdomens lacking prothoracic glands when analogue was applied (N = 25).
KIYOSHIHIRUMA
196
neck ligation apparently could not be stimulated to release ecdysone by the applied analogue. Thus, the prothoracic glands of day-3 larvae slowly gain competence to respond to the prothoracicotropic effect of juvenile hormone in the absence of the brain and corpora allata. This acquisition of competence to respond to juvenile hormone was found to occur also when day-3 glands were implanted into day-5 isolated abdomens as seen in Table 3. When the analogue was applied 5 days after the operation, nearly 30% of the abdomens subsequently pupated. Thus the prothoracic glands can acquire this competence whenever they are incubated in the presumed absence of hormones for 5 days. The lower percentage pupating in response to the analogue is most likely due to the poorer viability of transplanted glands due to lack of an adequate oxygen supply (FUKUDA, 1944). Importantly, those that pupated did so in a similar time course to the neckligatured larvae treated with analogue 5 days after ligation. The role of the brain in the change of prothoracic gland sensitivity to juvenile hormone
Brains of day-7 last-instar larvae are known to contain PTTH (HONDA, personal communication; HIRUMA and AGUI, 1977). Therefore, they were implanted into neck-ligatured day-3 larvae. Figure 2 shows that the prothoracic glands were sufficiently activated to cause pupation in 50% of the animals after 5 days exposure to the implanted brains. In these instances, 14-15 days was required for pupation. By
contrast, only 3 days exposure to implanted brains coupled with juvenile hormone analogue application caused 77% to pupate in an average of 11 days (Fig. 2). Without brain implantation, analogue application only stimulated the glands in 15% of the animals. Thus the analogue appeared to stimulate suboptimally 70
8 60
DAYS
PG
BEFORE
EXPOSED
TO
6
BRAINS
TRANSPLANTATION
Fig. 3. Activation of prothoracic glands (PG) of neckligatured larvae by the implantation of two day-7 larva1 brains as assayed by sensitivity to 1 fig juvenile hormone analogue after implantation into abdomens isolated from day 5 larvae. The controls (0 days exposure) were prothoracic glands removed from neck-ligatured larvae at various times after ligation: for day-0 larvae, on day 6: day-l larvae, on day 4; and day-2 and day-3 larvae, on day 3. Isolated abdomens with prothoracic glands to which acetone was applied never pupated. Ages of larvae at time of neck-ligature: 0 day 0, q day 1, .i day 2, n day 3. Numbers above bars indicate numbers of abdomens with implanted prothoracic glands (I pair per abdomen). activated glands and to enhance the output of ecdysone since pupation occurred earlier than with brains alone. None of the neck-ligatured day-3 larvae pupated in response to injections of homogenates of day-7 brains (Table 4). Yet these brain-injected larvae were subsequently more responsive to juvenile hormone application in that a higher percentage pupated. To determine if the hormone acted directly on the prothoracic glands previously exposed to PTTH, glands of neck-ligatured larvae were exposed to brain implants for varying lengths of time, then transplanted to isolated abdomens. Then juvenile hormone analogue was applied to the abdomens. Figure 3 shows that 50% of the abdomens pupated when the implanted glands from day-3 larvae had been exposed
to the brain and presumably PTTH for 3 days prior to transplantation. Only 15% of similar glands which had
$! 50 a’ 40 5 8 30 ;
4
3
0
20 IO 0 25
5
4
3
DAYS
EXPOSED JN yJyQ
2
I
0
TO BRAINS
CULTURE
Fig. 2. Effects of juvenile hormone analogue (JHA) on the pupation of neck-ligatured day-3 last-instar larvae after exposure to PTTH-secreting brains. Two brains from day-8 last&star larvae were implanted into a ligatured larvae. One pg of analogue ( n) or 1 ~1 of acetone (8) was applied topically once daily for 3 days after the brains were removed. All ligatured larvae pupated between in an average of 11 and 16 days. When two suboesophageal ganglia, instead of brains, were implanted into the n&k-lig&&d day-3 larvae for 3 days, only 17% (N = 12) ligates mmated after iuvenile hormonk anal&& &plicati&.(a) ,&&ogue was -applied daily from 3 days after neck-ligation without brain implantation for 3 days. Numbers above bars indicate numbers of larvae used.
not been exposed to brains (N = 34) responded to the analogue and caused pupation. None of the acetonetreated control abdomens with prothoracic glands (N = 15 for each time and treatment) ever pupated. Some of the glands from day 1 and day 2 larvae also gained a competence to respond to juvenile hormone analogue by prior exposure to brains for 3 days. The glands from day-0 larvae could not be activated by analogue even after 4-6 days culture with brains (N = 18). Thus, the brain apparently causes a suboptimal activation of larval day-l-3 prothoracic glands which allows them to respond subsequently to juvenile hormone analogue by increasing ecdysone production. This response to the analogue appears to be a direct one since it occurs in absence of neural connections. RoIe of ecdysteroids in the changing sensitivity prothoracic glands to juvenile hormone To ascertain whether brain or ecdysteroids
of the
it was substances from the resulting from stimulated
ecdysone secretion that caused the glands to change
Activation Table 4. Effects of juvenile
(2)
Brain homogenate
(2)
Brain homogenate Brain homogenate
analogue
gland
on the pupation of neck-ligatured homogenate
Treatment after injection of brain homogenate
Injection Number of 1 ( times 0.9” 0 NaCl solution*
hormone
of prothoracic
197 day-3 last-instar
No. of larvae used
Juvenile hormone analogue
larvae injected with brain
Average days required for pupation (Mean + SD.)
(‘I,) pupated
7
ot
Acetone
12
0
(2)
Juvenile hormone analogue
12
58t
(I)
Juvenile hormone analogue
II
0
Il.6 k 4.8
Injection of brain homogenate (once a day) was performed from a day of ligation. Brains from day-7 last-instar larvae were homogenized with 0.9:; NaCl solution and 10 brain equivalents of homogenate per larva were injected. One pg of analogue or I pl of acetone was applied topically daily from the day following brain homogenate injection for 3 days. *Ten ~1 of 0.9”, NaCl solution was injected. Experimental animals were observed for 25 days. t Significantly different at P > 0.01. Table
5. Effects
of juvenile
hormone
on the pupation of neck-ligatured ecdysteroids
Treatment after ecdysteroid injection
Injection Juvenile hormone 5 pg ecdysone 5 pg ecdysone 2.5 pg ecdysone 1 ptp ecdysone
analogue
analogue
5 pg 20-hydroxyecdysone 5 pg 20-hydroxyecdysone 2.5 pg 20-hydroxyecdysone IO”,, isopropanol Juvenile hormone analogue 5 pg 20-hydroxyecdysone IO’!,, isopropanol
No. of larvae used
or acetone application 24 hr after ecdysteroid injection Juvenile hormone analogue 29 Acetone 16 Juvenile hormone analogue 12 Juvenile hormone analogue 14 Juvenile Acetone Juvenile Juvenile
hormone
analogue
hormone hormone
analogue analogue
17 11 11 12
application 48 hr after 20-hydroxyecdysone Juvenile hormone analogue Juvenile hormone analogue
II 10
day-3
last-instar
(“,) pupated
24: 0 17 0 6t 0 9
larvae
injected
with
Average days required for pupation* (Mean :t S.D.)
8.4 + 1.3 6.5 If 0.5
6.0 5.0
01
18 20
6.5 + 2.5 5.0 + 1.0
Day-3 last-instar larvae were neck-ligatured at 12:O0. Immediately after ligation, injection of ecdysteroids was performed every 3 hr for 12 hr (5 injections). One pg ofjuvenile hormone analogue and 1 ~1 of acetone were applied topically 24 or 48 hr after ecdysteroid injection once a day for 3 days. *Calculated from the day of the end of treatment. Experimental animals were observed for 25 days. t Significantly different at P > 0.1. $Significantly different at P > 0.05. to juvenile hormone, ecdysone and 20hydroxyecdysone were injected into neck-ligatured day-3 larvae every 3 hr for 12 hr (injections of 5 times). Then the analogue was applied. Table 5 shows that about 259 of animals pupated when juvenile hormone analogue was applied 24 hr after ecdysone injection, whereas only one given 20-hydroxyecdysone pupated in response to the analogue. If analogue application was delayed until 48 hr, cu. 20% pupated, irrespective of whether given the 20-hydroxyecdysone or lop0 isopropanol. When 0.5 pg of 20-hydroxyecdysone was injected into larvae neck-ligatured on day 2 or day 3 of the last instar, 200 ng/ml of ecdysteroids remained in the haemolymph 3 hr after injection (HIRUMA and AGUI, unpublished). Therefore, injection every 3 hr maintained a low titre of this hormone. Perhaps this
their sensitivity
statement is too strong for a difference between 6 and 257& and I suggest the rewording as indicated that the ecdysteroids, especially ecdysone, can cause the change in juvenile hormone sensitivity of the prothoracic glands, but the action of ecdysone was weak compared with that of PTTH. It was concluded that both PTTH and ecdysone secreted from the prothoracic glands themselves caused the acquisition of their sensitivity to juvenile hormone. DISCUSSION In the final-larval instar of Mamestra, there are two peaks of ecdysteroid activity as determined by radioimmunoassay (AGUI and HIRUMA, 1982): The first occurs in day-3 larvae (30 ng/ml), the second in
KIYOSHIHIRUMA
198
day-7 larvae (1250 ng/ml). Presumably these two releases of ecdysone are preceded by PTTH release from the brain as has been shown for Manduca sexta (TRUMAN and RIDDIFORD, 1974: SAFRANEK and WILLIAMS, 1980) in which the two releases of ecdysone are somewhat closer, that is on day 3 and day 6 (BOLLENBACHER et al., 1975; WIELGUS et al., 1979).
Before and during the first release of ecdysone the prothoracic glands of Mamestra cannot be activated by juvenile hormone or its analogues (HIRUMA et al.. 1978: HIRUMA, unpublished) but immediately thereafter (on day 4) they can be.
The data presented in this paper show that the prothoracic glands from either day-2 or day-3 larvae can acquire this sensitivity to juvenile hormone activation by prolonged incubation in the absence of factors from the head. The acquisition of sensitivity occurs more rapidly in the presence of a factor from the brain, presumably PTTH. Since ecdysone can cause this acquisition in some animals (Table 5). I suggest that the acquisition of sensitivity in neckligatured larvae may be explained as follows: ecdysone slowly leaks from the prothoracic glands in the absence of juvenile hormone and acts back on the gland itself to cause a change in sensitivity to juvenile hormone. During larval life, when the JH titre is high, the glands are insensitive to the prothoracicotropic action ofjuvenile hormone. When the titre declines as in neck-ligatured larvae (FAIN and RIDDIFORD, 1976) or in intact feeding larvae (VARJAS et ul., 1976; YAGI, 1976). these glands gradually become competent to respond to juvenile hormone. Importantly, both the PTTH and the ecdysone release that appears to be at least partly a result of the decline of juvenile hormone (NIJHOUT and WILLIAMS, 1974) seem to promote switch in competence of the glands.
the
Although in most systems that have been studied itz vitro. 20-hydroxyecdysone, rather than ecdysone, has been found to be the most effective hormone (BERGAMASCO and HORN, 1980), DNA synthesis in Galleria mellonella wing discs (OBERLANDER, 1969, 1972) and mitosis in Drosophila melanogaster cells (COURGEON, 1972) are thought to be responses only to ecdysone. Possibly the prothoracic glands are more sensitive to ecdysone than to 20-hydroxyecdysone
(Table 5) so that they will undergo this change immediately that they begin to secrete in the absence of juvenile hormone and therefore are not dependent on the rate of metabolism of ecdysone to 20hydroxyecdysone. In Manduca the ratio of ecdysone: 20-hydroxyecdysone is I:1 at the time of the first ecdysone release (BOLLENBACHER et al., 1975) then changes to 15 (indicating a higher conversion rate) during the second release. Presumably the brain implants are more effective than ecdysone injection because they can activate the prothoracic glands to maintain a higher and/or longer ecdysone titre than the injections. However, since these are in vivo experiments. the difference may be due to other causes, for instance to a difference in the rate of metabolism or excretion of the two ecdysteroids. These findings are similar to those of SAFRANEK et al. (1980) for neck-ligatured Manduca. In this case they found that juvenile hormone delayed or prevented metamorphosis if given before the first release
of
ecdysone,
but
accelerated
pupation
if
applied after the first release. They hypothesized that juvenile hormone might intervene in ecdysone synthesis and secretion by the prothoracic glands as one possible explanation. The activation of transplanted glands in isolated abdomens by juvenile hormone (Table 3 and Fig. 3; HIRUMA et al., 1978) suggests very strongly that there is a direct effect on the glands themselves. But it does not rule out a possible effect on ecdysteroid metabolism. Only the direct measurement of the ecdysone output of prothoracic glands in vitro in the presence and the absence of juvenile hormone will answer this question. The finding that juvenile hormone caused pupation in sensitive larvae in less time (about 11 days) than did implanted brains (about 14-15 days) is puzzling. Likely it indicates that both PTTH and juvenile hormone are necessary to induce rapidly the large release of ecdysone required for pupation. Presumably the activation of the glands by the two hormones is through different mechanisms since PTTH is a peptide (ISHIZAKI et al., 1977: NAGASAWA et al., 1979) and juvenile hormone is a sesquiterpenoid. But whether different steps in ecdysone biosynthesis and/or secretion are affected by two hormones awaits further investigation. Acknow~ledgenzmts-I thank Professor LYNN M. RIDDIFORD, Department of Zoology, University of Washington. Dr. S. KIMURA. The Sericultural Experiment Station. and Dr. N. AGIJI. The National Institute of Health, for many discussions and for critical reading of the manuscript.
REFERENCES AGUI N. and HIRUMAK. (1982) Ecdysteroid titer and its critical period during larval and pupal ecdysis in the cabbage armyworm. Mamestra brassicae L. (Lepidoptera: Noctuidae). Appl. Enf. Zool. (in press). AGUI N., OGURAN. and OKAWARAM. (1975) Rearing of the cabbage armyworm. Mamestra brussicae L. (Lepidoptera: Noctuidae) and some lepidopterous larvae on artificial diets. Jup. J. uppl. Ent. Zoo/. 19, 91-96 (In Japanese with English summary). AGUI N., GRANCERN. A., GILBERTL. I. and BOLLENBACHER W. E. (1979) Cellular localization of the insect prothoracicotropic hormone: In vitro assay of a single neurosecretory cell. Proc. natn. Acad. Sci. U.S.A. 76, 5694-5698. BERGAMASCO R. and HORN D. H. S. (1980) The biological activities of ecdysteroids and ecdysteroid analogues. In Progress itz Ecdysone Research (Ed. by HOFFMANN J. A.) pp. 299-324, Elsevier/North-Holland, Amsterdam. BOLLENBACHERW. E., AGUI N., GRANGER N. A. and GILBERT L. I. (1979) In virro activation of insect prothoracic glands by the prothoracicotropic hormone. Proc. mtn. Acud. SC~. U.S.A. 76, 5148-5152. BOLLENBACHERW. E., VEDECKIS W. V., GILBERT L. 1. and O’CONNOR J. D. (1975) Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta. Devl. Biol. 44, 3345. COURGEON A.-M. (1972) Effects of G(-and /j’-ecdysone on in vitro diploid cell multiplication in Drosophila melanogaster. Nature, New Biol. 238, 250-251, CYMBOROWSKIB. and STOLARZ G. (1979) The role ofjuvenile hormone during larval-pupal transformation of Spodoptera lifforalis: Switchover in the sensitivity of the prothoracic gland to juvenile hormone. J. Insect Physiof. _ _ ^I ^ ^ ^ ‘3, Y3Y-Y4L
Activation
of prothoracic
FAIN M. J. and RIDDIFORD L. M. (1976) Reassessment of the critical periods for prothoracicotropic hormone and juvenile hormone secretion in the larval molt of the tobacco hornworm Manducasexta. Cert. romp. Endocr. 30, 131-141. FUKUDA S. (1944) The hormonal mechanism of larval molting and metamorphosis in the silkworm. J. Fat. Sci. Tokyo Univ. sec. IV. 6, 477-532. GILBERT L. I. and SCHNEIDERMANH. A. (1959) Prothoracic gland stimulation by juvenile hormone extracts of insects. Nature, Lond. 184, 171-173. HIRUMA K. (1980) Possible roles of juvenile hormone in the prepupal stage of Manwsrra hrmsicae. Gen. camp. Endow. 41, 392-399. HIRLIMA K. and Acut N. (1977) Relationship between histological changes and functions of the neurosecretorq ceils in the brain of the cabbage armyworm, Munlrsrra brassicae L. Appl. Ent. Zool. 12, 4249. H~RUMAK.. SHIMADAH. and YACI S. (1978) Activation ofthe prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae. J. Insect Physiol. 24. 215-220. ISHUAK~ H.. SUZUKI A.. ISOGA~ A., NAC;ASAWA H. and TAMURA S. (1977) Enzymatic and chemical inactivation of partially purified prothoracicotropic (brain) hormone of the silkworm. Bon~hy.v mori. J. hwect Ph.rsiol. 23. 1219-1222. KRISHNAKUMARAN A. and SCHNEIDERMAN H. A. (1965) Prothoracotrophic activity of compounds that mimic juvenile hormone. J. Insecr Physioi. 11, 1517-1532. MELTZER Y. (1971) Hormonal and Attractant Pesticrde Technology. Noyes Data, Park Ridge. New Jersey. NAGASAWA H.. ISOGAI A.. SUZUKI A., TAMURA S. and ISH~ZAKI H. (1979) Purification and properties of the prothoracicotropic hormone of the silkworm. Bon1bv.v tori. Devl. Grouth and Dijjer. 21, 29-38. NIJH~UT H. F. and WILLIAMS C. M. (1974) Control of moulting and metamorphosis in the tobacco hormworm, Munduco se~ta (L.): cessation of juvenile hormone secretion as a trigger for pupation. J. e.~p. Biol. 61, 493-501. OBERLANDERH. (1969) Effects of ecdysone, ecdysterone. and inokosterone on the in vitro initiation of metamorphosis of wing disks of G&via n~ellonellu. J. Insect Physiol. 15, 29 l-304. OBEKI.ANIIER H. (1972) a-Ecdysone induced DNA synthesis in cultured wing disks of Grdleriu me/lonellu: inhibition b) J. Insect Phj~siol. 20-hydroxyecdysone and 22-isoecdysone. 18. 233-228.
gland
199
RIDDIFOHI) L. M., CURTIS A. T. and KIGLII.HI K. (1979) Culture of the epidermis of the tobacco hornworm Manduccr .se.uu. Tissue Culture Association Man& 5, 975-985. ROLLER H. and DAHM K. H. (1968) The chemistr! and biology ofjuvenile hormone. Recent Prog. Harm. Res. 24. 65 1480. R~~LLERH.. BJERKE J. S., HOLTHAUS L. M.. NORGARIJ D. W. and M&HAN W. H. (1969) Isolation and biological properties of the juvenile hormone. J. lmrc~t Pl~~.sb?/.1.5. 379-389. SAFRANLX L. and WILLIAMS C. M. (19X0) Studies of the prothoracicotropic hormone in the tobacco hc>rnworm. Mandwa serta. Biol. Bull. 158, 141-l 53. SAFRANEK L.. CYMROROWSKIB. and WILUAMS C. M. ( 1980) Effects of juvenile hormone on ecdysone-dependent development in the tobacco hormworm, Manduca W.Yta Biol. Bull. 158, 248-156. TRUMAN J. W. and RIDIXFOKII L. M. (lY74) Physiology of insect rhythms. III. The temporal organization of the endocrine events underlying pupation of the tobacco hornworm. J. ex-p. Biol. 68, 37l-382. VARJAS L.. PAGKIIA P. and DE WI~.IX J. (1976) Juvenile hormone titers in penultimate and last instar larvae of Pieris hrassicae and Barurhra hross~ue, in relation to the effect of juvenoid application. E.vp-pcrientirr 32, 249-15 I. WIELGUSJ. 3.. BOLLENRA(.HERW. E. and GLLB~RT L. I. ( 1979) Correlations between epidermal DNA synthesis and haemolymph ecdysteroid titer during the last instar of Ihe tobacco hornworm. ,lfanduca SC\-tu.J. lnswr Ph~~siol.25, 9-16. WILLIAMS C. M. ( 1946) Physiology of insect diapduse: the role of the brain in the production and termination ot pupal dormancy in the giant silhworm. P/ut~wmru cecropia. Biol. Bull. 90, 234-243. WILLIAMS C. M. (1947) Physiology of insect diapause. Il. Interaction between the pupal brain and prothordcic glands in the metamorphosis of the gxmt silkworm. Plut~wmia cecropiu. Biol. Bull. 93, X9-9X. WILLIAMS C. M. (19521 Physiology of insect dtapause. IV. The brain and prothoracic glands as an endocrine s~stcm in the cecropia silkworm. Biol. Bu11. 103, 120-13X. WILLIAMS C. M. (1959) The juvenile hormone. I. Endocrine activit) of the corpora allata of the adult cecropia silkworm. Biol. Bull. 116, 323-338. YAU S. (1976) The role ofjuvenile hormone m diapause and phase variation in some lepidopterous insects. In The, Juvmilc Hormones (Ed. by GII BERT I I.) pp. 7xX-300. Plenum Press, New York.
1982.
0022-1910/82/020193~7$03.00/0 (0 1982 Pergamon Pws.s Ltd.
FACTORS AFFECTING CHANGE IN SENSITIVITY OF PROTHORACIC GLANDS TO JUVENILE HORMONE IN MAMESTRA BRASSICAE KIYOSHI HIRUMA Institute
01‘
Agriculture and Forestry.
University of Tsukuba. Ibaraki-ken 305, Japan
Sakura-mura.
Niihari-gun.
Abstract--The prothoracic glands of the early last-instar larva of Mamestra brassicae (day O-3) were found previously to be insensitive to stimulation by juvenile hormone, whereas those later in the instar (from day 4 on) were activated by this hormone. When neck-ligatured young larvae (day-l, day-2 and day-3) were given
juvenile hormone 5-10 days after ligation, pupation was induced. Similarly, juvenile hormone induced pupation of isolated abdomens which contained prothoracic glands taken from neck-ligatured day-3 larvae 5 days after ligation. If the glands were exposed to prothoracicotropic hormone (PTTH) from implanted brains before they were transplanted to isolated abdomens, their sensitivity to juvenile hormone activation was enhanced. Ecdysone but not 20-hydroxyecdysone given every 3 hr for 12 hr also slightly enhanced sensitivity. These results suggest that prothoracic glands from either day-l. day-2 or day-3 larvae can slowly acquire a sensitivity to juvenile hormone activation by prolonged incubation in the absence of factors from the head. The acquisition of sensitivity occurs more rapidly in the presence of both a factor from the brain. presumably PTTH. and ecdysone released from the prothoracic glands themselves.
INTRODUCTION WILLIAMS(1946. 1947. 1952) found that implantation of the chilled brain of Hvulophora cecropia activated the prothoracic glands of diapausing or brainless pupae and thereby induced adult development. Recently, the brain of Manduca sexta has been shown to secrete prothoracicotropic hormone (PTTH) which activates the prothoracic glands (BOLLENBACHERet al., 1979; AGUI et al., 1979). Juvenile hormone has also been shown to activate the pupal prothoracic glands of some lepidopterans (GILBERT and SCHNEIDERMAN, 19.59; WILLIAMS, lY5Y; KRISHNAKUMARAN and SCHNEIDERMAN. 1965; R~~LLER and DAHM, 1968: R~~LLER~~al., 1969). but its prothoracicotropic role in the larva is less clear. In Mamrstra brassicae. HIRUMA et ul. (1978) and HIRUMA (unpublished) found that juvenile hormone I, II or the analogue (methoprene) could not activate the prothoracic glands during the early part of the last-larval instar, but could activate the glands beginning about the time of cessation of feeding and during the prepupal period. At all times during the last-larval instar. PTTH could activate the glands. In Spodopteru littoralis, juvenile hormone analogue stimulated the prothoracic glands shortly before pupation (CYMBOROWSKIand STOLARZ, 1979). Similar effects have been reported for Manduca sexta (SAFRANEK et ul.. lY80). It has not, however. been reported that physiological concentrations ofjuvenile hormone can stimulate the prothoracic glands in vitro. The precise roles ofjuvenile hormone and of PTTH in the activation of the glands during the normal larval-pupal transformation in Lepidoptera however remain unclear. In Manzestra the level of juvenile hormone decreases rapidly l-2 days after ecdysis to the final instar, then increases again after the cessation
of feeding (VARJAS et al., 1976; Y AGI. 1976). This latter increase has a dual function: activation of the prothoracic glands and suppression of adult development (HIRUMA, 1980). Since, during the feeding period, the glands seem to be insensitive to the prothoracicotropic effect of juvenile hormone, the experiments reported here were done to determine how the prothoracic glands gain competence to respond to juvenile hormone. MATERIALS Insect reuring
AND METHODS
conditions
of the cabbage armyworm, Marnestru were reared on an artificial diet ( AGUI et al.. 1975) under aseptic conditions until the 5th instar. and then semi-aseptically during the 6th (last) instar. The larvae emptied their guts and entered the wandering stage 5 days after the last-larva1 ecdysis and developed into non-diapausing pupae under a long-day photoperiod of 16 hr light and 8 hr dark at 25°C. Larvae
brassicae,
Treatment
of hormones
The juvenile hormone analogue. isopropyl-l I methoxy-3,7-l l-trimethyl-2,4_dodecadienoate, (methoprene, provided by Otsuka Pharmaceutical Co.). Cl8juvenilehormone(JHI)(SigmaChemicalCo..Lot, 98(3-0109-l), Cl7-juvenile hormone (JHII) (Sigma, Lot, 108-0057-l) and C16-juvenile hormone (JHIII) (Sigma, Lot, 87C-02232) wereused for these experiments. One pg of hormone in I ~1 acetone was applied topically by microsyringe (Terumo Co.) to the dorsal region of larvae once a day for 3 days. As a control, 1.0 ~1 of acetone was applied in the same manner. 20-Hydroxyecdysone(RohtoPharmaceuticalCo.)was
193
KIYOSHIHIRUMA
194
dissolved in 107, isopropanol, and the concentration was checked spectrophotometrically (Earn = 12670; MELTZER, 1971) as described by RIDDIFORD et al. (1979). Ecdysone (Eco-Chemical Intermediate) was prepared in the same way as the 20-hydroxyecdysone (a 241 = 12300; MELTZER, 1971). The final hormone concentrations were adjusted to 0.05-0.1 pg/pl. Larvae were anaesthetized with CO,. and 5-10 ~1 of ecdysteroid solution was injected through the 4th abdominal proleg. The proleg was tied off with a silk thread ligature. implantation of brains and prothoracic glands Donor larvae were sterilized in 70;; ethyl alcohol for 2 min. then washed in sterilized distilled water. Both brains and prothoracic glands were removed under sterile 0.9’!, NaCl solution and washed thoroughly. These organs were implanted into a larval host through the 4th proleg, which was subsequently ligatured with a silk thread. Both dissection and implantation were performed under ethyl ether anaesthesia. To remove the implanted brains, the host larva was anaesthetized with ethyl ether, the ligature loosened, and the implants removed using forceps. The proleg was tied off again with a ligature. Injection of homogenized brains Brains from the day-7 last-instar larvae were used as a PTTH source. PTTH was prepared as follows: the brains were removed and stored in a 0.9% NaCl solution at -30°C. Before use the sample was reconstituted in water and homogenized. The homogenate was heated to 90°C for 3 min (to sterilize) and 10 ~1 containing 10 brain equivalents was injected into each larval abdomen as described above for 20-hydroxyecdysone. Ligations Ligations were performed either behind the head or thorax of the larvae with a silk thread ligature. The part anterior to the ligature was cut off to ensure complete removal of the brain or both the brain and prothoracic glands. All experimental animals were observed for 25 days after treatment.
RESULTS Initiation of pupation of neck-ligatured exogenous juvenile hormone
larvae by
When day-2 or day-3 final-instar Mamestra larvae were neck-ligatured, few pupated. Since 0.5 pgjuvenile hormone analogue had been found to activate the prothoracic glands at the time of the gut purge (HIRUMA et al., 1978) it was of interest to determine if this analogue could play a similar role in the neckligatured larvae of various stages. Therefore, 1 pg analogue was applied daily for 3 days to the neckligatured larvae beginning at various times after ligation. As seen in Fig. 1, day-2 larvae were most sensitive to juvenile hormone analogue application beginning 10 days after neck-ligation, whereas day-3 larvae became sensitive 3-5 days after ligation.
01
3
IO
5 DAYS JHA
AFTER APPLICATION
I5
25
50
LIGATION BEGUN
Fig. 1. Effects of the timing of juvenile hormone analogue (JHA) application on the pupation of last-instar larvae neckligatured on either day 2 or day 3. One pg of analogue in I pl of acetone was applied topically once daily for 3 days. The pupation rates of the ligates to which acetone was applied were 4.O”,d (in 21 days) for day-2 and 2.296 (in 20 days) for day-3 larvae. When days required for pupation were calculated from the day of the end of analogue treatment. day-2 ligates to which juvenile hormone analogue was applied 3, 5, IO and 17 days after ligation pupated in an average of 9.0,9.0,9.7 and 14.0 days, respectively. In day-3 larvae, the ligates to which analogue was applied I, 3. 5, 10 and 25 days after ligation pupated in 9.0, 8.4, 7.9, 10.8 and 15.3 days, respectively. Twenty-one to 46 ligates were scored per point. The data should not be compared with the data of the Tables and Figs since larvae from different batches were used.
Furthermore, twice as many day-3 larvae pupated in response to analogue application at the time of peak sensitivity as did day-2 larvae. Day-O and day-l larvae were excluded, because when neck-ligatured they did not survive for more than 10 days. To show that this effect was due to juvenile hormone rather than some other action of the analogue, juvenile hormone I, II and III were applied to neck-ligatured day-3 larvae for 3 days beginning either immediately after neck-ligation or 5 days later. Table 1 shows that both juvenile hormone I and II promoted pupation and as with the analogue were most effective 5 days after neck-ligation. Importantly. these 2 natural hormones caused pupation in 44.5 days as compared to the 8 days required after analogue treatment. In further experiments, the analogue has been utilized, since its effectiveness on the activation of prothoracic glands closely paralleled that of juvenile hormone 1 and II. Action of juvenile prothoracic glands
hormone
analogue
on isolated
To determine if juvenile hormone acted directly on the prothoracic glands, day-3 larvae were neekligatured, then the glands were removed either immediately or 5 days later and implanted into abdomens isolated from day-5 larvae which had emptied their guts. The abdomens were then given juvenile hormone analogue daily for 3 days. Table 2 shows that when the glands were removed 5 days after neck-ligation, most were responsive to the applied analogue and subsequently caused pupation of the abdomens. By contrast, those taken immediately after
Activation Table
I. Effects of naturally
Treatment
occurring
juvenile
of prothoracic
hormones
0 7 0 0 0
24 26 19 25
0 92 84 4
I II III
Acetone Juvenile Juvenile Juvenile
hormone hormone hormone
1 II III
5 5 5 5
One ng of each hormone in 1 ,Aof acetone was applied *Calculated from the day of the end of treatment.
hormone
hormone
analogue
topically
18.0
4.5 * 1.7 4.0 * 0.9 6.0
once a day for 3 days.
No. of abdomens
0 0 5 5
day 3 larvae Average days required for pupation* (Mean A- S.D.)
(“,) pupated
analogue on the pupation of isolated abdomens neck-ligatured day-3 final instar larvae* Days after neck-ligation of prothoracic gland removal
Treatment of isolated abdomen? analogue
of neck-ligatured
21 15 15 18 20
hormone hormone hormone
hormone
on the pupation
Days after ligation
0 0 0 0
Juvenile Acetone Juvenile Acetone
application
195
No. of larvae used
None Acetone Juvenile Juvenile Juvenile
Table 2. Effects of juvenile
gland
25 18 21 15
containing
prothoracic
(“,) pupated g: 0 62$ 0
glands
from
Average days required for pupation (Mean + SD.1 9.5 * 0.7 x.5 * I.3
* Day-3 last-instar larvae were neck-ligatured. Then either immediately or 5 days later, their prothoracic glands were removed. One pair was implanted into an abdomen isolated by ligature behind the thorax of a day-5 final-instar larvae. Experimental animals were observed for 25 days. Isolated abdomens without implanted prothoracic glands never pupated. even after analogue application (N = 23). t One pg of juvenile hormone analogue and 1 nl of acetone were applied topically once a day for 3 days. 1 Significantly different at P > 0.001.
Table 3. Effects ofjuvenile
analogue
on the pupation of isolated abdomens glands*
Time of application Days after ( implantation
Treatmentt Juvenile Acetone Juvenile Acetone
hormone
hormone
analogue
hormone
analogue
0 0 5 5
)
No. of abdomens 25 18 24 19
5 days after implantation
(I’,,) pupated 8+ 0+ 29: 0
of prothoracic
Average days required for pupation (Mean k SD.) 4.5 + (1.7 7.3 * 2.1
* Prothoracic glands were taken from day-3 last-instar larvae. One pair of prothoracic glands was implanted into each abdomen isolated from day-5 last-instar larvae. t One gg of juvenile hormone analogue or I ~1 of acetone was applied topically once a day for 3 days. $ Significantly different at P > 0.05. Experimental animals were observed for 25 days. There was no pupation of isolated abdomens lacking prothoracic glands when analogue was applied (N = 25).
KIYOSHIHIRUMA
196
neck ligation apparently could not be stimulated to release ecdysone by the applied analogue. Thus, the prothoracic glands of day-3 larvae slowly gain competence to respond to the prothoracicotropic effect of juvenile hormone in the absence of the brain and corpora allata. This acquisition of competence to respond to juvenile hormone was found to occur also when day-3 glands were implanted into day-5 isolated abdomens as seen in Table 3. When the analogue was applied 5 days after the operation, nearly 30% of the abdomens subsequently pupated. Thus the prothoracic glands can acquire this competence whenever they are incubated in the presumed absence of hormones for 5 days. The lower percentage pupating in response to the analogue is most likely due to the poorer viability of transplanted glands due to lack of an adequate oxygen supply (FUKUDA, 1944). Importantly, those that pupated did so in a similar time course to the neckligatured larvae treated with analogue 5 days after ligation. The role of the brain in the change of prothoracic gland sensitivity to juvenile hormone
Brains of day-7 last-instar larvae are known to contain PTTH (HONDA, personal communication; HIRUMA and AGUI, 1977). Therefore, they were implanted into neck-ligatured day-3 larvae. Figure 2 shows that the prothoracic glands were sufficiently activated to cause pupation in 50% of the animals after 5 days exposure to the implanted brains. In these instances, 14-15 days was required for pupation. By
contrast, only 3 days exposure to implanted brains coupled with juvenile hormone analogue application caused 77% to pupate in an average of 11 days (Fig. 2). Without brain implantation, analogue application only stimulated the glands in 15% of the animals. Thus the analogue appeared to stimulate suboptimally 70
8 60
DAYS
PG
BEFORE
EXPOSED
TO
6
BRAINS
TRANSPLANTATION
Fig. 3. Activation of prothoracic glands (PG) of neckligatured larvae by the implantation of two day-7 larva1 brains as assayed by sensitivity to 1 fig juvenile hormone analogue after implantation into abdomens isolated from day 5 larvae. The controls (0 days exposure) were prothoracic glands removed from neck-ligatured larvae at various times after ligation: for day-0 larvae, on day 6: day-l larvae, on day 4; and day-2 and day-3 larvae, on day 3. Isolated abdomens with prothoracic glands to which acetone was applied never pupated. Ages of larvae at time of neck-ligature: 0 day 0, q day 1, .i day 2, n day 3. Numbers above bars indicate numbers of abdomens with implanted prothoracic glands (I pair per abdomen). activated glands and to enhance the output of ecdysone since pupation occurred earlier than with brains alone. None of the neck-ligatured day-3 larvae pupated in response to injections of homogenates of day-7 brains (Table 4). Yet these brain-injected larvae were subsequently more responsive to juvenile hormone application in that a higher percentage pupated. To determine if the hormone acted directly on the prothoracic glands previously exposed to PTTH, glands of neck-ligatured larvae were exposed to brain implants for varying lengths of time, then transplanted to isolated abdomens. Then juvenile hormone analogue was applied to the abdomens. Figure 3 shows that 50% of the abdomens pupated when the implanted glands from day-3 larvae had been exposed
to the brain and presumably PTTH for 3 days prior to transplantation. Only 15% of similar glands which had
$! 50 a’ 40 5 8 30 ;
4
3
0
20 IO 0 25
5
4
3
DAYS
EXPOSED JN yJyQ
2
I
0
TO BRAINS
CULTURE
Fig. 2. Effects of juvenile hormone analogue (JHA) on the pupation of neck-ligatured day-3 last-instar larvae after exposure to PTTH-secreting brains. Two brains from day-8 last&star larvae were implanted into a ligatured larvae. One pg of analogue ( n) or 1 ~1 of acetone (8) was applied topically once daily for 3 days after the brains were removed. All ligatured larvae pupated between in an average of 11 and 16 days. When two suboesophageal ganglia, instead of brains, were implanted into the n&k-lig&&d day-3 larvae for 3 days, only 17% (N = 12) ligates mmated after iuvenile hormonk anal&& &plicati&.(a) ,&&ogue was -applied daily from 3 days after neck-ligation without brain implantation for 3 days. Numbers above bars indicate numbers of larvae used.
not been exposed to brains (N = 34) responded to the analogue and caused pupation. None of the acetonetreated control abdomens with prothoracic glands (N = 15 for each time and treatment) ever pupated. Some of the glands from day 1 and day 2 larvae also gained a competence to respond to juvenile hormone analogue by prior exposure to brains for 3 days. The glands from day-0 larvae could not be activated by analogue even after 4-6 days culture with brains (N = 18). Thus, the brain apparently causes a suboptimal activation of larval day-l-3 prothoracic glands which allows them to respond subsequently to juvenile hormone analogue by increasing ecdysone production. This response to the analogue appears to be a direct one since it occurs in absence of neural connections. RoIe of ecdysteroids in the changing sensitivity prothoracic glands to juvenile hormone To ascertain whether brain or ecdysteroids
of the
it was substances from the resulting from stimulated
ecdysone secretion that caused the glands to change
Activation Table 4. Effects of juvenile
(2)
Brain homogenate
(2)
Brain homogenate Brain homogenate
analogue
gland
on the pupation of neck-ligatured homogenate
Treatment after injection of brain homogenate
Injection Number of 1 ( times 0.9” 0 NaCl solution*
hormone
of prothoracic
197 day-3 last-instar
No. of larvae used
Juvenile hormone analogue
larvae injected with brain
Average days required for pupation (Mean + SD.)
(‘I,) pupated
7
ot
Acetone
12
0
(2)
Juvenile hormone analogue
12
58t
(I)
Juvenile hormone analogue
II
0
Il.6 k 4.8
Injection of brain homogenate (once a day) was performed from a day of ligation. Brains from day-7 last-instar larvae were homogenized with 0.9:; NaCl solution and 10 brain equivalents of homogenate per larva were injected. One pg of analogue or I pl of acetone was applied topically daily from the day following brain homogenate injection for 3 days. *Ten ~1 of 0.9”, NaCl solution was injected. Experimental animals were observed for 25 days. t Significantly different at P > 0.01. Table
5. Effects
of juvenile
hormone
on the pupation of neck-ligatured ecdysteroids
Treatment after ecdysteroid injection
Injection Juvenile hormone 5 pg ecdysone 5 pg ecdysone 2.5 pg ecdysone 1 ptp ecdysone
analogue
analogue
5 pg 20-hydroxyecdysone 5 pg 20-hydroxyecdysone 2.5 pg 20-hydroxyecdysone IO”,, isopropanol Juvenile hormone analogue 5 pg 20-hydroxyecdysone IO’!,, isopropanol
No. of larvae used
or acetone application 24 hr after ecdysteroid injection Juvenile hormone analogue 29 Acetone 16 Juvenile hormone analogue 12 Juvenile hormone analogue 14 Juvenile Acetone Juvenile Juvenile
hormone
analogue
hormone hormone
analogue analogue
17 11 11 12
application 48 hr after 20-hydroxyecdysone Juvenile hormone analogue Juvenile hormone analogue
II 10
day-3
last-instar
(“,) pupated
24: 0 17 0 6t 0 9
larvae
injected
with
Average days required for pupation* (Mean :t S.D.)
8.4 + 1.3 6.5 If 0.5
6.0 5.0
01
18 20
6.5 + 2.5 5.0 + 1.0
Day-3 last-instar larvae were neck-ligatured at 12:O0. Immediately after ligation, injection of ecdysteroids was performed every 3 hr for 12 hr (5 injections). One pg ofjuvenile hormone analogue and 1 ~1 of acetone were applied topically 24 or 48 hr after ecdysteroid injection once a day for 3 days. *Calculated from the day of the end of treatment. Experimental animals were observed for 25 days. t Significantly different at P > 0.1. $Significantly different at P > 0.05. to juvenile hormone, ecdysone and 20hydroxyecdysone were injected into neck-ligatured day-3 larvae every 3 hr for 12 hr (injections of 5 times). Then the analogue was applied. Table 5 shows that about 259 of animals pupated when juvenile hormone analogue was applied 24 hr after ecdysone injection, whereas only one given 20-hydroxyecdysone pupated in response to the analogue. If analogue application was delayed until 48 hr, cu. 20% pupated, irrespective of whether given the 20-hydroxyecdysone or lop0 isopropanol. When 0.5 pg of 20-hydroxyecdysone was injected into larvae neck-ligatured on day 2 or day 3 of the last instar, 200 ng/ml of ecdysteroids remained in the haemolymph 3 hr after injection (HIRUMA and AGUI, unpublished). Therefore, injection every 3 hr maintained a low titre of this hormone. Perhaps this
their sensitivity
statement is too strong for a difference between 6 and 257& and I suggest the rewording as indicated that the ecdysteroids, especially ecdysone, can cause the change in juvenile hormone sensitivity of the prothoracic glands, but the action of ecdysone was weak compared with that of PTTH. It was concluded that both PTTH and ecdysone secreted from the prothoracic glands themselves caused the acquisition of their sensitivity to juvenile hormone. DISCUSSION In the final-larval instar of Mamestra, there are two peaks of ecdysteroid activity as determined by radioimmunoassay (AGUI and HIRUMA, 1982): The first occurs in day-3 larvae (30 ng/ml), the second in
KIYOSHIHIRUMA
198
day-7 larvae (1250 ng/ml). Presumably these two releases of ecdysone are preceded by PTTH release from the brain as has been shown for Manduca sexta (TRUMAN and RIDDIFORD, 1974: SAFRANEK and WILLIAMS, 1980) in which the two releases of ecdysone are somewhat closer, that is on day 3 and day 6 (BOLLENBACHER et al., 1975; WIELGUS et al., 1979).
Before and during the first release of ecdysone the prothoracic glands of Mamestra cannot be activated by juvenile hormone or its analogues (HIRUMA et al.. 1978: HIRUMA, unpublished) but immediately thereafter (on day 4) they can be.
The data presented in this paper show that the prothoracic glands from either day-2 or day-3 larvae can acquire this sensitivity to juvenile hormone activation by prolonged incubation in the absence of factors from the head. The acquisition of sensitivity occurs more rapidly in the presence of a factor from the brain, presumably PTTH. Since ecdysone can cause this acquisition in some animals (Table 5). I suggest that the acquisition of sensitivity in neckligatured larvae may be explained as follows: ecdysone slowly leaks from the prothoracic glands in the absence of juvenile hormone and acts back on the gland itself to cause a change in sensitivity to juvenile hormone. During larval life, when the JH titre is high, the glands are insensitive to the prothoracicotropic action ofjuvenile hormone. When the titre declines as in neck-ligatured larvae (FAIN and RIDDIFORD, 1976) or in intact feeding larvae (VARJAS et ul., 1976; YAGI, 1976). these glands gradually become competent to respond to juvenile hormone. Importantly, both the PTTH and the ecdysone release that appears to be at least partly a result of the decline of juvenile hormone (NIJHOUT and WILLIAMS, 1974) seem to promote switch in competence of the glands.
the
Although in most systems that have been studied itz vitro. 20-hydroxyecdysone, rather than ecdysone, has been found to be the most effective hormone (BERGAMASCO and HORN, 1980), DNA synthesis in Galleria mellonella wing discs (OBERLANDER, 1969, 1972) and mitosis in Drosophila melanogaster cells (COURGEON, 1972) are thought to be responses only to ecdysone. Possibly the prothoracic glands are more sensitive to ecdysone than to 20-hydroxyecdysone
(Table 5) so that they will undergo this change immediately that they begin to secrete in the absence of juvenile hormone and therefore are not dependent on the rate of metabolism of ecdysone to 20hydroxyecdysone. In Manduca the ratio of ecdysone: 20-hydroxyecdysone is I:1 at the time of the first ecdysone release (BOLLENBACHER et al., 1975) then changes to 15 (indicating a higher conversion rate) during the second release. Presumably the brain implants are more effective than ecdysone injection because they can activate the prothoracic glands to maintain a higher and/or longer ecdysone titre than the injections. However, since these are in vivo experiments. the difference may be due to other causes, for instance to a difference in the rate of metabolism or excretion of the two ecdysteroids. These findings are similar to those of SAFRANEK et al. (1980) for neck-ligatured Manduca. In this case they found that juvenile hormone delayed or prevented metamorphosis if given before the first release
of
ecdysone,
but
accelerated
pupation
if
applied after the first release. They hypothesized that juvenile hormone might intervene in ecdysone synthesis and secretion by the prothoracic glands as one possible explanation. The activation of transplanted glands in isolated abdomens by juvenile hormone (Table 3 and Fig. 3; HIRUMA et al., 1978) suggests very strongly that there is a direct effect on the glands themselves. But it does not rule out a possible effect on ecdysteroid metabolism. Only the direct measurement of the ecdysone output of prothoracic glands in vitro in the presence and the absence of juvenile hormone will answer this question. The finding that juvenile hormone caused pupation in sensitive larvae in less time (about 11 days) than did implanted brains (about 14-15 days) is puzzling. Likely it indicates that both PTTH and juvenile hormone are necessary to induce rapidly the large release of ecdysone required for pupation. Presumably the activation of the glands by the two hormones is through different mechanisms since PTTH is a peptide (ISHIZAKI et al., 1977: NAGASAWA et al., 1979) and juvenile hormone is a sesquiterpenoid. But whether different steps in ecdysone biosynthesis and/or secretion are affected by two hormones awaits further investigation. Acknow~ledgenzmts-I thank Professor LYNN M. RIDDIFORD, Department of Zoology, University of Washington. Dr. S. KIMURA. The Sericultural Experiment Station. and Dr. N. AGIJI. The National Institute of Health, for many discussions and for critical reading of the manuscript.
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