848 T. Singtripop et al. Insect Biochemistry and Molecular Biology 30 2000 847–854
dehydroecdysone andor ecdysone. Such a decrease is caused by depletion of prothoracicotropic hormone
PTTH, a neuropeptide which is produced by two pairs of neurosecretory cells in the brain and which stimulates
the prothoracic glands. In larval diapause, a high juvenile hormone JH titer in the hemolymph is reported to be
involved in suppression of the brain–prothoracic glands axis, preventing the release of ecdysteroids for larval
growth and pupation Denlinger, 1985. In fact, removal of CA from diapausing larvae causes a decrease in JH
concentration, which induces an increase in hemolymph ecdysteroid,
thus terminating diapause Yagi
and Fukaya, 1974; Yin and Chippendale, 1979.
During the long larval diapause in O. fuscidentalis, the
hemolymph ecdysteroid
concentration is
low Singtripop et al., 1999. This indicates that JH might be
involved in maintaining the larval diapause of the bam- boo borer, as in other lepidopteran larvae Yin and Chip-
pendale, 1973. Application of JH analogue JHA, how- ever, terminated the larval diapause. In the present study,
we report that in O. fuscidentalis, JH is not involved in maintenance of the larval diapause, but rather stimulates
the prothoracic glands of the diapausing larvae.
2. Materials and methods
2.1. Animals Bamboo borer larvae were collected from bamboo,
Dendrocalamus membranaceus, in a forest in Amphur Maewang, Chiang Mai Province, Thailand and kept in
plastic containers 12 ×
14 ×
8 cm on wet paper towels at 25
° C in continuous dark Singtripop et al., 1999. Larvae
used in the present experiments were collected from November through to February.
2.2. Hormones S–methoprene .95 stereochemically pure; SDS
Biotech, Tokyo was dissolved in acetone at a concen- tration of 5 mgml and kept at 235
° C as a stock solution.
An aliquot of the stock solution was diluted to an appro- priate concentration with acetone, and a 5
µ l aliquot was
topically applied to the dorsal surface of each larva using a 50
µ l micro-syringe. Ecdysone and 20–hydroxyecdy-
sone 20E Sigma, St. Louis, MO was dissolved in dis- tilled water at 1 mgml and stored at 220
° C until the
used. The 20E stock solution was diluted with distilled water, and a 5
µ l aliquot was injected into each larva
through the first proleg. 2.3. Preparation of brain extract
A crude extract of brains was used as a PTTH sample. One hundred brains from diapausing larvae were homo-
genized in 500 µ
l Grace’s insect culture medium Life Technologies, Gland Island, NY and heated in boiling
water for 3 min. The solution was centrifuged at 10,000 g for 10 min, and the resulting supernatant was kept at
235 °
C. The brain extract was diluted with Grace’s medium to a concentration of one brain equivalent in 25
µ l medium, for use in incubations of prothoracic glands.
2.4. Measurement of hemolymph ecdysteroid concentration
Hemolymph 30 µ
l was combined with 270 µ
l meth-
anol and centrifuged at 10,000 g for 5 min. The super- natant was transferred to a small test tube and dried
under reduced pressure at room temperature. The residue was dissolved in water and an aliquot of the aqueous
solution was subjected to ecdysteroid radioimmunoassay RIA Sakurai et al., 1998. The cross-reactivity of the
antibody to ecdysone and 20E was 1:5 Yokoyama et al., 1996.
2.5. In vitro incubation of prothoracic glands Prothoracic glands were individually incubated in 25
µ l Grace’s insect culture medium, pH 6.5, adjusted with
1 N NaOH, at 25 °
C for 6 h. After incubation, the amount of ecdysteroid in the medium was determined by RIA.
3. Results
3.1. Response of diapausing larvae to 20– hydroxyecdysone
Larvae were injected with various doses of 20E and observed for six weeks thereafter for pupal cuticle for-
mation Table 1. Larvae injected with 1–4 µ
g 20E
Table 1 Response of diapausing larvae to 20-hydroxyecdysone
Dose No. of
No. of Mean
S.D. Range
µ g
larvae used larvae that
day
b
days responded
a
4 15
14 8.1
1.4 7–11
2 15
15 11.6
4.4 7–21
1 15
13 11.6
6.5 7–32
0.5 15
3 10
10 0.25
15 6
21 8.4
10–33 0.1
15 0.05
15
c
15
a
Larvae injected with 20E did not shed the old cuticle but produced a tanned pupal cuticle.
b
Mean day was calculated only for the larvae that produced pupal cuticle.
c
Water 5 µ
l was injected as a control.
849 T. Singtripop et al. Insect Biochemistry and Molecular Biology 30 2000 847–854
actively moved on the day of injection and also on the following day if touched, but became inactive two days
after the injection. They produced frosted frass and a partially ruptured hindgut was occasionally visible. After
becoming inactive, the larvae produced a tanned pupal cuticle beneath the larval cuticle but did not shed the
old larval cuticle. At higher doses 1–4
µ g, most larvae
deposited a pupal cuticle, but the day of pupal cuticle formation ranged from 7 to 32 days. The first day of
pupal cuticle formation was the same with doses of 1, 2 and 4
µ g, but the last day was delayed in proportion
to the dose injected. The mean day of pupal cuticle for- mation with 4
µ g was significantly earlier than that with
2 or 1 µ
g ANOVA, P =
0.026, while there was no stat- istical difference between 1 and 2
µ g P
= 0.387. Effects
of 20E were less at doses of 0.5 and 0.25 µ
g. These results showed that the diapausing larvae were com-
petent to respond to 20E and that the critical dose to induce a response fell between 0.5 and 1
µ glarva. It
should be noted that 20E injection did not induce a stationary molt nor produce larval–pupal intermediates.
3.2. Termination of larval diapause with JHA When the diapausing larvae were topically treated
with 1 µ
g JHA, they turned brown with a hard and pig- mented cuticle, which indicated pupation of these larvae
Fig. 1A. When 0.1 µ
g JHA was applied, the larvae became inactive prior to formation of the brown cuticle.
The body color then changed from creamy to light yel- low, and those larvae were designated as G1. On the
following day, the dorsal epidermis became light brown G2, due to the deposition of pigmented pupal cuticle
beneath the old larval cuticle. About one day later, the entire body became brown G3. The body color turned
darker and harder two days after G2, that stage was designated G4. At stage G5, the pupae were approxi-
mately three days after deposition of the pupal cuticle G2. None of the G5 larvae shed the old cuticle. If the
old cuticle was removed with forceps Fig. 1B, the ani- mals possessed evaginated appendages such as antennae,
compound eyes, and mandibles; forewings covered with tanned cuticle; hind wings with almost no tanned cuticle;
and legs with tanned cuticle. The prolegs with crochets disappeared data not shown. These morphological
characteristics indicate that the animals with tanned cuti- cle were complete pupae.
JHA effects were further examined by applying vari- ous doses of JHA to diapausing larvae, which were then
observed for six weeks. When larvae received 0.025 µ
g JHA or more, they eventually pupated, even though they
failed to shed the old cuticle. The JHA effect was dose– dependent, with the lowest effective dose between 0.05
and 0.025 µ
glarva Table 2. The effect of JHA was more pronounced with four applications every other day.
As shown in Fig. 1C, larvae occasionally shed the old cuticle and formed complete pupae.
3.3. Involvement of the brain in the termination of diapause by JHA
In order to determine whether JHA stimulated the brain to release PTTH in the diapausing larvae, brains
were removed from larvae 1, 4, 7 or 10 days after treat- ment with 1
µ g JHA. As shown in Fig. 2A, the day of
pupation was not altered by the day of brain removal, suggesting that the brain was not directly involved in the
termination of diapause by JHA. This possibility was further examined by application of various doses of JHA
on larvae whose brains were removed prior to JHA application. In the brainless larvae, a dose of JHA less
than 0.005
µ g was still effective Fig. 2B: nine of 15
larvae pupated, but the time period between JHA appli- cation and pupation was longer than in those treated with
0.05 µ
g or more. Control larvae treated with acetone did not pupate at all.
3.4. Changes in hemolymph ecdysteroid titer after JHA treatment
The hemolymph ecdysteroid titer was determined after treatment with 1
µ g JHA Fig. 3. For the first 12
days after JHA application, the titer remained at the same low level. Although the concentration was low, it
was at a measurable level and never declined below the detection limit 0.2 ngml. After day 12, the ecdysteroid
titer gradually increased to a peak of 10 ngml on day 16 after the application. The titer decreased on day 18
and then abruptly increased after day 20. During the 20- day period, the titer in control larvae remained low.
Since pupation occurred in some individuals after day 20, we employed the morphological indicators to deter-
mine the physiological age of the larvae, rather than using the actual age in days after JHA application see
Fig. 1. Larvae exhibited G1 morphology 20 days after JHA application. The titer increased to 154 ngml on the
day of pupation G2 and to 289 ngml in G4 animals. In G5 animals, the titer decreased to a level similar to
that of G1–3.
3.5. Secretory activity of prothoracic glands after JHA treatment
The secretory activity of the prothoracic glands was determined in vitro after treatment with 1
µ g JHA Fig.
4. Prothoracic glands of the diapausing larvae produced ecdysone in vitro, although the amount was small. The
secretory activity of the glands of JHA-treated larvae remained at a level similar to that of diapausing larvae
until four days after JHA application. On day 10, the
850 T. Singtripop et al. Insect Biochemistry and Molecular Biology 30 2000 847–854
Fig. 1. Pupal metamorphosis induced by S–methoprene JHA treatment of diapausing larvae of Omphisa fuscidentalis. Larvae were topically
treated once with 1 µ
g JHA A and B or four times with 0.1 µ
g every other day C. A, progression of pupal metamorphosis graded from 1 to 5 G1–G5 in text. B typical pupa produced after a single application of JHA. The old cuticle of a G3 pupa B1 removed to show evaginated
appendages B2. C complete pupa obtained after four applications of 0.1 µ
g JHA every other day. C1, dorsal view; C2, ventral view. For B and C: a, antennae; ce, compound eye; fw, forewing; hw, hindwing; hc, larval head capsule; ll, larval thoracic leg; pl, pupal leg: l in C2, pupal
leg; ls, larval spiracle;T1–T3, pro-, meta-, mesothoracic tergites, respectively.
activity was four to five times as much as that of the diapausing larvae.
3.6. Responsiveness of prothoracic glands to PTTH Four different JHA doses were applied to diapausing
larvae, and the prothoracic glands were cultured 1, 4, 7 or 10 days later. As shown in Fig. 5, the secretory
activity of the prothoracic glands increased seven days after JHA treatment with any dose applied. In order to
determine whether the glands became competent to respond to brain extracts, or PTTH, after the treatment
with four different JHA doses, one gland of a larva was cultured in plain medium while the contralateral gland
was cultured in the presence of brain extracts Fig. 5. Prothoracic glands of the diapausing larvae did not
respond to the brain extracts data not shown. At a dose of 0.1
µ g JHA, the glands did not respond to the brain
extracts 10 days after the treatment. At 0.25 and 0.5 µ
g, the glands responded to brain extracts on day 10. When
1 µ
g JHA was applied, the glands became competent to respond to brain extracts beginning on day seven. An
activation ratio Ar. see Bollenbacher et al., 1984 dem- onstrates that an effective dose to elicit a response to
851 T. Singtripop et al. Insect Biochemistry and Molecular Biology 30 2000 847–854
Table 2 Break of larval diapause with JHA
Single application 4 applications
c
Dose N
a
n
b
Mean SD
N
a
n
b
Mean SD
µ g
day day
d
15 15
0.0005 15
15 0.001
15 15
8 29.8
2.9 0.005
15 30
13 32.6
10.7 0.01
15 30
14 31.9
3.7 0.025
15 2
28.5 0.5
30 17
31.4 5.4
0.05 15
10 32.1
9.3 30
14 30.3
9.0 0.1
15 11
24.5 3.1
30 16
28.1 5.0
0.3 15
10 21.9
4.4 0.6
15 13
20.5 2.7
1.25 15
8 22.1
1.3
a
Number of larvae used.
b
Number of larvae that pupated within 6 weeks after JHA appli- cation.
c
4 applications of each dose indicated.
d
Acetone 5 µ
l was applied as a control.
brain extracts on day seven after treatment fell between 0.5 and 1
µ g, while a dose between 0.1 and 0.25
µ g
elicited a response on day 10. Thus, the sensitivity of the glands to brain extracts increased by about 2-fold
from seven and 10 days after JHA application. The responsiveness of the prothoracic glands to brain
extracts in vivo was tested by injecting diapausing larvae with five and 10
µ l of brain extact containing one and
two brain equivalents, respectively. The larvae were observed for six weeks after injection but did not exhibit
any change.
4. Discussion
