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
