Insect Biochemistry and Molecular Biology 30 2000 499–505 www.elsevier.comlocateibmb
Temporal analysis of ecdysteroidogenic activity of the prothoracic glands during the fourth larval instar of the silkworm, Bombyx
mori
Shi-Hong Gu
a
, Wen-Hsien Tsia
b
, Yien-Shing Chow
a, b,
a
Department of Zoology, National Museum of Natural Science, 1 Kuan Chien Road, Taichung 404-19, Taiwan, Republic of China
b
Institute of Zoology, Academia Sinica, Taipei, Taiwan 11529, Republic of China Received 25 August 1999; received in revised form 5 January 2000; accepted 1 February 2000
Abstract
The cellular mechanism underlying ecdysteroidogenesis during the fourth larval instar of the silkworm, Bombyx mori, was ana- lyzed by determining the in vitro ecdysteroid biosynthetic activity of the prothoracic glands, cAMP accumulation of the gland cells,
the in vitro release of prothoracicotropic hormone PTTH, etc. According to the differential responsiveness of prothoracic glands to PTTH, dibutyryl cAMP dbcAMP, and 1-methyl-3-isobutylxanthine MIX, the following different stages were classified and
changes in PTTH signal transduction were assumed. During the first stage between days 0 and 1, the glands showed low basal and PTTH-stimulated activities in both cAMP accumulation and ecdysteroidogenesis, and PTTH release in vitro was maintained
at low but detectable levels, implying that a low but sustained PTTH signal may be transduced to prothoracic gland cells. On day 1.5, when low basal ecdysteroid production of the prothoracic glands was being maintained, both the responsiveness of glands to
the stimulation of PTTH and PTTH release in vitro dramatically increased, indicating greatly increased PTTH transduction. On day 3 when the basal ecdysteroidogenesis became maximal and afterwards, high PTTH release in vitro was maintained, but the gland
showed no response to PTTH, implying that the refractoriness of gland cells to PTTH may occur at this stage. We assume that the development-specific changes in PTTH signal transduction during the penultimate larval instar may play a critical role in
regulating changes in ecdysteroidogenesis of the prothoracic glands.
2000 Elsevier Science Ltd. All rights reserved.
Keywords: Prothoracicotropic hormone; Ecdysteroidogenesis; cAMP; Signal transduction; Fourth larval instar; Silkworms
1. Introduction
Ecdysteroids, synthesized
by insect
prothoracic glands, play important roles in directing insect growth
and development, with the major ecdysteroid peaks elic- iting molting and metamorphosis Gilbert et al. 1988,
1996; Sehnal, 1989; Smith, 1985. In addition, subtle fluctuations exist in basal ecdysteroid levels during lar-
val stages of several insect species Sehnal, 1989. How- ever, their developmental significance has been little
examined. Recently, the physiological significance of differential basal ecdysteroid levels between the early
penultimate and last larval instars has been demonstrated
Corresponding author. Tel.: +
886-4-3226940 ext. 200; fax: +
886- 4-3232617.
E-mail address: chowmail.nmns.edu.tw Y.-S. Chow.
0965-174800 - see front matter
2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 2 6 - 6
Gu et al. 1992, 1995; Gu and Chow 1993, 1996. It was found that the relatively high ecdysteroid levels during
the early stages of the penultimate instar are important for the corpora allata CA to continue producing juven-
ile hormone JH Gu and Chow, 1996. When these ecdysteroid levels are artificially reduced to very low
levels, the CA stop producing JH, and therefore pre- cocious metamorphosis is induced at the end of the pen-
ultimate larval instar. In light of this, the low but con- tinued ecdysteroid production by the prothoracic glands
which is responsible for the relatively high ecdysteroid levels during the early penultimate larval instar is criti-
cal for ensuring that the next molting is an additional larval-larval molting. Unfortunately, very few studies
have been conducted on the cellular mechanism underly- ing sustained ecdysteroid biosynthesis by the prothoracic
glands during the fourth larval instar.
On the other hand, PTTH is the primary regulator of
500 S.-H. Gu et al. Insect Biochemistry and Molecular Biology 30 2000 499–505
ecdysteroidogenesis of prothoracic glands Bollenbacher and Granger, 1985; Gilbert et al. 1988, 1996; Ishizaki
and Suzuki, 1994; Kawakami et al., 1990; Smith, 1993. The cellular mechanism of PTTH action has been well
documented in prothoracic gland cells of the last instar larvae of both M. sexta Gilbert et al. 1988, 1996; Hen-
rich et al., 1999; Smith et al., 1984; Smith 1993, 1995 and B. mori Gu et al. 1996, 1997. PTTH appears to
stimulate ecdysteroid secretion through cell surface receptors,
resulting in
the stimulation
of a
calciumcalmodulin-sensitive adenylate
cyclase, increases in both cellular Ca
2 +
and cAMP levels, and enhanced protein phosphorylation and synthesis Gilbert
et al. 1988, 1996; Gu et al. 1996, 1997; Smith et al., 1984; Smith 1993, 1995. However, the developmental
changes in PTTH signal transduction during a larval-lar- val molting cycle, have not been extensively studied.
The purpose of the present study is to clarify changes in
PTTH signal
transduction during
the fourth
penultimate larval instar of the silkworm, B. mori. We looked at the developmental changes of the steroidog-
enic capability of the prothoracic glands and their responsiveness to PTTH, dibutyryl cAMP dbcAMP, as
well as 1-methyl-3-isobutylxanthine MIX during the penultimate larval instar. Changes in cAMP accumu-
lation by prothoracic gland cells in vitro after PTTH or MIX stimulation and in vitro PTTH release from brain-
corpus cardiacum-corpus allatum complexes BR-CC- CA were also examined. The role of PTTH release in
regulating changes in responsiveness of prothoracic glands to PTTH is discussed.
2. Materials and methods
