1080 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
included a requirement for extracellular Ca
2 +
Smith et al., 1985, increased activity of an adenylate cyclase
Smith et al., 1984 now known to be a Ca
2 +
-calmodulin activated adenylate cyclase Meller et al. 1988, 1990,
and the subsequent activation of the cAMP protein kin- ase PKA Smith et al., 1986; see review by Gilbert et
al., 1988. Further studies revealed that the final step in the cascade ended with the multiple phosphorylation of
the ribosomal protein S6 mediated by PKA and S6 kin- ase Song and Gilbert, 1995; see review by Henrich et
al., 1999.
The fourth critical event was the availability of Mand- uca
recombinant PTTH rPTTH produced by the Kataoka laboratory M. Shionoya, H. Matsubayashi, S.
Nagata, H. Kuniyoshi, M. Asahina, L.M. Riddiford and H. Kataoka, in preparation. This was of great impor-
tance for obvious reasons. In addition to being a probe for the study of the PTTH receptor, the utilization of
rPTTH in the in vitro system could establish whether it was PTTH or one or more other molecules in brain
extracts that elicited the transductory cascade and increased ecdysteroidogenesis in the prothoracic gland.
This was important in order to establish, or cast doubt on, the conclusion that PTTH is the active component
in the crude and semi-pure brain extracts utilized in the studies summarized above. Indeed, the use of crude
extracts was viewed quite negatively by at least one granting agency and even led to negative reviews of
manuscripts. Thus, the availability of Manduca rPTTH was vital to corroborate or not 20 years of research on
PTTH action as well as for future studies. For these reasons we decided to compare the action of rPTTH and
brain extracts using a variety of critical parameters, and present the data here.
2. Materials and methods
2.1. Animals Since the actual experiments described herein using
live Manduca larvae were conducted in two different laboratories Gilbert, Smith, there were slight variations
in the raising of the Manduca larvae. In all cases animals were timed precisely and the slight insect-rearing vari-
ations had no effect on the experimental data, so that one can make relative comparisons using animals from
both laboratories.
In the Gilbert and Smith laboratories, Manduca were reared on an artificial diet at 25
° C, .60 relative
humidity and under a photoperiod of 16 h light8 h dark with 2400 h Arbitrary Zeitgeber Time lights off set at
2200 h Eastern Standard Time. A synchronous popu- lation of animals was selected by routinely staging on
days 0 of the third larval instar, fifth larval instar, and at pupation.
2.2. Immunocytochemistry Rabbit antiserum against recombinant Manduca
PTTH rPTTH was generated by conventional methods with rPTTH as an immunogen. The antiserum obtained
was mixed with the same volume of saturated ammonium sulfate solution, and after centrifugation the
precipitate was dissolved in 0.1 M Tris–HCl buffer, pH 8.0. The IgG antibody in the solution was purified
using a protein-A-conjugated column HiTrap Protein A, Pharmacia.
Whole mount immunocytochemistry was performed by a modification of the method of O’Brien et al. 1988.
Heads of day 0 pupae and day 3 fifth-instar larvae were fixed in aqueous Bouin’s solution for 2 h, followed by
thorough washing with 50 mM of phosphate-buffered saline PBS; pH 7.4 containing 0.1 sodium azide. The
brain and retrocerebral complexes brain, corpora car- diaca, corpora allata: Br–CC–CA were dissected in the
same buffer and the neural sheath was partially removed. The Br–CC–CA were stored at 4
° C in PBS containing
2 Triton X-100 and 0.1 sodium azide PBS–Triton for 1 day and then incubated overnight at 4
° C with anti-
rPTTH rabbit antibody in PBS–Triton 2 µ
gml. The specimens were washed three times with PBS containing
0.05 Tween-20 PBS–Tween followed by a 2 h incu- bation at room temperature with horseradish peroxidase
HRP-conjugated sheep
anti-rabbit IgG
antibody Boehringer Mannheim and diluted 1:200 with PBS–
Triton without sodium azide. The specimens were washed three times with PBS–Tween and then incubated
for 10 min with 50 mM Tris-buffered saline, pH 7.6, containing 1.3 mM 3,39-diaminobenzadine tetrahydroch-
loride and 0.02 H
2
O
2
. Following three washes in PBS and one in distilled water, the tissues were dehydrated
through graded ethanol and cleared with methyl salicy- late.
Sections of Br–CC–CA were immunostained by mod- ifying the method described previously Mizoguchi et
al., 1987. Heads of day 3 fifth-instar larvae were fixed in aqueous Bouin’s solution for 4 h followed by washing
with 70 ethanol. The Br–CC–CA were dissected out in 70 ethanol and dehydrated through graded ethanol,
embedded in paraffin, and sectioned at 7
µ m. Specimens
were incubated first with anti-rPTTH rabbit antibody 1
µ gml for 3 h and then with 1:500 diluted HRP-conju-
gated second antibody for 1.5 h. As a substrate for HRP, 3-amidino-9-ethylcorbazole was used. The immunos-
tained specimens were counterstained with Mayer’s hematoxylin solution. All incubations were performed at
room temperature.
2.3. Measurement of cAMP Prothoracic glands were removed under lepidopteran
saline and held in Grace’s medium as described pre-
1081 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
viously Smith et al., 1984. To measure cAMP, glands were transferred to standing droplets of Grace’s medium
0.03 ml, with or without rPTTH or brain extract, for 5 min at room temperature. In all cases, matched pairs
of glands from the same animal were used to compare experimental and control treatments. After 5 min, glands
were flash-frozen on dry ice. Cyclic AMP was extracted and measured with the use of cAMP Enzyme Immunoas-
say Kit BIOMOL Res. Lab., Plymouth Meeting, PA. Briefly, cyclic AMP was extracted by addition of
0.21 ml of 0.1 M HCl, followed by vigorous vortexing. Standards and samples 0.1 ml were acetylated for
increased sensitivity, and the cAMP content determined according to kit instructions.
Standard and phosphate-free Grace’s insect culture media were obtained from Gibco Grand Island, NY.
Carrier-free [
32
P]O
4
10 mCiml was from Amersham. Brain extract containing only big PTTH was prepared as
described previously Song and Gilbert, 1995. 2.4. Stimulation of ecdysteroidogenesis
For dose–response experiments, the prothoracic glands from day 6 last-instar V
6
larvae were dissected under lepidopteran saline at room temperature. Individ-
ual glands were placed in cell culture wells 16 mm diameter, 2 ml maximum volume containing 50
µ l of
Grace’s insect tissue culture medium for at least 30 min at room temperature. The medium was then carefully
removed and 50 µ
l of fresh medium containing the indi- cated doses of rPTTH or brain extract was added to each
well. After incubation for 1 h, the medium was collected for ecdysteroid quantification by radioimmunoassay
RIA Warren and Gilbert 1986, 1988.
For the time-course study, prothoracic glands from V
3
and V
6
larvae were incubated with 0.25 nggland of rPTTH or 0.25 brain equivalentgland of brain extract
the predetermined doses from the dose–response study described above for the indicated period of time. The
medium was then collected for ecdysteroid quantification by RIA. For developmental comparisons, prothoracic
glands P
were also dissected and preincubated as above. The preincubation medium was replaced with
either 25 µ
l of Grace’s medium or with 25 µ
l of Grace’s medium containing either 0.1 ng or 1.0 ng rPTTH. After
a 90 min incubation, 20 µ
l of the medium was stored and analyzed as described above.
2.5. Effect of anti-rPTTH monoclonal antibodies on Manduca brain extract PTTH activity
Monoclonal 3H3 antibody 5 µ
g, which was gener- ated against the rPTTH and purified using a protein A
column BioRad MAPS II kit, was incubated with 25 µ
l 2.5 brain equivalents of P
brain extract Bollenbacher et al. 1979, 1984 for 1 h at room temperature, with
gentle shaking. During this period, five pairs of P pro-
thoracic glands were dissected out and preincubated for 30 min in 50
µ l of Grace’s medium. After the 1 h incu-
bation of brain extract with antibody, this mix was diluted with 100
µ l of Grace’s medium. The preincu-
bation medium was then removed from the glands and replaced with either 25
µ l of Grace’s medium or with
25 µ
l of the diluted antibodybrain extract 25 µ
l0.5 brain equivalent. This incubation was continued for
90 min, at which point 20 µ
l of the incubation mix was removed and frozen for eventual RIA analysis Warren
and Gilbert 1986, 1988 using the SHO 3 antibody courtesy of Dr Sho Sakurai; see Kiriishi et al., 1990.
The monoclonal 3H3 antibody as well as a polyclonal antibody against Manduca rPTTH both developed by A.
Mizoguchi recognize Manduca PTTH in Western blots.
2.6. Effect of rPTTH on general and specific protein synthesis
Early V
4
prothoracic glands larvae between 8.0 and 9.0 g live weight were dissected and preincubated for
30 min in 50 µ
l of low-methionine Grace’s medium 10 of the normal methionine content of Grace’s; Ryb-
czynski and Gilbert, 1994. The preincubation medium was removed and replaced with 25
µ l of low-methionine
Grace’s medium containing 10 µ
Ci of
35
S-methionine ICN: 1175 Cimmol with or without 0.25 ng of rPTTH
about 0.325 nM. After incubation periods of 30, 60 or 90 min, the prothoracic glands were removed, flash-
frozen and stored at 280 °
C. The analysis of newly syn- thesized, radiolabeled proteins was performed by
determining the TCA trichloroacetic acid-precipitable radioactivity per gland and by SDS sodium dodecyl
sulfate–polyacrylamide gel electrophoresis PAGE of gland proteins, as described by Rybczynski and Gil-
bert 1994.
2.7. Stimulation of protein phosphorylation To investigate the protein phosphorylation patterns
elicited by rPTTH, prothoracic glands from V
3
and V
6
larvae and P new pupae were prepared as described
above. Four glands as a group were placed in a culture plate well containing 150
µ l of phosphate-free Grace’s
medium for at least 30 min. After removal of the medium, 150
µ l of fresh phosphate-free medium, to
which carrier-free [
32
P]O
4
20 µ
Ci150 µ
l was added, was placed into each well and preincubated for 1 h to
label the endogenous adenosine triphosphate ATP pool Song and Gilbert, 1995. The glands were then incu-
bated for the indicated period of time in 150 µ
l of fresh phosphate-free medium containing 0.25 nggland of
rPTTH or 0.25 brain equivalentgland of brain extract. At the end of incubation, the glands were rinsed once
with phosphate-free Grace’s medium, transferred to a
1082 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
microtube containing 40 µ
l of 3X SDS gel sample buffer, heated to 100
° C for 5 min and subjected to SDS–
PAGE and autoradiography. 2.8. Two-dimensional PAGE 2D-PAGE analysis of
multiple S6 phosphorylation 2D-PAGE, as described previously Song and Gilbert,
1995, was used to analyze the multiple-site phosphoryl- ation of S6 stimulated by rPTTH. For purification of the
80S ribosomal proteins for 2D-PAGE analysis, 40 glands at a time were prelabeled for 1 h in 0.8 ml of fresh phos-
phate-free Grace’s medium to which carrier-free [
32
P]O
4
100 µ
Ci0.8 ml was added, followed by rPTTH challenge 0.25 nggland for 1 h. A total of 120 labeled
glands were collected for ribosomal protein purification as described previously Song and Gilbert, 1995. The
purified 80S ribosomal proteins were subjected to 2D- PAGE and autoradiography.
3. Results and discussion
