683 N. Audsley et al. Insect Biochemistry and Molecular Biology 30 2000 681–689
2.5. JH esterase activity The JH esterase activities of CA from last-day Vth
instar larvae were determined by incubating
3
H-JH III 606.8 GBqmmol, ca. 350 Bq per 5 CA pr; NEN Life
Sciences with CA under normal experimental con- ditions e.g., in medium-199 for 4 h at 30
° C. Corpora
allata were either dissected clean of surrounding tissue or with some tracheafat body attached. At the end of
the incubation period the medium was extracted and sub- jected to RP-HPLC as described above to assess the rela-
tive amounts of
3
H-JH III remaining and
3
H-JH III acid produced.
2.6. Tissue extraction and RP-HPLC separation Brains 100 were dissected from mixed-aged VIth
instar larval L. oleracea, sonicated Soniprep 150; MSE in ice-cold acetone and centrifuged at 13,200g for
20 min at 4 °
C. Acetone was discarded and the pellets were re-extracted 2
× in 75 ethanol0.2 M HCl. The
supernatants were then combined, diluted 10 times with 0.1 trifluoroacetic acid TFA and loaded on to a C
18
Sep-pak cartridge Waters, conditioned with bovine serum albumin. The cartridge was eluted stepwise with
5 ml of 20, 45 and 60 acetonitrile0.1 TFA. The 45 fraction was diluted to ,10 acetonitrile with
0.1 TFA and fractionated by RP-HPLC using the same system and column as described above, but eluted with
a linear gradient of 20–50 acetonitrile0.1 TFA over 30 min at a flow rate of 1 mlmin, and monitored at
214 nm. Thirty fractions 1 ml were collected using a RediFRAC fraction collector, and aliquots dried on to a
multiwell
plate for
enzyme-linked immunosorbent
assay ELISA. 2.7. ELISA for Manduca allatotropin
An indirect ELISA for Mas-AT was developed using primary antiserum raised against Mas-AT Veenstra and
Hagedorn, 1993 using similar methods to those reported for Mas-AS ELISA Audsley et al., 1998.
Briefly, HPLC fractions and Mas-AT were dried on to multiwell plates Sigma, UK; catalogue no. M4034
at 37 °
C and then incubated overnight at 4 °
C with 100 µ
l of 0.1 M bicarbonate coating buffer pH 9.6. Plates
were washed three times with 150 µ
l of 10 mmoll phos- phate
buffer0.1 TWEEN-20
[phosphate-buffered saline PBS], blocking solution 150
µ l; 2 non-fat
milk in PBS was added, and the plates incubated for 90 min at 37
° C. After a further PBS wash, 100
µ l of
primary antiserum 1:10,000 dilution was added to each well and the plates incubated for another 90 min at 37
° C.
Next, 100 µ
l of goat anti-rabbit antiserum conjugated with horseradish peroxidase 1:3000 dilution in PBS
was added as secondary antibody after washing three times with PBS. Plates were then incubated for 40 min
at 37 °
C. After a final PBS wash 3 ×
, 100 µ
l of substrate solution 10 mg O-phenylenediamine, 10
µ l H
2
O
2
in 25 ml citrate buffer, pH 5.0 was added to each well and
incubated for 40 min at 37 °
C. The reaction was stopped by addition of 50
µ l of 1.0 N H
2
SO
4
to each well and optical density read at 492 nm on a Labsystems Multi-
skan MCC340. 2.8. Synthetic Manduca sexta allatostatin and
allatotropin M. sexta allatostatin was custom-synthesised using
solid-phase methodology Fmoc procedure on an Applied Biosystems model 431A automatic peptide syn-
thesiser at the Advanced Biotechnology Centre, Charring Cross and Westminster Hospital Medical School, Lon-
don. M. sexta allatotropin was purchased from Sigma, UK.
2.9. Statistical treatment Results are presented as mean
± standard error SE.
Treatments were considered significantly different when a Student’s t-test indicated a P value of ,0.05.
3. Results
3.1. JH biosynthesis by larval CA in vitro The radiolabelled JH homologues synthesised by CA
from last-day Vth instar larvae in vitro are shown in [Fig. 1A] marked by arrows as disintegrations per minute
dpm in each HPLC fraction. Under control conditions, larval CA synthesise predominantly JH II, some of
which appears as JH II acid [Fig. 1A]. Very little radio- activity was detected in the JH I zone, although some
appeared as JH I acid [Fig. 1A]. The total amount of JH synthesised JH and JH acid was 184.7
± 14.9 fmolprh
mean ±
SE, n =
6, of which ca. 90 was JH II. In some incubations when CA were not cleared of
all contiguous tissue, all of the JH synthesised eluted as acid homologues [Fig. 1B]. Here the total amount of
JH acid produced was 162.3 ±
17.4 fmolprh mean ±
SE, n
= 8, of which ca. 80 was JH II acid.
3.2. JH esterase activity of larval corpora allata Fig. 2A shows the amount of radiolabel dpm and
elution position of
3
H-JH III under RP-HPLC con- ditions. The arrow shows the elution position of
3
H-JH III acid produced by the chemical conversion of
3
H-JH III. The total amount of radiolabel in all HPLC fractions
collected was 15,375 dpm, of which 13,502 dpm 88 eluted in the JH III position. In the presence of larval
684 N. Audsley et al. Insect Biochemistry and Molecular Biology 30 2000 681–689
Fig. 1. Separation by RP-HPLC of radiolabelled JH homologues syn-
thesised by CA from last-day Vth instar larval L. oleracea A, and the effect of tissue B on JH homologues produced. Hatched bars
show the amount of JH dpm measured in each fraction means ±
SE, n
= 6. Elution positions of JH I, JH II, JH I acid and JH II acid are
shown by arrows.
CA, 27.1 of
3
H-JH III was converted to
3
H-JH III acid with 41.7 remaining as JH III [Fig. 2B]. A further
27.3 was converted to an unidentified product [UP; Fig. 2B]. This conversion of
3
H-JH III to
3
H-JH III acid was virtually complete in the presence of CA with
tissue attached [Fig. 2C], with 84 of total dpm con- verted to JH III acid and only 3.7 remaining in the JH
III co-eluting fraction. It became clear that the esterolytic activity was dependent on both CA and the amount of
other tissue present.
3.3. The effect of Mas-AS on control rates of larval corpora allata JH biosynthesis
Under control conditions the incorporation of propi- onate into JH homologues by CA from last-day Vth
instar larvae was low, with most radioactivity eluting in the JH II acid position, and smaller amounts in a zone
corresponding to JH I acid Fig. 3. Virtually no radioac-
Fig. 2. Distribution of radioactivity dpm in RP-HPLC fractions
after separation of
3
H-JH III A in the absence of tissue B following incubation CA alone, and C CA
+ attached tissue. Corpora allata were
from last-day Vth instar larval L. oleracea. Elution positions of JH III, JH III acid and an unknown product UP are shown.
tivity was detected in the JH I and JH II zones. The estimated total rate JH I acid
+ JH II acid under control
conditions was 125.8 ±
14.7 fmolprh. On the addition of 10
µ M Mas-AS, JH I acid and JH II acid were signifi-
cantly reduced
by 60.8
P =
0.018 and
72.5 P
= 0.001, respectively, to a total of 36.6
± 5.6 fmolprh
means ±
SE, n =
6; Fig. 3. A lower dose 100 nM of Mas-AS resulted in rates
of JH biosynthesis not significantly different from con-
685 N. Audsley et al. Insect Biochemistry and Molecular Biology 30 2000 681–689
Fig. 3. RP-HPLC separation of JH homologues produced by last-day
Vth instar larval L. oleracea CA, showing inhibition of JH synthesis by Mas-AS. Open bars represent control levels of radioactivity in each
fraction and solid bars the amount in CA treated with 10 µ
M Mas- AS means
± SE, n
= 6. JH I, JH I acid, JH II and JH II acid
= juvenile
hormone homologues.
trol rates. Control rate =
142.8 ±
11.5 fmolprh; rate in the presence
of 100 nM
Mas-AS =
134.2 ±
4.2 fmolprh means
± SE, n
= 4.
3.4. Stimulation of JH biosynthesis in larval CA by MAS-AT
Fig. 4 shows the increase in radioactivity dpm detected in each RP-HPLC fraction under control and
Mas-AT 100 nM stimulated conditions in extracts of incubations of CA from last-day Vth instar larvae.
Radioactivity was distributed between the JH and the JH acid zones under control conditions in this series of
Fig. 4. RP-HPLC separation of JH homologues produced by last-day
Vth instar larval L. oleracea CA, showing stimulation of JH synthesis by Mas-AT. Solid bars represent control levels of radioactivity in each
fraction and open bars the amount in CA treated with 100 nM Mas- AT means
± SE, n
= 6–8.
Fig. 5. Dose–response relationship between concentration of Mas-
AT and measurements of total JH produced by last-day Vth instar larval L. oleracea CA means
± SE, n
= 4–10, represented as percentage
of maximum response.
experiments, and total radioactivity in the JH zones was significantly increased 183, P,0.001 in glands
treated with Mas-AT. The total JH and JH acid syn- thesised
under control
conditions was
181.6 ±
17.8 fmolprh; in the presence of Mas-AT it was 513.7
± 31.2 fmolprh means
± SE, n
= 6–8.
A dose-dependent relationship was observed for Mas- AT stimulation of total larval JH biosynthesis expressed
as a percentage of a maximum response achieved with 1
µ M Mas-AT which is about a threefold increase over
control values Fig. 5. The stimulation by Mas-AT is linear between 0.1 and 10 nM Mas-AT means
± SE,
n =
4–10. 3.5. Mas-AT-stimulated JH production by larval CA
and the effects of Mas-AS Last-day Vth instar larval CA incubated in the pres-
ence of Mas-AT 1 and 10 nM and Mas-AS 10 µ
M synthesised JH at levels that were not significantly dif-
ferent to those stimulated by Mas-AT alone Table 1. Inhibition of JH biosynthesis was only 6 1 nM Mas-
Table 1 The effect of Mas-AT and combined effect of Mas-AT
+ Mas-AS on
the rate of total JH biosynthesis by larval L. oleracea CA means ±
SE, n
= 4–6
Treatment JH biosynthesis fmolprh
No Mas-AT
a
1 nM Mas-AT 10 nM Mas-AT
No Mas-AS 125.8
± 14.7
464.4 ±
89.7 331.3
± 38.1
10 µ
M Mas-AS 36.6 ±
5.6 436.8
± 46.9
b
268.3 ±
13.5
b
P value ,0.02
0.42 0.17
a
Data from Section 3.3.
b
CA were incubated in the presence of both Mas-AS and Mas-AT.
686 N. Audsley et al. Insect Biochemistry and Molecular Biology 30 2000 681–689
AT +
10 µ
M Mas-AS or 19 10 nM Mas-AT +
10 µ
M Mas-AS compared with the stimulated rate with Mas-
AT alone. In contrast, inhibition of total JH synthesis by Mas-AS in the absence of Mas-AT was greater than 60
and significant P,0.02; Table 1, see also Section 3.3.
3.6. Mas-AT immunoreactivity in brain extract The Mas-AT-like immunoreactivity in individual
HPLC fractions from an extract of 100 brains from mixed-aged VIth instar larval L. oleracea measured by
ELISA assayed at 10 brain equivalents is shown in Fig. 6. The major immunoreactive fractions co-eluted with
synthetic Mas-AT indicated by arrow.
4. Discussion
