682 N. Audsley et al. Insect Biochemistry and Molecular Biology 30 2000 681–689
Lepidoptera, namely P. unipuncta Jansons et al., 1996, L. oleracea Audsley et al., 1998 and S. littoralis
Audsley et al., 1999a, but to date no inhibitory activity of Mas-AS has been demonstrated in any of these larvae.
Synthetic Mas-AS does, however, cause up to 60 inhi- bition of JH biosynthesis in CA of adult female P.
unipuncta Jansons et al., 1996 and 50–60 inhibition of CA output in adult male and female L. oleracea
Audsley et al., 1999a,b. Recently, Oeh et al. 1999 reported that Mas-AS is active on CA of adult female
Spodoptera frugiperda, but only on Mas-AT-stimulated levels of JH production.
Bhatt and Horodyski 1999 have demonstrated Mas- AT mRNA and immunoreactivity in M. sexta larval ner-
vous system, but this peptide does not stimulate JH biosynthesis in larval CA of M. sexta Kataoka et al.,
1989 or L. oleracea Audsley et al., 1999b. M. sexta allatotropin does, however, stimulate JH synthesis in CA
of adult female M. sexta Kataoka et al., 1989, L. olera- cea Audsley et al., 1999b and S. frugiperda Oeh et
al., 1999. Audsley et al. 1999a,b suggest that the low rates of JH biosynthesis observed in larval L. oleracea
and the apparent inactivity of Mas-AS and Mas-AT may be due to the assay procedures used to measure JH lev-
els, rather than to an actual lack of bioactivity.
This study investigates these possibilities and deter- mines that JHs produced by CA of larval L. oleracea
are converted to acid homologues by JH esterases, and reports for the first time the regulation of JH biosynthesis
in the CA of a larval lepidopteran by both an allatostatin and allatotropin first characterised from M. sexta
Kataoka et al., 1989; Kramer et al., 1991. In addition to the identification of Mas-AS in L. oleracea Audsley
et al., 1998, evidence is presented that a Mas-AT immu- noreactive peptide is present in this noctuid.
2. Materials and methods
2.1. Experimental animals L. oleracea were reared at 20
° C and 65 relative
humidity under a 16 h light8 h dark photoperiod. Larvae were fed on a maize-flour-based noctuid artificial diet
Korano Ltd, Montalieu, France, and adults on a sol- ution of 10 sucrose, 2 gl wv methyl-4-hydroxyben-
zoate and 2 vv of mixed vitamins.
2.2. JH biosynthesis assay Corpora allata were dissected from last-day Vth instar
larval L. oleracea Vth =
penultimate stadium, and the rate of JH biosynthesis determined by the incorporation
of a radiolabel using procedures derived from those developed by Pratt and Tobe 1974. Five pairs pr of
CA were incubated at 30 °
C for 4 h in 100 µ
l of methion- ine-free tissue-culture medium medium 199; Gib-
coBRL, catalogue no. 041-90009 H which contained Hanks salts and 25 mM HEPES buffer, pH 7.2, and to
which was added 2 Ficoll, 0.1 bovine serum albumin protease-free, 0.3 bacitracin, 5 mM calcium chloride,
and 30 mM sodium acetate and 200
µ M methionine. To
this was added 0.74 MBqml of [1-
14
C]-propionic acid sodium salt, 1.99 GBqmmol; NEN Life Sciences. It is
important to note that propionate does not incorporate into JH III Schooley and Baker, 1985, and hence this
homologue would not be measured by this method.
Synthetic peptides were added to individual incu- bation tubes in 1
µ l volumes of 70 acetonitrile Mas-
AS or water Mas-AT. Similar amounts of solvent alone were added to control CA incubations. To deter-
mine the effects of Mas-AS on Mas-AT-stimulated lev- els of JH, both peptides were added at the start of the
incubation period. At the end of the incubation period synthetic JH standards were added and samples extracted
with 2
× 500
µ l ethyl acetate. The organic phase was dried
under nitrogen and resuspended in 100 µ
l of 70 ace- tonitrile for analysis by liquid chromatography.
2.3. High-performance liquid chromatography of JHsJH acids
Reversed-phase high-performance liquid chromato- graphy RP-HPLC was performed using a Beckman
System Gold chromatographic system, utilising a dual- pump programmable solvent module 126. Samples were
loaded via a Rheodyne loop injector on to an Aquapore RP-300, 7
µ m analytical column 220 mm
× 4.6 mm;
Anachem fitted with a guard column 30 mm ×
4.6 mm of similar packing material. The column was eluted with
a linear gradient of 12–80 acetonitrile5 mM HEPES, pH 7.4, over 20 min at a flow rate of 1 mlmin, and elu-
tions were monitored at 245 nm by a System Gold diode- array detector module 168. Fractions 1 ml were col-
lected into 3 ml Ecoscint A National Diagnostics for counting on a Beckman LS 6000TA liquid-scintillation
spectrometer. Columns were routinely flushed with methanol to remove residual contaminants.
2.4. Chemical conversion of JH to JH acid The acid homologues of JH I, JH II and JH III were
produced using the methods of Goodman 1984. Juven- ile hormone was incubated in the dark at 40
° C for 4 h
in methanol1 N NaOH 1:1, vv. At the end of the incu- bation period 1 N HCl was added to reduce the pH to
ca. 5.0, and the mixture extracted with 2 ×
500 µ
l ethyl acetate. The organic phase was dried under N
2
and resus- pended in 70 acetonitrile for HPLC. The elution pos-
ition of each acid analogue was determined using the above HPLC conditions.
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
