720 U. Oeh et al. Insect Biochemistry and Molecular Biology 30 2000 719–727
vitro in the presence of radiolabelled methionine Range, unpublished data.
Levels of JH in haemolymph or tissues are affected by the rate at which the hormones are biosynthesized
by the CA and the rate at which these molecules are metabolized
or excreted.
Although JH-degrading
enzymes are known to play a role in determining physio- logical levels of JH, it is likely that the overall control
of the JH level is more intimately linked to changes in the rates of its synthesis Tobe and Stay, 1985; Weaver
et al., 1998. During the last decade, interest has focused on signals or factors that regulate JH biosynthesis by the
CA. Depending on the species and developmental stage, the regulatory signals may reach the glands via the hae-
molymph or via nervous connections. These signals may be
either stimulatory
allatotropic or
inhibitory allatostatic in nature Tobe and Stay, 1985; Goodman,
1990; Stay et al., 1994. So far, a large number of neuro- peptides that are potentially inhibitory on JH production
by the CA in vitro have been isolated from the brains of several insect species moths, cockroaches, locusts,
crickets, flies and bees; for reviews see Bendena et al., 1997; Ga¨de et al., 1997; Weaver et al., 1998; Hoffmann
et al., 1999. These allatostatins can be divided into three groups. One peptide family is characterized by a com-
mon C-terminal pentapeptide sequence YFXFGLI- amide allatostatin A family; allatostatin superfamily. In
some species these peptides have no effect on the CA of the source insect, but instead exhibit myo-inhibiting
properties Duve and Thorpe, 1994. The second group consists of peptides which were isolated from Gryllus
bimaculatus and which have a common amino acid Trp at positions 2 and 9 allatostatin B family; W
2
W
9
-peptide family Lorenz et al., 1995a. Manduca sexta allatosta-
tin Mas-AS is the only known representative of the third “allatostatin family”. Mas-AS shows no sequence
similarity to any other allatostatin Kramer et al., 1991 and is the only allatostatic neuropeptide isolated from a
lepidopteran species. It was first isolated from M. sexta Kramer et al., 1991, but in Pseudaletia unipuncta a
cDNA could be characterized that also encodes the 15- residue peptide Jansons et al., 1996. Audsley et al.
1998 identified an apparently identical peptide to Mas- AS in Lacanobia oleracea. Mas-AS strongly inhibits JH
biosynthesis in vitro by CA of fifth instar larvae and adult females of M. sexta. It also inhibits the CA of the
moth, Heliothis virescens, but had no effect on CA of two orthopteroid species, Periplaneta americana and
Melanoplus sanguinipes, or on CA of the beetle Teneb- rio molitor Kramer et al., 1991.
To date, only one allatotropin has been identified Kataoka et al., 1989. This allatotropin was isolated
from 10,000 heads of pharate adults of M. sexta and was called
M. sexta
allatotropin Mas-AT;
GFKNVEMMTARGF–NH
2
. Mas-AT stimulates JH biosynthesis in vitro in CA of adults of M. sexta and H.
virescens Kataoka et al., 1989, and also of the moth L. oleracea Audsley et al., 1999. No CA-activating
effect by Mas-AT was found in any non-lepidopteran species Hoffmann et al., 1999.
These results indicate an exceptional position of the lepidopteran order with respect to the regulation of CA
activity. In the fall armyworm, S. frugiperda, little is known about allatoregulating peptides or the control of
JH biosynthesis in general. In this paper, we report on the identification of a peptide which strongly stimulates
JH biosynthesis in vitro by the CA of adult females, and we demonstrate a novel mechanism of allatoregulation,
whereby JH biosynthesis is inhibited by Mas-AS only in those glands which had previously been activated by
the M. sexta allatotropin.
2. Materials and methods
2.1. Insects Larvae of S. frugiperda were reared at 27
° C and ca.
70 relative humidity under a L16:D8 photoperiod and raised on an artificial diet based on bean meal. To pre-
vent cannibalism, L4 larvae were maintained individu- ally in separate compartments of assortment boxes with
40 compartments 49
× 32
× 36 mm per compartment;
Licefa, Bad Salzuflen, Germany. Pupae and adults were kept under the same conditions in 20
× 20
× 10 cm plastic
boxes. Immediately after emergence, sexes were separ- ated and provided with water and saturated sucrose sol-
ution.
2.2. Radiochemical assay for allatoregulating activity Juvenile hormone biosynthesis was measured accord-
ing to the rapid partition assay Feyereisen and Tobe, 1981, a variant of the radiochemical assay of Tobe and
Pratt 1974 and Pratt and Tobe 1974. As CA of S. frugiperda release mainly JH III diol, and less amounts
of JH II diol Range, unpublished data, and up to 35 of the JH III diol could remain in the aqueous, non-iso-
octane phase when using the partition assay Share and Roe, 1988, our results may not represent absolute rates
of JH release by CA in vitro. The radiochemical assay was carried out as previously described Lorenz et al.,
1995a,b with some modifications: TC 199 incubation medium with Hanks’ salts and sodium bicarbonate, with-
out L-glutamine, buffered with 25 mM HEPES, sup- plemented with CaCl
2
to a final concentration of 10 mM and NaCl to a final concentration of 180 mM, fortified
with 1 Ficoll 400, was adjusted to pH 7.2 and sterilized by compression through a 0.2
µ m filter. L-[methyl-
3
H] methionine initial specific activity 75 Ci mmol
21
; Hart- mann Analytic, Braunschweig, Germany was used as
the radiolabelled precursor. The final specific activity of
721 U. Oeh et al. Insect Biochemistry and Molecular Biology 30 2000 719–727
the radiolabelled precursor was set to 200 mCi mmol
21
final L-methionine concentration ca. 0.11 mM. Ani- mals were dissected under modified cricket Ringer
Lorenz et al., 1997. CC and CA from one insect were incubated together as a complex. After a 1.5 h preincu-
bation in radioactive medium, to allow equilibration of the endogenous methionine with the radiolabelled meth-
ionine Tobe and Stay, 1985, the CC–CA-complexes were incubated for 2 h in the dark with gentle shaking
at 27
° C. The CC–CA-complexes were then transferred
to medium containing brain extracts and synthetic pep- tides, respectively, and were incubated for a second 2 h
period. At the end of each incubation period, media were analysed for JH release Feyereisen and Tobe, 1981.
Rates of release were determined for the first and second incubation, respectively, and the percentage change was
calculated second incubationfirst incubation21
× 100.
Inhibition rates are presented as negative values, stimu- lation rates as positive values. In other experiments,
three incubations each for 2 h were performed and either peptides were added or not, according to the
details in the figure legends.
2.3. Brain extracts and SEP-PAK purification Here, 5800 brains 100 per batch from 1- to 3-day-
old adult females were dissected and stored in extraction medium methanolwateracetic acid, 100101, vv at
225 °
C prior to purification. Each batch was homogen-
ized by sonication and centrifuged 10 min, 9000g, 2
° C
three times. The supernatants were pooled and the pellets resuspended in 500
µ l of extraction medium. Combined
supernatants from three batches representing 300 brains were dried down in a vacuum concentrator to ca.
200 µ
l, loaded onto a C
18
SEP-PAK cartridge Waters and rinsed three times with 500
µ l 0.1 TFA in water.
Cartridges were eluted with a stepwise gradient 4 ml each of 0.1 TFA in water, 0.1 TFA in 16 acetonitr-
ile CH
3
CN, 0.1 TFA in 60 CH
3
CN and 0.1 TFA in 100 CH
3
CN. The 16–60 SEP-PAK fractions, which contained the allatoregulating peptides Lorenz et
al., 1995b, were combined and two samples with 2800 and 3000 brain equivalents were obtained. Both samples
were dried down and stored at 225 °
C prior to further purification by HPLC.
2.4. HPLC purification Three reversed-phase high performance liquid chro-
matography HPLC steps were necessary to purify the bioactive peptide. The first two HPLC runs were perfor-
med on a Jasco HPLC system Jasco Labor- und Daten- technik GmbH, Großumstadt, Germany with the follow-
ing components: two PU-980 HPLC pumps, DG-980- 50 on-line degasser, UV-975 variable wavelength UV-
detector set to 214 nm, BFO-04 np column thermostat Jet-stream Peltier set to 25
° C and 7125 sample injector
Rheodyne Inc., Cotati, CA, USA. Data were processed using Borwin V 1.21 chromatographic software JMBS
Developpements, Grenoble, France with a personal computer.
2.4.1. First HPLC run The first purification step was performed using a
ReproSil-Pur C
18
-AQ column, 120 A ˚ , 5
µ m, 250
× 4.6
mm, with guard column, 10 ×
4.6 mm same material; Maisch, Ammerbuch, Germany and 2000
µ l sample
loop Sykam GmbH, Gilching, Germany. Solvent A: 0.115 TFA in water; solvent B: 0.1 TFA in CH
3
CN; gradient: 0–4 min 5 B, 4–94 min 5–50 B linear
gradient, 0.5 per min, 94–97 min 50–100 B, 97– 107 min 100 B, 107–112 min 100–5 B; flow rate: 1
mlmin. Both samples 2800 and 3000 brain equivalents were resolved in 1 ml 5 B and separately loaded onto
the HPLC column. Peaks were collected according to their UV signal. Fractions from one HPLC run 2800
brain equivalents were tested in the radiochemical bioassay for allatoregulating activity 30 brain equiva-
lents per assay. A fraction eluting between 54.5 and 55.3 min showed distinct allatotropic activity and, there-
fore, was used for further purification.
2.4.2. Second HPLC run Column: Capcell SG 120 C
18
, 120 A ˚ , 3
µ m, 150
× 3
mm, with guard column, 10 ×
3 mm same material; Grom, Herrenberg-Kayh, Germany; 500
µ l sample loop
Sykam GmbH, Gilching, Germany; solvent A: 0.13 HFBA in water, solvent B: 0.13 HFBA in CH
3
CN; gradient: 0–45 min 10–55 B linear gradient, 1 per
min, 45–48 min 55–80 B, 48–53 min 80 B, 53–55 min 80–10 B; flow rate: 300
µ lmin. The sample from
the first HPLC run was dried down to ca. 50 µ
l, diluted with 10 B to a volume of 250
µ l and loaded onto the
column. Peaks were collected and assayed for allatoreg- ulating activity 50 brain equivalents per assay. A frac-
tion eluting between 53.0 and 54.8 min showed high allatotropic activity and, therefore, was chosen for the
next HPLC purification step.
2.4.3. Third HPLC run This purification step was carried out on a micro
HPLC system with the following components: high pressure gradient HPLC pump Eldex Micro Pro, column
thermostat Spark Mistral set to 37 °
C with built-in Rheodyne 8125 injector, 200
µ l sample loop and an UV-
detector Spectra Flow 505 set to 214 nm equipped with a 35 nl ZU-flow cell SunChrom GmbH, Friedrichsdorf,
Germany. Operating conditions were as follows: col- umn ReproSil-Pur C
18
-AQ, 120 A ˚ , 5
µ m, 250
× 1.5 mm,
with guard column, 10 ×
1.5 mm same material; Maisch, Ammerbuch, Germany; solvent A: 0.113 TFA in 5
CH
3
CN, solvent B: 0.1 TFA in 80 CH
3
CN; gradient:
722 U. Oeh et al. Insect Biochemistry and Molecular Biology 30 2000 719–727
0–6 min 20 B, 6–46 min 20–36 B linear gradient, 0.4 B per min that means 0.3 CH
3
CN per min, 46– 52 min 36–100 B, 52–57 min 100 B, 57–65 min
100–20 B; flow rate: 100 µ
lmin. The sample from the second HPLC run was reduced to a volume of ca. 10
µ l, filled up with 20 B to an injection volume of 100
µ l and loaded onto the column. Peaks were collected and
tested for allatoregulating activity 50 brain equivalent per assay. The most significant peak eluting between
26.6 and 27.4 min showed allatotropic activity and was pure enough to be analysed by mass spectrometry and
Edman degradation.
2.5. Mass spectrometry and Edman degradation For mass spectrometry and Edman degradation 1
µ l
of the purified sample was dried down and dissolved in 3
µ l waterCH
3
CN 11, vv. A 1 µ
l sample was ana- lysed by mass spectroscopy using a MALDI-TOF instru-
ment Voyager DE-STR, PerSeptive Biosystems, Fram- ingham, MA, USA. Peptide samples were prepared
using dihydroxybenzoic acid as a matrix. For external molecular weight calibration a mixture of four synthetic
peptides was applied.
Edman sequencing was performed using the auto- mated protein sequencer 494 cLC from ABI-Perkin-
Ellmer Perkin Ellmer Biosystems, Warrington, UK. The Fast A software program was used to search for
sequence similarities in SwissProt and PIR protein data- bases.
2.6. Coelution of synthetic and native peptides Synthetic Mas-AT was purchased from Bachem
Bubendorf, Switzerland and coeluted with native pep- tide using the micro HPLC system as in the third HPLC
purification step, with the following modifications and chromatographic conditions: 10
µ l sample loop; column:
YMC-Pack ODS-AQ, 120 A ˚ , 5
µ m, 150
× 0.5 mm; sol-
vent A: 0.113 TFA in 5 CH
3
CN, solvent B: 0.1 TFA in 80 CH
3
CN; gradient: 0–6 min 20 B, 6–46 min 20–36 B linear gradient, 0.4 B per min that
means 0.3 CH
3
CN per min, 46–52 min 36–100 B, 52–57 min 100 B, 57–65 min 100–20 B; flow rate:
10 µ
lmin. After initial runs with synthetic Mas-AT and native peptide separately, both peptides were coinjected
in equal amounts of about 20 pmol.
3. Results
