712 M.W. Lorenz et al. Insect Biochemistry and Molecular Biology 30 2000 711–718
physiology of the parthenogenetic stick insect Carausius morosus. Previous studies had revealed that, although JH
is apparently not required for normal vitellogenesis Pflugfelder, 1937; Bradley et al., 1995, the CA from
adult egg-carrying stick insects do synthesise and release JH III in vitro Lorenz et al., 1999b. In pilot studies we
were also able to extract and partially purify fractions from the brain of adult stick insects, which inhibited JH
biosynthesis of CA from G. bimaculatus in vitro Lorenz and Hoffmann, 1994; Ga¨de et al., 1997. The present
investigation was designed to purify and identify these factors, so that synthetic peptides could be obtained for
more intensive studies of their effects on the repro- ductive cycle of the stick insect.
2. Materials and methods
2.1. Insects Indian
stick insects,
C. morosus
Brunner Phasmatodea, Lonchodinae, were collected in the field
around Cape Town or obtained from a laboratory-bred colony that was reared on ivy leaves under short-day
conditions 12 h light12 h dark at ambient temperature 18–22
° C. Mediterranean field crickets, G. bimaculatus
de Geer Ensifera, Gryllidae, were reared at 27 °
C under long-day conditions as described elsewhere Lorenz et
al., 1997a. 2.2. Rapid partition assay for allatostatic activity
Release of JH III and allatostatic activity of the frac- tions purified by high-performance liquid chromato-
graphy HPLC on single cricket CA were determined by a rapid partition assay Feyereisen and Tobe, 1981
with some modifications Lorenz et al., 1997a. Tests for allatostatic activity of the synthetic peptides on the CA
of G. bimaculatus and C. morosus were performed in the same way, except that in the case of C. morosus a
pair of CA was used.
2.3. Tissue extraction and solid-phase prepurification Brains dissected from adult egg-laying C. morosus
were transferred into 500 µ
l of ice-cold extraction medium
methanolwateracetic acid;
100101 in
batches of 50. The procedures of extraction and solid- phase prepurification have been described elsewhere
Ga¨de et al., 1997.
2.4. High-performance liquid chromatography The first three HPLC runs were performed on a Jasco
series 900 high-pressure-gradient HPLC system con- sisting of two PU-980 pumps, a DG-980-50 on-line
degasser, a UV-975 variable-wavelength ultraviolet UV detector set to 214 nm, a Jet-stream Peltier column
thermostat set to 25 °
C, and a Rheodyne 7125 injector with a 2 ml sample loop. The system was run with Bor-
win Chromatography Software 1.21. The chromato- graphic conditions are shown in Table 1.
In the first HPLC run, lyophilised samples 1175 and 1100 brain equivalents from the 40 acetonitrile
MeCN solid-phase prepurification step were resus- pended each in 1 ml of water and injected onto the col-
umn. Peak fractions that eluted between 16 and 60 min were collected and tested for allatostatic activity on
cricket CA 30 brain equivalents per CA.
For the second HPLC run, active fractions pooled from the two identical first HPLC runs equivalent to
2185 brains were reduced in volume to approximately 500
µ l, diluted with 500
µ l of 0.13 heptafluorobutyric
acid HFBA in 10 MeCN and injected onto the col- umn. Peak fractions were collected and tested on cricket
CA 40 brain equivalents per CA. In the third HPLC run, active fractions from the
second HPLC run equivalent to 2065 brains were reduced in volume to approximately 500
µ l, diluted with
500 µ
l of 20 mM ammonium acetate NH
4
Ac and injected onto the column. Peak fractions were collected
and tested on cricket CA 50 brain equivalents per CA. The fourth HPLC run was performed on a Jasco series
900, low-pressure-gradient HPLC system consisting of a PU-980 pump, an LG-980-02 ternary gradient unit, a
DG-980-50 on-line degasser, an MD 910 photodiode- array detector 195–650 nm, a Jet-stream Peltier column
thermostat set to 30
° C, and a Rheodyne 7125 injector
with a 200 µ
l sample loop. The system was run with Borwin PDA software. The chromatographic conditions
are shown in Table 1. Active peaks from the third HPLC run equivalent to 1915 brains were reduced in volume
to approximately 50 µ
l, diluted with 50 µ
l of 0.1 tri- fluoroacetic acid TFA in 10 MeCN and injected onto
the column. Single pure peaks were collected and sub- jected to automated sequencing and mass determination.
Co-elution of native and synthetic peptides was per- formed on a micro HPLC system consisting of an Eldex
MicroPro high-pressure-gradient HPLC pump, a Spark Mistral column thermostat set to 37
° C with built-in
Rheodyne 8125 injector and 10 µ
l sample loop, and a Spectra Flow 505 UV detector equipped with a 35 nl
UZ-flow cell
SunChrom GmbH,
Friedrichsdorf, Germany. Peaks were detected at 214 and 280 nm sim-
ultaneously. Chromatographic conditions were as fol- lows: column — YMC-Pack ODS-AQ, 120 A
˚ , 5 µ
m, 150 mm
× 0.5 mm; solvent A — 0.1 TFA in 5 MeCN;
solvent B — 0.115 TFA in 80 MeCN; gradient — 0–50 min, 12–54 solvent B linear gradient, 0.84 sol-
vent B per min =
0.63 MeCN per min, followed by a 5 min rinse at 100 solvent B; flow rate — 10
µ lmin.
Between 5 and 10 pmol of the native peptides was co-
713 M.W. Lorenz et al. Insect Biochemistry and Molecular Biology 30 2000 711–718
Table 1 Chromatographic conditions of the four HPLC runs employed for the isolation of stick insect allatostatins
HPLC run Column
Solvents
a
Gradient Flow rate mlmin
1st LiChroCART Superspher 100
A: 0.115 TFA in water 0–5 min: 0 B
1 RP-18, 100 A
˚ , 4 µ
m, B: 0.1 TFA in MeCN
5–8 min: 0–20 B 124 mm
× 4 mm with guard
8–51 min: 20–33 B linear column 4 mm
× 4 mm Merck,
gradient, 0.3 MeCN per min Darmstadt, Germany
2nd Shiseido CAPCELL PAK C
18
A: 0.13 HFBA in water 0–2 min: 5 B
1 SG 300, 300 A
˚ , 5 µ
m, B: 0.13 HFBA in MeCN
2–52 min: 5–60 B linear 250 mm
× 4.6 mm with guard
gradient, 1.1 MeCN per min column 10 mm
× 4.6 mm Grom,
Herrenberg-Kayh, Germany 3rd
Shiseido CAPCELL PAK C
8
SG A: 20 mM NH
4
Ac in water 0–40 min: 6–63 B linear
1 300, 300 A
˚ , 5 µ
m, pH 7.0
gradient, 1.14 MeCN per min 150 mm
× 4.6 mm with guard
B: 20 mM NH
4
Ac in 80 MeCN column 10 mm
× 4.6 mm Grom
4th Vydac 218TP, 300 A
˚ , 5 µ
m, A: 0.115 TFA in water
0–2 min: 5 B 0.25
250 mm ×
2.1 mm MZ B: 0.1 TFA in MeCN
2–17 min: 5–20 B linear Analysentechnik, Mainz,
gradient, 1 MeCN per min Germany
17–57 min: 20–40 B linear gradient, 0.5 MeCN per min
a
HFBA, heptafluorobutyric acid; MeCN, acetonitrile; NH
4
Ac, ammonium acetate; TFA, trifluoroacetic acid.
injected with an equal amount of the corresponding syn- thetic peptide after having run native and synthetic pep-
tides separately on the same HPLC system.
2.5. Sequence analysis The allatostatic material from the final HPLC separ-
ations was loaded onto a polybrene-coated glass-fibre filter and sequenced by automated Edman degradation
using a model 477A sequenator connected to a model 120A on-line phenylthiohydantoin analyser Applied
Biosystems, Weiterstadt, Germany.
2.6. Mass spectrometry analysis Mass spectra were aquired using a matrix-assisted
laser desorptionionisation spectrometer Bruker Reflex, Bruker Franzen, Bremen, Germany. The acceleration
voltage was set to 30 kV for the linear mode. The matrix was a saturated solution of
α -cyano-4-hydroxycinnamic
acid dissolved in waterMeCN 7:3, vv. Peptide sol- utions 0.5
µ l, ca. 1 pmol were mixed on target with the
matrix solution 1:1, vv and left to dry. Each spectrum was the average of ca. 50–200 single-shot spectra
acquired in sets of five shots.
2.7. Peptide synthesis Peptide synthesis was performed on a model 9050
peptide synthesiser Milligen, Eschborn, Germany using FmocHOBt chemistry. Peptides were synthesised in the
amide form using an Fmoc-peptide amide linker poly- ethylene
glycol–polystyrene resin
Milligen. Syn- thesised peptides were purified after cleavage from the
resin by reversed-phase HPLC and checked by mass analysis. Peptide synthesis was kindly performed by N.
Weidner Mainz.
3. Results
