Eur. J. Entorno!. 96: 267-274, 1999 ISSN 1210-5759

Allatostatins inGryllus bimaculatus (Ensifera: Gryllidae): New structures and physiological properties*

M atthias W. LORENZ’, R oland KELLNER2 and K laus H. HOFFMANN1

'Department of Animal Ecology 1, University of Bayreuth, 95440 Bayreuth, Germany; e-mail:[email protected] 2Biomed Fo/GBT, Merck KGaA, 64271 Darmstadt, Germany

Key words. Allatostatin, juvenile hormone, farnesol, peptide sequence, cricket,Gryllus bimaculatus, Gryllidae

Abstract. Four peptides with allatostatic activity were isolated from brains of the Mediterranean field cricket, Gryllus bimaculatus. Three of them (Grb-AST A3: AGMYSFGL-NH2; Grb-AST A4: SRPFGFGL-NH2; Grb-AST A5: GPDHRFAFGL-NH2) belong to the wide-spread family of Y/FXFGL/I-amide peptides, the fourth (Grb-AST B5: AWDQLRPGW-NH2) is a member of the W2W9- amide family of neuropeptides. All of these peptides are potent inhibitors of juvenile hormone (JH) biosynthesis by cricket corpora allata in vitro, causing 50% inhibition of JH biosynthesis at 0.4-3 x 10~8 M. The two peptides Grb-AST A5 and Grb-AST B5 have virtually the same potency and efficacy in inhibiting JH biosynthesis in vitro. No synergistic effect of the two peptide families with respect to the inhibition of JH biosynthesis could be observed. Peptides of both families decrease the accumulation of methylfarneso- ate, the direct precursor of JH, within CA that have been incubated in farnesol-rich medium. This suggests an involvement of these ASTs in the late steps of JH biosynthesis.

INTRODUCTION tion of vitellogenin release from the periovaric fat body of Development and reproduction of insects are regulated the German cockroach,Blattella germánica (Martín et ah, to a large extent by juvenile hormone (JH) and ecdyster­ 1996) and the inhibition of myogenic contractions of the oids (Hardie, 1995; Hoffmann & Lorenz, 1997). During foregut (Duve et ah, 1995), the hindgut (Duve et ah, the last decade, interest has focused on the isolation, puri­ 1996), and the oviduct (Schoofs et ah, 1997). Further­ fication and identification of factors that regulate the bio­ more, they can act as antimyotropins (Hertel & Penzlin, synthesis and release of these glandular hormones, since 1992; Lange et ah, 1995). it is hoped that such compounds may help in designing In addition to the Y/FXFGL/I-amide allatostatins (A- safer and more specific insecticides (Couillaud & Peype- allatostatins, Lorenz et ah, 1995a) a fourth group of alla­ lut, 1995; Hoffmann & Lorenz, 1998). Factors that stimu­ tostatic neuropeptides, members of the W2W9-amide late (allatotropins) or inhibit (allatostatins) the activity of peptide family, has been found in brain extracts of the the JH-producing corpora allata (CA) have been isolated cricket Gryllus bimaculatus (Lorenz et ah, 1995b). These from a variety of insects (for review see Gade et al., 1997; peptides have been designated B-allatostatins or W2W9- Hoffmann et ah, 1999). The allatoregulating peptides can amide allatostatins and are closely related to myoinhibit- be classified into four groups. The first two groups are an ing peptides isolated from Locusta migratoria (Schoofs et allatotropin (Mas-AT) isolated from the tobacco horn- ah, 1991) and M. sexta (Blackburn et ah, 1995). Members worm, Manduca sexta (Kataoka et ah, 1989) and an alla­ of this peptide family have also been found in brain ex­ tostatin (Mas-AST), isolated from the same species (Kra­ tracts of the Indian stick insect Carausius morosus (Lo­ mer et ah, 1991), that both seem to act as allatoregulators renz et ah, 1998a). However, their allatostatic function only in lepidopterans (Weaver et ah, 1998). seems to be restricted to crickets (Lorenz et ah, 1997a). In The third and most prominent group of allatoregulating addition to their myoinhibiting function in locusts and neuropeptides is the Y/FXFGL/I-amide allatostatin (AST) moths and their allatostatic effect on cricket CA, they superfamily, originally isolated from brains of the Hawai­ were shown to inhibit ovarian ecdysteroid biosynthesis in ian beetle roach, Diploptera punctata (Woodhead et ah, crickets (Lorenz et ah, 1997b) and they are capable of af­ 1989). Although these peptides have been isolated from fecting JH-, ecdysteroid-, and vitellogenin-titres in vivo various cockroaches (Belles et ah, 1994; Weaver et ah, when injected into adult crickets (Lorenz et ah, 1998b). 1994), flies (Duve et ah, 1993), mosquitoes (Veenstra et In the present study, we report the isolation and identi­ ah, 1997), crickets (Lorenz et ah, 1995a), locusts (Vee- fication of additional members of both the Y/FXFGL/I- laert et ah, 1996), and moths (Davis et ah, 1997; Duve et amide and the W2W9-amide allatostatin family from ah, 1997a, b), they seem to inhibit JH biosynthesis only in cricket brains, and on one of their possible target sites cockroaches and crickets. However, additional functions within the pathway leading to the formation of JH III. of this peptide family have been found, such as the inhibi­

* This paper is based on a lecture presented at the 6th European Congress of Entomology held in České Budějovice, Czech Repub­ lic, August 1998.

267 MATERIAL AND METHODS ments to obtain maximally stimulated CA. In order to investi­ gate effects of allatostatins under such stimulated conditions, the Insects and tissue dissection CA were preincubated in radioactive medium without farnesol, Mediterranean field crickets Gryllus bimaculatus de Geer transferred into medium with 200 pM farnesol for the 1st incu­ (Ensifera: Gryllidae) were reared as described (Lorenz et al., bation and transferred again after 2 h, this time into medium ei­ 1997b). Brains were dissected from 2-4 day-old virgin females, ther containing 200 pM farnesol alone (control) or 200 pM immediately transferred into ice-cold extraction medium farnesol plus the allatostatins to be tested (in concentrations of (methanol/water/acetic acid, 100/10/1, v/v/v), and stored at 10~'° to 10^ M). After the second incubation period the glands -75°C. Single CA from 3 day-old virgin females were used to were transferred into 20 pi of HPLC-grade water. Medium and test chromatographic fractions for allatoregulating activity and glands were then extracted separately with isooctane. The isooc­ to generate dose-response curves with the synthetic peptides. tane phase from the extracted medium containing JH III that had Single CA from 1 day-old unpaired adult males were used in the been released into the medium was directly pipetted into 2.5 ml experiments employing farnesol. of scintillation cocktail Rotiszint 11 (Roth, Karlsruhe, Radiochemical assay for allatostatic activity Germany). The isooctane phase from the CA extract that con­ tained unreleased JH III that had been produced by the CA, as Juvenile hormone biosynthesis and the effects of chroma­ well as the immediate JH III precursor, methylfarnesoate (MF), tographic fractions and synthetic peptides on CA activity were was subjected to thin-layer chromatography on Silica gel 60 F254 determined by the rapid partition assay (Feyereisen & Tobe, plates (Merck, Darmstadt, Germany) using xylol : ethylacetate 1981) as described (Lorenz et al., 1997b). In the standard ex­ (80/20, v/v) as the solvent system. Unlabelled JH III (Fluka, periments, single CA were preincubated in radioactive medium Neu-Ulm, Germany) that had been added to the extract served to allow the precursor (L-[methyl-l4C]methionine) to equilibrate as a tracer to identify the JH III band. MF was identified on the with the endogenous methionine within the glands, resulting in basis of its Rf-value. Bands were visualised by uv-illumination consistent rates of JH biosynthesis during the following incuba­ at 254 nm and scraped into 3 ml of scintillation cocktail Rotisz­ tions. After 90 min, the CA were transferred to fresh radioactive int 2211 (Roth). Samples were measured using a TriCarb 2100 medium without any additives and incubated for 120 min (first TR liquid scintillation counter (Canberra Packard, Frankfurt, incubation) to establish basal rates of JH release. Then, the CA Germany). were transferred for the second incubation (120 min) either to fresh radioactive medium without any additions (controls) or to Tissue extraction and peptide purification medium containing the HPLC fractions/synthetic peptides to be Extraction and SEP-PAK purification of the brain material tested. Incubations were stopped by removing the glands from were carried out as described (Lorenz et al., 1995b). Since only the medium. Inhibition of CA activity was calculated as the per­ the 40% acetonitrile (MeCN) SEP-PAK fraction showed consis­ centage change in JH release between the first and the second tent allatostatic activity, this fraction was chosen for further pu­ incubation. rification by a four step reversed-phase high performance liquid In some experiments, the glands were incubated in medium chromatography (HPLC) procedure. The first HPLC step was containing the allatostatins to be tested without preincubation. carried out on a Shimadzu HPLC system (LC-9A V2.2 HPLC The CA were repeatedly transferred in hourly or two-hour inter­ pump; SPD-6A UV-VIS detector, set to 215 nm; FCV-9AL low- vals into fresh medium containing allatostatins; the total incuba­ pressure flow control valve; DGU-2A helium degassing unit; tion time in the presence of allatostatins was 5 h in these experi­ Shimadzu Europa GmbH, Duisburg, Germany), the other HPLC ments. Then the CA were repeatedly transferred into medium steps were carried out on a Jasco series 900 high-pressure gradi­ without allatostatins in hourly intervals to follow the recoveryent HPLC system (Jasco, Groß-Umstadt, Germany), consisting from inhibition. of two pumps PU-980, an on-line degasser DG-980-50, a vari­ Farnesol, a late precursor of JH biosynthesis that stimulates able wavelength UV-detector UV-975 (set to 214 nm), a column the last steps of JH formation was employed in some experi­ thermostat Jet-stream Peltier (set to 25°C), and a Rheodyne

T able 1. Chromatographic conditions of the four HPLC runs employed for the isolation of cricket allatostatins. HPLC Flow Column Solvents Gradient run rate 1st LiChroCART Superspher 100 RP-18, A: 0.115% TFA in water 0-5 min 0% B 1 ml/min 100 A, 4 pm, 124 x 4 mm with guard B: 0.1% TFA in MeCN 5-8 min 0-20% B column 4 x 4 mm (Merck, Darmstadt, 8-51 min 20-33% B (linear Germany) gradient, 0.3% MeCN per min)

2nd Shiseido CAPCELL PAK C,8 SG 300, A: 0.13% HFBA in water 0-2 min 5% B 1 ml/min 300 Ä, 5 pm, 250 x 4.6 mm with guard B: 0.13% HFBA in MeCN 2-52 min 5-60% B (linear column 10 x 4.6 mm (Grom, gradient, 1.1% MeCN per min) Herrenberg-Kayh, Germany)

3rd Shiseido CAPCELL PAK C8 SG 300, A: 20 mM NH4Ac in water (pH 7.0) 0-40 min 6-63% B (linear 1 ml/min 300 Ä, 5 pm, 150 x 4.6 mm with guard B: 20 mM NH4Ac in 80% MeCN gradient, 1.14% MeCN per min) column 10 x 4.6 mm (Grom);

4th Kromasil 100 C4, 100 A, 5pm, 125 x 4 A: 0.115% TFA in water 0^1 min 10% B 0.5 mm (MZ Analysentechnik, Mainz, B: 0.1% TFA in MeCN 4^12 min 10-52% B (linear ml/min Germany) gradient, 1.1% MeCN per min) HFBA - heptafluorobutyric acid; MeCN - acetonitrile; NH4Ac - ammonium acetate; TFA - trifluoroacetic acid.

268 7125 injector with a 2 ml sample loop. The system was coupled Coelution of native and synthetic peptides to a PC equipped with BORWIN chromatographic software. In order to further confirm the identity of the synthetic pep­ The chromatographic conditions of the HPLC runs are shown in tides with their native counterparts, native and synthetic pep­ Table 1. tides were coeluted using a micro HPLC system consisting of a First HPLC run: In total, 2,700 brain equivalents from the high pressure gradient HPLC pump Eldex MicroPro, a column 40% MeCN SEP-PAK. fractions were processed in 5 runs. Frac­ thermostat Spark Mistral (set to 37°C) with built-in Rheodyne tions eluting between 13 and 50 min were tested at a concentra­ 8125 injector and 10 pi sample loop, and a UV-detector Spectra tion of 20 brain equivalents per CA in the rapid partition assay. Flow 505 equipped with a 35 nl UZ-flow cell (SunChrom The fractions eluting at 19, 20, 23 and 24 min showed the high­ GmbH, Friedrichsdorf, Germany). Peaks were detected at 214 est allatostatic activity and led to the isolation of the allato- and 280 nm simultaneously. Chromatographic conditions were statins Grb-AST AI, A2 (Lorenz et al., 1995a), Bl, B2, B3 and as follows: column: YMC-Pack ODS-AQ, 120 A, 5 pm, 150 x B4 (Lorenz et al., 1995b). In this paper, we report the further 0.5 mm; solvent A: 0.1% TFA in 5% MeCN; solvent B: 0.115% purification of the fractions eluting between 25 and 28 min (cor­ TFA in 80% MeCN; gradient: 0-50 min 12-54% solvent B (lin­ responding to 25.1-26.3% MeCN) from the first HPLC run, that ear gradient, 0.84% solvent B per min = 0.63% MeCN per min); also showed consistent allatostatic activity. followed by a 5 min rinse at 100% solvent B; flow rate: 10 Second HPLC run: Active fractions (25 + 26 and 27 + 28) pl/min. Between 5 and 10 pmol of the native peptides were co­ pooled from the five identical first HPLC runs (equivalent to injected with an equal amount of the corresponding synthetic 2,600 brains) were dried down to approximately 500 pi (Speed- peptide after having run native and synthetic peptides separately Vac concentrator), diluted with 500 pi of 0.13% heptafluorobu- on the same HPLC system. tyric acid (HFBA) in 10% MeCN and injected separately onto the column. Peak fractions were collected and tested in the rapid Statistics partition assay (20 brain equivalents per CA). All results are means ± S.E. for the number of individual Third HPLC run: Active fractions from the second HPLC run measurements indicated in the legend of each figure. The Welch (equivalent to 2,500 brains) were dried down to approximately t-test was used in the statistical treatment of the data. 500 pi, diluted with 500 pi of 20 mM ammonium acetate (NH4Ac) and injected onto the column. Peak material was col­ RESULTS lected and tested in the rapid partition assay (40 brain equiva­ Peptide purification and sequence elucidation lents per CA). Fourth HPLC run: Active fractions from the third HPLC run The purification of the four 1-min fractions from the (equivalent to 2,340 brains) were dried down to approximately first HPLC run reported in this paper led to the sequence 500 pi, diluted with 500 pi of 0.1% trifluoroacetic acid (TFA) in elucidation of four peptides with marked allatostatic ac­ 10% MeCN and injected onto the column. Single pure peaks tivity. Sequence and mass data unequivocally identified were collected and tested in the rapid partition assay (60 brain three of these peptides as novel members of the equivalents per CA). The remaining material (equivalent to Y/FXFGL/I-amide family and one peptide as a novel 2,160 brains) was subjected to sequence and mass analysis. member of the W2W9-amide family. A flow chart show­ Sequence and mass analysis, peptide synthesis ing the purification of the peptides is presented in Fig. 1. For sequence analysis by Edman degradation, the active ma­ All the synthetic peptides had the same retention times as terial from the final HPLC separations was loaded onto a their native counterparts in all the four chromatographic polybrene-coated glass-fiber filter and introduced into a Model extraction 2700 Grvllus bimaculatus brains in methanol/water/acetic acid (100/10/11 477A sequenator connected to a Model 120A on-line phenyl- thiohydantoin analyzer (Applied Biosystems, Weiterstadt, Ger­ many). prepurification 40% acetonitrile fraction Mass spectra were aquired using a matrix-assisted laser de­ (SEP-PAK C18) sorption ionisation time-of-flight (MALDI-TOF) spectrometer (Bruker Reflex, Bruker Franzen, Bremen, Germany). The accel­ eration voltage was set to 30 kV for the linear mode. The matrix was a saturated solution of a-cyano-4-hydroxycinnamic acid 1st HPLC run dissolved in water/MeCN (7/3, v/v). Peptide solutions (0.5 pi, ~1 pmol) were mixed on target with matrix solution (1/1, v/v) and left to dry. Each spectrum was the average of -50-200 sin­ 2nd HPLC run 40.5% 41.9% 41.9% 43.0% 44.5% gle shot spectra acquired in sets of five shots. Calibration was achieved by subjecting the peptide calibration mixture from Per- septive Biosystems (Wiesbaden, Germany) to an external two- 3rd HPLC run 27.5% 26.8% 26.8% 28.0% 27.5% point calibration. Peptide synthesis was performed on a Model 9050 peptide synthesizer (Milligen, Eschborn, Germany) using Fmoc/HOBt chemistry. Peptides were synthesised in the amide form using an 4th HPLC run 46.1% 45.3% 45.2% 46.1% 45.9% Fmoc-peptide amide linker polyethylene glycol-polystyrene resin (Milligen). Synthetic peptides were purified after cleavage " from the resin by reversed-phase HPLC. The mass of the syn­ peptide Grb-AST A3 Grb-AST B5 Grb-AST A4 Grb-AST A5 thetic peptides was determined by MALDI-TOF to confirm identity with the native peptides. Fig. 1. Flow chart showing percentage of acetonitrile at which allatostatins eluted from different chromatographic steps during purification.

269 T able 2. Sequence, molecular mass, calculated content per brain (losses during purification not taken into account; overall pep­ tide recovery was 50-60%, as calculated from the peak areas) and IC50 values (concentration required for 50% inhibition of JH III release by CA of 3 day-old virgin female Gryllus bimaculatus) of the isolated peptides. Monoisotopic (M+H)+ Measured Calculated peptide Peptide Amino acid sequence IC50 [M] for the am ¡dated peptide (M+H)+ content [pmol per brain] Grb-AST A3 AGMYSFGL-NH2 844.38 844.4 0.11 7 x 10-9

Grb-AST A4 SRPFGFGL-NH2 879.46 880.1 0.31 4 x 10"9

Grb-AST A5 GPDHRFAFGL-NH2 1,115.55 n.d. 0.16 3 x 10-8

Grb-AST B5 AWDQLRPGW-NH2 1,127.55 n.d. 1.49 3 x 10“8 n.d. - not determined. systems employed for the peptide purification, indicating (i.e. binding affinity to the receptor) and efficacy (i.e. the identity of the native and the synthetic peptide. Fur­ maximum effect at saturated conditions) to inhibit JH re­ thermore, the coelution of the native and the synthetic lease. The inhibition of JH release was reversible for all peptides revealed single pure peaks for all four peptides peptides: glands recovered completely from inhibited (chromatograms not shown). The isolated peptides were rates after the allatostatin had been withdrawn from the designated Grb-AST A3, A4, A5, and B5, according to incubation medium (not shown). their biological function and peptide family affiliation, The peptides Grb-AST A5 and B5 were also tested for using the widely accepted nomenclature for invertebrate a longer duration by transferring freshly dissected CA di­ peptides proposed by Raina & Gade (1988). Peptide se­ rectly into medium containing the allatostatins. Therefore, quences and approximate peptide content per brain are the CA did not have the possibility to produce radioactive shown in Table 2. JH III at basal rates during a radioactive incubation in Bioassays on corpora allata from 3 day-old virginmedium without the addition of allatostatins. Conse­ female crickets quently, the rates of inhibition were higher (Fig. 3) com­ pared to the standard testing procedure. The highest pep­ Synthetic peptides were tested in concentrations rang­ tide concentration tested (10-7 M) caused an almost com­ ing from 10“10 to 10 5 M using the standard testing proce­ plete inhibition of JH III release (more than 98% inhibi­ dure (90 min preincubation plus first and second tion). However, even after 5 h exposure to 10-7 M allato­ incubation for 120 min each). Fig. 2 presents the typical statin, the glands recovered to control rates of JH release sigmoidal dose-response curves that were generated, within 2 h after the allatostatin had been withdrawn from showing highly significant inhibition of JH release at con­ the medium (Fig. 3). centrations of 10-8 M and higher. Maximum inhibition To investigate whether allatostatins of the two different (60-70%) of JH III release was reached with an allatosta- peptide families may have a synergistic effect on JH re­ tin concentration of about 10“7 M, 50% inhibition of JH lease, CA were incubated in the presence of a mixture of release (IC50 ) was obtained with 0.4-3 x 10“8 M (Table 2). Grb-AST A5 and B5 (1(F8 M each). Compared to the ef­ The two allatostatins Grb-AST A5 and B5 that belong to fect of 2 x 10-8 M Grb-AST A5 or B5 alone, respectively, different peptide families had virtually the same potency

-log peptide concentration [M] Fig. 3. Effect of allatostatins on CA from 3 day-old adult vir­ Fig. 2. Dose response for inhibition of JH III release by Grb- gin females that had not been preincubated in radioactive me­ AST A3 (solid circles), A4 (solid squares), A5 (open circles), dium before allatostatin treatment. Controls (solid columns) and B5 (open squares) for CA from 3 day-old adult virgin fe­ without allatostatin during all incubation periods, Grb-AST A5 males. Means ± S.E. of 15 (10~8 M) or 10 (all other concentra­ (crosshatched columns) and B5 (open columns) at 10~7 M dur­ tions) determinations, respectively, ns - not significantly ing the first four incubation periods. Means ± S.E. of 10 deter­ different; *** p < 0.001. JH III release by untreated glands was minations. ns - not significantly different; ** p < 0.01; *** p < ca. 42-48 pmol x CA-1 x h"1 in this experiment. 0.001.

270 Table 3. Effect of allatostatins A5 and B5 on JH III release and JH III content within the CA. Means ± S.E. of20 (10 8 M) or 10 (all other peptide concentrations) replicates, respectively. Control (no peptide during Peptide Grb-AST B5 2nd incubation) Grb-AST A5 concentra- tion (during JH III JH III JH III in CA JH III JH III JH III in CA JH III JH III JH III in CA 2nd incuba- release release (after 2nd release release (after 2nd release release (after 2nd tion) [M] (1st incub.) (2nd incub.) incub.) (1st incub.) (2nd incub.) incub.) (lstincub.) (2nd incub.) incub.) [pmol-h-1] [pmol-h"1] [pmol] [pmol-h-1] [pmol-h"1] [pmol] [pmol-h"1] [pmol-h ’] [pmol]

1 0 - 10 6.44 ± 0.92 6.10 ±0.78 2.74 ± 0.67 7.01 ±0.88 7.31 ±0.92 3.00 ± 1.45 6.08 ±0.77 6.46 ±0.78 2.58 ±0.94 lO-9 5.46 ±0.84 5.94 ± 0.93 2.23 ± 0.48 6.82 ± 0.64 7.66 ±0.70 2.51 ±0.92 6.21 ±0.39 6.74 ±0.41 2.70 ± 1.05

1 0 - 8 6.16 ± 0.51 6.78 ±0.57 2.68 ± 0.40 6.17 ±0.42 5.58 ±0.43 1.81 ±0.80 5.99 ±0.48 6.18 ±0.43 2.38 ±0.52 lO'7 7.14 ± 1.24 7.55 ± 1.13 2.99 ± 0.94 6.98 ± 0.98 7.44 ± 1.10 2.53 ±0.65 6.74 ±0.57 7.56 ±0.62 2.44 ± 0.80

1 0 -* 6.28 ±0.71 6.74 ± 0.68 2.50 ±0.73 5.70 ±0.71 5.81 ±0.53 2.18 ± 0.41 6.10 ±0.60 5.45 ±0.59 1.78 ±0.64 Single CA were taken from 1 day-old unpaired males and incubated in medium containing 200 pM farnesol. No significant differ­ ences with respect to JH III release and JH III content within the CA between control and peptide-treated CA could be observed. no change in the rate of inhibition of JH release was ob­ four novel allatostatic neuropeptides. Three of the pep­ served (Fig. 4), indicating that both types of allatostatic tides belong to the wide-spread family of Y/FXFGL/I- peptides do not act synergistically on cricket CA. amide peptides, but are not identical to any of the known Bioassays on corpora allata from 1 day-old unpairedY/FXFGL/I-amides. The fourth of the isolated allato­ statins is a member of a family of peptides that seem to be male crickets in the presence of farnesol active as allatoregulators only in crickets (Lorenz et al., When allatostatins were tested on CA from 1 day-old 1997a), the W2W9-amide peptide family, that had origi­ unpaired males in the presence of 200 pM farnesol, no ef­ nally been isolated from locusts (Schoofs et al., 1991) and fect of the peptides on JH release or JH content within the moths (Blackburn et al., 1995), using a bioassay that CA could be observed, even at the highest allatostatin measured their myoinhibiting action. The peptide Grb- concentrations employed (Table 3). The accumulation of AST A4 seems to be a homologue of the peptides MF within the CA, however, was significantly reduced in helicostatin/cydiastatin 3 isolated from moths (Duve et the allatostatin-treated glands, indicating that these pep­ al., 1997a, b), from which it differs only in positions 4 tides can also inhibit the late steps of JH biosynthesis and 5, as well as of the cockroach peptides Dip-/Pea-AST (Fig. 5). The peptide Grb-AST B5 was somewhat more 6 (Stay et al., 1994), from which it differs in positions 1, potent in reducing the MF accumulation (IC50 = 2.5 x 10 s 4 and 5. The other isolated A-allatostatins are unique M) compared to Grb-AST A5 (IC50 = 5 x 10"8 M). structures that do not closely resemble any of the known DISCUSSION allatostatins, except in their C-terminal pentapeptide se­ quence. The peptide Grb-AST B5 differs from the other Our search for allatoregulating peptides from cricket W2W9-amide peptides isolated from cricket brains in hav- brain extracts has led to the isolation and identification of

70 n

A5 B5 A5 + B5 control a a a [2 x 10 8 M] [2 x 10'8 M) [10 ® M each] -log peptide concentration [M] Fig. 4. Comparison of the allatostatic effect of Grb-AST A5 Fig. 5. Dose response for inhibition of MF accumulation and B5, respectively, and of a combination of the two peptides within the CA from 1 day-old adult unpaired males by Grb-AST on CA from 3 day-old adult virgin females. Means ± S.E. of 10 A5 (solid circles) and B5 (open circles). Means ± S.E. of 20 determinations. Inhibition of JH release was significant at p < (10~8 M) or 10 (all other concentrations) determinations, respec­ 0.001 for all treatments. No significant difference was observed tively. ns - not significantly different; * p < 0.05; ** p < 0.01; between the treatments. JH III release by untreated glands was *** p < 0.001. Accumulation of MF within the control CA was ca. 42—46 pmol x CA 1 x h“1 in this experiment. ca. 16 pmol x CA-1.

271 ing the amino acid Pro in position 7, where the other four ber of closely related peptides occurring in one species. In B-allatostatins have the amino acid Gly. A common fea­ crickets, however, we have never found such biphasic re­ ture of the W2W9-amide isolated so far is that many of sponses to any allatostatin (or combinations thereof) them have the N-terminal amino acid sequence tested. A/GWQDL [all the W2W9-amides isolated from C. moro- The inhibiting effect of the peptides Grb-AST A5 and sus (Lorenz et al., 1998a), the Lom-MIP fromL. migrato­ B5 on the accumulation of MF, the direct JH III ria (Schoofs et al., 1991), the Grb-AST B1 from G. precursor, within the CA was quite surprising. It is known bimaculatus (Lorenz et al., 1995b), and the peptides Mas- from D. punctata that brain extract (containing allato­ MÍP 1 and 2 from M. sexta (Blackburn et al., 1995)]. The statins), forskolin (an activator of the adenylate cyclase) peptide Grb-AST B5 resembles this structure in so far and high potassium concentrations inhibit JH biosynthesis that it has almost the same sequence, except that the posi­ in vitro, but these inhibiting effects can be antagonised by tions of the amino acids Gin3 and Asp4 are changed to precursors of JH biosynthesis such as mevalonic acid and Asp3 and Gin4. Taken together, the only common features famesoic acid, implicating an early biosynthetic step as of all the peptides of the W2W9-amide family isolated so the target of inhibition (Feyereisen & Farnsworth, 1987). far are the amino acid Trp in positions 2 and 9 and the Sutherland & Feyereisen (1996) were able to demonstrate amino acids Ala or Gly in position 1. Changes in the that the inhibition of JH biosynthesis by Dip-AST 7 oc­ other amino acid positions apparently do not have a dra­ curs at a very early step of JH formation, i.e. the transfer matic effect on the biological activity of these peptides. of C2-units from mitochondria to the cytoplasm. L. In mi­ In general, the activity of the B-allatostatins is almost gratoria, the stimulating principle from the brain also acts one order of magnitude lower compared to the A- at the earlier steps of JH biosynthesis (Couillaud et al., allatostatins (Lorenz et al., 1995a, b; this study). On the 1988; Couillaud, 1991; Gadot & Applebaum, 1986). other hand, most of the B-allatostatins occur in markedly However, there seems to be one principal difference with higher concentrations in the brain (Lorenz et al., 1995a, b; respect to the CA physiology ofL. migratoria and D. unpubl.). However, the peptides Grb-AST A5 and B5 had punctata on the one hand and G. bimaculatus (and also the same potency and efficacy in inhibiting JH release of Periplaneta americana and B. germánica) on the other cricket CA (Grb-AST A5 is the least active of the cricket hand: the stimulation of CA activity with late precursors Y/FXFGL/I-amides, whereas Grb-AST B5 is a moder­ of JH biosynthesis normally leads to an accumulation of ately active member of the W2W9-amides of the cricket), MF within the CA of G. bimaculatus (Klein et al., 1993; which makes them ideal tools for comparative studies. this study), P. americana (Pratt et al., 1975), and B. ger­ One reason for the presence of multiple allatostatic neu­ mánica (Bellés et al., 1989), indicating that the methyl ropeptides (either all of them members of one peptide famesoate epoxidase (EC. 1.14.14) can be a rate-limiting family as is the case in cockroaches, or even belonging to enzyme in these species, at least under the experimental two completely different peptide families as in the conditions employed. In other insect species such asL. cricket) within one species may be the necessity for a migratoria (Couillaud et al., 1988), D. punctata (Feyere­ joint action of different allatostatins to obtain maximal re­ isen et al., 1981), and C. morosus (Lorenz et al., 1999) no sponse at the target tissue. Therefore, the two peptides accumulation of MF within the CA has been observed. Grb-AST A5 and B5 were used to investigate the possi­ Therefore, in these species, the methyl famesoate epoxi­ bility that allatostatins of the different peptide families dase is not a rate-limiting enzyme of JH biosynthesis, may act synergistically. However, no such synergistic ac­ even under conditions of maximal stimulation of the late tion could be observed. This finding indicates that both steps of JH formation. These differences in the physiol­ groups of allatostatic peptides either may bind to the same ogy of the CA may also require different mechanisms of receptor, which is rather unlikely, or, if discrete receptors regulation. exist for each allatostatin family, the pathways of signal Our results on the reduction of MF accumulation in CA transduction from the different receptor types into the cell that had been treated with 200 pM famesol plus allato­ seem to be completely independent from each other. Dif­ statins clearly indicate that there is an inhibiting mecha­ ferent receptor subtypes for allatostatic peptides of the nism that acts on the late steps of JH biosynthesis inG. Y/FXFGL/I-amide family have been proposed in D. bimaculatus. Also in corpora cardiaca-corpora allata com­ punctata because a biphasic response of the CA to Dip- plexes from 3 day-old virgin females of G. bimaculatus AST 7 was observed, which could be resolved into two treated with 200 pM famesol and 10"6 M Dip-AST 7 a re­ quasi-additive high-dose and low-dose components (Pratt duction of MF accumulation, but no reduction of JH III et al., 1991). The same phenomenon was found with ana­ biosynthesis had been observed (Neuhauser et al., 1994). logues of the peptide Dip-AST 2 (Pratt et al., 1997). The same phenomenon occurred when single CA from 3 Biphasic dose-response curves were also generated with day-old virgin crickets were incubated in the presence of CAST 8, but not with CAST 5, when these peptides were 200 pM famesol and 10"* M octopamine (Woodring & tested for their ability to inhibit spontaneous contractions Hoffmann, 1994). In D. punctata the activity of the of the hindgut of Calliphora vomitoria (Duve et al., methyl famesoate epoxidase can be significantly inhibited 1996). These distinct dose-response curves for different by allatostatins, however, since no accumulation of MF members of the Y/FXFGL/I-amide peptide family may be occurred in control glands, no effect of the allatostatin on significant in providing an explanation for the huge num­ the MF accumulation could be measured (Wang et al.,

272 1994). Taken together, these results strongly support the D uve H., Johnsen A.H., M aestro J.-L., Scott A.G., W instan ­ assumption that the late steps of JH biosynthesis can also ley D., D avey M., East P.D. & T horpe A. 1997b: Lepidop- be targeted by inhibiting factors. Whether this mechanism teran peptides of the allatostatin superfamily. Peptides 18: plays an important role under normal physiological condi­ 1301-1309. F eyereisen R. & Farnsworth D.E. 1987: Comparison of the in­ tions in vivo remains unclear. hibitory effects of brain extract, high K+ and forskolin on ju­ ACKNOWLEDGEMENTS. This work was in part supported finan­ venile hormone synthesis by Diploptera punctata corpora al­ cially by the Deutsche Forschungsgemeinschaft (Ho 631/15-2). lata. Insect Biochem. 17: 939-942. We thank C.-J. Goebel and F. Ramming for technical assistance F eyereisen R. & T obe S.S. 1981 : A rapid partition assay for rou­ and N. 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