Eur. J. Biochem.253, 8102816 (1998) FEBS1998

Growth-blocking peptide expressed in the insect nervous systemCloning and functional characterization

Yoichi HAYAKAWA and Hirofumi NOGUCHI

Biochemical Laboratory, Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan

(Received 5 January1998) 2 EJB 98 0008/3

Growth-blocking peptide (GBP) is an insect biogenic peptide that retards larval growth and, conse-quently, delays the onset of pupation of lepidopteran larvae. Transcription of the GBP gene has beenproven to be regulated in a tissue-dependent manner by an approximately 0.8-kb mRNA transcript in fatbody and 2.5-kb transcript in the brain central nervous system (CNS) [Hayakawa, Y., Ohnishi, A., Yama-naka, A., Izumi, S. & Tomino, S. (1995) FEBS Lett. 367, 1852189]. The GBP cDNA in the brain CNSwas isolated, sequenced and found to be almost identical with that in the fat body, except that it encodesa long 5′-untranslated region (approximately1.1 kb), thus indicating that the brain CNS is synthesized asa polyprotein precursor and released through posttranslational processing in the same manner as thatpredicted in the fat body. Sequence analyses of the genomic GBP gene fragments demonstrated that the5′-untranslated region contains an intron encoding a sequence element, TGATAA, which has been re-ported to be important for expression in the insect larval fat body, suggesting that this element contributesto a tissue-specific expression of the shorter GBP mRNA in fat body. Ribonuclease protection assaysshowed that parasitization by a parasitic wasp elevated the expression of GBP mRNA in brain CNS. Theparasitization-induced increase of GBP precursor and GBP concentrations was also demonstrated in brainCNS. Brain CNS dopamine was increased eightfold by incubating the tissue with GBP for 8 h throughthe elevation of dopa decarboxylase activity by a transcriptional enhancement perhaps due to GBP-induced increase of cytoplasmic Ca21 concentration. Dopamine elevation reducedin vitro incorporationof methionine into proteins synthesized in brain CNS.

Keywords:nervous system; growth-blocking peptide; dopamine; dopa decarboxylase ; parasitization.

Many endoparasitic wasps disrupt the metamorphosis of ho- fourfold. Based on these results, it was proposed that GBP mightlometabolous host insects. In many cases, the host is develop- be a hormone-like peptide which coordinates, along with juve-mentally arrested in the larval stage and never initiates metamor-nile hormone, the regulation of larval characteristics in lepidop-phosis [1]. Last instar larvae of the armywormPseudaletia sepa- teran insects [7].rata parasitized with the parasitic waspCotesia kariyaido not Recently, we have determined the sequence of the GBPinitiate metamorphosis to pupae and, eventually, wasp larvaecDNA in the fat body of the armyworm larvae and demonstratedemerge from the larval hosts [2]. Studies on the mechanismsthat GBP is first synthesized as a polyprotein precursor [8].whereby parasitism suppresses metamorphosis of the host in-Furthermore, it has been proven that the GBP mRNA is tran-sects indicate that a novel peptide, growth-blocking peptidescribed in both the fat body and the brain central nervous system(GBP), occurs in the hemolymph of parasitized last instar larvae(CNS) and that the mRNA (approximately 2.5 kb) expressed inof the host armyworm [3, 4]. Injection of the isolated GBP intobrain CNS is much longer than that (approximately 0.8 kb) innonparasitized host insects during the early part of the last larvalfat body. Estimations of the absolute amount of GBP mRNA ininstar clearly retards larval development and causes a delay infat body and brain CNS indicate that it is about100 times morepupation through repression of hemolymph juvenile hormone es-abundant in the former than the latter, suggesting that hemo-terase activity [5, 6]. Further studies demonstrated that GBP islymph GBP is secreted primarily from fat body.a biogenic peptide of the host insect. Hemolymph levels of GBP It has been demonstrated that hemolymph GBP elevated byare much higher in the penultimate instar larvae than in the lastparasitization causes a rise in integumental dopa decarboxylaseinstar larvae, whereas parasitization induces an elevation ofactivity which increases dopamine levels in the integument [9].plasma GBP levels during the last larval instar approximatelyThe dopamine-elevated integument secretes dopamine into the

hemolymph, by which larval development is retarded [10].Correspondence toY. Hayakawa, Biochemical Laboratory, InstituteTherefore, it can reasonably be assumed that hemolymph GBP,of Low Temperature Science, North-19, West-8, Kita-Ku, Sapporo, Ja-

pan 060 which is abundantly synthesized and released from the fat body,Fax: 181 11 706 7142. plays an important role in the retardation of larval developmentAbbreviations.CNS, central nervous system; GBP, growth-blockingof the host insect by parasitism. However, neither the function

peptide; dopa, 3,4-dihydroxy-L-phenylalanine. of GBP in nervous tissue nor the mRNA transcript structureEnzyme.Dopa decarboxylase (EC 4.1.1.28).are known. We report here the molecular cloning and functionalNote. The novel DNA sequence reported here has been depositedcharacterization of GBP in brain CNS, and show that it serves aswith the DDBJ database and is available under accession number

980320133405.10276. a secretory peptide which stimulates dopa decarboxylase activity

811Hayakawa and Noguchi (Eur. J. Biochem. 253)

leading to increased dopamine levels in brain CNS similar to Thermal asymmetric interlaced-PCR mixture (50µl) con-those in the integument. Furthermore, we show the effect of ele- sisted of PCR buffer, 0.2 mM dNTPs, 0.01% gelatin,1 U Am-vated dopamine on the rate of protein synthesis in isolated brain pliTaq polymerase (Perkin-Elmer), 0.15 µM specific primerCNS. (5′-AAGGATCCGCACACAAAGTCACTAATGAGATTC) and

5 µM of an arbitrary degenerate primer (5′-AAGAATTCNN-NAGGGTAAG). The thermal cycling conditions are the sameas those described by Liu and Whittier [15].MATERIALS AND METHODS

Ribonuclease protection assay.The GBP cDNA fragmentAnimals. P. separatalarvae were reared on an artificial diet(428 bases) and dopa decarboxylase cDNA fragment (1490

at 2561 °C with a photoperiod of16-h light/8-h dark. Parasit- bases), mainly protein-coding reagions, were subcloned intoization by C. kariyai was carried out by exposing prospectivepBluescript II vector and, after linearization with certain restric-hosts (day 0 last instar larvae ofP. separata) to female wasps. tion enzymes, antisense probes were generated by T3 RNA poly-After a single oviposition was observed, the parasitized larvamerase with [A-32P]UTP using a kit purchased from Stratagene.was immediately removed in order to avoid superparasitizationThe 649-base controlβ-tubulin probe was generated using theand then reared on an artificial diet. Penultimate instar larvaesame procedure. All the riboprobes were purified by gel electro-undergoing ecdysis between 424.5 h after starting the light pe- phoresis and eluted in ribonuclease-free elution buffer (Am-riod were designated as day 0 last instar larvae. Adult waspsbion). For each assay, 2µg RNA was hybridized to104 cpm ofwere maintained with honey. each riboprobe, basically according to the manufacturer’s in-

Chemicals. Radioactively labelled reagents were obtainedstructions (Ambion).from NEN Research Products (Dupont). Oligonucleotides were cDNA synthesis and cloning.Total RNA was prepared bygamma radiolabelled using T4 polinucleotide kinase withγ- homogenizing brain CNS of day 2 last instar larvae of the army-32P]ATP [11] ; cDNA fragments were labelled using the randomworm 2 days after parasitization.prime labelling method with [A-32P]dCTP [12]. GBP was synthe- Polyadenylated mRNA was purified from the total RNAsized by the solid-phase procedure using an Applied Biosystemusing the Quick Prep mRNA purification system (Pharmacia).model 430A peptide synthesizer (Perkin-Elmer). All otherSynthesis of cDNA from 2µg poly(A)-rich RNA was achievedchemicals were reagent grade. by the method of Gubler and Hoffman [16] with a cDNA synthe-

Isolation of RNA and genomic DNA. Brain, CNS and fat sis kit (Amersham). The cDNA was size fractionated to removebody were dissected and washed well with NaCl/Pi (8 mM fragments, 0.5 kb ligated toλgt10 arms and packaged into cap-Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, sids using a commercial kit,λgt10 cDNA cloning system (Amer-pH 7.2). Immediately after isolation, the tissues were frozen insham). For initial screening, the recombinant phages were platedliquid nitrogen and total RNA was isolated by the method ofon Escherichia coliNM 514 (Amersham) at about 3000 plaque-Chomczynski and Sacchi [13]. forming units/90-mm petri dish, and plaques were transferred to

P. separatagenomic DNA was isolated from hemocytesHybond N1 nylon membranes (Amersham). A32P-labelled fatusing DNA ZOL reagent (Gibco BRL) according to the manu-body GBP cDNA fragment corresponding to the coding regionfacturer’s instructions. was used to screen the cDNA library. The cDNA inserts were

Polymerase chain reaction.Oligonucleotide primers were subcloned into pBluescript II phagemid vector using a commer-designed from the sequence of brain CNS GBP cDNA as fol-cial ligation kit (Takara).lows: primer N1, 5′-AAGGATCCGCCAGTGTTGACGAAGT- DNA sequencing.For nucleotide sequencing of the cDNAGAG; primer N2, 5′-AAGAGCTCCTTCAGTCAAGAGAAA- insert, serial deletion derivatives were prepared using exo-ATTATCTG; primer C1, 5′-AAGAGCTCCACGCATACAAA-

nuclease III and mung bean nuclease [17]. The deleted cDNACTACAACAACCC; primer C2, 5′-AAGGATCCCCATCTGC-fragments and PCR-amplified DNA fragments were sequenced

ACCGTTATATTGCAGTG; primer C3, 5′-AAGAATTCAGTA-by a Taq dye primer cycle sequencing kit (Perkin-Elmer). Com-ATTAATTGCTAGAAGGTGGG.puter-assisted sequence analysis was performed with GEN-Oligonucleotide primers for dopa decarboxylase cDNA wereETYX-MAC Ver 6.2.0 (Software Development Co.).designed from the sequence of itsP. separatacDNA (sequenced

Northern blot hybridization. 10 µg total RNA was dena-recently in our laboratory) as follows: sense primer, 5′-AAGA-tured with glyoxal and dimethyl sulfoxide and separated byATTCATGGAGGCCGGAGATTTCAAAG; antisense primer,1.0% (mass/vol.) agarose gel electrophoresis in10 mm sodium5′-AAGTCGACTTATGAAGTTGTTAGCAATCGC.phosphate (pH 7.0). After the gel electrophoreses, the RNAsOligonucleotide primers forβ-tubulin cDNA were designedwere transferred to Hybond N1 nylon membrane. Hybridizationfrom the consensus sequences reported forDrosophila melano-and washing were performed according to the protocols of Sam-gasterand chicken brainβ-tubulin as follows: sense primer, 5′-brook et al. [18].AAGAGCTCTGGAGCCMGGYACSATGGACTCKGT;

Dopamine and dopa decarboxylase assay.Dopamineantisense primer, 5′-AAGAGCTCGTCTTCACGTTGTTGGG-levels were determined by HPLC with coulometric electrochem-GATCCA.ical detection system (ESA) as described previously [19]. DopaThe PCR amplification reaction was conducted according todecarboxylase activity was assayed according to the procedurethe procedure slightly modified from the method of Strub andof Noguchi and Hayakawa [9].Walter [14]. Standard PCR was performed in a 50-µl volume

Ca2+ influx assay.Synaptosomes prepared from10 isolatedcontaining 50 ng cDNA (or 500 ng genomic DNA), PCR buffer,brain CNS, using a series of differential centrifugations basically100 µM dNTPs, 0.01% gelatin,1 U AmpliTaq polymerase (Per-according to the procedure as described by Cotman [20], werekin-Elmer) and1 µM of each oligonucleotide primer set. Tem-suspended in100 µl NaCl/Pi containing 20µM Fluo-3 (Dojindoperature-cycling profiles were generally based on the followinglaboratories) and incubated at 37°C for 1 h. After centrifugingmodel : 35 cycles at 95°C for 1min, 63°C for 2 min and 72°Cat 800g for 3 min, the pellet was resuspended with 500µl NaCl/for 3 min. Some of the PCR products were ligated withPi and centrifuged again at 800g for 3 min. The Fluo-3-loadedpBluescript II phagemid vector (Stratagene) for DNA sequenc-synaptosome pellet was resuspended in 5 ml Grace’s insect me-ing and preparing RNA probes after digestion with the appropri-

ate restriction enzyme(s). dium. Immediately after adding1 µl GBP solution into1ml of

812 Hayakawa and Noguchi (Eur. J. Biochem. 253)

study this possibility more substantially, it was necessary tocharacterize brain CNS GBP cDNA and gene structures.

The cloned cDNA encoding fat body GBP [8] was used toscreen aλgt10 cDNA library constructed from mRNA of larvalbrain CNS. Four clones encoding GBP were independently iso-lated, bearing the cDNA inserts ranging over1.522.0 kb inlength. These four clones were sequenced100% in both direc-tions and found to have various lengths of 5′ and 3′ noncodingregions although all of them were identical to each other in thecoding sequence. The nucleotide sequence of the longest 2.0-kbcDNA insert is shown in Fig. 2. The brain CNS cDNA is com-prised of an approximately1.1-kb 5′-untranslated region and thefollowing portion, open reading frame and 3′-untranslated re-gion, which is almost identical with fat body GBP cDNA. Theopen reading frame encoding the GBP precursor polypeptide isidentical with that in fat body.

PCR primers N1 and C1 were designed from the sequencesclose to both ends of the cloned cDNA to amplify a GBP gene.PCR amplification on genomic DNA prepared from the army-worm hemocytes gave approximately 2.1 kb of the productwhich was bigger than expected, approximately1.8 kb, thus in-dicating that the GBP gene may have 0.3 kb of intron(s). Se-quencing of the product amplified by the second PCR using

Fig. 1. Expression of the gene encoding the GBP precursor.(A) Brain primers N1 and C2 revealed that the 5′-untranslated region con-(B), central nervous system (N) and fat body (F) were dissected fromtains a single 0.3-kb intron whose intron/exon boundaries matchday 2 penultimate instar larvae of nonparasitized armyworm.10 µg totalthe consensus sequence for splice sites [23]. Furthermore, thisRNA was electrophoresed on each lane and analyzed by northern hybrid-intron contains a typical GATA motif (TGATAA) and two cog-ization using fat body GBP cDNA as a probe. (B) The same membranenates (KGATAW) that have been reported to be important forwas probed again with actin cDNA as a loading control.expression in the insect larval fat body [24, 25], thus suggestingthat the region downstream following this intron should be tran-scribed in the fat body. The 5′-flanking domain of the GBP genethe synaptosome suspension, the fluorescence of the mixturewas amplified by a thermal asymmetric interlaced PCR and se-was measured to calculate the Ca21 concentration.quenced. Inspection of the nucleotide sequence immediately up-GBP assay.GBP was assayed by the procedure based onstream of the 5′ end of the cDNA sequence revealed a TATA-ELISA as described previously [21].like sequence (TATAAA) and the consensus capsite initiator se-Analyses of protein synthesisin vitro. Four brain CNS dis-quence (TCAGT) [26] as shown in Fig. 2.sected from larvae in various conditions (parasitized, nonparasit-

ized and dopa-fed) were incubated in Grace’s medium contain-Expression of the GBP gene in brain CNS.To examine ex-ing 10 µCi L-[35S]methionine at 37°C for 4 h. After washingpression of GBP mRNA in brain CNS of the host armywormthree times with NaCl/Pi, the tissues were lightly blotted with alarvae after parasitization, a ribonuclease protection assay wasfilter paper, immediately transferred to a1.5-ml polypropyleneconducted. Previous studies demonstrated that the hemolymphmicrotest tube containing 50µl homogenizing buffer containingGBP titer was increased within one day after parasitization but,50 mM Tris/HCl pH 7.5, 30 mM dithiothreitol,1 mM (p-during this period, there was no difference in the levels of GBPamidinophenyl)methanesulfonyl fluoride hydrochloride (WakomRNA in the fat body of parasitized and nonparasitized larvaePure Chemical Ind.) and sonicated by an Artek sonic dismem-[8]. The brain CNS GBP mRNA level was, however, increasedbrator (20 pulses at 50 W). The homogenate was centrifuged atwithin 1 day after parasitization and this elevated level of ex-20 000g for 10 min at 4°C; 20µl of the supernatant was mixedpression maintained through at least 3 days after parasitizationwith 0.4 M perchloric acid and the radioactivity of the superna-(Fig. 3).tant after centrifugation at 20000g for 10 min at 4°C was mea-

From these results, it is reasonable to suggest that biosynthe-sured by a scintillation counter (Aloka LSC-5100). The remain-sis of GBP in brain CNS is increased by parasitization. Thising 30µl was prepared for SDS/PAGE by incubating with 80idea was supported by the finding that GBP precursor and GBPmM Tris/HCl pH 8.8 containing10% SDS and 2.5% 2-mercap-concentrations were elevated in brain CNS within16 h after par-toethanol in boiling water for 5 min and developed by theasitization and a part of GBP elevated in the tissue was secretedmethod of Laemmli [22]. The gel was stained with Coomassieinto Grace’s insect medium at the end of the12-h incubationbrilliant blue R-250 and, after drying, exposed to X-Omat ARperiod (Table1). Therefore, we expected that GBP secreted fromfilm (Kodak) for autoradiography.brain CNS cells might affect the nervous cells themselves inbrain CNS as a local mediator, such as autocrine and paracrinefactors. To confirm this hypothesis, we focused our attention onRESULTSdopamine levels in the dissected brain CNS after incubation withGBP, because previous studies showed that GBP elevated theCharacterization of brain CNS GBP cDNA and gene.A

northern hybridization was carried out on RNA extracted from integumental dopamine level through an increase in dopa decar-boxylase activity in integumentin vitro [9].brain, central nervous system (CNS) and fat body using fat body

GBP cDNA as a probe. The positive band in both brain andCNS was much longer (approximately 2.5 kb) than that (approx-Dopamine level and dopa decarboxylase activity in brain

CNS. To determine whether dopamine in brain CNS could beimately 0.8 kb) in fat body (Fig.1), suggesting that transcriptionof the GBP gene is regulated in a tissue-dependent manner. To increased by GBP, the dissected tissues were incubated with

813Hayakawa and Noguchi (Eur. J. Biochem. 253)

Fig. 2. Growth-blocking peptide cDNA and genomic nucleotide sequence.The sequences of brain CNS GBP cDNA are depicted by capitalletters. The sequence almost identical with fat body GBP cDNA is underlined. The coding region is double underlined. The TATA box and theconsensus capsite initiator sequences are boxed. The GATA motif is denoted by thick line.

Table 1. Effect on the concentrations of GBP and GBP precursorprotein in isolated brain nerve cord. GBP and GBP precursor concen-trations represent the mean6SD for 4 determinations;10 brain CNSwere isolated for each determination. GBP concentration secreted fromisolated brain CNS represents the mean6SD for 4 determinations. Foreach determination,15 brain CNS were incubated in 50µl Grace’s insectmedium at 37°C for 10 h.

Protein Concn in brain nerve cord

nonparasitized parasitized

pmol/tissueFig. 3. Ribonuclease protection assay of GBP mRNA in parasitizedP. separata.Nonpara D023, total RNA prepared from brain CNS of

GBP 0.02860.005 0.08160.010nonparasitized last instar larvae at days 023. Para D123, total RNAGBP precursor 0.03260.013 0.18460.024prepared from last instar larvae at days123 after parasitization on dayGBP secreted into medium 0.01260.004 0.02260.0060 (4 h after start of light period).β-Tubulin cDNA fragment was ana-

lyzed as a control.

GBP and the dopamine contents in the tissues were monitored. The elevation of the dopamine level in brain CNS is likelyto be due to changes in the activity of enzymes involved inThe dopamine level in brain CNS was increased 8-fold at 8 h

following incubation with GBP but only after a lag period of at dopamine metabolism such as tyrosine hydroxylase and dopadecarboxylase. Previous studies demonstrated that GBP did notleast 4 h (Fig. 4A). GBP showed a dose-dependent capacity to

elevate the dopamine level in brain CNS (Fig. 4B). enhance the former activity but enhanced the latter in the integu-

814 Hayakawa and Noguchi (Eur. J. Biochem. 253)

Fig.6. Ca2+ influx into brain CNS synaptosomes by GBP.Fluores-cence was measured immediately after adding various concentrations of

Fig. 4. GBP effect on dopamine level in brain CNSin vitro. (A) Effect GBP into 1 ml Fluo-3-loaded synaptosome suspension prepared fromof incubation time on dopamine content in brain CNS. Brain CNS wasbrain CNS (two brains). Each point represents the mean6SD for sixdissected from day 2 last instar larvae ofP. separataand incubated in determinations.Grace’s medium containing 0.8µM GBP at 37°C. (B) Effect of variousconcentrations of GBP. Dissected brain CNS was incubated in the me-dium containing indicated concentrations of GBP at 37°C for 8 h.

centration levels in brain CNS synaptosomes were measured af-ter mixing with various concentrations of GBP using Ca21 fluo-rescence indicator, Fluo-3. Ca21 influx was clearly induced byTable 2. Dopa decarboxylase activity in brain CNS of armywormGBP in a concentration-dependent manner (Fig. 6) with a maxi-larvae. Assays were performed on day 2 last instar larvae. Each valuemum at 0.4 pmol/ml GBP in the incubation medium.represents the mean6SD of ten independent determinations.

Sample Dopa decarboxylase Global protein synthesis.In vitro incorporation of [35S]methio-nine into protein synthesized by brain CNS was examined

pmol · min21 · µg protein21(Fig. 7). First, the radioactivity incorporation by parasitized andnonparasitized larval brain CNS was compared. As shown in theControl 0.8160.05

GBP 1.6660.21 histogram, parasitization almost halved the incorporation. A50% decrease in the radioactivity incorporation was also ob-served in brain CNS, dissected from the larvae whose nervoustissue dopamine concentration had been elevated about fourfoldby feeding with dopa for1 day before dissection. This decreasein the radioactivity incorporation was reproduced by incubatingthe dissected brain CNS with GBP for 8 h prior to the incubationwith [35S]methionine. The gel autoradiography presented inFig. 7 shows a typical case SDS/PAGE protein analysis, indicat-ing that there are no specific proteins which decreased concomi-tantly with the rate of synthesis induced by parasitization, dopa-mine or GBP.

Fig. 5. Ribonuclease protection assay of dopa decarboxylase (DDC)DISCUSSIONmRNA in brain CNS. Brain CNS was dissected from day 2 last instar

larvae. Control 026, total RNA prepared from brain CNS at 0, 3 h or In the present study we used fat body GBP cDNA to isolate6 h after incubation in Grace’s insect medium containing 0.8µM BSA.GBP cDNAs from armyworm brain CNS cDNA libraries. TheGBP 026, total RNA prepared from brain CNS at 0, 3 h or 6 h afterbrain CNS GBP cDNA encodes a1.1-kb 5′-untranslated se-incubation in the medium containing 0.8µM GBP. Other explanationsquence followed by an almost identical sequence of the fat bodyas in Fig. 3.GBP cDNA. The function of the long 5′-untranslated region isuncertain at present, although similar regions have been reportedto contribute to the stability of mRNAs in various kinds ofment, thereby causing the increase in dopamine concentrations

[9]. Based on this observation, dopa decarboxylase activity was mRNA such asBacillus subtilis succinate dehydrogenasemRNA [27], E. coli outer membrane protein A mRNA [28] andmeasured in brain CNS following incubation with GBP for 6 h

and found to be approximately double that in the control (Ta- glutamyl-tRNA synthetase mRNA [29]. It has been reported thatthe glutamyl-tRNA synthetase mRNA might be stabilized by theble 2). The results of the ribonuclease protection assay showed

that the relative abundance of dopa decarboxylase gene expres- stable hairpin at its 5′ end [29]. Computer-assisted analyses pre-dicted that the 5′-untranslated region of the brain CNS GBPsion was enhanced in brain CNS by incubation with GBP, thus

indicating that the enzyme was transcriptionally enhanced by mRNA can adopt at least three stable stem-loop structures atposition 8452868, 111421133 and144621474, thereby indi-GBP (Fig. 5).cating that the long 5′ region of the GBP mRNA might contrib-ute substantially to this stability.Ca2+ influx into brain CNS synaptosomes. To examine

whether intracellular Ca21 concentration could be related to the The majority of eukaryotic mRNAs have a short 5′-un-translated region of 202100 nucleotides with no upstream AUGactivation of the dopa decarboxylase gene expression, Ca21 con-

815Hayakawa and Noguchi (Eur. J. Biochem. 253)

Fig. 7. SDS/PAGE and autoradiography of radiolabelled protein synthesisin vitro by brain CNS. Brain CNS was dissected from day 2 lastinstar larvae. Para and Nopara, parasitization was carried out on day 0 last larval instar ; DOPA and Control, the test larvae were fed an artificialdiet containing1% (by mass) dopa and BSA for 24 h before dissection, respectively; GBP and Control, brain CNS dissected from the larvae wasincubated with 0.8µM GBP and BSA at 37°C for 8 h prior to the incubation with [35S]methionine, respectively. Right, electropherogram stainedwith Coomassie brilliant blue R-250. Left, autoradiogram of the SDS/PAGE gel. Each value on the upper histogram is the mean of three separateassays. The radioactivity incorporated by brain CNS dissected from nonparasitized day 2 last instar larvae is defined as100%.

codons, enabling efficient translation, whereas 5210% of verte- andPhormia terraenovae, about 300 neurons displayed dopa-mine and tyrosine hydroxylase immunoreactivity in the brainbrate mRNAs have long, highly structured 5′-untranslated re-

gions that usually contain upstream AUG codons [30]. Such and subesophageal ganglion [37]. The high basal level of dopa-mine and its widespread distribution, suggest that dopaminetranscripts primarily encode regulatory proteins such as growth

factors, transcription factors, proto-oncogenes, cytokine recep- might be capable of influencing the rate of protein synthesisin the nervous tissue directly or indirectly. It is proposed thattors and proteins in signal transduction and the immune response

[31235]. Certainly the brain CNS GBP mRNA seems to be one parasitism- or GBP-induced reductions in incorporation of radio-active methionine into newly synthesized proteins may be dueof these examples. Based on these reports together with the fact

that GBP is actively expressed in brain CNS of the parasitized mainly to the elevation of dopamine levels in brain CNS.Recently we have demonstrated that the dopamine level isinsect, it is reasonable to expect that the long 5′-untranslated

region of brain CNS GBP mRNA might contribute to regulatory significantly higher in brain CNS of diapause-destined pupaethan in non-diapause-destined pupae of the cabbage armywormmechanisms involving nervous-tissue-specific and stress-sensi-

tive expression of GBP. Mamestra brassicae[38]. Furthermore, elevation of the dopa-mine level accomplished by feeding dopa to last instar larvaeThe cDNA sequence of brain CNS GBP indicates that it is

a secretory peptide just as in fat body GBP; thus, GBP is first induced a diapause-like state in more than 50% of the pupaeunder long day lengths [38]. These results suggest that dopaminesynthesized as a pre-pro-peptide and secreted outside of the ner-

vous secretory cells; a part of GBP might be released into the might act on brain CNS to inhibit the normal neurosecretoryprocess and then change the developmental program in the cab-surrounding hemolymph through exocytosis. Although we can-

not deny the possibility that GBP secreted from brain CNS has bage armyworm. Therefore, dopamine may play a crucial rolein insect development as a neurotransmitter or neuromodulator.a target organ other than brain CNS itself, the previous observa-

tion that GBP is secreted into hemolymph more abundantly from The present study shows that GBP increases the brain CNS do-pamine level in the armyworm, thereby suggesting that GBPfat body than from brain CNS allows us to speculate that brain

CNS GBP acts as a local mediator. This speculation was partly might serve as a key neuromodulator, possibly linking environ-mental stimuli to the induction of diapause in the cabbage army-supported in the present study by the observation that GBP is

secreted into the culture medium more abundantly from the worm.As demonstrated recently, the parasitism-induced elevationbrain CNS of parasitized larvae than from that of nonparasitized

larvae. Furthermore, dopamine concentration in brain CNS is of the hemolymph GBP level is mostly attributable to anincrease in the level of GBP precursor protein in the fat body ofelevated eightfold after incubation with 0.8µM GBP. Accord-

ingly, it is reasonable to expect that GBP secreted from brain the armyworm, perhaps through translational enhancement [39].The present experiments showed a parasitism-induced elevationCNS exerts a potent stimulatory effect on neurosecretory cells

to produce dopamine in the nervous tissue. The GBP-induced of the GBP and GBP precursor concentrations in brain CNS ofthe armyworm through transcriptional activation. Therefore, thedopamine was demonstrated to be due to an increase in dopa

decarboxylase activity through enhancement of transcriptional two tissues, fat body and brain CNS, seemed to be affected dif-ferently by parasitization. Although we have not characterizedlevels of this enzyme perhaps by the increase of cytoplasmic

Ca21 concentration. the promoter regions of the GBP gene in the present study, theintron in 5′-untranslated region of brain CNS GBP mRNA mayDopamine is the most abundant monoamine in the insect

nervous system [36]. In the blowfliesCalliphora erythrocephala contribute toward the differences in expression patterns between

816 Hayakawa and Noguchi (Eur. J. Biochem. 253)

19. Hayakawa, Y., Bodnaryk, R. P. & Downer, R. G. H. (1987) Effectbrain CNS and fat body. Further analysis of the promoter regionof taurine on excitation-induced changes in brain and hemolymphof the GBP gene will undoubtedly advance our understandingoctopamine levels in Periplaneta americana,Arch. Insect Bio-on the mechanisms through which different species of GBPchem. Physiol. 5, 1292137.mRNA are expressed in brain CNS and fat body in response to

20. Cotman, C. W. (1974) Isolation of synaptosomal and synapticenvironmental stimuli.plasma membrane fractions,Methods Enzymol. 31, 4452452.

21. Ohnishi, A., Hayakawa, Y., Matsuda, Y., Kwon, K. W., Takahashi,We thank Professor Roger G. H. Downer of the Asian Institute ofT. A. & Sekiguchi, S. (1995) Growth-blocking peptide titer duringTechnology (Thailand) for his critical reading of the manuscript. Thislarval development of parasitized and cold-stressed armyworm,work was supported by Grant-in-aid for research on Priority Area (No.Insect Biochem. Mol. Biol. 25, 112121127.08276101) from the Ministry of Science, Education and Culture of Ja-

22. Laemmli, U. K. (1970) Cleavage of structural proteins during thepan.assembly of the head of bacteriophage T4,Nature 227, 6802685.

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