Regulation of insect steroid hormone biosynthesisby innervating peptidergic neuronsNaoki Yamanaka*, Dusan Zitnan†, Young-Joon Kim‡, Michael E. Adams‡, Yue-Jin Hua§, Yusuke Suzuki¶, Minoru Suzuki¶,Akemi Suzuki¶, Honoo Satake�, Akira Mizoguchi**, Kiyoshi Asaoka††, Yoshiaki Tanaka‡‡, and Hiroshi Kataoka*§§

*Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan; †Institute of Zoology, SlovakAcademy of Sciences, Dubravska cesta 9, 84206 Bratislava, Slovakia; ‡Departments of Entomology and Cell Biology and Neuroscience, University ofCalifornia, 5429 Boyce Hall, Riverside, CA 92521; §Institute of Nuclear-Agricultural Science, Zhejiang University, Zhejiang 310029, China; ¶Frontier ResearchSystem, RIKEN, Saitama 351-0198, Japan; �Suntory Institute for Bioorganic Research, Osaka 618-8503, Japan; **Division of Biological Science, GraduateSchool of Science, Nagoya University, Nagoya 464-8602, Japan; and Laboratories of ††Insect Neurobiology and ‡‡Insect Growth Regulation, National Instituteof Agrobiological Science, Ibaraki 305-8634, Japan

Edited by Bruce D. Hammock, University of California, Davis, CA, and approved March 31, 2006 (received for review December 27, 2005)

In insects, steroid hormones named ecdysteroids elicit molting andmetamorphosis. The prothoracic gland (PG) is a predominantsource of ecdysteroids, where their biosynthesis (ecdysteroidogen-esis) is regulated by several neuropeptides. Here, we report thatFMRFamide-related peptides (FaRPs) regulate ecdysteroidogenesisthrough direct innervation of the PG in the silkworm Bombyx mori.We purified a previously uncharacterized Bombyx FaRP, DPSFIRF-amide, and identified the corresponding Bombyx FMRFamide gene(Bommo-FMRFamide, BRFa), which encodes three additional FaRPs.All BRFa peptides suppressed ecdysteroidogenesis in the PG byreducing cAMP production by means of the receptor for Bommo-myosuppressin, another FaRP we have previously shown to act asa prothoracicostatic factor. BRFa is predominantly expressed inneurosecretory cells of thoracic ganglia, and the neurons in theprothoracic ganglion innervate the PG to supply all four peptidesto the gland surface. Electrophysiological recordings during devel-opment confirmed the increased firing activity of BRFa neurons instages with low PG activity and decreased ecdysteroid levels in thehemolymph. To our knowledge, this study provides the first reportof peptides controlling ecdysteroidogenesis by direct innervation.

ecdysteroid � FMRFamide-related peptides � metamorphosis � molting �prothoracic gland

S teroid hormones constitute a very important class of com-pounds that regulate development and homeostasis in most

animals. In insects, steroid hormones named ecdysteroids directdevelopment and elicit molting and metamorphosis (1). They actby means of the nuclear ecdysone receptor�ultraspiracle complexes,which mediate ecdysteroid-induced gene expression (2, 3).

The activity of the prothoracic gland (PG), a principal endo-crine organ producing ecdysteroids, is regulated by a cerebralneuropeptide called prothoracicotropic hormone (PTTH). It iswidely accepted that PTTH stimulates the PGs to synthesize andrelease ecdysteroids before each molting and during early meta-morphosis (1, 4, 5). However, various reports on proprioceptiveand environmental inputs that affect molting and metamorpho-sis suggest the existence of additional regulators of PG activity(6, 7). In fact, there is growing evidence that the CNS exerts aprothoracicostatic effect in addition to the tropic effect, thusregulating the complex pattern of the ecdysteroid titer in thehemolymph during development (8–10). It is important toelucidate these prothoracicostatic factors and their modes ofaction to understand the complexities of insect morphogenesis aswell as the peptide regulatory mechanisms of steroid hormonebiosynthesis, which exist in both invertebrates and vertebrates (11).

We previously identified Bommo-myosuppressin (BMS),which is an FMRFamide-related peptide (FaRP), as a previouslyunidentified prothoracicostatic factor in the silkworm Bombyxmori (12). BMS was shown to suppress ecdysteroidogenesis in thePG, where the BMS receptor (BMSR) is highly expressed.Interestingly, BMSRs responded to two other FaRPs from

another lepidopteran species (Manduca sexta) that also exertinhibitory effects on Bombyx PGs. These results suggest thatsome other endogenous FaRPs in Bombyx may also act asprothoracicostatic factors.

Here, we report the purification and identification of fourBombyx extended FMRFamides (Bommo-FMRFamides,BRFas) as previously uncharacterized prothoracicostatic fac-tors. These neuropeptides are encoded by the same gene. Weshow that BRFa peptides are produced in the CNS neurons thatsuppress PG activity by direct innervation. Although the impor-tance of the PG-innervating neurons in the control of ecdys-teroidogenesis has been well documented (13–18), studies re-vealing molecular basis of the PG regulation have been restrictedto hormonal substances throughout the last century (1). To ourknowledge, this study is the first report of peptides controllingecdysteroidogenesis by direct innervation, which may shed lighton the regulatory mechanisms of insect development.

ResultsPurification and Identification of Bombyx FMRFamides. We devel-oped an ELISA system to purify endogenous FaRPs in Bombyx,in addition to BMS. Competitive ELISA revealed that someHPLC fractions, originally prepared from Bombyx pupal brainsfor the purification of BMS (12), show FMRFamide-like immu-noreactivity (Fig. 1A). Three more steps were required to purifyone of the immunoreactive fractions (no. 19) to homogeneity,sufficient for MALDI-TOF MS analysis (Fig. 1B). Two otherFaRPs were purified from fraction no. 16 (unpublished data).

An aliquot of the isolated fraction was subjected to MALDI-TOF MS, resulting in the detection of a monoisotopic mass of880.5 ([M�H]�). Further analysis of this putative peptide withpostsource decay (PSD) yielded the sequence DPSF(L�I)RFamide (Fig. 6A, which is published as supporting informa-tion on the PNAS web site). Because a glycine residue is requiredfor the amidation at the C terminus, the sequence DPSFLRFGwas used as a query for the TBLASTN search against Bombyxwhole-genome shotgun sequence contigs. This search resulted inidentification of contig 105432 with a putative exon sequencecoding for DPSFIRFG flanked by basic arginine residues. Usingthis putative coding sequence and its f lanking regions, RACEwas performed to obtain the nucleotide sequence of the corre-

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: FaRP, FMRFamide-related peptide; PG, prothoracic gland; PTTH, protho-racicotropic hormone; BMS, Bommo-myosuppressin; BMSR, BMS receptor; BRFa, Bommo-FMRFamide; BRFaR, BRFa receptor; NS-VTL1,2, ventrolateral neurosecretory cells 1 and 2;PBST, PBS containing 0.05% (vol�vol) Tween 20.

Data deposition: The sequences reported in this paper have been deposited in the GenBankdatabase (accession nos. AB234100 and AB253536).

§§To whom correspondence should be addressed. E-mail: kataoka@k.u-tokyo.ac.jp.

© 2006 by The National Academy of Sciences of the USA

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sponding cDNA encoding the peptide precursor. Based on thededuced amino acid sequence of this cDNA (Fig. 1C), the fifthresidue of the purified peptide was confirmed to be an isoleu-cine. The PSD profile and the HPLC elution times of the purifiedpeptide and the synthetic DPSFIRFamide were completelyidentical (Fig. 6 and data not shown).

The identified cDNA encodes a 177-residue protein with aputative signal peptide at its N terminus (Fig. 1C). The propep-tide sequence contained four putative mature peptides withMIRFamide, FVRFamide, FIRFamide, and FIRLamide at theirC termini. Although none of them had the canonical FMRF-amide C terminus, the three latter sequences can also be foundin the cockroach FMRFamides (19). Moreover, a BLASTP searchfor the homolog of the prepropeptide in the SwissProt proteindatabase showed highest similarity with snail (Lymnaea stagnalisand Helix aspersa) and fly (Drosophila melanogaster and Dro-sophila virilis) FMRFamides. From these features, we concludedthat the Bombyx cDNA encodes FMRFamide homologs, and thegene, therefore, was named Bommo-FMRFamide (BRFa). Theputative mature peptides were named BRFa-1 to BRFa-4 insequential order from the N terminus of the propeptide.

BRFa Peptides Are Prothoracicostatic Factors. To elucidate theprothoracicostatic activity of BRFa, cAMP and ecdysteroidassays were performed by using PGs of the fifth larval instar on

day 4, as described for BMS in ref. 12. As shown in Fig. 2 A andB, BRFa peptides inhibited both basal and PTTH-stimulatedcAMP accumulation in the PGs in a dose-dependent manner. Inthe ecdysteroid assay, BRFa peptides inhibited PTTH-stimulated ecdysteroidogenesis, although the effects were lesspronounced (Fig. 2C). These results suggest that BRFa peptides,like BMS, inhibit ecdysteroidogenesis through activation ofBMSR to lower the cAMP level in the PGs.

To clarify that the prothoracicostatic effects of BRFa areactually mediated by BMSR, Ca2� imaging analysis was per-formed by using a heterologous expression system. Humanembryonic kidney (HEK) 293 cells expressing both BMSR andG�15 responded to BRFa peptides in a dose-dependent manner(Fig. 2D). Moreover, the relative responsiveness of BMSR tofour BRFa peptides corresponded well to that in the cAMP assayand the ecdysone assay, demonstrating that BMSR actuallymediates the prothoracicostatic effects of BRFa.

As for FMRFamides in Drosophila, a highly specific receptorthat is different from the myosuppressin receptor has beenreported (20). Using this receptor sequence, we identified ahomologous gene coding a putative BRFa receptor (BRFaR) inthe Bombyx genome and elucidated its high responsiveness toBRFa peptides (Fig. 7 A–C, which is published as supportinginformation on the PNAS web site). However, no expression ofBRFaR was detected in the PG (Fig. 7D), further demonstratingthat BMSR is the functional receptor for BRFa in the PG.

Although these results indicate that BRFa peptides actuallyact as prothoracicostatic factors by means of BMSR, theireffective concentrations were always much higher than those ofBMS. Whereas BMS has been suggested to work as a humoralinhibitory factor, BRFa peptides appear to suppress the PG

Fig. 1. Purification and identification of BRFa. (A and B) The first (A) and thefourth (B) HPLC separation steps are shown. In each step, the solid line denotesUV absorbance; the dashed line denotes the acetonitrile gradient. One of theimmunoreactive fractions (fraction no. 19, arrowhead in A) was subjected tothree additional purification steps, which resulted in a homogeneous peak(arrowhead in B). Fraction no. 22 in A was originally subjected to purificationof BMS, and FaRP content in this fraction was presented as the amount ofpurified BMS. (C) The deduced amino acid sequence of the BRFa precursor. Themost likely site of the signal peptide cleavage is indicated with an arrowhead.The purified peptide (BRFa-3) is underlined with a black bar, and putativemature peptides are underlined with gray bars. For each mature peptide, theC-terminal glycine residue is necessary for amidation.

Fig. 2. Prothoracicostatic activity of BRFa. (A and B) cAMP assays without (A)or with (B) 10�9 M PTTH. The prothoracicostatic activity is expressed as aninhibition�activation (I�A) index (n � 5). (C) Ecdysteroid assays in the presenceof 10�9 M PTTH (n � 8). Statistically significant inhibitions compared with thecontrol (the amount of ecdysteroids secreted without BMS or BRFa) areindicated (*, P � 0.05). (D) Ca2� imaging analysis using HEK293 cells expressingBMSR. Relative response was expressed as a percentage of the highest re-sponse induced by 10�6 M BMS. For each ligand, each datum point wascalculated from the responses of 100–200 cells in three independent experi-ments. Color codes are as follows: dark blue, BMS; green, BRFa-1; yellow,BRFa-2; light blue, BRFa-3; red, BRFa-4. The vertical bars represent SE.

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activity via a different pathway, possibly by direct innervation,because the local concentrations of neuropeptides can be muchhigher if they are released in close proximity of the target organ.This possibility was further verified by experiments describedbelow.

BRFa Is Predominantly Expressed in Thoracic Ganglia. The expressionpattern of BRFa was determined by RT-PCR analysis. As shownin Fig. 3A, BRFa was expressed only in the CNS and predomi-nantly in the thoracic ganglia of wandering fifth-instar larvae onday 6. A similar pattern was observed in fifth-instar larvae on day2 (data not shown).

The relative expression level of BRFa in the prothoracicganglion was determined throughout the last instar to the firstday of the pupal stage by using quantitative RT-PCR (Fig. 3B).BRFa is expressed throughout this developmental period, withhighest expression levels observed in the early half of the fifthinstar, similar to those of BMS.

BRFa Neurons Innervate the PG. In situ hybridization with the DNAprobe revealed strong BRFa expression in two pairs of largeneurons in each thoracic ganglion (Fig. 4A). Only a few othersmall neurons were stained in the CNS. Subsequent doubleimmunohistochemical staining of the same CNS showed thatlarge thoracic neurons are ventrolateral neurosecretory cells 1and 2 (NS-VTL1,2) (Fig. 4B), which project their axons throughtransverse nerves into the periphery. Axons originating in BRFaneurons of the prothoracic ganglion innervate the PG (Fig. 4C).Moreover, these axons and axon terminals contain numerousvaricosities, suggesting that BRFa is released in close proximityof the PG (Fig. 4D).

To further confirm that all four BRFa peptides are actuallydelivered to the gland surface, direct MS analysis of the axonsrunning on the gland surface was performed. This analysis resultedin the detection of four major signals, which corresponded totheoretical masses of the four BRFa peptides (Fig. 4E).

Developmental Profile of the BRFa Neuron Activity. To investigatethe physiological function of BRFa neurons, their temporal

patterns of activity were investigated electrophysiologically.Using a suction electrode, the firing frequency of BRFaneurons was measured throughout the last instar (Fig. 5). In allof the larval stages tested, the recordings showed a combina-tion of regular firing patterns, ref lecting the signals frommultiple neurosecretory cells (Fig. 5A). However, the spikesrarely overlapped with each other (arrowhead in Fig. 5A),which made it possible to monitor the firing frequency. Therecorded spikes exhibited a slow time course and a waveformtypical of neurosecretory cells (Fig. 5B) (21, 22). Moreover,the time course of recorded action potentials was highlyuniform in all of the experiments, suggesting that the activityemanated only from NS-VTL1,2 (Fig. 5B). Ultrastructure of thenerve at the location from which recordings were madeindicates that only four neurosecretory axons exist, further

Fig. 3. BRFa expression. (A) Standard RT-PCR analysis of BRFa expression invarious tissues from day-6 fifth-instar larvae. RpL3 was used as a control gene.BR, brain; SOG, suboesophageal ganglion; TG1, prothoracic ganglion; TG2,mesothoracic ganglion; TG3, metathoracic ganglion; AG, unfused abdominalganglia; TAG, terminal abdominal ganglia; MG, midgut; SG, salivary gland; FB,fat body; MT, Malpighian tubule; OV, ovary; TE, testis. (B) Quantitative RT-PCRanalysis of BRFa expression during development in the prothoracic ganglion(n � 3). V0–V9 indicates fifth-instar days 0–9, and P0 indicates pupal day 0. Therelative expression level of each stage was normalized against that of V0.

Fig. 4. Projection of BRFa neurons. (A) In situ hybridization analysis of themesothoracic ganglion with the BRFa probe. (B) Subsequent double stainingof the same ganglion with RFamide and myoinhibitory peptide (MIP) I anti-bodies. Green, RFamide; red, MIP I. Arrows in A and B indicate BRFa-expressingNS-VTL1,2. The same neurons were also stained in the pro- and metathoracicganglia. (C) RFamide-immunoreactive axons of the NS-VTL1,2 on the surface ofthe PG. (D) Numerous stained varicosities along RFamide-immunopositiveaxons and in axon terminals on the PG surface. (Scale bars: B–D, 100 �m.) (E)Direct MS analysis of the NS-VTL1,2 axons on the PG. The theoretical monoiso-topic masses ([M�H]�) of BRFa peptides are as follows: BRFa-1, 1,194.6;BRFa-2, 812.4; BRFa-3, 880.5; BRFa-4, 1,025.6.

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confirming that all of the recorded spikes are from four BRFaneurons (data not shown).

The activity of BRFa neurons is high during the feedingperiod of the last instar, when the hemolymph ecdysteroid titeris low. Activity decreases remarkably on the day before larvaestart wandering, in line with the gradual increase of theecdysteroid titer. The firing frequency recovers after the onsetof wandering, followed again by a marked decrease to almostcomplete inactivation. The overall f luctuation pattern ofBRFa neuron activity is shown in Fig. 5C, with the schematic

illustration of the hemolymph ecdysteroid titer (based on refs.5 and 23).

DiscussionIn this study, we identified four BRFa peptides as previouslyunidentified prothoracicostatic factors and elucidated theirmode of action on the PG. Although the importance of PGinnervation has been well documented (13–18), to our knowl-edge, this study is the first report on the molecular basis forneural regulation of ecdysteroidogenesis.

BRFa peptides were shown to regulate ecdysteroidogenesis bymeans of BMSR, the receptor previously reported to mediate theprothoracicostatic effect of BMS (12). Although effective con-centrations of BRFa were much higher than BMS, this differencereflects their different modes of action on the PGs. It is wellknown that hormonal factors are usually effective at nanomolarconcentrations, whereas peptides delivered by direct innervationact at micromolar concentrations (24–26). The even highereffective concentrations of BRFa in the heterologous expressionsystem compared with those in PG incubated in vitro (cAMP andecdysteroid assays) can be explained by different sensitivities ofthese two systems. As for BMSR, the EC50 for BMS in the cAMPassay (0.089 nM) was �400 times lower than that in theheterologous expression system (32 nM) (12). However, thisrelatively high value is comparable with the EC50 of Drosophilamyosuppressin (DMS) (40 nM) that is necessary for the activa-tion of two DMS receptors expressed in a similar heterologoussystem (27). Taking these relative differences into consideration,it appears that the responsiveness of BMSR to BRFa corre-sponds well to the effective concentrations in the cAMP andecdysteroid assays (see Fig. 2). The absence of highly responsiveBRFaR in the PG further suggests that BMSR is the functionalreceptor for BRFa in the PG (Fig. 7).

Why does Bombyx use two different factors for the BMSR-mediated signaling? Although much needs to be clarified toanswer this question, we propose that insects use BMSR-mediated signaling for both general and specific inactivation.Our previous results indicate that BMS is secreted into thehemolymph from the brain neurosecretory cells and works as amultifunctional neuropeptide. We showed expression of BMSRin various tissues, indicating that blood-borne BMS simulta-neously acts on a number of organs (12). On the other hand,BRFa is delivered directly to the PG surface by the innervatingneurons to act as direct and tissue-specific inhibitors of ecdys-teroidogenesis. In Manduca, the BMS homolog (F10) was thedominant FaRP in the hemolymph of feeding larvae, whereasBRFa homologs (F7D and F7G) were not detected as circulatinghormones (28), in line with our hypothesis.

The overall developmental f luctuation of BRFa neuron firingfrequency showed an inverse relationship with the hemolymphecdysteroid titer (Fig. 5C), in accordance with the prothoraci-costatic activity of BRFa. BRFa neuron activity is high duringthe feeding period of last-instar larvae (days 0–4), correspond-ing to the low ecdysteroid titer in the hemolymph. The activitydecreases on the day before larvae start wandering (day 5), whenthe ecdysteroid titer increases gradually. BRFa neurons arecompletely inactive when the ecdysteroid titer reaches its highestlevel on the day before pupation (day 9). It is interesting thatBRFa neurons transiently recover their activity after the hemo-lymph ecdysteroid titer begins to increase (days 6–8). Thispossible brief inactivation of the PG may prevent the suddenincrease of the ecdysteroid titer, giving the larvae enough timeto prepare for metamorphosis. This neural regulatory mecha-nism of ecdysteroidogenesis is summarized in Fig. 5 D and E.

It is important to note that the general f luctuation of thehemolymph ecdysteroid titer can basically be explained by thedevelopmental profile of the basal and PTTH-stimulated PGactivity (5, 29, 30), although slight discrepancies still remain. For

Fig. 5. Developmental profile of BRFa neuron activity. (A) Frequent (uppertrace, from fifth-instar day 0) and sparse (lower trace, from fifth-instar day 5)firing patterns of BRFa neurons. The arrowhead indicates overlapping spikes.(Scale bar: 1 s.) (B) Eight independent spikes recorded in the same experiment.(Scale bar: 100 ms.) (C) Developmental changes of the firing frequency of BRFaneurons innervating the PG. The blue dashed line is the schematic illustrationof the hemolymph ecdysteroid titer (n � 8–16). V0–V9 indicates fifth-instardays 0–9, and P0 indicates pupal day 0. The onset of wandering is indicated byan arrowhead. The vertical bars represent SE. (D and E) Neural regulatorymechanism of ecdysteroidogenesis. When the BRFa neurons in the prothoracicganglion (TG1) are firing frequently (D), the PG activity is down-regulated.When the BRFa neurons are inactive (E), the PG is active and secretes highamounts of ecdysteroids.

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example, a surge of basal activity of isolated PG during thefeeding period has been reported in Bombyx and Manduca, whichis not reflected by the increased hemolymph ecdysteroid titer inintact animals (29, 30). This discrepancy is presumably due to theremoval of active neural inhibition from isolated PGs. As westated above, BRFas may thus function as fine regulators ofecdysteroidogenesis, especially during feeding and wanderingperiods, when slight fluctuations of the ecdysteroid titer playimportant roles in insect development (23).

In addition to PTTH and FaRPs, there are a few more factorsthat are known to regulate the PG (1, 10, 31). Insects may usethese factors in response to different signals that are importantfor regulation of ecdysteroidogenesis. These signals are thenintegrated as PG activity, thus coordinating the proper timing formolting and metamorphosis. Until now, model systems forpeptide regulation of steroidogenesis by identified innervatingneurons and their neuropeptides have rarely been reported, evenin vertebrates (32, 33). This BRFa system could serve as a goodmodel for investigating how organisms integrate neural andhormonal inputs to regulate their biosynthesis of steroid hor-mones in response to multiple developmental signals (34).

Materials and MethodsExperimental Animals. B. mori racial hybrids were fed on theartificial diet called Silkmate (Nihon Nosan Kogyo, Yokohama,Japan) at 25°C under a 16-h light�8-h dark photoperiod andstaged after the final (fourth) larval ecdysis. Most larvae startedwandering behavior on day 6 of the fifth instar and pupated onday 10.

FMRFamide ELISA. Equal amounts of neuropeptides DVDHVFL-RFamide, DVVHSFLRFamide, DVGHVFLRFamide, andGQERNFLRFamide were coupled to human serum albumin(HSA) as reported in ref. 35. ELISA plate wells were coated withthe HSA-peptide conjugates overnight at 4°C and washed withPBS containing 0.05% (vol�vol) Tween 20 (PBST) just beforeuse. Wells were blocked with 0.5% (wt�vol) skim milk in PBSTfor 1 h, followed by rinsing with PBST. Sample or standard in 50�l of PBST was then added to each well, followed by 50 �l ofantiserum (anti-FMRFamide rabbit polyclonal antiserum,Bachem) diluted 1:2,000 with 0.5% skim milk in PBST. The platewas incubated for 3 h with shaking. After rinsing with PBST, 100�l of enzyme-labeled secondary antibody (anti-rabbit IgG alka-line phosphatase conjugate, Promega) diluted 1:5,000 in PBSTwas added to each well and incubated further for 2 h. Substrate(100 �g of p-nitrophenyl phosphate, Sigma-Aldrich) was addedto each well in 100 �l of substrate buffer [50 mM sodiumcarbonate buffer containing 1 mM MgCl2 (pH 9.6)] after rinsingwith PBST, and the color was allowed to develop for 100 min.The reaction was stopped by adding 10 �l of 1 M NaOH to eachwell, and the absorbance was measured at 405 nm with a Model550 Microplate Reader (Bio-Rad). Because BMS was used as astandard, the RFamide content of each sample was expressed inBMS equivalents.

Purification of BRFa-3. Residual fractions of the first purificationstep of Bombyx pupal brain extracts, which were previously usedfor the purification of BMS (12), were applied to the FMRF-amide ELISA. One of the immunoreactive fractions (no. 19 inFig. 1 A) was loaded onto a PEGASIL-300 C4P column (4.6 �250 mm, Senshu Kagaku, Tokyo) using a Waters 2695 Separa-tions Module. Elution was performed with a linear gradient of10–40% acetonitrile in 0.1% trif luoroacetic acid (TFA) over 60min at a flow rate of 1 ml�min. The FMRFamide-positivefraction was then applied onto a PEGASIL-300 ODS-II column(4.6 � 250 mm, Senshu Kagaku). The column was eluted with alinear gradient of 10–40% acetonitrile in 0.1% TFA over 60 minat a flow rate of 0.5 ml�min. The immunoreactive fraction was

further purified by passing over a PEGASIL-300 ODS-II column(4.6 � 250 mm), eluting it with a linear gradient of 10–40%acetonitrile in 0.1% heptafluorobutyric acid over 60 min at aflow rate of 0.5 ml�min.

MALDI-TOF MS. Mass spectra and postsource decay mass spectraof peptides were obtained with an AXIMA-CFR (Shimadzu)mass spectrometer. Recrystallized �-cyano-4-hydroxycinnamicacid was used as the matrix with a concentration of 5 mg�ml ofacetonitrile�water (1:1 by volume) containing 0.1% trif luoro-acetic acid. All spectra were measured in reflector mode.

Synthetic and Recombinant Peptides. BMS and BRFa peptideswere all custom-synthesized and further purified by HPLC. Therecombinant PTTH was prepared as described in ref. 36.

Cloning of BRFa. Using the amino acid sequence of the purifiedpeptide as a query, BLAST analysis (TBLASTN) was performedagainst Bombyx whole-genome shotgun sequence contigs byusing KAIKOBLAST. Based on the putative coding sequence of theobtained contig (contig 105432; GenBank accession no.BAAB01002501), RACE primers were designed as follows:sense primer, 5�-CGATTTGGTCGCGACCCTAGCTT-TATCC-3�; sense nested primer, 5�-GACCCTAGCTTTATC-CGGTTCGGAAGG-3�; antisense primer, 5�-TGAAGTGAT-TGCGAGCTCTGTGCCTCC-3�; antisense nested primer, 5�-CCGTTGGGGGTAAGTGTTGGTTTCAGC-3�. To obtainthe whole cDNA sequence of BRFa (GenBank accession no.AB234100), RACE was performed by using the above primersand the SMART RACE cDNA Amplification Kit (BD Bio-sciences), according to the manufacturer’s instructions. First-strand cDNA prepared from larval brain total RNA was used asa template.

cAMP Assay and Ecdysteroid Assay. The cAMP assay and theecdysteroid assay were performed as described in ref. 12. For thecAMP assay, prothoracicostatic activity of a sample was ex-pressed as an inhibition�activation index calculated with thefollowing formula: inhibition�activation index � (cAMP contentof PG incubated with sample�cAMP content incubated withoutsample).

HEK293 Cell Expression and Ca2� Imaging Analysis of BMSR. BMSRcloned into the pME18S mammalian expression vector wastransfected into HEK293 cells with the promiscuous G proteinG�15 as described in ref. 37. Ca2� imaging analysis was per-formed as reported in ref. 37.

Cloning and Characterization of BRFaR. BRFaR (GenBank accessionno. AB253536) was annotated from the genome sequence andcloned into the pME18S vector. The expression analysis wasperformed by using RT-PCR. See Supporting Materials andMethods, which is published as supporting information on thePNAS web site, for detailed information.

RT-PCR Analyses of BRFa. For standard RT-PCR analysis, cDNAsfrom various tissues of wandering fifth-instar larvae were syn-thesized as described in ref. 38. Specific primers used were asfollows: sense primer, 5�-AAGCTCGTCGTCGCAGTGC-TATCGACC-3�; antisense primer, 5�-GCGCAATCATTG-CAAGGGTCAACAACGG-3�. RpL3 was used as a controlgene. Thirty cycles (94°C for 30 s, 65°C for 30 s, and 72°C for 40 s)were carried out for the amplification.

For quantitative RT-PCR analysis, cDNAs were preparedfrom prothoracic ganglia of each stage larvae and pupae. Foreach stage, three batches of total RNAs were prepared inde-pendently from 10–20 ganglia, each of which was then convertedinto cDNA. Quantitative RT-PCR was performed on the Smart

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Cycler System (Cepheid, Sunnyvale, CA) essentially as describedin ref. 39. After 1 min at 95°C, 40 cycles (95°C for 10 s and 68°Cfor 20 s) were carried out for the amplification. BRFa specificprimers used were as follows: sense primer, 5�-CGATTTG-GTCGCGACCCTAGCTTTATCC-3�; antisense primer, 5�-TGAAGTGATTGCGAGCTCTGTGCCTCC-3�. Serial dilu-tions of plasmids containing cDNAs of BRFa and RpL3 wereused for standards, and the transcript levels of BRFa werenormalized with RpL3 levels in the same samples.

In Situ Hybridization and Immunohistochemistry. For detection ofBRFa expression in the CNS, we used a digoxigenin-labeledsingle-stranded DNA probe. Dig-11-dUTP (Roche AppliedScience, Basel) was incorporated into single-stranded antisenseDNA by asymmetric PCR according to a modified procedure(40). See Supporting Materials and Methods for the modified insitu hybridization procedure used in this study. After staining ofneurons expressing the BRFa mRNA, the CNS was rinsed andincubated with a mixture of rabbit polyclonal antiserum toRFamide and mouse monoclonal antibody to myoinhibitorypeptide I for 2 days at 4°C. The tissue was then washed withPBST and incubated with a mixture of Alexa Fluor 488-labeledgoat anti rabbit IgG and Alexa Fluor 555-labeled goat anti-mouse IgG (Invitrogen). BRFa expression was observed under aconfocal laser microscope (510, Zeiss) using Nomarski DICoptics and FITC and rhodamine filters.

Direct MS Analysis. Direct MS analysis was performed based onprevious reports (19, 41), which were slightly modified. Larvaewere anesthetized in water for 10–20 min, and the PG-innervating nerve was dissected rapidly from the PG surface insterile saline [0.85% NaCl (wt�vol)]. The dissected piece of the

nerve was transferred onto a MALDI sample plate, and thedroplet of saline transferred along with the nerve was blottedwith cellulose paper. The tissue was then washed on the sampleplate with 1 �l of pure water, which was again completelyremoved with cellulose paper. After drying the sample com-pletely, it was covered with �100 nl of matrix solution (saturatedsolution of �-cyano-4-hydroxycinnamic acid in methanol�water)by using a fine pipette. The whole preparation was again driedcompletely, and spectra were obtained with a Voyager-DE STRmass spectrometer (Applied Biosystems).

Electrophysiology. Efferent electrical activity of the BRFa neu-rons was recorded in situ throughout the last instar. Larvae wereanesthetized briefly under CO2 and immobilized in silicon tubes.After the prothoracic region was opened ventrally, the BRFanerve was cut just posterior to the prothoracic ganglion. Thenerve was then drawn rapidly into a suction electrode filled withinsect Ringer’s solution (154.8 mM NaCl�12.3 mM KCl�4.0 mMMgCl2�4.5 mM CaCl2�2.1 mM NaHCO3�0.1 mM Na2HPO4�67.1 mM glucose�10.0 mM Hepes, pH 6.8). Spike activities of theefferent neurons were recorded with respect to an indifferentelectrode placed in the hemolymph and amplified with aDAM50 biological amplifier (WPI Instruments, Waltham, MA)for extracellular recordings. Signals were digitized with ananalog�digital converter (IDAC, Syntech, Hilversum, The Neth-erlands), and the number of spikes during the first 3 min of eachrecording was counted by using computer software (AUTO-SPIKE32, Syntech).

We thank Dr. Toshio Ichikawa for technical support and advice. Thiswork was supported by grants from the Research for the Future Programof the Japan Society for the Promotion of Science and National Institutesof Health Grant AI40555.

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