Structural characterization and expression analysis of prothoracicotropic hormone in the corn earworm, Helicoverpa zea

Peptides 24 (2003) 1319–1325

Structural characterization and expression analysis of prothoracicotropic hormone in the corn earworm, Helicoverpa zea夽 Wei-Hua Xu a,b , Joseph P. Rinehart a , David L. Denlinger a,∗ b

a Department of Entomology, Ohio State University, 318 West 12th Avenue, Columbus, OH 43210-1242, USA Department of Molecular and Cell Biology, University of Science and Technology of China, Hefei 230027, China

Received 10 July 2003; received in revised form 1 August 2003; accepted 5 August 2003

Abstract The cDNA encoding prothoracicotropic hormone (PTTH), the brain neuropeptide that stimulates the prothoracic glands to synthesize ecdysone, was cloned from the corn earworm Helicoverpa zea (Hez). The amino acid sequence deduced from the cDNA indicates a molecular structure that is distinct from the PTTH’s reported in other Lepidoptera, but all contain an identical proteolytic cleavage site and the seven cysteine residues that are essential for activity. Northern hybridization shows a single mRNA present in the brain–subesophageal ganglion complex. Using RT–PCR, we observed constant amounts of PTTH mRNA during larval development but large fluctuations at pupation and prior to adult eclosion. © 2003 Elsevier Inc. All rights reserved. Keywords: Prothoracicotropic hormone; cDNA structure; Developmental expression; Helicoverpa zea

1. Introduction Classic experiments by Williams elegantly demonstrated that a brain hormone is required to stimulate the insect’s prothoracic glands to produce the ecdysone needed to stimulate molting and metamorphosis [16]. This brain hormone, known as prothoracicotropic hormone (PTTH), has now been characterized from several species of Lepidoptera. Molecular cloning of PTTH from the silkworm, Bombyx mori (Bom) revealed Bom-PTTH to be a 30 kDa glycoprotein consisting of two identical subunits linked by a disulfide bond. Each monomeric PTTH subunit is generated by proteolytic cleavage of a precursor molecule, PTTH preprohormone [5]. Several additional PTTHs have been cloned and show cDNA structures quite similar to Bom-PTTH. Thus far, all of the PTTH cDNAs that have been cloned are from a single Lepidoptera superfamily, the Bombycoidea: B. mori [5], Antheraea pernyi [9], Hyalophora cecropia [10], Manduca sexta [11], and Samia cynthia ricini (GenBank accession no. L25668). 夽 The sequence has been deposited in the GenBank data base under accession no. AY172670. ∗ Corresponding author. Tel.: +1-614-292-6425; fax: +1-614-292-7865. E-mail address: [email protected] (D.L. Denlinger).

0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.08.001

In this study, we examine PTTH from the corn earworm, Helicoverpa zea (Hez), an agriculturally important noctuid moth that is phylogenetically distinct from the other species of Lepidoptera that have been examined. We report the isolation and characterization of Hez-PTTH cDNA, the tissue distribution of the corresponding mRNA in pupae, and the developmental expression of this cDNA during the larval and pupal stages. We provide evidence that Hez-PTTH has an amino acid sequence rather distinct from the others that have so far been identified. In addition, we suggest that PTTH present in the hemolymph may be regulated at the level of transcription at specific times during metamorphosis.

2. Materials and methods 2.1. Insects Eggs of H. zea were kindly provided by Dr. Roger Meola of Texas A&M University, College Station, Texas. Larvae were reared on an artificial diet (Bio-Serv, US) at 25 ◦ C and a light–dark cycle of L15: D9. New pupae were collected daily, and adults were fed a 10% sucrose solution. Brains, brain–subesophageal ganglion (Br–SG) complexes, and other tissues were dissected in saline (0.75%

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NaCl) and stored frozen at −70 ◦ C until used to extract total RNA. 2.2. Isolation and sequencing of the initial PTTH cDNA clone Total RNA from Br–SG complexes of pharate adults was prepared using TRIzol reagent (Invitrogen). Degenerate primers DFP1, DRP1, and DRP2 (Fig. 1) were synthesized by Invitrogen based on the sequences from PTTH cDNAs of B. mori [5], A. pernyi [9], H. cecropia [10], M. sexta [11], and S. c. ricini. Primed with DRP1, 1.5 ␮g of total RNA

was reverse transcribed at 37 ◦ C for 1 h in 1 × RT buffer (50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 ), 10 mM DTT, 0.5 mM dNTPs, and 200 ␮ of M-MLV reverse transcriptase (Invitrogen) in a total volume of 10 ␮l. The cDNA was amplified using the degenerate primers DFP1 and DRP1 under the following conditions: three cycles of 94 ◦ C, 45 s; 45 ◦ C, 1 min; 72 ◦ C, 45 s, and then 30 cycles of 94 ◦ C, 30 s; 50 ◦ C, 1 min; 72 ◦ C, 45 s. A weak DNA band corresponding to approximately 400 bp of the expected size was cut from the agarose gel and purified using Ultrafree-DA (Millipore). The second PCR was performed with the DNA fragment purified above as the template,

Fig. 1. Nucleotide and deduced amino acid sequences of a cDNA encoding PTTH. The suggested start ATG, stop codons TAA, and polyadenylation signal (AATAAA) are shown in bold letters. Predicted proteolytic cleavage sites are printed in bold and underlined. The active hormone portion of PTTH is shown in italics. The possible N-linked glycosylation site is shown by a box. Arrows below the nucleotide sequences represent the position of the different synthetic primers used in PCR. Degenerate primers are DFP1 (5 -TT/G GATTATGA/C A/T AAC/T ATGA-3 ), DRP1 (5 -TAA/G TCCCTC/G GTA/G CAC/T AA/T/C ACA-3 ), and DRP2 (5 -TATAAA/G C/G TTTCC/T TTGCA-3 ). Specific primers, SFP1, SRP1, and SRP2 were used in RACE and PCR analyses. The nucleotide sequence is available in GenBank under accession no. AY172670.

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using primers DFP1 and DRP2; the PCR conditions were the same as above. The amplified DNA was separated on an agarose gel using Ultrafree-DA, and the DNA fragment was subcloned into pCR® 2.1 cloning vector using a TOPO TA cloning kit (Invitrogen), and recombinant DNA from positive clones was sequenced. 2.3. Rapid amplification of cDNA ends (RACE) for PTTH For 5 - and 3 -RACE experiments, cDNAs were synthesized from 1 ␮g of total RNA with primers 5 -CDS and SMART II for 5 -RACE and with primer 3 -CDS for 3 -RACE at 42 ◦ C for 1.5 h with PowerScriptTM reverse transcriptase using the SMARTTM RACE cDNA amplification kit (Clontech). 5 -RACE amplification was performed on 2.5 ␮l of 5 -cDNA with primers SRP1 (Fig. 1) and Universal Primer Mix (UPM, Clontech). 3 -RACE was carried out on 2.5 ␮l of 3 -cDNA with primers UPM and SFP1 (Fig. 1). The PCR conditions were modified from the protocol (Clontech) as follows: after 2 min at 94 ◦ C, 25 cycles of 30 s at 94 ◦ C, 30 s at 60 ◦ C, and 50 s at 72 ◦ C, then 7 min at 72 ◦ C. 2.4. Cloning and sequencing The 5 - and 3 -RACE products were purified after agarose gel electrophoresis and ligated into plasmid vectors as described above. DNA from positive clones was isolated using an SV Minipreps kit (Promega) and digested with EcoRI to screen for the presence of inserts after transformation. These insert DNAs were sequenced by the Plant-Microbe Genomics Facility, Ohio State University. 2.5. Northern hybridization 25 ␮g of total RNA from various tissues was separated by electrophoresis in a 1.2% agarose gel containing 0.22 M formaldehyde and ethidium bromide. RNAs in each lane were checked under UV light after electrophoresis and then transferred onto a Magnacharge + nylon membrane (Osmotics), by downward capillary action using an alkaline transfer buffer (Schleicher and Schull). Transferred nucleic acids were cross-linked to the membrane by UV (1200 ␮). Blotted RNAs were prehybridized for 2 h at 68 ◦ C in a hybridization buffer (0.5 M NaCl, 0.1 M NaH2 PO4 , 6 mM EDTA, 1% SDS), and then hybridized overnight at 68 ◦ C with the same buffer containing biotin-labeled Hez-PTTH cDNA as a probe. After hybridization, the filter was washed with 2×SSC/0.1% SDS at room temperature for 10 min, then twice with 0.1 × SSC/0.1% SDS for 15 min at 68 ◦ C. Hybridization signals were visualized using a PhototopeTM -Star Detection Kit for Nucleic Acids (New England BioLabs) according to standard protocol. Finally, X-ray film (Fuji) was exposed to the filters in the presence of an intensifying screen at room temperature for 2–3 h.

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2.6. RT–PCR and Southern blots RT–PCR was used to investigate the developmental expression of the PTTH gene according to the method of Xu et al. [17]. Total RNA was extracted from brain (fifth instar larvae and early stage pupae) or brain–SG complexes (middle-late stage pupae, pharate adults). 0.5–2 ␮g of total RNA (equaling the amount in one brain or Br–SG complex) from fifth instar larvae to pharate adults was reverse transcribed in a volume of 15 ␮l and 1 ␮l of the cDNA was added to 25 ␮l of PCR reaction buffer consisting of 100 mM Tris–HCl (pH 8.4), 250 mM KCl, 7.5 mM MgCl2 , 100 ␮M dNTPs, 0.2 ␮M of primers SFP1 and SRP2, and 1.25 units of Taq polymerase (Invitrogen). The reaction mixture was subjected to 18 cycles consisting of 94 ◦ C, 30 s; 57 ◦ C, 30 s; 72 ◦ C, 30 s. After electrophoresis, PCR products were denatured with a denaturing buffer (3 M NaCl, 0.4 M NaOH) and transferred onto a Magnacharge + nylon membrane (Osmotics) as described above. Hybridization with the biotin-labeled PTTH cDNA probe and signal detection were the same as described for Northern blot analysis. 2.7. Phylogenetic inference A neighbor-joining (NJ) and most-parsimonious (MP) tree based on the amino acid sequences were constructed by ClustealX [8,13]. 3. Results 3.1. Isolation and sequence analysis of Hez-PTTH Using degenerate primers based on the nucleotide sequences conserved among B. mori, A. pernyi, H. cecropia, M. sexta [5,9–11], and S. c. ricini (GenBank accession no. L25668), we failed to isolate any bands by PCR amplification with cDNA that had been reverse transcribed with oligo dT, even after 35 PCR cycles. We then changed our strategy to elevate the proportion of PTTH cDNA by carrying out reverse transcription with primer DRP1. The use of degenerate primers DFP1 and DRP1 yielded a weak band of approximately 400 bp. A second PCR was performed with degenerate primers DFP1 and DRP2, and this yielded a product of approximately 260 bp that showed 55–64% identity at the amino acid level with the corresponding regions of PTTH from M. sexta, B. mori and A. pernyi. To obtain the full-length PTTH cDNA, 5 -and 3 -RACE were performed with specific primers SRP1 for the 5 end and SFP1 for the 3 end, based on the sequence of the 260 bp cDNA fragment sequenced above. Approximately 550 bp at the 5 end and 500 bp at the 3 end were amplified by PCR, and these DNA fragments were subcloned into vectors and sequenced (Fig. 1). The cDNA revealed a large open reading frame of 226 amino acids that encodes prepro-PTTH, a precursor molecule for PTTH (Fig. 2). The

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BNP1 Hez Hvi Bom Scr Anp Hyc Mas

MVTRPLLCVI MITRPLVCVI MITRPIILVI MISRSIVILL MISRSIVILL MISRPIVILL MKPLRTIILC

VCFGLFILIQ VCFGFIILIQ LCYAILMIVQ VCIGALIIIQ ACSGVLIIME VCIGALIIIQ SCIFMVIQFL

SLVPKVMAVK SLVPKVMAMK SFVPKAVALK SLMPKTMAMR ALMPRTMAMK SLMPKTMAMK APTAMAIKRT

HSN VDEYMLE HSN VDEYMLE RKPDVGGFMVE NTRNIDEFMIE STRNIHEFMIE NTRNVDEFMIE SNINEYTVENE

DQRTRKRKNY DQRTRKRKNY DQRTHKSHNY DQRTRKKHNY DQRTRKKHNY DQRTRKRHNY –-RTRKKQNY

Hez Hvi Bom Scr Anp Hyc Mas

LGKPGNVGTN LDKPGNVGTN LGDKENVRPN LLRKKNYDLM L-RNKNNGLM LRNKNNYDLM -RDKDNFGLM

ANPDELSAFI ANPEELSAFI TSPEELSALI NPEEF-SNLL SPEDY-SNLV NPEEF-SNLL NPEEL-SAFI

Hez Hvi Bom Scr Anp Hyc Mas

EKYNNQALPD EKYNNQALPD E---NQAIPD EKH-NQAIQD Q---N--IPD EKH-NQAIQD EEY-NQAIPD

YDTDSFQLEP YDTDSFQVEP PYYTEPFDPD YNMEASDLDS YDMESLEIDS YNMESFDLDS YKTEPFEPDA 1 2 PPCACKFSPN PPCACKSSPN PPCTCKYKKE PPCSCGYTQT PPCSCEYTNE PPCTCGYTQT PPCSCEYKKG

RTDLGENTYP RTDLGENTYP IEDLGENSVP LLDFGKNAFP TVDFGENAFP LLDFGKNAFP FINLGENVFP

VDYANMIRND VDYANMIRND VDYANMIRND LDYDNMKKNN MDYANMKKND MDYDNMKKND VDYANMIRND 3 RYIETRNCSQ RYVETRNCSQ RFIETRNCNK RHVVTRNCSD RHVESRNCSE RHVVTRNCSD SNIETINCST

Hez Hvi Bom Scr Anp Hyc Mas

IIRRKEFQNQ IIRRKEFQNQ ILKRRETKSQ ILKRRETSTQ VLKRRQSTTQ ILKRRESATQ ILRKRKSMAE

ATLEDIPHDL ATLDDMPHDL ESLEIPNELK ISEEVPRELK PSEKVPNELK ISEEVPRELK KSLARPTDLE

EFRWVAENYP KFRWIAEYYP Y-RWVAESHP F-RWIGEKWQ F-RWIAEKWQ F-RWIGEKWQ I-GWVAESLP

6 7 VSVGCVCTRDYYATER 226 VSVGCVCTRDYYATEK VSVACLCTRDYQLRYNNN ISVGCMCTRDYRNSTEDYQPRLLTKIIQQRDLS ISVGCVCTRDYRDTINQD ISVGCMCTRDYRNTTEDYQPRLLTKIVQQRDLS ISVGCICTRDYVI

VILLDKSVET VILLDKSVET VILLDNSVET VVLLDNSIET VFLLDNSIET VILLDNSIET VILLDNSVET 4 ARQQSCRPPY ARQQSCRLPY TQQPTCRPPY QQQS-CLFPY LRQSSCLFPY LQQ-SCLFPY NQQQSCPPPY

VVRLAR DSEI 60 VVRLAR DSEI MMKRARNDV-VLQRPRNNE-MFQRDRNND-I VLQKPRNNDMLLHRNKNSF--

RTRKRGNIKV RTRKRGNIKV RTRKRGNIQV RTRKRGDLRR RTRKRGNIKR RTRKRGDIRR RTRKRGNIKV 5 VCRENYYNIT VCRENYYNIT ICKESLYSIT VCKETLYDVN VCKETLYDIS VCKETLYDVN ICKESIYEIK

120

180

Fig. 2. Sequence alignment of the PTTH preprohormone proteins. PTTH amino acid sequences are from seven species, Helicoverpa zea (Hez), Bombyx mori (Bom, D90082), Samia cynthia ricini (Scr, L25668), Antheraea pernyi (Anp, U62535), Hyalophora cecropia, (Hyc, AF288695), H. virescens(Hvi, AY172671) and Manduca sexta (Mas, AY007724). The putative proteolytic cleavage sites are indicated in bold letters. The active hormone portion of PTTH is boxed. Numbers 1–7 represent the positions of the cysteines; the first cysteine (Cys18 ) and other six cysteine residues are predicated in intrachain and interchain disulfide bond(s), respectively as reported in B. mori [4]. BNP1, Brain neuropeptide 1.

identity of the Hez-PTTH ORF is 92% for its close noctuid relative Heliothis virescens (GenBank AY172671), but the identity is much lower for members of the Bombycoidea: 56% for B. mori, 55% for M. sexta, 51% for H. cecropia, 48% for S. c. ricini, and 46% for A. pernyi. The mature PTTH molecular identity is 92% for H. virescens, 59% for B. mori, 55% for M. sexta, 49% for A. pernyi, and 48% for H. cecropia and S. c. ricini. Although the identity is not high between H. zea/H. virescens and other known species, Hez-PTTH shares several important PTTH structural features with the other species. First, there is a proteolytic cleavage site (R113 -K114 -R115 ) present in Hez-PTTH that is common to all the PTTH preprohormones; this site is thought to liberate the active hormone subunit. Second, all seven cysteine residues thought to be necessary for prothoracicotropic activity are present in Hez-PTTH [4] (Fig. 2).

Based on phylogenetic inference, Hez-PTTH, along with PTTH from H. virescens, forms a cluster that is distinct from the other known PTTHs (Fig. 3). 3.2. Tissue distribution of Hez-PTTH The PTTH cDNA was used as a hybridization probe in Northern blot analysis of various tissues. A hybridization signal was observed from the brain–SG complex of pharate adults (12 days after pupation, 1 day prior to adult eclosion) and Day 1 adults (Fig. 4). The signal was detected as a single band of approximately 0.9 kb, indicating that the characterized cDNA represents full-length mRNA. No hybridization signal was found using RNA from the midgut or Malpighian tubules of Day 6 pharate adults. By the more sensitive method of RT–PCR, we reexamined tissue

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(B)

(A)

He. virescen s He. virescen s B. mori

He. zea

He. zea M. sexta 100

100 NT = 1 TL = 328 CI = 0.936 RI = 0.865 RC = 0.810

56

B. mori

69 100

100

A . pernyi

100

100

S. cynthia

A . pernyi

M. sexta

0.1

Hy. cecropia

Hy. cecropia S. cynthia

Fig. 3. Phylogenetic analysis constructed by (A) most-parsimonious (MP) and (B) neighbor-joining (NJ) trees. The analysis is based on PTTH amino acid sequences (ORF) reported in Fig. 2. Bootstrap values (percentage of 1000 pseudoreplicates) are shown. In the MP tree, the number of most-parsimonious trees recovered (NT), total tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency index (RC) are also shown. Branch lengths are proportional to the scale given in substitutions per nucleotide for the NJ tree.

specificity of PTTH gene expression using the brain and SG of Day-1 adults, at which time the brain and SG can be dissected separately. The results indicated that PTTH was expressed in the brain but not in the SG (data not shown). 3.3. Developmental expression of Hez-PTTH

Fig. 4. Northern blot analysis of PTTH mRNA. Total RNA was extracted from brains of pharate adults (12 days after pupation) and Day-1 adults. The midgut and Malpighian tubule are from Day 6 pupae. 25 ␮g of total RNA was loaded on each lane, and checked under UV light. Hybridization then was performed with biotin-labeled PTTH cDNA as a probe. P, pharate adult; A, adult; Mg, midgut; Mt, Malpighian tubules.

We measured developmental changes in the amount of PTTH mRNA present during larval and pupal/pharate adult stages using RT–PCR combined with Southern blot analysis. The PTTH mRNA content in the brain of fifth instar larvae persisted at a high level from Day 0 to Day 3, and then decreased dramatically when larvae entered the wandering stage (Fig. 5A). At pupation, the PTTH mRNA was low, it then increased to a fairly constant level from Day 2 through Day 10, and then again increased to its highest level on Day 12, the day before adult eclosion (Fig. 5B).

Fig. 5. Developmental changes of PTTH mRNA. RNA was extracted from 20 brains or brain–SG complexes of fifth instar larvae, pupae and pharate adults. 0.5–2 ␮g of RNA from fifth instar larvae to pharate adults (equaling amounts of the brain or Br–SG complex) was reverse transcribed, subjected to RT–PCR amplification with 20 cycles for fifth instars, and 18 cycles for pupae to ensure that the PCR products would linearly increase (based on our preliminary PCR results), and then Southern blotted with Hez-PTTH cDNA as a probe. (A) fifth instar larvae. (B) pupal/pharate adult stages. The Arabic numeral represent the day of fifth instar larval and pupal/pharate adult development; W, wandering larvae.

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4. Discussion From the brain of H. zea, we have isolated and characterized an 878 bp cDNA that encodes a precursor polyprotein containing the PTTH sequence. The deduced amino acid sequence further verifies that we have cloned the authentic Hez-PTTH cDNA. Hez-PTTH preprohormone shares the key structural features of known PTTH cDNAs, including the proteolytic cleavage sites and the seven cysteines that are known from the same region in other species. The cysteine residues are involved in establishing disulfide bonds between and within chains, a characteristic that is essential for the full expression of prothoracicotropic activity [4]. Phylogenetic analysis indicates that Hez-PTTH does not belong to any of the other PTTH clusters that have been identified, thus indicating that it is a new member of the PTTH family having molecular characteristics distinct from those of other species that have been reported thus far. This, along with the description of PTTH from H. virescens[19], is the first characterization of a PTTH cDNA from a noctuid moth. PTTH sequences for all other Lepidoptera thus far reported are from members of the Bombycoidea superfamily. Northern hybridization revealed a single band of approximately 900 bp which is similar in length to the PTTH cDNA, thus suggesting that the cDNA we report is full length, or nearly so. We predict that at least two peptides, PTTH and BNP1 (brain neuropeptide) are released from the precursor through posttranslational processing, as indicated in A. pernyi, H. cecropia, and M. sexta [9–11]. A comparison of the proteolytic cleavage sites among known PTTH cDNAs suggests that the last cleavage site (R113 -K114 -R115 ) immediately preceding the mature PTTH subunit is conserved among all species. All precursors have only one additional cleavage site (R45 -K46 -R47 ), except B. mori which has two cleavage sites (K30 -R31 -K32 and K54 -R55 ) [5], a feature that may be due to the rapid evolution promoted by artificial selection in this highly domesticated species [18]. Numerous experiments have documented bursts of PTTH in the hemolymph during the final larval instar, e.g. M. sexta [2] and B. mori [6,12], but levels of PTTH mRNA during this time do not appear to change dramatically [1]. Immunocytochemical results also indicate that PTTH levels in the neurosecretory cells do not undergo much variation around the time of the larval, pupal and adult molts [3,7,9,14,15]. It thus appears that the primary regulation of PTTH levels in the hemolymph is at the site of synaptic release [9]. Our observations with H. zea, showing that PTTH mRNA is present throughout development, are consistent with this conclusion. Yet, we do note several distinct fluctuations in PTTH mRNA. One conspicuous drop is evident during the wandering phase of the fifth instar. The amount of PTTH mRNA remains low shortly after pupation, rises again during pharate adult development and reaches a peak on the day before adult eclosion. This suggests that PTTH titers in the hemolymph, especially during metamorphosis, may be

regulated not only by release of the hormone but also by regulation of transcription.

Acknowledgments We thank Dr. Roger Meola for providing H. zea, and Dr. Shin Goto, Osaka City University, Japan for assistance with the phylogenetic analysis. This work was supported in part by USDA-NRI grant 98-35302-6659 and the Major State Basic Research Development Program of the P.R. China (G20000162). References [1] Adachi-Yamada T, Iwami M, Kataoka H, Suzuki A, Ishizaki H. Structure and expression of the gene for the prothoracicotropic hormone of the silkmoth Bombyx mori. Eur J Biochem 1994;220: 633–43. [2] Bollenbacher W, Granger NA, Katahira EJ, O’Brien MA. Developmental endocrinology of larval molting in the tobacco hornworm, Manduca sexta. J Exp Biol 1987;128:175–92. [3] Dai J, Mizoguchi A, Satake S, Ishizaki H, Gilbert LI. Developmental changes in the prothoracicotropic hormone content of the Bombyx mori brain-retrocerebral complex and hemolymph: analysis by immunogold electron microscopy, quantitative image analysis, and time-resolved fluoroimmunoassay. Dev Biol 1995;171:212–23. [4] Ishibashi J, Kataoka H, Isogai A, Kawakami A, Saegusa H, Yagi Y, et al. Assignment of disulfide bond location in prothoracicotropic hormone of the silkworm, Bombyx mori: a homodimeric peptide. Biochemistry 1994;33:5912–9. [5] Kawakami A, Kataoka H, Oka T, Mizoguchi A, Kimura-Kawakami M, Adachi T, et al. Molecular cloning of the Bombyx mori prothoracicotropic hormone. Science 1990;247:1333–5. [6] Mizoguchi A, Ohashi Y, Hosoda K, Ishibashi J, Kataoka H. Developmental profile of the changes in the prothoracicotropic hormone titer in hemolymph of the silkworm Bombyx mori: correlation with ecdysteroid secretion. Insect Biochem Mol Biol 2001;31:349–58. [7] O’Brien MA, Katahira EJ, Flanagan TR, Arnold LW, Haughton G, Bollenbacher WE. A monoclonal antibody to the insect prothoracicotropic hormone. J Neurosci 1988;8:3247–57. [8] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406– 25. [9] Sauman I, Reppert SM. Molecular characterization of prothoracicotropic hormone from the giant silkmoth, Antheraea pernyi: developmental appearance of PTTH expressing cells and relationship to circadian clock cells in central brain. Dev Biol 1996;178:418– 29. [10] Sehnal F, Hansen I, Scheller K. The cDNA–structure of the prothoracicotropic hormone of the silkmoth Hyalophora cecropia. Insect Biochem Mol Biol 2002;32:233–7. [11] Shionoya M, Matsubayashi H, Asahina M, Kuniyoshi H, Nagata S, Riddiford LM, et al. Molecular cloning of the prothoracicotropic hormone from the tobacco hornworm, Manduca sexta. Insect Biochem Mol Biol 2003;33:795–801. [12] Shirai Y, Aizono Y, Iwasaki T, Yanagida A, Mori H, Sumida M, et al. Prothoracicotropic hormone is released five times in the 5th-larval instar of the silkworm, Bombyx mori. J Insect Physiol 1993;39: 83–8. [13] Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTER-X windows interface: flexible strategies for multiple

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