Identification and expression profiles of neuropeptides and their G 3 ...

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Receptor. Close family. Likely ligands. Neuropeptide receptor A1. CG7285, CG13702, BmA1. AstC. Neuropeptide receptor A2. BmA2. ITP. Neuropeptide ...
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Supplementary Information:

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Identification and expression profiles of neuropeptides and their G

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protein-coupled receptors in the rice stem borer Chilo suppressalis

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Gang Xu1, Gui-Xiang Gu1, Zi-Wen Teng1, Shun-Fan Wu1, 2, Jia Huang1, Qi-Sheng Song3,

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Gong-Yin Ye1, and Qi Fang1*

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State Key Laboratory of Rice Biology & Key Laboratory of Agricultural Entomology of Ministry

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of Agriculture, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China

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2

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Local Joint Engineering Research Center of Green Pesticide Invention and Application

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College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; State &

Division of Plant Sciences, Missouri University, Columbia, MO 65211, USA

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*Corresponding author: Qi Fang, Institute of Insect Sciences, Zhejiang University, Hangzhou

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310058, China, Tel: 86-0571-88982696, E-mail: [email protected]

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This file includes:

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Tables S1-S3

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Figures S1-S10

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Table S1 G protein-coupled receptors with likely ligands in insects. Receptor Neuropeptide receptor A1 Neuropeptide receptor A2 Neuropeptide receptor A3 Neuropeptide receptor A4 Neuropeptide receptor A5 Neuropeptide receptor A6-A Neuropeptide receptor A6-B Neuropeptide receptor A7 Neuropeptide receptor A8 Neuropeptide receptor A9 Neuropeptide receptor A10 Neuropeptide receptor A11 Neuropeptide receptor A12 Neuropeptide receptor A13 Neuropeptide receptor A14 Neuropeptide receptor A15 Neuropeptide receptor A16 Neuropeptide receptor A17 Neuropeptide receptor A18 Neuropeptide receptor A19 Neuropeptide receptor A20 Neuropeptide receptor A21 Neuropeptide receptor A22 Neuropeptide receptor A23 Neuropeptide receptor A24 Neuropeptide receptor A25 Neuropeptide receptor A26 Neuropeptide receptor A27 Neuropeptide receptor A28 Neuropeptide receptor A29 Neuropeptide receptor A30 Neuropeptide receptor A31 Neuropeptide receptor A32 Neuropeptide receptor A33 Neuropeptide receptor A34 Neuropeptide receptor A35 Adipokinetic hormone receptor Allatostatin A receptor Diapause hormone receptor FMRFamide receptor Myosuppressin receptor Pheromone biosynthesis activating neuropeptide receptor Sex peptide receptor SIFamide receptor Neuropeptide receptor B1 Neuropeptide receptor B2 Neuropeptide receptor B3 Neuropeptide receptor B4 Diuretic hormone receptor Leucine-rich repeat G protein-coupled receptor 1 Leucine-rich repeat G protein-coupled receptor 2

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Close family CG7285, CG13702, BmA1 BmA2 CG13229, BmA3 CG1147, BmA4 BmA5 CG5911-A, BmA6-A CG5911-B, BmA6-B CG7395, BmA7 CG13229, BmA8 CG6881, CG6857, BmA9 CG7395, BmA10 CG7395, BmA11 BmA12 BmA13 CG14593, BmA14 CG14484, BmA15 BmA16 CG13995, BmA17 CG6986, BmA18 CG5811, BmA19 CG13229, BmA20 CG10698, BmA21 CG5811, BmA22 CG10626, BmA23 CG7887, BmA24 CG14575, BmA25 CG6111, BmA26 CG14575, BmA27 CG11325, BmA28 CG11325, BmA29 CG6111, BmA30 CG14003, BmA31 CG6515, BmA32 CG6515, BmA33 CG30340, BmA34 BmA35 CG11325, BmAKHR CG2872, CG10001, BmBAR CG9918, BmDpHR CG2114, BmRFaR CG8985 CG13802 BmMSR CG8784, CG8795 BmPBANR

Likely ligands AstC ITP Orphan NPF Orphan ETH ETH Orphan Orphan SK sNPF sNPF Orphan MS CCH2 CCH1 AT Orphan CNMa RY Orphan Crz RY LK TK/ITPL CAPA-PVK CCAP CAPA-PVK ACP ACP CCAP TR NTL (FXXXRa) NTL (YXXXRa) ITP Orphan AKH1, AKH2 AstA CAPA-PK-1 FMRF MS PK-2

CG16752, BmSPR CG10823, BmSIFR CG17415, BmB1 CG13758, BmB2 BmB3 CG4395, BmB4 CG8422, CG12370, BmDHR CG8930

SP/AstB SIF DH31 PDF Orphan Orphan DH41 GPA2/GPB5

CG7665

Bursicon

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Table S2 Primers used for the qRT-PCR analysis of neuropeptide genes. Primers

Forward primer (5’-3’)

Reverse primer (5’-3’)

AKH1 AKH2 AKH3 AstA AstB AstC AstCC AT ITG Burα Burβ CAPA CCH1 CCH2 Crz CCAP DH/PBAN DH31 DH41 DH34 DH45 EH ETH FMRF GPA2 GPB5 ILP ITP LK MS NTL NP NPF1 NPF2 NPLP1 OKA OKB PDF Pro PTTH RY sNPF SIF IMF SK TK TR EF-1

CGAGTCTCAACTTCAAACAGCA GACGCCCAGTTGACCTTCAG GATCAGGTGTAGGGGCGTGA CTGGCCCAGTTTACGAAGAG GCGAACTTCCACGGATCTTG CGGGCAACGCTTACTTTATC CGCTCCCGTTACTGTTGGTG ACCACCAGGGCTGAGCTGTA TACCGCCACTACGCTTACGG CTTCGTCATCCCGGGTGTAA GCGGGGAAGTCAGTGTCAAC AGTCATCGCCAGCGCATATC GCCAGCTGCGAACAAAGAAC GGCAAACGATCTGGCGATAC CGAACCTAAGCCTGCTGCTG CAGGTTGTGGTCGCAAGAGA CGAACAGTTGCCTGCGTATG TGCTCGTCGTACCCACTTCA CAACTACGGTGTGCCACCAA CAACTACGGTGTGCCACCAA CAACTACGGTGTGCCACCAA TCGTGCTGTTCGCTGTCTTC GCGTTAGTCGCATATTGGCTGT CGCAATCGATCGCAGTATGA CCTGGCTGTCATCGAATAGGTC GCAGACGGACCAGAATGGAC CCAGATGCTGGGAGAGATCG GCATCTGCGACGACTGCTAC CATGGGGAGGCAAAAGATCA GAGCACCGAAACCCGTGTAG TGAGGGGCAGACGTGATTCT GTCCCGTGCTGCAAAGAATG TGAACAAACGCATCGCAGTC GTTGCTGTCCGCCATCTTGT AAAATGGCCAACTCCCGACT AATTTCGTGCGCAAGAGGAA ACCGCTGATTCGCTACAGGA TGCCCAAAGTCTGGCTTGAT CCAAAGCAAGGAACGACAGC ACGGTCATCATGTGCTGTGG GGCGTTGATGACGCTAGTGG TCAGCGTCAGCGGACAGTAG TGCGTGCAGTATTCGCCATA TCGCTGGTGCAAGTAGGAGA CTATCTTCGCGTTGGCAGTG TGGGCAAGAGGTGCAGAAAC CCTGTGGCAGTGAGTGCTTG AAATCGGCGGTATTGGTACG

AGCCGCTATAGCTTCGTCGT TTTTGCCACCATCTCTACACGA TGTCGACAAAGCCTGGTGAA AAATTGAAACGGTGCAGGTC AGGGCTGACCATGCCTTCTT ATCCGGACGAACTACAATCG GTCGACCTCCATCTGCTCGT ACTGACCGGGTGAAATCAGG CGCCCTCCGTCATCTGTATC GGGGCAAAAGAGGACTACCG TCTTCCAGCCTCAAGCCATC CTGACGTTTGGCTGTCATCG TCCAACATCGGTTTGGCTTC GCTTCCCTGGCGCTATCTTC GCCTGCCTTCCAACAAGTTC CTGCGCTTGCATCTTGAATG TGATCCGGTTGTGTGACTGC CTGAGGCCAAGATCCAATGC CCTCTCCTTCTCCAGGCTGA CTCTGAAGCACGTCCACAGC GCACCTCCATTGGGTTGTTG ACAGGATTCTGCGCATAGGG CTCCCCATTCGTGGGATGT GGGCTGTCGTATTCGTCGTC GTTGCAGCACTGACCCAATG CACTACTCCTGGGTCGCAATG TGACGTAGCAAGCCTTGTGG TTCCCGACGAAGTCGATCAT GCTTGTCCAGCGAGGGTATG GCTCGAGGAAGGTGTTGAGC TTCTTCTTCCCGCCTTCCTC CCAGCCACCGTCTACCCTTA CGGTCTAGCAGCCTGTGTGT CATCTGCCTCTTGCCGTACC CTACCGACGCGGTAACCATC TGGAACCAATGGGAAACGAC GATGCCAGCGAGGTTTCTTC ATCACCATCCGTCCAGTTGC CGCCGCGAACTTTCTTAAAATC TCCAATGCCATCTTCTGCAC TGCCCATGAAGAACCTGTCA AGCTCGTCCATTTCGTCCAA ATGCCTGGCAGGTTTCTGAG TGGGAACCAAGCTTGACAGG AGATGGCCGTAGTCGTCGAA GCATGCCTACGAAGCCCATA TACCACGGTTCAGCCACAGA AAGGGGAGGGAATTCTTGGA

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Table S3 Primers used for the qRT-PCR analysis of the neuropeptide receptor genes. Primers

Forward primer (5’-3’)

Reverse primer (5’-3’)

A1 A2 A3 A4 A5 A6-A A6-B A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24 A25 A26 A27 A28 A29 A30 A31 A32 A33 A34 A35 AKHR AstAR DpHR RFaR MSR PBANR SIFR SPR B1 B2 B3 B4 DHR LGR1 LGR2 EF-1

GCTTCGCTACTCCAAAATGC TTGGCGAAGTAGGATGTCGTC TTAACCGTCGTGGGATACGTC TTCTCGCAGAACAGGAAGGT GTGGCGGATTTCATGGTGAT AATGAGAAGCGAAGGGCACA ATCGCCAAGAACCTCATGGA GCTGGAACAGCTAGGAGTGG ATGTCGGTTCTACGGGAGCA GGGAATGCGCCATGAGATAC TTTGTTGCGCATTTCATAGC CGTACGTCCAAGGAACCAGT GGATCTTGCTGCTGCTTGCT TGGACATGTTGGCGTTGTTC CCTGGTTCCGGTAATGTTCG TGTGTTCGTGAGGCACAAGG GAGAAAAGCTGCCAAAATGC CCTCGGCAACGTCCTCATAG TCATCGTGTCGTCCGTGTTC TCAGCCAGGGACAACAGGAT TTACGGCACACAACGAATGC GTGGTCTGCGGGAAACGTAG TGATCGTGGTGGCTGTATGG CCACATCGAGACGAATGCAG TGCCCATAATGTCCATGTCG TGCTTAGCCTCCGTGCTAGA ATAGCTGCCCTGCTCTGCAC CCGAGCGTTGAGGATAGTGG GTTTGGCGATTGCGAGAAAG TTGTAGCCTGCCACAGAGCA CTCGGCAAGAGAGCCACTGT CTCGTGGTGACGTTGTCGAG ACGTACGCGGTTCCAATGTT GAAATGGGCGCACCTTAACA TTACTGGAGGGACCGGTTGA CACGCTGCTCATCTTCATCG CGCCTGGATGTACTCCTCAT AGTATCAGCGGAAAGCAGGA GGCTTGGCATCAGAAACGTC CGTGTACCTGACGCTCATCG ACGGGATTAGCAGTCGCTGA TTGTGGACATACCGCCAGTG CTGGATCTTCGCCGTACTCG TCGTGGATCGGCAGTATGTG GCCCTGTCCAGACTTTGTGC TGCCGTATCTTTGCGAACTC GCAACGCTGTGTTCGTATGC GTCATCGGGTTCGACTCCAG GCGATGAGGTGGAAGAATGC GTGTACTCCGAAGCCGAACG AGGCGCATAACAACCCACAC AAATCGGCGGTATTGGTACG

GGTGGCAGACCGCTATGTAT AATGCCACAGCCCAGATCAC CGACGGTTGTGTGAATGTCG GTAGAGGCCAGTGCTTCGTC GGAAGCAGATGGCGTACCAG ATCAAAACGCTTCGCCTCAA CGCTTTAAATGGCAGCAAGC CCACAGTCAGCAAGTCCTCA AGTAGCCATGGATGCGGTTG CGGAGTCCAGCTTCTCCTCA AACGGTTTCATTACCGTTGC TGACCTATCTGAGGGCCAAC TAGCGGAGTTTGCATGACCA TTGTGGGCTCCATCTTGGTC CGGCACGCATATCAAGATCA CACTGACACCCCAATGCTGA TGATCGCTGAGTTGGCATAG CCAGTATGCCACTGCGTTCA GCGCAAAGTTTATGCCGAAG TGACTCGCGAACCACACGTA AAGTCTCGCCGAGTCAGCAC GACCAATTTGCCGCTTCTTG AGCGCAAACTGGAGAGCAAG GGCAGAAAGGGCACATGAAG ACCGCGAATATCACCACCAC ACTACAGCCCCATCCTGACG ATCACCACAGCCTGGAGGAA CAAGCTGCTCAGCTCGCATA CGCATAGGCAGAACACGTTG TCTCGCAGAAGATGCACACG TACGGCGACCAGCATAGGAC GTAGCTGAGCGAGTGCACGA GCCAGCAGATGCCGAATATC GAGCGTGTTTCCCAAGATGG AACAGCACCAAGAGCCGAAC CCTCGCCGAAGTACCAGATG GCTATGCAAATCAGCACGAA TCTGAAAGCGACCCTGAAGT GCTTGTGGTAGTGCCAGTGC TCGACGCTCACGTACGTCTC GGGTGATTGTCAACCAGATCG CGACGCAGGTGCTTATGTTG CCGTGGGGAGGATGTAACAG ACAGGGGATCAAGTGGACGA CAGCGACACGCTGTAACCTG CCCACCCCACAACGTAGTTT AGCACGAGTGGTCGTTGTGA AAGCAGTGCTGCCACAGACA GGGTGGCATTTTGTGTGTCA GAGGTGGCACATGAGGAAGC AACACGAGGTCGCTGAGTCC AAGGGGAGGGAATTCTTGGA

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Figure S1 Alignment of natalisins (NTLs) predicted from C. suppressalis (Cs), B. mori (Bm), and

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D. melanogaster (Dm). Identities are highlighted in red, and similarities are indicated by gray. The

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red asterisks indicate the characteristics of NTL (FXXXRa, YXXXRa).

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Figure S2 Sequence alignment of CAPA (PK1) (A) and DH/PBAN (PK2) (B) from C. suppressalis

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(Cs), B. mori (Bm), N. lugens (Nl), T. castaneum (Tc), and D. melanogaster (Dm). Identities are

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highlighted in dark red, and similarities are indicated by gray. The solid boxes indicate the mature

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peptides. PVK, periviscerokinin; CPPB, CAPA precursor peptide B; PK, pyrokinin.

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Figure S3 Protein alignment of diapause hormone/PBAN (DH/PBAN) precursor sequences in

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moths, including C. suppressalis (Cs), B. mori (Bm), Plutella xylostella (Px), M. sexta (Ms), H.

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zea (Hz), H. armigera (Ha), S. exigua (Se), and Agrotis ipsilon (Ai). SGNP: subesophageal

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ganglion neuropeptide. Identities are highlighted in dark red, and similarities are indicated by gray.

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Figure S4 Schematic representation of the highly conserved ITP gene structures that contains

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exons arranged in tandem and lead to derived alternative mRNA splice forms that encode the

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common and the distinctive parts of short (yellow, ITP) and long (red, ITPL) peptide isoforms.

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The red arrow marks the stop codon of ITPL. SP, signal peptide; IPRP, ITP precursor-related

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peptide.

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Figure S5 Protein alignment of the ITP and ITPL precursor sequences from C. suppressalis (Cs), B.

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mori (Bm), M. sexta (Ms), T. castaneum (Tc), A. mellifera (Am), S. gregaria (Sg), A. aegypti (Aa),

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and D. melanogaster (Dm). Identities are highlighted in dark red, and similarities are highlighted

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in light color. The signal peptide, ITP precursor-related peptide (IPRP), and mature peptide are

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separated by dashed lines. The red asterisks mark the conserved cysteine residues, and three

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disulfide bridges are indicated.

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Figure S6 Sequence alignment of the allatostatin double C (AstCC) from C. suppressalis (Cs), B.

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mori (Bm), N. vitripennis (Nv), A. mellifera (Am), N. lugens (Nl), A. pisum (Ap), T. castaneum

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(Tc), and D. melanogaster (Dm). Identities are highlighted in dark red, and similarities are

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indicated by gray. The solid box indicates the mature peptide, and the dashed boxes indicate Lys

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and Arg residues which may form convertase cleavages sites or be removed by carboxypeptides.

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Figure S7 Protein alignment of novel alternative splicing variants of three neuropeptide precursor

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genes from C. suppressalis. (A) AstCC; (B) CCH1; (C) sNPF. The dashed boxes indicate the

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signal peptides, the solid red boxes indicate the mature peptides, and the solid blue box indicates

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one more mature peptide of sNPF. The red texts represent the differences between two splicing

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variants.

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Figure S8 Alignment of CCHamides predicted from C. suppressalis (Cs), B. mori (Bm), N. lugens

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(Nl), A. mellifera (Am), T. castaneum (Tc), and D. melanogaster (Dm). Identities are highlighted

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in dark red, and similarities are indicated by gray. The red asterisks mark the conserved residues

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explaining the etymology of CCHamide.

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Figure S9 Alignment of short neuropeptide F (sNPF) peptides from C. suppressalis (Cs), B. mori

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(Bm), A. mellifera (Am), and D. melanogaster (Dm). Identities are highlighted in dark red, and

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similarities are indicated by gray.

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Figure S10 Protein alignment of proctolin (Pro) precursor sequences from C. suppressalis (Cs), B.

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mori (Bm), N. lugens (Nl), T. castaneum (Tc), and D. melanogaster (Dm). Identities are

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highlighted in dark red, and similarities are indicated by gray. The solid boxes indicate the mature

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peptides. The arrows indicate potential cleavage sites.