a 467 bp 1 482 2 93 3 321 4 7 281 6 21 7 66 8 176 19 12 13 212 113 16 8 b ATG TCA GGA CAT GTA ATG GAG GAA TGT GTA GTT CAC GGT ACG TTA GCG GCA GTA TTG CGT TTA ATG GGC GTA GTG M S G H V M E E C V V H G T L A A V L R L M G V V GGA ATG ATG CCA GTG CGA TTG GTG TCA CTC TCA GGC GCA TTC GTT GTT CGT TAT TCG TTC GCA AAA GAC GTC ATT G M M P V R L V S L S G A F V V R Y S F A K D V I GCT CGT TTG TTT TAT AGC TGT TTC TGT CTC CTG CTG TTG ACA TGG GAA TTG GTA TAT CTC GTC CCT AAA ATT TTG A R L F Y S C F C L L L L T W E L V Y L V P K I L TCC AAG AAC AAT ACT TCA CAG AAA CGC CTA ATC TTT TTG TTA TCA ATC ATT GGT GTA ATT GTT CGG ATG GCA GTC S K T S Q K R L I F L L S I I G V I V R M A V ATA TTT TTT GCA GTT TTA AAG GGA TAC CAA CGG ACA AAT AAC TTA TGT AGA AAT CTC AAT AAA ATT CAA AAG CTT I F F A V L K G Y Q R T L C R L K I Q K L AAC ATA GAA GGC GTT GAT GAT GCC GCA GTT GCT AAC ATA GAA AAA AGA AGA TTT AAA TTA TTT ATT ATT CTT TTA I E G V D D A A V A I E K R R F K L F I I L L GTA ATG TCA CCG GTA ATT GTT ATT GCT GAA TTT ATA ATA GGT GGG GAA CGC ATT ACG CAT GTT AAA AAT TTC CAC V M S P V I V I A E F I I G G E R I T H V K F H TTT TTA ATC ATC AGG AAA AAC ATG TAT CAG TTT GCA TAT TTT GTA ATA TTA ACA TTG CAA GCG CAA TTC TCG TTC F L I I R K M Y Q F A Y F V I L T L Q A Q F S F ACA ATA CTC AGC GTG CAC ACC GCG CTT CAG GCA GTC AAC CGT CGT CTT GGG AAG TTG TTA ACA GAT TTC AAA AAA T I L S V H T A L Q A V R R L G K L L T D F K K AAC CAG GAA AGA AGC AAT GAA AGA GCG CAT AAG ACT CTC AGA CAA TTG GCA ACA TCG TAT AGC ACT CTC TGT GAT Q E R S E R A H K T L R Q L A T S Y S T L C D GTT ATG AGG ACT TTG GAT AAG GAG AAT GGA TTG ATT ATT TTA CTG TCA TTT ATC TCA ATG TTG GTG TAC GCT GTA V M R T L D K E G L I I L L S F I S M L V Y A V CAA TGT ATA TTC TTC CTA GTC GTC TTT TGG AGT GTA AAT CCT TTC ACG AAC TTG CCT TAT ACA GGA ATC GAG TTG Q C I F F L V V F W S V P F T L P Y T G I E L GAA AAA TTA AAC TTA AAT AGA ATA ATG CAA TGT TGT CAT TGT TTA CAT ATA TTT ATA AAA ATA ATG TTT TTA CTG E K L L R I M Q C C H C L H I F I K I M F L L GAA CCC TGC CAA TGG ACA TAT AAT GGG ATG GAG GTA ACA AGG CTG TTT GTG ACC AGA TTG ACG CAC TAC ACG CCA E P C Q W T Y G M E V T R L F V T R L T H Y T P ACT ACT AGC GGG CCA ATT GTC AAT GAG CTG GAG ATG TTC TAT CGT CTT GTC TTC ACA AAC ATT CCC CCG TAC TCG T T S G P I V E L E M F Y R L V F T I P P Y S CCA TTT CAA TTG TTT ACT CTA AGG CGA TCA ATT ATA CTG GAG TTG TCG GGA GCA CTT ACT ACA TGG TTG CTT GTC P F Q L F T L R R S I I L E L S G A L T T W L L V GTT TTA CGT GAT AAA AGC CCA TAA V L R D K S P * TM1 TM2 TM3 TM4 TM TM6 TM7 Supplementary Figure S1: Structure of the PxutGr1 gene and nucleotide and amino acid sequences of the coding region. a: Schematic representation of the genomic structure of PxutGr1. The bold line shows the length in nucleotides from the initiation to the termination codons shown in B. White boxes indicate exon regions 1-8. The numbers of nucleotides (bp) for each exon and intron are denoted. b: ucleotide and amino acid sequences of the PxutGr1 coding region. Gray boxes (TM1-7) show transmembrane regions predicted by the TMHMM algorithm. Arrowheads indicate intron junctions.
a 23 Gr 8. 963 r 1 998 DmelGr8c DmelGr1b DmelGr2a HvirR3 Dmelr43a Dmelr49b Dmelr3a HvirR8 HvirR1 HvirR17 Dmelr7a DmelGr98a DmelGr9e DmelGr77a PxutGr1 DmelGr43a HvirR4 Dmelr67b Dmelr63a Dmelr6a HvirR18 HvirR2 HvirR9 HvirR7 HvirR12 Dmelr8e Dmelr83a DmelGr93a DmelGr93d DmelGr93c DmelGr93b DmelGr92a DmelGr23aB DmelGr23aA DmelGr98b DmelGr98d DmelGr98c Dmelr74a Dmelr3a Dmelr13a Cquir83b Agamr83b Dmelr8d Ccapr83b Dmelr8c Dmelr83b Dmelr8b Dpser83b Dmelr67a Dmelr94b Dmelr94a Dmelr71a Dmelr46aB Dmelr46aA Dmelr19b Dmelr19a Dmelr2a Dmelr9a Dmelr33c Dmelr33b Dmelr33a Dmelr23a DmelGr89a DmelGr9a DmelGr8a DmelGr39b DmelGr63a DmelGr21a DmelGr9f DmelGr47a DmelGr8a DmelGr8b DmelGr8a DmelGr47b DmelGr7a DmelGr39aB DmelGr39aA DmelGr39aD DmelGr39aC DmelGr32a DmelGr68a Dmelr4b Dmelr24a Dmelr22c Dmelr1a Dmelr82a Dmelr98b Dmelr47a Dmelr9a Dmelr4a Dmelr1a Aperr83b Bmorr2 Hzear83b Mbrar83b Sexir83b HvirR2 HvirR19 HvirR21 Dmelr43b Dmelr42a Dmelr22b Dmelr22a Dmelr8a Dmelr9c Dmelr98a Dmelr9b Dmelr42b DmelGr28a DmelGr28bE DmelGr28bC DmelGr28bB DmelGr28bD DmelGr28bA HvirR HvirR1 DmelGr64e DmelGr64f DmelGra DmelGr64d DmelGr64c DmelGr64b DmelGr64a DmelGr61a DmelGr33a DmelGr66a DmelGr9b DmelGr9a DmelGr9c DmelGr9d DmelGr36b DmelGr36c DmelGr36a DmelGr1a DmelGr97a DmelGr94a DmelGr22c DmelGr22f DmelGr22b DmelGr22a DmelGr22e DmelGr22d Dmelr88a Dmelr69aB Dmelr69aA Dmelr92a Dmelr67c HvirR11 HvirR14 HvirR1 HvirR6 HvirR16 Bmorr3 Bmorr1 HvirR13 Bmorr Bmorr4 Bmorr6 Dmelr47b Dmelr6c Dmelr6b Dmelr6a Dmelr8f Dmelr49a Dmelr83c Dmelr67d b DmelGr93a DmelGr93d DmelGr93c DmelGr93b DmelGr92a DmelGr1b DmelGr8c DmelGr39b DmelGr63a DmelGr21a DmelGr89a DmelGr9a DmelGr8a DmelGr2a DmelGr98a DmelGr28a DmelGr28bE DmelGr28bC DmelGr28bB DmelGr28bD DmelGr28bA DmelGr23aB DmelGr23aA DmelGr98b DmelGr98d DmelGr98c DmelGr9f DmelGr47a DmelGr8a DmelGr8b DmelGr8a DmelGr9e HvirR HvirR1 DmelGr64e DmelGr64f DmelGra DmelGr64d DmelGr64c DmelGr64b DmelGr64a DmelGr61a DmelGr47b DmelGr7a DmelGr39aB DmelGr39aA DmelGr39aD DmelGr39aC DmelGr32a DmelGr68a DmelGr77a PxutGr1 DmelGr43a HvirR4 DmelGr66a DmelGr33a DmelGr9b DmelGr9a DmelGr9c DmelGr9d DmelGr36b DmelGr36c DmelGr36a DmelGr1a DmelGr97a DmelGr94a DmelGr22c DmelGr22f DmelGr22b DmelGr22a DmelGr22e DmelGr22d. r Supplementary Figure S2: A dendrogram of putative insect chemoreceptors. a: verview of the dendrogram. Gr: gustatory receptor. r: olfactory receptor. Dmel: Drosophila melanogaster16. Hvir: Heliothis virescens4. Pxut: Papilio xuthus (AB2611). Agam: Anopheles gambiae (XP_312379). Cqui: Culex quinquefasciatus (ABB2931). Ccap: Ceratitis capitata (AAX1477). Aper: Antheraea pernyi (CAD882). Bmor: Bombyx mori (CAI4391, P_1376, P_13692, P_136927, P_116227, P_136926). Hzea: Helicoverpa zea (AAX14773). Mbra: Mamestra brassicae (AAS4992). Sexi: Spodoptera exigua (AAW283). b: Gr-centric view of the dendrogram as in (a).
a 3 3 2 2 1 1 addition 3 6 9 12 Time (s) b 4 Synephrine ctopamine 3 2 1 66 9 114 66 9 114 Growth period (hr) Supplementary Figure S3: Effect of culture age of Sf9 cells on the response to synephrine and octopamine. a: Sf9 cells were infected with baculovirus containing Aequorin and PxutGr1 for various periods, and stimulated by synephrine as in Methods (orange: 66hr, red: 9hr, blue: 114hr.). The arrow indicates the time of pulse (2 seconds) of synephrine solution (3 mm). b: Experiments in (a) are repeated 6-8 times and presented as histograms with error bars. Stimulation by octopamine (3mM) was performed as with synephrine. Error bars represent Standard Deviation.
H + - synephrine chiro-inositol stachydrine rutinosyl rutin H bufotenine rutinosyl H naritutin H -hydroxy-ω-methyltryptamine rutinosyl H 2 hesperidin glucosyl ribosyl adenosine glucosyl vicenine-2 Supplementary Figure S4: Structure of ten oviposition stimulants.
3 3 3 3 2 2 1 1 3 2 2 1 1 3 2 2 1 1 Seconds 3 6 9 12 3 6 9 12 3 6 9 12 chiro-inositol stachydrine adenosine 3 3 3 3 2 2 1 1 3 2 2 1 1 3 2 2 1 1 Seconds 3 6 9 12 3 6 9 12 3 6 9 12 rutin naritutin hesperidin 3 3 3 2 2 1 1 3 2 2 1 1 3 2 2 1 1 Seconds 3 6 9 12 3 6 9 12 3 6 9 12 bufotenine -hydroxy-ω-methyltryptamine Supplementary Figure S: Response of Sf9 (PxutGr1/Aeq) cells to various oviposition stimulants. The calcium imaging assay was performed as described in Methods using cultures 9 hrs after the viral infection. Arrowheads indicate addition of each compound for 2 seconds. o activity was observed for at least 12 seconds in any compounds tested. ther eight oviposition stimulants other than synephrine. Mixture is the mixture of the 8 compounds in this figure
H 2 H 2 H 2 octopamine tyramine dopamine H 2 H 2 H L-tyrosine α-(methylaminomethyl) benzyl alcohol α-(1-aminoethyl)-4- hydroxybenzyl alcohol H (R)-(-)-Phenylephrine mianserin Supplementary Figure S6: Structure of compounds structurally related with synephrine and an antagonist, mianserin.
3 3 3 3 2 2 1 1 3 2 2 1 1 3 2 2 1 1 Seconds 3 6 9 12 3 6 9 12 3 6 9 12 tyramine dopamine L-tyrosine 3 3 3 3 2 2 1 1 3 2 2 1 1 3 2 2 1 1 Seconds 3 6 9 12 3 6 9 12 3 6 9 12 α-(methylaminomethyl) benzyl alcohol α-(1-aminoethyl)-4- hydroxybenzyl alcohol (R)-(-)-Phenylephrine Supplementary Figure S7: Response of Sf9 (PxutGr1/Aeq) cells to various chemicals structurally related to synephrine. Arrowheads indicate addition of each compound for 2 seconds. o activity was observed for at least 12 seconds in any compounds tested.
tarsus th tarsomere foreleg bristle sensillum wax electrode Ringer's solution 1 µm stimulant electrode amplifier Supplementary Figure S8: A diagram of electrophysiological recording system with a SEM picture of th tarsomere. The system is described in Methods.
a Synephrine 1mM no injection Synephrine 1mM GFPdsRA Synephrine 1mM PxutGr1BdsRA Synephrine 1mM PxutGr1BdsRA % of spikes per s % of spikes per s % of spikes per s 1 6 2.4 1.6 2.8 1 6 2.4 1.6 2.8 1 6 2.4 1.6 2.8 mv 1 mv b Stachydrine 2mM no injection ms % of spikes per s 1 6 2.4 1.6 2.8 Stachydrine 2mM GFPdsRA % of spikes per s 1 6 2.4 1.6 2.8 Stachydrine 2mM PxutGr1BdsRA % of spikes per s 1 6 2.4 1.6 2.8 mv c chiro-inositol 2mM no injection chiro-inositol 2mM GFPdsRA chiro-inositol 2mM PxutGr1BdsRA 1 mv ms 1 mv ms % of spikes per s % of spikes per s % of spikes per s 1 6 2.4 1.6 2.8 1 6 2.4 1.6 2.8 1 6 2.4 1.6 2.8 mv Supplementary Figure S9: Recordings of electrophysiological responses to various stimulants: Effects of injection of dsra of PxutGr1 and GFP (control). The response of each sensillum was determined for seconds and analyzed as described in Methods. a: Responses to 1mM synephrine. o variance was observed in non-injected and GFPdsRA injected individuals. Since the responses by sensillum from PxutGr1dsRA injected individuals were variable from sensillum to sensillum, typical two examples are shown. b: Responses to 2mM stachydrine. o effects were observed by injections within the statistic variations. c: Responses to 2mM chiro-inositol. o effects were observed by injections.
3 Expression level 2 1-3 -2-1 1 2 3 7 Time course (days) Supplementary Figure S1: Developmental time course of the expression of PxutGr1 in pupae and adults. RA was prepared from a single foreleg tarsi cut off from pupae and adults and relative expression levels of PxutGr1 were measured by quantitative RT-PCR as described in Methods. Each bar represents mean value of relative mra expression level with SD error bar from 3-7 experiments. The mean value of day (the day of eclosion) was used as a reference to normalize.
a Vessel for Ringer s solution with or without mianserin Vessels for compound solutions Cocks 3-way cock Low level luminescent detector Petri dish (Sf9 cells) Peristaltic pump b Supplementary Figure S11: Instrument used for the calcium imaging assay. a: An overall picture of the instrument. Parts showing their functions are indicated. b: The Petri-dish containing 2ml cell culture and a perfusion device are shown
Supplementary Figure S12: Injection of water or dsra into pupae. 1µl (1µg/µl) solution was injected using a capillary syringe into the ventral side of female pupae, at the junction between the thorax and abdomen.
Supplementary Table S1 The list of primers. RT-PCR PxutGr1 full length ATGTCAGGACATGTAATGGAGGAATG TTATGGGCTTTTATCACGTAAAACGAC Ribosomal protein S9 TGAAGATCATCGGTGAATATGGTCTC TCTTCTTCGTCATCGTTAGCAGCAC Baculovirus expression PxutGr1 GGATCTCTAGACCACCATGTCAGGAC ATGTAATGGAGGAATG GTTTATTAGGCCTTTAGTGGTGGTGGT GGTGGTGTGGGCTTTTATCACGTAAA ACGAC Aequorin GATCCCCGGGCCACCATGACAAGCAA ACAATACTCAGTCAAGCTTACACC TTTATTACTAGTTTAGGGGACAGCTCC ACCGTAGAGCTTTTCGCAAGCAGG PxutGr1dsA sense CAGATCTGAATTCCCACCGGAGGAAT GTGTAGTTCACGGTACG TTATTATCCTCAGGTTTCTATGTTAGC AACTGCGGCATC PxutGr1dsA anti-sense CTTTTATTACTAGTGGAGGAATGTGTA GTTCACGGTACG CACAGATCCCCGGGCCACCTTTCTAT GTTAGCAACTGCGGCATC PxutGr1dsB sense CAGATCTGAATTCCCACCGCAGTTTTA AAGGGATACCAACGGAC TTATTATCCTCAGGCGCTCTTTCATTG CTTCTTTCCTGG PxutGr1dsB anti-sense CTTTTATTACTAGTGCAGTTTTAAAGG GATACCAACGGAC CACAGATCCCCGGGCCACCCGCTCTT TCATTGCTTCTTTCCTGG PxutGr1dsC sense CAGATCTGAATTCCCACCTGGCAACA TCGTATAGCACTCTCTG TTATTATCCTCAGGTAAACAATTGAAA TGGCGAGTACGG PxutGr1dsC anti-sense CTTTTATTACTAGTTGGCAACATCGTA TAGCACTCTCTG CACAGATCCCCGGGCCACCTAAACAA TTGAAATGGCGAGTACGG GFPds sense CAGATCTGAATTCCCACCCATGACTTT TTCAAGTCTGCCATGC TTATTATCCTCAGGACCATGTGGTCTC TCTTTTCGTTTG GFPds anti-sense CTTTTATTACTAGTCATGACTTTTTCAA GTCTGCCATGC CACAGATCCCCGGGCCACCACCATGT GGTCTCTCTTTTCGTTTG dsra preparation PxutGr1 region A TAATACGACTCACTATAGGGAGAGCA GTTTTAAAGGGATACCAACGGAC TAATACGACTCACTATAGGGAGACGC TCTTTCATTGCTTCTTTCCTGG PxutGr1 region B TAATACGACTCACTATAGGGAGAGGA GGAATGTGTAGTTCACGGTACG TAATACGACTCACTATAGGGAGATTTC TATGTTAGCAACTGCGGCATC Quantitative RT-PCR PxutGr1 GACAAGGCTGTTTGTGACC CACGTAAAACGAGAAGCAACC ribosomal protein S17 GTAATAAAATTGCCGGATTTGC GAAGCCCATTGATGTTGTTAAAG