Table S6 List of the primers used for the Q-RT-PCR analysis. Found at: doi:10.1371/journal.pone.0003048.s006 (0.03 MB XLS) Figure S1 Postsource decay mass spectra of TPVRLRFamide (A) and SMRLRFamide (B). For each peptide, upper panel is the purified sample and the lower panel is the synthetic sample. The b- and y-type ion signals are indicated at the top. Signals of the indicated mass range are magnified 10 times to facilitate comparison. The final confirmation of the leucine residue was carried out based on the cDNA sequence. Found at: doi:10.1371/journal.pone.0003048.s007 (0.39 MB TIF) Figure S2 Direct MS analysis of CC-CA complex. The identified peaks in (A) are summarized in (B). Asterisks in (A) denote sodium adducts for the peptides 5, 9, 12 and 13. Found at: doi:10.1371/journal.pone.0003048.s008 (0.97 MB TIF) References 1. Hewes RS, Taghert PH (2001) Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome. Genome Res 11: 1126–1142. 2. Hauser F, Williamson M, Cazzamali G, Grimmelikhuijzen CJ (2006) Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data. Brief Funct Genomic Proteomic 4: 321–330. 3. Kataoka H, Toschi A, Li J, Carney RL, Schooley DA, et al. (1989) Identification of an allatotropin from adult Manduca sexta. Science 243: 1481–1483. 4. Gilbert LI, Granger NA, Roe RM (2000) The juvenile hormones: historical facts and speculations on future research directions. Insect Biochem Mol Biol 30: 617–644. 5. Riddiford LM (1994) Cellular and molecular actions of juvenile hormone. I.General considerations and premetamorphic actions. Advances in Insect Physiology 24: 213–274. 6. Wyatt GR, Davey KG (1996) Cellular and molecular actions of juvenile hormone. II. Roles of juvenile hormone in adult insects. Advances in Insect Physiology 26: 1–155. 7. Truman JW, Riddiford LM (2007) The morphostatic actions of juvenile hormone. Insect Biochem Mol Biol 37: 761–770. 8. Wu Q, Wen T, Lee G, Park JH, Cai HN, et al. (2003) Developmental control of foraging and social behavior by the Drosophila neuropeptide Y-like system. Neuron 39: 147–161. 9. Kim YJ, Zitnan D, Galizia CG, Cho KH, Adams ME (2006) A command chemical triggers an innate behavior by sequential activation of multiple peptidergic ensembles. Curr Biol 16: 1395–1407. 10. Kim YJ, Spalovska-Valachova I, Cho KH, Zitnanova I, Park Y, et al. (2004) Corazonin receptor signaling in ecdysis initiation. Proc Natl Acad Sci U S A 101: 6704–6709. 11. Yamanaka N, Hua YJ, Mizoguchi A, Watanabe K, Niwa R, et al. (2005) Identification of a novel prothoracicostatic hormone and its receptor in the silkworm Bombyx mori. J Biol Chem 280: 14684–14690. 12. Yamanaka N, Zitnan D, Kim YJ, Adams ME, Hua YJ, et al. (2006) Regulation of insect steroid hormone biosynthesis by innervating peptidergic neurons. Proc Natl Acad Sci U S A 103: 8622–8627. 13. Mita K, Kasahara M, Sasaki S, Nagayasu Y, Yamada T, et al. (2004) The genome sequence of silkworm, Bombyx mori. DNA Res 11: 27–35. 14. Xia Q, Zhou Z, Lu C, Cheng D, Dai F, et al. (2004) A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science 306: 1937–1940. 15. Vassilatis DK, Hohmann JG, Zeng H, Li F, Ranchalis JE, et al. (2003) The G protein-coupled receptor repertoires of human and mouse. Proc Natl Acad Sci U S A 100: 4903–4908. 16. Metpally RP, Sowdhamini R (2005) Cross genome phylogenetic analysis of human and Drosophila G protein-coupled receptors: application to functional annotation of orphan receptors. BMC Genomics 6: 106. 17. Fredriksson R, Schioth HB (2005) The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol Pharmacol 67: 1414–1425. 18. Hauser F, Cazzamali G, Williamson M, Blenau W, Grimmelikhuijzen CJ (2006) A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera. Prog Neurobiol 80: 1–19. 19. Bjarnadottir TK, Gloriam DE, Hellstrand SH, Kristiansson H, Fredriksson R, et al. (2006) Comprehensive repertoire and phylogenetic analysis of the G protein- coupled receptors in human and mouse. Genomics 88: 263–273. 20. Brody T, Cravchik A (2000) Drosophila melanogaster G protein-coupled receptors. J Cell Biol 150: F83–88. 21. Iversen A, Cazzamali G, Williamson M, Hauser F, Grimmelikhuijzen CJ (2002) Molecular identification of the first insect ecdysis triggering hormone receptors. Biochem Biophys Res Commun 299: 924–931. 22. Iversen A, Cazzamali G, Williamson M, Hauser F, Grimmelikhuijzen CJ (2002) Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2. Biochem Biophys Res Commun 299: 628–633. Bombyx Neuropeptide GPCRs Figure S3 In situ hybridization analysis of BNGR-A6-A on the CC-CA. Scale bar, 100 micrometer. Found at: doi:10.1371/journal.pone.0003048.s009 (7.87 MB TIF) Acknowledgments We thank Drs. Yusuke Suzuki, Minoru Suzuki and Akemi Suzuki for MS analysis, Dr. Michael E. Adams for providing synthetic ETH and PETH, Drs. Ivana Daubnerova´, Laco Roller and Young-Joon Kim for designing and preparing in situ probes, and Dr. Michael B. O’Connor for critical reading of the manuscript. Author Contributions Conceived and designed the experiments: NY KH YT TS HK. Performed the experiments: NY SY D KW YK KH YT TS. Analyzed the data: NY. Contributed reagents/materials/analysis tools: TK HS. Wrote the paper: NY D KH TS. 23. Egerod K, Reynisson E, Hauser F, Cazzamali G, Williamson M, et al. (2003) Molecular cloning and functional expression of the first two specific insect myosuppressin receptors. Proc Natl Acad Sci U S A 100: 9808–9813. 24. Cazzamali G, Hauser F, Kobberup S, Williamson M, Grimmelikhuijzen CJ (2003) Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide. Biochem Biophys Res Commun 303: 146–152. 25. Ha SD, Kataoka H, Suzuki A, Kim BJ, Kim HJ, et al. (2000) Cloning and sequence analysis of cDNA for diuretic hormone receptor from the Bombyx mori. Mol Cells 10: 13–17. 26. Secher T, Lenz C, Cazzamali G, Sorensen G, Williamson M, et al. (2001) Molecular cloning of a functional allatostatin gut/brain receptor and an allatostatin preprohormone from the silkworm Bombyx mori. J Biol Chem 276: 47052–47060. 27. Staubli F, Jorgensen TJ, Cazzamali G, Williamson M, Lenz C, et al. (2002) Molecular identification of the insect adipokinetic hormone receptors. Proc Natl Acad Sci U S A 99: 3446–3451. 28. Hull JJ, Ohnishi A, Moto K, Kawasaki Y, Kurata R, et al. (2004) Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor from the silkmoth, Bombyx mori. Significance of the carboxyl terminus in receptor internalization. J Biol Chem 279: 51500–51507. 29. Jorgensen LM, Hauser F, Cazzamali G, Williamson M, Grimmelikhuijzen CJ (2006) Molecular identification of the first SIFamide receptor. Biochem Biophys Res Commun 340: 696–701. 30. Homma T, Watanabe K, Tsurumaru S, Kataoka H, Imai K, et al. (2006) G protein-coupled receptor for diapause hormone, an inducer of Bombyx embryonic diapause. Biochem Biophys Res Commun 344: 386–393. 31. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305: 567–580. 32. Marchler-Bauer A, Anderson JB, Cherukuri PF, DeWeese-Scott C, Geer LY, et al. (2005) CDD: a Conserved Domain Database for protein classification. Nucleic Acids Res 33: D192–196. 33. Oda Y, Uejuma M, Iwami M, Sakurai S (2000) Involvement of adipokinetic hormone in the homeostatic control of hemolymph trehalose concentration in the larvae of Bombyx mori. Arch Insect Biochem Physiol 45: 156–165. 34. Audsley N, Kay I, Hayes TK, Coast GM (1995) Cross reactivity studies of CRF- related peptides on insect Malpighian tubules. Comp Biochem Physiol A Physiol 110: 87–93. 35. Kramer SJ, Toschi A, Miller CA, Kataoka H, Quistad GB, et al. (1991) Identification of an allatostatin from the tobacco hornworm Manduca sexta. Proc Natl Acad Sci U S A 88: 9458–9462. 36. Kreienkamp HJ, Larusson HJ, Witte I, Roeder T, Birgul N, et al. (2002) Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology. J Biol Chem 277: 39937–39943. 37. Nassel DR (2002) Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones. Prog Neurobiol 68: 1–84. 38. Liu F, Baggerman G, D’Hertog W, Verleyen P, Schoofs L, et al. (2006) In silico identification of new secretory peptide genes in Drosophila melanogaster. Mol Cell Proteomics 5: 510–522. 39. Zitnan D, Kim YJ, Zitnanova I, Roller L, Adams ME (2007) Complex steroid- peptide-receptor cascade controls insect ecdysis. Gen Comp Endocrinol 153: 88–96. 40. De Loof A, Baggerman G, Breuer M, Claeys I, Cerstiaens A, et al. (2001) Gonadotropins in insects: an overview. Arch Insect Biochem Physiol 47: 129–138. PLoS ONE | www.plosone.org 11 August 2008 | Volume 3 | Issue 8 | e3048