Cell and Tissue Research

, Volume 364, Issue 3, pp 499–512 | Cite as

Molecular cloning, expression and identification of the promoter regulatory region for the neuropeptide trissin in the nervous system of the silkmoth Bombyx mori

  • Ladislav Roller
  • Daniel Čižmár
  • Zuzana Gáliková
  • Branislav Bednár
  • Ivana Daubnerová
  • Dušan ŽitňanEmail author
Regular Article


Trissin has recently been identified as a conserved insect neuropeptide, but its cellular expression and function is unknown. We detected the presence of this neuropeptide in the silkworm Bombyx mori using in silico search and molecular cloning. In situ hybridisation was used to examine trissin expression in the entire central nervous system (CNS) and gut of larvae, pupae and adults. Surprisingly, its expression is restricted to only two pairs of small protocerebral interneurons and four to five large neurons in the frontal ganglion (FG). These neurons were further characterised by subsequent multiple staining with selected antibodies against insect neuropeptides. The brain interneurons innervate edges of the mushroom bodies and co-express trissin with myoinhibitory peptides (MIP) and CRF-like diuretic hormones (CRF-DH). In the FG, one pair of neurons co-express trissin with calcitonin-like diuretic hormone (CT-DH), short neuropeptide F (sNPF) and MIP. These neurons innervate the brain tritocerebrum and musculature of the anterior midgut. The other pair of trissin neurons in the FG co-express sNPF and project axons to the tritocerebrum and midgut. We also used the baculovirus expression system to identify the promoter regulatory region of the trissin gene for targeted expression of various molecular markers in these neurons. Dominant expression of trissin in the FG indicates its possible role in the regulation of foregut–midgut contractions and food intake.


Neuropeptide Neuron Gut In situ hybridisation Immunohistochemistry 



This study was supported by Slovak grant agencies, Agentúra na podporu výskumu a vývoja (APVV-0827-11) a Vedecká grantová agentúra (VEGA 2/0162/13; VEGA 2/0121/13). We are grateful to C.J.P. Grimelikhuijzen, A. Mizoguchi, Y. Tanaka and J.A. Veenstra for providing valuable antisera.


  1. Audsley N, Duve H, Thorpe A, Weaver RJ (2000) Morphological and physiological comparisons of two types of allatostatin in the brain and retrocerebral complex of the tomato moth, Lacanobia oleracea, (Lepidoptera: Noctuidae). J Comp Neurol 424:37–46CrossRefPubMedGoogle Scholar
  2. Ayali A (2004) The insect frontal ganglion and stomatogastric pattern generator networks. Neurosignals 13:20–36CrossRefPubMedGoogle Scholar
  3. Blackburn MB, Wagner RM, Kochansky JP, Harrison DJ, Thomas-Laemont P, Raina AK (1995) The identification of two myoinhibitory peptides, with sequence similarities to the galanins, isolated from the ventral nerve cord of Manduca sexta. Regul Pept 57:213–219CrossRefPubMedGoogle Scholar
  4. Cavanaugh DJ, Geratowski JD, Wooltorton JR, Spaethling JM, Hector CE, Zheng X, Johnson EC, Eberwine JH, Sehgal A (2014) Identification of a circadian output circuit for rest:activity rhythms in Drosophila. Cell 157(3):689–701CrossRefPubMedPubMedCentralGoogle Scholar
  5. Copenhaver PF, Truman JW (1986) Metamorphosis of the cerebral neuroendocrine system in the moth Manduca sexta. J Comp Neurol 249:186–204CrossRefPubMedGoogle Scholar
  6. Daubnerová I, Roller L, Žitňan D (2009) Transgenesis approaches for functional analysis of peptidergic cells in the silkworm Bombyx mori. Gen Comp Endocrinol 162:36–42CrossRefPubMedPubMedCentralGoogle Scholar
  7. Davis NT, Velleman SG, Kingan TG, Keshishian H (1989) Identification and distribution of a proctolin-like neuropeptide in the nervous system of the gypsy moth, Lymantria dispar, and in other Lepidoptera. J Comp Neurol 283(1):71–85CrossRefPubMedGoogle Scholar
  8. Davis NT, Veenstra JA, Feyereisen R, Hildebrand JG (1997) Allatostatin-like immunoreactive neurons of the Tobacco hornworm, Manduca sexta, and isolation and identification of a new peptide related to cockroach allatostatins. J Comp Neurol 385:265–284CrossRefPubMedGoogle Scholar
  9. Davis NT, Blackburn MB, Golubeva EG, Hildebrand JG (2003) Localization of myoinhibitory peptide immunoreactivity in Manduca sexta and Bombyx mori, with indications that the peptide has a role in molting and ecdysis. J Exp Biol 206:1449–1460CrossRefPubMedGoogle Scholar
  10. Dus M, Lai JS, Gunapala KM, Min S, Tayler TD, Hergarden AC, Geraud E, Joseph CM, Suh GS (2015) Nutrient sensor in the brain directs the action of the brain-gut axis in drosophila. Neuron 87:1–13CrossRefGoogle Scholar
  11. Duve H, Thorpe A (1994) Distribution and functional significance of Leu-callatostatins in the blowfly Calliphora vomitoria. Cell Tissue Res 276:367–379CrossRefPubMedGoogle Scholar
  12. Duve H, Thorpe A (2003) Neuropeptide co-localisation in the lepidopteran frontal ganglion studied by confocal laser scanning microscopy. Cell Tissue Res 311(1):79–89CrossRefPubMedGoogle Scholar
  13. Duve H, Johnsen AH, Maestro JL, Scott AG, Crook N, Winstanley D, Thorpe A (1997) Identification, tissue localisation and physiological effect in vitro of a neuroendocrine peptide identical to a dipteran Leu-callatostatin in the codling moth Cydia pomonella (Tortricidae: Lepidoptera). Cell Tissue Res 289:73–83CrossRefPubMedGoogle Scholar
  14. Duve H, East PD, Thorpe A (1999) Regulation of lepidopteran foregut movement by allatostatins and allatotropin from the frontal ganglion. J Comp Neurol 413(3):405–416CrossRefPubMedGoogle Scholar
  15. Duve H, Audsley N, Weaver RJ, Thorpe A (2000) Triple colocalisation of two types of allatostatin and an allatotropin in the frontal ganglion of the lepidopteran Lacanobia oleracea (Noctuidae): innervation and action on the foregut. Cell Tissue Res 300(1):153–163CrossRefPubMedGoogle Scholar
  16. Dyrløv JB, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795CrossRefGoogle Scholar
  17. Emery SB, Ma MC, Wong WKR, Tips A, De Loof A (1994) Immunocytochemical localization of a diuretic peptide Manduca diuresin (Mas DPII) in the brain and suboesophageal ganglion of the tobacco hawkmoth Manduca sexta (Lepidoptera: Sphingidae). Arch Insect Biochem Physiol 27:137–152CrossRefGoogle Scholar
  18. Gonzalez R, Orchard I (2009) Physiological activity of neuropeptide F on the hindgut of the blood-feeding hemipteran, Rhodnius prolixus. J Insect Sci 9:57CrossRefPubMedCentralGoogle Scholar
  19. Grimmelikhuijzen CJP, Spencer AN (1984) FMRFamide immunoreactivity in the nervous system of the medusa Polyorchis penicillatus. J Comp Neurol 230:361–371CrossRefPubMedGoogle Scholar
  20. Hewes RS, Taghert PH (2001) Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome. Genome Res 11:1126–1142CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hummon AB, Richmond TA, Verleyen P, Baggerman G, Huybrechts J, Ewing MA, Vierstraete E, Rodriguez-Zas SL, Schoofs L, Robinson GE, Sweedler JV (2006) From the genome to the proteome: uncovering peptides in the Apis brain. Science 314:647–649CrossRefPubMedGoogle Scholar
  22. Ida T, Takahashi T, Tominaga H, Sato T, Kume K, Yoshizawa-Kumagaye K, Nishio H, Kato J, Murakami N, Miyazato M, Kangawa K, Kojima M (2011) Identification of the endogenous cysteine-rich peptide trissin, a ligand for an orphan G protein-coupled receptor in Drosophila. Biochem Biophys Res Commun 414(1):44–48CrossRefPubMedGoogle Scholar
  23. Jiang H, Ikhagva A, Daubnerová I, Chae H, Šimo L, Jung S-H, Yoon Y-K, Seong J-Y, Žitňan D, Park Y, Kim Y-J (2013) Natalisin, a new tachykinin-like signaling system, regulates sexual activity and fecundity in insects. Proc Natl Acad Sci U S A 110(37):3526–3534CrossRefGoogle Scholar
  24. Kim YJ, Žitňan D, Cho KH, Schooley DA, Mizoguchi A, Adams ME (2006) Central peptidergic ensembles associated with organization of an innate behavior. Proc Natl Acad Sci U S A 103:14211–14216CrossRefPubMedPubMedCentralGoogle Scholar
  25. Lee K-M, Daubnerová I, Isaac RE, Zhang C, Choi S, Chung J, Kim Y-J (2015) A neuronal pathway that controls sperm ejection and storage in female Drosophila. Curr Biol 25:790–797CrossRefPubMedGoogle Scholar
  26. Li B, Predel R, Neupert S, Hauser F, Tanaka Y, Cazzamali G, Williamson M, Arakane Y, Verleyen P, Schoofs L, Schachtner J, Grimmelikhuijzen CJ, Park Y (2008) Genomics, transcriptomics, and peptidomics of neuropeptides and protein hormones in the red flour beetle Tribolium castaneum. Genome Res 18:113–122CrossRefPubMedPubMedCentralGoogle Scholar
  27. Li C, Song X, Chen X, Liu X, Sang M, Wu W, Yun X, Hu X, Li B (2014) Identification and comparative analysis of G protein-coupled receptors in Pediculus humanus humanus. Genomics 104(1):58–67CrossRefPubMedGoogle Scholar
  28. Matsumoto S, Brown MR, Crim JW, Vigna SR, Lea AO (1989) Isolation and primary structure of neuropeptides from the mosquito, Aedes aegypti, immunoreactive to FMRFamide antiserum. Insect Biochem Mol Biol 19:277–283Google Scholar
  29. Miles CI, Booker R (1994) The role of the frontal ganglion in foregut movements of the moth Manduca sexta. J Comp Physiol 174:755–767CrossRefGoogle Scholar
  30. Mita K, Morimyo M, Okano K, Koike Y, Nohata J, Kawasaki H, Kadono-Okuda K, Yamamoto K, Suzuki MG, Shimada T, Goldsmith MR, Maeda S (2003) The construction of an EST database for Bombyx mori and its application. Proc Natl Acad Sci U S A 100:14121–14126CrossRefPubMedPubMedCentralGoogle Scholar
  31. Nässel DR, Homberg U (2006) Neuropeptides in interneurons of the insect brain. Cell Tissue Res 326:1–24CrossRefPubMedGoogle Scholar
  32. Nässel DR, Winther AM (2010) Drosophila neuropeptides in regulation of physiology and behavior. Prog Neurobiol 92(1):42–104CrossRefPubMedGoogle Scholar
  33. Oh Y, Yoon SE, Zhang Q, Chae HS, Daubnerová I, Shafer OT, Choe J, Kim YJ (2014) A homeostatic sleep-stabilizing pathway in drosophila composed of the sex peptide receptor and its ligand, the myoinhibitory peptide. PLoS Biol 12(10):e1001974CrossRefPubMedPubMedCentralGoogle Scholar
  34. Park D, Veenstra JA, Park JH, Taghert PH (2008) Mapping peptidergic cells in Drosophila: where DIMM fits in. PLoS ONE 3:e1896CrossRefPubMedPubMedCentralGoogle Scholar
  35. Roller L, Yamanaka N, Watanabe K, Daubnerova I, Zitnan D, Kataoka H, Tanaka Y (2008) The unique evolution of neuropeptide genes in the silkworm Bombyx mori. Insect Biochem Mol Biol 38:1147–1157CrossRefPubMedGoogle Scholar
  36. Sadd BM, Barribeau SM, Bloch G et al. (2015) The genomes of two key bumblebee species with primitive eusocial organization. Genome Biol 16(1): Article 76Google Scholar
  37. Schoofs L, Clynen E, Cerstiaens A, Baggerman G, Wei Z, Vercammen T, Nachman R, De Loof A, Tanaka S (2001) Newly discovered functions for some myotropic neuropeptides in locusts. Peptides 22:219–227CrossRefPubMedGoogle Scholar
  38. Schooley DA, Horodyski FM, Coast GM et al. (2012) Hormones controlling homeostasis in insects. In: Gilbert, LI (ed) Insect Endocrinology. Elsevier, Oxford, pp 366–429Google Scholar
  39. Shiomi K, Kajiura Z, Nakagaki M, Yamashita O (2003) Baculovirus-mediated efficient gene transfer into the central nervous system of the silkworm, Bombyx mori. J Insect Biotechnol Sericol 72:149–155Google Scholar
  40. Stay B, Chan KK, Woodhead AP (1992) Allatostatin-immunoreactive neurons projecting to the corpora allata of adult Diploptera punctata. Cell Tissue Res 270:15–23CrossRefPubMedGoogle Scholar
  41. Tanaka Y, Suetsugu Y, Yamamoto K, Noda H, Shinoda T (2013) Transcriptome analysis of neuropeptides and G-protein coupled receptors (GPCRs) for neuropeptides in the brown planthopper Nilaparvata lugens. Peptides 53:125–133CrossRefPubMedGoogle Scholar
  42. Te Brugge VA, Miksys SM, Coast GM, Schooley DA, Orchard I (1999) The distribution of a CRF-like diuretic peptide in the blood-feeding bug Rhodnius prolixus. J Exp Biol 202:2017–2027Google Scholar
  43. Te Brugge VA, Schooley DA, Orchard I (2008) Amino acid sequence and biological activity of a calcitonin-like diuretic hormone (DH31) from Rhodnius prolixus. J Exp Biol 211:382–390CrossRefPubMedGoogle Scholar
  44. Veenstra JA (2000) Mono- and dibasic proteolytic cleavage sites in insect neuroendocrine peptide precursors. Arch Insect Biochem Physiol 43:49–63CrossRefPubMedGoogle Scholar
  45. Veenstra JA (2014) The contribution of the genomes of a termite and a locust to our understanding of insect neuropeptides and neurohormones. Front Physiol 5: Article 454Google Scholar
  46. Veenstra JA (2015) The power of next-generation sequencing as illustrated by the neuropeptidome of the crayfish Procambarus clarkii. Gen Comp Endocrinol. doi: 10.1016/j.ygcen.2015.06.013 PubMedGoogle Scholar
  47. Veenstra JA, Rombauts S, Grbić M (2012) In silico cloning of genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors in a spider mite. Insect Biochem Mol Biol 42(4):277–295CrossRefPubMedGoogle Scholar
  48. Yamanaka N, Yamamoto S, Žitňan D, Watanabe K, Kawada T, Satake H, Kaneko Y, Hiruma K, Tanaka Y, Shinoda T, Kataoka H (2008) Neuropeptide receptor transcriptome reveals unidentified neuroendocrine pathways. PLoS ONE 3:e3048CrossRefPubMedPubMedCentralGoogle Scholar
  49. Yamanaka N, Hua Y-J, Roller L, Spalovská-Valachová I, Mizoguchi A, Kataoka H, Tanaka Y (2010) Bombyx prothoracicostatic peptides activate the sex peptide receptor to regulate ecdysteroid biosynthesis. Proc Natl Acad Sci U S A 107(5):2060–2065CrossRefPubMedPubMedCentralGoogle Scholar
  50. Yamanaka N, Roller L, Žitňan D, Satake H, Mizoguchi A, Kataoka H, Tanaka Y (2011) Bombyx orcokinins are brain-gut peptides involved in the neuronal regulation of ecdysteroidogenesis. J Compara Neurol 519:238–246CrossRefGoogle Scholar
  51. Žitňan D, Endo Y, Sehnal F (1989) Stomatogastric nervous system of Galleria mellonella L. (Lepidoptera: Pyralidae): changes during metamorphosis with special reference to FMRFamide neurons. Int J Insect Morphol Embryol 18(4):227–237CrossRefGoogle Scholar
  52. Žitňan D, Šauman I, Sehnal F (1993) Peptidergic innervation and endocrine cells of insect midgut. Arch Insect Biochem Physiol 22(1–2):113–132Google Scholar
  53. Žitňan D, Kingan TG, Kramer SJ, Beckage NE (1995) Accumulation of neuropeptides in the cerebral neurosecretory system of Manduca sexta larvae parasitised by the braconid wasp Cotesia congregata. J Comp Neurol 356(1):83–100CrossRefPubMedGoogle Scholar
  54. Žitňan D, Hollar L, Spalovská I, Takáč P, Žitňanová I, Gill SS, Adams ME (2002) Molecular cloning and function of ecdysis-triggering hormones in the silkworm Bombyx mori. J Exp Biol 205:3459–3473PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Ladislav Roller
    • 1
  • Daniel Čižmár
    • 1
  • Zuzana Gáliková
    • 1
  • Branislav Bednár
    • 1
  • Ivana Daubnerová
    • 1
  • Dušan Žitňan
    • 1
    Email author
  1. 1.Institute of ZoologySlovak Academy of SciencesBratislavaSlovakia

Personalised recommendations