Eur. J. Entomol. 106 (3): 335-345, 2009 | DOI: 10.14411/eje.2009.041

Apoptosis of neuronal cells in the brains of postembryonic silkworms Bombyx mori (Lepidoptera: Bombycidae)

Mi Young KIM*,1, Kang Kim MIN*,1, Chang Ok CHOI1, Hwa Young SONG1, Chang Lee SEOK1, Bong Woo KIM1, Pil Don KANG2, Soung Hoo JEON3, Byung Pil CHO4, Yeon Jae BAE1, Young-Gyo KO1, Bong Lee HEE1,3,**
1 School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea
2 Department of Agricultural Biology, National Institute of Agricultural Science and Technology, Suwon 441-100, Korea
3 Korea Entomological Institute, Korea University, Seoul 136-701, Korea
4 Department of Anatomy, Yonsei University Wonju College of Medicine, Wonju 220-701, Korea

The pattern and signal transduction of neuronal apoptosis in the brain of the silk moth, Bombyx mori, during postembryonic life, were characterized. Peak numbers of apoptotic neurons were detected in 4 day old 4th instar larvae, 9 day old 5th instar larvae and 4 day old pupae, indicating three waves of neuronal apoptosis during postembryonic development. Most of the apoptotic neurons were in the lateral portions of the brain. No apoptotic neurons were detected in 1 day old 1st instar larvae or in 7 day old pupae to 1 day old adults. Injection of 20-hydroxyecdysone (20E) into larvae resulted in a substantial increase in the brain in both neuronal apoptosis and cleavage of procaspases-8 and -3 into caspases-8 and -3. However, the injection of larvae with actinomycin D or cycloheximide inhibited death of pre-apoptotic neurons. Both the cleavage of procaspases-8 and -3 and death of pre-apoptotic neurons were inhibited by a general caspase inhibitor and caspase-8 and -3 inhibitors injected into larvae. These results suggest that 20E triggered the synthesis of a new protein that, in turn, induces cleavage of procaspases-8 and -3 into caspases-8 and -3. These caspases are prerequisites for neuronal apoptosis in postembryonic brains.

Keywords: Silk moth, postembryonic life, brain, neuronal apoptosis, ecdysone, actinomycin, caspase

Received: November 27, 2008; Accepted: March 3, 2009; Published: July 24, 2009

Download citation

References

  1. BAEHRECKE E.H. 2000: Steroid regulation of programmed cell death during Drosophila development. Cell Death Differ. 7: 1057-1062 Go to original source...
  2. BINDER L.I., FRANKFURTER A., KIM H., CACERES A., PAYNE M.R. & REBHUN L.I. 1984: Heterogeneity of microtubule-associated protein 2 during rat brain development. Proc. Natl. Acad. Sci. U.S.A. 81: 5613-5617 Go to original source...
  3. BUREK M.J. & OPPENHEIM R.W. 1996: Programmed cell death in the developing nervous system. Brain Pathol. 6: 427-446 Go to original source...
  4. CACERES A., BINDER L.I., PAYNE M.R., BENDER P., REBHUN L. & STEWARD O. 1984: Differential subcellular localization of tubulin and the microtubule-associated protein MAP2 in brain tissue as revealed by immunocytochemistry with monoclonal hybridoma antibodies. J. Neurosci. 4: 394-410
  5. CHINZEI Y. 1975: Biochemical evidence of DNA transport from the silk gland to the fat body of the silkworm, Bombyx mori. J. Insect Physiol. 21: 163-171 Go to original source...
  6. DAI J.D. & GILBERT L.I. 1997: Programmed cell death of the prothoracic glands of Manduca sexta during pupal-adult metamorphosis. Insect Biochem. Mol. Biol. 27: 69-78 Go to original source...
  7. DAI J.L., MIZOGUCHI A., SATAKE S., ISHIZAKI H. & GILBERT L.I. 1995: 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. 171: 212-223 Go to original source...
  8. DORSTYN L., COLUSSI P.A., QUINN L.M., RICHARDSON H. & KUMAR S. 1999: DRONC, an ecdysone-inducible Drosophila caspase. Proc. Natl. Acad. Sci. U.S.A . 96: 4307-4312 Go to original source...
  9. DRAIZEN T.A., EWER J. & ROBINOW S. 1999: Genetic and hormonal regulation of the death of peptidergic neurons in the Drosophila central nervous system. J. Neurosci. 38: 455-465
  10. ENARI M., SAKAHIRA H., YOKOYAMA H., OKAWA K., IWAMATSU A. & NAGATA S. 1998: A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391: 43-50 Go to original source...
  11. FERRI K.F. & KROEMER G. 2001: Organelle-specific initiation of cell death pathways. Nat. Cell Biol. 3: E255-263 Go to original source...
  12. FRASER A.G. & EVAN G.I. 1997: Identification of a Drosophila melanogaster ICE/CED-3-related protease, drICE. EMBO J. 16: 2805-2813 Go to original source...
  13. FUJIWARA H. & OGAI S. 2001: Ecdysteroid-induced programmed cell death and cell proliferation during pupal wing development of the silkworm, Bombyx mori. Dev. Genes Evol. 211: 118-123 Go to original source...
  14. GANESHINA O., SCHAFER S. & MALUN D. 2000: Proliferation and programmed cell death of neuronal precursors in the mushroom bodies of the honeybee. J. Comp. Neurol. 417: 349-365 Go to original source...
  15. GAVRIELI Y., SHERMAN Y. & BEN-SASSON S.A. 1992: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119: 493-501 Go to original source...
  16. GUO M. & HAY B.A. 1999: Cell proliferation and apoptosis. Curr. Opin. Cell Biol. 11: 745-752 Go to original source...
  17. HENKART P.A. 1996: ICE family proteases: mediators of all apoptotic cell death? Immunity 4: 195-201 Go to original source...
  18. HOFFMAN K.L. & WEEKS J.C. 1998: Programmed cell death of an identified motoneuron in vitro: temporal requirements for steroid exposure and protein synthesis. J. Neurobiol. 35: 300-322 Go to original source...
  19. HOFFMAN K.L. & WEEKS J.C. 2001: Role of caspases and mitochondria in the steroid-induced programmed cell death of a motoneuron during metamorphosis. Dev. Biol. 229: 517-536. Go to original source...
  20. IZANT J.G. & MCINTOSH J.R. 1980: Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons. Proc. Natl. Acad. Sci. U. S. A. 77: 4741-4745 Go to original source...
  21. JACOBSON M.D. WEIL M. & RAFF M.C. 1997: Programmed cell death in animal development. Cell 88: 347-354 Go to original source...
  22. JIANG C., BAEHRECKE E.H. & THUMMEL C.S. 1997: Steroid regulated programmed cell death during Drosophila metamorphosis. Development 124: 4673-4683
  23. KIM J.H., PARK C.W., SONG H.Y., KIM M.Y., CHOI C.O. & LEE B.H. 2004: Effects of brain-derived neurotrophic factor on neurite growth of deutocerebral neurons in brain of the silk moth Bombyx mori. Entomol. Res. 34: 113-122 Go to original source...
  24. KINCH G., HOFFMAN K.L., RODRIGUES E.M., ZEE M.C. & WEEKS J.C. 2003: Steroid-triggered programmed cell death of a motoneuron is autophagic and involves structural changes in mitochondria. J. Comp. Neurol. 457: 384-403 Go to original source...
  25. KRAFT R., LEVINE R.B. & RESTIFO L.L. 1998: The steroid hormone 20-hydroxyecdysone enhances neurite growth of Drosophila mushroom body neurons isolated during metamorphosis. J. Neurosci. 18: 8886-8899
  26. LEE C.Y., COOKSEY B.A. & BAEHRECKE E.H. 2002: Steroid regulation of midgut cell death during Drosophila development. Dev. Biol. 250: 101-111 Go to original source...
  27. LEVINE R.B. & TRUMAN J.W. 1985: Dendritic reorganization of abdominal motoneurons during metamorphosis of the moth, Manduca sexta. J. Neurosci. 5: 2424-2431
  28. LOCKSHIN R.A. & WILLIAMS C.M. 1965: Programmed cell death. V. Cytolytic enzymes in relation to the breakdown of the intersegmental muscles of silkmoths. J. Insect Physiol. 11: 831-844 Go to original source...
  29. MANCINI M., NICHOLSON D.W., ROY S., THORNBERRY N.A., PETERSON E.P., CASCIOLA-ROSEN L.A. & ROSEN A. 1998: The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling. J. Cell Biol. 140: 1485-1495 Go to original source...
  30. MARTIN D.N. & BAEHRECKE E.H. 2004: Caspases function in autophagic programmed cell death in Drosophila. Development 131: 275-284 Go to original source...
  31. MEHMET H. 2000: Caspases find a new place to hide. Nature 403: 29-30 Go to original source...
  32. MILLER D.T. & CAGAN R.L. 1998: Local induction of patterning and programmed cell death in the developing Drosophila retina. Development 125: 2327-2335
  33. MIZOGUCHI A., OHASHI Y., HOSODA K., ISHIBASHI J. & KATAOKA H. 2001: 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. 31: 349-358 Go to original source...
  34. NARKILAHTI S., PIRTTILA T.J., LUKASIUK K., TUUNANEN J. & PITKANEN A. 2003: Expression and activation of caspase 3 following status epilepticus in the rat. Eur. J. Neurosci. 18: 1486-1496 Go to original source...
  35. OBERHAMMER F., WILSON J.W., DIVE C., MORRIS I.D., HICKMAN J.A., WAKELING A.E., WALKER P.R. & SIKORSKA M. 1993: Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12: 3679-3684
  36. PARK C.W., KIM J.H., KIM K.M., HWANG J.S., KANG S.W., KANG H.S., CHO B.P., YU C.H., KIM H.R. & LEE B.H. 2004: Evidence for brain-derived neurotrophic factor-like neuropeptide in brain of the silk moth Bombyx mori during postembryonic periods. Peptides 25: 1891-1897 Go to original source...
  37. PRUGH J., CROCE K.D. & LEVINE R.B. 1992: Effects of the steroid hormone, 20-hydroxyecdysone, on the growth of neurites by identified insect motoneurons in vitro. Dev. Biol. 154: 331-347 Go to original source...
  38. RAY S.K., SCHAECHER K.E., SHIELDS D.C., HOGAN E.L. & BANIK N.L. 2000: Combined TUNEL and double immunofluorescent labeling for detection of apoptotic mononuclear phagocytes in autoimmune demyelinating disease. Brain Res. Protoc. 5: 305-311 Go to original source...
  39. ROBINOW S., DRAIZEN T.A. & TRUMAN J.W. 1997: Genes that induce apoptosis: transcriptional regulation in identified, doomed neurons of the Drosophila CNS. Dev. Biol. 190: 206-213 Go to original source...
  40. SANDSTROM D.J. & WEEKS J.C. 1996: Novel dual innervation of a larval proleg muscle by two similar motoneurons in the tobacco hornworm, Manduca sexta. J. Exp. Biol. 199: 775-791
  41. SCHMECHEL D., MARANGOS P.J. & BRIGHTMAN M. 1978: Neuronespecific enolase is a molecular marker for peripheral and central neuroendocrine cells. Nature 276: 834-836 Go to original source...
  42. SCHUBIGER M., WADE A.A., CARNEY G.E., TRUMAN J.W. & BENDER M. 1998: Drosophila EcR-B ecdysone receptor isoforms are required for larval molting and for neuron remodeling during metamorphosis. Development 125: 2053-2062
  43. SCHWARTZ L.M. & TRUMAN J.W. 1982: Peptide and steroid regulation of muscle degeneration in an insect. Science 215: 1420-1421 Go to original source...
  44. STREICHERT L.C. & WEEKS J.C. 1995: Decreased monosynaptic sensory input to an identified motoneuron is associated with steroid-mediated dendritic regression during metamorphosis in Manduca sexta. J. Neurosci. 15: 1484-1495
  45. STREICHERT L.C., PIERCE J.T., NELSON J.A. & WEEKS J.C. 1997: Segment-specific programmed cell death of identified motoneurons triggered directly by steroid hormones in vitro. Dev. Biol. 183: 95-107 Go to original source...
  46. TERASHIMA J., YASUHARA N., IWAMI M., SAKURAI S. & SAKURAI S. 2000: Programmed cell death triggered by insect steroid hormone, 20-hydroxyecdysone, in the anterior silk gland of the silkworm, Bombyx mori. Dev. Genes Evol. 210: 545-558 Go to original source...
  47. TRUMAN J.W. 1983: Programmed cell death in the nervous system of an adult insect. J. Comp. Neurol. 216: 445-452 Go to original source...
  48. TRUMAN J.W. & SCHWARTZ L.M. 1984: Steroid regulation of neuronal death in the moth nervous system. J. Neurosci. 4: 274-280
  49. TRUMAN J.W., THORN R.S. & ROBINOW S. 1992: Programmed neuronal death in insect development. J. Neurobiol. 23: 1295-1311 Go to original source...
  50. TRUMAN J.W., TAYLOR B.J. & AWAD T. 1993: Formation of the adult nervous system. In Bate M. & Martinez-Arias A. (eds): The Development of Drosophila melenogaster. Cold Spring Harbor Laboratory Press, New York, pp. 1245-1276
  51. TRUMAN J.W., TALBOT W.S., FAHRBACH S.E. & HOGNESS D.S. 1994: Ecdysone receptor expression in the CNS correlates with stage-specific responses to ecdysteroids during Drosophila and Manduca development. Development 120: 219-234
  52. TSUZUKI S., IWAMI M. & SAKURAI S. 2001: Ecdysteroid-inducible genes in the programmed cell death during insect metamorphosis. Insect Biochem. Mol. Biol. 31: 321-331 Go to original source...
  53. VULLINGS H.G., LEGERSTEE-OOSTVEEN G.G., DIEDEREN J.H. & JANSEN W.F. 1989: Immunocytochemical demonstration of neuron-specific enolase in neuronal cell bodies of an insect. Neurosci. Lett. 104: 48-52 Go to original source...
  54. WANG X. 2001: The expanding role of mitochondria in apoptosis. Genes Dev. 15: 2922-2933
  55. WEEKS J.C. & DAVIDSON S.K. 1994: Influence of interganglionic interactions on steroid-mediated dendritic reorganization and death of proleg motor neurons during metamorphosis in Manduca sexta. J. Neurobiol. 25: 535-554. Go to original source...
  56. WEIL M., JACOBSON M.D., COLES H.S., DAVIES T.J., GARDNER R.L., RAFF K.D. & RAFF M.C. 1996: Constitutive expression of the machinery for programmed cell death. J. Cell Biol. 133: 1053-1059 Go to original source...
  57. YAKOVLEV A.G., OTA K., WANG G., MOVSESYAN V., BAO W.L., YOSHIHARA K. & FADEN A.I. 2001: Differential expression of apoptotic protease-activating factor-1 and caspase-3 genes and susceptibility to apoptosis during brain development and after traumatic brain injury . J. Neurosci. 21: 7439-7446
  58. YOUNG C., KLOCKE B.J., TENKOVA T., CHOI J., LABRUYERE J., QIN Y.Q., HOLTZMAN D.M., ROTH K.A. & OLNEY J.W. 2003: Ethanol-induced neuronal apoptosis in vivo requires BAX in the developing mouse brain. Cell Death Differ. 10: 1148-1155 Go to original source...
  59. YU S.Y., YOO S.J., YANG L., ZAPATA C., SRINIVASAN A., HAY B.A. & BAKER N.E. 2002: A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye. Development 129: 3269-3278
  60. ZEE M.C. & WEEKS J.C. 2001: Developmental change in the steroid hormone signal for cell-autonomous, segment-specific programmed cell death of a motoneuron. Dev. Biol. 235: 45-61 Go to original source...