, Volume 20, Issue 1, pp 1–9 | Cite as

Pro-apoptotic gene regulation and its activation by gamma-irradiation in the Caribbean fruit fly, Anastrepha suspensa

  • Xavier Nirmala
  • Marc F. Schetelig
  • Grazyna J. Zimowska
  • Lei Zhou
  • Alfred M. Handler
Original Paper


Transcriptional activation of pro-apoptotic genes in response to cytotoxic stimuli is a conserved feature of the cell death pathway in metazoans. However, understanding the extent of this conservation in insects has been limited by the lack of known pro-apoptotic genes in non-drosophilids. Recently, we described the pro-apoptotic genes, Asrpr and Ashid, from the tephritid, Anastrepha suspensa, that now allow us to explore the conservation of pro-apoptotic gene regulation between a tephritid and drosophilids. In this study, we determined the developmental profiles of Asrpr and Ashid transcripts during embryogenesis and in embryos exposed to γ-irradiation. Transcript levels of both genes determined by qRT-PCR were low throughout embryogenesis, with strong Ashid expression occurring during early to mid-embryogenesis and Asrpr expression peaking in late embryogenesis. This correlated to acridine orange stained apoptotic cells first appearing at 17 h and increasing over time. However, when irradiated at 16 h post-oviposition embryos exhibited significant levels of apoptosis consistent with strong induction of Asrpr and Ashid transcript levels by γ-irradiation in young embryos <24 h post-oviposition. Furthermore, embryos irradiated <24 h post-oviposition failed to hatch, those irradiated between 24 and 32 h had increased hatching rates, but between 48 and 72 h irradiation had no effect on egg hatching. This indicates a transition of embryos from an irradiation-sensitive to irradiation-resistance stage between 24 and 48 h. Throughout post-embryonic development, the two pro-apoptotic genes share similar patterns of up-regulated gene expression, which correlate to ecdysone-induced developmental events, especially during metamorphosis. Together these results provide the first direct evidence for a conserved molecular mechanism of the programmed cell death pathway in insects.


Ashid Asrpr Irradiation Ecdysteroids Cell death Tephritids 



We thank Dr. Chris Geden for assistance with irradiation protocols. Funding from the USDA-NIFA-Agriculture and Food Research Initiative and the Biotechnology Risk Assessment Program (Grant #2011-39211-30769) (AMH), Emmy Noether program SCHE 1833/1 of the German Research Foundation (MFS), and the LOEWE Center for Insect Biotechnology & Bioresources (MFS) are gratefully acknowledged.

Conflict of interest

All authors declare no conflict of interest.


Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.


  1. 1.
    Chinnaiyan AM (1999) The apoptosome: heart and soul of the cell death machine. Neoplasia 1:5–15PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    White K, Grether ME, Abrams JM, Young L, Farrell K, Steller H (1994) Genetic control of programmed cell death in Drosophila. Science 264:677–683PubMedCrossRefGoogle Scholar
  3. 3.
    Zhou L, Jiang G, Chan G, Santos CP, Severson DW, Xiao L (2005) Michelob_x is the missing inhibitor of apoptosis protein antagonist in mosquito genomes. EMBO Rep 6:769–774PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Zhang Y, Lin N, Carroll PM, Chan G, Guan B, Xiao H et al (2008) Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos. Dev Cell 14:481–493PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Metcalf R (1995) The need for research on exotic pests in California. In: Morse JG, Metcalf L, Carey JR, Dowell RV (eds). The mediterranean fruit fly in California: defining critical research. University of California, Riverside, pp 5–40Google Scholar
  6. 6.
    Schetelig MF, Nirmala X, Handler AM (2011) Pro-apoptotic cell death genes, hid and reaper, from the tephritid pest species, Anastrepha suspensa. Apoptosis 16:759–768PubMedCrossRefGoogle Scholar
  7. 7.
    Beverley SM, Wilson AC (1984) Molecular evolution in drosophila and the higher diptera, II: a time scale for dipteran evolution. J Mol Evol 21:1–13PubMedCrossRefGoogle Scholar
  8. 8.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  9. 9.
    Abrams JM, White K, Fessler LI, Steller H (1993) Programmed cell death during Drosophila embryogenesis. Development 117:29–43PubMedGoogle Scholar
  10. 10.
    Campos-Ortega JA, Hartenstein V (1985) The embryonic development of Drosophila melanogaster. Springer-Verlag, Berlin/Heidelberg/New-York/Tokyo. p 227Google Scholar
  11. 11.
    Goyal L, McCall K, Agapite J, Hartwieg E, Steller H (2000) Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function. EMBO J 19:589–597PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Zhou L, Steller H (2003) Distinct pathways mediate UV-induced apoptosis in Drosophila embryos. Dev Cell 4:599–605PubMedCrossRefGoogle Scholar
  13. 13.
    Jiang C, Lamblin AF, Steller H, Thummel CS (2000) A steroid-triggered transcriptional hierarchy controls salivary gland cell death during Drosophila metamorphosis. Mol Cell 5:445–455PubMedCrossRefGoogle Scholar
  14. 14.
    Bryant B, Zhang Y, Zhang C, Santos CP, Clem RJ, Zhou L (2009) A lepidopteran orthologue of reaper reveals functional conservation and evolution of IAP antagonists. Insect Mol Biol 18:341–351PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Urness LD, Thummel CS (1990) Molecular interactions within the ecdysone regulatory hierarchy: DNA binding properties of the Drosophila ecdysone-inducible E74A protein. Cell 63:47–61PubMedCrossRefGoogle Scholar
  16. 16.
    Denissova NG, Tereshchenko IV, Cui E, Stambrook PJ, Shao C, Tischfield JA (2011) Ionizing radiation is a potent inducer of mitotic recombination in mouse embryonic stem cells. Mutat Res 715:1–6PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Monje ML, Mizumatsu S, Fike JR, Palmer TD (2002) Irradiation induces neural precursor-cell dysfunction. Nat Med 8:955–962PubMedCrossRefGoogle Scholar
  18. 18.
    Beltrán-Pardo E, Jönsson KI, Wojcik A, Haghdoost S, Harms-Ringdahl M, Bermúdez-Cruz RM et al (2013) Effects of ionizing radiation on embryos of the Tardigrade Milnesium cf. tardigradum at different stages of development. PLoS ONE 8:e72098PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Nascimento JC, de Oliveira AK (1996) Embryogenesis in Anastrepha fraterculus (Diptera: Tephritidae). Interciencia 21:158–165Google Scholar
  20. 20.
    Brodsky MH, Weinert BT, Tsang G, Rong YS, McGinnis NM, Golic KG et al (2004) Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage. Mol Cell Biol 24:1219–1231PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Lee JH, Lee E, Park J, Kim E, Kim J, Chung J (2003) In vivo p53 function is indispensable for DNA damage-induced apoptotic signaling in Drosophila. FEBS Lett 550:5–10PubMedCrossRefGoogle Scholar
  22. 22.
    Jiang C, Baehrecke EH, Thummel CS (1997) Steroid regulated programmed cell death during Drosophila metamorphosis. Development 124:4673–4683PubMedGoogle Scholar
  23. 23.
    Lawrence PO, Hagedorn HH, Wheelock G (1984) Ecdysteroid levels and integument changes in post-embryonic stages of Anastrepha suspensa. J Insect Physiol 30:713–719CrossRefGoogle Scholar
  24. 24.
    Sato K, Hayashi Y, Ninomiya Y, Shigenobu S, Arita K, Mukai M et al (2007) Maternal Nanos represses hid/skl-dependent apoptosis to maintain the germ line in Drosophila embryos. Proc Natl Acad Sci 104:7455–7460PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Brennecke J, Hipfner DR, Stark A, Russell RB, Cohen SM (2003) Bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113:25–36PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York (outside the USA) 2014

Authors and Affiliations

  • Xavier Nirmala
    • 1
    • 2
  • Marc F. Schetelig
    • 1
    • 3
  • Grazyna J. Zimowska
    • 1
  • Lei Zhou
    • 4
  • Alfred M. Handler
    • 1
  1. 1.United States Department of AgricultureAgricultural Research Service, Center for Medical, Agricultural and Veterinary EntomologyGainesvilleUSA
  2. 2.Department of Entomology and NematologyUniversity of FloridaGainesvilleUSA
  3. 3.Justus-Liebig-University GiessenGiessenGermany
  4. 4.Department of Molecular Genetics and Microbiology & UF Shands Cancer Center, College of MedicineUniversity of FloridaGainesvilleUSA

Personalised recommendations