International Journal of Legal Medicine

, Volume 127, Issue 1, pp 243–249 | Cite as

Differential gene expression during metamorphosis: a promising approach for age estimation of forensically important Calliphora vicina pupae (Diptera: Calliphoridae)

  • Petra BoehmeEmail author
  • Philipp Spahn
  • Jens Amendt
  • Richard Zehner
Original Article


Necrophagous blow fly larvae can provide accurate estimates of the minimum postmortem interval in death investigations. During larval development, predictable morphological changes occur and measurements of weight, length, and width are compared to species-specific growth curves for reliable age estimates. However, aging blow fly pupae is more challenging because morphological and anatomical changes are not visible with the naked eye. Thus, delicate preparation of the pupae or rearing to the adult stage seems unavoidable. Conversely, metamorphosis evokes a remodelling of the larval shape to adult structures, and gene expression analysis potentially serves as a molecular tool to mirror the ageing process of a pupa. The present study focuses on the differential expression of two newly described, arbitrarily named genes (15_2, 2014192) and two previously identified genes (actin, arylphorin receptor) during Calliphora vicina (Diptera: Calliphoridae) metamorphosis. Quantification through real-time PCR revealed significant up- and downregulation of these transcripts found to be temperature dependent and age specific, hence, a new possibility to age forensically important blow fly pupae.


Forensic entomology Postmortem interval Calliphora vicina Differentially expressed genes Metamorphosis 



This project was financially supported by the Deutsche Forschungsgemeinschaft (project number: ZE 501/2-1).

Supplementary material

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ESM 1 (DOCX 12 kb)
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  1. 1.
    Smith KGV (1986) A manual of forensic entomology. Cornell University Press, LondonGoogle Scholar
  2. 2.
    Villet MH, Amendt J (2011) Advances in entomological methods for death time estimation. In: Turk EE (ed) Forensic Pathology Reviews doi: 10.1007/978-1-61779-249-6_11.
  3. 3.
    Amendt J, Richards CS, Campobasso CP, Zehner R, Hall MJR (2011) Forensic entomology: applications and limitations. Forensic Sci Med Pathol doi: 10.1007/s12024-010-9209-2
  4. 4.
    Forbes SI, Dadour I (2010) The soil environment and forensic entomology. In: Byrd JH and Castner JL (eds) Forensic entomology—The utility of arthropods in legal investigations, 2nd edn. CRC, Boca Raton, p 416Google Scholar
  5. 5.
    Richards CS, Crous KL, Villet M (2009) Models of development for blowfly sister species Chrysomya chloropyga and Chrysomya putoria. Med Vet Entomol 23:56–61PubMedCrossRefGoogle Scholar
  6. 6.
    Grassberger M, Reiter C (2001) Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen- and isomorphen-diagram. Forensic Sci Int 120:32–36PubMedCrossRefGoogle Scholar
  7. 7.
    Wells JD, LaMotte LR (1995) Estimating maggot age from weight using inverse prediction. J Forensic Sci 40:585–590Google Scholar
  8. 8.
    Greenberg B (1991) Flies as forensic indicators. J Med Entomol 28:565–577PubMedGoogle Scholar
  9. 9.
    Reiter C (1984) Zum Wachstumsverhalten der Maden der blauen Schmeißfliege Calliphora vicina. Zeitschrift für Rechtsmedizin, 91:295–308CrossRefGoogle Scholar
  10. 10.
    Marchenko MI (2001) Medicolegal relevance of cadaver entomofauna for the determination of the time of death. Forensic Sci Int 120:89–109PubMedCrossRefGoogle Scholar
  11. 11.
    Zajac BK (2011) Morphologische und histologische Methoden zur Bestimmung des Alters forensisch relevanter Fliegenpuppen. Diplomarbeit. Goethe-Universität Frankfurt am MainGoogle Scholar
  12. 12.
    Bainbridge SP, Bownes M (1981) Staging the metamorphosis of Drosophila melangaster. J Embryol exp Morph 66:57–80PubMedGoogle Scholar
  13. 13.
    Robertson CW (1936) The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological changes. J Morphol 59:351–399.CrossRefGoogle Scholar
  14. 14.
    Beckstead RB, Lam G, Thummel CS (2005) The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis. Genome Biol 6:R99PubMedCrossRefGoogle Scholar
  15. 15.
    Thummel CS (1996) Flies on steroids—Drosophila metamorphosis and the mechanisms of steroid hormone action. Trends Genet 12:306–310PubMedCrossRefGoogle Scholar
  16. 16.
    Buszczak M, Segraves WA (2000) Insect metamorphosis: out with the old, in with the new. Curr Biol 10:R830–R833PubMedCrossRefGoogle Scholar
  17. 17.
    White KP, Rifkin SA, Hurban P, Hogness DS (1999) Microarray analysis of Drosophila development during metamorphosis. Science 286:2179–2184PubMedCrossRefGoogle Scholar
  18. 18.
    Bowen ID, Mullarkey K, Morgan SM (1996) Programmed cell death during metamorphosis in the blow-fly Calliphora vomitoria. Microsc Res Tech 34:202–217PubMedCrossRefGoogle Scholar
  19. 19.
    Tarone AM, Jennings KC, Foran DR (2007) Aging blow fly eggs using gene expression: a feasibility study. J Forensic Sci 52:1350–1354PubMedCrossRefGoogle Scholar
  20. 20.
    Tarone AM, Foran DR (2011) Gene expression during blow fly development: improving the precision of age estimates in forensic entomology. J Forensic Sci 56:S112–S122.PubMedCrossRefGoogle Scholar
  21. 21.
    Ames C, Turner B, Daniel B (2006) Estimating the post-mortem interval (II): the use of differential temporal gene expression to determine the age of blowfly pupae. International Congress Series 1288:861–863CrossRefGoogle Scholar
  22. 22.
    Gaudry E, Blais C, Annick M, Dauphin-Villemant C (2006) Study of steroidogenesis in pupae of the forensically important blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae). Forensic Sci Int 160:27–34PubMedCrossRefGoogle Scholar
  23. 23.
    Mösch SA (2005) Molekularbiologische Altersbestimmung an Puppen der forensisch relevanten Schmeißfliege Lucilia sericata. Diplomarbeit, Fachhochschule Aachen.Google Scholar
  24. 24.
    Kim YJ, Kwak CI, Gu YY, Hwang IT, Chun JY (2004) Annealing control primer system for identification of differentially expressed genes on agarose gels. BioTech 36:424–434Google Scholar
  25. 25.
    Hwang IT, Kim YJ, Kim SH, Kwak CI, Gu YY, Chun JY (2003) Annealing control primer system for improving specificity of PCR amplification. BioTech 35:2–6Google Scholar
  26. 26.
    Rognes K (1991) Blowflies (Diptera, Calliphoridae) of Fennoscandia and Denmark. E.J. Brill/Scandinavian Science, LeidenGoogle Scholar
  27. 27.
    GeneFishing™ DEG Premix Kit, User Manual, SeegeneGoogle Scholar
  28. 28.
    Beckmann B (2004) Studien der Genexpressionsänderung während der Entwicklung von Drosophila melanogaster mittels DNA-Microarrays. Dissertation. Ruprecht-Karls-Universität HeidelbergGoogle Scholar
  29. 29.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta (CT)) method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  30. 30.
    Pfaffl MW (2001) A new mathematical model for relative quantificationin real-time RT-PCR. Nucleic Acids Res 29: e45PubMedCrossRefGoogle Scholar
  31. 31.
    Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3: research0034.1-research0034.11Google Scholar
  32. 32.
    Ramakers C, Ruijter JM, Lekanne Deprez RH, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66PubMedCrossRefGoogle Scholar
  33. 33.
    Burmester T, Scheller K (1997) Developmentally controlled cleavage of the Calliphora arylphorin receptor and posttranslational action of the steroid hormone 20-hydroxyecdysone. Eur J Biochem 247:695–702PubMedCrossRefGoogle Scholar
  34. 34.
    Tarone AM, Picard CJ, Spiegelman C, Foran DR (2011) Population and temperature effects on Lucilia sericata (Diptera: Calliphoridae) body size and minimum developmental time. J Med Entomol 48:1062–1068.PubMedCrossRefGoogle Scholar
  35. 35.
    Gallagher MB, Sandhu S, Kimsey R (2010) Variation in developmental time for geographically distinct populations of the common green bottle fly, Lucilia sericata (Meigen). J Forensic Sci 55:438–442PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Petra Boehme
    • 1
    • 2
    Email author
  • Philipp Spahn
    • 3
  • Jens Amendt
    • 1
  • Richard Zehner
    • 1
  1. 1.Institute of Forensic MedicineGoethe-University FrankfurtFrankfurt am MainGermany
  2. 2.Department of Aquatic EcotoxicologyGoethe-University FrankfurtFrankfurt am MainGermany
  3. 3.Division of Animal GeneticsUniversity of TuebingenTuebingenGermany

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