Research Article| Volume 15, P137-146, March 2015

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De novo transcriptome analysis and highly sensitive digital gene expression profiling of Calliphora vicina (Diptera: Calliphoridae) pupae using MACE (Massive Analysis of cDNA Ends)

Published:November 21, 2014DOI:


      • We analyzed the transcriptome of pupae of the blow fly Calliphora vicina.
      • MACE provided high-resolution gene expression profiles of 15 pupal development stages.
      • 53,539 distinct transcripts were detected, 7548 (14.1%) were annotated to known genes.
      • Genes of Interest (GOI) for each of the 15 pupal stages were identified and support molecular age estimation in forensic entomology.


      Determining a post-mortem interval using the weight or length of blow fly larvae to calculate the insect's age is well established. However, to date, there are only a handful studies dealing with age estimation of blow fly pupae, in which weight or length cannot be used as a relevant parameter. The analysis of genetic markers, which indicate a certain developmental stage, can extend the period for a successful post-mortem interval determination. In order to break new ground in the field of age determination of forensic relevant blow flies, we performed a de novo transcriptome analysis of Calliphora vicina pupae at 15 different developmental stages. Obtained data serve as base to establish molecular age determination techniques. We used a new, deeper, and more cost-effective digital gene expression profiling method called MACE (Massive Analysis of cDNA Ends). We generated 15 libraries out of 15 developmental stages, with 3–8 million reads per library. In total, 53,539 distinct transcripts were detected, and 7548 were annotated to known insect genes. The analysis provides high-resolution gene expression profiles of all covered transcripts, which were used to identify differentially expressed genetic markers as candidates for a molecular age estimation of C. vicina pupae. Moreover, the analysis allows insights into gene activity of pupal development and the relationship between different genes interesting for insect development in general.


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        • Greenberg B.
        Flies as forensic indicators.
        J. Med. Entomol. 1991; 28: 565-577
        • Smith K.G.
        A Manual of Forensic Entomology.
        Cornell University Press, London1986
        • Arnaldos M.I.
        • Garcia M.
        • Romera E.
        • Presa J.
        • Luna A.
        Estimation of postmortem interval in real cases based on experimentally obtained entomological evidence.
        Forensic Sci. Int. 2005; 149: 57-65
        • Villet M.H.
        • Amendt J.
        Advances in entomological methods for estimating time of death.
        in: Turk E.E. Forensic Pathology Reviews. Humana Press, Heidelberg2011: 213-237
        • Amendt J.
        • Richards C.S.
        • Campobasso C.P.
        • Zehner R.
        • Hall M.J.R.
        Forensic entomology: applications and limitations.
        Forensic Sci. Med. Pathol. 2011; 7: 379-392
        • Forbes S.I.
        • Dadour I.
        The soil environment and forensic entomology.
        in: Byrd J.H. Castner J.L. Forensic Entomology: The Utility of Arthropods in Legal Investigations. 2nd ed. Taylor & Francis, Boca Raton/New York2009: 416
        • Richards C.S.
        • Crous K.L.
        • Villet M.H.
        Models of development for blow fly sister species Chrysomya chloropyga and Chrysomya putoria.
        Med. Vet. Entomol. 2009; 23: 56-61
        • Grassberger M.
        • Reiter C.
        Effect of temperature on development of the forensically important holarctic blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae).
        Forensic Sci. Int. 2002; 128: 177-182
        • Grassberger M.
        • Reiter C.
        Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen- and isomorphendiagram.
        Forensic Sci. Int. 2001; 120: 32-36
        • Wells J.D.
        • LaMotte L.R.
        Estimating maggot age from weight using inverse prediction.
        J. Forensic Sci. 1995; 40: 585-590
        • Reiter C.
        Zum Wachstumsverhalten der Maden der blauen Schmeißfliege Calliphora vicina.
        Z. Rechtsmed. 1984; 91: 295-308
        • Tomberlin J.K.
        • Benbow M.E.
        • Tarone A.M.
        • Mohr R.M.
        Basic research in evolution and ecology enhances forensics.
        Trends Ecol. Evol. 2011; 26: 53-55
        • Zajac B.
        • Amendt J.
        Bestimmung des Alters forensisch relevanter Fliegenpuppen: Morphologische und histologische Methoden.
        Rechtsmedizin. 2012; 22: 456-465
        • Fraenkel G.
        • Bhaskaran G.
        Pupariation and pupation in cyclorrhaphous flies (Diptera) terminology and interpretation.
        Ann. Entomol. Soc. Am. 1973; 66: 418-422
        • Davies K.
        • Harvey M.L.
        Internal morphological analysis for age estimation of blow fly pupae (Diptera: Calliphoridae) in postmortem interval estimation.
        J. Forensic Sci. 2013; 58: 79-84
        • Richards C.S.
        • Simonsen T.J.
        • Abel R.L.
        • Hall M.J.
        • Schwyn D.A.
        • Wicklein M.
        • Hall M.J.
        Virtual forensic entomology: improving estimates of minimum post-mortem interval with 3D micro-computed tomography.
        Forensic Sci. Int. 2012; 220: 251-264
        • Marchenko M.I.
        Medico-legal relevance of cadaver entomofauna for the determination of the time since death.
        Acta Med. Leg. Soc. 2001; 120: 89-109
        • Amendt J.
        • Campobasso C.P.
        • Gaudry E.
        • Reiter C.
        • LeBlanc H.N.
        • Hall M.
        Best practice in forensic entomology—standards and guidelines.
        Int. J. Leg. Med. 2007; 121: 90-104
        • Bainbridge S.P.
        Staging the metamorphosis of Drosophila melanogaster.
        J. Embryol. Exp. Morphol. 1981; : 57-80
        • Boehme P.
        • Spahn P.
        • Amendt J.
        • Zehner R.
        Differential gene expression during metamorphosis: a promising approach for age estimation of forensically important Calliphora vicina pupae (Diptera: Calliphoridae).
        Int. J. Leg. Med. 2013; 127: 243-249
        • Tarone A.M.
        • Foran D.R.
        Gene expression during blow fly development: improving the precision of age estimates in forensic entomology.
        J. Forensic Sci. 2011; 56: S112
        • Zehner R.
        • Amendt J.
        • Boehme P.
        Gene expression analysis as a tool for age estimation of blow fly pupae.
        Forensic Sci. Int.: Genet. Suppl. Ser. 2009; 2: 292-293
        • Tarone A.M.
        • Jennings K.C.
        • Foran D.R.
        Aging blow fly eggs using gene expression: a feasibility study.
        J. Forensic Sci. 2007; 52: 1350-1354
        • Sze S.-H.
        • Dunham J.P.
        • Carey B.
        • Chang P.L.
        • Li F.
        • Edman R.M.
        • Fjeldsted C.
        • Scott M.J.
        • Nuzhdin S.V.
        • Tarone A.M.
        A de novo transcriptome assembly of Lucilia sericata (Diptera: Calliphoridae) with predicted alternative splices, single nucleotide polymorphisms and transcript expression estimates.
        Insect Mol. Biol. 2012; 21: 205-221
        • Farncombe K.
        • Beresford D.
        • Kyle C.
        Characterization of microsatellite loci in Phormia regina towards expanding molecular applications in forensic entomology.
        Forensic Sci. Int. 2014; 240: 122-125
        • Vera J.C.
        • Wheat C.W.
        • Fescemyer H.W.
        • Frilander M.J.
        • Crawford D.L.
        • Hanski I.
        • Marden J.H.
        Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing.
        Mol. Ecol. 2008; 17: 1636-1647
        • Hahn D.A.
        • Ragland G.J.
        • Shoemaker D.D.
        • Denlinger D.L.
        Gene discovery using massively parallel pyrosequencing to develop ESTs for the flesh fly Sarcophaga crassipalpis.
        BMC Genomics. 2009; 10: 234
        • Wang X.-W.
        • Luan J.-B.
        • Li J.-M.
        • Bao Y.-Y.
        • Zhang C.-X.
        • Liu S.-S.
        De novo characterization of a whitefly transcriptome and analysis of its gene expression during development.
        BMC Genomics. 2010; 11: 400
        • Torres T.T.
        • Metta M.
        • Ottenwalder B.
        • Schlotterer C.
        Gene expression profiling by massively parallel sequencing.
        Genome Res. 2007; 18: 172-177
        • Asmann Y.W.
        • Klee E.W.
        • Thompson E.A.
        • Perez E.A.
        • Middha S.
        • Oberg A.L.
        • Therneau T.M.
        • Smith D.I.
        • Poland G.A.
        • Wieben E.D.
        • Kocher J.-P.A.
        3’ tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer.
        BMC Genomics. 2009; 10: 531
        • Lenz T.L.
        • Eizaguirre C.
        • Rotter B.
        • Kalbe M.
        • Milinski M.
        Exploring local immunological adaptation of two stickleback ecotypes by experimental infection and transcriptome-wide digital gene expression analysis.
        Mol. Ecol. 2013; 22: 774-786
        • Yakovlev I.A.
        • Lee Y.
        • Rotter B.
        • Olsen J.E.
        • Skrøppa T.
        • Johnsen Ø.
        • Fossdal C.G.
        Temperature-dependent differential transcriptomes during formation of an epigenetic memory in Norway spruce embryogenesis.
        Tree Genet. Genomes. 2014; 10: 355-366
        • Zawada A.M.
        • Rogacev K.S.
        • Müller S.
        • Rotter B.
        • Winter P.
        • Fliser D.
        • Heine G.H.
        Massive Analysis of cDNA Ends (MACE) and miRNA expression profiling identifies proatherogenic pathways in chronic kidney disease MACE and miRNA profiling in CKD.
        Epigenetics. 2014; 9
        • Folmer O.
        • Black M.
        • Hoeh W.
        • Lutz R.
        • Vrijenhoek R.
        DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.
        Mol. Marine Biol. Biotechnol. 1994; 3: 294-299
        • Winnebeck E.C.
        • Millar C.D.
        • Warman G.R.
        Why does insect RNA look degraded?.
        J. Insect Sci. 2010; 10: 159
        • Benjamini Y.
        • Hochberg Y.
        Controlling the false discovery rate: a practical and powerful approach to multiple testing.
        J. R. Stat. Soc. Ser. B: Methodol. 1995; 57: 289-300
        • Rivals I.
        • Personnaz L.
        • Taing L.
        • Potier M.-C.
        Enrichment or depletion of a GO category within a class of genes: which test?.
        Bioinformatics. 2007; 23: 401-407
      1. M. Baqué, R. Zehner, M.A. Verhoff, J. Amendt, Descriptive analyses of differentially expressed genes during larval development of Calliphora vicina (Diptera: Calliphoridae), Int. J. Leg. Med., submitted (2014).

        • Gaudry E.
        • Blais C.
        • Maria A.
        • Dauphin-Villemant C.
        Study of steroidogenesis in pupae of the forensically important blow fly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae).
        Forensic Sci. Int. 2006; 160: 27-34
        • Foran D.
        Generating More Precise Post Mortem Interval Estimates With Entomological Evidence: Reliable Patterns of Gene Expression Throughout Calliphorid Larval and Pupal Development.
        Michigan State University, National Institute of Justice/NCJRS, 2007
        • Boehme P.
        • Spahn P.
        • Amendt J.
        • Zehner R.
        The analysis of temporal gene expression to estimate the age of forensically important blow fly pupae: results from three blind studies.
        Int. J. Leg. Med. 2014; 128: 565-573
        • Tarazona S.
        • Garcia-Alcalde F.
        • Dopazo J.
        • Ferrer A.
        • Conesa A.
        Differential expression in RNA-Seq: a matter of depth.
        Genome Res. 2011; 21: 2213-2223