Detection of fly artifacts from four species of necrophagous flies on household materials using immunoassays


An immunoassay was previously developed as a technique to improve methods for detection and analysis of fly artifacts found at crime scenes. The dot blot assay utilized a polyclonal antiserum (anti-md3) based on a unique digestive cathepsin D found in cyclorrhaphous Diptera. In this study, artifacts produced by adults of Calliphora vicina, Cynomya cadaverina, Sarcophaga bullata, and Protophormia terraenovae were examined using the immunoassay to determine if insect-derived stains could be distinguished from a range of human body fluid stains. A lift technique was developed which permitted transfer of fly artifacts from test materials to filter paper for dot blot analyses. All species readily deposited artifacts on all test household materials regardless of diet consumed. Despite differences in texture and porosity of the household materials, artifacts of all species transferred to the filter paper. With all fly species, anti-md3 serum bound to artifacts produced after feeding on semen, blood, feces, urine, and saliva. By contrast, anti-md3 serum did not react with any of the human fluids tested, nor with any of the lifts from household materials not exposed to flies. There was no evidence of false positives with any of the fly species tested, regardless of diet consumed. There was also no indication of false negatives with any of the dot blot assays. These observations suggest that immunoassays using anti-md3 serum performed on a simple lift of suspected fly artifacts can be used effectively as a confirmatory assay to distinguish fly regurgitate and fecal stains from human body fluids.

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  1. 1.

    Barton Browne L (1993) Physiologically induced changes in resource-oriented behavior. Annu Rev Entomol 38:1–25

    Article  Google Scholar 

  2. 2.

    Durdle A, Mitchell R, van Oorschot RAH (2016) The food preferences of the blow fly Lucilia cuprina offered human blood, semen, and saliva, and various nonhuman foods sources. J Forensic Sci 61:99–103

    Article  Google Scholar 

  3. 3.

    Archer MS, Elgar MA (2003) Effects of decomposition on carcass attendance in a guild of carrion-breeding flies. Med Vet Entomol 17:263–271

    CAS  Article  Google Scholar 

  4. 4.

    Browne LB, Gerwen AV (1992) Volume of protein meals taken by females of the blowfly, Lucilia cuprina: ovarian development-related and direct effects of protein ingestion. Physiol Entomol 17:9–18

    Article  Google Scholar 

  5. 5.

    Belzer WR (1978) Patterns of selective protein ingestion by the blowfly Phormia regina. Physiol Entomol 3:169–175

    CAS  Article  Google Scholar 

  6. 6.

    Stoffolano JG Jr, Li M-F, Sutton JA Jr, Yin C-M (1995) Faeces feeding by adult Phormia regina (Diptera: Calliphoridae): impact on reproduction. Med Vet Entomol 9:388–392

    Article  Google Scholar 

  7. 7.

    Wessels FJ, Jordan DC, Hahn DA (2010) Allocation from capital and income sources to reproduction shift from first to second clutch in the flesh fly, Sarcophaga crassipalpis. J Insect Physiol 56:1269–1274

    CAS  Article  Google Scholar 

  8. 8.

    Kulstein G, Amendt J, Zehner R (2015) Blow fly artifacts from blood and putrefaction fluid on various surfaces: a source for forensic STR typing. Entomol Exp et Appl 157:255–262

    Article  Google Scholar 

  9. 9.

    Parker MA, Benecke M, Byrd JH, Hawkes R, Brown R (2010) Entomological alteration of bloodstain evidence. In: Byrd JH, Castner JL (eds) Forensic entomology: the utility of using arthropods in legal investigations, 2nd edn. CRC Press, Boca Raton, pp 539–580

    Google Scholar 

  10. 10.

    Smith KGV (1986) A manual of forensic entomology. London and Cornell University Press, London

    Google Scholar 

  11. 11.

    Viero A, Montisci M, Pelletti G, Vanin S (2019) Crime scene and body alterations caused by arthropods: implications in death investigation. Int J Legal Med 133:307–316

    CAS  Article  Google Scholar 

  12. 12.

    Rivers DB, Geiman T (2017) Insect artifacts are more than just altered bloodstains. Insects: Adv Forensic Entomol.

  13. 13.

    Durdle A, van Oorschot RAH, Mitchell RJ (2013) The morphology of fecal and regurgitation artifacts deposited by the blow fly Lucilia cuprina fed a diet of human blood. J Forensic Sci 58:897–903

    Article  Google Scholar 

  14. 14.

    Peters W (2003) Ernährung und Verdauung. In: Dettner K, Peters W (eds) Lehrbuch der Entomologie Spektrum. Verlag, Munich, pp 91–126

    Google Scholar 

  15. 15.

    Rivers DB, Cavanagh G, Greisman V, McGregor A, Brogan R, Schoeffield A (2019) Immunoassay detection of fly artifacts produced by several species of necrophagous flies following feeding on human blood. Forensic Sci Int: Synergy.

  16. 16.

    Bevel T, Gardner R (2008) Bloodstain pattern analysis: with an introduction to crime scene reconstruction, 3rd edn. CRC Press, Boca Raton

    Google Scholar 

  17. 17.

    Striman B, Fujikawa A, Barksdale L, Carter DO (2011) Alteration of expirated bloodstain patterns by Calliphora vicina and Lucilia sericata (Diptera: Calliphoridae) through ingestion and deposition of artifacts. J Forensic Sci 56:S123–S127

    Article  Google Scholar 

  18. 18.

    Benecke M, Barksdale L (2003) Distinction of bloodstain patterns from fly artifacts. Forensic Sci Int 137:152–159

    Article  Google Scholar 

  19. 19.

    James SH, Nordby JJ, Bell S (2014) Forensic science: an introduction to scientific and investigative techniques, 4th edn. CRC Press, Boca Raton

    Book  Google Scholar 

  20. 20.

    Fujikawa A, Barksdale L, Higley LG, Carter DO (2011) Changes in the morphology and presumptive chemistry of impact and pooled bloodstain patterns by Lucilia sericata (Meigen)(Diptera: Calliphoridae). J Forensic Sci 56:1315–1318

    CAS  Article  Google Scholar 

  21. 21.

    Rivers DB, McGregor A (2018) Morphological features of regurgitate and defecatory stains deposited by five species of necrophagous flies are influenced by adult diets and body size. J Forensic Sci 63:154–161

    Article  Google Scholar 

  22. 22.

    Durdle A, Mitchell RJ, van Oorschot RA (2013) The human DNA content in artifacts deposited by the blowfly Lucilia cuprina fed human blood, semen and saliva. Forensic Sci Int 233:212–219

    CAS  Article  Google Scholar 

  23. 23.

    Durdle A, Mitchell RJ, van Oorschot RAH (2011) The change in human DNA content over time in the artefacts of the blowfly Lucilia cuprina (Meigen) (Diptera: Calliphoridae). Forensic Sci Int 3:e289–e290

    Google Scholar 

  24. 24.

    Durdle A, Mitchell RJ, van Oorschot RAH (2015) The use of forensic tests to distinguish blowfly artifacts from human blood, semen, and saliva. J Forensic Sci 60:468–470

    CAS  Article  Google Scholar 

  25. 25.

    Pelletti G, Mazzotti MC, Fais P, Martini D, Ingra L, Amadasi A, Palazzo C, Falconi M, Pelotti S (2019) Scanning electron microscopy in the identification of fly artifacts. Int J Legal Med.

  26. 26.

    Langer SV, Illes M (2015) Confounding factors of fly artefacts in bloodstain pattern analysis. J Can Soc Forensic Sci 48:215–224

    Article  Google Scholar 

  27. 27.

    Rivers DB, Acca G, Fink M, Brogan R, Chen D, Schoeffield A (2018) Distinction of fly artifacts from human blood using immunodetection. J Forensic Sci 63:1704–1711

    CAS  Article  Google Scholar 

  28. 28.

    Marshall SA, Whitworth T, Roscoe L. (2011) Blow flies (Diptera; Calliphoridae) of eastern Canada with a key to Calliphoridae subfamilies and genera of eastern North America, and a key to the eastern Canadian species of Calliphorinae, Luciliinae and Chrysomyiinae. Can J Arthr Ident No. 11: doi:

  29. 29.

    Denlinger DL (1972) Induction and termination of pupal diapause in Sarcophaga (Diptera: Sarcophagidae). Biol Bull 142:11–24

    Article  Google Scholar 

  30. 30.

    de Castro TC, Taylor MC, Kieser JA, Carr DJ, Duncan W (2015) Systematic investigations of drip stains on apparel fabrics: the effects of prior-laundering, fibre content and fabric structure on final stain appearance. Forensic Sci Int 250:98–109

    Article  Google Scholar 

  31. 31.

    Oldfield C, Morgan RM, Miles HF, French JC (2017) The efficacy of luminol in detecting bloodstains that have been washed with sodium percarbonate and exposed to environmental conditions. Aust J Forensic Sci.

  32. 32.

    LaLonde W, Millman JS (2019) Case study: loss of Kastle-Meyer test specificity on jeans. Sci Justice 59:359–361

    Article  Google Scholar 

  33. 33.

    Fujikawa A, Barksdale L, Carter DO (2009) Calliphora vicina (Diptera: Calliphoridae) and their ability to alter the morphology and presumptive chemistry of bloodstain patterns. J Forensic Ident 59:502–512

    Google Scholar 

  34. 34.

    Padilha MHP, Pimentel AC, Ribeiro AF, Terra WR (2009) Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut. Insect Biochem Mol Biol 39:782–791

    CAS  Article  Google Scholar 

  35. 35.

    Chang JYM, Michielsen S (2016) Effect of fabric mounting method and backing material on bloodstain patterns of drip stains on textiles. Int J Legal Med 130:649–659

    CAS  Article  Google Scholar 

  36. 36.

    Cho Y, Springer F, Tulleners FA, Ristenpart WD (2015) Quantitative bloodstain analysis: differentiation of contact transfer patterns versus spatter patterns on fabric via microscopic inspection. Forensic Sci Int 249:233–240

    CAS  Article  Google Scholar 

  37. 37.

    de Castro TC, Nickson T, Carr DJ, Knock C (2013) Interpreting the formation of bloodstains on selected apparel fabrics. Int J Legal Med 127:251–258

    Article  Google Scholar 

  38. 38.

    Knecht DA, Mierendorf RC, Dimond RL (1983) Immunological recognition of modifications on functionally related proteins. Methods Enzymol 98:159–166

    CAS  Article  Google Scholar 

  39. 39.

    Tamaki FK, Padilha MHP, Pimentel AC, Ribeiro AF, Terra WR (2012) Properties and secretory mechanism of Musca domestica digestive chymotrypsin and its relation with Drosophila melanogaster homologs. Insect Biochem Mol Biol 42:482–490

    CAS  Article  Google Scholar 

  40. 40.

    Leyria J, Fruttero LL, Ligabue-Braun R, Defferrari MS, Arrese EL, Soulages JL, Settembrini BP, Carlini CR, Canavoso LE (2018) DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae): purification, bioinformatics analyses and the significance of its interaction with lipophorin in the internalization by developing oocytes. J Insect Physiol 105:28–39

    CAS  Article  Google Scholar 

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The authors thank Brian Martin from Carpets by Martin (Shrewsbury, Pennsylvania, USA) for generously providing some of the carpet samples used in this study. This project was supported by Award No. 2016-DN-BX-0181, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of the Department of Justice.

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Correspondence to David B. Rivers.

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Rivers, D.B., Cavanagh, G., Greisman, V. et al. Detection of fly artifacts from four species of necrophagous flies on household materials using immunoassays. Int J Legal Med 134, 1239–1253 (2020).

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  • Insect stains
  • Digestive artifacts
  • Bloodstains
  • Immunodetection
  • Forensic entomology