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The transfer of human DNA by Lucilia cuprina (Meigen) (Diptera: Calliphoridae)

  • Annalisa Durdle
    Correspondence
    Corresponding author at: Department of Genetics, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora, Melbourne, Victoria 3086, Australia. Tel.: +61 3 9479 2194.
    Affiliations
    Department of Genetics, Faculty of Science, Technology and Engineering, La Trobe University, Melbourne, Victoria 3086, Australia

    Biology Division, Forensic Services Department, Victoria Police, MacLeod, Victoria 3085, Australia
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  • Roland A.H. van Oorschot
    Affiliations
    Biology Division, Forensic Services Department, Victoria Police, MacLeod, Victoria 3085, Australia
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  • Robert John Mitchell
    Affiliations
    Department of Genetics, Faculty of Science, Technology and Engineering, La Trobe University, Melbourne, Victoria 3086, Australia
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Published:November 04, 2009DOI:https://doi.org/10.1016/j.fsigss.2009.08.095

      Abstract

      Blowflies leave deposits, termed artefacts, through the processes of excretion and regurgitation. To date, little consideration has been given to the possibility of adult blowflies consuming biological material and subsequently acting as vectors of human DNA through these artefacts. In this study, Lucilia cuprina (Meigen) (Diptera: Calliphoridae) were fed either human blood or human semen ad libitum and their artefacts were analysed for human DNA content. Samples containing 1, 10, 30 and 50 artefacts were tested. Quantifiable and typeable levels of human DNA were found in samples derived from both food sources, and even in samples containing a single artefact. Semen-derived artefacts contained significantly more human DNA than artefacts produced after a blood meal. Consequently a smaller number of artefacts was required to collect sufficient DNA for genotyping. These findings are forensically important as it provides investigators with another potential source of DNA at a crime scene where a body has been moved, or an attempt has been made to clean up biological material. They also highlight how fly artefacts could potentially contaminate and compromise evidence.

      Keywords

      1. Introduction

      Flies leave spots that are termed ‘fly artefacts’ through the mechanisms of regurgitation and excretion [
      • Brown R.
      • Hawkes R.
      • Anderson Parker M.
      • Byrd J.
      Entomological alteration of bloodstain evidence.
      ]. These artefacts may vary greatly in colour, shape and size due to the different processes involved in their creation [
      • Zuha R.
      • Supriyani M.
      • Omar B.
      Fly artefact documentation of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae)—a forensically important blowfly species in Malaysia.
      ]. Spots may also be deposited by blowflies walking through liquid [
      • Brown R.
      • Hawkes R.
      • Anderson Parker M.
      • Byrd J.
      Entomological alteration of bloodstain evidence.
      ] but for the purposes of this paper ‘artefacts’ refer to faecal or regurgitation spots.
      Fly artefacts can often be found at crime scenes where blood is present [
      • Brown R.
      • Hawkes R.
      • Anderson Parker M.
      • Byrd J.
      Entomological alteration of bloodstain evidence.
      ]. In some instances, it can be difficult to distinguish between these artefacts and blood spatter patterns, due to the similar morphology and high number of artefacts [
      • Brown R.
      • Hawkes R.
      • Anderson Parker M.
      • Byrd J.
      Entomological alteration of bloodstain evidence.
      ]. Consequently, it would not be unexpected for an investigator to sample fly artefacts in the mistaken belief that it is blood spatter. For this reason, it is important to know if blowflies can deposit a typeable amount of DNA as there is the potential for inadvertently collected DNA to interfere with the reconstruction of events or the evidentiary value of DNA profiles. Conversely, if fly artefacts are found to contain sufficient human DNA, they could prove to be a valuable source of DNA for investigators where a body has been moved or an offender has attempted to clean up biological material. Spitaleri et al. demonstrated it was possible to obtain an almost complete human DNA profile from a single mosquito blood meal stain, evidence which contributed to a second degree murder conviction [
      • Spitaleri S.
      • Romano C.
      • Di Luise E.
      • Ginestra E.
      • Saravo L.
      Genotyping of human DNA recovered from mosquitoes found on a crime scene.
      ]. Human DNA has also been successfully extracted and profiled at two loci from the excreta of adult human crab louse [
      • Replogle J.
      • Lord W.
      • Budowle B.
      • Meinking T.
      • Taplin D.
      Identification of host DNA by amplified fragment length polymorphism and lysis: preliminary analysis of human crab louse (Anoplura: Pediculidae) excreta.
      ].
      This research aims to determine if human DNA can be extracted from artefacts deposited by the forensically relevant Lucilia cuprina blowflies after they have fed on human blood or human semen, and the number of artefacts required to obtain a quantifiable and typeable amount of human DNA.

      2. Materials and methods

      2.1 Fly experiments

      Live blowfly pupae of L. cuprina were allowed to hatch in decontaminated, well-ventilated containers. In addition to water, the adult flies were supplied with human blood or human semen ad libitum. Biological material was stored at 4 °C until required and supplied to the flies within a week of extraction or ejaculation. Only a single source of blood and a single source of semen were used.
      Once each replicate experiment had been set up, it was left for three days. This allowed time for sufficient feeding, digestion and excretion to occur to generate a large number of fly artefacts. The blowflies were then removed from the container.
      A single cotton swab moistened with sterile water was used to collect 1, 10, 30 or 50 fly artefacts. No distinction was made between regurgitation and faecal artefacts when collecting the samples. The swabs were allowed to air-dry at room temperature for at least 24 h before DNA analysis.

      2.2 Human DNA analysis

      Within three weeks of the artefacts being deposited, DNA extraction was conducted using the DNA IQ™ System Kit (Promega Corporation, Madison, WI, USA) and a MagnaRack (Invitrogen Corporation, Carlsbad, CA, USA), human-specific quantification was performed using the Quantifiler™ Human DNA Quantification kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM® 7500 real-time PCR instrument (AB), and amplification and profiling was performed using the AmpFlSTR Profiler Plus® multiplex PCR Amplification kit (AB), a GeneAmp® PCR System 9700 thermal cycler (AB), an ABI3100 genetic analyser (AB) and GeneMapper™ ID software (AB). A DNA amount of 0.1 ng was considered the threshold amount from which good profiles could be obtained.

      3. Results

      3.1 Blood meals

      The threshold of 0.1 ng DNA to achieve full profiles was reached in 35%, 57%, 57% and 64% of samples containing 1, 10, 30 or 50 blood-derived artefacts respectively (Fig. 1). The median amount of DNA in the single-artefact group was below the detection limits of the Quantifiler™ kit. The 10, 30 and 50 groups had median DNA amounts of 0.60 ng, 0.47 ng and 0.72 ng respectively (Fig. 1). Regression analysis showed only a very weak correlation between the median amounts and the number of artefacts collected (R2 = 0.382). The maximum amount of DNA extracted in each group decreased as the number of artefacts collected increased (Fig. 1).
      Figure thumbnail gr1
      Fig. 1The median and range of DNA amounts (ng) in samples containing 1, 10, 30 or 50 fly artefacts derived from either human blood or human semen meals.

      3.2 Semen meals

      The semen-derived artefacts contained significantly more DNA than the artefacts deposited after blood meals in all artefact-number groups (P < 0.05, Mann–Whitney U-test). All samples tested in the 10, 30 and 50 artefact-number groups contained sufficient DNA to reach the threshold of 0.1 ng for profiling (Fig. 1). Only 2 of the 10 samples in the single-artefact group did not meet the threshold. Regression analysis shows there is a strong correlation between the median amount and the number of artefacts collected (R2 = 0.998). The minimum amounts of DNA in each sample group increased with the increase in number of artefacts, but the maximum amounts did not consistently increase with increasing artefact number (Fig. 1).

      3.3 DNA profiling

      The DNA from one randomly selected sample containing ≥0.1 ng from each group was genotyped. The DNA profiles from all samples tested corresponded to the profile of the biological material donor.

      4. Discussion

      The results indicate that human DNA can be extracted from fly artefacts in sufficient quantities to provide a full profile of the donor.
      The data suggest the amount of DNA in artefacts can be dependent on the meal type. Artefacts derived from semen contained significantly higher amounts of DNA than artefacts derived from blood. This is not surprising given the considerably higher content of DNA in semen when compared to blood [
      • Committee on DNA Technology in Forensic Science, National Research Council
      DNA Technology in Forensic Science.
      ]. Consequently, fewer semen artefacts are required to obtain a typeable amount of DNA than blood artefacts. A minimum of 10 semen-derived artefacts would be recommended for collection. Further research is being conducted to determine the optimum amount of artefacts from blood-derived meals that should be collected.
      The variation observed in the ranges and median values of the amounts of DNA extracted, and the lack of a linear trend in some artefact-number groups, may be due to a number of factors including variability in DNA degradation [
      • Lindahl T.
      Instability and decay of the primary structure of DNA.
      ] and the amount of biological material consumed. This variability also suggests that certain types of digestive processes may generate artefacts with more or less DNA content than others, depending on the level of DNA breakdown occurring. Therefore, further research is being undertaken to enable investigators to target those artefacts likely to contain the most human DNA.
      Of particular interest are the DNA profiles obtained from single artefacts. This raises the possibility of a single blowfly transferring human DNA between locations at levels that could prove problematic in terms of contaminated profiles or crime scene interpretation. These findings may provide lawyers with the opportunity to raise doubt in the minds of a jury in regard to DNA evidence. Therefore it would be very useful to be able to comment on the likelihood of a crime scene being contaminated with DNA from an innocent person through the movement and activity of blowflies.
      The forensic implications of the presence of human DNA in fly artefacts make it important to conduct further research into this area. The finding of human DNA in samples derived from various biological materials shows that fly artefacts can be a valuable source of DNA at crime scenes, in addition to being a potential contaminant.

      Role of funding

      Funding for this study was provided by La Trobe University, Australia.

      Conflict of interest

      None.

      Acknowledgements

      The author would like to thank the Institute of Environmental Science and Research (ESR), Auckland, New Zealand, and the New Zealand Police for funding and support during pilot studies. Further thanks go to Merilyn Geary of the Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia for supplying the blowflies.

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