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The effect of substrates and time of deposition on molecular analysis of fly artifacts

Published:October 25, 2022DOI:https://doi.org/10.1016/j.fsigss.2022.10.062

      Abstract

      The activity of animals and insects at the crime scene can provide useful elements to reconstruct the dynamics of the event. Any insect that interacts with wet body fluids can produce artifacts which can be confused with human bloodstains. Considering that flies are the early colonizers of the crime scene and first players of the process of contamination, the problem is to distinguish stains produced by fly regurgitation or fecal elimination on a crime scene. Actually, fly artifacts are morphologically very similar to impact, projected, sneezed, and expirated bloodstains and cannot be reliably distinguished using presumptive or confirmatory tests for identification of human blood. Several techniques have been proposed to differentiate insect-derived artifacts based on morphological approaches and immunological assay. Recently, a DNA-based method by the analysis of the cytochrome c oxidase subunit I (COI) gene has been designed for the distinction of fly artifacts from human bloodstains on spots characterized by different morphological features and deposited by fly on a glass surface.
      Here, we present a study to assess the feasibility of the molecular analysis of fly artifacts deposited on different substrates as glass, paper, plexiglass and cotton and to evaluate the COI amplification success at different time intervals up to two years after fly artifacts deposition. Our results showed that the deposition time seems do not affect the detection of fly artifacts DNA, while a substrate interference was demonstrated.

      Keywords

      1. Introduction

      Bloodstains yielded by the victim or by the perpetrator at the scene could provide countless information for the crime reconstruction even when the body is no longer present on the crime scene. However, bloodstains are subjected to modifications due to the interaction with flies, which are attracted from sources of food and colonize corpses feeding on their biological fluids [
      • Rivers D.B.
      • Geiman T.
      Insect artifacts are more than just altered bloodstains.
      ]. The fly artifacts are defined as “bloodstains resulting from insect activity” by SWGSTAIN [

      Scientific Working Group on Bloodstain Pattern Analysis: Guidelines for the Minimum Educational and Training Requirements for Bloodstain Pattern Analysis (SWGSTAIN). Forensic Science Communications, Jan 2008 - Vol 10 - N. 1 https://archives.fbi.gov/archives/about-us/lab/forensic-science-communications/fsc/jan2008/standards/2008_01_standards01.htm.

      ] and include digestive artifacts, translocation, tarsal tracks and castoff stains. They could mislead the investigations being deposited even distant from the body [
      • Durdle A.
      • van Oorschot R.A.
      • Mitchell R.J.
      The morphology of fecal and regurgitation artifacts deposited by the blow fly Lucilia cuprina fed a diet of human blood.
      ], but they could be the only available source of human DNA required for identification of the victim or assailant [
      • Kulstein G.
      • Amendt J.
      • Zehner R.
      Blow fly artifacts from blood and putrefaction fluid on various surfaces: a source for forensic STR typing.
      ].
      Recently, a DNA-based method has been proposed to detect fly DNA in fly artifacts deposited on glass surface by Calliphora vomitoria after feeding on human blood [
      • Bini C.
      • Giorgetti A.
      • Iuvaro A.
      • et al.
      A DNA-based method for distinction of fly artifacts from human bloodstains.
      ].
      The present study aims to investigate the feasibility of the molecular analysis of fly artifacts deposited on different substrates and to evaluate the COI amplification success at different time intervals up to two years after fly artifacts deposition.

      2. Materials and methods

      The study was conducted following procedures in accordance with the ethical standards and was approved by the Bioethical Committee of the University of Bologna (Approval number: 34339).
      Ten ml of fresh human blood collected under informed consent from a male volunteer was placed in a fly box consisting of 5 walls covered with white cotton and one glass wall room. Adults of Musca domestica were inserted in the fly box and left free to feed ad libitum (Supplementary Fig. A and B). After 72 h the flies were released and artifacts were collected at time 0, 1 month and 2 months after flies’ deposition.
      The analysis of aged stains was performed on fly artifacts stored at room temperature for 2 years-old and deposited by Calliphora vomitoria on plexiglass and paper as reported in [
      • Pelletti G.
      • Mazzotti M.C.
      • Fais P.
      • et al.
      Scanning electron microscopy in the identification of fly artifacts.
      ]. Fly artifacts deposited on cotton and paper were considered together and both as porous surfaces and, similarly, plexiglass and plastic were considered as non-porous surfaces.
      A total of 72 fly artifacts were sampled and grouped as shown in Supplementary Table A. The thirty-six samples from non-porous surfaces were collected by 4N6FLOQSwabs™ (Copan) and 36 fly artifacts were sampled from porous surfaces by cutting about 4 mm2 of cotton and paper. Flies and pupae from Musca domestica and Calliphora vomitoria were collected as fly DNA reference.
      The molecular analysis on both pupae and fly artifacts was performed as described in [
      • Bini C.
      • Giorgetti A.
      • Iuvaro A.
      • et al.
      A DNA-based method for distinction of fly artifacts from human bloodstains.
      ]. Sequencing was performed on a SeqStudio Genetic Analyzer (Applied Biosystems) and analyzed by Sequencing Analysis 7 v7.0 software (Applied Biosystems). Fly species were identified by comparing the nucleotide sequences of the samples to sequences deposited in Genebank and the basic local alignment search tool (BLAST). A 97% or greater concordance rate of the nucleotide sequences was considered for species identification.
      Beside descriptive statistics, fresh and aged fly artifacts, as well as porous and non-porous surfaces comparisons were performed by a Chi square test using Stata (StataCorp LLC, Texas, USA, v. 15.1) (p < 0.05 for significance). Figures were realized with Prism (GraphPad software LLC, San Diego, USA v. 8.4.3).

      3. Results

      Fly artifacts of different morphology, from circular and elliptical stains to artifacts with sperm-like tail or a tear-shaped body were produced on the walls of the fly box (Supplementary Fig. C). The mitochondrial cytochrome c oxidase subunit I (COI) gene was successfully amplified for 76.4% (n = 55) of the whole samples (Supplementary Table B).
      Considering the substrate type, 33 out of 36 fly artifacts sampled from paper and cotton were amplified for the COI region of fly DNA, against 22 positive amplifications obtained out of the 36 fly artifacts taken from glass and plexiglass (Fig. 1A). A statistically significant association between the successfulness of amplification of the COI region and porous vs non -porous substrates was demonstrated. The BLAST search for identification purposes reported a significant alignment of 99–100% identity with Musca domestica species for flies and fresh fly artifacts and Calliphora vomitoria for aged fly artifacts.
      Fig. 1
      Fig. 1Influence of type of substrate and chronological factor on the success rate of the COI amplification. T0: time zero; M1: month 1; M2: month 2; Y2: year 2.
      Comparing fresh at different sampling time vs 2 years old fly artifacts based on the chronological factor, no statistically significant difference in the frequency of positively amplified samples was shown. The influence of chronological factor also tested non-significant by considering positive fresh fly artifacts all together vs aged fly artifacts (Fig. 1B). The same result was obtained taking into consideration the different type of substrates (Supplementary Fig. D).

      4. Discussion

      Insects are known to be attracted to crime scene and they can modify it producing artifacts in the environment on different surfaces. Fly artifacts may be located on lampshades, windows, walls, ceiling and on any other objects composed of absorbent and non-absorbent surfaces.
      In this study, fly artifacts sampled from absorbent surfaces, such as cotton or tissue paper, provided a greater success rate of amplification for fly DNA than non-absorbent surfaces, as glass and plexiglass. The higher negative result rate of molecular analysis of fly artifacts sampled from non-porous surfaces may be due to the feeding behavior and interaction of the flies with dried bloodstains [
      • Rivers D.B.
      Differential responses of adult Calliphora vicina to dry bloodstains on porous versus non-porous surface materials.
      ]. Likewise, also the adopted sampling technique might be the reason causing the reduced DNA retrieval. Indeed, the collection of fly artifacts from glass and plexiglass was carried out with swabs slightly soaked with sterile water to prevent flaking and dislodging of dried bloodstains, but increasing the risk to spread the stain on the surface. On the contrary, from porous surfaces the evidence is collected cutting the piece of fabric which is directly submitted to extraction thus preventing the loss of material.
      Regarding the evaluation of the chronological factor, our results showed that the non-significant decrease of the cytochrome c oxidase (COI) gene amplification occurred already after one month from fly artifacts deposition remained constant also after two years. These observations suggest that dated crime scenes could still provide information about the reconstruction events by molecular analysis of presumptive fly artifacts together with the fly species identification useful for post-mortem interval evaluation.

      5. Conclusion

      Although further research is needed to assess the effect of other variables as different food sources, environmental conditions and insects’ behaviors which could affect the DNA recovery and genotyping success, the fly artifacts identification could be also helpful to understand if human DNA transfer event occurred via insect vector addressing the activity level propositions.

      Conflict of interest statement

      The authors declare that they have no conflict of interest.

      Acknowledgments

      Authors would like to kindly thank Luigi Ruggeri for breeding and supplying pupae and adult flies of Calliphora vomitoria and Musca domestica.

      Appendix A. Supplementary material

      References

        • Rivers D.B.
        • Geiman T.
        Insect artifacts are more than just altered bloodstains.
        Insects. 2017; 8: 37
      1. Scientific Working Group on Bloodstain Pattern Analysis: Guidelines for the Minimum Educational and Training Requirements for Bloodstain Pattern Analysis (SWGSTAIN). Forensic Science Communications, Jan 2008 - Vol 10 - N. 1 https://archives.fbi.gov/archives/about-us/lab/forensic-science-communications/fsc/jan2008/standards/2008_01_standards01.htm.

        • Durdle A.
        • van Oorschot R.A.
        • Mitchell R.J.
        The morphology of fecal and regurgitation artifacts deposited by the blow fly Lucilia cuprina fed a diet of human blood.
        J. Forensic Sci. 2013; 58: 897-903
        • Kulstein G.
        • Amendt J.
        • Zehner R.
        Blow fly artifacts from blood and putrefaction fluid on various surfaces: a source for forensic STR typing.
        Èntomol. Exp. Appl. 2015; 3: 255-262
        • Bini C.
        • Giorgetti A.
        • Iuvaro A.
        • et al.
        A DNA-based method for distinction of fly artifacts from human bloodstains.
        Int. J. Leg. Med. 2021; 135: 2155-2161
        • Pelletti G.
        • Mazzotti M.C.
        • Fais P.
        • et al.
        Scanning electron microscopy in the identification of fly artifacts.
        Int. J. Leg. Med. 2019; 133: 1575-1580
        • Rivers D.B.
        Differential responses of adult Calliphora vicina to dry bloodstains on porous versus non-porous surface materials.
        Forensic Sci. Int. 2021; : 328