Distinction of Fly Artifacts from Human Blood using Immunodetection

Abstract

Insect stains produced by necrophagous flies are indistinguishable morphologically from human bloodstains. At present, no diagnostic tests exist to overcome this deficiency. As the first step toward developing a chemical test to recognize fly artifacts, polyclonal antisera were generated in rats against three distinct antigenic sequences of fly cathepsin D‐like proteinase, an enzyme that is structurally distinct in cyclorrhaphous Diptera from other animals. The resulting rat antisera bound to artifacts produced by Protophormia terraenovae and synthetic peptides used to generate the polyclonal antisera, but not with any type of mammalian blood tested in immunoassays. Among the three antisera, anti‐md3 serum displayed the highest reactivity for fly stains, demonstrated cross‐reactivity for all synthetic peptides representing antigenic sequences of the mature fly enzyme, and bound artifacts originating from the fly digestive tract. Further work is needed to determine whether the antisera are suitable for non‐laboratory conditions.

A DNA-based method for distinction of fly artifacts from human bloodstains

“…Since Rivers et al [10][11][12] has reported that only artifacts derived from alimentary canal of the adult flies reacted positively to the immunoassay test, we have chosen to analyze fly artifacts more likely originated from regurgitation or fecal elimination processes, excluding transfer pattern stains produced by tarsi or other body parts, because fly touch DNA was not expected.…”

“…In the last years, Rivers et al [10,11] tested a polyclonal antiserum (anti-md3 serum) generated toward a unique cathepsin D proteinase that has been shown to react with regurgitate and defecatory fly artifacts produced by different species of blowflies, but not with transfer patterns or blood controls from humans or other animals. Recently, the study was extended to other stains produced by different species of flies following the consumption of semen, saliva, feces, and urine, showing that more than 94% of fly artifacts reacted positively with anti-md3 [12].…”

A review of Forensic Entomology literature in the Northeastern United States

“…A sizable amount of work has been done on fly artifacts, including various detection techniques (Rivers et al, 2018; Rivers et al, 2019; Rivers, Cavanagh, Greisman, Brogan, & Schoeffield, 2020) and analyses of their stains (Rivers, Dunphy, Hammerschmidt, & Carrigan, 2020; Rivers & Geiman, 2017; Rivers & McGregor, 2018). Other work relating to entomotoxicology found that both opiates (Introna, Dico, Caplan, & Smialek, 1990) and barbiturates (Levine, Golle, & Smialek, 2000) could be obtained from larvae feeding on remains that had ingested these drugs.…”

Insects as vectors of DNA in a forensic context

“…The authors report that the antiserum is able to detect the synthetic peptide created for the development of the assay to a dilution of 1:500,000. While the occasional false negative has occurred, the test shows real promise (Rivers et al, ; D. B. Rivers et al, 2019). Strong positive reactions have been obtained from artifacts stored for 3, 5, and 7 years at 25°C.…”

“…Strong positive reactions have been obtained from artifacts stored for 3, 5, and 7 years at 25°C. However, the group is yet to test whether chemical alteration of the artifacts through application of cleaning products or extended exposure to environmental conditions such as ultraviolet light, might impact on the efficacy of the test, and whether the antisera would be effective on fly artifacts deposited after feeding on human biological material other than blood, or artifacts deposited in the field (Rivers et al, ; D. B. Rivers et al, 2019).…”