Regardless of the mechanism of artifact production used by necrophagous flies, there is no method for reliably distinguishing between insect-generated artifacts and human bloodstains. The only recourse has been to attempt to identify fly artifacts based on unique visual characteristics of the insect stains coupled with contextual analysis (i.e., location of stains in relation to the crime scene).

There are several reasons why this approach to insect stain identification is not appropriate, but the most concerning is that any method of visual and/or contextual analysis of fly artifacts relies almost exclusively on the experience and opinion of the analyst rather than on standardized and reproducible methodology. The inability to consistently and reliably distinguish fly artifacts from bloodstains and other body fluids represents a major limitation with respect to analyses of entomological contaminants at crime scenes.

A New Diagnostic Tool

One avenue that has not been explored with respect to insect artifacts is the development of confirmatory tests based on the chemical composition of insect-derived stains. Recent research by our lab demonstrated that regurgitate stains deposited by Protophormia terraenovae Robineau-Desvoidy (Diptera: Calliphoridae) possessed at least three digestive enzymes (trypsin-like, chymotrypsin-like, and pepsin-like) that were also found in the crop of the adult fly, independent of the food source.

The pepsin-like enzyme appears to be a cathepsin D-like proteinase adapted for functionality in the strongly acidic midgut environment of larvae and adult flies. Sequence analyses of digestive cathepsin D-like proteinase from the common house fly Musca domestica L. (Diptera: Muscidae) show that the enzyme lacks the proline loop (of motif DxPxPx(G/A)P)) typical of other insects as well as vertebrates, yielding a functional protein similar to vertebrate pepsin. Thus, the cathepsin D-like proteinase in M. domestica is functionally similar to vertebrate pepsin but is structurally a cathepsin D aspartic proteinase.

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. Additional testing of anti-md3 serum has shown that the antiserum reacts positively with digestive artifacts produced from 27 species of necrophagous flies representing 9 families of Diptera.

The utility of the antiserum is not restricted to just distinction of bloodstains, as fly artifacts have also been shown to be identified from human semen, saliva, feces and urine. Further work is continuing to determine whether the antisera are suitable for non-laboratory conditions.