Proteolytic enzyme activity in the foregut region of adults of Protophormia terraenovae and implications for identification of fly spots

Several species of flies can hinder investigation at a crime scene by leaving artifacts (spots or speaks) that can be easily mistaken for bloodstains.  Presently, there is no diagnostic test for the identification of these fly artifacts, leaving subjective interpretation as the only means available to distinguish fly evidence from bloodstains.  The current approach is unsatisfactory because it is not quantifiable and very few individuals are qualified to make the distinctions between the two types of stains.  In this research, we tested the hypothesis that fly regurgitate (i.e., digestive fluid at least partially composed of crop contents), contains a fly-specific enzyme that can be detected in fly spots but not in human blood.   To do so, proteolytic enzymes were characterized in the foregut of adult males and females of Protophormia terraenovae, enzymatic activity in fly regurgitate was compared to adult flies, and polyclonal antibodies were generated against synthetic peptides based on cathepsin D-like proteinase from larvae of Musca domestica   The antibodies were used for western blot and dot blot analyses of blood stains and fly regurgitate spots.    Based on substrate specificity, pH optima, and use of specific protease inhibitors, all adults tested displayed enzyme activity in the foregut consistent with pepsin, trypsin and chymotrypsin.  Chymotrypsin-like and trypsin-like enzyme activity were detected in all gut fluids and tissues tested, with chymotrypsin displaying the highest activity in saliva and salivary gland tissue, whereas maximal trypsin activity was evident in the crop.  Pepsin-like activity was only evident in crop fluids and tissues.   Pepsin-like activity was presumed to be cathepsin D-like proteinase based on antibody binding, enzymatic analyses, and molecular mass.  The enzyme was detectable in fly regurgitate but not in human or bovine blood.