Germ-Line Transformation of Forensically Important Flies

Determining when death occurred is important for a homicide investigation. One common way to do this is to estimate the age of an insect associated with the victim based on the extent of its development (e.g., size and life stage). Because some insects begin their life when deposited as an egg on a corpse, an estimate of that insect's age provides a minimum time the person must have been dead. A fundamental legal standard of acceptance of forensic evidence in court is that the analytical method should have been empirically validated. That is, its accuracy and reliability should have been demonstrated under conditions closely resembling those of a crime scene. However, until now it has not been possible to assess the accuracy of carrion insect age estimation methods, which are derived from laboratory data, when they are applied to real cases. This is because the true age of an insect on a corpse cannot be known, so the accuracy of an estimate of its age cannot be known. We propose to overcome that problem by genetically transforming carrion insects so that all life stages will contain a protein that glows when illuminated by a particular wavelength of light. Both red and green proteins are available, and will be used for each experimental insect species. Genetically transformed insects must then be tested to be sure that the transformation process did not alter the insects development rate. Once such modified insects are available, and as part of future research, eggs of known age from a lab colony, added to an experimental corpse, can be located on the corpse at a later time because they fluoresce. These specimens of known age that grew under realistic conditions can be used to test the accuracy of methods currently employed to estimate a minimum time since death. For this project we propose to: 1) use germ-line transformation to produce laboratory colonies of two species of forensically important flies, so that all life stages will contain fluorescent protein in their bodies, and 2) compare laboratory growth and development rates of the transformed flies to the original strains of untransformed flies, to be sure that this procedure did not alter the forensically important aspects of their biology. Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). CA/NCF