Proteomic and microRNA markers of fly development: Stable correlates of insect age to improve forensic entomology casework

As submitted by the proposer: Insect evidence can provide valuable information during death investigations. Since carrion-feeding flies often colonize remains soon after death, estimates of insect age derived from immature insects collected from a body can provide information regarding the timing of a death, including the postmortem interval itself if some assumptions are satisfied. Predictions of insect age rely on temporal correlates of age (traditionally developmental stage or size) on or in an insect specimen and recently mRNA and cuticular hydrocarbons have been promoted as molecular markers of insect age that can provide more precise estimates of insect age as compared to traditional physical markers of age. However, both of these types of molecules have limitations. Cuticular hydrocarbons are known to differ among populations of insects that can be found in different thermal environments, as one function of these molecules is to limit insect desiccation and it is unknown if they are affected by storage in ethanol or other common preservatives of immature insects. Further, mRNA is much more unstable than DNA as evidence – a feature that limits its usefulness in real-world settings as casework samples are often not ideal for molecular biology applications and cultural shifts in preservation practices are difficult to implement. This project is designed to identify molecular markers (microRNAs and proteins) that are more likely to be preserved during the normal collection of insect evidence and provide similar types of precise information regarding the developmental progress of immature insects of forensic importance. The project will target a stage of development associated with imprecise estimates of age and evaluate the usefulness of microRNA and proteins in developing more precise estimates of insect age through the application of high throughput sequencing on the HiSeq platform and 2D-DIGE proteomic experiments coupled with mass spectrographic identification of differentially expressed protein spots. This process will be done in four separate species of forensic importance, including one that is common in casework in the Houston area but has little developmental data associated with it. The next step of the proposal will be to confirm the genetic and environmental stability of identified markers and to validate the usefulness of these analyses by doing blind validations of the methods with mock casework samples. The successful completion of the project should move the field toward doing actual casework with molecular markers of age - improving the precision of age estimates compared to traditional methods. This project contains a research and/or development component, as defined in applicable law. ca/ncf