1st Place: Generating fluorescent blow fly strains to aid in validation of postmortem interval estimates

The age of a blow fly larva is commonly used in forensic investigations to estimate the minimum postmortem interval (PMImin). In forensic entomology, a maggot of unknown age is compared to experimental reference data to predict its age adjusted for temperature. However, typical reference data illustrate development rate under artificial conditions. Current practices do not satisfy an important criterion for expert testimony: that a technique has a known error rate under realistic conditions. This would require empirical validation, but such experiments have not been performed in forensic entomology. Under realistic conditions, the true age of a maggot is unknown other than it cannot be older than when the corpse was first exposed. If a larva could be persistently marked, eggs of known age could be added to the wild population, and would have developed under casework conditions with a known age. We microinjected 1391 pre-blastoderm Phormia regina embryos with a piggyBac donor plasmid containing a marker gene, DsRed-Express2, driven by a heterologous constitutive promoter. The survival rate post-injection was 33%. Surviving adults were outcrossed to wild type and the G1 offspring were screened for fluorescence. We found 8 positive larvae from one injected cross and the estimated transformation rate was 1.8%. The stable transgenic line expresses DsRed across all life stages. The insertion(s) will be characterized by next generation sequencing and assessed for any alteration of development. This marks a step forward in the field as they will facilitate the validation of growth rate models used for PMImin predictions.