LARVAL AGE DETERMINATION IN FORENSICALLY IMPORTANT INSECT SPECIES USING BIOCHEMICAL AND FLUORESCENCE MICROSCOPY TECHNIQUES
Caitlyn Tobita, Helen Turner, Danielle Flores, Amy Miller-Weber, Angelique Showman, Lori Shimoda, Carl Sung, David Carter.
Chaminade University of Honolulu, Honolulu, HI.
Entomological methodologies for analyzing fly larvae for estimating the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analyzing fly larvae differ, most rely on light microscopy or electron microscopy. The former is low resolution but tractable, the latter is resource intensive and suitable for only small sample sets. This study served as a proof of concept in the forensically important Chrysomya rufifacies, where we sought to combine high-content fluorescence miscopy and biochemical measures of developmental marker proteins to improve resolution of larval age. We applied confocal microscopy and observed robust, non-bleaching, autofluorescence of appropriately prepared larval posterior sections. Here we describe a pilot study that established fixation and mounting protocols, defined a set of measurable morphometric criteria, and captured developmental transitions of second instar to third instars using both fluorescence microscopy and anti-ecdysone receptor western blot analysis. The data show that these instar stages can be distinguished on the basis of epi-fluorescent microscopy and confocal microscopy. High content imaging techniques using confocal microscopy, combined with morphometry and biochemical techniques, may therefore aid forensic entomologists in determining estimated TOC.