title: Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically Important Chrysomya rufifacies (Diptera: Calliphoridae).
abstract: Entomological protocols for aging blowfly (Diptera: Calliphoridae) larvae to estimate 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, genetic analysis, or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically important blowfly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, nonbleaching, autofluorescence of larval posterior spiracles. High-content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC.
authors.affiliation: Laboratory of Immunology and Signal Transduction, Division of Natural Sciences and Mathematics, Chaminade University, 3140 Waialae Avenue, Honolulu, HI, 96816.|Graduate Program in Molecular Biosciences and Bioengineering, University of Hawaii at Mānoa, 1650 East-West Road, Honolulu, HI, 96816.
authors.forename: Danielle
authors.initials: D
authors.lastname: Flores
authors.affiliation: Graduate Program in Molecular Biosciences and Bioengineering, University of Hawaii at Mānoa, 1650 East-West Road, Honolulu, HI, 96816.
authors.forename: Amy L
authors.identifier: http://orcid.org/0000-0003-4521-2763
authors.initials: AL
authors.lastname: Miller
authors.affiliation: Laboratory of Immunology and Signal Transduction, Division of Natural Sciences and Mathematics, Chaminade University, 3140 Waialae Avenue, Honolulu, HI, 96816.|Laboratory of Forensic Taphonomy, Division of Natural Sciences and Mathematics, Chaminade University, 3140 Waialae Avenue, Honolulu, HI, 96816.
authors.forename: Angelique
authors.initials: A
authors.lastname: Showman
authors.affiliation: Laboratory of Immunology and Signal Transduction, Division of Natural Sciences and Mathematics, Chaminade University, 3140 Waialae Avenue, Honolulu, HI, 96816.
authors.forename: Caitlyn
authors.initials: C
authors.lastname: Tobita
authors.affiliation: Laboratory of Immunology and Signal Transduction, Division of Natural Sciences and Mathematics, Chaminade University, 3140 Waialae Avenue, Honolulu, HI, 96816.
authors.forename: Lori M N
authors.initials: LM
authors.lastname: Shimoda
authors.affiliation: Laboratory of Immunology and Signal Transduction, Division of Natural Sciences and Mathematics, Chaminade University, 3140 Waialae Avenue, Honolulu, HI, 96816.
authors.forename: Carl
authors.initials: C
authors.lastname: Sung
authors.affiliation: Laboratory of Experimental Medicine, John A. Burns School of Medicine, University of Hawaìi, 651 Ilalo Street, Honolulu, HI, 96813.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Stokes
authors.affiliation: Department of Entomology, Texas A&M University, College Station, TX, 77843.
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Tomberlin
authors.affiliation: Graduate Program in Molecular Biosciences and Bioengineering, University of Hawaii at Mānoa, 1650 East-West Road, Honolulu, HI, 96816.
authors.forename: David O
authors.initials: DO
authors.lastname: Carter
authors.affiliation: Laboratory of Immunology and Signal Transduction, Division of Natural Sciences and Mathematics, Chaminade University, 3140 Waialae Avenue, Honolulu, HI, 96816.
authors.forename: Helen
authors.initials: H
authors.lastname: Turner
mesh_terms: D000818:Animals; D004175:Diptera; D004772:Entomology; D005453:Fluorescence; D007814:Larva; D008853:Microscopy; D010865:Pilot Projects; D011180:Postmortem Changes; D013997:Time Factors
keywords: calliphoridae; fluorescence microscopy; forensic entomology; forensic science; postmortem interval