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Committee Approval Form.Indd UNIVERSITY OF CINCINNATI Date:___________________November 19, 2008 Trevor I. Stamper I, _______________________________________________________________, hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy in: Biological Science It is entitled: Improving the Accuracy of Postmortem Interval Estimations Using Carrion Flies (Diptera: Sarcophagidae, Calliphoridae and Muscidae) This work and its defense approved by: Chair: _______________________________Ronald W. DeBry _______________________________Theresa Culley _______________________________George Uetz _______________________________Gregory Dahlem _______________________________Anthony Perzigian Improving the Accuracy of Postmortem Interval Estimations Using Carrion Flies (Diptera: Sarcophagidae, Calliphoridae and Muscidae) A dissertation submitted to the Graduate School Of the University of Cincinnati In partial fulfillment of the requirements for the degree of Doctor of Philosophy In the Department of Biological Sciences Of the McMicken College of Arts and Sciences By Trevor I. Stamper M.A., Anthropology, New Mexico State University at Las Cruces, January 2002 B.A., Anthropology, New Mexico State University at Las Cruces, May 1997 Committee chair: Ronald W. DeBry Abstract The use of flies in forensic entomology in postmortem interval estimations is hindered by lack of information. For accurate postmortem interval estimations using flies, the single most important information is the species identity of the immature flies found upon a corpse. One of the three major groups that associate with carrion, the fleshflies (Diptera: Sarcophagidae), are difficult to identify in almost all life stages, especially in the larval stages commonly found upon carrion sources. Additionally, behavioral information on nocturnal oviposition activity amongst carrion flies in general is needed to resolve a time window of up to twelve hours in postmortem interval estimations. The two major goals of this study are: 1) to resolve the phylogenetic relationships of several major genera within the sub-family Sarcophaginae and 2) to investigate the behavioral patterns of nocturnal oviposition for carrion flies. Relationships of one of the major lineages of Sarcophagidae, the Sarcophaginae, remain unresolved. Most forensically important fleshfly species fall within the Sarcophaginae and so resolving this group can help identify unknown forensically important specimens. I analyzed the complete cytochrome oxidase I (COI) and cytochrome oxidase II (COII) mtDNA genes, along with portions of the dehydrogenase subunit four (ND4) mtDNA gene and elongation factor one-alpha nDNA gene from 21 species of fleshfly representing 11 genera, and four representatives from other closely related dipteran families. I confirm the monophyly of the family Sarcophagidae and the generic-level organization within the Sarcophaginae of those genera supported by multiple exemplars included in this study (Helicobia, Sarcophaga, Ravinia, Peckia and Boettcheria). The behavioral patterns of nocturnal oviposition represent a window of time that potentially has a large impact on postmortem interval estimations. I investigated the behavioral patterns of carrions flies at night by exposing euthanized rats between sunset and sunrise to see if carrion flies oviposited upon the carrion over three subsequent summers. I encountered significant methodological problems that I corrected. I also investigated urban and rural locations, in both lit and unlit conditions. In the end, I found that nocturnal ovipositing did not occur in the Cincinnati metropolitan area. ii iii Acknowledgements The molecular portion of this work was supported by Grant No. 2005-DA-BX-K102 awarded by the National Institute of Justice, Office of Justice Programs, US Department of Justice to Ronald W. DeBry. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the Department of Justice. The behavioral portion of this work was funded by multiple Wiemenn/Wendel/Benedict Summer Fellowships to Trevor Stamper and from the kind donations of previously euthanized rodents from Dr. Rebecca German and the Genome Research Institute. I have been blessed to have a wonderful committee to work with while developing my ideas and testing them. I thank my advisor, Ronald W. DeBry, for his support and trust that eventually, I would produce something to justify his time. Ron allowed an errant Anthropologist to come into his lab with precious little Biology experience, but a whole lot of interest in dead people and molecules. Without Ron’s unfailing support, my life would be very different than it is today. Much to his credit, he didn’t even complain when I started breeding flies in odd corners of the lab, kept asking for equipment that no self-respecting molecular biologist should need (who needs dress zippers?), or decided that hand sewing an outrageous number of collapsible traps was not that bad of an idea, especially since we couldn’t find a trap to do what I wanted mine to do. Ron has tried his best to keep me focused and it worked sometimes. I am also deeply indebted to Dr. Gregory Dahlem, who spent an awful lot of time teaching me about flies and how they shouldn’t all be ground up for DNA. We don’t see eye-to-eye that ALL flies should be preserved carefully, but I grind up far fewer than I used to, and have even dedicated a portion of my (as of yet unfinished) house to their proper storage and preservation! Greg went out of his way to help me learn about teaching in academics, mentoring me through the PFF program and providing me with opportunities to teach when our interests aligned. I owe special thanks to Dr. Theresa Culley, who listened to and talked me through organizing large portions of the projects detailed herein and many that are not. Theresa spent countless hours editing manuscripts for me, often with little notice and was always available to listen to my latest crazy idea on how to use biology in postmortem interval estimations. She is a dedicated educator who is always willing to listen to a student and has really good insights. I am a better researcher and teacher for knowing and learning from her. Dr. George Uetz was willing to step iv outside the life of spiders for a while to help me investigate fly behavior (or lack thereof) on carcasses at night, and his insight into insect behavior was often dead-on despite my protests. Dr. Anthony Perzigian kindly agreed to come over from Anthropology and lend his insight into Jurisprudence and Forensic Anthropology to my work. While our time discussing these topics was necessarily limited by his busy schedule, his understanding of how science and the justice system work hand-in-hand was invaluable. I would also like to thank the other members of the department who have proved so helpful. Specifically, Dr. Carl Huether, who directed the Preparing Future Faculty program until recently, helped me navigate that program’s requirements in addition to my normal lab commitments. Finally, to the office staff: without you, this department wouldn’t function. Thanks a lot for helping me navigate the esoteric byways of the University of Cincinnati graduate system. I am indebted to my colleagues in the DeBry lab. First, there is Dr. Hector Miranda, who helped me get going on molecular biology and patiently explained many of the common mistakes I routinely made. Second, to Mr. David Hauber, who retired from the crime lab system in Kentucky to escape forensics only to find himself in a lab with someone who wanted to talk about nothing but forensics (and dead people). David has been a nearly constant companion my many years in the DeBry lab, and his advice and expertise in many things, professional and personal, has always been helpful. Finally, to Dr. Alicia Timm, who came into the lab late in the game, but nevertheless helped me with the final push to finish sequencing a seemingly endless number of species and who saved my bacon with a few problematic specimens. Several undergraduate students came into the DeBry lab during my tenure who have helped with these projects. Colin White studied under me while learning molecular lab techniques. Colin helped characterize some of the PCR settings used in the molecular portion of my work, and helped me cut out net trap templates during 2004. Paula Davis came into the lab under the auspices of the WISE program and helped with both some of the last of the molecular work and spent a summer driving around Cincinnati dropping off and picking up dead rodents. Without Paula’s help, running four sites at the same time for nocturnal ovipositing would have never happened! Outside of the obvious influences from work, others have helped me with this research. I would like to thank Mathew Klooster, Brian and Laura Gilkison, Christine and Brian Moskalik, Colin White and v my wife, Christina, all of whom let me place dead rats in their yards just after sunset for several years, in the name of science. This is a sacrifice not common to most people, who get a bit squimish around dead rats. Thank-you all, and I hope I didn’t wake anyone up when I came to collect the poor little dead rats before dawn! I extend special thanks to me friends and family, almost all of whom have had to deal with my little obsession for collecting “forensically important” flies using decomposing chicken in mason jars for the past half decade, and many of whom have had to mail pupae back to me because my stay was too short to accommodate the fly maturation process and those poor fly maggots just needed “a few more days” eating the bacteria rotting chicken to grow up big and strong. Special attention needs to be paid to the following debts in preparing this manuscript. Michael P. Cosentino spent many hours editing drafts of various portions of this work (and others). There is passive voice left in these chapters, and it is my fault, not his.
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