The author(s) shown below used Federal funding provided by the U.S. Department of Justice to prepare the following resource: Document Title: DNA-based Identification of Forensically Important Diptera Author(s): Ronald W. DeBry Document Number: 253080 Date Received: July 2019 Award Number: 2005-DA-BX-K102 This resource has not been published by the U.S. Department of Justice. This resource is being made publically available through the Office of Justice Programs’ National Criminal Justice Reference Service. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Final Technical Report 2005-DA-BX-K102 DNA-based identification of forensically important Diptera, 2005-DA-BX-K102, Ronald W. DeBry Abstract Flies (Diptera) from three families (Calliphoridae, Sarcophagidae, Muscidae) are normally the first insect visitors to an exposed body. Many species of dipterans either lay eggs or deposit live larvae into a body within hours of death. By determining both the species identity and the age of larvae present at a human death scene at the time of discovery, it is possible to estimate the post-mortem interval. With regard to species identity, common practice requires collection of live larvae that are maintained until they achieve adulthood, at which time they can be identified to species using morphological characteristics. Purpose: The purpose of this project is to develop a reference database of DNA sequences that is suitable for species-level identification of forensically relevant Diptera from the 48 contiguous United States. We focus here on flies in the families Calliphoridae and Sarcophagidae. This reference database will be suitable for querying DNA sequences obtained from larvae collected at a death scene. Goals and Objectives: Goals of this project were to: 1) Collect reference adult dipteran specimens from across the 48 contiguous states. Specific attention was to be given to collecting specimens from widely spread geographic localities, with an emphasis on the margins of the relevant area. 2) Identify a genetic locus that is suitable for discrimination of relevant species in a phylogenetic analysis. 3) Develop a reference DNA database. The database will have associated with it all original physical specimens preserved as vouchers, with sufficient morphological features retained so as to allow future re-examination of the specimens. Research subjects: The research subjects for this project are flies (Diptera) of the families Calliphoridae, Sarcophagidae, and Muscidae. Research Design: The project is divided into two phases. In Phase I, we examine a range of possible genetic loci from a limited number of species and individuals, in order to assess the relative performance of the different loci (and regions therein) at discriminating among species. In Phase II, data are collected from a large number of species and individuals for the locus determined in Phase I to be best for discrimination among forensically important species. Methods: Adult flies were collected both using traps baited with meat and by sweep netting. Specimens were identified using morphological characteristics. Those flies from the forensically relevant families were identified to the species level. A small portion of each specimen (usually 1-3 legs) was removed and used for DNA extraction. Loci of interest were amplified by the polymerase chain reaction, and the DNA sequence was determined for the resulting product. The utility of the final reference database was assessed by phylogenetic analysis with bootstrap resampling using 1000 pseudoreplicates. Results: Over 2,500 flies were collected and identified from 84 different counties in 20 different states. In Phase I, it was determined that any region of the mitochondrial genome was likely to be sufficient for discrimination among most forensically relevant species, but that a region within the mitochondrially encoded gene Cytochrome oxidase I was best for discrimination between a particularly closely related pair of species. In Phase II, DNA This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. sequence for the chosen locus was obtained for a total of 504 individual flies representing 106 distinct species. Conclusions: The reference DNA database is suitable (based on bootstrap support values >95%) for correct species identification of 16 of the 18 species that are most important for forensic investigations. One pair (Lucilia coeruleiviridis and Lucilia mexicana) are indistinguishable, apparently for any mitochondrial locus. A further 16 of 20 species represented in our database that are deemed to be of lesser forensic utility can be unambiguously identified using phylogenetic analysis of the reference database, with two species pairs (both in the genus Ravinia) being indistinguishable. Table of Contents: Abstract 1 Executive Summary 2 Introduction 8 Review of relevant literature 9 Statement of rationale 16 Methods 16 Results 20 Remaining issues 29 Tables 29 Figures 186 Conclusions 193 References 196 Dissemination of research findings 200 Executive Summary Synopsis of the problem. A fundamental task in death investigation is determination of the post-mortem interval (PMI). One tool that can sometimes be used for PMI estimation is the developmental stage of carrion-feeding insects (forensic entomology). During the relatively early stages of decomposition, the primary entomological evidence is obtained by determining the developmental stage and species identification of larval flies (Diptera), primarily of the families Calliphoridae, Sarcophagidae, and Muscidae. Species identification is crucial, because different species develop at different rates under the same environmental conditions. Species identification by gross morphology requires adult flies in a large proportion of cases, because larvae of many species at many age ranges often do not carry sufficient distinguishing characteristics. Existing practice requires collection of live larvae, with (generally) transfer of those larvae to an established forensic entomologist, who will rear the larvae to adulthood (ca. 1-2 weeks in most cases) and then make the species determination. Possibly in large part because of this step, entomological evidence is generally under-utilized in death investigation in the United States. For a number of years, an important goal in forensic entomology has been to This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. develop a method capable of providing accurate species identification directly from the dipteran larvae. Purpose. The goal of this project is to provide the tools needed for quick, reliable species identification of forensically important flies directly from larvae. To accomplish this goal, we have developed a database of DNA sequences from a known-identity reference collection. The original reference collection consists of adult flies that have been identified to species by an expert in the taxonomy of the group, and is maintained as vouchers for future morphological study, should the need arise. The functional part of the identification database is the set of DNA sequences generated from those vouchers. It is presented as an appendix to this report. We have further developed PCR primers that are useful for amplification of a DNA segment that is appropriate for discrimination among the large majority of the species of Diptera that are likely to be encountered in an early-stage forensic setting in the contiguous United States. Research design. Development of a list of the primary forensically important species of Diptera: We examined a large variety of relevant literature for lists of dipteran species that had been identified either as being encountered in a forensic investigation or as having a life cycle that is associated with large animal carrion, with the focus being on flies that are likely to be among the earliest colonizers of a corpse. That process led us to develop the list of forensically most important species, which are shown in red boldface in Table 1 of the main body of this report. In total, we identified 11 species of Calliphoridae (blow flies), 6 species of Sarcophagidae (flesh flies), and 2 species of Muscidae (house flies) as being the dipterans most likely to be relevant for a forensic death investigation in the contiguous United States. Sampling strategy: Our sampling strategy had the following goals: 1. Maximize the probability that we will obtain samples for all of the species of primary forensic importance in the contiguous United States. 2. Ensure that we also sample species that, while not of primary importance, have been reported as being associated with large-animal carrion. 3. Maximize the geographic coverage for those species of primary forensic importance that have large geographic ranges. 4. Maximize the total species coverage of the reference database. A reference database for species identification should include as many species as possible that both are found in the relevant geographic area and are phylogenetically related to the species