A Three‐Locus,PCR‐based Method for Forensic Identification of Plant Material

Plant residue is currently an underutilized resource in forensic investigations despite the fact that many crime scenes, as well as suspects and victims, harbor plant‐derived residue that could be recovered and analyzed. Notwithstanding the considerable skill of forensic botanists, current methods of species determination could benefit from tools for DNA‐based species identification. However, DNA barcoding in plants has been hampered by sequence complications in the plant genome. Following a database search for usable barcodes, broad‐spectrum primers were designed and utilized to amplify and sequence the rbcL, trnL‐F, and rrn18 genetic loci from a variety of household plants. Once obtained, these DNA sequences were used to design species‐targeted primers that could successfully discriminate the source of plant residue from among the 21 species tested.     

Molecular Identification of Necrophagous Dermestes Species in South Korea Using Cytochrome c Oxidase Subunit I Nucleotide Sequences (Genus Dermestes)

Species identification of necrophagous insects found on a dead body is an essential key in applying medicolegal entomology to the estimation of postmortem interval (PMI). Due to limited morphological identification of insect evidence, several studies have identified species using molecular information such as DNA markers. While considerable cytochrome c oxidase subunit I (COI) gene sequence data of necrophagous fly species have been collected and annotated, those of necrophagous beetle species have not. Since necrophagous beetles such as Dermestes species have a larval period longer than that of flies, beetles are useful in even the late decomposition phase in estimating minimum PMI. To obtain the full-length COI gene sequences of six Dermestes species collected from South Korea, we designed primers for polymerase chain reaction amplification and sequencing. The obtained full COI nucleotide sequences were used for performing phylogenic analysis and comparison with previously reported sequences. The results demonstrated that the COI gene sequences could be used to identify forensically important Dermestes species in South Korea.     

Forensic Entomology and the Estimation of the Minimum Time Since Death in Indoor Cases

Eight cases that occurred indoors in which the insects played an important role in the mPMI estimation are presented. The bodies of socially isolated people and old people living alone were discovered in central Italy between June and November. mPMI ranged from a few days to several weeks. Insects were collected during the body recovery and the postmortem. Climatic data were obtained from the closest meteorological stations and from measurements performed on the site. Sarcophagidae and Calliphoridae species were present in 75% of the cases with Lucilia sericata and Chrysomya albiceps collected in 50% of the cases. Chrysomya albiceps was always found in association with Lucilia species. Scuttle flies (Phoridae) were found in 37.5% of the cases, confirming the ability of these species in indoor body colonization. We show that if sealed environment may delay, the insect arrival dirty houses may create the environment where sarcosaprophagous insects are already present.     

DNA Barcoding Identifies all Immature Life Stages of a Forensically Important Flesh Fly (Diptera: Sarcophagidae)

Carrion‐breeding insects, such as flesh flies (Diptera: Sarcophagidae), can be used as evidence in forensic investigations. Despite their considerable forensic potential, their use has been limited because morphological species identification, at any life stage, is very challenging. This study investigated whether DNA could be extracted and cytochrome oxidase subunit I (COI) barcode sequences obtained for molecular identification of each immature life stage of the forensically important Australian flesh fly, Sarcophaga (Sarcorohdendorfia) impatiens (Walker). Genomic DNA extracts were prepared from all larval instars and puparia. Amplification of the barcoding region was successful from all extracts, but puparia amplicons were weak. All sequences were identified as S. impatiens with 99.95% confidence using the Barcoding of Life Database (BOLD). Importantly, crop removal was necessary to eliminate PCR inhibition for specimens from late second and early third instars. Similar results are expected for immatures of other carrion‐breeding species, enhancing the use of evidence from immature flies in forensic investigations.     

Identification and Individualization of Lophophora using DNA Analysis of the trnL/trnF Region and rbcL Gene

Lophophora williamsii (peyote) is a small, spineless, greenish‐blue cactus found in Mexico and the southwestern United States. Ingestion of the cactus can result in hallucinations due to its content of mescaline. In the United States, L. williamsii is classified as a Schedule I controlled substance. In this study, we use DNA analysis of the chloroplast trnL/trnF region and chloroplast rbcL gene to identify the individuals of Lophophora. Using the rbcL gene, Lophophora specimens could be distinguished from outgroups, but species within the genus could not be distinguished. The trnL/trnF region split the Lophophora genus into several groups based on the length and substructure of an AT‐rich segment of the sequence. Our results indicate that the genetic variability at the trnL/trnF locus is greater than previously recognized. Although DNA structures at the trnL/trnF region and rbcL gene do not align with the classification of Lophophora species, they can be used to aid in forensic analysis.     

Validation of the barcoding gene COI for use in forensic genetic species identification

The application of forensics to wildlife crime investigation routinely involves genetic species identification based on DNA sequence similarity. This work can be hindered by a lack of authenticated reference DNA sequence data resulting in weak matches between evidence and reference samples. The introduction of DNA barcoding has highlighted the expanding use of the mtDNA gene, cytochrome c oxidase I (COI), as a genetic marker for species identification. Here, we assess the COI gene for use in forensic analysis following published human validation guidelines. Validation experiments investigated reproducibility, heteroplasmy, mixed DNA, DNA template concentration, chemical treatments, substrate variation, environmental conditions and thermocycling parameters. Sequence similarity searches using both GenBank BLASTn and BOLD search engines indicated that the COI gene consistently identifies species where authenticated reference sequence data exists. Where misidentification occurred the cause was attributable to either erroneous reference sequences from published data, or lack of primer specificity. Although amplification failure was observed under certain sample treatments, there was no evidence of environmentally induced sequence mutation in those sequences that were generated. A simulated case study compared the performance of COI and cytochrome b mtDNA genes. Findings are discussed in relation to the utility of the COI gene in forensic species identification.     

Species Identification of Cannabis sativa Using Real‐Time Quantitative PCR (qPCR),

Most narcotics‐related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3′ exon‐trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real‐time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa.     

Molecular Identification of Three Indian Snake Species Using Simple PCR–RFLP Method*

Abstract: Three endangered Indian snake species, Python molurus, Naja naja, and Xenochrophis piscator are known to be significantly involved in illegal trade. Effective authentication of species is required to curb this illegal trade. In the absence of morphological features, molecular identification techniques hold promise to address the issue of species identification. We present an effective PCR–restriction fragment length polymorphism method for easy identification of the three endangered snake species, Python molurus, Naja naja, and Xenochrophis piscator. A 431‐bp amplicon from cytochrome b gene was amplified using novel snake‐specific primers following restriction digestion with enzymes Mbo II and Fok I. The species‐specific reference fragment patterns were obtained for the target species, which enabled successful identification of even highly degraded shed skin sample confirming the utility of the technique in case of poor‐quality DNA. The assay could be effectively used for forensic authentication of three Indian snake species and would help strengthen conservation efforts.     

A Grass Molecular Identification System for Forensic Botany: A Critical Evaluation of the Strengths and Limitations*

Abstract: Plant material is frequently encountered in criminal investigations but often overlooked as potential evidence. We designed a DNA‐based molecular identification system for 100 Australian grasses that consisted of a series of polymerase chain reaction assays that enabled the progressive identification of grasses to different taxonomic levels. The identification system was based on DNA sequence variation at four chloroplast and two mitochondrial loci. Seventeen informative indels and 68 single‐nucleotide polymorphisms were utilized as molecular markers for subfamily to species‐level identification. To identify an unknown sample to subfamily level required a minimum of four markers or nine markers for species identification. The accuracy of the system was confirmed by blind tests. We have demonstrated “proof of concept” of a molecular identification system for trace botanical samples. Our evaluation suggests that the adoption of a system that combines this approach with DNA sequencing could assist the morphological identification of grasses found as forensic evidence.     

Comparison of Decomposition Rates and Faunal Colonization of Carrion in Indoor and Outdoor Environments

Abstract: Decomposition and insect colonization of pig (Sus scrofa L.) carcasses were observed over a 42‐day period inside and outside a house in a suburban region of Edmonton, Alberta. Three freshly killed pig carcasses were placed outdoors on grass and three carcasses were placed in separate rooms inside a house in a suburban residential area. The carcasses were examined and sampled regularly. Outside carcasses were colonized rapidly by Calliphora vicina (R‐D), Lucilia sericata (Meigen), Lucilia illustris (Meigen), Eucalliphora latifrons (Hough), Cynomya cadaverina (R‐D), Phormia regina (Meigen), and Protophormia terraenovae (R‐D). There was a delay of 5 days before inside carcasses were first colonized but all species except E. latifrons and L. illustris readily colonized at this time. Many more insects colonized the outside carcasses, and these were rapidly skeletonized. Inside, much fewer insects were present and decomposition was slowed and colonization extended. Dispersal patterns of postfeeding larvae inside the house on hard substrate were greatly expanded compared with those from carcasses on grass.     

Forensic Identification of Indian Snakeroot (Rauvolfia serpentina Benth. ex Kurz) Using DNA Barcoding

Indian snakeroot (Rauvolfia serpentina) is a valuable forest product, root extracts of which are used as an antihypertensive drug. Increasing demand led to overharvesting in the wild. Control of international trade is hampered by the inability to identify root samples to the species level. We therefore evaluated the potential of molecular identification by searching for species‐specific DNA polymorphisms. We found two species‐specific indels in the rps16 intron region for R. serpentina. Our DNA barcoding method was tested for its specificity, reproducibility, sensitivity and stability. We included samples of various tissues and ages, which had been treated differently for preservation. DNA extractions were tested in a range of amplification settings and dilutions. Species‐specific rps16 intron sequences were obtained from 79 herbarium accessions and one confiscated root, encompassing 39 different species. Our results demonstrate that molecular analysis provides new perspectives for forensic identification of Indian snakeroot.     

Molecular Identification of Indian Crocodile Species: PCR‐RFLP Method for Forensic Authentication*

Abstract: South East Asian countries are known for illegal poaching and trade of crocodiles clandestinely, to be used in skin, medicinal, and cosmetic industries. Besides crocodiles being listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, India has its Wildlife Protection Act, 1972 for conservation of crocodile species. Hitherto, lack of any rapid and reliable technique for examinations of crocodile‐based crime exhibits such as skin, bones, etc. has been a major problem for an effective promulgation of law on illegal trade. DNA‐based identification of species using PCR‐RFLP technique for an apt identification of all the three Indian crocodile species namely, Crocodylus porosus, Crocodylus palustris and Gavialis gangeticus is presented here. A 628 bp segment of cytochrome b gene was amplified using novel primers followed by restriction digestion with three enzymes i.e., HaeIII, MboI, and MwoI, separately and in combination. The technique has produced a species‐specific pattern for identifying the three crocodile species individually, which fulfills the requirement for its forensic application. It is expected that the technique will prove handy in identification of all the three Indian crocodile species and strengthen conservation efforts.     

Arthropods of Forensic Importance on Pig Carrion in the Coahuilan Semidesert,Mexico

Abstract: This is the first report of an ongoing research to establish a sarcosaprophagous arthropod database in the Coahuilan semidesert. Seven pigs (Sus scrofa L.) were used as human models to determine succession in an open urban area during the 2007 winter–spring period. Arthropods were collected manually and from pitfall traps. Carcass biomass loss, as well as arthropod colonization, was recorded during 71 days postmortem. Five decomposition stages were identified during which most abundant orders were found to be Diptera, Coleoptera, and Hymenoptera. Lucilia sericata (Meigen), Chrysomya rufifacies (Macquart), Necrobia rufipes (DeGeer), Dermestes maculatus (DeGeer), Pheidole hyatti Emery, and Pogonomyrmex rugosus Emery stood out as dominant species.     

Forensic Identification of CITES Protected Slimming Cactus (Hoodia) Using DNA Barcoding

Slimming cactus (Hoodia), found only in southwestern Africa, is a well‐known herbal product for losing weight. Consequently, Hoodia extracts are sought‐after worldwide despite a CITES Appendix II status. The failure to eradicate illegal trade is due to problems with detecting and identifying Hoodia using morphological and chemical characters. Our aim was to evaluate the potential of molecular identification of Hoodia based on DNA barcoding. Screening of nrITS1 and psbA‐trnH DNA sequences from 26 accessions of Ceropegieae resulted in successful identification, while conventional chemical profiling using DLI‐MS led to inaccurate detection and identification of Hoodia. The presence of Hoodia in herbal products was also successfully established using DNA sequences. A validation procedure of our DNA barcoding protocol demonstrated its robustness to changes in PCR conditions. We conclude that DNA barcoding is an effective tool for Hoodia detection and identification which can contribute to preventing illegal trade.     

Identification of Indian Crocodile Species Through DNA Barcodes

The biodiversity of India includes three crocodile species, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, whose status is threatened due to bushmeat crisis and illegal hunting. The crocodilian conservation management requires novel techniques to help forensic analysts to reveal species identity. DNA barcoding is a species identification technique, where a partial cytochrome c oxidase subunit 1 gene is used as a marker for species identification. Herein, the DNA barcoding technique is evaluated for three Indian crocodiles by analyzing an approximately 750‐bp barcode region. The alignment result shows interspecific variations between sequences for discrimination of the three Indian crocodiles leading to species identification. The phylogenetic analyses also substantiate the established crocodilian relationships, which add further advantage to use this DNA barcoding approach for Indian crocodiles. This study provides preliminary evidences for the use of DNA barcoding technique in the identification of Indian crocodile species.     

DNA Barcoding in Forensic Entomology – Establishing a DNA Reference Library of Potentially Forensic Relevant Arthropod Species,

Throughout the years, DNA barcoding has gained in importance in forensic entomology as it leads to fast and reliable species determination. High‐quality results, however, can only be achieved with a comprehensive DNA barcode reference database at hand. In collaboration with the Bavarian State Criminal Police Office, we have initiated at the Bavarian State Collection of Zoology the establishment of a reference library containing arthropods of potential forensic relevance to be used for DNA barcoding applications. CO1‐5P’ DNA barcode sequences of hundreds of arthropods were obtained via DNA extraction, PCR and Sanger Sequencing, leading to the establishment of a database containing 502 high‐quality sequences which provide coverage for 88 arthropod species. Furthermore, we demonstrate an application example of this library using it as a backbone to a high throughput sequencing analysis of arthropod bulk samples collected from human corpses, which enabled the identification of 31 different arthropod Barcode Index Numbers.     

应用mtDNA COI基因序列鉴别常见嗜尸性蝇类种属

目的探讨mtDNA基因序列对常见嗜尸性蝇类的种属鉴别应用价值。方法收集不同区域2科4属6个种30个蝇类样本,提取样本线粒体DNA后扩增COI基因序列,以琼脂糖电泳检测扩增产物并测序,以DNAMAN6.0分析软件分别截取498bp序列,用MEGA5.2软件分别进行序列分析,然后构建系统发育树,比较各地区不同种属样本的序列差异。结果 6个种属的嗜尸性蝇类30个样本mtDNA的COI基因具有一定的序列差异,种内进化分歧均数在0.1%~1.6%之间,种间进化分歧均数在2.2%~11.2%之间,6个种属通过系统发育树均可明确区分。结论 COI基因序列分析和系统发育树对嗜尸性蝇类的种属检验具有重要帮助作用,可用于现场样本的准确、快速种属鉴定。     

Variation in Developmental Time for Geographically Distinct Populations of the Common Green Bottle Fly,Lucilia sericata (Meigen)*

Abstract: Time between death and discovery of remains, or postmortem interval (PMI), can be assessed using blow fly maggot age. Forensic entomologists rely on published, often nonlocal, species‐specific developmental tables to determine maggot age. In a series of common garden experiments, we investigated the developmental rate variation between populations of Lucilia sericata collected from Sacramento, CA, San Diego, CA, and Easton, MA at 16°C, 26°C, and 36°C. For the 16°C trial the time measurement started at egg hatch, while for the higher temperatures the experiment began at oviposition; the wandering stage signified the endpoint for all experiments. The distribution of developmental times differed significantly (ANOVA, p < 0.001) between the three populations within each temperature treatment. We discovered that regional variation of developmental times within a blow fly species exists. This study demonstrates the importance of assembling local population‐specific developmental tables when estimating larval age to determine PMI.     

Internal Morphological Analysis for Age Estimation of Blow Fly Pupae (Diptera: Calliphoridae) in Postmortem Interval Estimation*

Abstract: Larvae and pupae of blow fly species are frequently used in postmortem interval estimation, their age indicating minimum time since death. Most studies have considered age estimation of larvae, neglecting study of pupae. Relative development of external pupal features is useful, but there are also internal changes during metamorphosis that may be indicators of age, utilizing histological techniques. This study aimed to optimize preservation and histological analysis of blow fly pupae, specifically Calliphora vicina Robineau‐Desvoidy and Lucilia sericata (Meigen), and to examine internal features with potential for age estimation. Effect of hot‐water‐killing and different preservatives were examined. It was determined that blow fly pupae should be pierced through the three tagma, hot‐water‐killed, and preserved in 80% ethanol as the optimal preservation for subsequent analyses. Hematoxylin and eosin stained pupal sections revealed differences in brain and thoracic muscle development throughout the pupal stage with potential for age estimation.     

An Evaluation of Differentially Spliced Genes as Markers of Sex for Forensic Entomology,,

Blow flies (Calliphoridae) are important medically and economically and are commonly used in forensics as temporal markers in death investigations. While phenotypic traits in adult flies can be sexually dimorphic, sex identification in immatures is difficult. Consequently, little is known about how sex may result in developmental disparities among sexes even though there are indications that they may be important in some instances. Since genetic mechanisms for sex are well studied in model flies and species of agricultural and medical importance, we exploit the sex-specifically spliced genes transformer (tra) and doublesex (dsx) in the sex determination pathway to optimize a sex identification assay for immatures. Using known primer sets for tra and with a novel one for dsx, we develop PCR assays for identifying sex in four forensically relevant Calliphoridae species: Lucilia sericata (Meigen), Lucilia cuprina (Wiedemann), Cochliomyia macellaria (Fabricius), and Chrysomya rufifacies (Macquart) and evaluated their performance. Band detection rates were found to range from 71 to 100%, call rates ranged from 90 to 100%, and no error was found when bands could be called. Such information is informative for purposes of testimony and in preparation for development studies. The developed assays will assist in further differentiating sexually dimorphic differences in development of the Calliphoridae and aid in more accurately estimating insect age when age predictive markers (size, development time, molecular expression) are sexually dimorphic.