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.     

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.     

Validation of a multiplex PCR assay for the forensic identification of Indian crocodiles

A dependable and efficient wildlife species identification system is essential for swift dispensation of the justice linking wildlife crimes. Development of molecular techniques is befitting the need of the time. The forensic laboratories often receive highly ill-treated samples for identification purposes, and thus, validation of any novel methodology is necessary for forensic usage. We validate a novel multiplex polymerase chain reaction assay, developed at this laboratory for the forensic identification of three Indian crocodiles, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, following the guidelines of Scientific Working Group on DNA Analysis Methods. The multiplex PCR was tested for its specificity, reproducibility, sensitivity, and stability. This study also includes the samples treated with various chemical substances and exposed to various environmental regimes. The result of this validation study promises this technique to be an efficient identification tool for Indian crocodiles and therefore is recommended for forensic purposes.     

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.     

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.     

Species identification from dried snake venom

Illegal trade in snake parts has increased enormously. In spite of strict protection under wildlife act, a large number of snakes are being killed ruthlessly in India for venom and skin. Here, an interesting case involving confiscation of crystallized dried snake venom and subsequent DNA-based species identification is reported. The analysis using the universal primers for cytochrome b region of the mitochondrial DNA revealed that the venom was extracted from an Indian cobra (Naja naja). On the basis of this report, the forwarding authority booked a case in the court of law against the accused for illegal hunting of an endangered venomous snake and smuggling of snake venom. This approach thus has immense potential for rapid identification of snake species facing endangerment because of illegal trade. This is also the first report of DNA isolation from dried snake venom for species identification.     

Preventing Wildlife Crimes: Solutions That Can Overcome the ‘Tragedy of the Commons’

The 'tragedy of the commons' dilemma occurs when individuals working independently of one another, will overuse a common-property resource for short-term benefits while decimating the resource for long-term use (Hardin 1968). This is often found in the field of wildlife crimes where species become overexploited to increase short-term profits while endangering and eliminating a natural resource for future users. Wildlife crimes suffering from the ‘tragedy’ need to be prevented in order for species to avoid extinction while also conserving a natural resource that monetarily benefits numerous people and their respective communities. Current approaches to the illegal wildlife trade include implementing trade bans or regulatory schemes at the national and international level, yet their effectiveness of reducing the trade is unknown. Perhaps, a better approach in reducing the illegal wildlife trade is a combination of making it more difficult to poach (i.e. situational crime prevention) and incentivizing locals to abstain from poaching. This paper will first review the literature on wildlife crimes and then use a case study approach that will examine the literature on the illegal parrot trade, the market for wildlife skins, and over-fishing. Through these case studies, a comprehensive review of the problem will be detailed as well as innovative conservation solutions that show promise in reducing the poaching and exploitation of species. Amongst these solutions will be the use of situational crime prevention that has shown immediate reductions in crime when tailored towards highly-targeted areas and crimes.     

Phylogenetic Evidence for a Case of Misleading Rather than Mislabeling in Caviar in the United Kingdom

Sturgeons and paddlefish are freshwater fish which are highly valued for their caviar. Despite the fact that every single species of sturgeon and paddlefish is listed under CITES, there are reports of illegal trade in caviar where products are deliberately mislabeled. Three samples of caviar purchased in the United Kingdom were investigated for accurate CITES labeling using COI and cyt b sequencing. Initial species identification was carried out using BLAST followed by phylogenetic analyses using both maximum parsimony and maximum likelihood methods. Results showed no evidence for mislabeling with respect to CITES labels in any of the three samples, but we observed clear evidence for a case of misleading the customer in one sample.     

Constructing STR Multiplexes for Individual Identification of Hungarian Red Deer

Red deer is the most valuable game of the fauna in Hungary, and there is a strong need for genetic identification of individuals. For this purpose, 10 tetranucleotide STR markers were developed and amplified in two 5‐plex systems. The study presented here includes the flanking region sequence analysis and the allele nomenclature of the 10 loci as well as the PCR optimization of the DeerPlex I and II. LD pairwise tests and cross‐species similarity analyses showed the 10 loci to be independently inherited. Considerable levels of genetic differences between two subpopulations were recorded, and F_ST was 0.034 using AMOVA. The average probability of identity (PI_ave) was at the value of 2.6736 × 10^?15. This low value for PI_ave nearly eliminates false identification. An illegal hunting case solved by DeerPlex is described herein. The calculated likelihood ratio (LR) illustrates the potential of the 10 red deer microsatellite markers for forensic investigations.     

DNA‐Based Identification of Forensically Important Lucilia (Diptera: Calliphoridae) in the Continental United States*

Abstract: Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA‐based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100‐bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA‐based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA‐based identification is likely to be successful for the other seven species.     

Specters of Indigeneity in British‐Indian Migration, 1914

Colonial legal histories of indigeneity and British‐Indian migration have not often been placed in conversation with one another. This article pursues such a project by tracing indigeneity as a spectral presence that emerged with uneven regularity in juridico‐political conflicts over British‐Indian migration. Specifically, I focus on the 1914 journey of the Komagata Maru, a Japanese steamship carrying 376 Punjabi migrants that sailed from Hong Kong to Shanghai, Moji to Yokohama, and across the Pacific, eventually arriving in Vancouver, Canada. Crisscrossing continents and approaching law in its broadest sense, I explore three struggles over the ship and its passengers: a satirical cartoon published in the Hindi Punch (Bombay), a legal test case heard by the British Columbia Court of Appeal (Vancouver), and a public debate on the racial meanings of Imperial subjecthood that ensued among Indian middle‐class supporters of the ship and unfolded in English newspapers in various Indian cities. In each moment of struggle, I examine the changing conceptions of indigeneity that were strategically appropriated, never by indigenous peoples themselves or on their own terms, but by the Dominion of Canada and by British Indians, each deploying indigeneity to its own advantage and to achieve particular effects. Ultimately, this article considers the political and legal work that the spectral figure of indigeneity performed, the conceptions of time that underwrote its recurrence, and the temporalities that it sustained and called into question.     

Application of species-specific polymerase chain reaction in the forensic identification of tiger species

Globally, tigers are considered to be endangered, and are listed on Appendix I of CITES. A simple test, using a species-specific primer pair, was developed to identify tiger meat, faeces and dried skin, and provide forensic evidence of illegal wildlife trade. The specific fragment of mitochondrial cytochrome b gene was also successfully amplified from raw DNA products extracted from single tiger hairs. This PCR-based approach opens a new avenue to forensic identification of less-than-optimal samples.     

Single Nucleotide Polymorphism Assay for Genetic Identification of Lophophora williamsii*

Lophophora is a member of the Cactaceae family, which contains two species: Lophophora williamsii and L. diffusa. Lophophora williamsii is an illegal plant containing mescaline, a hallucinogenic alkaloid. In this study, a novel method based on a single nucleotide polymorphism (SNP) assay was developed for identifying L. williamsii; this assay reliably detects SNPs within chloroplast DNA (rbcL, matK, and trnL-trnF IGS) and was validated for identifying Lophophora and L. williamsii simultaneously. The chloroplast DNA sequences from four L. williamsii and three L. diffusa plants were obtained and compared using DNA sequence data from approximately 300 other Cactaceae species available in GenBank. From this sequence data, a total of seven SNPs were determined to be suitable for identifying L. williamsii. A multiplex assay was constructed using the ABI PRISM^® SNaPshot™ Multiplex Kit (Applied Biosystems, Forster City, CA) to analyze species-specific SNPs. Using this multiplex assay, we clearly distinguished the Lophophora among 19 species in the Cactaceae family. Additionally, L. williamsii was distinguished from L. diffusa. These results suggest that the newly developed assay may help resolve crimes related to illegal distribution and use. This multiplex assay will be useful for the genetic identification of L. williamsii and can complement conventional methods of detecting mescaline.     

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.     

Mitochondrial DNA Profiling of Illegal Tortoiseshell Products Derived from Hawksbill Sea Turtles

The hawksbill sea turtle (Eretmochelys imbricata) is a highly endangered species, commonly poached for its ornate shell. “Tortoiseshell” products made from the shell are widely, although illegally, available in many countries. Hawksbills have a circumglobal distribution; thus, determining their origin is difficult, although genetic differences exist geographically. In the research presented, a procedure was developed to extract and amplify mitochondrial DNA from tortoiseshell items, in an effort to better understand where the species is being poached. Confiscated tortoiseshell items were obtained from the U.S. Fish and Wildlife Service, and DNA from 56 of them was analyzed. Multiple mitochondrial haplotypes were identified, including five not previously reported. Only one tortoiseshell item proved to be of Atlantic origin, while all others corresponded to genetic stocks in the Indo‐Pacific region. The developed methodology allows for unique, and previously unattainable, genetic information on the illegal poaching of sea turtles for the decorative tortoiseshell trade.     

Counterfeit Adderall Containing Aceclofenac from Internet Pharmacies

A nontargeted approach based on liquid chromatography equipped with a quadrupole time‐of‐flight mass detector (LC‐MS Q‐TOF) joined to nuclear magnetic resonance (NMR) analysis allowed rapid identification and quantification of the anti‐inflammatory drug aceclofenac in illegal Adderall tablets. The largest chromatographic peak had m/z = 354.030 and m/z = 376.012 matching, respectively, the ionic structures (M + H)⁺ and (M + Na)⁺ of a molecule M. The accurate mass data generated the molecular formula C₁₆H₁₃Cl₂NO₄. A screening of the pharmaceutical active substances having that molecular formula together with the MS/MS fragmentation pattern suggested aceclofenac. Aceclofenac structure was unambiguously confirmed by ¹H and ¹³C NMR experiments. The aceclofenac content was 90 mg/tablet (RSD 2%) as detected by quantitative NMR. Information on the identity and content of illegal drugs is required for legal purposes; it supports in evaluating the effective impact on users safety, and it is useful for control laboratories using a targeted approach in their analytical activities.     

CITES,wild plants,and opportunities for crime

The illegal trade in endangered plants damages both the environment and local communities by threatening and destroying numerous species and important natural resources. There is very little research which systematically addresses this issue by identifying specific opportunities for crime. This article presents the results of an interdisciplinary study which brings together criminological and conservation science expertise to identify criminal opportunities in the illegal wild plant trade and suggest strategies in order to prevent and mitigate the problem. Methodologically, the study adapts a crime proofing of legislation approach to the UN Convention on the International Trade in Endangered Species of Wild Fauna and Flora and is based on documentary and interview data. Situational crime prevention is used as a framework to provide points for effective intervention.     

Doing Green,Critical Criminology with an Auto-Ethnographic,Feminist Approach

In this article, I employ an auto-ethnographic methodology as a point of departure in order to explore my path into research on the legal and illegal trade in wild animals which, over the years, has consisted of interviews with experts and enforcement agencies in Brazil, Colombia and Norway including offenders (in Norway), and analysis of verdicts, interrogation reports, and custom seizure reports from Norway. I argue that research not only may, but should be value-driven and that a researcher’s personal biography can (1) provide additional insight into a research area; (2) serve to create rapport with informants; and (3) forge an important foundation for the formulation of research questions and the analysis of empirical data. Values also provide a platform for the choice of theoretical framework that is applied and which enhance and further knowledge within the field—in this case, perspectives of harm and justice, particularly species justice and eco-justice. The article calls for a stronger interdisciplinary approach in green criminology—one that includes feminist care ethics, philosophy and compassionate conservation perspectives, and which offers a more radical critique of human exploitation of ‘wildlife’ specifically and other animals more generally.     

Can Stable Isotopes and Radiocarbon Dating Provide a Forensic Solution for Curbing Illegal Harvesting of Threatened Cycads?

Cycads in South Africa are facing an extinction crisis due to the illegal extraction of plants from the wild. Proving wild origin of suspect ex situ cycads to the satisfaction of a court of law is difficult, limiting law enforcement efforts. We investigated the feasibility of using multiple stable isotopes to identify specimens removed from the wild. Relocated and wild specimens from two species in the African genus Encephalartos (E. lebomboensis and E. arenarius) were sampled. ¹⁴C analysis indicated that a ± 30‐year chronology could be reliably obtained from the cycads. For E. arenarius, pre‐relocation tissue was consistent with a wild origin, whereas tissue grown post‐relocation was isotopically distinct from the wild for ⁸⁷Sr/⁸⁶Sr and δ¹⁵N. For E. lebomboensis, δ³⁴S, δ¹⁸O, and ⁸⁷Sr/⁸⁶Sr were different between relocated and control plants, consistent with the >30 years since relocation. Our findings demonstrate the potential for a forensic isotope approach to identify illegal ex situ cycads.     

An Assessment of the Utility of Universal and Specific Genetic Markers for Opium Poppy Identification

Abstract: The proper identification of illicit plants such as Papaver somniferum L (opium poppy) is important for law enforcement agencies. The identification of opium poppy was presently tested using 10 genetic markers that are universal for all plants or specific to a few poppy plants. The genetic distances of universal markers such as nuclear internal transcribed spacer (ITS), 18S rRNA, plastid rbcL, and trnL‐trnF intergenic spacer (IGS) of 14 species included in the Papaveraceae and Fumariaceae family were acquired by sequence comparisons. Both the ITS region and trnL‐trnF IGS showed high levels of interspecific divergence. Six Papaver genera‐specific markers were developed from coding regions involved in morphine biosynthesis. Three markers (TYDC, NCS, and BBE) produced amplicons only in opium poppy, providing a presence/absence test for opium poppy, while three additional markers (CYP80B1, SAT, and COR) were genus specific. These 10 markers might be useful for the forensic DNA analysis of opium poppy.