Wildlife Forensics Research Articles

Sequence Characterization of Mitochondrial 12S rRNA Gene in Mouse Deer (Moschiola indica) for PCR-RFLP Based Species Identification

Mitochondrial 12S rRNA has proven to be a useful molecular marker for better conservation and management of the endangered species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the mitochondrial 12S rRNA gene has proven to be a reliable and efficient tool for the identification of different Indian deer species of family cervidae. In the present study, mitochondrial 12S rRNA gene sequence of mouse deer (Moschiola indica) belonging to the family Tragulidae was characterized and analysed in silico for its use in species identification. Genomic DNA was isolated from the hair follicles and mitochondrial 12S rRNA gene was amplified using universal primers. PCR product was cloned and sequenced for the first time. The sequence of mouse deer showed 90.04, 90.08, 90.04, 91.2, 90.04, and 90.08% identities with sika deer, sambar, hog deer, musk deer, chital, and barking deer, respectively. Restriction mapping in Lasergene (DNAstar Inc., Madison, WI, USA) revealed that mouse deer mitochondrial 12S rRNA gene sequence can be differentiated from the other deer species in PCR-RFLP using RsaI, DdeI, BsrI, and BstSFI. With the help of predicted pattern, mouse deer can be identified using genomic DNA from a variety of biomaterials, thereby providing molecular aid in wildlife forensics and conservation of the species.

Book Review: Wildlife Forensic Investigation: Principles and Practice

Book Review: Wildlife Forensic Investigation: Principles and Practice

Bomb-curve radiocarbon measurement of recent biologic tissues and applications to wildlife forensics and stable isotope (paleo)ecology

Above-ground thermonuclear weapons testing from 1952 through 1962 nearly doubled the concentration of radiocarbon ((14)C) in the atmosphere. As a result, organic material formed during or after this period may be radiocarbon-dated using the abrupt rise and steady fall of the atmospheric (14)C concentration known as the bomb-curve. We test the accuracy of accelerator mass spectrometry radiocarbon dating of 29 herbivore and plant tissues collected on known dates between 1905 and 2008 in East Africa. Herbivore samples include teeth, tusks, soft tissue, hair, and horn. Tissues formed after 1955 are dated to within 0.3-1.3 y of formation, depending on the tissue type, whereas tissues older than ca. 1955 have high age uncertainties (>17 y) due to the Suess effect. (14)C dating of tissues has applications to stable isotope (paleo)ecology and wildlife forensics. We use data from 41 additional samples to determine growth rates of tusks, molars, and hair, which improve interpretations of serial stable isotope data for (paleo)ecological studies. (14)C dating can also be used to calculate the time interval represented in periodic histological structures in dental tissues (i.e., perikymata), which in turn may be used as chronometers in fossil teeth. Bomb-curve (14)C dating of confiscated animal tissues (e.g., ivory statues) can be used to determine whether trade of the item is legal, because many Convention of International Trade of Endangered Species restrictions are based on the age of the tissue, and thus can serve as a powerful forensic tool to combat illegal trade in animal parts.

REFERENCE: Huffman JE, Wallace JR. Wildlife forensics: methods and applications. Chichester, West Sussex, UK: John Wiley & Sons Ltd, 2012, 370 pp.

REFERENCE: Huffman JE, Wallace JR. Wildlife forensics: methods and applications. Chichester, West Sussex, UK: John Wiley & Sons Ltd, 2012, 370 pp.

Fungal palaeodiversity revealed using high‐throughput metabarcoding of ancient DNA from arctic permafrost

Environmental MicrobiologyVolume 15, Issue 7 p. 2146-2146 ErratumFree Access Fungal palaeodiversity revealed using high-throughput metabarcoding of ancient DNA from arctic permafrost This article corrects the following: Fungal palaeodiversity revealed using high-throughput metabarcoding of ancient DNA from arctic permafrost Eva Bellemain, Marie L. Davey, Håvard Kauserud, Laura S. Epp, Sanne Boessenkool, Eric Coissac, Jozsef Geml, Mary Edwards, Eske Willerslev, Galina Gussarova, Pierre Taberlet, Christian Brochmann, Volume 15Issue 4Environmental Microbiology pages: 1176-1189 First Published online: November 22, 2012 First published: 04 July 2013 https://doi.org/10.1111/1462-2920.12175AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Eva Bellemain, Marie L. Davey, Håvard Kauserud, Laura S. Epp, Sanne Boessenkool, Eric Coissac, Jozsef Geml, Mary Edwards, Eske Willerslev, Galina Gussarova, Pierre Taberlet and Christian Brochmann In the article by Bellemain et al., published as part of the April 2013 issue of Environmental Microbiology, James Haile should be included as a co-author. The correct list of authors appears below. Eva Bellemain, Marie L. Davey, Håvard Kauserud, Laura S. Epp, Sanne Boessenkool, Eric Coissac, Jozsef Geml, Mary Edwards, Eske Willerslev, Galina Gussarova, Pierre Taberlet, James Haile and Christian Brochmann Haile's affiliation is as follows: Ancient DNA and Wildlife Forensics Laboratory, Murdoch University, 90 South Street, Murdoch, Perth 6150, Australia The Publisher apologizes for this error. Reference Bellemain, E., Davey, M.L., Kauserud, H., Epp, L.S., Boessenkool, S., Coissac, E., et al. (2013). Fungal palaeodiversity revealed using high-throughput metabarcoding of ancient DNA from arctic permafrost. Environ Microbiol 15: 1176– 1189. Volume15, Issue7Thematic Issue: Invertebrate-Microbe InteractionsJuly 2013Pages 2146-2146 ReferencesRelatedInformation

Advances in sex determination in bats and its utility in wind‐wildlife studies

We developed a simple and reliable genetic method to determine sex in bats from the Vespertilionidae and Molossidae families. Polymerase chain reaction was used to amplify a portion of the introns within the zinc-finger-X (Zfx) and zinc-finger-Y (Zfy) genes. We designed primers to produce size variation between the Zfx and Zfy products that could be visualized using gel electrophoresis. Using an example from our wind-wildlife research, we show how sex data generated using this method are superior to sex data based on external morphology. Our method allows for the generation of sex data across a wide range of bats that can be used to address key questions in wildlife forensics, behavioural ecology, conservation and evolutionary biology.

Identification of sarcosaprophagous Diptera species through DNA barcoding in wildlife forensics

In recent years, forensic entomology has been applied in wildlife crimes, such as neglect cases, animal cruelty and illegal poaching. Likewise in human death investigations, in which insects can help to provide information about postmortem interval (PMI) and corpse transfer, entomology may be an important source of information in animal murder suspicion. The use of insects in forensic context relies primarily on its identification at the species level. To overcome some problems of morphological determination, molecular identification has gained relevance and has been applied frequently in forensic areas. Cytochrome c oxidase I (COI) gene was adopted in DNA barcoding approach. This methodology intends to unify the DNA-based identification using a specific region of mitochondrial DNA. COI sequences have been collected into the BOLD online database, allowing the molecular identification of sequences from unknown specimens. Nonetheless, to achieve a correct identification of an unknown sample, it is necessary that sequences from species under study exist, for comparison, in online databases. Due to the geographic differences, it is of huge importance to have samples from a certain species from its distribution range. In that sense, the aim of this research is to contribute to the potential and accuracy improvement of such databases in identification of species commonly found in wildlife carcasses. A portion of COI was sequenced from 95 specimens of seven species belonging to two families of Diptera (Calliphoridae and Muscidae) found in wildlife carcasses-baited traps in Serra da Estrela (Portugal). All specimens were identified at species level with a high specimen similarity and maximum identity percentage (through BOLD Systems and GenBank online databases, respectively). We also demonstrate the correct discrimination of all species through phylogenic and sequence divergence analyses proposed in DNA barcoding studies, reinforcing the suitability of this marker.

Stable hydrogen and oxygen isotopes in aquatic food webs are tracers of diet and provenance

Summary The stable hydrogen and oxygen isotope (δ2H and δ18O) composition of animal tissues are well‐established tracers for terrestrial migration ecology and wildlife forensics. However, the behaviour of these isotopes in aquatic ecosystems and their potential as tracers of diet and provenance are complicated because of inputs from ambient H2O and diet. We conducted controlled aquaria‐based experiments to quantify the mechanisms that drive the H and O isotopic flow within and among aquatic species. The isotopic composition of water and diet of two aquatic species (Chironomus tentans and Poecilia reticulata), representing two trophic levels, was varied in six isothermal treatments. Both species were raised from juvenile to adult to ensure that tissues were in isotopic equilibrium with their dietary and environmental conditions (ambient water, food, dissolved oxygen). We measured water, dissolved O2, diet, tissue protein and lipids for δ2H or δ18O. The flows of H and O isotopes for tissue formation in aquatic organisms were parameterized using a steady‐state multi‐pool mass‐balance model. The ambient H2O contribution to tissue protein H and O isotopes in both species was significant (30–50% for 2H and >80% for 18O). An apparent trophic effect for δ2H and isotopic discrimination between water and protein for δ18O (c. 15 ‰) were identified. Our isotopic data and model revealed potential applications and cautions in using δ2H and δ18O measurements for ecological studies in aquatic food webs. Tissue δ2H values may be a complementary trophic tracer in aquatic food webs, but only when the main controlling mechanisms are properly accounted for (i.e., H isotopic exchange with water during protein synthesis and influence of metabolic water). Measurements of δ18O, on the other hand, reflect that of water and so can be used for predicting isotopic assignment to origin of aquatic organisms as there is no complicating trophic effect, but more δ18O field data and improved analytical precision may be required to better establish the strengths to ecological applications.

A multiplex PCR method to identify bushmeat species in wildlife forensics

Airports are important hubs for bushmeat trade, that is, the illegal commercialization of wildlife meat, often from endangered species. While the identification of bushmeat species is sometimes possible morphologically, in most cases genetic characterization would be necessary for precise taxonomic identification. We validated a mtDNA-based species identification method for its application to bushmeat species imported into Switzerland. This method consists of a multiplex-PCR-setup with 8 primers varying in their specificity to amplify a region of the mitochondrial cytochrome b (cytb) gene in different animal classes (mammals, fishes and birds). We tested the method on meat (cooked, dried, smoked or raw) and blood samples from 24 different species, obtained from museums and butchers. Following successful PCR and sequencing, we identified the species of origin using BLAST alignments to the cytb entries in the NCBI nucleotide database. Our method also passed all validation criteria, including repeatability, reproducibility, and robustness. We then applied this method to identify the taxonomic origin of 250 samples of putative bushmeat confiscated at the airports of Zurich and Geneva between September 2011 and January 2013. For 224 samples, there was sufficient DNA to allow successful sequencing. The samples belonged to one of four vertebrate groups (mammals, birds, reptiles and fishes): approximately two thirds of the analyzed samples originated from wild animals, one third of which are listed in the CITES appendices. Our validated DNA method offers a practical and easy approach for the identification of bushmeat at airports and in general in wildlife forensics.

Identification of Galliformes through Forensically Informative Nucleotide Sequencing (FINS) and its Implication in Wildlife Forensics

Galliformes are hunted for the demand of their attractive feathers and to supply a cheap animal food for the rural communities. In such cases, species identification through visual inspection of the meat or based on feather morphometrics is a challenging task for the law enforcement agencies to enforce the Wildlife Protection Act (WPA). Here, we extracted DNA from the individual feathers of unknown species encountered during field surveys and two mitochondrial genes (12S rRNA and Cytochrome b) were amplified using universal primers for species identification. Most homologous sequences were retrieved using NCBI-BLAST for each generated sequence of both the genes. Neighbor-Joining trees based on Kimura 2 parameter distance matrices in FINS analysis identified the species from the individual feather with strong bootstrap support. Nine species specific polymorphic sites were found in the partial sequence of Cytochrome b gene that differentiated Pavo cristatus to Pavo muticus imperator. Our study highlighted the importance of feathers in identifying the species and their applicability in wildlife offence cases using FINS approach.

Sifting through cycads: A guide to identifying the stem fragments of six South African medicinal Encephalartos species

Sixty-three percent of the world's cycad taxa are threatened with extinction. In South Africa, 25 of the 38 indigenous species are categorised as Threatened on the IUCN Red List, primarily due to illegal collecting. Three of the country's Encephalartos species are already Extinct in the Wild, while 10 Critically Endangered endemic Encephalartos species are undergoing rapid local extirpations across their ranges. Nearly two-thirds of South Africa's Encephalartos species are used for traditional medicine, some of which are sold in traditional medicine markets in the form of bark strips and stem fragments. The leafless Encephalartos stems encountered in the medicinal and horticultural trades are generally difficult to identify to species and, since species identification of South African Encephalartos using DNA barcoding is not yet feasible, alternative methods of identification are required. To determine the identity of the species sold in South Africa's two largest traditional medicine markets in KwaZulu-Natal (KZN) and Gauteng, surveys were conducted in the Warwick and Faraday markets respectively. Stem fragment samples purchased from the vendors in the markets were identified to species using stem and leaf base morphological characters in tandem with harvesting locality records. Six KZN Encephalartos species were successfully identified viz. E. ferox, E. ghellinckii, E. natalensis, E. ngoynaus, E. senticusos and E. villosus. Photographs of stem fragments and leaf bases of each species are presented here with supplementary quantitative and descriptive stem anatomical data. This paper therefore presents an identification guide to assist researchers and law enforcement officers in the identification of Encephalartos species unlawfully harvested for the traditional medicine and horticultural trades. The guide is thus also useful in the emerging discipline of wildlife forensics, and offers a unique approach to determining difficult-to-identify Encephalartos stem material.

Use of DNA barcoding in wildlife forensics: a study of sambar deer (Rusa unicolor)

This paper reports a case in which a wildlife range officer had suspected that some people had killed a sambar deer (Rusa unicolor). Cooked meat and dried skin were seized from the suspects and forwarded to our laboratory for DNA testing to resolve the identity of the animal. Mitochondrial cytochrome C oxidase subunit 1 (CO1) analysis revealed that the cooked meat and skin of the animal were of a sambar deer by comparing the sequences with the reference sequence of R. unicolor generated in our laboratory. The study illuminates the use of barcode sequences and reference databases in wildlife forensic applications.

Cytochrome b based genetic differentiation of Indian wild pig (Sus scrofa cristatus) and domestic pig (Sus scrofa domestica) and its use in wildlife forensics

The Indian wild pig (Sus scrofa cristatus) is a protected species and listed in the Indian Wildlife (Protection) Act, 1972. The wild pig is often hunted illegally and sold in market as meat warranting punishment under law. To avoid confusion in identification of these two subspecies during wildlife forensic examinations, we describe genetic differentiation of Indian wild and domestic pigs using a molecular technique. Analysis of sequence generated from the partial fragment (421bp) of mitochondrial DNA (mtDNA) cytochrome b (Cyt b) gene exhibited unambiguous (>3%) genetic variation between Indian wild and domestic pigs. We observed nine forensically informative nucleotide sequence (FINS) variations between Indian wild and domestic pigs. The overall genetic variation described in this study is helpful in forensic identification of the biological samples of wild and domestic pigs. It also helped in differentiating the Indian wild pig from other wild pig races. This study indicates that domestic pigs in India are not descendent of the Indian wild pig, however; they are closer to the other wild pig races found in Asia and Europe.

DNA species surveillance: Monitoring bushmeat poaching and trading in Kenya using partial cytochrome b gene

DNA species identification has applications in such areas as forensic science, systematics, conservation genetics and agriculture. One key anthropogenic activity threatening large wildlife fauna is illegal exploitation. In Kenya, species identification of raw and processed meat products remains a constraint to effective enforcement of illegal trade in game meat (bushmeat) and products. We tested the reliability of a 321 bp mitochondrial cytochrome b (cyt b) region as a species identification tool for application in wildlife forensics. Query sequences were generated from known specimens of 14 Eastern African wildlife species, 13 representing commonly poached ungulates, and three domesticated species. These were compared, using Basic Local Alignment Search Tool (BLAST) algorithm, with NCBI GenBank reference sequences for species identity. These query sequences were subsequently deposited on Genbank. They represent a contribution to a diagnostic internal East African Wildlife reference cyt b database. The test species comprised: Cape buffalo, bushbuck, Guenther’s dik-dik, common duiker, common eland, Grant’s gazelle, hartebeest, impala, lesser kudu, plains zebra, Thomson’s gazelle, common warthog, wildebeest, Maasai ostrich, cattle, goat and sheep. Additionally, cooked beef and pork samples were analyzed. The results show that, when conspecific sequences were available in the database, species discrimination was 100%. Phylogeny clustering of the species by maximum likelihood supported the species determination by BLAST. The second part of the study carried out a preliminary survey of the prevalence of illegal game meat sold in the dispersal area of Tsavo National Park, Kenya. Sixty two raw meat samples were randomly collected from small roadside retail outlets along the Nairobi-Mombasa highway (A109), a major transnational highway that transverses Tsavo National Park. The results indicate a 9.7% (n = 6) illegal game meat sale, comprising five Guenther’s dik-diks and a Beisa oryx. A 2 km radius hotspot, with 83% (n = 5) of the bushmeat sales was identified just south of Tsavo East National Park. Key words: East Africa, Kenya, bushmeat, poaching, wildlife conservation, species identification, mitochondrial cytochrome b gene.

Comparison of established methods for quantifying genotyping error rates in wildlife forensics

Several methods have been applied to calculate genotyping error rates (GER) for non-invasive population size estimations. However, there is a lack of comparability between these methods. Here we focused on the comparison of methods for determination of GER within one study using faeces samples of wild boars (Sus scrofa). Error rates were calculated by (1) comparison of reference tissue samples and rectum faeces samples (2) the number of deviations between replicates and the assumed consensus genotypes, (3) re-analysis of a subsample interpreted by allelic and genotype comparisons, and (4) a blind-test of anonymously subdivided faecal samples. The error rates differed widely between these four methods (0–57.5 %) and underline the need of a consensus approach. The blind-test resulted in a GER of 4.3 %. We recommend conducting such a blind-test for estimating realistic GER when starting a pilot study in wildlife forensics.

Conservation of endemic and threatened wildlife: Molecular forensic DNA against poaching of the Cypriot mouflon (Ovis orientalis ophion, Bovidae)

Molecular DNA techniques in combination with appropriate reference population database and statistical methods are fundamental tools to forensic wildlife investigations. This is even more relevant when taxa with uncertain systematics are involved, as is the case of the genus Ovis (Bovidae), whose evolution has been influenced by multiple events of domestication. The Cypriot mouflon, Ovis orientalis ophion, a protected subspecies endemic to Cyprus, is threatened by poaching. This study deals with a case of alleged poaching that occurred in Cyprus (September, 2010). A car did not stop at a checkpoint and when finally blocked by the police, several bloodstained exhibits (n=12) were recovered. Three recently deceased mouflons were found by game wardens at the roadside. The Cyprus Veterinary Services established that these animals had been killed by gunshot. As part of the investigation, DNA testing was performed to establish if there was a link between the dead mouflons and the bloodstained exhibits. The mitochondrial Cytochrome-b gene (Cyt-b) and 12 loci of microsatellite DNA were used as markers. The Cyt-b sequences were obtained from 11 exhibits. They were the same as each other and the same as the single haplotype obtained from the three dead mouflons and all the investigated wild Cypriot mouflons (20 individuals). A database of wild mouflons (47 individuals) from which the unknown samples may have originated was generated. The probability of identity (PID) of the microsatellite panel, computed by genotyping all 47 wild mouflons (10 selected loci, PID=10−5), allowed us to assign nine exhibits to two out of the three carcasses (seven with very strong support: Likelihood Ratio, LR>3000 and Random Match Probability, RMP, <10−3). This study represents the first genetic reference for the Cypriot mouflon and the first published material of forensic wildlife investigations in Cyprus.

Different Animal Species Hairs as Biological Tool for the Forensic Assessment of Individual Identification Characteristics from Animals of Zoological Park, Pragti Maidan, New Delhi, India

Ongoing research shows that how hair varies-between species and even on the same animals-and how he developed a database of mammal hair that can be used by wild life forensic scientist to identify hair in cases of illegal trafficking of the world’s most endangered animals. Hair samples from some domestic and wild animals were evaluated in this study. The main hair structures (cuticle, cortex and medulla) were investigated. The guard hair diameter, colour, shaft, cuticle pattern, the medulla, the scale patterns and, root were examined using a light microscope. The current research on morphologic characteristics of animal hair can be performed to provide more information for wild life forensics and allowing for the identification of illegal transport of animals, poaching or wildlife crime, fraud in textile and fur industry, and dentification of their victims, etc. In the present study, different animal hairs collected from National Zoological Park, Pragati Maidan, New Delhi, India were microscopically examined. Wild life animal species like Baby sheep hair, baby goat hair, bear hair, buffalo hair, cattle hair, deer hair, Doberman dog hair, dog hair, emu hair, flamingo feather, goat hair, Labrador dog hair, sheep hair, tiger hair identification /characterization using microscopic hair characteristics is discussed here. As part of a research project, this paper provides data that allow rapid and low cost animal species identification based on the hair microscopic features because hair is strongly resistant from decomposition and this property makes hair a nearly ideal type of physical evidence.

DNA Wildlife Forensics: Present and Future

Wildlife crime investigation is one of the important facets of forensics science. DNA typing is one of the effective protocols for dealing such issues. The availability of conserved and universal primers has enormous scope in DNA wildlife forensics. These universal primers are designed based on the various conserved regions located on the genes of mitochondrial genome. This study explains the use of DNA typing and genetic profiling in dealing the wildlife crime cases.

Wildlife forensics using mitochondrial DNA sequences: Species identification based on hairs collected in the field and confiscated tanned Felidae leathers

To identify species based on samples without recognizable morphological characteristics, DNA-based approaches are the best option. Here, we describe two cases of the determination of species and geographical origin of wildlife specimens under the regulation of international treaties and domestic laws related to wildlife management in South Korea. First, hairs of suspected wild or reared endangered Asiatic black bears were analyzed using cytochrome oxidase I and the control region. Confiscated Felidae leathers were also investigated using cytochrome b, but they were proven to be fabricated canine leathers. These results were used as scientific evidence for wildlife-related law enforcement. Our results suggest that unrecognizable wildlife specimens can be identified efficiently using DNA sequence-based analysis. Finally, this study shows that conservation genetics research and its applications can be incorporated into wildlife forensic studies.

Individual identification of fox ( Vulpes vulpes) in forensic wildlife investigations

In recent casework the relatedness of a group of foxes ( Vulpes vulpes) was questioned, as the manner of their relatedness might indicate the intentional release of several individuals. To this extent a fox-genotyping technique was pursued. 21 microsatellite markers (STRs) designed for individual identification of dogs ( Canis lupus familiaris) were tested on foxes to determine which markers could be amplified reliably and scored unambiguously. For this subset of markers the possibility of multiplex amplification was tested, as were the variability and allele range of these markers. Approximately two third of the tested STRs of which some were combined in multiplex reactions, were amplified and scored readily in foxes. Both the number of alleles and allele range were observed to differ between dogs and foxes for most markers. To understand these differences and their possible effect on the use of these markers the sequence of several alleles was determined. Changes in both flanking regions and repeat structure were identified, however these changes did not influence the use of the STRs for identification purposes. After genotyping the case samples with this subset of markers a conclusion was formulated expressing the likelihood of a familial relationship of all individual foxes. The obtained genotypes are stored to compare with biological material found if a suspect of an intentional release were to be found.