Egyptian Journal of Forensic Sciences (2016) 6, 524–533
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Egyptian Journal of Forensic Sciences
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CASE REPORT Illegal trade of Indian Pangolin (Manis crassicaudata): Genetic study from scales based on mitochondrial genes
Ved Prakash Kumar a,e, Ankita Rajpoot b, Mukesh c, Malay Shukla d, Dhyanendra Kumar e, Surendra Prakash Goyal a,* a Wildlife Institute of India, Post Box # 18, Chandrabani, Dehradun 248001, Uttarakhand, India b Zoological Survey of India, NRC, Kaulagarh, Dehradun 248001, Uttarakhand, India c Amity Institute of Wildlife Sciences, Amity University, Sector 125, Noida 201 313, Uttar Pradesh, India d Gujrat Forensic University, Gandhinagar 382007, Gujrat, India e Veer Kunwar Singh University, Arrah 802301, Bihar, India
Received 2 November 2015; revised 26 March 2016; accepted 21 June 2016 Available online 3 August 2016
KEYWORDS Abstract Indian Pangolin (Manis crassicaudata), an endangered mammalian species under IUCN DNA forensic science; red list. In India, small population of Chines Pangolin (Manis pentadactyla) are also present in the Manis crassicaudata; eastern part and protected under the Wildlife (Protection) Act, 1972 as a schedule I species along Species identification; with Indian Pangolin. In national and international market the Indian Pangolin are highly in trade Illegal trades and mitochon- principally for its meat and scales. In curbing illegal wildlife, trade of Indian Pangolin on national drial gene and international level wildlife DNA forensics plays an important role in the identification of species from the seized material. We received n = 15 wildlife seizures assumed to be the scales of Pangolin from different state forest and the custom department for molecular analysis. The seizures sequenced for two mitochondrial genes i.e. Cyt b and 16S rRNA, which are often used in wildlife DNA forensic to species identification. The sequences thus generated from these seizures compared to the NCBI database through blast search tool and our laboratory-generated references, yielding 100% similarity with Indian Pangolin. We identified twelve (n = 12) Cyt b and five (n = 5) 16S rRNA species specific SNPs, fixed in Indian Pangolin. This study shows, the importance of authen- ticated references, DNA sequence data availability, highlighting the application of DNA forensics in identifying species from scales. Ó 2016 The International Association of Law and Forensic Sciences (IALFS). Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
* Corresponding author at: Wildlife Forensic and Conservation Genetic Cell, Wildlife Institute of India, Post Box # 18, Chandrabani, Dehradun 248001, Uttarakhand, India. Fax: + 91 135 2640115. E-mail addresses: [email protected] (V.P. Kumar), [email protected] (S.P. Goyal). Peer review under responsibility of The International Association of Law and Forensic Sciences (IALFS). http://dx.doi.org/10.1016/j.ejfs.2016.06.008 2090-536X Ó 2016 The International Association of Law and Forensic Sciences (IALFS). Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Illegal trade of Indian Pangolin 525
1. Introduction distributed in South Asia from parts of eastern Pakistan through much of India, south of the Himalayas, Southern 2,3 Pangolin is a mammalian species classified under the order Nepal, Bangladesh and Sri Lanka, while the Chines Pangolin present in the Himalayan foothills of Nepal, southern Pholidota which includes only family Manidae. There are eight 3–5 species of Pangolin distributed predominantly in Asia and Bhutan and northeastern India. Africa. These eight species are divided into three genus, Manis, In India, the wildlife is protected under the Wildlife Phataginus and Smutsia. Among these three genus, Manis has (Protection) Act 1972 (WPA), therefore due to rapidly four species and they present in the Asian subcontinent while declining Pangolin population in India, this species is kept in Phataginus and Smutsia have two-two species and are present schedule I species under the WPA and also considered as 1 endangered species under the International Union for Conser- in African subcontinent. Among four species of Asian 6 Pangolin, two species are found in the Indian subcontinent vation of Nature and Natural Resources (IUCN). Due to i.e. Indian Pangolin (Manis crassicaudata) and Chines high demand for Indian Pangolin in international market it Pangolin (Manis pentadactyla). The Indian Pangolin is is listed in Appendix II of a Convention on International
Figure 1 Distribution of Indian Pangolin (Manis crassicaudata) and Chinese Pangolin (Manis pentadactyla), red circle represent seizure location. 526 V.P. Kumar et al.
Table 1 Detail of used reference species Cyt b and 16S ribosomal RNA sequences retrieve from GenBank data base and detail of wildlife seizures. Species/Individual Common name Origin Accession No. Cyt b Accession Conservation status No. 16S rRNA References detail Manis crassicaudata/ Indian Pangolin Asian/India KU9258021 to KU925803 to Endangered n =4 KU9258024 KU925806 Manis pentadactyla/ Chinese Pangolin Asian/India- JN411577.1 JN411577.1 Critically Endangered n =1 Chinese Manis tetradactyla/ Black-bellied Pangolin African KJ193388 AJ421454.1 Vulnerable n =1 Manis tricuspis/n = 1 White-bellied Pangolin African KP306514.1| KP306514.1 Vulnerable Manis javanica/n = 1 Malayan Pangolin Asian KP306515.1 X Critically Endangered Manis gigantea/n = 1 Giant ground Pangolin African KJ193383.1 KJ193120.1 Vulnerable Manis temminckii/ Temminck’s ground African X KP125951.1 Vulnerable n=1 Pangolin Specimen Suspected Origin Accession No. Cyt b Accession No. 16S rRNA Seizures detail Seizure-1 Pangolin India/Haryana KU9258025 KU925807 Seizure-2 to 4 Indian Pangolin India/ KU9258026 to KU9258027 KU925808 to Uttarakhand KU925809 Seizure-5 Pangolin India/Uttar KU9258028 KU9258010 Pradesh Seizure-6 Pangolin India/Assam KU9258029 KU9258011 Seizure-7 and 8 Indian Pangolin India/Madhya KU9258030 to KU9258031 KU9258012 to Pradesh KU9258013 Seizure-9 and10 Indian Pangolin India/ KU9258032 to KU9258033 KU9258014 to Chhattisgarh KU9258015 Seizure-11 Indian Pangolin India/Andhra KU9258034 KU9258016 Pradesh Seizure-12 and Indian Pangolin India/Karnataka KU9258035 to KU9258038 KU9258017 to Seizure-15 KU9258020
Table 2 (A) Most similarities in the Cyt b and 16S rRNA based on NCBI GenBank Blast search and (B) similarity based on ClustalW (http://www.ebi.ac.uk/clustalw) compared with India Pangolin reference. 2A Specimen Species with the highest similarity Query coverage (%) Similarity (%) Cyt b 16S rRNA Cyt b 16S rRNA Seizure-1–15 Manis pentadactyla 98 91 92 96 Seizure-1–15 Manis javanica 98 91 90 96
2B Specimen Species with the highest similarity Len(nt) bp Similarity (%) Cyt b 16S rRNA Cyt b 16S rRNA Seizure 1–15 Manis crassicaudata 331 477 100 100 Seizure 1–15 Manis pentadactyla 331 477 88 96 Seizure 1–15 Manis javanica 331 477 88 X Seizure 1–15 Manis tetradactyla 331 477 81 90 Seizure 1–15 Manis tricuspis 331 477 83 90 Seizure 1–15 Manis gigantea X 477 X 90 Seizure 1–15 Manis temminckii 331 477 82 90 Illegal trade of Indian Pangolin 527
Trade in Endangered Species of Wild Fauna and Flora states, which poses a serious threat to the species5 while the (CITES).7 Chines Pangolin in India, also is completely pro- flesh of the species is relished, by some tribal communities.17 tected being listed in Schedule I of the Wildlife (Protection) Scales are the one of the most characteristic features of Act 1972,8,9 critically endangered Pangolin species under the Pangolin and of the reported eight species of Pangolin, there IUCN red data book and Appendix II specie in CITES. has been very constricted knowledge of their morphological Historically, Indian Pangolin was distributed across examination at species level. However, these eight species are southwest China10,11 and in Myanmar12 but due to hunting, morphologically distinct, but somehow the scales of Pangolin poaching13–15 and habitat destruction they are now extinct in do not show clear distinction based on visual examination.18 some regions and now only present in South Asia (Fig. 1). Conventional taxonomist can identify the seized Pangolin spe- The Pangolin has been hunted for the last four decades for cies based on morphological characteristics but this will not the consumption of its meat as a protein source, and for its help further, once the Pangolin scales are removed from trade scales for use in traditional medicines among other spiritual in the market. Consequently, a DNA based approach for spe- and ritualistic uses.16 It also continues to be hunted ritualisti- cies identification may prove to be a powerful and reliable tool cally, for example, during ‘Shikar Utsav’ in Eastern Indian for wildlife law enforcement agencies.
Table 3A Similarity of seizure with Asian and African Pangolin reference sequences data. The number in table represents the variable site position with available Bos taurus (AY676873.1). Alignment of Cyt b mtDNA gene and fixed SNPs sites in Indian Pangolin represent in highlight.
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Table 3A (continued)
In recent years, numbers of DNA based approaches have ing region i.e. low level of mutation, high copy number in cell, been developed for species identification, like DNA hybridiza- maternally inheritance, etc. 12S ribosomal RNA (12S rRNA), tion, restriction enzyme digestion, random PCR amplification, 16S ribosomal RNA (16S rRNA), cytochrome b (Cyt b), and species-specific PCR primer and DNA sequencing.19–24,17,25 cytochrome oxidase I (COI) gene are the mostly used mito- The DNA sequencing is one of the most reliable and robust chondrial gene for species identification in wildlife foren- techniques to identify species from the unknown sample in sic.26–29 Species identification depends on the reliable wildlife forensic. Mitochondrial gene is best suited for the iden- reference sequences availability.30 At present most of time tification of species due to multiple characteristics in their cod- NCBI (http://www.ncbi.nlm.nih.gov/database) or BoLD Illegal trade of Indian Pangolin 529
(http://www.barcodinglife.org) database that are publicly and referred to Wildlife Forensic and Conservation Genetic available is used for species comparison. In the present study, Cell (WFCG), Wildlife Institute of India (WII), Dehradun for the first time in the world, we attempted to address the for genetic study. molecular identification of Indian Pangolin from scales seized under the wildlife offence case and its application in wildlife 2.1. DNA extraction forensics and to frame a conservation action plan and strate- gies for Pangolin conservation in India. The samples undertaken for the study i.e. scales were subjected for DNA extraction, which were initially cleaned with ethanol 2. Materials and methods and 10X Phosphate buffered saline (PBS) two times, outer sur- face (0.5 mm) of these samples was removed using a surgical Included fifteen independent consignments of scale samples blade to remove possible surface contaminants and again seized in India (Fig. 1) during year 2011–2014. The samples washed with 10X PBS. A small section of scales part was cut were seized under Wildlife (Protection) Act 1972, of India out, transferred to a 2 ll eppendorf tube using published
Table 3B Similarity of seizure with Asian and African Pangolin reference sequences data. The number in table represents the variable site position with available Bos taurus (AY676873.1). Alignment of 16S rRNA mtDNA gene and fixed SNPs sites in Indian Pangolin represent in highlight.
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Table 3B (continued)
Seizure-1 Seizure-2 Seizure-3 Seizure-4 Seizure-5 Seizure-6 Seizure-7 Seizure-8 Seizure-9 100 Seizure-10 Seizure-11 Asian pangolin Seizure-12 Seizure-13 Seizure-14 99 Seizure-15 Manis crassicaudata-1 Manis crassicaudata-2 Manis crassicaudata-3 Manis crassicaudata-4 Manis pentadactyla 99 Manis javanica Manis temminckii Manis tetradactyla African Pangolin 100 Manis tricuspis
0.02
Figure 2 Cytochrome b based phylogenetic similarity between the seizures and other pangolins, shaded box represent Indian pangolin similarity with seizures. Illegal trade of Indian Pangolin 531
Seizure-1 Seizure-2 Seizure-3 Seizure-4 Seizure-5 Seizure-6 Seizure-7 Seizure-8 Sizure-9 100 Seizure-10 Asian pangolin Seizure-11 Seizure-12 Seizure-13 Seizure-14 100 Seizure-15 Manis crassicaudata 1 Manis crassicaudata 2 Manis crassicaudata 3 Manis crassicaudata 4 Manis pentadactyla
76 Manis tetradactyla Manis tricuspis African pangolin Manis gigantea 98 Manis temminckii) (A)
0.01
Figure 3 16S ribosomal RNA based phylogenetic similarity between the seizures and pangolin, shaded box represent Indian pangolin similarity with seizure. ancient bone extraction protocol for further process.31 The algorithm implemented in BioEdit version 7.0.5.335 was used DNA concentration was quantified by absorbance measure- in the Multiple Sequence Alignments (MSA).The sequences ment at 260 nm. Absorbance measurements at 260 nm were obtained from the fifteen different specimens were compared done using NanoDrop ND-1000 (Thermo Fisher Scientific with other Pangolin sequences available on public domain at Inc., Waltham, USA) with 1 ll of samples. GenBank (http://blast.ncbi.nlm.nih.gov/) and wildlife forensic reference databases (Table 1). For the species diagnosis, we 2.2. PCR amplification considered the percentage similarity between query and refer- ence sequence pairs. To conform MSA result, we compare Partial fragments of the Cyt b gene (ca.350 bp) and 16S rRNA our data with phylogenetic analysis conducted using MEGA 36 gene (ca. 511 bp) were amplified.32,33 All PCR reactions were v6 for Cyt b and 16S rRNA using the neighbor-joining carried out on a Thermal Cycler 2720 (Applied Biosystem, method. USA) were set up in 15 ll of the reaction volume containing 7.5 llof2Â Qiagen Multiplex PCR Master Mix, 0.30 llof 3. Result and discussion 10 lM of each primer pair, 1 ll of Q solution (supplied with kit), 2 ll of DNA elutant (approx. 20 ng) and remaining The obtained genomic DNA concentration in all seizures was RNase-free water to make the final reaction volume of 15 ll. good and ranged from 10.90 ng/lL to 12.84 ng/lL. In PCR, Primers, PCR conditions, and sequencing protocol were as used mitochondrial genes i.e., Cyt b and 16S rRNA genes were described in Kumar et al. (2014).34 Positive and negative con- successfully amplified in all seizures. The seizures yielded read- trols were performed throughout all DNA extraction and PCR able sequences for Cyt b and 16S rRNA genes of length ca. amplifications. 331 bp and ca. 477 bp, respectively. The sequences obtained were submitted as independent entries in BLAST analysis, 2.3. DNA sequencing and analysis Cyt b gene indicated that the all seizures were 92% similar to the Chinese Pangolin (Manis pentadactyla) and 91% with The PCR products were purified using ExoSAP and finally Malayan Pangolin (Manis javanica)(Table 2A) where the sequenced on an ABI 3130 genetic analyzer using a Big Dye 16S rRNA mitochondrial gene sequences showed 96% similar- Terminator v 3.1 Kit (Applied Biosystem, USA). Quality of ity with Chinese and Malayan Pangolin. In public domain, sin- sequences was determined using Sequence Analysis v5.2 soft- gle short Cyt b sequence of Indian Pangolin was available. ware (Applied Biosystem). Apart from that CLUSTAL W Therefore these fifteen sequences of both mitochondrial genes 532 V.P. Kumar et al. show higher similarity with Chinese and Malayan Pangolins Ethical approval but, when we are comparing the gene sequences of all seizures with the reference sequence, available at WFCG,WII refer- Necessary ethical approval was obtained from the institute ences repository, the seizures showed high similarity (100%) ethics committee. to Indian Pangolin (Manis crassicaudata) (Table 2B). In a Cyt b gene, twelve (n = 12) species specific fixed SNPs were Acknowledgments detected in Indian Pangolin, while in 16S rRNA gene, five (n = 5) species specific fixed SNPs were detected The authors are thankful to the Director, Dean and Research (Table 3A & 3B).The neighbor-joining phylogenetic tree Coordinator, WII, Dehradun, for their strong support along showed that CLUSTAL W analysis is similar in Cyt b gene with the Nodal Officer of Wildlife Forensic and Conservation and 16S rRNA (Figs. 2 & 3). All generated sequences were Genetics, Cell. We would like to thank all the researchers and submitted in GenBank and the assigned accession number is staff of the Wildlife Forensic and Conservation Genetics Cell, given in Table 1 for future use. To find out the nucleotide posi- WII for providing scientific and technical assistance while tion of variable sites we align our data with available complete undertaking the work. mitochondrial genome of Bos taurus (AY676873.1).
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