Midst Of Richest Biodiversity Nations

Published Date: 02 Nov 2017

Researchers from diverse fields counting veterinary sciences, taxonomy, wildlife biology, forensic wildlife, the textile industry and forensic medicine, have used the micromorphological characterization of different types of hairs for investigating crime and suspected samples[7]. Determination keys for getting accurate identification are based on macroscopic features such as size, shape, profile and colour of the hairs, scale pattern similarly microscopic characteristics of the cortex medulla and cuticle structure[8]. Although, the identification is problematical by the occurrence of significant discrepancy caused by differences of species, breed and gender of the animals, by environmental conditions (climate, habitat, nutrition) and possibly also by the body region from which the hairs are derived[9].

Now a day, the crime related to animals of high economic impact (Scheduled Under Wildlife Protection Acts) has gained specific focus where hair can be used for identification of the animals in issues related to species identification,[10] but this demands reference samples for the examination. Reference sample collection for each case from zoos, wildlife rescue centers and museums is a lengthy task to perform examination based on microscopy between closely associated species like tiger, lion, and leopard since they belongs to the same family. The animals are mostly poached for their meat, skin, bones, claws, nails and ivory etc where hair can be used to identify the species[11]. Hair plays a vital role as the key evidence found in cases related to wildlife crimes of these species[12]. This evidence will lead to the identification of species with the help of Scanning Electron Microscope (SEM) and can be utilized for the identification of the animals[13]. Scanning electron microscopy provides higher range of magnification and coupled Energy Dispersive Spectra(EDS) which will leads to the identification of geographical region by elemental analysis can be used further for other examinations[14]. A comprehensive microscopic hair characteristic atlas of indigenous mammals of Australia have been reported earlier in which the hair scale pattern of different species have been incorporate[15]. Similarly, a key atlas on microscopic hair characters of west European mammals has been published. Examination hair have been widely used for taxonomic identification has been reported for few species. Hair sample can be used for genomic and mitochondrial DNA analysis and other chemical tests can also be subjected to go for individual identification. But till date, not so much validated methods for hair examination regarding intra-family species identification are available using just a fraction of hair. As growth pattern and life span of the animals from same family varies significantly[16]. Based on the growth of the animals, hair scale pattern and its growth also varies from species to species[17]. Current study shows that how hair varies-between species and how he developed a database of mammal hair that can be used by wild life forensic scientists in investigating cases of illegal trafficking of the world’s most endangered animals[18]. Hair samples from some wild animals were evaluated in this study. SEM of the scale pattern was investigated for identification of scale pattern difference in three closely related species from the family Felidae including Panthera leo persica, panthera pradus fusca and panthera tigris tigris. The ongoing research on microscopic characteristics of hair can be used to offer more information for wildlife forensics and provide vital information for the identification of prohibited transfer of animals, poaching or wildlife crime, fraud in textile and fur industry, and recognition of their victims, etc.

Materials and Methods

Reagents: Absolute alcohol, SDS, HNO3, H2O2, Rectified Spirit used were analytical grade reagents procured from Hi-Media and Merck specialties. Carbon tape, aluminum adhesive and gold-palladium coating material was taken from ZEISS SEM supplies.

Sample collection : Hairs of lion, leopard and tigers were collected from Indroda nature park, Gandhinagar (23011’32.70"N, 720 39’03.04"E); Kamala Nehru Zoological Garden Kankaria, Ahmedabad (23000’34.11"N, 72035’58.17"E); Sakkarbaug Zoo (21032’27.05"N, 70028’06.53"E), Wildlife Rescue Center Junagadh (21032’56.82"N, 70028’23.44"E) wide letter No. WLP/28/B/4651-54/2012-13 from the Office of the Principle Chief Conservator of Forests and Chief Wildlife Warden, Gujarat State. 10 hair of each of 90 animals were taken from the cage and enclosure using forceps with gloves to avoid contamination and were packed in zip-lock bags followed by paper envelops. Soil and water samples of the same were collected from 30 different sites from the area selected for the present study. Geographical locations were identified by "Bhaskaracharya Institute of Space Applications and Geo-Informatics", Gandhinagar, INDIA.

Sample preparation: Hair samples were washed with absolute alcohol followed by washing in 2% SDS, rinsed, repeated these steps three times, dried at room temperature for about 24 hours followed by packing in fresh zip-lock bags and stored at 00 C till further analysis. For Scanning electron microscopy hair samples were dissected in pieces of 5 mm size leaving 3 mm from root side and placed on sample holder followed by plasma Gold-Palladium coating at 5 milibar vacuum and 5 mA current for 105 seconds.

SEM-EDS analysis: the samples were examined under Scanning Electron Microscopy [SEM] using Zeiss EVO 18 Special edition for imaging and elemental analysis. Examination was done with working distance (WD) of 7 mm and Electron Gun frequency at 10kV for imaging at a line integral of n=50 and 24 bit imaging software mode provided with SEM Workstation, and at WD of 9.5 mm Gun Beam on 20kV using Energy dispersive Spectrum detector provided by Oxford Instruments for EDS using Dot-Id setup for the elements S, Cu, Fe, K, Ca and Ba.

For getting the clarity in the results 10 hair samples from 70 different animals were tested using SEM for getting accurate scale differentiation between each layer of the scales for all the three species and difference was measured at various magnifications i.e. at 500 X, 1000 X, 2000X and raised up to 7500 X for crosschecking the accuracy using annotations present in the software EM Server. The systematic examination using various instruments resulted in detection of the elements with their corresponding quantities. These have shown the constituent concentrations of the elements that are present in hair samples.

Results

The measurements obtained during current study are shown in table no. 1.

There is a significant difference in scale layers between all three species of the animals have been observed and shown a close variation between intra-species from all the samples. The variation between intra-species were tested statistically and p<0.01 while it is observed with a significant difference in inter-species comparison where the distance between each layer of hair scale was observed in 30 animals from 10 samples of each animal for all three species and observed the scale difference between each layer ranges from 6.367 µmeter to 6.948 µmeter in Asiatic Lion, from 7.758 µmeter-7.992 µmeter for tiger while it ranges from 8.63-9.884 µmeter in case of leopard. The scale pattern differences have observed at different magnifications i.e. 500X and 200X and also magnified more than 7500X for verifying the accuracy. Elemental profiling have observed under SEM-EDS .

The scale pattern difference of Panthera leo persica have been observed at three different magnifications i.e. 500X, 2000X and 7.58KX as shown in figure no. 1-3 where the constant differences in the scales were observed with minor variations from 6.367-9.948 µmeter from all the sides of the body hairs fallen naturally from the body, inside the enclosure of the animal for all 30 animals.

The differences between scale layers of Panthera pradus fusca using SEM have revealed the differences with a significant variation from all the remaining two species of Felidae family under study that ranges between 8.63-9.884 µmeter, shown in figure 4 while the difference among animals of species panthera tigris tigris were observed to have a range of 7.758-7.992 µmeters shown in figure no. 5-6.

Elemental analysis using SEM-EDS have revealed about the percentage of Sodium, Sulfur, Calcium and Potassium on the basis of weight% and Atomic % respectively as shown in table no 2 and figures no. 7-8 that revealed the metal concentration in panthera leo among all samples of the species where p<0.01. SEM-EDS study of other species of the Felidae family have revealed about the weight and Atomic percentage of sodium, Sulfur, Potassium, and Calcium in Panthera pradus Fusca and Panthera tigris tigris have been observed as shown in table no. 3, figure no 9-10 for Panthera pradus fusca and table no. 4, figure no. 11-12 for Panthera tigris tigris where P<0.01.

Discussion

The use of scanning electron microscopy in wildlife forensic cases has been described for species identification [19]. The surface cuticular pattern, cross section and medullary index provides the information regarding the species [20]. Researchers have revealed the scale architecture of regular mosaic with smooth margins of shahtoosh wool. But when the hair evidence from different species from the same family were found to be blended together than the investigation becomes quite tough and typical so the present study which incorporates the scale layer difference between closely related species can provide the information regarding its species to the forensic expert. Elemental analysis through EDS provides significant information of the hair sample of specific sample which can further be used as a geographical region and species identification tool. SEM and SEM-EDS requires just a fraction of hair sample and provides vital information regarding the sample which leads to the criminal identification as in detection of Species, occupational exposure which is present inside the hair sample due to environmental exposure and sometimes the geographical area if the soil particles were recovered from SEM and SEM-EDS analysis. Although there is a vast range of literature available regarding medullary index, scale pattern, pigmentation and growth there is a need to develop the database of the differentiation in scale layers which does not only provide the information regarding species[21-26] but also give slue regarding his disease history as well as environmental exposure with the help of techniques like SEM. EDXRF, EDS etc. This study will work as a tool / primary source in forming the database of the scale layer pattern for the identification of closely related species from the same family where the examiner faces problems regarding identification.

Conclusion

Although our studies were performed with a small sample size, our results are based on the morphology, elemental analysis and cuticular scale pattern of the hair obtained using most advanced investigational methods clearly shows the new finding previously not reported in identification of hair. Our study provides a clear and significant research results for the identification of closely related species on the bases of their scale layer difference and elemental analysis profile pattern.

The second major conclusion of work is that it provides a vital information regarding identification of three different species from the Felidae family on the bases of their scale layer differentiation pattern whereas it was quite difficult to identify them on the bases of previously reported literature and provides a new area for the identification of hairs.