Tiger (Panthera tigris corbetti) hair analysis from Uttarakhand, by S.K. Gupta, Scientist from Wildlife Institute of India, Wildlife Forensic Lab, Dehradun 2010.

A report on similarity test of two hair samples of Tiger (Panthera tigris)

Abstract:

Two tiger hair samples were sent by Frederique Lacraz, Society for Mahseer Conservancy, Ramnagar, Uttarakhand to Wildlife Institute of India. Those two tiger hair samples were collected in the same area (Tera village) where there has been two attacks on ladies by tigers. The reason for a DNA analysis was to identify, whether both the sample belonged to the same individual. Such issue can be dealt based on commonly used genotyping based genetic analysis. We describe here similarity test between two hair samples of Tigers (Panthera tigris). There are genetic markers used in similarity analysis are called Microsatellites. We used 5 fluorescent labelled microsatellite loci for analysis. After the comparison of all loci, the DNA typing indicates that in both hair samples alleles tested were not the same. We conclude the both the hair samples have different genotype i.e. Hair sample marked as F-1553 (174176, 130130, 182182, 124126, 166166) and Hair sample marked as R-1944 (174176, 132144, 182182, 124126, 162162) are not from the same individual.

Introduction:

DNA marker technologies have revolutionized molecular genetic techniques as they can contribute to the management of threatened species. However, despite a myriad of potential application, the usefulness of these techniques for making informed management decisions is generally underappreciated. Molecular genetic technique can be used to identify same individuals, parents, offspring and close relatives in captive and wild population (Liu and Cordes, 2004). Everyone, except identical twins, have a one-of-a-kind DNA genome. The DNA molecule is made of monomers called nucleotides, and the specific order in which nucleotides occur in a DNA molecule represents each individual’s unique genetic code. The analysis tests the DNA isolated to locate certain regions of chromosomes that are known to vary in length between individuals. These sites are tested; each site is called a “locus”, (“loci” – plural). Analysis of these sites in a large population reveals many different sized versions associated with each site.

Methodology

DNA fingerprinting determines whether two biological samples belong to same individual or not.

 Extraction of DNA

Both the hair samples marked as F-1553 and R-1944 were subjected to DNA extraction. Commercially available DNeasy Tissue Kit (QIAGEN, Germany) was used to extract DNA from these samples.

 PCR Amplification and electrophoresis

Samples were analysed using 5 polymorphic mcrosatellite loci developed for tiger. These primers (markers) are C6, F1, D6, FCA304 and Ple23.

Versions of a DNA sequence or a gene are called “alleles”. At one locus, there are two alleles, one come from father and other from mother. Genetic markers used in individual identification and parentage analysis are called Microsatellites, or Simple Sequence Repeats (SSRs), there are polymorphic loci present in nuclear and organelle DNA that consist of repeating units of 1-6 base pairs in length. They are typically neutral, co-dominant and are used as molecular markers which have wide-ranging applications in the field of genetics, including kinship and population studies.

Because each individual has two of each type of chromosome, one inherited from each parent and therefore has two alleles at each locus. These two alleles are sometimes identical called homozygous, but sometimes are not the same size called heterozygous. During similarity test on the basis of DNA fingerprinting, the analysis identifies the length of the two alleles found at each locus, by comparing the DNA profiles of the two different samples. In this report we document the similarity test undertaken between two hair samples of tiger sent by a NGO from Ramnagar, Uttarakhand and marked as F-1553 and R-1944.

Methodology

DNA fingerprinting determines whether two biological samples belong to same individual or not.

Extraction of DNA

Both the hair samples marked as F-1553 and R-1944 were subjected to DNA extraction. Commercially available DNeasy Tissue Kit (QIAGEN, Germany) was used to extract DNA from these samples.

PCR Amplification and electrophoresis

Samples were analysed using 5 polymorphic mcrosatellite loci developed for tiger. These primers (markers) are C6, F1, D6, FCA304 and Ple23.

PCR amplifications were performed in ABI thermal cycler GeneAmp® PCR System 2700, Applied Biosystems, Singapore) in a final volume of 10 μl, containing 25-50 ng of genomic DNA, 1X PCR buffer (applied biosystem), 2.0 mM MgCl2, 0.2 mM of each dNTP, 5 pmol of each primer and 1 units of AmpliTaq Gold DNA polymerase. Amplification conditions were 94°C for 10 min followed by 35 cycles at 94°C for 45s, 55 °C for 45s and 72°C for 1 min, with a final extension of 72°C for 20 min. Distilled water was taken as negative control to ensure there was no contamination in reaction mixtures. PCR products were genotyped using ABI Prism 3130 genetic analyzer.

Results

All the 5 fluorescent microsatellite were amplified successfully. Test in Table1 and Fig. 1 indicate multilocus 5 loci genotype obtained from both the hair samples. After the comparison of all loci, the genotype of both the samples is different at 2 loci. Therefore it is concluded that the hair sample marked as F-1553 having multilocus genotype (174176, 130130, 182182, 124126, 166166) is not the exactly same as the hair sample marked as R-1944 (174176, 132144, 182182, 124126, 162162).

Conclusion:

Five different loci were used as genetic markers in similarity test concluded to determine whether two tiger hair sample sent by Society for Mahseer Conservancy, Ramnagar, Uttarakhand are derived from the same individuals. The multilocus genotype data obtained from both the hair samples indicate that these hair are having the different allele at two loci out of five loci, therefore; both the hair samples are the derivative of two different individual. Genetic id or multilocus genotyping of both the hairs are as follows.

Hair I (F-1553): 174176, 130130, 182182, 124126, 166166

Hair II (R-1944): 174176, 132144, 182182, 124126, 162162

Neema

In the village of Ringora, dist. Nainital, a girl named Neema is making artefact using local raw material, the “saan”. Her products are the only local creation that can be found in and around Corbett, making it priceless.

But Neema is not only a brand, it represents a whole history of Kumaoni women who work incredibly hard every day. Their daily life starts early morning, where they go inside the jungle to collect firewood and grass, to cook and heat the houses, to feed the cattle but also to collect this particular grass that is much needed to repair the roofs and that can be sold at a good price.

Ringora woman coming back from grass cutting

Those women are putting their lives in danger, since every venue inside the jungle can be matched with potential wildlife encounter. Tiger are the best known mammals to attack villagers, but they are not the only ones. A list of many animals including leopard, bear, elephant and snake can turn the whole excursion into a sad day. But they have no choice. The government is given almost no alternative to this wood collection, and for many villagers – not only in Corbett, but everywhere in India – the free wood and grass that forests are providing is the assurance of the next meals.

When they come back from the jungle, women have to prepare food, take care of the children, work in the fields, do the family laundry, go to collect water from the water source, they hardly rest. And the most amazing thing among all those women is that they never complain.

From my European point of view, I find them to be amazingly brave, physically and mentally strong. I love spending time with them since everything is a pretext to have a laugh, they enjoy life as it is and they would give quite a lecture to many people’s behaviour and way of thinking.

Neema bag

Neema doll

Neema’s products, dexterously made, comprise hand bags for a classy touch and also a range of dolls, the “Neema dolls” that are the representation of a Kumaoni women, carrying a load of wood or grass on their heads. This girl is the ambassador of many women, and by making those artefacts, she definitely gives a shout that those women are important in so many ways and people should never forget that they exist and they are ready to stand for their rights.  By purchasing the Neema’s handicrafts, people are directly improving the lives of the girls who are making them, contributing to the girls and women empowerment of forgotten villages, like Ringora.

Neema making dolls in her Ringora house

If you want to buy some Neema artefacts, please contact us at mahseerconservancy@gmail.com

Women collecting wood

In India, a high density of people is dependant on forest products in order to cook, to heat the houses and to feed the cattle. This has been a tradition for decades if not centuries among Indian villagers. Wood is indeed a privileged energy source since it is free of cost and is, for now on, still available. But this wood collection is step by step destroying the ecosystems of the forests. The forest products collected which are bark, dead and green wood and grass all participate in the balance to keep forests in good health. The barks are protecting the trees from any aggression; the green wood is vital for trees and especially for young trees to grow; the dead wood, by decomposing, plays a major role in the richness of the soil, creating micro ecosystems and the grass gives wild herbivores a source of food. A degradation of any of those elements is putting the others in danger.

Smokeless chulah

But the villagers can not be blamed so easily. The government is given almost no alternative to this wood collection. Liquefied Petrol Gas can be purchased at low price (≈200INR) but for poor villagers, this alternative is still too much expensive. The only way to get energy supply and moreover for free is by collecting wood. The best approach to reduce the wood consumption and therefore the wood collection is to provide sustainable alternatives to the villagers for cooking like smokeless chulah and solar ovens. The introduction of those alternatives may be hard at the beginning but they are necessary to change the wood consumption pattern and reduce the impact of the villagers on the forests. Another way to reduce the wood consumption is to give the opportunity to the villagers to raise their monthly income and make them able to afford energy supplies else than wood. The Lantana furniture making project serves this aim.

Woman making wood bundle

The wood collection is, one must never forget, a hard job. Villagers (mostly women) go early morning and walk sometime several kilometres inside the jungle in order to find wood supply. They spend around 2 hours collecting and cutting branches with a dharati. After that, they make wood bundles and tie them with green barks. They walk then back home with loads of dozens of kilograms on their heads. Sometime, they go twice a day in the jungle to get more wood if the weather is good, if more wood is needed or if children do not go to school. When asked, the women often say that they do not like going inside the jungle. First because they are afraid of tigers but also because it enable them to do work at home.

Wood bundles are to be sold

The collection of wood serves two purposes. One is for the use in the houses, the other one is to have it sold. For the second one, the problem is even bigger since the collection of wood can be seen as an usual job, and the forests as money suppliers. The more you take from the forests, the more money you will get. This excessive collection can only be stopped or at least reduced if an economic alternative is proposed to those villagers.

Woman attacked by a tiger in June 2009

Those ladies are incredibly brave, especially considering the fact that the collection of wood happens in areas where potentially lethal animals (tiger, leopard, elephant, bears, snakes, etc) can be met. Since December 2008, four attacks on villagers by tigers have been encountered, one leading to death. Those attacks are mostly due to the fact that villagers, by going inside the jungle, are stepping on big cats territories. That intrusion is sometime severely punished. When they are moving in groups (more than 4 persons), the villagers are safe from tiger attacks. But when they separate on the collection point, they are much more vulnerable and can not always look around for their safety since they have to search for wood.

In this time of rising Man-animal conflict it is hard to see a way that these magnificent creatures will find a way to survive, it is surely only with local support and their respect of the wildlife that my grand children may visit here and experience what I have been privileged enough to.

Snakes have always held that ‘Fear” factor in man, just the mere mention of the word “Snake” can drive masses into panic, in India in particular there reputation as man Killers holds some truth to it with many thousands of people a year losing their lives to snake bites. It is therefore, understandable if the villagers here would first look to kill snakes out of self preservation.

However, yesterday when a snake entered a lady’s room and decided to rest there, no ones first thought was to kill the snake instead they reached for the phone in search of help to remove this snake so that it could carry on with its life in the jungle.  The snake in question was no ordinary snake either it was the King of Kings the King Cobra.  A snake so large and with such a fearsome reputation it would have been no surprise if the immediate reaction was to kill it out of self preservation.  Instead their reaction of reaching out for help to preserve this magnificent and rare snake is the first sign of conservation and a huge encouragement that the wildlife in this area will be saved.

Sanjay Chhimwal a renowned naturalist of the area stepped forward to rescue the King and although there was a lot of excitement and fear amongst the by standers, typified by many of them scurrying for safety atop the nearby roof, to look into their eyes was where the real hope lay, there was no malice no intention to kill this lost soul, only a fascination and awe of this hugely impressive King and all bar none gave it the respect it so richly deserves.

For me the most pleasing aspect of this whole episode was to see the amount of people who I had originally involved in the snake conservation project that I started last year.  Many of them like Hem Bahuguna, Khalidatt Kargetti, came from Ramnagar to lend their support, Sumantha Ghosh dropped everything to rush over and lend a hand, and looking around at the faces of people I now call friends it was easy to see that any of them if wouldn’t have hesitated in rescuing the snake.

When it finally freed from its confines to the nearby river the joyous hand and back slapping typified everyone’s wish to see the King back in the Wild where he belongs.

I can only hope that this sort of reaction will be long lasting and will lead to conservation of all species in the area because India without the Majestic and Mystifying Creatures that roam it is just another country and India so much more than that.