Molecular characterization by using 12SrRNA and Cytochrome b for identification of species of genus Ratufa (Rodentia: Scuiridae) including Ratufa indica, endemic species of India.

Oriental giant squirrels are tree squirrels classified under family Ratufinae. In India, there are three species of genus Ratufa, i.e. Ratufa bicolor, Ratufa macroura and Ratufa indica. They are also distributed in South and Southeast Asia. However Ratufa indica is endemic to India. The fourth species Ratufa affinis is restricted to Maritime Southeast Asia (East Malaysia, Indonesia, Brunei and Thailand) and probably in Singapore. The species is near threatened .The species R.macroura is endemic to South Asia. Forests of South and Southeast Asia are hotspots of squirrel diversity but at the same time they are at a high risk of extinction because of high deforestation rate and habitat fragmentation. The present molecular study is the first study of the species of Ratufa for their identification. In this study old taxidermy samples were used for amplification of 12SrRNA and Cytochrome b genes. Maximum likelihood and neighbor-joining methods were used to delineate the species by using MEGA 6 and also for molecular analysis for variable, conserved, parsimony and singleton sites. Similarities between species through BLAST indicated 92.21-89.57% between R.macroura vs. R. bicolour; 93.22-90% Ratufa macroura vs. Ratufa indica; 96-92% Ratufa indica vs. Ratufa bicolour, 93.88% between Ratufa affinis vs. Ratufa indica, 93.5% R. affinis vs. R. bicolor, 90.5%. R. affinis vs. R. macroura. Ratufa bicolor is noted to be genetically closer to R. indica as inferred by using both markers. The BLAST result indicated that the obtained sequences matched 99-100% with their respective species. It was also noted that R. bicolor of Jalpaiguri, West Bengal are genetically closer to that of Bhutan. The study also revealed the evolution of R. indica and R. macroura from a single population.

Araniella cucurbitina: the first molecular evidence of a Palearctic species of genus Araniella inhabiting India.

The present study represents first genetic record of single spider species Araniella cucurbitina, genus Araniella from Uttarakhand, India. There are 12 identified species under Araniella genus and they are distributed in Palearctic region. Here, we used known N = 47 (2 from present study and 45 from GenBank) cytochrome oxidase 1 (CO1) sequences of A. cucurbitina representing seven different geographical groups, additionally 23 sequences of eight Araniella species were used for phylogenetic relationship. The CO1 (561 bp) sequences of A. cucurbitina consisted of n = 14 haplotypes, where haplotype 14 (Hap 14) represents Indian species, while all 13 haplotypes (Hap1-13) shared between six A. cucurbitina groups. The overall 'h' and 'π' diversities among seven groups of A. cucurbitina were 0.85291 and 0.00888, respectively, while overall evolutionary divergence was 0.04. The Indian Hap 14, showed minimum sequence divergence (0.02) from Italy and Czech Republic haplotypes (Hap 6), it means it is the closest group compared to others. Evolutionary divergence among eight species of Araniella ranges from 0.003 to 0.114. The maximum likelihood (ML) topology based on 14 haplotypes of A. cucurbitina was divided into two major clades and further two subclades. Furthermore, ML topology between eight species of Araniella was divided into three major clades, where A. cucurbitina and A. proxima clustered together in clade 'A', while six others were present together in clade 'B' and 'C'. This study helps to identify the Indian species from the rest of Araniella species and other cucurbitina population across the world. This study further needs to be on a large scale to know the exact status distribution and molecular phylogeography of this single species of genus Araniella from India.

Forensically informative nucleotide sequencing (FINS) for species and subspecies of genus Prionailurus (Mammalia: Carnivora: Felidae) through mitochondrial genes (12SrRNA and cytochrome b) by using old taxidermy samples.

Genus Prionailurus comprises four species, i.e. Leopard cat, Rusty spotted cat, Fishing cat, Flat-headed cat, listed under IUCN as threatened species except P. bengalensis. In U.S.A., P. bengalensis is listed as Endangered. Subspecies of P. bengalensis, i.e. iriomotensis is listed as Critically Endangered under IUCN since 2008. The present study describes the use of two markers 12SrRNA and cytochrome b genes to differentiate the three species and three subspecies of Prionailurus. Old taxidermy samples (three skin samples) of P. viverrinus and P.b. horsfieldi were used from India for the study. The study done by using DNAsp v5, MEGA 6.0, and Network 5.0.0.1, proved that both gene markers are useful for differentiating the species and subspecies of Prionailurus. This study is also the first study to present forensically informative nucleotide sequence (FINS) for three species and three subspecies of Prionailurus.

Wildlife forensics in battle against veneration frauds in Uttarakhand, India: identification of protected Indian monitor lizard in items available in the local market under the name of Hatha Jodi.

Certain articles of worship are commonly sold in Uttarakhand, India by the name Hatha Jodi, a root of a rare plant found only in a few parts of central India. The present work provides genetic proof that the Hatha Jodi sold in three local markets of Uttarakhand contained material from the Varanus species, species protected under the Indian Wildlife (Protection) Act, 1972. A total of eight samples were bought, two each from the local markets in Haridwar and Rishikesh, three from Dehradun and one from an online source (Amazon). The initial inspection confirmed that two of the samples were made of plastic material. Therefore only the other six samples were subjected to DNA analysis. DNA sequences were successfully obtained and matched with reference sequences available in NCBI Genbank database through BLAST search tool for species identification. All the six samples matched 100% with the Indian monitor lizard. The findings indicate how commercialization and the wildlife trade are playing a role in decline of the population of the Indian monitor lizard. If strong protection measures are not taken as soon as possible, the Indian monitor lizards will go Extinct very soon. Therefore, we suggest that the Government and Wildlife enforcement agencies take serious action against the illegal articles available in the local markets of Uttarakhand under the name Hatha Jodi. Further, the government needs to take legal action against offenders in other states in which the product is available for sale.

The preliminary molecular study of four skink species in Rajaji Tiger Reserve (RTR), Uttarakhand, using 12S rRNA mitochondrial locus.

Skinks are present under the Scincidae family, widely distributed species in Indian subcontinent. Uttarakhand is one of the hotspot where number of identified and unidentified skink species reported. Herein, we first time provided the 12S rRNA genetic reference database of four skink species, i.e. Eutropis macularia, Eutropis carinata, Asymblepharus himalayanus and Lygosoma punctata, in Rajaji Tiger Reserve (RTR), Uttarakhand (India). The identified four species belong to three different genera, where Eutropis carinata and Asymblepharus himalayanus listed Least Concern and Vulnerable in IUCN, respectively. Here, we collected tissue samples of four different skink species from Rajaji Tiger Reserve during field survey. After successful laboratory procedure, we compared obtained sequences with publically available genetic database and we observed four sequences matched with respective species. Furthermore, the evolutionary sequence divergence result revealed that the Eutropis carinata and Eutropis macularia are close to each other with 0.11 genetic distance. The present study indicates that the exact number and population distribution of skink species are unidentified; therefore, herein we suggest the proper screening of Uttarakhand population around should be investigated, further genetic study in combination with a good sampling strategy to investigate species biology and status for conservation program.

Forensically informative nucleotide sequencing (FINS) for the first time authentication of Indian Varanus species: implication in wildlife forensics and conservation.

Monitor lizards are Varanus species widely distributed, endangered reptile in the IUCN red data list. In India, based on the morphological and ecological characteristic, it is divided into four species viz. Bengal monitor lizard, Yellow monitor lizard, Desert monitor lizard and Water monitor lizard. These four species listed as Schedule I species in Indian Wildlife (Protection) Act 1972. This paper first attempt to present Forensically Informative Nucleotide Sequencing (FINS) for the Indian Varanus based on three mitochondrial genes. The molecular framework will be useful for the identification of Indian Varanus species and trade products derived from monitors and as such, have important applications for wildlife management and conservation. Here, we used known 14 individual skin pieces of four species of monitor lizards; the partial fragment of three mitochondrial genes (Cyt b, 12S rRNA, and 16S rRNA) were amplified for genetic study. In Cyt b, 12S rRNA and 16s rRNA, we observed, 5, 5 and 4 Haplotypes; 71, 69, and 43 Variables sites; 90, 89, and 50 Parsimony Informative sites within four species of Indian monitor lizards, respectively. Despite it, the nucleotide composition was T 26.4, C 32.8, A 29.2 and G11.6; T 18.8, C 29.7, A 34.0 and G 17.5; T 21.7, C 27.3, A 32.5 and G 18.5 in Cyt b, 12S rRNA and 16S rRNA, respectively. The neighbor joining phylogenetic tree and maximum parsimony tree of three mitochondrial genes, showed similar results and reveal that, there are two major clades are present in Indian monitor lizards.

Testicular protein profile (SDS-PAGE) study of zinc deficient Wistar albino rat.

Present study has revealed that zinc plays important role in regulating the production and secretion of proteins at transcriptional or translational level. Study has firmly depicted the change in the levels of polypeptide of 70 kDa in zinc deficient group. The protein pattern in pair fed group has been affected mainly to combat the insult due to low food intake.