The German Primate Center and Deforestation

1.Introduction


The German Primate Center (DPZ), was founded in 1977 as an independent research institute with a service character for German science in Göttingen. The center conducts biological and biomedical research on non-human primates in three main research topics; Infection Research, Neuroscience, and Primate Biology. Research in the Primate Genetics covers studying the immune function of lymphocytes, investigate the biological diversity in different ecosystems, and reconstruct phylogenetic relationships among primates. “The evolutionary history of primates can be traced back 65million years”. Primates have two distinct suborders, Strepsirrhini (lemurs, and lorises) and Haplorrhini (tarsiers, monkeys, apes, and humans). These primates can be found throughout Africa, Madagascar, India, and Southeast Asia. About 60% of primate species are threatened with extinction including: 87% of species in Madagascar, 73% in Asia, 37% in Africa, and 36% in South and Central America. Additionally, as stated by Estrada et al (2017) 75% of primate species are decreasing population due to: deforestation, forest fragmentation, and hunting (Grubb, 1998).
“The capped langur (Trachypithecus pileatus) is a species of primate in the family Cercopithecidae, found in Bangladesh, Bhutan, India, and Myanmar (https://www.iucnredlist.org/species/22041/ 9350087)”. As shown by Groves (2005) Trachypithecus pileatus has four recognized subspecies: Trachypithecus pileatus pileatus, T. pileatus durga, T. pileatus brahma and T. pileatus tenebricus. Molecular determination of sex and species is one of the key tools for determining and monitoring the social structure of wild and endangered primates. Developing markers that simultaneously detect Y- and X-chromosome specific sequences and work across many species is highly desirable. Moreover, Palle and Tina (2006) conducted a study on fast and non-invasive PCR sexing of primates and they identified UTX/UTY locus as the best candidate for a universal primate sexing marker.
Similarly, for better conservation and management of the endangered species, mitochondrial 12S rRNA has proven to be a useful molecular marker. According to Siddappa et al (2013), mitochondrial genes like 12S and 16S rRNA are extensively used in mammalian species identification. In the same way, sequencing the complete mitochondrial genome of a species is crucial for further genetic researches and conservation. Accordingly, Shi et al (2015) determined the complete mitochondrial genome of the Trachypithecus pileatus with the total sequence length of 16,526 bp and 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and D-loop. However, detailed information on the complete mitochondrial genome of Trachypithecus pileatus tenebricus subspecies is limited. Thus, this work aims, on the molecular determination of sex and species of different primates, and to sequence the complete mitochondrial genome of Trachypithecus pileatus tenebricus.

2. Materials and methods


2.1. Sample


Eight fecal samples putatively from primates from diverse locations in India namely Trachypithecus pileatus tenebricus from Manas National Park, Trachypithecus pileatus pileatus (Kaziranga National Park), T.Pileatus pileatus (Hoollongapar Gibbon sanctuary), Hoolock hoolock (Hoollongapar Gibbon sanctuary), and four samples of Trachypithecus geei from Kakoijana reserve forest were collected and conserved with 80% ethanol and transported to DPZ for analysis.

2.2. DNA extraction


Genomic DNA from stool samples was isolated using the NucleoSpin® DNA stool kit protocol. For this, 180-220mg of stool sample was transferred into the NucleoSpin® Bead Tube Type A and diluted with 850µl of ST1 buffer and lysis sample through incubating NucleoSpin® Bead Tubes for 5min at 70oC. Precipitating contaminants via centrifuge for 3minutes at 13,000 x rpm. Then, transfer 600µl of supernatant to 2mL microcentrifuge tube and aliquoted with 100µl of ST2 buffer and vortex for 5 second and incubate at 4oc for 5 minutes. Afterwards centrifuge for 3min at 13,000 x rpm. To filter lysate 550µl of clear lysate was transferred into the NucleoSpin® Inhibitor Removal column (red ring) with a 2mL collection tube and centrifuge for 1min at 13,000 x rpm. The NucleoSpin® Inhibitor Removal column was discarded and 200µl of ST3 buffer was added and vortex for 5seconds.
To bind DNA, place a NucleoSpin® DNA stool column (green ring) in a collection tube and 700µl of the sample was loaded and centrifuged for 1min at 13,000 x rpm. Discard the flow-through and place back the column and wash silica membrane, 1st wash, add 600µl of ST3 buffer and centrifuged for 1min at 13,000 x rpm. Second wash, discard flow-through and add 550µl of ST4 buffer into NucleoSpin® DNA stool column and centrifuge, discard flow-through and add 700µl of ST5 buffer into the NucleoSpin® DNA stool column, vortex, and centrifuged (3rd & 4th wash). To dry silica membrane centrifuged for 2min at 13,000 x rpm. To elute DNA, place NucleoSpin® DNA stool column into a new 1.5mL microcentrifuge tube and add 30µl of SE buffer and centrifuge for 1min at 13,000 x rpm and discard the column. Lastly, for Qubit Fluorometer measurement 199µl of Qubit Fluorometer working solution and 1µl of DNA was diluted and DNA concertation was quantified through Qubit Fluorometer and NanoDrop Spectrophotometer.

2.3. Molecular identification of sex


Universal sexing primer pairs 8260-F/8261-R that simultaneously bind X (180bp) and Y (140bp) at pseudo-autosomal regions were used. PCR reaction was set up in 25µL volume consisting: 2.5µl buffer, 0.5µl of dNTP, 1µl of each primer, 0.25µl of high-fidelity Tag polymerase, 19.75µl of HPLC water and 100ng/µl of template DNA. Amplification was carried out in a thermal cycler at 94 oC 2’, 50 cycles with 95 30’’, 57oC 30’’, 72 oC 30’’ and 72 5’ and the amplicon was visualized at 2.5% agarose gel electrophoresis.

2.4. Molecular identification of species


Mitochondrial 12S rRNA gene with ca450bp was amplified using universal primers Egel-F/Egel-R that amplify homologous mitochondrial genomic sequences from a wide variety of mammal species at 95oC 2’, 50 cycles with 95 30’’, 50oC 30’’, 72oC 30’’ and 72 5’. The PCR products were separated via 1.5% agarose gel electrophoresis and the excised PCR products were purified using-Monarch DNA Gel extraction kit protocol and the gel was diluted with 340µl of Monarch ®Gel Dissolving buffer and incubated at 50oC for 10 minutes and transfer to the column with collection tube and spin for 1minute with 13,000rpm then the flow-through had discarded and re-insert the column to collection tube and 200µl of Monarch® DNA wash buffer was added and spin for 1minute (2x). Purified DNA was eluted using14µl of HPLC water and 3µl of loading dye,3x was aliquoted with 1µl of DNA sample and three standards (10, 25 & 50ng/µl of plasmid DNA) were used as an internal control and the concentration was measured under UV-light and Sanger sequenced.

2.5. Complete mitochondrial genome sequencing


Twenty different overlapping PCR products were used to sequence the complete mitochondrial genome of Trachypithecus Pileatus tenebricus from Manas National Park, India. PCR reaction was set up in 25µL volume consisting: 2.5µl buffer, 0.5µl of dNTP, 1µl of each primer, 0.25µl of high-fidelity Tag polymerase,18.75µl of HPLC water and 1µl (100ng/µl) of template DNA. PCR reactions were run with the following program: 94oC 2’, 50 cycles with 94oC 1’, 58oC 1’, 72oC 1.5’, and 72oC 5’. The PCR products were separated in 1.5% agarose gels and then excised and purified using-Monarch DNA Gel extraction kit protocol as stated before and 10, 25 and 50ng/µl of plasmid DNA were used as an internal control. Later, the concentration was measured under UV-light and Sanger sequenced.

3. Results

3.1. Molecular determination of sex


Universal sexing primer pairs 8260-F/8261-R that simultaneously bind X (180bp) and Y(140bp) at pseudo-autosomal region were used and PCR products were then visualized under standard gel electrophoresis, revealing females as homozygotes (XX) and males as heterozygotes (XY). The results of the present study (Fig.1) revealed that Trachypithecus pileatus tenebricus (1) from Manas national Park, Hoolock hoolock (4) from Hoollongapar Gibbon sanctuary and Trachypithecus geei (8) from Kakoijana reserve forest showed a strong double band (Male) and the remaining five samples had a single band (Female).

Fig1.Molecular determination of sex
Selected male (M) and female (F) primate samples amplified with primer pair 8260-F/8261-R and run on 2.5% agarose gel. SS = size standard (100 bp ladder). The males 1,4, and 8 shown are heterozygous-(XY). The females 2,3,5,6, & 7 are homozygous-(XX). No band on PCR products with negative control (NK) was observed.


3.2. Molecular determination of species


Genomic DNA was isolated from the eight stool samples and the mitochondrial 12S rRNA gene was amplified using universal primers. PCR product was subjected to Sanger sequencing and an online Basic Local Alignment Search Tool (BLAST) at NCBI was used to compare the nucleotide sequences. In the current study, PCR amplification has provided mitochondrial 12S rRNA gene of nearly 500bp (Figure 2) long nucleotide sequence which was visualized by gel electrophoresis.
The result revealed that the mitochondrial 12S rRNA gene sequence of Trachypithecus pileatus tenebricus from Manas National Park showed 100% identity with T.pileatus. Similarly, mitochondrial 12S rRNA gene sequence of T.pileatus from Kaziranga National Park, T.pileatus (Hoollongapar Gibbon sanctuary), and Hoolock hoolock from Hoollongapar Gibbon sanctuary showed 99.73, 99.75, and 99.23 % identity with T.pileatus, Muntiacus muntjak, and Hoolock hoolock respectively. The result also suggested that the mitochondrial 12S rRNA gene sequence of four Trachypithecus geei species (Table 2.) from the Kakoijana reserve forest showed, 99.74, 99.49, 99.24, and 98.49% identity with Trachypithecus pileatus species. Indeed, for 7 samples the species identity was confirmed, while for one sample, putatively from a T. p. pileatus was actually from Muntiacus. \nFig2. Analysis of the PCR amplicon on 1.5% agarose gel electrophoresis. Samples 1-8 have ca.500bp, NK; negative control.
Fig3. PCR amplification of universal primers Egel-F/Egel-R for species determination. Internal standards:50, 25, and 10ng/µl of the plasmid. Samples 1, 2,4,5,6,7, and 8 had 50ng/µl of DNA concentration but sample 3 had 25ng/µl of DNA. NK; negative control.


3.3. Complete mitochondrial genome sequence of Trachypithecus pileatus tenebricus


In the present study, we used 20 sequencing primers to sequence overlapping fragments to generate the complete mitochondrial genome. The result revealed that the PCR amplification of 20 overlapping sequencing primers provides a DNA molecular size of 1kb base pair (bp). Besides, majority of primer pairs had DNA concentration of 50ng/µl, but small number of primer pairs showed 25 and 10ng/µl of DNA (Fig 4). \n
Fig4. PCR amplification of 20 overlapping sequencing primers. Visualized on 1.5% gel electrophoresis, *SS; the size of ladder 1kb bp, NC; negative control \n
Fig5. DNA concentration of 20 overlapping PCR products. *Standards as an internal control;50, 25 and 10ng/µl,1kb base pair (bp) product length with 10ng/µl of sample concentration with the volume of 15µl for sequencing

4. Discussion


4.1. Molecular determination of sex and species


Molecular identification of sex is one of the key tools for determining and monitoring the social structure of wild and endangered primates. The results of the present study revealed that three samples are identified as males and the remaining five samples are derived from females. The result showed that small PCR product size hence can be applied to degraded DNA as extracted from feces and these primers can be universally used for sexing mammals like muntijak.
The 12S rRNA gene of the mitochondrial genome is necessary for the translation of messenger RNAs into mitochondrial proteins. The result revealed that the mitochondrial 12S rRNA gene sequence of Trachypithecus pileatus tenebricus from Manas National Park showed 100% identity with T.pileatus. Similarly, mitochondrial 12S rRNA gene sequence of T.pileatus from Kaziranga National Park, T.pileatus (Hoollongapar Gibbon sanctuary), and Hoolock hoolock from Hoollongapar Gibbon sanctuary showed 99.73, 99.75, and 99.23 % identity with T.pileatus, Muntiacus muntijak, and Hoolock hoolock respectively. The study conducted by Yang et al. (2014) suggested that for commercially obtained tissues including cow, rabbit, chicken, pig, fish and shrimp identities with the corresponding 12S rRNA mitochondrial genomes were 100% (cow, rabbit, chicken, pig, and eel) or 99% (fish and shrimp). The present result supports the explanation of Gupta et al (2008) who stated that universal mitochondrial 12S rRNA gene primers can amplify corresponding regions from a wide variety of organisms, including birds and insects.

4.2. Complete mitochondrial genome sequence


The result revealed that the PCR amplification of 20 overlapping PCR products provides a DNA molecular size of 1kb base pair (bp). Besides, majority of PCR products had DNA concentrations of 50ng/µl, but some showed only 25 and 10ng/µl of DNA. Sequencing results of mitochondrial genome of Trachypithecus pileatus tenebricus has been pending.