DNA Barcoding: An Effective Tool For Shark Species Identification (essay).

Convention of International Trade in Endangered Species (CITES) and Sustainable Shark Trade

Question:

Discuss about the Philosophical Transactions of Royal Society.

The ceratotrichia, collagenous protein present inside the fins of shark are the main constituent that serves as the delicacy in the Shark fin soup. There is an increased activity of shark fishing that drives the soup market which is a very unsustainable. In light of the declining shark population the Convention of International Trade in Endangered Species (CITES) have listed eight species of shark which are Cetorhinus maximus, Lamna nasus, Sphyrna lewini, Carcharhinus longimanus, Carcharodon carcharias and S. zygaena as well as S mokarran (Shivji et al. 2005). It is to note that any kind of international trade involving the listed species should be accompanied with special permit that certifies that the trade is not detrimental to the population of the shark species. Therefore, there is a common need for both the exporting and the importing countries to understand and validate which species of shark are they trading.

Though shark species can be determined by looking at morphological features, processed samples are difficult to be identified. Using DNA barcoding methods can help in such situations. The PCR amplification of cytochrome c oxidase I gene is a standard barcoding technique that is used. However, this process may be limited by extensive processing of the samples with chemicals that can damage the DNA making it literally impossible to obtain intact DNA and sequences for amplification to aid in genetic identification.

DNA barcoding is a technique that aids in identification of species through sections of DNA from a specified standard region of the genome. It is given the term barcoding as every species has a unique sequence that is conserved throughout the species and is not different among members of the same species. Therefore establishing a specified sequence as a DNA barcode will eventually help in species identification with ease. The most often used gene region is the 648 base pair cytochrome C oxidase 1 gene (Moritz & Cicero 2004). The COX1 gene has been extensively used to establish species identification among many oranisms such as birds (Hebert et al. 2004), butterflies (Wiemers & Fiedler 2007), fishes (Weigt et al. 2012) etc. the region is extensively used for species barcoding because it is long enough to be sequenced and yet get a good result as well as small enough to be easily amplified using a PCR. However, the COX1 gene is not so useful in identification of plant species (Chase et al. 2005).

In order to establish a barcode the obtained specimen is lysed and DNA is isolated. The isolated DNA is then amplified for the COX1 gene and sequenced. The sequence is then called a barcode that is deposited in the barcode of Life Data Systems (BOLD) database. Thus the BOLD database is a repository of DNA barcodes and can be used to identify unknown species using DNA sequence from the COX1 gene (Ratnasingham & Hebert 2007). Similarly, though not a barcode repository the sequence database NCBI GenBank stores sequences so far identified of organisms. It is an extensive repository of sequences and all sequences known till date is updated in the database. Therefore searching the database for similarity in the obtained sequence of a specimen will also help in establishing the proximity of phylogenetic relationship between two organism. The two approach BOLD and GenBank search using BLAST (basic Local Alignment and Search Tool) will help in proper identification of unknown sample based on their DNA sequence.

Understanding DNA Barcoding and COX1 Gene

There are other methods that employs other segments of DNA  (Pinhal et al. 2008)other than the cytochrome oxidase gene present in the mitochondria (Naylor et al. 2012). However, COX 1 gene remains the most exploited DNA sequence. The present technique aims to identify shark specimen based on their COX 1 gene obtained by PCR amplification of DNA obtained from tissue samples.

DNA from the shark fin samples were isolated after pulverization of the hard tissues using a kitchen blender to yield single cells. The shark tissues collected were stored at 70% ethanol before pulverization. 2 mm³ of the pulverized cells were cells were lysed with proteinase K (20µg/mL) and 580 µL of TNES (50mM Tris, 400mM NaCl, 100mM EDTA, 0.5% SDS) buffer followed by incubation at 55^oC for 3 hours in a waterbath. After incubation the tube is transferred to room temperature and 170 µL of 5mM NaCl was added followed by vortexing for 15 seconds. The sample was then centrifuged at 14000 rpm for 10 minutes to remove cell debris. The supernatant is recovered to which 770 µL of ice cold 100% ethanol was added. The mixture was inverted 5 times to allow precipitation of DNA followed by centrifugation at 14000 rpm for 5 minutes. The pellet was collected followed by washing with 70% ethanol to remove residual salt followed by centrifugation at 14000 rpm for 5 minutes. The supernatant was removed and pellet was dried at 55^oC for 10 minutes followed by resuspension in 30 µL of sterile water. The quality of DNA was determined by measuring absorbance at 260 and 280 nm and taking the ratio of the two. The amount is determined by measuring absorbance at 260 nm.

DNA isolated from the shark tissue samples were subjected to PCR amplification using cytochrome oxidase I gene forward primer 5’ TCA ACC AAC CAC AAA GAC ATT GGC AC 3’ and reverse primer 5’TAA ACT TCA GGG TGA CCA AAA AAT CA3’ was used. 500ng of the template was taken in a PCR tube with 100pM of primers and mixed with premixed BYBR green mastermix (KAPA SYBR FAST

qPCR Master Mix 2X, KAPABiosystems). Final volume of 20µL was used for the PCR set up. PCR was conducted at 95^oC denaturation, 60^oC annealing for 30 seconds and amplification at 72 ^oC for 30 Seconds. The cycle was repeated for 30 cycles followed by a final extension step of 72^oC for 5 minutes. The product was then analyzed on an agarose gel to validate the size of the product. The PCR product was then subjected to DNA sequencing.

The sequence obtained was subjected to alignment using NCBI BLAST and Clustal W  on Bioedit software. The sequence alignment at NCBI BLAST was also used for establishment of species identity and drawing a phylogenetic tree to identify the species of the shark to which the sample belong to. In BLAST alignment the most similar sequence to the query sequence was considered as the species. Further the species was confirmed using the barcode of life website BOLDSYSTEMS.

PCR Amplification for COX1 Gene and DNA Sequencing

During alignment using ClustalW segments spanning 150 bp without any missing nucleotides in all the six sequence provided were used for in depth analysis of similarity and difference in the nucleotides.

Around 20µg of DNA was recovered from the tissue sample provided which was estimated by measuring absorbance at 260nm using a UV spectrophotometer. 500ng of DNA was used as a template for PCR amplification with the 100pM of forward and reverse primer as described in materials and methods. The PCR yielded around 650 bp product which was the expected size of the product (Figure 1).  The amplified product showed a good amplification but was also seen with some non-specific amplified product that needs to be cleaned up before setting up a sequencing experiment.

The PCR product obtained was excised from the agarose gel and purified using a spin column to obtain the specific PCR product and to negate the non-specific amplification that was observed along with the PCR amplicon. The sequence yielded on analysis of the PCR product (Figure 2) was subjected to alignment using BLAST and Clustal W.

The sequence alignment of the DNA sequence obtained from the PCR product yielded a very similar hit on BLAST analysis. The shark species was identified as Carcharhinus obscurus. The phylogenetic tree was also established that showed their evolutionary relationship with other shark species (Figure 3). The sequence that was obtained after PCR amplification when aligned with already established sequences of species revealed a 100% match with the shark species Carcharhinus obscurus and other subspecies of the same. We also found a close phylogenetic relationship of the identified species with Carcharhinus leucas.

The barcode assay that we conducted using the COX 1 gene led to successful amplification of the target with the expected product size. The primers used may be used as a common or universal primer for identification of other shark species as it will amplify the COX 1 genes of most species. However, a distinction between species can only be made after realizing the actual difference in the sequence. Therefore, the PCR amplification is not the only step that can identify the species but obtaining a sequence of the PCR product is a must.

The sequence that was obtained after PCR amplification when aligned with already established sequences of species revealed a 100% match with the shark species Carcharhinus obscurus. Other shark species that were closely related to the identified species were sequences identified as Carcarhinus obscurus 2, 3 and 4. These may be subspecies of the identified Carcharhinus obscurus species. The identified species is closely related to Carcharhinus leucas and prionace glauca as seen from the phylogenetic report.

The identified sequence was also similar to Carcharhinus sps. Indicating a close phylogenetic relationship. GenBank and BOLD sequence alignment is helpful in identification of species that are listed in CITES. We observed a high sequence similarity between CITES listed species and the closest relative was as similar in sequence as a difference of only 2 bp as observed earlier (Fields et al. 2015). A more thorough identification strategy may be formulated to discriminate between closely related Carcharhinus obscurus species. A combination of BOLD and BLAST identification is recommended for such work.

Identification of Shark Species: Examples of NCBI BLAST and ClustalW Alignment

The data thus obtained will help in establishing species of sharks that are being commercially exploited and will help conserve endangered species from over exploitation (Baum, Kehler & Myers 2005). The combination of BOLD and BLAST strategy is highly efficient but may need visual inspection of short nucleotide sequences. Using the barcoding assay many applications can be formulated which are: law enforcement agencies can be confident of the identification technique and use it as a legal tool to present evidence of species identification, the method can be employed to both processed and unprocessed samples. The sequence can also be used to establish stock records of shark species and may be helpful in understanding origin of the stock and migration pattern (Ward, Hanner & Hebert 2009; Ward et al. 2005). The protocol may also be useful to quantify types of species that are being extensively marketed and used as soup ingredients.

Nevertheless, there are shortcomings that need to be addressed as we have tried the amplification with a sample that is already available with us. We may try to collect sample from various sources in both processed and unprocessed forms to validate our result and show its applicability in different sample forms. However, we are confident that the protocol will eventually help in identification of endangered species that has been overexploited and help in enforcing conservation strategies to save declining population of some species of sharks.

References:

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Fields, AT, Abercrombie, DL, Eng, R, Feldheim, K & Chapman, DD 2015, 'A novel mini-DNA barcoding assay to identify processed fins from internationally protected shark species', PloS one, vol. 10, no. 2, p. e0114844.

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