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Material and Methods

A short DNA sequence from a standardized region also called a DNA barcode has been upgraded as the best identifier of unknown species (Rach et al. 2017). The mitochondrial CO1 gene region also called cytochrome oxidase 1 has been crowned as the universal DNA barcoding for the metazoan organisms (Herbert et al. 2003). The CO1 gene has some features that make it of primary importance in evolutionary studies (Rach et al. 2017). The feature is that the size and structure of the CO1 gene is conserved in all respiratory (aerobic) organisms (Saraste 1990). Secondly, the 1600 bp gene contains a range of different functional domains that show heterogeneous substitution arrangements (patterns) (Erpenbeck, Hooper & Worheide 2006) (Lunt & Hyman 1997). The CO1 is easy to amplify, the evolves faster than the codding regions due to a lack of proofreading mechanisms (Avise 2004) (Hoy 2003). Sharks are fished across the globe both as targeted species or as bycatch (Brautigam et al. 2015). There is a wide variety of shark products such as cartilage, oil, fins, and meat on the world market (Clarke 2004). Sharks are vulnerable to overfishing because they take a longer time to mature, long gestation period, and have low fecundity (Brautigam et al. 2015). Nearly 20% of shark species are categorized as critically endangered, endangered, or vulnerable and another 12% are considered threatened (Brautigam et al. 2015). The standard accepting DNA barcoding for metazoans is approximately 650 pb region for the CO1 gene. The campaigns such as fish barcodes of life (Sabrina 2011) have resulted in DNA barcoding being supported by a large database of sequences that help in species identification. DNA barcoding has have been used to reveal mislabelled shark products that are being hidden, sporting out trade of threatened and endangered species (Asis, Lacsamana & Santos 2016). However, due to processed shark products in the market, it is difficult to obtain full DNA barcodes from shark products because would be highly degraded (Fields et al. 2015). In this paper, the main objectives were to use the CO1 gene as a DNA barcode (generated from MEGA X software) to help in identifying 16 unknown shark species, distinguish one critically endangered species of shark from the identified species, create a phylogenetic tree, keep the barcode for the critically endangered species for determining illegally traded shark products that are for the critically endangered shark.  The hypotheses are:

  1. Because the shark species are closely related (are all sharks) most of their base pairs would match in MEGA X software.
  2. There is at least one critically endangered species from the 16 sequences provided because a higher percentage of sharks are endangered.  
  • More than 1 sequences are likely to identify the same type of shark because some sequences appear to have the same pattern of base pairs.
  1. The phylogenetic tree will depict the 16 unknown sharks to stem from a common ancestor because the base pairs of each of the 16 sharks are closely related in size.

Tissue samples were extracted from fragments of white mascles of sharks. 170 µL of 5Ml of NaCl was added to the fragments the mixture was vortexed for 15 seconds to mix them. The resultant mixture was then exposed to a 14000-rpm centrifuge for 10 minutes to precipitate and remove the proteins. A new centrifuge 1.5 ml tube was labeled with the sample number. 770 µL ice-cold 100% ethanol at -20oC was added to the new tube and the tube was placed back on ice. After removing the proteins, precipitation of DNA followed. The tube was collected from the centrifuge the top liquid containing the DNA was poured into a new tube that contained ice-cold ethanol. The previous tube with unwanted proteins at the bottom was discarded. The tube with DNA was then inverted 5 times to clump the DNA, then centrifuged at 14000-rpm for 5 minutes. After the DNA was precipitated, purification followed. The supernatant liquid from the previous step was poured and the DNA pellet was kept. The DNA pellet was then washed with 300 µL of 70% ethanol. Centrifuge at 14000-rpm for 5 minutes followed and the supernatant was poured. Without touching the pellet, the pipette was used to remove as much supernatant as possible.  The pellet was then air-dried in a heat blook at 55oC for 10 minutes. The final product was resuspended in 30 µL of water and slowly, the pellet was dislodged by pipetting the liquid in and out of the tube. Gel electrophoresis by using 2% agarose gel (2g in 1000ml of TBE buffer) was used to determine if the DNA extraction process was a success.  10 µL of DNA and 5µL of loading dye and the gel was run at 110 V for 30 minutes for gel electrophoresis process.  

Results

 Figure one indicates that there were about 8 base pairs

Figure one indicates that there were about 8 base pairs that were visible or had a sufficient amount of DNA after DNA extraction.

After gene exaction and conforming it was a success, the next step was to carry out a polymerase chain reaction (PCR) to amplify the DNA.  To carry out PCR the following ingredients were used. The extracted DNA template, deoxynucleotides, Taq, Buffer + magnesium chloride, and primers. The preparation of the DNA for the PCR machine. Added 5µL of stock DNA and 95µL of sterile water in a labeled tube. Then added 2µL of distillate DNA sample, then added 18µL of master mix, and then placed the tube in the PC machine.

  placed the tube in the PC machine.

The PCR process was performed under the following conditions: initial heating at 95oC for 1 minute, denaturing at 94oC for 30 seconds, annealing at 52oC for 30 seconds, extension at 72oC for 1 minute, and final extension at 72o for 10 minutes. And then the result from the PCR machine was held at 4oC.

After PCR, gel electrophoresis was run again to confirm the successfulness of PCR. The PCR product was then sent for sequencing using the Sager sequencing method. The ingredients to run the DNA synthesis were; DNA template, dNTPs, ddNTPs, and DNA polymerase. The electrophoresis was performed on each of the 4 tubes having one type of ddNTP for sequencing.

The sequences were loaded into MEGA X software to come up with CO1 barcodes to blast and determine the unknown species.

In this paper, the experiment, mitochondrial DNA from unknown 16 shark species was extracted and gel electrophoresis was performed to confirm the successfulness of DNA extraction. The extracted DNA (CO1 gene) was then amplified through PCR and gel electrophoresis was performed again to confirm the success of PCR. A wholistic list of all nucleotides for all the chromosomes of each shark species was determined using the "Sangar Sequencing" method and 16 sequences for each unknown shark species were determined. All of the 16 sequences were loaded into the MEGA (Molecular Evolutionary Analysis) software for alignment and cropping the 16 DNA sequences. After alignment, the cropped sequence (barcode) of each unknown shark species was identified using blast websites, "NCBI" and "boldsystems". Then the phylogenetic tree was created in MEGA software for all the identified 16 shark species.

Figure 2, below left most image indicates the success of DNA extraction with the larger sized allele with a size greater than 1000 base pairs. The rightmost image shows the success of PCR with the smaller allele sized at approximately 650 base pairs. The PCR product indicates the success of amplification because the bands under PCR are larger and brighter than the bands under the DNA samples. This means that the extracted DNA was indeed amplified.

 shows the alleles for each of the 16 shark species.

Figure 3 shows the alleles for each of the 16 shark species. The bands for sharks 1, 3, 4, 5, 11, 13, and 15 show a high density of the same allele size, and thus these bands are brighter. The bands are also distributed within a small range of band size. This means that sequences for the 16-shark species have patterns with a small difference in the base-pair arrangement.

  shows that some sequences have a perfect march meaning

 Figure 4 shows that some sequences have a perfect march meaning that they belong to the same shark species.

 below shows the identification of each of the unknown 16 shark species

Table 2 below shows the identification of each of the unknown 16 shark species by scientific name and common name. The names of the shark were chosen from the top row of the blast website with 100% percentage identity. There are only 8 unknown shark species represented by the 16 barcodes; Bull shark, Dusky shark, smooth hammerhead shark, shortfin mako shark, Australian blacktip shark, great white shark, and Australian angel shark. Shark 7 which is the shark of interest in this report is the Australian angle shark or simply, the angel shark.

 

  shows that the shark Carcharhinus obscurus 

 Figure 5 shows that the shark Carcharhinus obscurus was identified 4 times using 4 sequences. The tree also shows that the confidence of getting the same results for Carcharhinus o. is 100%. Carcharhinus o. is more closely related to Prionace glauca than any other shark species. Carcharhinus leucas, Sphyrna zygaena, Galeocerdo cuvier, and Carcharodon carcharias were all identified twice.

The process of DNA extraction from the tissues of the shark was successful. The gel electrophoreses successfully separated the alleles in the DNA extracted DNA by size figure 2. From figure 2. PCR run and gel electrophoresis on the PCR products revealed 16 bands for each of the 16 unknown shark species. The Bands for each of the 16 sharks revolve around nearly the same base pair. This is a clear indication that most of the base pairs in the 16 CO1 gene sequences for each of the 16 unknown shark sequences provided match, this supports the hypothesis (i) which states that "Because the shark species are closely related (are all shark) most of their base pairs would match in MEGA X software". However, at this point, the hypothesis is speculative. Another hypothesis "More than 1 sequences are likely to identify the same type of shark because some sequences appear to have the same pattern of base pair" is also supported by the results from figure 2. This is because there is a possibility that more than one sequence is likely to have base pairs of equal size. From figure 3, most base pairs in 16 unknown sharks indeed matched when ran through the MEGA X, confirming the hypothesis (i), "Because the shark species are closely related (are all shark) most of their base pairs would match in MEGA X software". Table 2, shows the identification of all the 16 unknown shark species, and some shark species are indeed identified by more than one sequence, this confirms hypothesis (iii), "More than 1 sequences are likely to identify the same type of shark because some sequences appear to have the same pattern of base pairs" The results from both NCBI and barcode of life home page produced the same results shown in table 2. From table 2, the critically endangered species is shark 7, Squatina australis commonly called the Australian angel shark (Unsupported source type (InternetSite) for source Ric20.). Figure 5 shows that sharks 2,9,10 and 13 are distantly related to sharks 7. Shark 7 is closely related to sharks 12, 5, and 6. Figure 5 confirms hypothesis (iv), " The molecular phylogenetic tree will depict the 16 unknown sharks as stemming from a common ancestor because the base pairs of each of the 16 sharks are closely related in size”. From Figure 5 critically endangered species (S. australis) is the most less evolved species with the least number of branches from the common ancestor. This means that S. australis lacks survival features to help it escape evolving predators, technology, and climate. Hence, S. australis can easily be caught by fishermen, it experiences a hostile environment to global warming and pollution, and it easily is threatened by other evolved species. The barcode for the S. australis “

The sequences of the 16 sharks had a sequence of the critically endangered species of shark (shark 7), S. australis. Using the barcode for S. australis the products of S. australis can be identified in the market and come up with ways to save, S. australis. The governments and all concerned bodies should put more effort to conserve S. australis. Fishing of S. australis should be burned and all fishermen educated on how to identify S. australis and shy away from fishing it.  

References

Unsupported source type (InternetSite) for source Ric20.

Asis, AM, Lacsamana, JK & Santos, MD 2016, 'Illegal trade of regulated and protected aquatic species in the Philippines detected by DNA barcoding', Mitochondrial DNA Part A, pp. 659-666.

Avise , JC 2004, Molecular markers, natural history, and evolution, Sinauer Associates, Massachusetts.

Brautigam, A, Callow , M, Campell, IR, Camhi, MD, Cornish, AS, Dulvy, NK, Fordham, SV, Fowler, SL, Hood, AR, McCleannen, C, Reuter, EL, Sant, G, Simpfendorfer, CA & Welch, DJ 2015, 'Global Priorities for Conserving Sharks and Rays: A 2015–2025 Strategy'.

Clarke, S 2004, 'Shark Product Trade in Hong Kong and Mainland China and Implementation of the CITES Shark Listings', Hong Kong, China: TRAFFIC East Asia.

Erpenbeck, D, Hooper , JN & Worheide, G 2006, 'CO1 phylogenies in diploblasts and the’Barcoding of Life’—are we sequencing a suboptimal partition?', Molecular Ecology Notes, pp. 550-553.

Fields , AT, Albercrombie, 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.

Herbert, PD, Cywlnska, A, Ball, SL & deWaard, JR 2003, 'Biological identifications through DNA barcodes', Proc Sci, pp. 313-321.

Hoy, M 2003, Insect Molecular Genetics, Academic Press, San Diego.

Lunt, JC & Hyman, BC 1997, 'Animal mitochondrial DNA recombination', Nature.

Rach, J, Bergmann, T, Paknia, O, Desalle, R, Schierwatter, B & Hadrys, H 2017, 'The marker choice: Unexpected resolving power of an unexplored CO1 region for layered DNA barcoding approaches', Plos One, pp. 1-14.

Sabrina, IP 2011, beSpefic , <https://www.bespacific.com/fish-barcode-of-life-campaign-fish-bol/>.

Saraste , M 1990, 'Structural features of cytochrome oxidase', Q Rev Biophys, pp. 331-366.

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