The exploitation of shark species to satisfy the high market demand is now becoming a worldwide concern since the numbers for many species of these apex predators are now declining at an alarming rate. Shark DNA extracted from shark samples collected from the Australian fisheries management, amplified and used for identification. Although there are different methods of identifying a species, they proved to be insignificant at times of need. A dire need for species identification method that is both accurate and reliable was needed. It was established that the mitochondrial gene cytochrome oxidase I (COI) could provide a viable way for bio identification for animals. This study was performed with the aim to better understand the rate of illicit shark catches and more importantly stop illegal fishing of endangered shark species and secondly to check the reliability of the DNA barcode method using the COI gene. The result obtained showed that the samples were properly identified to the species level, with profound accuracy.
The increase in the popularity of shark fin soup worldwide also increases the threats of illegal fishing of some endangered species of these cartilaginous fish. Two other factors can also be an indirect threat to the shark species which is, mislabelling of shark meat and unnecessary shark fishing. The overfishing of sharks is specifically tricky due to the fact that these predators present a key function in the marine ecosystem, and, therefore, their population dynamics can also have an effect on all local marine diversity (Bonfil, 1994).
And previous studies reveal that the captured shark is usually found without their fins and their head removed and the body is sold in the market without proper labelling or just as ‘Sharks’. This inhibits the tracing of the structure of the species and recognition of the sharks which are protected by the laws of fisheries. The great white, grey nurse & Bull Shark has been marked as a near threatened or endangered shark species by IUCN (International Union for Conservation of Nature) (Diaz-Jaimes et al., 2014). In numerous situations, sharks have also been the focus of risk reduction programs owing to attacks on humans (Dudley and Simpfendorfer, 2006). Although sharks have been used commercially for its skin, liver oil and flesh, at present its fins are the major product that increases the demand for this and many other species. To stop this illicit shark fishing for the fin and other shark by-product and save the near-threatened or endangered species the identification of species presents a vital opening in stopping these businesses (Bornatowski, Braga and Vitule, 2014).
It was necessary for a quick yet effective testing process to specifically identify shark species. And so, the development of DNA barcoding revolutionized the way of species identification. The cytochrome Oxidase I (COI) gene, of the mitochondrial DNA (mtDNA), is a consistent gene section that allowed researchers a quick and exact identification of species (Hebert, Cywinska, Ball and deWaard, 2003) (Holmes, Steinke and Ward, 2009). Earlier methods of testing included the use of polymerase chain reaction (PCR) assays and species-specific primer (Magnussen et al., 2007) (Clarke et al., 2006) (Shivji, Chapman, Pikitch and Raymond, 2005).
Materials & Methods
The unidentified shark tissue sample for this study was collected from the apprehended illegal catches, received from the Australian fisheries management, these samples were either seized from poachers’ boat or found as finless and headless shark carcasses by the sea-shore or at sea by the management. An approximate of 16 tissue samples were collected (Table-1). The tissues were properly labelled and stored in 90% ethanol at a temperature of -20oC. The DNA extraction for each sample was carried out using the salting out method and the extracted DNA was stored at a cool temperature until further use (Armani et al., 2015).
For DNA extraction 2 mm3 of the tissue from every sample was used. The tissues were lysed in a 1.5 ml microcentrifuge tube using 20µl Proteinase K (Sigma Aldrich Australia) and 580µl TNES buffer and left at 55oC for 3 hours. The proteins were precipitated with 170 µl 5M NaCl and vortexed for 15 seconds before centrifuging for 10 minutes at 14000 rpm. The supernatant liquid containing the DNA was transferred to a new microcentrifuge tube containing 770 µl of ice cold 100% ethanol. The precipitated DNA were extracted, washed, air dried and diluted with 30μl sterile water & kept at a cold temperature prior to PCR (Aljanabi, 1997) (Sunnucks and Hales, 1996). The final product contained 30μl of resuspended DNA stocks from which, DNA fragments were evaluated by gel electrophoresis on 2% agarose gel and 10 µl of the DNA was stained with GelRed (Sigma Aldrich Australia) and DNA extraction success was examined (Figure-1). The gel was run at 110V for 30 minutes.
PCR amplification and sequencing
For amplifying the COI gene, instructions by the manufacturer were followed for PCR, where 5μL of the DNA stock was added to 95μL of sterile water in a 1.5 ml microcentrifuge tubes to get a 20-fold dilution. In a 0.2 ml PCR tube, 2μL of the diluted sample was added to the master mix. The content of the master mix includes 10µl qPCR mix Kappa SYBR fast (Kapa Biosystems, United States) containing dNTPs, MgCl2, and Taq polymerase and forward primer and reverse primer of 0.5µl each with 7µl of sterile water for PCR. The COI fragment would be 650 bp long. The cycling conditions were set to 35 cycles having 950C for 1 min, 940c for 30 sec, 520c for 30 sec, 720c for 1 min and a final extension of 720C for 10 min (Ward et al., 2005). The amplified COI genes were identified using Sanger Sequencing and the instrument used was Macrogen Inc. (South Korea). The chain-terminating inhibitors dideoxynucleotide (ddNTP’s; defective A, T, C, G) were used for the sequencing (Sanger, Nicklen and Coulson, 1977). Prior to PCR analysis 5μL of the PCR products were run into 2% agarose gel and compared with a 100bp DNA ladder to assess the presence of expected amplicons which were later amplified (Armani et al., 2015). The gels are viewed using the BIO-RAD Gel Doc EZ imager.
The sequence obtained from the PCR were aligned using ClustalW integrated into MEGA X (Kumar et al., 2018) (Tamura et al., 2013). The sequences were trimmed manually in the MEGA X after visual inspection and they were used to run a BLAST analysis and sequences that showed 100% perfect similarity on GenBank were selected for possible species identification (Barbuto et al., 2010). The sequences were deposited into the BOLD system (Barcode of Life Data System) for identification (RATNASINGHAM and HEBERT, 2007). A phylogenetic tree (Figure-4) was constructed using the MEGA X with neighbour-joining method using the bootstrap option to calculate node statistics (Zhang and Sun, 2008) (The neighbor-joining method: a new method for reconstructing phylogenetic trees., 1987).
Discussion and Conclusion
The use of the COI gene produces more reliable results and allowed the identification of all the shark samples at the specific species level. However, the success of this study is dependent on factor such as DNA quality, as a result, some samples that had poor DNA quality was reprocessed before PCR amplification and sequencing was performed.
There are other molecular methods for shark species identification. One of them includes the design of species-specific primers to identify species of sharks. Advantage of this method includes identifying multiple species even in a processed or degraded sample, but the shortcoming of this process is the insufficient collection of species-specific primers and the probability that the primers may also not be one hundred percent specific to the sample DNA (Abercrombie, Clarke and Shivji, 2005) (Hoelzel, 2001).
The results from the study suggest alarming outcomes about how demanding shark products are which is becoming the driving force for these illegal fishing businesses. These situations were already reported in other studies (Ward et al., 2005), and reported as shark carcasses being misidentified and incorrectly labelled. The results from table-1 indicate how the popularity of shark product globally causing fishers to extensively and illegally hunt sharks without proper controls and possibly bringing an upset to the marine ecosystem by
Identification using the distinct DNA barcodes can be a practical way to monitor mislabelled shark carcasses and prevent the extinction of these apex predators who play a major role in the marine ecosystem. The worldwide exhaustion of the shark population that has been documented universally (Ward et al., 2005) indicates that these species require critical attention and conservation. Additionally, proper monitoring of their genetic stock is particularly important to secure the chances of the full recovery and maintenance of the genetic diversity.
It is conclusive that DNA barcoding can be applied to identify shark species and is a reliable and rapid method that fisheries management authorities can utilize to collect data from legal and illegal shark fisheries and manage species conservation.
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