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One of the problems that arose from the meetings of the Barcode in Mexico project was the urgency of having a method in which Mexican authorities could trust for detecting shark finning. This study examined DNA barcoding as a method to identify 14 dried shark fins confiscated by the Mexican Government in two exportation shipments in Mazatlán and Manzanillo ports. Fins were DNA barcoded using the COI mitochondrial gene and provided matching sequences of six species: Prionace glauca, Carcharhinus falciformis, Carcharhinus limbatus, Alopias pelagicus, Mustelus henlei and Rhizoprionodon longurio. There is no information on DNA barcoding sharkfin trade in the Mexican Pacific and this is the first group effort with Mexican Government Agencies for the conservation of sharks. Keywords: Barcode, conservation. finning, shark, DNA, COI, 1 Introduction Sharks are cartilaginous fishes that belong to the class Chondrichthyes, which includes sharks, rays and chimaeras. Sharks comprise over 500 known species [1,2] and there are about 100 shark species of commercial importance that are exploited by artisanal and industrial fisheries. Sharks are usually exploited to obtain its meat, liver oil, cartilage, jaws, skin and fins, as well as for tourism; *Corresponding author: Héctor Espinosa, Colección Nacional de Peces, Instituto de Biología, Universidad Nacional Autónoma de México. Distrito Federal. 04510. México, Email: email@example.com Christian Lambarri, Armando Martínez, Andrea Jiménez, Colección Nacional de Peces, Instituto de Biología, Universidad Nacional Autónoma de México. Distrito Federal. 04510. México although meat has a low commercial price. Finning consists in slicing off sharks fins and discarding the body at sea . Fins are a highly prized commodity in many Asian cultures and are used in soups served at important occasions . It is estimated that fins of 26 to 73 million sharks enter the global trade each year  either coming from legal fisheries, by-catch or illegal, unreported and unregulated fisheries . Shark finning includes tens of shark species such as Whale Shark, Hammerhead, Megamouth, Blue and Thresher Sharks. These and other shark species mature slowly, have low fecundity, long gestation periods and are top predators in the food chain, meaning that shark finning (and consequent death) of an adult individual directly affects population and ecosystem health. In situations where species are hard to identify, molecular analysis and databases such as BOLD and GenBank facilitate the identification of species known only from parts or remains . Ward et al.  conducted the first study that included sharks dried or fresh parts, and expanded it to identifying putative new species . Likewise other authors  proved DNA barcodes to be capable of identifying both teleost and chondrichthyan species in 98-99% of the examined fish species. 2 Methods Unidentified fin samples (n = 14) collected from two exportation vessels were received from the Mexican Government Agency Procuraduría Federal de Protección al Ambiente (PROFEPA). Six fins (Batch 1) were confiscated in Mazatlán, Sinaloa and eight fins (Batch 2) from Manzanillo, Colima (Figure 1). Voucher tissues and DNA are stored at the Colección Nacional de Peces of the Instituto de Biología of the Universidad Nacional Autónoma de México (CNPEIBUNAM). Dried tissues were washed in distilled water before the extraction and stored in ethanol 96%. EZ-10 Spin Column Animal DNA Mini-Preps Kit (Bio Basic Canada Inc.) © 2015 Héctor Espinosa et al. licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Figure 1. Shark fins seized by the PROFEPA (Procuraduría Federal de Protección al Ambiente) and deposited in the Colección Nacional de Peces of the IBUNAM in Mexico City. PROFEPA 01 to 06 are Sample Ids Pr T-01 to Pr T-06 of the Table 1 respectively. and DNeasy Tissue Kit (QIAGEN Inc.) were used for batch 1 and 2, respectively, to perform the genomic DNA extraction following the instructions of the manufacturer. We assembled the first six samples (Batch 1) with a negative control. We ran the PCR with a total volume of 25 µL. PCR reaction included 16.375 µL of ultra-pure water, 2.5 µL 10x PCR buffer, 0.5 µL dNTPs, 1.5 µL MgCl2, 0.125 µL of TaqDNA, 1 µL of each COI primer (FishR1- FishF1 or FishR2- FishF2) , and 2.0 µL of pure DNA sample. We conducted two PCR reactions with each set of primers. The thermo cycle profile for COI consisted in a temperature of 94°C for 2 min, 35 cycles of 94°C for 30 s, 52°C for 40 s, and 72°C for 1 min, with an extension of 72°C for 10 min and a final lowering to 4°C. H. Espinosa, et al. The last eight samples (Batch 2) were also assembled with a negative control. We ran the PCR with a total volume of 15 µL. PCR reaction included 10.4 µL of ultrapure water, 3.0 µL 5x PCR buffer, 0.12 µL of TaqDNA, 1 µL of COI primers: VF1d_t1 and VR1d_t1 , and 1.0 µL of pure DNA sample. Sequencing was done at the Sequencer of the Instituto de Biología of the Universidad Nacional Autónoma de Mexico and we searched and aligned the sequences with the GenBank and BOLD databases. 3 Results The confiscated samples revealed the exploitation for finning purposes of six different species in the Ports of Mazatlán and Manzanillo in Mexico (Table 1). The most common species were Prionace glauca (Blue Shark) and Alopias pelagicus (Pelagic Thresher Shark) with 28.57% of occurrence, followed by Rhizoprionodon longurio (Pacific Sharpnose Shark) and Carcharhinus limbatus (Blacktip Shark) with 14.29% of occurrences. The least common species were Carcharhinus falciformis (Silky Shark) and Mustelus henlei (Brown Smoothhound Shark), both with 7.14% of occurrence. Four of these species are listed in the International Union for Conservation of Nature (IUCN) Red List, with their current status ranging from near threatened through vulnerable (Table 2), despite they are not included in the CITES list (Convention on International Trade in Endangered Species of Wild Fauna and Flora) as are other shark species in Mexico. All of these species are common pelagic species in the Mexican Pacific, caught mainly with bottom trawls and Table 1. Shark fin sequences and their respective percentage of similitude with BOLD and GenBank records. Sample PR T-01 PR T-02 PR T-03 PR T-04 PR T-05 PR T-06 PR 01 PR 02 PR 03 PR 04 PR 01' PR 02' PR 03' PR 04' BOLD Probability Carcharhinus falciformis (Müller & Henle 1839) Prionace glauca (Linnaeus 1758) Prionace glauca (Linnaeus 1758) Prionace glauca (Linnaeus 1758) Mustelus henlei (Gill 1863) Prionace glauca (Linnaeus 1758) Alopias pelagicus Nakamura 1935 Rhizoprionodon longurio (Jordan & Gilbert 1882) Unable to match to species level Alopias pelagicus Nakamura 1935 Unable to match to species level Rhizoprionodon longurio (Jordan & Gilbert 1882) Carcharhinus limbatus (Müller & Henle 1839) Alopias pelagicus Nakamura 1935 98.81% 100.00% 100.00% 100.00% 98.43% 100.00% 100.00% 100.00% 100.00% 99.30% 100.00% 100.00% GenBank Probability Carcharhinus falciformis (Müller & Henle 1839) Prionace glauca (Linnaeus 1758) Prionace glauca (Linnaeus 1758) Prionace glauca (Linnaeus 1758) Mustelus henlei (Gill 1863) Prionace glauca (Linnaeus 1758) Alopias pelagicus Nakamura 1935 Rhizoprionodon terraenovae (Richardson, 1836) Carcharhinus limbatus (Müller & Henle 1839) Alopias pelagicus Nakamura 1935 Alopias pelagicus Nakamura 1935 Rhizoprionodon terraenovae (Richardson, 1836) Carcharhinus limbatus (Müller & Henle 1839) Alopias pelagicus Nakamura 1935 99.00% 99.00% 98.00% 100.00% 100.00% 99.00% 98.00% 91.00% 99.50% 99.60% 99.00% 91.00% 100.00% 99.00% Table 2. Shark species common names and risk categories according to the IUCN Red List (2015). Species Prionace glauca (Linnaeus 1758) Alopias pelagicus Nakamura 1935 Rhizoprionodon longurio (Jordan & Gilbert 1882) Carcharhinus limbatus (Müller & Henle 1839) Carcharhinus falciformis (Müller & Henle 1839) Mustelus henlei (Gill 1863) Common name Blue Shark Pelagic Thresher Pacific Sharpnose Shark Blacktip Shark Silky Shark Brown Smoothhound IUCN 2015 Near Threatened Vulnerable Data Deficient Near Threatened Near Threatened Least Concern lines in commercial and artisanal fisheries, where the true identity of the landed species is not known. Particularly the Brown Smoothhound Shark are frequently caught as by catch when fishing tuna, salmon, pollock, mackerel and other shark species . 4 Discussion Shark finning is more frequent in communities of low economic resources, because the fin sale represents a much greater income than the average salary. In several countries where finning happens, drastic measures have been placed on the conditions in which shark fishing is reported, especially as for the presentation of the complete individuals onshore. The 2010 meeting of the Fish Stocks Agreement Review Conference underscored support for keeping fins naturally attached and recommended strengthening enforcement of prohibitions on shark finning . Many fished sharks are unreported or reported only as bycatch and there is a need to identify their fins with a 100% certainty in order to initialize legal actions against it. In Mexico the Project Barcode of Life highlighted the urgency of having a method in which the Mexican authorities could trust for detecting shark finning. This is the first study using genetic barcodes to identify shark fins in Mexico that yielded the Government Agency PROFEPA and the CNPE-IBUNAM in order to prevent shark fin illegal trade. Barcoding of animal tissues has proved to be an important technique to identify species from animal remains that cannot be identified by conventional morphological means and therefore cannot be adequately traced. Rapid identification of these samples will help to the conservation of species, easing and accelerating the identification of their illegal trade in Mexico. Acknowledgements: We thank the project Red Temática CONACYT Mex-Bol for support and funding. We also thank A. Hernández for her active help in the processing of samples and L. Márquez from the laboratory of DNA Sequencing of the Instituto de Biología of the Universidad Nacional Autónoma de México. We thank Dr. M. ValdezMoreno and M. Elias for their support and invitation to participate in the Second International Conference on DNA Barcoding of Fishes held in Chetumal city in September 2014. Finally we want to thank the reviewers for their important contributions and improvement of the manuscript. Conflict of interest: Authors declare nothing to disclose.
DNA Barcodes – de Gruyter
Published: Jan 1, 2015
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