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DNA barcoding
Cyt B

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Doğdu, S. A., & Turan, C. (2021). AUTHENTICATION AND TRACEABILITY OF PUFFERFISH ‎SPECIES USING DNA SEQUENCING. Pakistan Journal of Marine Sciences, 30(1), 1–11. Retrieved from


Pufferfish is known a powerful toxin in its skin and organs called Tetrodotoxin. Validation of food authenticity is usually based on the analysis of proteins or DNA sequences. DNA based methods are more effective and can also be applied to different food matrices. DNA barcoding is a rapidly rising global initiative in which species are characterized using mtDNA sequence. This is based on the premise that species are generally well delineated by a species-specific sequence. In this study, we revealed DNA barcoding of three pufferfish species Lagocephalus sceleratus, Sphoeroides pachygaster and Torquigener flavimaculosus using the mtDNA cytochrome b gene region. There were 122 variable and 266 conservative nucleotides of which 110 were parsimony informative over 388 bp sequences. The mean nucleotides composition was found as %28.0 thymine (T), %30.7 cytosine (C), %24.8 adenine (A) and %16.6 guanine (G). Twelve haplotypes were found out of 18 sequences and mean haplotype diversity was observed at 0.9346. This study has strongly authenticated and traceability the efficacy of Cty b in identifying L. sceleratus, S. pachygaster and T. flavimaculosus species with designated barcodes. This is the recorded of L. sceleratus, S. pachygaster and T. flavimaculosus cytochrome b gene sequence from the eastern Mediterranean coast of Turkey.

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Akbora, H.D., İ. Kunter, T. Erçeti̇n, A.M. Elagöz and B.A. Çi̇çek, 2020. Determination of ‎tetrodotoxin (TTX) levels in various tissues of the silver cheeked pufferfish ‎‎(Lagocephalus sceleratus (Gmelin, 1789)) in Northern Cyprus Sea (Eastern ‎Mediterranean). Toxicon. 175: 1-6. ‎

Bautista, J.M., V. Verrez-Bagnis and N. Kourti, 2006. Genetic catalogue, biological ‎reference collections and online database of European marine fishes.‎

Cantatore, P., M. Roberti, G. Pesole, A. Ludovico, F. Milella, M.N. Gadaletal and C. ‎Saccone, 1994. Evolutionary analysis of cytochrome b sequences in some ‎Perciformes: evidence for a slower rate of evolution than in mammals. J. ‎Molecul.Evolut. 39(6): 589-597.‎

Chang, C.H., H.Y. Lin, Q. Ren, Y.S. Lin and K.T. Shao, 2016. DNA barcode ‎identification of fish products in Taiwan: Government-commissioned authentication ‎cases. Food Control. 66: 38-43.‎

Doğdu, S.A., C. Turan and D. Ayas, 2019. Isolation and characterization of collagen and ‎gelatin from skin of silver cheeked pufferfish Lagocephalus sceleratus for ‎pharmaceutical and biomedical applications. Natur. Engineer. Sci. 308-314.‎

El-Ganainy, A.A., M.M. Sabrah and M.A. Zaky, 2006. Biology and toxicity of the ‎pufferfish Lagocephalus sceleratus (Gmelin, 1789) from the Gulf of Suez. Egypt. J. ‎Aquat. Res. 32(1): 283-297.‎

Farrag, M., A.A. El-Haweet and M.A. Moustafa, 2016. Occurrence of pufferfishes ‎‎(Tetraodontidae) in the eastern Mediterranean, Egyptian coast-filling in the gap. ‎BioInvas. Rec. 5(1): 47-54.‎

Filonzi, L., S. Chiesa, M. Vaghi and F.N. Marzano, 2010. Molecular barcoding reveals ‎mislabelling of commercial fish products in Italy. Food Res. Int. 43(5): 1383-1388.‎

Galimberti, A., F. De Mattia, A. Losa, I. Bruni, S. Federici, M. Casiraghi and M. Labra, ‎‎2013. DNA barcoding as a new tool for food traceability. Food Res. Int. 50(1): 55-‎‎63.‎

Ghouri, M.Z., M. Ismail, M.A. Javed, S.H. Khan, N. Munawar, A.B. Umar and A. ‎Ahmad, 2020. Identification of edible fish species of Pakistan through DNA ‎barcoding. Front. Mar. Sci. 7: 554183.‎

Giusti, A., E. Ricci, M. Guarducci, L. Gasperetti, N. Davidovich, A Guidi and A. Armani, ‎‎2018. Emerging risks in the European seafood chain: Molecular identification of ‎toxic Lagocephalus spp. in fresh and processed products. Food Control. 91: 311-‎‎320.‎

Guardone, L., Maneschi, A., Meucci, V., Gasperetti, L., Nucera, D., and Armani, A. 2020. ‎A global retrospective study on human cases of tetrodotoxin (TTX) poisoning after ‎seafood consumption. Food Rev. Int. 36(7): 645-667.‎

Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and ‎analysis program for Windows 95/98/NT. In: Nucleic acids symposium series (Vol. ‎‎41, No. 41, pp. 95-98). [London]: Information Retrieval Ltd., c1979-c2000.‎

Hebert, P.D., S. Ratnasingham and J.R. De Waard, 2003. Barcoding animal life: ‎cytochrome c oxidase subunit 1 divergences among closely related species. ‎Proceedings of the Royal Society of London. Series B: Biological Sciences, 270(1): ‎S96-S99.‎

Hsieh, C.H., W.T. Chang, H.C. Chang, H.S. Hsieh, Y.L. Chung and D.F. Hwang, 2010. ‎Puffer fish-based commercial fraud identification in a segment of cytochrome b ‎region by PCR–RFLP analysis. Food Chem. 121(4): 1305-1311.‎

Hsieh, Y.W. and D.F. Hwang, 2004. Molecular phylogenetic relationships of pufferfish ‎inferred from partial sequences of cytochrome b gene and restriction fragment ‎length polymorphism analysis. J. Agri. Food Chem. 52(13): 4159-4165.‎

Islam, Q.T., M.A. Razzak, M.A. Islam, M.I. Bari, A. Basher, F.R. Chowdhury and D. ‎Mebs, 2011. Pufferfish poisoning in Bangladesh: clinical and toxicological results ‎from large outbreaks in 2008. Transactions of the Roy. Soc. Trop. Med. Hygien. ‎‎105(2): 74-80.‎

Kasapidis, P., P. Peristeraki, G. Tserpes and A. Magoulas, 2007. First record of the ‎lessepsian migrant Lagocephalussceleratus (Gmelin1789) (Osteichthyes: ‎Tetraodontidae) in the Cretan sea (Aegean, Greece). Aquat. Invas. 2(1): 71-73.‎

Kosker, A.R., F. Özogul, D. Ayas, M. Durmus, Y. Ucar, J.M. Regenstein and Y. Özogul, ‎‎2019. Tetrodotoxin levels of three pufferfish species (Lagocephalus sp.) caught in ‎the North-Eastern Mediterranean sea. Chemosphere. 219: 95-99.‎

Larkin, M.A., G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan, H. McWilliam ‎and D.G. Higgins,. 2007. Clustal W. and Clustal X version 2.0. bioinformatics. ‎‎23(21): 2947-2948.‎

Mafra, I., I.M. Ferreira and M.B.P. Oliveira, 2008. Food authentication by PCR-based ‎methods. Euro. Food Res. Technol. 227(3): 649-665.‎

Maralit, B.A., R.D. Aguila, M.F.H. Ventolero, S.K.L. Perez, D.A. Willette and M.D. Santos, ‎‎2013. Detection of mislabeled commercial fishery by-products in the Philippines ‎using DNA barcodes and its implications to food traceability and safety. Food ‎Control. 33(1): 119-125.‎

Nehal, N., B. Choudhary, A. Nagpure and R.K. Gupta, 2021. DNA barcoding: a modern-‎age tool for detection of adulteration in food. Critic. Rev. Biotechnol. 45(1): 767-‎‎791.‎

Oyaizu, M., Y. Fujimoto, F. Takenaga and S. Itoh, 2000. Fatty acid composition of total ‎lipids in pufferfish meat. Food Preserv. Sci. 26(6): 333-338.‎

Santini, F., M.T. Nguyen, L. Sorenson T.B. Waltzek, J.W. Lynch Alfaro, J.M. Eastman ‎and M.E. Alfaro, 2013. Do habitat shifts drive diversification in teleost fishes? An ‎example from the pufferfishes (Tetraodontidae). J. Evolut. Biol. 26(5): 1003-1018.‎

Sato, K.I., S. Akai, H. Shoji, N. Sugita, S. Yoshida, Y. Nagai and J. Yoshimura, 2008. ‎Stereoselective and efficient total synthesis of optically active tetrodotoxin from D-‎glucose. J. Organ. Chem. 73(4): 1234-1242.‎

Tuney, I. 2016. Molecular identification of pufferfish Lagocephalussceleratus (Gmelin, ‎‎1789) and Lagocephalus spadiceus (Richardson, 1845) from Eastern ‎Mediterranean, Turkey. Fresen. Environ. Bull. 25(5): 1428-1436.‎

Turan, C., M. Gürlek, N. Başusta, A. Uyan, S.A. Doğdu and S. Karan, 2018. A checklist ‎of the non-indigenous fishes in Turkish marine waters. Nat. Engineer. Sci. 3(3): 333-‎‎358.‎

Turan, C., M. Gürlek, D. Ergüden, A. Uyan, S. Karan and S.A. Doğdu, 2017. Assessing ‎DNA Barcodes for Identification of Pufferfish Species (Tetraodontidae) in Turkish ‎Marine Waters. Natur. Engineer. Sci. 2(3): 55-66.‎

Turan, C., A. Uyan, M. Gürlek and S.A. Doğdu, 2020. DNA Barcodes for Identifications ‎of Two Lionfish Species Pterois miles (Bennett, 1828) and Pterois volitans ‎‎(Linnaeus, 1758) in the Mediterranean. FishTaxa. 16, 29-36.‎

Yamanoue, Y., M. Miya, K. Matsuura, S. Miyazawa, N. Tsukamoto, H. Doi and H. ‎Sakai, 2009. Explosive speciation of Takifugu: another use of fugu as a model ‎system for evolutionary biology. Molecul. Biol. Evol. 26(3): 623-629.‎

Zhu, S., Y. Yang, Z. Fan, Q. Zhou and X. Fan, 2021. A fatal case of severe pufferfish ‎poisoning: A case report. Medicine: Case Reports and Study Protocols. 2(1): e0036.‎

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Copyright (c) 2021 Servet Ahmet Doğdu, Cemal Turan