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Italian Journal of Food Safety 2014; volume 3:4521 Application of DNA barcoding oxidase (COI) gene. This gene codes for one part of the terminal enzyme of the mithocondr- Correspondence: Tiziana Civera, Dipartimento di for controlling of the species ial respiratory chain. The COI gene has been Scienze Veterinarie, Università degli Studi di from Octopus genus chosen as a universal molecular target since it Torino, largo Braccini 2, 10095 Grugliasco (TO), allows the design of universal, robust and Italy. Francesco Debenedetti,1 functional primers for almost all the members Tel. +39.011.67092 - Fax: +39.011.6709224. E-mail: [email protected] Alessandra Dalmasso,1 of the animal Phyla (Folmer et al., 1994). The Maria Teresa Bottero,1 effectiveness of this fragment for the identifi- Key words: DNA barcoding, Octopus spp., Maurizio Gilli,2 Stefano Gili,2 cation of species has been demonstrated for Commercial frauds. Valentina Tepedino,3 Tiziana Civera1 several animal species, from vertebrates to invertebrates (Waugh, 2007; Ward and Conflict of interests: the authors declare no 1Dipartimento di Scienze Veterinarie, Holmes, 2007). Recently, this technique was potential conflict of interests. Università degli Studi di Torino, recognised as the best method in forensics for 2 Received for publication: 8 July 2014. Grugliasco (TO); ASLTO1, Torino; species identification, and was proposed by the 3Eurofishmarket, Bologna, Italy Revision received: -. United States Food and Drug Administration Accepted for publication: 15 September 2014. (USFDA) as the methodology for the authenti- cation of commercial fish products (Dawnay et This work is licensed under a Creative Commons al., 2007). The US agency has also the inten- Attribution 3.0 License (by-nc 3.0). Abstract tion of introduce the DNA barcoding data in the Regulatory Fish Encyclopedia (RFE) to sup- ©Copyright F. Debenedetti et al., 2014 Licensee PAGEPress, Italy port the investigations of mislabeling and the The DNA barcoding proposes the use of a Italian Journal of Food Safety 2014; 3:4521 particular sequence from a single genomic substitution of the fish species (Yancy et al., doi:10.4081/ijfs.2014.4521 region as the base for an identifying system 2008). In May 2004, the Consortium for the capable to determine all animal species. This Barcoding of Life (CBOL) was formed, com- methodology comprises the analysis of a 655 prising several international patterns. Since base-pair region from the mithocondrial the beginning, its mission has been the explo- cephalopodsonly and octopus. cytochrome C oxidase gene (COI). Its applica- ration and the development of the potentiality In this study we test the DNA barcoding for tion in the species identification of fishery of the DNA barcoding as a practical research the identification of some octopus species of products has been very promising. However, in tool for species identification. The use of this greatest commercial interest (Octopus mem- the last years some doubts about its usage methodology for species identification ofuse fish- branaceus , Octopus vulgaris, Octopus aegina, have emerged. In this work, we make use of ery products has been very promising from the Octopus cyanea) focusing the attention on the the DNA barcoding for the identification of outset, given the large number of species reliability and completeness of the available some of the octopus species with higher com- already identified. It has been shown that 98 databases. mercial interest (Octopus membranaceus, and 93% of the marine and fresh water Octopus vulgaris, Octopus aegina, Octopus species, respectively, can be differentiated cyanea) focusing the attention on the reliabil- using barcodes (Savolainen et al., 2005; Ward ity and completeness of the available informa- et al., 2009). Materials and Methods tion on the databases. The study looked over 51 This favourable outcome as well as the need individuals apparently belonging to the of a complete and reliable instrument for iden- The sampling involved the collection of 51 Octopus genus. For the identification of O.aegi- tification of the species, have led to the forma- specimens belonging to the genus Octopus: 1 na, O.cyanea, O.vulgaris species no particular tion of the initiative Fish Barcode of Life O.dollfusi, 2 O.aegina, 2 O.cyanea, 24 O.vul- problems were found. On the other hand, most (FISH-BOL)commercial (www.fishbol.org). This cam- garis and 22 O.membranaceus. All samples, col- of the samples of O.membranaceus, though paign, launched in 2005, has as main objective lected in 2012 and 2013 from different suppli- they clearly presented the morphological char- the collection of DNA barcodes of all the fish in ers, were morphologically identified and kept acteristics of the species, were not identified the world, equivalent to 31,000 species approx- at -20°C. The DNA extraction was done using with the biomolecular analyses. imately. It endorses the FishBase (www.fish- the DNeasy Tissue and Blood Kit (Qiagen, Nonbase.org) as the taxonomic authority, and the Valencia, CA, USA) according to the manufac- BOL database (BOLD) as the working bioinfor- turer’s instructions for animal tissue. The matic platform. FISH-BOL represents one of extracted DNA was quantified with NanoDrop Introduction the most complete resources for the species 1000 (Thermo Scientific, Waltham, MA, USA). identification of the fishery products (Ward et For all samples, segments of the COI genes The DNA barcoding puts forward the use of al., 2009). were amplified following the protocol proposed a sequence from a single genomic region However, in the past years, some doubts by Folmer et al. (1994). Since there were no (defined as barcode), as the base of a recogni- regarding the use of DNA barcoding have available reference sequences for O.mem- tion system capable to identify all animal emerged because of the difficulties to discrim- branaceus in FISH-BOL, a second sequencing species (Hebert et al., 2003). This biomolecu- inate recently spread species, or species with a protocol was introduced. It contemplated the lar methodology consists in the identification wide spatial differentiation; as well as the usage of cytochrome b (cytb), a historical tar- of the belonging species through the sequenc- inability to differentiate new and hybrid get used for species identification (Espineira ing of a fragment of mitochondrial genes, its species (Moritz and Cicero, 2004; Hickerson et et al., 2010). alignment and comparison with the informa- al., 2006; Rubinoff, 2006). Furthermore, this Amplicons were visualised by electrophore- tion available on the databases. In particular, technique has been extensively evaluated for sis on a 2% agarose gel and coloured with the barcoding approach comprises the analysis the identification of fish species, still there are Eurosafe Nucleic Acid Stain. The amplified of a 655 bp region located at the 5’ end of the few data supporting the applicability in the products were purified with the enzyme subunit I of the mithocondrial cytochrome C identification of the most common species of ExoSap-IT (USB) and sequence with the [page 196] [Italian Journal of Food Safety 2014; 3:4521] Article BigDye terminator kit (Applied Biosystems, for the COI gene and 651 bp for the cytb gene. lack of reference sequences of O.mem- Carlsbad, CA, USA) according to the manufac- The sequencing results are presented in Table branaceus in both GenBank and BOLD data- turer’s instructions. The extension products 1. For the species O.aegina, O.cyanea, O.vul- bases (Figure 2). were purified using the DyeEx 2.0 spin kit garis and O.dollfusi there were no identifica- (Qiagen), denatured in formamide and tion problems. We were able to identify 27 analysed with the ABI Prism 310 Genetic samples, out of 29, with a similarity of 99- Analyzer (Applied Biosystems). The obtained 100%. Out of these, 22 samples were in accor- Discussion sequences were examined, corrected and dance with the labels of the products, while 5 analysed with the MEGA5 software and sub- did not correspond to what was declared in the The expansion in food preservation, pro- jected to identification with BOLD-IDS for the product description. Finally, for 2 samples cessing technologies and market liberalisation COI gene, and to BLASTN (GenBank) for both labeled as O.vulgaris it was not possible to has contributed significantly to the globalisa- genes. obtain the biomolecular identification, since tion of fish trade, both in terms of species and Finally phylogenetic relationships among they presented a low identity (91%) with products. In a globalised market, the eagerness the studied samples were investigated with Amphioctopus rex and Amphioctopus margina- to offer leading products appreciated and differences method (Nei and Kumar, 2000). tus, species not reported in the FAO catalogue. recognised to derive maximum revenue, can For the distance matrix, phylogenetic trees Further difficulties were encountered in the introduce the habit of replacing valuable were constructed using the Neighbour-Joining identification of samples labelled as O.mem- species with others very similar but economi- method. The bootstrap method (500 replica- branaceus. One sample only was identified cally or qualitatively inferior, thus incurring tes) was used to obtain the support of different from both databases in a non-ambiguous way into commercial fraud (Jacquet and Pauly, groups included in the phylogenetic (100% similarity) as O.aegina, while for the 2008). (Felsenstein, 1985). other 21 samples it was not possible to obtain The identification of species in fisherystock a certain identification. In particular, regard- products, essential at the moment of the com- ing the cytb, the similarity obtained values mercialisation, has been based for a long time ranged from 93-94% with O.membranaceus, on morphological characteristics solely, and it Results 93% with O.aegina and 90% with Cistopus tai- isonly still the official method for taxonomic classi- wanicus (Figure 1). The results were even fication. However, in the last years this mor- All samples originated an amplicon of 655 bp more complex for the COI gene because of the phological classification has been supported by use commercial Non Figure 1.

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