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A Barcode for the Authentication of the Snappers (Lutjanidae) of the Western Atlantic: Rdna 5S Or Mitochondrial COI?

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A Barcode for the Authentication of the Snappers (Lutjanidae) of the Western Atlantic: Rdna 5S Or Mitochondrial COI? Food Control 38 (2014) 116e123 Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont A barcode for the authentication of the snappers (Lutjanidae) of the western Atlantic: rDNA 5S or mitochondrial COI? Ivana Veneza a, Bruna Felipe a, Joiciane Oliveira a, Raimundo Silva a, Iracilda Sampaio b, Horacio Schneider b, Grazielle Gomes a,b,* a Laboratório de Genética Aplicada, Instituto de Estudos Costeiros, Universidade Federal do Pará, Campus Universitário de Bragança, Alameda Leandro Ribeiro s/n, Aldeia, Bragança, Pará, Brazil b Laboratório de Genética and Biologia Molecular, Instituto de Estudos Costeiros, Universidade Federal do Pará, Campus Universitário de Bragança, Brazil article info abstract Article history: The increasing demand for fishery resources in recent years has stimulated a growth in the output of Received 5 February 2013 processed products, which has made the fraudulent substitution of species a common practice. In the Received in revised form present study two different protocols were evaluated for the molecular authentication of lutjanid species, 8 October 2013 one based on the banding pattern of the nuclear rDNA 5S gene, and the other on the sequences of the Accepted 9 October 2013 mitochondrial Cytochrome Oxidase subunit I (COI) gene. A total of 132 samples were analyzed from specimens identified previously as belonging to seven lutjanid species (Lutjanus purpureus, Lutjanus Keywords: synagris, Lutjanus vivanus, Lutjanus jocu, Lutjanus analis, Ocyurus chrysurus,andRhomboplites aurorubens), COI fi fi rDNA 5S as well as unidenti ed individuals. The results indicate the absence of a species-speci c rDNA 5S banding fi Snappers pattern in lutjanids. However, the 1131 bp fragment of the COI gene not only discriminated the identi ed Barcode lutjanid species systematically, but also defined the species of the unidentified specimens, identifying Lutjanids another two species from the database, Lutjanusbucanella and Lutjanuscyanopterus. The species were represented by well-defined consensual clades in the phylogenetic trees, supported by the interspecific distances and the mutations characteristic of each species. This segment of the COI gene proved to be a robust tool for the molecular authentication of lutjanid species. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction processing can remove distinguishing features, and impede the diagnosis of species recognized solely on the basis of morphological The trade in fishery products has expanded significantly in traits (Carvalho, Neto, Brasil, & Oliveira, 2011; Céspedes et al., 1999; recent years, with a total worldwide harvest of 154 million tons in Filonzi, Chiesa, Vaghi, & Marzano, 2010; Sales, Rodrigues-Filho, 2011, including both wild-caught and farmed produce (FAO, 2012). Haimovici, Sampaio, & Schneider, 2011; Sotelo, Piñeiro, Gallardo, At the same time, there has been an ever-increasing tendency for & Pérez-Martín, 1993). the diversification of the products being marketed, including fillets Inadequate product labeling can have serious consequences in and steaks, smoked fish and canned goods, derived from a wide terms of public health, as well as having ecological and economic range of different fish species. implications. In addition to entailing potential risks for the con- This growth in trade has been accompanied by an increase in the sumer (Van Leeuwen et al., 2009), including financial costs e as fraudulent substitution of more valuable species by inferior ones shown by Marko et al. (2004) in the case of the snappers e it may (Ward, 2000). In addition to the marked morphological similarities impact management programs designed for the conservation of the of species of some fish families, such as the Sciaenidae, Mugilidae, stocks of certain species (Ward, 2000). and Lutjanidae (Allen, 1985; Cervigón, 1993; Cervigón et al., 1993), The lutjanids fishes known as snappers represent an important fishery resource in all the regions where they occur (Allen, 1985; Cervigón, 1993; Matos-Caraballo, 2000; Mendoza & Larez, 1996; Prescod, Oxenford, & Taylor, 1996; Zhang & Liu, 2006). These me- * Corresponding author. Instituto de Estudos Costeiros, Universidade Federal do dium to large-sized fishes are widely distributed in the Atlantic, Pará e Bragança, Alameda Leandro Ribeiro s/n, Aldeia, Bragança, CEP: 68.600-000 fi PA, Brazil. Tel.: þ55 091 3425 1593. Indian, and Paci c oceans. The family is composed of approximately E-mail addresses: [email protected], [email protected] (G. Gomes). 108 species distributed among 17 genera, organized in four 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.10.012 I. Veneza et al. / Food Control 38 (2014) 116e123 117 subfamilies, the Etelinae, Apsilinae, Paradicichthyinae, and Lutja- cases (see Céspedes et al., 1999; Rodrigues-Filho et al., 2010; Sales ninae (Allen, 1985; Cervigón, 1993; Froese & Pauly, 2012; Moura & et al., 2011). Lindeman, 2007; Nelson, 2006). Given the marked morphological similarities among the Many lutjanids, such as the red snappers (Lutjanus vivanus, L. different lutjanid species and the fact that snappers are typically purpureus, L. campechanus, L. bucanella, and L. peru), are highly marketed in fillet form, which facilitates the illicit substitution of similar morphologically (Cervigón, 1993; Cervigón et al., 1993; species, an effective molecular species identification protocol Nelson, 2006) and are difficult to identify reliably based on which is both fast and inexpensive is urgently needed. In an external characteristics. This problem is exacerbated by the in- attempt to provide an appropriate approach to this problem, the dustrial processing of catches, which typically involves the removal present study evaluated two different molecular methods e one of the fillets. based on the banding patterns of the amplified rDNA 5S gene Based on the analysis of a fragment of the mitochondrial Cyto- resolved on an agarose gel, and the other, a more conventional chrome b gene, Marko et al. (2004) discovered that approximately approach, which has been shown to be effective in snappers (see 80% of the fillets sold in the United States as red snapper Victor, Hanner, Shivji, Hyde, & Caldow, 2009), based on the analysis (L. campechanus) were actually derived from other lutjanid species, of the sequences of a fragment of the mitochondrial COI gene. This presumably as a result of errors in the identification of specimens comparative analysis will provide an initial step towards the during the production process and/or the intentional substitution development of rapid and low-cost molecular protocols that pro- with less popular and/or cheaper species. vide an unambiguous identification of snapper species. Molecular studies based on nucleotide sequences have become increasingly popular for the identification of fishes and/ 2. Material and methods or fishery products (Brown et al., 1996; Carrera et al., 2000; Chiu, Su, Pai, & Chang, 2012; De Salle & Birstein, 1996; Filonzi et al., 2.1. Samples 2010; Mackie et al., 1999; Rasmussen & Morrissey, 2008; Wen, Hu, Zhang, & Fan, 2011). One genomic region that has been A total of 132 lutjanid specimens (Table 1) were analyzed in the used successfully for molecular diagnosis is the Cytochrome present study. The samples were obtained from the Lutjanidae Oxidase subunit I (COI) gene, which is considered to be a “bio- tissue bank held by the Applied Genetics Laboratory at the Coastal logical barcode” (Hebert, Cywinska, Ball, & de Waard, 2003). A Studies Institute of the Federal University of Pará in Bragança, number of studies have demonstrated the usefulness of different Brazil. In all, 37 of the specimens had been identified previously fragments of this gene for the identification of fish species and (Allen, 1985; Cervigón, 1993; Cervigón et al., 1993; Menezes & the products derived from them (Carvalho, Neto, et al., 2011; Figueiredo, 1980), representing seven species, belonging to three Carvalho, Oliveira, et al., 2011; Filonzi et al., 2010; Haye, genera e Lutjanus (L. purpureus, L. synagris, L. jocu, L. analis, and Segovia, Vera, Gallardo, & Gallardo-Escárate, 2012; Rasmussen, L. vivanus), and the monotypic Rhomboplites aurorubens and Morrissey, & Hebert, 2009; Silva-Oliveira et al., 2011; Ward, Ocyurus chrysurus. The remaining 95 specimens, which were Zemlak, Innes, Last, & Hebert, 2005; Yang, Huang, Hsieh, Huang, collected at a number of different locations around the Brazilian & Chen, 2012). coast, were considered to be “unidentified snappers” or UISs for In addition to DNA sequences, a number of alternatives have this analysis. A number of additional specimens were included in been tested, based on faster and more practicable approaches, the analysis to provide a comparative perspective of rDNA 5S which allow for the analysis of PCR (Polymerase Chain Reaction) banding patterns at the family level. These specimens include three products directly in agarose gels (see Rodrigues-Filho et al., 2010), individuals representing two haemulidae species (Conodon nobilis such as the banding pattern provided by the amplification of the and Genyatremus luteus), a scombridae (Scomberomorus brasi- rDNA 5S gene. This marker is a multigenic family composed of liensis), a centropomidae (Centropomus undecimalis), and a sciae- repeated units in a conserved coding region with approximately nidae, Cynoscion sp. (Table 1). 120 base pairs arranged in tandem, separated by non-transcribed The approach adopted here
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