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1 Application of DNA barcoding for the control of the species from the Octopus genus. 2 3 Francesco Debenedetti1, Alessandra Dalmasso1, Maria Teresa Bottero1, Maurizio Gilli3, Stefano Gili3, 4 Valentina Tepedino2, Tiziana Civera1* 5 6 7 1 8 (TO), Italy 9 2 Eurofishmarket, Piazza Sergio Vieira De Mello 4, 40128 Bologna, Italy 10 3 ASLTO1, Via della Consolata 10, 10122 Torino Department of Veterinary Sciences, Università degli Studi di Torino, largo Braccini 2, 10095 Grugliasco 11 12 Correspondence: * Tiziana Civera, Department of Veterinary Sciences, Università degli Studi di Torino, 13 largo Braccini 2, 10095 Grugliasco (TO), Italy. 14 Phone:+39. 011.67092 - Fax: +39.011.6709224 15 16 Key words: DNA barcoding; Octopus spp.; commercial frauds 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Conflict of interests: the authors declare no potential conflict of interests. 38 Abstract 39 The DNA barcoding proposes the use of a particular sequence from a single genomic region as the base for 40 an identifying system capable to determine all animal species. This methodology comprises the analysis of a 41 655 base-pair region from the mithocondrial cytochrome C oxidase gene (COI). Its application in the species 42 identification of fishery products has been very promising, however in the last years some doubts about its 43 usage have emerged. 44 In this work, we make use of the DNA barcoding for the identification of some of the octopus species with 45 higher commercial interest (Octopus membranaceus, Octopus vulgaris, Octopus aegina, Octopus cyanea) 46 focusing the attention on the reliability and completeness of the available information on the databases. 47 The study looked over 51 individuals apparently belonging to the Octopus genus. 48 For the identification of O.aegina, O.cyanea, O.vulgaris species no particular problems were found. On the 49 other hand most of the samples of O.membranaceus, despite they clearly presented the morphological 50 characteristics of the species, were not identified with the biomolecular analyses. 51 52 Riassunto 53 Il DNA barcoding propone l'utilizzo di una sequenza di una singola porzione genica come base di un sistema 54 identificativo valido per l’identificazione di tutti gli animali. Questa tecnica prevede l’analisi di un 55 frammento di 655 bp della subunità I della citocromo C ossidasi (COI). La sua applicazione nel campo 56 dell’identificazione delle specie ittiche si è rilevata molto promettente fin dall'inizio, tuttavia sono emersi, nel 57 corso degli anni, alcuni dubbi sul suo utilizzo. 58 In questo lavoro abbiamo voluto impiegare il DNA barcoding per l'identificazione di alcune specie di polpi 59 di maggior interesse commerciale (Octopus membranaceus, Octopus vulgaris, Octopus aegina, Octopus 60 cyanea) focalizzando l’attenzione sull'attendibilità e la completezza delle banche dati 61 Sono stati analizzati 51 soggetti di appartenenti al genere Octopus. 62 Per le specie 63 nell’identificazione. Viceversa la maggior parte dei campioni di O.membranaceus, nonostante presentasse 64 inequivocabilmente le caratteristiche morfologiche della specie, non è stata identificata con le analisi 65 biomolecolari. 66 67 68 69 70 71 72 73 74 O.aegina , O.cyanea , O.vulgaris non sono state riscontrate particolari difficoltà 75 Introduction 76 The DNA barcoding puts forward the use of a sequence from a single genomic region (defined as 77 “barcode”), as the base of a recognition system capable to identify all animal species (Hebert et al., 2003). 78 This biomolecular methodology consists on the identification of the belonging species through the 79 sequencing of a fragment of mitochondrial genes, its alignment and comparison with the information 80 available on the databases. 81 In particular, the barcoding approach comprises the analysis of a 655 bp region located at the 5’ end of the 82 subunit I of the mithocondrial cytochrome C oxidase gene (COI). This gene codes for one part of the 83 terminal enzyme of the mithocondrial respiratory chain. The COI gene has been chosen as a universal 84 molecular target since it allows the design of universal, robust and functional primers for almost all the 85 member of the animal Phyla (Folmer et al., 1994). 86 The effectiveness of this fragment for the identification of species. has been demonstrated for several animal 87 species, from vertebrates to invertebrates (Waugh, 2007; Ward and Holmes, 2007). 88 Recently, this technique was recognized as the best method in Forensics for species’ identification, and was 89 proposed by the United States Food and Drug Administration (USFDA) as the methodology for the 90 authentication of commercial fish products (Dawnay et al., 2007). 91 The US agency has also the intention of introduce the DNA barcoding data in the Regulatory Fish 92 Encyclopedia (RFE) to support the investigations of mislabeling and the substitution of the fish species 93 (Yancy et al., 2008). In May 2004, the Consortium for the Barcoding of Life (CBOL) was formed, 94 comprising several international patterns. Since the beginning, its mission has been the exploration and the 95 development of the potentiality of the DNA barcoding as a practical and research tool for the species’ 96 identification. The use of this methodology for the species identification of fishery products has been very 97 promising from the outset, given the large number of species already identified. Actually it has been shown 98 that 98% and 93% of the marine and fresh water species, respectively, can be differentiated using barcodes 99 (Savolainen et al., 2005; Ward et al., 2009). 100 This favorable outcome as well as the need of a complete and reliable instrument for identification of the 101 species, have derived into the formation of the initiative Fish Barcode of Life (FISH-BOL) 102 (www.fishbol.org). 103 This campaign, launched in 2005, has as main objective the collection of DNA barcodes of all the fish in the 104 world, equivalent to 31,000 species approximately. It endorses the FishBase (www.fishbase.org) as the 105 taxonomic authority, and the BOL database (BOLD) as the working bioinformatic platform. FISH-BOL 106 represents one of the most complete resources for the species identification of the fishery products (Ward et 107 al., 2009). 108 However, in the past years, some doubts regarding the use of DNA barcoding have emerged because of the 109 difficulties to discriminate recently spread species, or species with a wide spatial differentiation; as well as 110 the inability to differentiate new and hybrid species (Moritz and Cicero, 2004; Hickerson et al., 2006; 111 Rubinoff et al., 2006). 112 Furthermore, if on one hand this technique has been extensively evaluated for the identification of fish 113 species, there are still few data supporting the applicability in the identification of the most common species 114 of cephalopods and octopus. 115 In this work we test the DNA barcoding for the identification of some octopus species of greatest 116 commercial interest (Octopus membranaceus, Octopus vulgaris, Octopus aegina, Octopus cyanea) focusing 117 the attention on the reliability and completeness of the available databases. 118 119 Materials and Methods 120 The sampling involved the collection of 51 specimens belonging to the genus Octopus: 1 O.dollfusi, 2 121 O.aegina, 2 O.cyanea, 24 O.vulgaris and 22 O.membranaceus. All samples, collected in 2012 and 2013 122 from different suppliers, were morphologically identified and kept at -20°C. 123 The DNA extraction was done using the DNeasy Tissue and Blood Kit (Qiagen) according to the 124 manufacturer’s instructions for animal tissue. The extracted DNA was quantified with NanoDrop 1000 125 (Thermo Scientific). 126 For all samples, segments of the COI genes were amplified following the protocol proposed by 127 Folmer et al., (1994). Since there were no available reference sequences for O.membranaceus in FISH-BOL, 128 a second sequencing protocol was introduced. It contemplated the usage of cytochrome b (cytb), a historical 129 target used for the species identification (Espineira et al., 2010). 130 Amplicons were visualized by electrophoresis on a 2% agarose gel and colored with Eurosafe Nucleic Acid 131 Stain. 132 The amplified products were purified with the enzyme ExoSap-IT (USB) and sequence with the BigDye 133 terminator kit (Applied Biosystems) according to the manufacturer’s instructions. The extension products 134 were purified using the DyeEx 2.0 spin kit (Qiagen), denatured in formamide and analyzed with the ABI 135 Prism 310 Genetic Analyzer (Applied Biosystems). 136 The obtained sequences were examined, corrected and analyzed with the MEGA5 software and subjected to 137 identification with BOLD-IDS (Bold) for the COI gene, and to BLASTN (GenBank) for both genes. 138 Finally phylogenetic relationships among the studied samples were investigated with differences method 139 (Nei and Kumar, 2000). For the distance matrix, phylogenetic trees were constructed using the Neighbour- 140 Joining method. The bootstrap method (500 replicates) was used to obtain the support of different groups 141 included in the phylogenetic (Felsenstein, 1985). 142 143 Results 144 All samples originated an amplicon of 655 bp for the COI gene and of 651 bp for the cytb gene. 145 The sequencing results are presented on Table 1. 146 For the species O.aegina, O.cyanea, O.vulgaris and O.dollfusi there were no identification problems. 147 Actually we were able to identify 27 samples, out of 29, with a similarity of 99-100%. Out of these, 22 148 samples were in accordance with the labels of the products, while 5 didn’t correspond to what was declared 149 in the product description. 150 Finally, for 2 samples labeled as O.vulgaris it was not possible to obtain the biomolecular identification, 151 since they presented a low identity (91%) with Amphioctopus rex and Amphioctopus marginatus, species not 152 reported in the FAO catalogue. 153 Further difficulties were encountered in the identification of samples labeled as O.membranaceus. 154 Actually only one sample was identified from both databases in a non ambiguous way (100% similarity) as 155 O.aegina, while for the other 21 samples it was not possible to obtain a certain identification. In particular, 156 regarding the cytb, the similarity obtained values ranged from 93-94% with O.membranaceus, 93% with 157 O.aegina and 90% with Cistopus taiwanicus (Fig.1). 158 The results were even more complex for the COI gene because of the lack of reference sequences of 159 O.membranaceus in both GenBank and BOLD databases (Fig.2). 160 161 Discussion 162 The expansion in food preservation, processing technologies and market liberalization has contributed 163 significantly to the globalization of fish trade, both in terms of species and products. 164 In a globalized market, the eagerness to offer leading products appreciated and recognized to derive 165 maximum revenue, can introduce the habit of replacing valuable species with others very similar but 166 economically or qualitatively inferior, incurring into commercial fraud (Jacquet and Pauly, 2008). 167 The identification of species in fisherystock products, essential at the moment of the commercialization, has 168 been based for a long time solely on morphological characteristics, and is still the official method for the 169 taxonomic classification. However, in the last years this morphological classification has been supported by 170 biomolecular laboratory techniques based on the sequencing of several mitochondrial genes (cytb, 16S 171 rRNA, 12SrRNA, NADH). 172 Particularly, the DNA barcoding, based on the sequencing of the COI gene, might be a reliable tool used in 173 the daily practice for the control of commercial fraud. 174 The ability of barcoding to provide unambiguous designation of species from a complete specimen or part of 175 it, has important implications in several fields: in the retail (accidental or intentional replacement, consumer 176 protection and the regulation of the commerce); in fisheries management (monitoring of fishing stocks, 177 sustainable fishing); in the conservation of fish stock (identification of endangered species, protected, 178 damaged or parts of them) (Costa and Carvalho, 2007). 179 Given the limited applications of DNA barcoding in the field of cephalopods, the present work evaluated the 180 possibility of using it to control the Octopus genus and the most commonly found species in the Italian fish 181 markets. 182 In fact octopus phylogeny has been subjected to confusion and controversy throughout the past years (Carlini 183 et al., 2001). 184 The Octopus genus is the largest within the family Octopidae, it comprises more than 200 species. Of these, 185 90% were inserted just in the above-mentioned genus, for this reason it is called "catch-all" (Lü et al., 2013). 186 Several taxonomic studies based on morphology, have recognized more complexity within the genus 187 Octopus spp. ("Species group"). At the beginning there were identified 9 groups of species, many of which 188 are under review. For example, the O.aegina complex was classified as Amphioctopus spp.. Currently many 189 species, previously classified as Octopus spp., were included in the Amphioctopus genus (Huffard and 190 Hochberg, 2005). Furthermore, the analysis of morphological parameters does not seem sufficient to 191 describe a so complex variability, confirmed also by the data obtained in this work. 192 If for the species O.aegina, O.vulgaris and O.cyanea there were no identification problems, the same cannot 193 be said for O.membranaceus, important species of commercial value, fished in the Indo-Pacific area and 194 widely spread in Italian fisheries. 195 From the analysis of the COI gene, the majority of the O.membranaceus samples, despite they clearly 196 presented the morphological characteristics of the species (a conspicuous, dark, ringed ocellus on web at 197 base of arms II, enteroventral to the eyes) (FAO Fisheries synopsis), were not identified with molecular 198 analyses. In particular regarding the gene COI, there were no reference sequences, while in the case of cytb 199 the samples showed values of similarity with O.membranaceus, unexpectedly low (93-94%). 200 From the alignments of the sequences of the O.membranaceus samples, both the COI and cytb presented a 201 great variability not only with respect to the sequences deposited in GenBank, but also between the same 202 samples. The only exception, represented by a group of homologous samples (identity of 99-100%) is 203 justified by the fact that the 12 specimens were from the same batch (Fig. 1 and 2). 204 Moreover this considerable variability was also found in O. vulgaris species: in fact from the alignment of 205 the sequences of the COI gene a range of similarity between the 96% and 100% was observed. As example, 206 in table 2, ten representative sequences of this gene were aligned, highliting the variable sites between them. 207 There were found show 35 SNPs out of 655 bp, which demonstrate a higher variability. 208 These results showed, on one hand that the extreme genotypic variability is not correlated with the 209 phenotypic variability, and on the other hand are confirmed by the literature related to the Octopus genus. 210 Actually the polyphyletic of the Octopus genus has been demonstrated by a number of molecular studies (Lü 211 et al., 2013). The reconsideration of generic names and the major revision of these taxa have been proposed 212 by some authors (Guzik et al., 2005;) 213 214 Conclusion 215 The DNA barcoding applied in the control of Octopus spp. highlights the need to enlarge the information in 216 the database by increasing the number of species and the number of sequences available for each one. In 217 addition, from this works emerges too the complexity in the interpretation of the data, pointing out the 218 importance of the training and experience of the operator to avoid making mistakes. 219 220 221 References 222 Carlini DB, Young RE, Vecchione M, 2001. A molecular phylogeny of the Octopoda (Mollusca: 223 Cephalopoda) evaluated in light of the morphological evidence. Mol Phylogenet Evol 21:388–97. 224 225 Costa FO, Carvalho GR, 2007. The barcode of life initiative: synopsis and prospective societal impacts of 226 DNA barcoding of fish. Genomics, Society and Policy 3:29–40. 227 228 Dawnay N, Ogden R, McEwing R, Carvalho GR, Thorpe RS, 2007. Validation of the barcoding gene CO1 229 for use in forensic genetic species identification. Forensic Sci Int 173:1–6. 230 231 Espiñeira M, Vieites JM, Santaclara FJ, 2010. Species authentication of octopus, cuttle fish, bobtail and 232 bottle squids (families Octopodidae, Sepiidae and Sepiolidae) by FINS methodology in seafoods. Food 233 Chem 121:527-32. 234 235 FAO Fisheries Synopsis No. 125, Volume 3 FAO Species Catalogue Vol. 3. Cephalopods of The World - An 236 Annotated and Illustrated Catalogue of Species of Interest to Fisheries. 237 238 Felsenstein J, 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783- 239 91. 240 241 Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R, 1994. DNA primers for amplification of mitochondrial 242 cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotech 3:294–9. 243 244 Guzik, M.T., Norman, M.D., Crozier, R.H. (2005) Molecular phylogeny of the benthic shallow-water 245 octopuses (Cephalopoda: Octopodinae). Mol Phylogenet Evol 37:235–48. 246 247 Hebert PDN, Cywinska A, Ball SL, de Waard JR, 2003. Biological identifications through DNA barcodes. 248 Proc. R. Soc. Lond. B 270:313–22. 249 250 Hickerson MJ, Meyer CP, Moritz C, 2006. DNA barcoding will often fail to discover new animal species 251 overbroad parameterspace. Syst Biol 55:729–39. 252 253 Huffard CL, Hochberg FG, 2005. Description of a new species of the genus Amphioctopus (Mollusca: 254 Octopodidae) from the Hawaiian islands. Mollusc Res 23:113-28. 255 256 257 Jacquet J, Pauly D, 2008. Trade secrets: renaming and mislabeling of seafood. Mar Policy 32:309-18. 258 Lü ZM, Cui WT, Liu LQ,Li HM, Wu CW, 2013. Phylogenetic relationships among Octopodidae species in 259 coastal waters of China inferred from two mitochondrial DNA gene sequences. Genet Mol Res 12: 3755-65. 260 261 Moritz C, Cicero C, 2004. DNA barcoding: promise and pitfalls. PLoS Biology 2: 1529-31. 262 263 Nei M and Kumar S, 2000. Molecular Evolution and Phylogenetics. Oxford University Press, New York. 264 265 Rubinoff D, 2006. Utility of mitochondrial DNA barcodes in species conservation. Conserv Biol 20:1026– 266 33. 267 268 Savolainen V, Cowan RS, Vogler AP, Roderick GK, Lane R, 2005. Towards writing the encyclopedia of 269 life: an introduction to DNA barcoding. Philos T Roy Soc B 360:1805–11. 270 271 Ward RD, Hanner R, Hebert PDN, 2009. The campaign to DNA barcode all fishes, FISH-BOL. J Fish Biol 272 74: 329-56. 273 274 Ward RD, Holmes BH 2007. An analysis of nucleotide and amino acid variability in the barcode region of 275 cytochrome c oxidase I (cox1) in fishes. Mol Ecol 7:899–907. 276 277 Waugh J, 2007. DNA barcoding in animal species: progress, potential and pitfalls. BioEssays 29:188–97. 278 279 Yancy HF, Zemlak TS, Mason JA, Washington JD, Tenge BJ, Nguyen N-LNT, Barnett JD, Savary WE, Hill 280 WE, Moore MM, Fry FS, Randolph SC, Rogers PL, Hebert PDN, 2008. Potential use of DNA Barcodes in 281 Regulatory Science: Applications of the Regulatory Fish Encyclopedia. J Food Prot 71: 210–17. 282 283 284 285 286 287 288 289 290 291 Table 1: Results of sequencing identification 292 Number of samples Labeled Species Similarity (%) 2 O.aegina O.aegina 99 2 O.cyaneus O.cyanea 99-100 1 O. dollfusi O.aegina 100 4 O.vulgaris O.cyanea 99-100 18 O.vulgaris O.vulgaris 99-100 2 O.vulgaris n.a. n.a. 1 O.membranaceus O.aegina 99-100 21 O.membranaceus n.a. n.a. 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 n.a.: identification not available 310 Figure 1 311 Phylogenetic tree showing the relationship among the studied samples carried from the alignment of the cytb 312 gene 313 314 The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are 315 shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the 316 evolutionary distances used to infer the phylogenetic tree. The distances were computed using the number of 317 differences method and are in the units of the number of base differences per sequence. 318 319 320 Figure 2 321 Phylogenetic tree showing the relationship among the studied samples carried fron the alignment of the COI 322 gene. 323 324 325 326 The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are 327 shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the 328 evolutionary distances used to infer the phylogenetic tree. The distances were computed using the number of 329 differences method and are in the units of the number of base differences per sequence. 330 Table 2: Example of variable sites in COI gene from the alignment of ten O.vulgaris representative sequences. SNPs position (referred to GenBank acc. n° HQ908427) 331 332 Octopus vulgaris 1 Octopus vulgaris 2 Octopus vulgaris 3 Octopus vulgaris 4 Octopus vulgaris 5 Octopus vulgaris 6 Octopus vulgaris 7 Octopus vulgaris 8 Octopus vulgaris 9 Octopus vulgaris 10 1 0 7 T . . . . . . . . C 1 5 4 T C C C C . . . . . 1 7 5 T C C C C . . . . . 1 9 6 A . G . . . . . . . Dots indicate homolog nucleotides 2 1 2 A G G G G G G G G G 2 4 7 C T T T T . . . . . 2 6 2 C . . . . A A A . . 2 7 7 C T T T T . . . . . 3 0 1 T A A A A . . . . . 3 1 0 A . . . . . . . T . 3 1 3 T . C C C . . . . . 3 4 0 A G G G G . . . . . 3 4 2 C T . . . . . . . . 3 6 7 A T . . . . . . . . 3 7 3 C T T T T . . . . . 3 8 3 C . . . . T . . . . 3 8 5 A T . . . . . . . . 3 8 8 A C C C C . . . . . 4 0 6 A T . . . . . . . . 4 1 5 T . . C . . . . . . 4 2 4 T C C C C . . . . . 4 3 9 C . . . . . . . T . 4 8 1 A T . . . . . . . . 4 9 3 T . . . . C C C . . 5 0 5 G A A A A A A A . . 5 1 4 T A A A A . . . . . 5 3 9 T C C C C C C C C . 5 6 2 A . . . . G G G . . 5 8 0 A T . . . . . . . . 6 0 1 C T T T T . . . . . 6 1 6 A T T T T . . . . . 6 1 7 A G . . . . . . . . 6 1 8 G A . . . . . . . . 6 3 1 C T T T T . . . . . 6 5 0 T . . . . . . . . C