Genetic traceability of canned fish products manufactured in Cape Verde

Genetic traceability of canned fish products manufactured in Cap Genetic traceability of canned fish products manufactured in Cape Verde e Verde Nilson Brás 2 , Isilda Fortes 2 , Javier Quinteiro 3 , Manuel Rey-Méndez 3 , Corrine Almeida 2 , Pablo Manent 1 , Nieves González 1 1.- Departamento de Biología. Universidad de Las Palmas de Gran Canaria (ULPGC). 2.- Departamento de Engenharias e Ciências do Mar. Universidade de Cabo Verde. 3.- Departamento de Bioquímica y Biología Molecular. Universidade de Santiago de Compostela. INTRODUCTION Genetic traceability has been defined as the ability to identify the geographic origin and/or the essence of ingredients used in the elaboration of a food product or ingredient. The production of canned fish in the Cape Verde archipelago is supported by the local captures of the mackerel scad, Decapterus macarellus (Cuvier, 1833), and a number of scombrid species, including Thunnus albacares (Bonnaterre, 1788), Auxis thazard (Lacepède, 1800) and Katsuwonus pelamis (Linnaeus, 1758). In canned food, morphological features that allow the identification of taxa used in the elaboration process are removed. In this context, the genetic identification of tissues is a tool useful within the industry traceability standards, to verify the authenticity of seafood, and to resolve questions about fraud or accidental substitution with less valuable raw material. MATERIALS AND METHODS Two sets of samples were analysed. The first one contains locally captured fish including Decapterus macarellus (Cuvier, 1833), Thunnus albacares (Bonnaterre, 1788), Auxis thazard (Lacepède, 1800) and Katsuwonus pelamis (Linnaeus, 1758). The second set includes a collection of canned products, and a variable number of replicates from each one can. DNA was isolated in all cases using Speedtools Food DNA Extraction kit (Bio-Tools). PCR was elaborated using GoTaq (Promega) and standard PCR cycling conditions. Concretely, the melting temperature was set at 50ºC and MgCl2 ranged from 1.5mM-3.5mM. The primers set used for partial COX1 amplification were FISHF2/FISHR2 (Ward et al. 2005), whereas for Cytochrome b amplifications were L14725/H15149 and L15424/H15573 (Kocher et al. 1989; Pääbo 1990) and L15998/CSBDH (Alvarado-Bremer 1994) and DCANF/DCANR (Quinteiro 2011) for the control region (CR). Sequences were obtained following ordinary sequencing protocols including PCR product purification with ExoSAP-IT (Amersham-Biosciences), sequencing with BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and separation of extension products in an ABI3500 sequencer (Life Technologies). Chromatograms were revised and aligned in BioEdit (Hall 1999). Species identification for obtained sequences was performed by i) BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and detection of 99-100% similarity match in GenBank records, ii) COX1 barcoding analysis en BOLD systems (http://www.boldsystems.org/index.php/IDS_OpenIdEngine) and iii) by clade assignation after phylogenetic reconstruction (Quinteiro et al. 1998) with MEGA software (Kumar et al. 2008). RESULTS AND DISCUSSION Barcoding of species captured in caboverdian waters The locally sampled fishes in Cape Verde, theoretically belonging to the species considered in the present study and including Decapterus macarellus (Cuvier, 1833), Thunnus albacares (Bonnaterre, 1788), Auxis thazard (Lacepède, 1800) and Katsuwonus pelamis (Linnaeus, 1758) were genetically diagnosed using COX1 barcoding with FISHF2/FISHR2 primers. The sequences (N=5) belonging to the mackerel scad, “cavala preta” match (100% similarity) with Decapterus macarellus GENBANK records using BLAST algorithm and BOLD Systems. In particular the Cape Verde sample closely grouped with Caribean specimens (Figure 2) . Similarly, the tuna samples were genetically identified as Thunnus albacares, displaying >99% similarity values. Within the smallest tunas species were identified the bullet tuna, Auxis rochei (Risso, 1810) (Figure 1) and the frigate tuna Auxis thazard (Lacepède, 1800). Consequently, these genetically authenticated samples agree with the morphological diagnosis of captured fish and provide of reference tissues and sequences for the canned products traceability analysis. 0.003 Auxis rochei|[35]|' Auxis rochei|[31]|Philippines.Manila| Sarda orientalis|[34]|India.Kerala| Auxis rochei|[23]|' Auxis rochei|[2]|' Auxis rochei|[19]|Malaysia.Sabah| Auxis rochei rochei|[15]|Indonesia.Jawa Barat| Auxis rochei|[46]|Mexico| Auxis rochei|[33]|Mexico| Auxis thazard|[10]|Australia.Tasmania| Auxis rochei|[25]|' Auxis rochei|[43]|Belize| Auxis rochei|[22]|Malaysia.Sabah| Auxis rochei|[14]|' Auxis rochei|[49]|' Auxis rochei|[24]|' Auxis rochei|[26]|' Auxis rochei rochei|[50]|Italy| Auxis rochei|[20]|Malaysia.Sabah| Auxis rochei|[45]|Belize| Auxis rochei|[16]|Indonesia.Jawa Barat| Auxis rochei|[48]|' Auxis rochei|[38]|Indonesia.Jawa Barat| Sarda orientalis|[40]|India.Kerala| Auxis rochei|[30]|' Auxis thazard thazard|[29]|Taiwan.Penghu County| Auxis rochei|[7]|Philippines.Manila| Auxis rochei rochei|[27]|Taiwan.Hualien City| Auxis rochei|[1]|Mexico| Unknown Specimen|' Auxis rochei|[44]|' Auxis rochei rochei|[39]|Indonesia.Jawa Barat| Auxis thazard|[47]|Cape Verde| Auxis rochei|[11]|South Africa| Auxis rochei rochei|[41]|Portugal| Auxis thazard Auxis rochei|[13]|' Auxis rochei|[5]|' Auxis rochei|[3]|South Africa| Auxis rochei|[9]|Philippines| Auxis thazard thazard|[28]|Taiwan.Penghu County| Auxis rochei|[51]|' Auxis rochei rochei|[18]|Indonesia.Jawa Barat| Auxis rochei|[36]|Malaysia.Sabah| Auxis rochei rochei|[17]|Taiwan.Hualien City| Auxis rochei|[32]|' Auxis rochei|[21]|Malaysia.Sabah| Auxis rochei|[6]|' Auxis rochei|[8]|Philippines.Manila| Auxis rochei|[12]|Indonesia.Bali| Auxis rochei|[4]|Belize| Auxis rochei|[37]|Turkey|' Auxis rochei|[42]|' Figure 1. Tree based identification using BOLD Systems (http://www.boldsystems.org/). From an specimen (red), concretely ATHA07VV, it was obtained a partial COX1sequence and compared with the records in BOLD repository. This sequence shows a match of 100% similarity with Auxis rochei records. A midpoint rooted neighbor-joining tree displays graphically this similarity and the phylogenetic relationships respect to other taxa. All Auxis rochei records are monophyletically included in a weel supported clade, being A. thazard the sister clade. 0.009 Decapterus macarellus|[92]|Bermuda| Decapterus macarellus|[83]|Indonesia.Jawa Barat| Decapterus maruadsi|[15]|Malaysia.Sarawak| Decapterus russelli|[13]|Israel| Decapterus macarellus|[87]|South Africa| Decapterus muroadsi|[71]|Australia.Western Australia| Decapterus macarellus|[90]|Bermuda| Decapterus russelli|[5]|South Africa| Decapterus russelli|[18]|Australia.Queensland| Decapterus russelli|[4]|South Africa.KwaZulu-Natal| Decapterus macarellus|[85]|South Africa| Decapterus macarellus|[99]|Bermuda| Decapterus russelli|[8]|Israel| Decapterus sp.|[79]|Taiwan.Pingtung County| Decapterus sp.|[80]|Taiwan.Pingtung County| Decapterus maruadsi|[11]|Malaysia.Johor| Decapterus macarellus|[75]|Mozambique| Decapterus sp.|[81]|Taiwan.Pingtung County| Decapterus maruadsi|[16]|Malaysia.Johor| Decapterus macarellus|[88]|Philippines.Aurora| Decapterus tabl|[97]|Belize| Decapterus russelli|[9]|Israel| Decapterus russelli|[7]|Israel| Decapterus tabl|[98]|Belize| Decapterus macarellus|[86]|Indonesia.Jawa Barat| Decapterus maruadsi|[20]|' Decapterus russelli|[14]|Mediterranean Sea| Decapterus muroadsi|[72]|Australia.Western Australia| Decapterus russelli|[6]|Mozambique| Decapterus macarellus|[82]|Indonesia.Jawa Barat| Decapterus muroadsi|[70]|Australia.Western Australia| Decapterus macarellus|[73]|South Africa.KwaZulu-Natal| Decapterus maruadsi|[19]|' Decapterus russelli|[10]|Israel| Decapterus russelli|[3]|South Africa.KwaZulu-Natal| Decapterus macarellus|[95]|Bermuda| Unknown Specimen|' Decapterus macarellus|[89]|Philippines| Decapterus macarellus|[94]|Belize| Decapterus russelli|[2]|South Africa| Decapterus macarellus|[78]|Mozambique| Decapterus macarellus|[76]|Mozambique| Decapterus macarellus|[74]| Decapterus tabl|[93]|Cayman Islands| Decapterus macarellus|[96]|Bermuda| Decapterus russelli|[1]|South Africa.KwaZulu-Natal| Decapterus maruadsi|[17]|Malaysia.Sarawak| Decapterus macarellus|[84]|Mozambique| Decapterus macarellus|[77]|Mozambique| Decapterus macarellus|[91]|Bermuda| Decapterus maruadsi|[12]|Malaysia.Johor| Figure 2. Tree based identification using BOLD Systems (http://www.boldsystems.org/). From an specimen (red), concretely DMAC29vB, it was obtained a partial COX1sequence and compared with the records in BOLD repository. This sequence shows a match of 100% similarity with Decapterus macarellus records. A midpoint rooted neighbor-joining tree displays graphically this similarity and the phylogenetic relationships respect to other taxa. Species Identification of canned products The samples of canned products were identified by standard procedures of genetic traceability (Mackie et al. 1999; Quinteiro 2011; Quinteiro et al. 2008; Quinteiro et al. 1998). Within the majority of samples containing tuna it was identified the skipjack, Katsuwonus pelamis based on CYTB and CR sequences. The yellowfin, T. albacares, is absent of the complete set of analysed products. Two products contained the frigate tuna Auxis thazard. An unexpected result was the presence, in diverse canned products, of the Atlantic mackerel, Scomber scombrus and Atlantic chub mackerel, Scomber colias (Table 1). Only two products, with two replicates (4SU1-3, 5SU1-3), labelled as containing D. macarellus were successfully analysed by amplification with the L14524 and H15573 primers. Based in the sequencing results and the comparison with reference data from CytB, in these case a consistent result suggest the presence of D. macarellus tissue and the congruence with label indications. In addition, a PCR product located in the control region (DCANF/DCANR) was obtained from other 3 products (1SU1, 3SU3 and 4SU1). Although this set is not designed to this specie, the data also suggest the presence of D. macarellus in these products (Table 1). However, the analysis involving the canned products putatively containing D. macarellus and the standard primers sets consistently failed. In order to solve this amplification difficulties in food products, it was designed a new set of primers located in both the cytb and control region sequences. REFERENCES The phylogenetic handbook. A practical Approach to Phylogenetic Analysis and Hypothesis Testing, 2 edn. Cambridge University Press. Alvarado-Bremer JR (1994) Assessment of morphological and genetic variation of the swordfish ( Xiphias gladius Linnaeus): Evolutionary implications of the allometric growth and of the patterns of nucleotide substitution in the mitochondrial genome, University of Toronto. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 95-98. Kocher TD, Thomas WK, Meyer A, et al. (1989) Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers. Proc.Natl.Acad.Sci.USA 86, 6196-6200. Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences. Briefings in Bioinformatics 9, 299-306. Mackie IM, Pryde SE, Gonzales-Sotelo C, et al. (1999) Challenges in the identification of species of canned fish. Trends in Food Science & Technology 10, 9-14. Pääbo S (1990) Amplifying ancient DNA. In: PCR Protocols: A Guide to Methodsand Applications (eds. Innis MA, Gelfand DH, Sninsky JJ, White TJ), pp. 159-166. Academic Press, San Diego. Quinteiro J (2011) Filogenia molecular, estructura poblacional y trazabilidad genética de escómbridos (Pisces: Scombridae), Universidad de Santiago de Compostela. Quinteiro J, Santaclara FJ, Rehbein H (2008) Authenticity of Canned Seafood. In: Quality Parameters in Canned Seafoods (eds. Cabado AG, Vieites JM), pp. 135-158. Nova Science Publishers, Inc, New York. Quinteiro J, Sotelo CG, Rehbein H, et al. (1998) Use of mtDNA direct polymerase chain reaction (PCR) sequencing and PCR-restriction fragment length polymorphism methodologies in species identification of canned tuna. Journal of Agricultural and Food Chemistry 46, 1662-1669. Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PD (2005) DNA barcoding Australia's fish species. Philosophical Transactions of the Royal Society of London, Series B: Biological Sciences 360, 1847-1857. Species Identification of canned products The samples of canned products were identified by standard procedures of genetic traceability (Mackie et al. 1999; Quinteiro 2011; Quinteiro et al. 2008; Quinteiro et al. 1998). Within the majority of samples containing tuna it was identified the skipjack, Katsuwonus pelamis based on CYTB and CR sequences. The yellowfin, T. albacares, is absent of the complete set of analysed products. Two products contained the frigate tuna Auxis thazard. An unexpected result was the presence, in diverse canned products, of the Atlantic mackerel, Scomber scombrus and Atlantic chub mackerel, Scomber colias (Table 1). Only two products, with two replicates (4SU1-3, 5SU1-3), labelled as containing D. macarellus were successfully analysed by amplification with the L14524 and H15573 primers. Based in the sequencing results and the comparison with reference data from CytB, in these case a consistent result suggest the presence of D. macarellus tissue and the congruence with label indications. In addition, a PCR product located in the control region (DCANF/DCANR) was obtained from other 3 products (1SU1, 3SU3 and 4SU1). Although this set is not designed to this specie, the data also suggest the presence of D. macarellus in these products (Table 1). However, the analysis involving the canned products putatively containing D. macarellus and the standard primers sets consistently failed. In order to solve this amplification difficulties in food products, it was designed a new set of primers located in both the cytb and control region sequences. Decapterus macarellus (Cavala preta, Cabo Verde) Product Code Replicate Gene Primers set Species KP 1SU 1 CYTB L15424/H15573 Katsuwonus pelamis 2 Katsuwonus pelamis 3 Katsuwonus pelamis KP 4FR 1 CYTB L15424/H15573 Katsuwonus pelamis 2 Katsuwonus pelamis 3 Katsuwonus pelamis 4 Katsuwonus pelamis KPEL 02SU 1 CYTB L15424/H15573 Katsuwonus pelamis 2 Katsuwonus pelamis 3 Katsuwonus pelamis KPEL 03SU 1 CYTB L15424/H15573 Katsuwonus pelamis 2 Katsuwonus pelamis 3 Katsuwonus pelamis KPEL 09FR 1 CYTB L15424/H15573 Katsuwonus pelamis 2 Katsuwonus pelamis 3 Katsuwonus pelamis 4 Katsuwonus pelamis 5 Katsuwonus pelamis 6 Katsuwonus pelamis 7 Katsuwonus pelamis 8 Katsuwonus pelamis 9 Katsuwonus pelamis 10 Katsuwonus pelamis KPEL 01SU 2 Control Region DCANF/DCANR Katsuwonus pelamis KPEL 08FR 3 Control Region DCANF/DCANR Thunnus obesus 5 Thunnus obesus KPEL 09FR 6 Control Region DCANF/DCANR Thunnus obesus 7 Thunnus obesus 8 Thunnus obesus 9 Thunnus obesus ATHA 01SU 3 CYTB L15424/H15573 Auxis thazard ATHA 02SU 3 CYTB L15424/H15573 Auxis thazard 1SU 1 Control Region DCANF/DCANR Decapterus macarellus 3SU 3 Control Region DCANF/DCANR Decapterus macarellus 4SU 1 Control Region DCANF/DCANR Decapterus macarellus 2FR 1 Control Region DCANF/DCANR Scomber scombrus 2 Scomber scombrus 3 Scomber scombrus 4FR 3 Control Region DCANF/DCANR Scomber scombrus 8FR 8 Control Region DCANF/DCANR Scomber japonicus 9FR 3 Control Region DCANF/DCANR Scomber japonicus Caracterization of partial mitochondrial sequences in Decapterus macarellus and primer design. For the mackerel scad, Decapterus macarellus (Cuvier, 1833), a set of (N=16) sequences from the mitochondrial cytochrome b gene was obtained and flanked by the L14725 and H15573 primers. The alignment was 800 pb length from the initiation codon of the CytB gene. This sequence shows a 99-100% with partial cytochrome b haplotypes deposited in GenBank (EU349422, EU349423, EU349424). The alignment allows to design two set of primers in locations without intra-specific variability. The primers set Dmac-CB-1F/Dmac-CB-2R delimitated a 198bp length fragment (CB12) within the 5’ portion of the Cytochrome b. Alternatively, the Dmac-CB3F/Dmac-CB-4R primers flanks a 181 bp length fragment (CB34). Similarly, a 386 bp length alignment was elaborated for the control region, from 23 sequences amplified with the L15998 and CSBDH primers. Within this fragment the primers Dmac-CR1F and Dmac-CR-2R were designed to amplify a 159 bp length fragment. The design take in consideration the high intra and inter-especific variability around this region, being considered highly specific for D. macarellus after ”in silico” analysis. Design of a PCR-RFLP analysis for species identification In accord with this preliminary results about the species presence in canned products within tunas and relatives the main problematic question is to discriminate among T. albacares/T.obesus, K. pelamis and Auxis spp. and Scomber spp. The digestion of the B126 fragment produces a highly species-specific RFLP pattern for tuna (Quinteiro 2011) (Table 2). In the case of D. macarellus, a digestion strategy of the CB12 and CB34 fragments was selected to produce a different RFLP pattern from those expected in related species (Table 2). These PCR protocol will be evaluated for consistency in the available sample set. Constitutes a rapid and easy to perform protocol for species identification without the need of DNA sequencing (Quinteiro et al. 1998). HphI, AsuHPI GGTGA AciI, BspACII, SsiI CCGC MboI, Sau3AI ^GATC MnlI CCTC NlaIII ^CATG Fragment CB12 Decapterus macarellus 57/141 71/127 Fragment CB34 Decapterus macarellus 115/66 157/24 Fragment B126 L15424/H15573 T. albacares 47/129 21/25/35/43/52 176 T. obesus 176, 47/129 1 21/25/35/43/52 135+41 K. pelamis 176 60/49/43/24 135+41 Auxis thazard 146/30 103/52/21 135+41 Auxis rochei 146/30 78/73/25 135+41 1 Atypical pattern Table 2. Expected size fragments after enzymatic digestion of diverse PCR products from canned products manufactured in Cape Verde. Table 1. Samples of canned products RESULTS AND DISCUSSION