JFS:

Food Chemistry and Toxicology

Genetic Identification of Lophius budegassa and L. piscatorius by PCR-RFLP Analysis of a Mitochondrial tRNAGlu/Cytochrome b Segment
A. SANJUAN, J. RAPOSO-G UILLN AND A.S. COMESAA

Food Chemistry and Toxicology

ABSTRACT: Identification of Lophius budegassa (black-bellied angler) and L. piscatorius (angler) (Lophiiformes) was carried out on the amplification of a 486 bp tRNAGlu /cytochrome b segment using the polymerase chain reaction (PCR). Direct DNA sequencing of 6 PCR products was carried out. Six restriction endonucleases (AluI, CfoI, HaeIII, HinfI, MaeI, and ScrFI) with different species-specific restriction fragment length polymorphism (RFLP) were selected. Digestions of PCR products from 30 individuals showed no intraspecific polymorphism. Double digestions (CfoI and HinfI, and HaeIII and ScrFI) were simpler and more rapid than single digestions. This technique is suitable for distinguishing tails of both Lophius species. Keywords: anglerfishes, authentication, cytochrome b, Lophiiformes, PCR-RFLP

Introduction

HE IDENTIFICATION OF COMMERCIAL FISH SPECIES becomes a problem when the distinguishing external features of the fish are removed by processing into portions of flesh. The anglerfishes Lophius budegassa Spinola, 1807 (black-bellied angler) and L. piscatorius L. 1758 (angler) (Order Lophiiformes, Family Lophiidae) occur in the northeastern Atlantic (Caruso 1983), where they are important fishing resources with captures in 1999 of 876 and 53322 metric tons for L. budegassa and L. piscatorius, respectively (FAO 2001). Tails without external characteristics from these species are morphologically indistinguishable (Leiva Moreno 1998). Fresh, refrigerated, or frozen tails of L. piscatorius are sometimes labeled and marketed as L. budegassa, owing to the greater popularity and higher consumer demand for this latter species. Moreover, Storelli and Marcotrigiano (2001) have found that 23% of L. budegassa and 62.5% of L. piscatorius samples from the South Adriatic Sea showed mercury concentrations exceeding the tolerable peak value of 1 mg kg-1 wet wt set by the European Commission. The mercury concentration can be studied in tails of anglerfishes and identification of the species can be useful for comparative purposes. Identification of those products using nonmorphological methods is needed to avoid species substitution and to contribute for the estimate of the quality of these products from a hygienicsanitary standpoint. Several analytical methods have been developed for fish species authentication, including electrophoretic techniques, highperformance liquid chromatography, and immunoassays (Sotelo and others 1993; Andrews 1998; Mrtlbauer 1998; Etienne and others 2000, and references therein). Moreover, several of these approaches applied for different species of Lophius allow the identification of them (Lundstrom 1981; Crozier 1988; Grant and Leslie 1993). These methods rely upon the analysis of proteins, many of which are heat labile, lose their biological activity soon after death, and are subjet to modification in different cell types. Moreover, the above methods do not detect all the amino acid differences that may occur between different proteins (Bartlett and Davidson 1992; Davidson 1998; Mackie and others 1999; Wolf and others 2000). Advances in molecular biology techniques have enabled the devel-

opment of DNA-based methods for the identification of species origin in food products, as DNA is the same in every cell type of an individual, its information content is higher than that of proteins, and it is a remarkably stable molecule (Davidson 1998; Mackie and others 1999; Wolf and others 2000, and references therein). Most of these methods are based on amplification of a region of mitochondrial or nuclear DNA using the polymerase chain reaction (PCR) because of the simplicity, specificity, and sensitivity of this technique (Palumbi 1996). One of the PCR-based methodologies used has been the PCR direct sequence analysis, where the nucleotide sequence obtained of an amplified segment from a sample of an unknow species is compared with standard DNA sequences in a data base using a phylogenetic approach (Bartlett and Davidson 1991, 1992; Davidson 1998; Mackie and others 1999). Restriction Fragment Length Polymorphism (RFLP) of PCR products constitutes a simpler alternative than sequencing for the identification of species (Cspedes and others 1998, 2000; Davidson 1998; Mackie and others 1999; Cocolin and others 2000; Russell and others 2000; Wolf and others 2000; Sanjuan and Comesaa 2002, and references therein). The mitochondrial encoded cytochrome b (cyt b) gene has several advantages to be considered a useful genetical marker to identify the species origin of a fish sample: the high abundance of mitochondrial DNA in the total cellular nucleic acid preparations; the presence of only 1 allele, because of the maternal inheritance of mitochondria; the higher mutation rate compared to nuclear genes, which means considerable inter- and intra-species nucleotide variability but with the amount of variation within a species less than between species; and its pattern of nucleotide substitutions is known (for details see Carr and Marshall 1991; McVeigh and others 1991; Bartlett and Davidson 1992; Davidson 1998). Moreover, cyt b is among the most extensively sequenced genes to date across the vertebrates (Davidson 1998; Johns and Avise 1998), and in the authenticity methodologies one of the problems is the comparison of sample results with those of standards. These sequences can be found in international data banks, and can be used to compare sequences and to obtain restriction patterns. At present, a single sequence of a segment of the cyt b gene for Lophius species has
2002 Institute of Food Technologists

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Table 1Inferred restriction patterns from the DNA sequence of the tRNA Glu/cytochrome b segment for each Lophius budegassa and L. piscatorius using single and double digestions of six 4- and 5-bp cutting REs showing the expected length of the fragments. RE name Restriction site AGCT GCGC GGCC GANTC CTAG CCNGG

Lophius budegassa
5, 199, 30, 252 486 486 280, 206 159, 42, 285 414, 72 280, 206 414, 72

Lophius piscatorius
5, 229, 252 164, 322 309, 177 486 159, 42, 213, 72 486 164, 322 309, 177

been published (GenBank accession number AF165074; Wolf and others 2000), but, unfortunately, the identity of the studied species is not clear (Lophius sp.), and the DNA sequence has 16 nucleotide indeterminations. The main objective of this work was to develop a simple method for the identification of refrigerated and frozen tails from commercial Lophius budegassa (black-bellied angler) and L. piscatorius (angler) species using PCR/RFLP analysis of a 486-bp fragment in the mitochondrial tRNAGlu /cyt b region. This methodology can be applied to the detection of fraudulent or unintentional mislabeling of these species in the market of processed products and as a complement to estimate the quality of Lophius tails.

Cleanup, quantification, and sequencing of PCR products

Double-stranded DNA from PCR reactions with sufficient amounts of amplified DNA were cleaned using the Qiaquick PCR purification kit (Qiagen GmbH, Hilden, Germany), according to the manufacturers instructions. The DNA was eluted in 30 to 40 mL of the elution buffer provided with the kit. The concentration of the purified PCR product was estimated by agarose gel electrophoresis using a standard (Mass Ruler, BioRad Laboratories, Richmond, Calif., U.S.A.) as reference marker. Purified PCR products were directly sequenced in a Beckman CEQ2000 sequencer (Beckman Instruments, Bucks, U.K.) at the Facultade de Ciencias, Univ. de Vigo. DNA sequencing was accomplished using 4 ddNTPs dye terminators, dNTP mix, 10X sequencing reaction buffer and DNA polymerase enzyme provided in the CEQ Dye Terminator Cycle Sequencing Kit (Beckman Instruments, Bucks, U.K.), as well as 0.25 mM of one of the primers used in the initial PCR amplification. For each sequencing reaction, approximately 30 ng (about 100 fmoles) of purified PCR product were added. To substantiate correct sequentiation of PCR fragments, both strands of PCR products were separately sequenced using each of the primers used in the initial PCR amplification. These complementary sequences were then compared and combined to produce the complete sequence for at least 2 individuals of each Lophius species. Sequences were analyzed and prepared for publication with the help of the ESEE (The Eyeball Sequence Editor) computer program (Cabot and Beckenbach 1989). All different sequences are given as their nontemplate strand equivalents (Figure 1) and have been deposited in GenBank (accession numbers AY091553 to AY091555).

Materials and Methods

Samples and DNA extraction
Fifteen individuals of each Lophius budegassa Spinola, 1807 (black-bellied angler) and L. piscatorius L. 1758 (angler) (Order Lophiiformes) from Atlantic and Mediterranean Iberian waters were collected and analyzed. Whole individuals were obtained at different fish markets and different times from northwestern (Vigo) and southern Spain (Caleta de Vlez). Every specimen was morphologically identified following Caruso (1983) and Whitehead and others (1986). Total cellular DNA was extracted from muscle of each individual according to DeSalle and others (1993). Approximately 100 mg of muscle tissue was digested with 500 mL of extraction buffer (50 mM Tris, pH 8.0, 25 mM EDTA, 1% SDS) and 10 mL of 20 mg mL-1 proteinase K (Roche Diagnostic GmbH, Mannheim, Germany). The mixtures were incubated 16 to 18 h at 37 C. DNA was extracted once with 600 mL of phenol-chloroform-isoamylalcohol (25:24:1) and once with 550 mL of chloroform, and then precipitated 16 to 18 h with 1 mL of 95% ethanol at 20 C. The tubes were centrifuged at 6000 x g for 15 min, and, subsequently, the ethanol was discarded and the pellets were dried for 10 min in a vacuum concentrator system (SpeedVac plus; Savant Instruments, Farmingdale, N.Y., U.S.A.). The dried pellets were resuspended in 200 mL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

PCR amplification of the tRNAGlu/cytochrome b region

A 486-bp portion of the mitochondrial genome at the tRNAGlu / cyt b region was amplified by PCR. The following universal primers were employed: M14725, 5'-C GAA GCT TGA TAT GAA AAA CCA TCG TTG-3' (28 mer; Pbo 1990) (forward primer), and H15149, 5'A AAC TGC AGC CCC TCA GAA TGA TAT TTG TCC TCA-3' (34 mer; Kocher and others 1989) (reverse primer). The primers were synthesized by Amersham-Pharmacia-Biotech (Buckinghamshire, U.K.).

Restriction site analysis of PCR products

For the restriction site analysis of the tRNAGlu /cyt b region, DNA sequences were compared, including others previously reported. Several restriction enzymes (REs) were selected for species-specific restriction patterns using the DNASIS computer program (Hitachi Software Engineering Europe, Ardon, France).
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Food Chemistry and Toxicology

AluI CfoI HaeIII HinfI MaeI Scr FI CfoI + HinfI HaeIII + ScrFI

Double-stranded (symmetric) amplifications were carried out in a final volume of 50 mL containing 9 mM Tris HCl, pH 9.0, 2 mM MgCl2, 45 mM KCl, 0.09% Triton X-100; 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Amersham-Pharmacia-Biotech, Buckinghamshire, U.K.); 0.06 mM of each primer; 1.5 U of Taq DNA polymerase (Promega, Southampton, U.K.), and 3 mL of the above extracted genomic DNA as template. A negative control reaction, without extracted DNA, was also included for each set of PCR to test possible contamination. DNA amplification was performed in a Master Gradient thermal cycler (Eppendorf, Hamburg, Germany). An initial denaturation at 95 C for 5 min and, subsequently, 30 cycles were carried out with the following step-cycle profile: strand denaturation at 93 C for 60 s, primer annealing at 45 C for 60 s, and primer extension at 72 C for 120 s, with a final extension at 72 C for 10 min. The results of PCR amplifications were examined by agarose gel electrophoresis. The PCR products and a molecular weight marker (100 bp ladder, Amersham Pharmacia Biotech, Buckinghamshire, U.K.) were loaded in a 1.5% SeaKem LE agarose gel (FMC BioProducts, Rockland, Maine, U.S.A.), containing 1.2 mL of an ethidium bromide solution (10 mg mL-1), in TBE buffer (89 mM Tris base, 89 mM boric acid, 2.5 mM ethylenediaminetetraacetic acid, pH 7.5). Electrophoretic migration was carried out at 100 V for 45 min, and DNA fragments were visualized by UV transillumination and photographed with a Polaroid camera.

PCR-RFLP of Lophius species...

The purified PCR products were separately digested with 6 selected REs with 4-bp or 5-bp recognition sequences (Table 1): AluI, CfoI, HaeIII, HinfI, MaeI, and ScrFI (Roche Diagnostics GmbH, Mannheim, Germany). Satisfactory results were also obtained without previous purification of the amplified DNA fragments. Consequently, if sufficient amounts of amplified DNA were present, PCR products were digested directly with REs. A 10-mL aliquot of PCR product, with about 100 to 150 ng of the amplified DNA, was directly digested with 5 U of a RE in the incubation buffer provided with the RE for 16 to 18 h at 37 C. Moreover, double digestions using CfoI and HinfI, and ScrFI and HaeIII were carried out in a similar way, but using 4 U of each RE and the incubation buffer provided with the

Food Chemistry and Toxicology

Figure 1Aligned DNA sequences of the amplified tRNAGlu/cytochrome b region from 2 individuals of Lophius budegassa (CV03, from Caleta de Vlez, and V33, from Vigo) and 1 of Lophius piscatorius (V03, from Vigo), as well as the previously reported sequence of Lophius spp.(GenBank accession number, AF165074). In the 1st DNA sequence, the position of the primers M14725 and H15149 used for PCR amplification and sequencing is indicated in capital types. The DNA coding sequence of L. budegassa (CV03) for the cytochrome b gene is indicated in bold types and its protein sequence in capital letters at the bottom of the DNA sequences. A dot (.) indicates identity with the 1st DNA sequence. Restriction recognition sites for 6 REs are underlined. For the GenBank sequence of Lophius sp., a dash (-) indicates no data, k is g or t, m is a or c, n is any nucleotide (a, c, g, t), and y is c or t. 2646

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1st RE of each pair of REs, which was compatible for both REs (for example, double digestion with CfoI and HinfI was performed in the incubation buffer provided with CfoI, where CfoI and HinfI have 100% and 50 to 75% activity, respectively). The resulting fragments were separated by electrophoresis on a 3.0% SeaKem LE and Nusieve GTG agarose (2:1) gel (FMC BioProducts, Rockland, ME, USA), containing ethidium bromide in TBE buffer at 80 V for 1 h, or 1h 30 min, depending on the length of the fragments. Sometimes subsequently to the electrophoresis, gels were incubated in an ethidium bromide solution (1 mg mL-1) for 10 min. The restriction fragments were visualized by UV transillumination, photographed with a Polaroid camera, and sized by comparison with a 100-bp ladder using 1D Manager DNA analysis software (TDI, Alcobendas, Madrid, Spain). Only fragments larger than 70 bp were considered in the RFLP analysis as smaller fragments being beyond the detection limit of this technique. is a c and for L. piscatorius a t. Moreover, the differences and those indeterminations, which could be translated for amino acids, did not mean changes in the inferred amino acids with regard to L. piscatorius (data not shown). Consequently, the published DNA sequence of Lophius sp. probably corresponds to a L. piscatorius individual.

Identification of anglerfishes by RFLP

The restriction maps for REs with 4-bp or 5-bp recognition sites in the different DNA sequences were obtained using DNASIS computer program in search for REs that could distinguish and identify the PCR products of both Lophius species. Six REs were selected for screening to distinguish L. budegassa from L. piscatorius, and expected sizes for the restriction fragments inferred from the sequence analysis are shown in Table 1. Agarose electrophoresis following digestion of PCR products from 15 individuals of each L. budegassa and L. piscatorius showed band sizes in agreement with the expected sizes. No intraspecific restriction polymorphism was found for any RE. The REs AluI and MaeI cleaved the PCR products of both Lophius species (Table 1; Figure 2a), but the others cleaved the PCR products of a single species, either of L. budegassa (HinfI and ScrFI) or of L. piscatorius (CfoI and HaeIII) (Table 1). For these 4 latter REs, double digestions, as CfoI and HinfI, and HaeIII and ScrFI, could unambiguously identify both Lophius species (Table 1; Figure 2b), and at the same time guarantee a positive control of the enzymatic reactions. Consequently, each Lophius species could be identified with specific patterns for the 6 REs (Table 1 and Figure 2).

Results and Discussion

DNA sequence analysis
Fragments of 486 bp of the tRNAGlu /cyt b region of L. budegassa and L. piscatorius were consistently amplified by PCR using primers M14725 (Pbo 1990) and H15149 (Kocher and others 1989). PCR products of 4 individuals of L. budegassa (2 from Vigo and 2 from Caleta de Vlez) and 2 of L. piscatorius (from Vigo) were directly sequenced for both strands using the same primers. Alignment for different sequences showed 3 different haplotypes (Figure 1). Of the 4 analyzed sequences of L. budegassa, 1 (individual V33) showed a different nucleotide in the 177 position, which implied a different amino acid (amino acid 43, threonine instead of valine). The most common haplotype of L. budegassa and that of L. piscatorius showed 20 different nucleotides, 18 in the 3rd codon position (synonymous substitutions), and 2 in the 1st codon position coding different amino acids (missense substitutions) for each Lophius species (the amino acids 2 and 115 were threonine and valine for L. piscatorius instead of alanine and isoleucine). The GenBank data base was searched for cyt b sequences in Lophius species, and a sequence for Lophius sp. with 16 nucleotide indeterminations was available (accession number AF165074; Wolf and others 2000). Published sequence was aligned with sequences of this work (Figure 1), and of the 20 different nucleotides between L. budegassa and L. piscatorius, the published sequence of Lophius sp. showed 19 nucleotides as L. piscatorius and 1 indetermination as y (c or t) in the position 368 of the presente work, which for L. budegassa

Intraspecific restriction polymorphism

One of the problems of this technique is the existence of intraspecific genetic variability and, consequently, the loss or addition of restriction recognition sites within a species (Ram and others 1996; Mackie and others 1999; Sanjuan and Comesaa 2002). Intraspecific polymorphism was not found for 15 individuals of each Lophius species using the 6 studied enzymes, but the number of analyzed individuals was not very high. Alternatively, the use of genetic data base and population studies on DNA variation for each species may establish the degree of confidence for different REs. Results of PCR-RFLP analysis of a 464-bp fragment of cyt b (practically the same region of this work, but 14 bp and 8 bp short in the 5' and 3' end, respectively) for fish species, including 1 individual of Lophius sp., have recently been published (Wolf and others 2000). Restriction patterns were inferred from the published sequence (accession number AF165074; Wolf and others 2000), assuming that it belongs to a L. piscatorius individual (see above) and that the 39 nucleotides at the 5' end (including 28 pb of the primer sequence) and the 34 pb of the 3' end primer are the same as the present sequence (Figure 1). The inferred restriction pattern from this modified DNA sequence for AluI, showed fragments of 5, 159, 70, 183, and 69 bp, where the fragments of 159 and 70 bp appeared as a consequence of an indetermination (n) in the position 163 (Figure 1), given a putative recognition site for AluI (agct). If this recognition site is not considered, the inferred fragments would be of 5, 229, 183, and 69 bp, which are in accordance with data from their Table 2 (5- and 229-bp fragments are equivalents to the 220bp fragment from their Table 2 plus the 14-bp added fragment, and the 69-bp fragment to the 61-bp fragment from their Table 2 plus the 8-bp extra fragment). However, this restriction pattern is different from present results (Table 1), because of a different nucleotide (c) for the published sequence in the position 418, which means a recognition site for AluI (the 252 fragment of the present work corresponds to the sum of 183- and 69-bp fragments). In any case, this restriction pattern for this putative L. piscatorius is different from that of L. budegassa in the present work, and it must
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Figure 2Electrophoretic restriction patterns of the PCR products of the 486-bp tRNAGlu/cytochrome b region for 2 individuals of each L. budegassa (Lb) and L. piscatorius (Lp). a) Single digestions with AluI and MaeI. b) Double digestions using CfoI and HinfI, and HaeIII and ScrFI. U = undigested PCR product; L = 100-bp ladder.

Food Chemistry and Toxicology

PCR-RFLP of Lophius species...

be considered for authentication analyses. Published restriction pattern for HaeIII (Table 2 from Wolf and others 2000) was similar to present results: the published 295- and 169-bp fragments correspond to the 309- ( = 295 + 14) and 177-bp fragments ( = 169 + 8) of the present work, respectively. However, the inferred restriction pattern from the modified DNA sequence for HaeIII showed fragments of 309, 37, 6, and 134 bp, because of indeterminations in the positions 347 (m = a or c) and 354 (y = c or t) (Figure 1). This means, by comparison with results from their Table 2 (Wolf and others 2000), that the actual nucleotide for both positions was not c, which would give a recogniton site for HaeIII (ggcc; Table 1), and, consequently, the nucleotides for positions 347 and 354 were a and t, respectiveley, the same nucleotides as the present L. piscatorius sequence (Figure 1). Restriction patterns reported in their Table 2, or inferred from the published modified DNA, were concordant with our results for CfoI, HinfI, and ScrFI (Table 1, Figure 1), but MaeI showed a different inferred pattern because of an indetermination (y = c or t) in the position 102 (Figure 1). In this latter case, if the nucleotide were c, it would give a recognition site for MaeI (ctag; Table 1) and, consequently, a different restriction pattern with regard to the present results, but if it were t, it would be the same as the present L. piscatorius sequence (Table 1, Figure 1). Note that MaeI was not studied by Wolf and others (2000), and it was not possible to compare the present inferred restriction pattern with their results, as in the above case for HaeIII. Nucleotide indeterminations can be a problem for authentication and comparative purposes, and, consequently, DNA sequences must be published without indeterminations, and more than a single individual must be analyzed to obtain a high level of confidence. Of the 6 restriction enzymes studied (AluI, CfoI, HaeIII, HinfI, MaeI, and ScrFI), not one showed polymorphism for 15 analyzed individuals per species, and only 1 (AluI), or perhaps 2 (AluI and MaeI), showed different restriction patterns when compared with published results (Wolf and others 2000). In the last 2 cases, the different patterns did not affect the diagnosis for differentiation of L. budegassa and L. piscatorius. Consequently, for authentication purposes in inspection programs, 2 or more REs must be used to circumvent the problem of previously nondetected intraspecific genetic variability and to obtain an unambiguous identification. Nevertheless, because of the existence of previously nondetected variability, as shown above, studies of more individuals from different origins belonging to these Lophius species are needed to determine the genetic variability of each species in their entire distribution range, and, consequently, to increase the confidence level.
mens. BioTechniques 12:408-411. Cabot EL, Beckenbach AT. 1989. Simultaneous editing of multiple nucleic acid sequences with ESEE. Comput Appl Bio Sci 5:233-234. Caruso JH. 1983. The systematics and distribution of the Lophiid anglerfishes: II. Revisions of the genera Lophiomus and Lophius. Copeia 1983:11-30. Carr SM, Marshall HD. 1991. A direct approach to the measurement of genetic variation in fish populations: applications of the polymerase chain reaction to studies of Atlantic cod, Gadus morhua L. J Fish Biol 39:101-107. Cspedes A, Garca T, Carrera E, Gonzlez I, Sanz B, Hernndez PE, Martn R. 1998. Identification of flatfish species using polymerase chain reaction (PCR) and restriction analysis of the cytochrome b gene. J Food Sci 63:206-209. Cspedes A, Garca T, Carrera E, Gonzlez I, Fernndez A, Asensio L, Hernndez PE, Martn R. 2000. Genetic differentiation between sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) by PCR-RFLP analysis of a 12S rRNA gene fragment. J Sci Food Agric 80:29-32. Cocolin L, DAgaro E, Manzano M, Lanari D, Comi G. 2000. Rapid PCR-RFLP method for the identification of marine fish fillets (seabass, seabream, umbrine and dentex). J Food Sci 65:1315-1317. Crozier WW. 1988. Comparative electrophoretic examination of the 2 European species of angler fish (Lophiidae): Lophius piscatorius (L.) and Lophius budegassa (Spinola) and assessment of their genetic relationship. Comp Biochem Physiol B 90:95-98. Davidson WS. 1998. DNA/PCR techniques. In: Ashurst PR, Dennis MJ, editors. Analytical methods of food authentication. London: Blackie Academic and Professional, Thomson Science. p 182-203. DeSalle R, Williams AK, George M. 1993. Isolation and characterization of animal mitochondrial DNA. Methods Enzymol 224:176-234. Etienne M, Jrme M, Fleurence J, Rehbein H, Kndinger R, Mendes R, Costa H, Prez-Martn R, Pieiro-Gonzlez C, Craig A, Mackie I, Yman IM, Ferm M, Martnez I, Jessen F, Smelt A, Luten JB. 2000. Identification of fish species after cooking by SDS-PAGE and urea IEF: a collaborative study. J Agric Food Chem 48:2653-2658. FAO 2001. FAO yearbook. Fishery statistics. Capture production 1999. Vol 88/1. Rome: FAO. 752 p. Grant WS, Leslie RW. 1993. Biochemical divergence and biogeography of anglerfish of the genus Lophius (Lophiiformes). J Zool Lond 231:465-485. Johns GC, Avise JC. 1998. A comparative summary of genetic distances in the vertebrates from the mitochondrial cytochrome b gene. Mol Biol Evol 15:1481-1490. Kocher TD, Thomas WK, Meyer A, Edwards SV, Pbo S, Villablanca FX, Wilson AC. 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc Natl Acad Sci USA 86:61966200. Leiva Moreno J. 1998. Estudio diferencial de la cola de rape (Lophius piscatorius) y de tamboril (Sphoeroides cutaneus). Product Mar mayo-junio 1998:25-30. Lundstrom RC. 1981. Rapid fish species identification by agarose gel isoelectric focusing of sarcoplasmic proteins. J Assoc Off Anal Chem 64:38-43. Mackie IM, Pryde SE, Gonzales-Sotelo C, Medina I, Prez-Martn R, Quinteiro J, Rey-Mendez M, Rehbein H. 1999. Challenges in the identification of species of canned fish. Trends Food Sci Technol 10:9-14. Mrtlbauer E. 1998. Antibody techniques. In: Ashurst PR, Dennis MJ, editors. Analytical methods of food authentication. London: Blackie Academic and Professional, Thomson Science. p 241-268. McVeigh HP, Barlett SE, Davidson WS. 1991. Polymerase chain reaction/direct sequence analysis of the cytochrome b gene in Salmo salar. Aquaculture 95:225233. Pbo S. 1990. Amplifying ancient DNA. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols. A guide to methods and applications. San Diego, CA : Academic Press. p 159-166. Palumbi, SR. 1996. Nucleic acid II: The polymerase chain reaction. In: Hillis DM, Moritz C, Mable BK, editors. Molecular systematics. 2nd ed. Sunderland, MA: Sinauer Associates. p 205-247. Ram JL, Ram ML, Baidoun F. 1996. Authentication of canned tuna and bonito by sequence and restriction site analysis of polymerase chain reaction products of mitochondrial DNA. J Agric Food Chem 44:2460-2467. Russell VJ, Hold GL, Pryde SE, Rehbein H, Quinteiro J, Rey-Mendez M, Sotelo CG, Prez-Martn R, Santos AT, Rosa C. 2000. Use of restriction length polymorphism to distinguish between salmon species. J Agric Food Chem 48:21842188. Sanjuan A, Comesaa AS. 2002. Molecular identification of 9 commercial flatfish species by polymerase chain reaction-restriction fragment length polymorphism analysis of a segment of the cytochrome b region. J Food Prot 65:10161023. Sotelo CG, Pieiro C, Gallardo JM, Prez-Martn RI. 1993. Fish species identification in seafood products. Trends Food Sci Technol 4:395-401. Storelli MM, Marcotrigiano GO. 2001. Fish for human consumption: risk of contamination by mercury. Food Addit Contam 17:1007-1011. Whitehead PJP, Bauchot ML, Hureau JC, Nielsen J, Tortonese E. 1986. Fishes of the North-eastern Atlantic and the Mediterranean. Vol. 3. Paris: UNESCO. Clofnam. 1473 p. Wolf C, Burgener M, Hbner P, Lthy J. 2000. PCR-RFLP analysis of mitochondrial DNA: Differentiation of fish species. Lebensm Wiss uTechnol 33:144-150. MS 20020036 Submitted 1/18/02, Accepted 4/3/02, Received 7/1/02
This work was supported by an equipment grant for a DNA sequencer (Xunta de Galicia, Spain) and a contract (Centro de Investigacin y Control de la Calidad, Instituto Nacional de Consumo, Ministerio de Sanidad y Consumo, Spain).

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Conclusions
CR-RFLP OF A FRAGMENT OF 486 BP FOR THE tRNAGlu /cyt b region provided a powerful method to detect mislabeling or fraudulent substitution of Lophius budegassa and L. piscatorius. This method provides a simpler alternative to direct sequencing of PCR products or conventional mitochondrial DNA analysis for routinely studying large numbers of samples, as in inspection programs, for species identification. However, the new present DNA sequences of both Lophius species could also be used as standards for identification purposes using comparison of DNA sequences.

References
Andrews AT. 1998. Electrophoretic methods. In: Ashurst PR, Dennis MJ, editors. Analytical methods of food authentication. London: Blackie Academic and Professional, Thomson Science. p 204-239. Bartlett SE, Davidson WS. 1991. Identification of Thunnus tuna species by the polymerase chain reaction and direct sequence analysis of their mitochondrial cytochrome b genes. Can J Fish Aquat Sci 48:309-317. Bartlett SE, Davidson WS. 1992. FINS (Forensically Informative Nucleotide Sequencing): a procedure for identifying the animal origin of biological speci-

The authors are with Xentica Evolutiva Molecular, Facultade de CienciasBioloxa, Univ. de Vigo, E-36200 Vigo, Spain. Author Sanjuan is with Xentica Evolutiva Molecular, Facultade de Ciencias-Bioloxa, Univ. de Vigo, E-36200 Vigo, Spain; Direct inquiries to author Sanjuan (E-mail: asanjuan@uvigo.es).

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