Journal of Fish Biology (2001) 58, 1190–1194 doi:10.1006/jfbi.2000.1518, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION Genetic markers for identification of Patagonian toothfish and Antarctic toothfish P. J. S*¶, P. M. G† M. P‡ *National Institute of Water and Atmospheric Research Ltd, P.O. Box 14 901, Wellington, New Zealand; †College of Marine Studies, University of Delaware, Lewes, DE 19958, U.S.A. and ‡Sea Fisheries Research Institute, Private Bag X2, Rogge Bay 8012, Cape Town, South Africa (Received 14 July 2000, Accepted 20 November 2000) Fillet samples of the toothfish Dissostichus eleginoides and D. mawsoni can be distinguished readily by muscle proteins revealed by isoelectric focusing and mitochondrial DNA markers. The proteins also distinguish toothfish from other species marketed under similar trade names. Key words: Southern Ocean; toothfish; Dissostichus eleginoides; D. mawsoni; isoelectric focusing; mitochondrial DNA.
The toothfish fishery in the Southern Ocean has grown rapidly in recent years. Most of the catch is believed to be the northern Patagonian toothfish Dissostichus eleginoides Smitt, but as fishing activities move into more southern waters the Antarctic toothfish D. mawsoni Norman is being caught also. There is considerable unregulated fishing and it is estimated that the annual catch maybe c. 80 000 t, ten times the authorized total allowable catch (TAC) set by the Commission for the Conservation of Antarctic Marine Living Resources CCAMLR (Anon., 1998). The product is marketed under several different trade names, including common names applied to other species such as bass and hake (Anon., 1998). Identification of whole specimens of toothfish is relatively easy, based on keys (Fischer & Hureau, 1985). However, most toothfish are trunked at sea; the head, guts, and tail are removed and the product blast- or block-frozen. Further processing takes place on land and the product is sold as either trunks or fillets. Thus, a robust test is needed that will allow identification of trunked and filleted products that may have been frozen and thawed several times. Protein electrophoresis provides a method for discriminating closely related species. Allozymes have been used extensively to resolve taxonomic problems in fishes (Lacson & Bassler, 1992; Dayton et al., 1994) and have revealed cryptic species (Smith & Robertson, 1981; Lacson, 1994). Isoelectric focusing (IEF) of muscle proteins has been the preferred method for identification of fish fillets and products (Rehbein, 1990). This method is used for legal cases involving the identification of mis-labelled fish products and has been adopted by the U.S. Food and Drug Administration for identification of fish products (Tenge et al., 1993). IEF provides a finer separation of proteins than conventional starch and cellulose acetate electrophoresis, and typically the muscle proteins exhibit little intraspecific variation. In addition the muscle proteins are stable, withstanding repeat freezing and thawing, and provide a protein profile in one gel without the need to stain for many specific enzymes to identify a range of fillets. However, some closely related species, tuna (Bartlett & Davidson, 1991), tarakihi (Smith et al., 1996) share protein ¶Author to whom correspondence should be addressed. Tel.: +64 4 3860 300; fax: +64 4 3860 574; email:
[email protected] 1190 0022–1112/01/041190+05 $35.00/0
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T I. Common and scientific names of fish species used in the IEF analyses Common name Patagonian toothfish
Other common names
Hake Hake
Toothfish, mero, hake, Chilean sea-bass Toothfish, giant toothfish, Mawson codfish English hake Gemfish
Bass
Bass-groper
Hoki
Blue grenadier, whiptail hake
Antarctic toothfish
Scientific name Dissostichus eleginoides Smitt Dissostichus mawsoni Norman Merluccius australis Hutton Rexea solandri Cuvier and Valenciennes Polyprion americanus Bloch and Schneider Macruronus novaezelandiae Hector
profiles and thus IEF is not always suitable for distinguishing closely related species. DNA-based methods are used increasingly for identification of fish products (Bartlett & Davidson, 1992) and offer the advantage that the analysis may be conducted with minute or poorly preserved tissue samples. The present study was undertaken to provide a quick and robust molecular method to distinguish fillets of the two species of toothfish, from commercial vessels or the market place, and to allow the identification of mis-labelled toothfish products sold under different common or local names. Muscle tissue samples from 34 specimens of D. eleginoides and 50 specimens of D. mawsoni were collected and frozen in liquid nitrogen aboard a commercial vessel in the Ross Dependency. Whole specimens were identified by the use of keys (Fischer & Hureau, 1985). Muscle tissue samples from 50 specimens were collected from each of three fisheries by observers on commercial vessels, fishing legally for toothfish in the Southern Ocean around Heard Island, Macquarie Island and Prince Edward Island. Tissues were collected from 12 specimens from the South Atlantic by British Antarctic Survey staff onboard the ‘ FV Argos Galicia ’. Control samples from other marine fishes were collected aboard the ‘ RV Tangaroa ’ and ‘ RV Kaharoa ’ in the New Zealand Exclusive Economic Zone (Table I). All samples were stored at 30 C until tested. Toothfish fillets were purchased from a retail outlet on the east coast of the U.S.A. IEF followed the standard methods used at NIWA (Benson & Smith, 1989). About 0·5 g of white muscle was removed from each frozen sample and homogenized individually in two volumes of cold (4 C) deionized water and centrifuged at 12 000 g for 5 min at 4 C. The clear supernatants were pipetted onto filter paper wicks and placed directly onto agarose gels on a Pharmacia flat bed IEF system. The 1 mm 1% agarose gels were made up in wide range pharmalyte, pH 3–10, and focused at 1500 V for 90 min. After focusing the proteins were fixed, washed and stained with 0·2% Coomassie blue, destained and photographed. Muscle tissue from two specimens of D. eleginoides and two specimens of D. mawsoni were each sub-sampled four times and the sub-samples subject to (a) five repeat cycles of freezing and thawing, (b) ten repeat cycles of freezing and thawing, (c) 5 days at 4 C and (d) 10 days at 4 C. The samples from the two toothfish species produced different protein fingerprint patterns (Fig. 1), demonstrating that the IEF technique is an appropriate tool for distinguishing specimens of D. eliginoides and D. mawsoni. The IEF protein fingerprints of D. eleginoides and D. mawsoni were different from those from species with similar common trade names such as bass and hake (Fig. 1). No differences were found between samples of D. eleginoides from the Atlantic, Indian and Pacific Ocean sectors of the Southern Ocean (Fig. 1). No differences were found between frozen samples and sub-samples that had been subject to five and ten repeat cycles of thawing and freezing, and other sub-samples that had been held at 4 C for 5 and 10 days. Thus the IEF muscle protein markers are stable to the fish handling procedures used in the marketplace. Six fillets of Chilean sea-bass purchased from a retail outlet on the east coast of the U.S.A. were identified as
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F. 1. Photograph of an IEF plate showing the protein fingerprints of two species of toothfish and the other species. 1, Hake Rexea solandri; 2, hake Merluccius australis; 3, bass Polyprion moeone; 4, 5, D. mawsoni, 6–10, D. eleginoides from the Atlantic, Indian and Pacific Ocean sectors of the Southern Ocean.
D. eleginoides. Samples from commercial vessels showed that some of the catch labelled as D. eleginoides was D. mawsoni: of 50 specimens sampled around Prince Edward Island two were D. mawsoni, and of 50 specimens sampled around Heard Island one was D. mawsoni. DNA was extracted from 50–100 mg of muscle tissue, stored frozen or in 95% ethanol, using a standard phenol-chloroform method. For polymerase chain reaction (PCR) amplification, individual DNA extracts were diluted with TE buffer pH 8·0 to c. 50 ng l 1 after DNA concentrations had been quantified using a Hoefer DynaQuant fluorometer. Target regions from both mitochondrial and nuclear genes were amplified by PCR. A variety of universal primer sets taken from the literature or developed by one of the present authors (PMG) was tested; only those targets that amplified well in both species and yielded species-diagnostic restriction fragment length polymorphism (RFLPs) are reported here. Several mtDNA regions are useful for species identification. The 16S ribosomal RNA gene, exhibits species-specific digestion patterns that can be resolved in an agarose mini-gel. The universal primers 16SAR/16SBR (Palumbi et al., 1991) amplify a c. 630 bp product in the two species. Partial sequence data available from GenBank (Accessions AF145410 and Z32726 for D. eleginoides and D. mawsoni, respectively) suggested that several restriction enzymes (Hae III, Taq I, Tsp509 I) would provide species-specific fragment profiles. This proved to be the case (Fig. 2). No intraspecific polymorphism was observed in either species. Another target mtDNA region, NADH dehydrogenase subunit 2 (ND2), was amplified in both species using the forward primer t-Met of Park et al. (1993), coupled with a new primer targeted to the tRNATrp gene (Table II). Digestion of the PCR product from the two species yielded diagnostic RFLP patterns easily resolved on standard agarose gels. The D. mawsoni product was cut once, into fragments c. 700 and 450 bp. PCR products from D. eleginoides showed two distinctive RFLP patterns: type A (c. 900, c. 200 bp) and type B (c. 500, c. 400, c. 200 bp). Alu I digests were diagnostic also, but resolved less clearly on agarose gels. A portion of the mitochondrial control region was amplified using universal primers L16498 and 12SAR-H. The PCR product differed visibly in size on agarose gels: in D. mawsoni, the product was several hundred bp smaller than in D. eleginoides, which was
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F. 2. Restriction fragment profiles of the mitochondrial 16S region in D. eleginoides (De) and D. mawsoni (Dm). Lanes 1–3: Hae III digests, Dm from Ross Dependency, De from Argentina, De from Heard Island; lanes 4–6: Tsp509 I digests, Dm from Ross Dependency, De from Argentina, De from Heard Island); lanes 7–9: Taq I digests, Dm from Ross Dependency, De from Argentina, De from Heard Island); lane 10: 100 bp ladder (from bottom: 80, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp). T II. PCR primer sequences used to amplify toothfish nuclear and mitochondrial targets Primer 16SAR 16SBR t-Met Mt-76 GC-L16498 12SAR-H
Sequence
Reference
CGC CTG TTT ATC AAA AAC AT CCG GTC TGA ACT CAG ATC ACG T AAG CTA TCG GGC CCA TAC CC CCG CTT AGY GCT TTG AAG GC CGC CGC CGC CGC CGC CGC ATC TGG TTC CTA CTT CAG G ATA GTG GGG TAT CTA ATC CCA GTT
Palumbi et al., 1991 Palumbi et al., 1991 Park et al., 1993 P. M. Gaffney, unpubl. data P. M. Gaffney, unpubl. data Palumbi et al., 1991
typically c. 1·2 kb, with size variation and occasional apparent heteroplasmy. This product allows a rapid species diagnosis without restriction enzyme analysis. The distinct IEF patterns in D. eleginoides and D. mawsoni demonstrate that the IEF method is an appropriate tool for identifying fresh and frozen product from the fishery and fillets in the marketplace. The technique is relatively fast and one operator can process and identify more than 100 suspect samples per day. DNA methods also provide a ready means of identifying tissue samples. Their advantage is that they can be applied to minute pieces of poorly preserved tissue. One possible disadvantage is that species other than toothfish fortuitously may possess a RFLP pattern similar to one of the Dissostichus species, whereas IEF profiles tend to be species-specific fingerprints. However, it is very unlikely that any other species would possess Dissostichus-like RFLP profiles for several mitochondrial and nuclear target regions. In order to test for toothfish product in the market place the authors can make available control samples from the two Dissostichus species for researchers to test in their own laboratories; alternatively small frozen or ethanol fixed samples from suspect toothfish product can be sent to New Zealand for identification. This research was supported by a New Zealand Foundation for Research Science & Technology contract to PJS, and a European Union Grant to PMG. We thank M. White (U.K.) and R. Stanley (Australia) for supplying tissue samples from vessels fishing legally in the Southern Ocean. 2001 Government of New Zealand
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. . . References
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