Fish Species Substitution
Flatfish are a group of bottom-dwelling finfish that belong to the order Pleuronectiformes. Some common families within this order include flounders, halibuts, plaices, soles, and turbots. Flatfish are an important food fish: in 2004, U.S. commercial landings of flatfish were valued at U.S. $380 million (Voorhees 2008). Halibut ranked among the most valuable of the group, contributing a commercial landing value of U.S. $227 million for that year. Some commercial uses for flatfish include fresh/frozen fillets and processed products such as fish sticks and portions (Rodger 2006). When the external morphological features of the fish have been removed, species identification of similar flatfish can become difficult. These products are sometimes mistakenly or intentionally mislabeled (Sanjuan and Comesana 2002). In cases of intentional fish fraud, cheaper species are often substituted for more expensive, popular species. For this reason, numerous studies have been carried out to apply current DNA-based methods to species identification of flatfish (Table 1).
Table 1—. Genetic targets and methods used in flatfish authentication research.
|Sole, European plaice, and flounder||PCR-RFLP (3 enzymes)||mt cytochrome b||359 bp||Fresh/frozen||Cespedes and others 1998a|
|Sole, European plaice, flounder, and Greenland halibut||PCR-RFLP (4 enzymes)||mt cytochrome b||201 bp||Fresh/frozen||Cespedes and others 1998b|
|Sole and Greenland halibut||PCR with species-specific primers||Nuclear 5S rRNA||Species-specific||Frozen||Cespedes and others 1999|
|Sole and Greenland halibut||PCR-RFLP (2 enzymes)||mt 12S rRNA||321 bp||Frozen||Cespedes and others 2000|
|21 flatfish species||PCR-RFLP (3 enzymes)||mt cytochrome b||464 bp||Fresh/frozen||Sotelo and others 2001|
|9 flatfish species (halibut, 4-spotted scaldfish, megrim, flounder, European plaice, Greenland halibut, turbot, brill, and sole)||PCR-RFLP (5 enzymes)||mt tRNAGlu-cytochrome b||486 bp||Fresh/frozen||Sanjuan and Comesana 2002|
|5 flatfish species (megrim, turbot, sole, halibut, and flounder)||PCR-RFLP (2 to 3 enzymes)||mt 12S rRNA||433 bp||Fresh/frozen||Comesana and others 2003|
One example of potential fraud is the substitution of Greenland halibut fillets (aka Greenland turbot, Reinhardtius hippoglossoides) for other flatfish species with higher consumer demand, such as the common sole (Solea solea) (Cespedes and others 2000). PCR-RFLP analysis of a fragment of the mitochondrial cytochrome b (mt cyt b) gene of 4 species of flatfish allowed for the differentiation of common sole, European plaice (Pleuronectes platessa), and flounder (Platichthys flesus) (Cespedes and others 1998a). However, this method did not prove effective for identification of Greenland halibut. In subsequent research, differentiation of all 4 species was achieved by amplifying a different region of the mt cyt b gene using a specifically designed primer and then using 4 restriction enzymes for PCR-RFLP analysis (Cespedes and others 1998b). Additionally, differentiation of Greenland halibut from common sole has been reported to be successful with either PCR-RFLP (Cespedes and others 2000) or PCR-SSCP (Cespedes and others 1999).
By providing a number of methods for the DNA-based detection of mislabeled sole products, the above studies may be used by regulatory agencies to enforce the authentication of common sole. However, limitations arise when the products being analyzed contain additional flatfish species. To successfully differentiate numerous fish species simultaneously, a gene fragment must be selected that exhibits sufficient interspecies polymorphism and minimal intraspecies polymorphism. In this regard, Sotelo and others (2001) were able to differentiate 21 species of fresh/frozen flatfish by carrying out PCR-RFLP on a 464 bp fragment of the mt cyt b gene. Analysis of this gene fragment also allowed for the identification of a catfish species (Sutchi catfish, Pangasianodon hypophthalmus) that has been known to be substituted for certain types of flatfish sold at higher prices, such as sole or flounder (Rehbein 2008). A later study reported the identification of 9 species of flatfish using a 486 bp fragment of the mt transfer ribonucleic acid (tRNA)Glu-cyt b region (Sanjuan and Comesana 2002). However, a minimum of 5 restriction enzymes were required for PCR-RFLP analysis. Refrigerated and frozen fillets of 5 commercial flatfish were differentiated using PCR-RFLP analysis on a 433 bp region of the mt 12S rRNA gene (Comesana and others 2003). Although these studies have been successful in differentiating numerous flatfish species, the samples that were analyzed were fresh/frozen, but not cooked or further processed. Since DNA is known to degrade during processing, further work in this area may be directed toward differentiation based on smaller DNA fragments from common flatfish products, such as precooked fish sticks.
Hakes, pollock, and codfish
Hakes, pollock, and codfish belong to the order Gadiformes, which includes the taxonomic families Gadidae, Phycidae, Lotidae, and Merlucciidae (Bakke and Johansen 2005). Fish belonging to these 4 families are under the suborder Gadoidei, and are commonly known as gadoids. Many important commercial species can be found within the gadoids and a number of studies have been carried out to apply DNA-based methods to gadoid species identification (Table 2). For a review of gadoid species identification methods and their economic impact on the Czech Republic seafood market, see Hubalkova and others (2007).
Table 2—. Genetic targets and methods used in the authentication of hakes, pollocks, and codfish.
|11 Merluccius species||PCR-RFLP (4 enzymes) and sequencing||mt control region||197 bp||Frozen, ethanol-preserved, or thermally processed||Quinteiro and others 2001|
|Cod, haddock, and whiting||Multiplex real-time PCR (TaqMan)||ATPase 6 and 8||103 bp||Raw tissue and ethanol-preserved eggs||Taylor and others 2002|
|Gadoids (16 species)||PCR-RFLP (3 enzymes) and FINS||mt cytochrome b||464 bp||Fresh, frozen, and salted||Calo-Mata and others 2003|
|European hake, silver hake, and Argentinean hake||PCR and analysis of allele sizes and frequencies||Microsatellite loci: Mmer UEAW01, Mmer Hk3b||Allele sizes vary with species||Ethanol-preserved, frozen, and precooked (fish fingers)||Castillo and others 2003|
|9 Merlucciidae species (deep-water Cape hake, Senegalese hake, Southern hake, South Pacific hake, silver hake, Argentinean hake, Patagonian grenadier, European hake, and shallow-water Cape hake)||PCR amplification and PCR-RFLP (1 enzyme)||Nuclear 5S rRNA and mt cytochrome b||Species-specific and 354 to 404 bp (cyt b)||Ethanol-preserved muscle or gill tissue and commercial samples (fish fingers, frozen fillets, frozen slices, and pre-grilled hake)||Perez and Garcia-Vazquez 2004|
|South Atlantic hake species (Argentinean and Southern)||PCR-RFLP (1 enzyme)||mt cytochrome b||122 bp (PCR test) 464 bp (PCR-RFLP)||Ultra-frozen hake tails, precooked panned fillets, hake broth, and fish cakes||Perez and others 2004|
|9 Gadidae and Merlucciidae species (European hake, Atlantic cod, Alaska pollock, Pacific cod, Greenland cod, Argentine hake, Halobatrachus didactylus, Opsanus pardus, and shallow-water Cape hake)||PCR and sequence analysis||mt cytochrome b||359 bp||Frozen, smoked, cooked, salted, and dried||Pepe and others 2005|
|Alaska pollock, Pacific cod, and Atlantic cod||PCR-RFLP (2 enzymes)||mt cytochrome b||558 bp||Frozen||Aranishi and others 2005a|
|Alaska pollock roe and gray cod roe||PCR-RFLP (1 enzyme)||mt cytochrome b||558 bp||Spicy pollock or codfish roe products||Aranishi and others 2005b|
|10 white fish species (Atlantic cod, Pacific cod, saithe, haddock, European hake, deep-water Cape hake, European Plaice, whiting, Alaska pollock, and blue grenadier)||PCR-RFLP and lab-on-a-chip CE (3 enzymes)||mt cytochrome b||464 bp||Frozen, ethanol-preserved, and dried||Dooley and others 2005b|
|8 species (Pacific cod, Atlantic cod, Alaska pollock, Greenland cod, haddock, ling, saithe, and Brosme brosme)||PCR-RFLP (7 enzymes), PCR-SSCP, and DGGE||mt cytochrome b||<400 bp||Muscle tissue||Comi and others 2005|
|Haddock||Real-time PCR (Taqman)||Nuclear transferrin gene||n/a||Fresh||Hird and others 2005|
|Variety of species (Gadidae, Lotidae, and Merlucciidae families)||PCR with species-specific primers||Nuclear 5S rRNA||Species-specific||Fresh, frozen, and processed||Moran and Garcia-Vazquez 2006|
|9 cod fish species (Pacific cod, Alaska pollock, Saffron cod, Arctic cod, Southern blue whiting, Southern hake, South Pacific hake, longfin codling, and blue grenadier)||PCR-RFLP (4 enzymes)||mt tRNAGlu-cytochrome b||460 bp||Frozen, ethanol-preserved, and dried||Akasaki and others 2006b|
|7 species (Atlantic cod, Alaska pollock, blue whiting, Spanish ling, greater forkbeard, and 2 subspecies of poor cod)||PCR-RFLP (5 enzymes) and sequencing||mt 12S and 16S rRNA||430 bp (12S rRNA) 630 bp (16S rRNA)||Fresh and unprocessed||Di Finizio and others 2007|
|22 species (20 from the Gadidae, Lotidae, and Merlucciidae families; 2 from the Pomacentridae and Sparidae families)||PCR, sequencing and phylogenetic analysis||mt cytochrome b||359 bp||Uncooked fish muscle and 19 surimi products||Pepe and others 2007|
|6 gadoids (Alaska pollock, blue whiting, whiting, saithe, Atlantic cod, and hake species)||Species-specific monoplex and quadruplex PCR||pantophysin I||201 to 737 (species-specific)||Fresh/frozen fillets and compressed blocks of fish meat||Hubalkova and others 2008|
Hakes Hakes belong to the genus Merluccius, which has been reported to contain between 12 and 15 different hake species distributed throughout the Atlantic and Pacific Oceans (Castillo and others 2003; Froese and Pauly 2006). These fisheries experience a combined annual global harvest of approximately 2 million metric tons, with the majority of the landings taking place in countries such as Argentina, Uruguay, Namibia, and the United States (Castillo and others 2003). Europe holds high commercial value for certain species of hakes, which they import from Africa and the Americas in fresh, frozen, and processed (fish fingers) forms. Many of these fish are morphologically similar and often mistakenly captured within the same fishery, leading to species mislabeling (Perez and others 2004). Therefore, DNA-based identification of hakes has become an important area of research (Table 2).
Due to the variety of commercial products that are sold as Merluccius, many studies have focused on the detection of DNA fragments in processed hake samples. For example, Quinteiro and others (2001) demonstrated the ability to successfully identify species origin of hake samples from heat-sterilized baby food. The authors first carried out phylogenetic sequence analysis in 11 species of hake to uncover suitable restriction sites in a short fragment (197 bp) of the mt control region. They then performed PCR-RFLP analysis with 4 restriction enzymes on frozen or ethanol-preserved samples to develop unique restriction profiles for each species, which were used to identify the hake species in food samples. Despite the promising results of this study, others have suggested that there may be potential problems with reproducibility due to the high intraspecies polymorphism of the mt control region (Perez and others 2004). In a later study, a method using PCR and microsatellite loci was also reported to be successful in species identification of 3 different Atlantic hakes: European hake (M. merluccius), silver hake (M. bilinearis), and Argentinean hake (M. hubbsi) (Castillo and others 2003). Microsatellite loci with species-specific polymorphisms in size and frequency were identified in ethanol-preserved tissue samples. These loci were then used to correctly identify samples of processed hake (fish fingers and frozen hake fillets) and a number of “blind” samples of ethanol-preserved hakes.
Species diagnosis of processed hake samples has also been achieved with PCR-RFLP analysis on a fragment of the mt cyt b gene (Perez and others 2004). First, the authors amplified a 122 bp fragment of the gene to verify the presence of hake in processed food samples. In samples that contained hake, a 464 bp fragment was then amplified and analyzed with RFLP using only 1 restriction enzyme. This allowed for the identification of samples of 2 different hake species: Southern hake (M. australis) and Argentinean hake. PCR amplification of the nuclear 5S rRNA gene resulted in the ability to differentiate 7 species of Merlucciidae, including Southern hake, South Pacific hake (Merluccius gayi gayi), silver hake, Argentinean hake, and Patagonian grenadier (Macruronus magellanicus) (Perez and Garcia-Vazquez 2004). Two additional species, the European hake and shallow-water Cape hake (Merluccius capensis), showed the same DNA band length for the 5S rRNA gene; however, a further PCR-RFLP analysis on a region of the mt cyt b gene allowed for the identification of these species. These DNA markers were then used to analyze commercial samples of processed hake products, including fish fingers and precooked slices. Several of the products were found to be mislabeled: Argentinean hake was substituted with Patagonian grenadier, shallow-water Cape hake was substituted with deep-water Cape hake and a sample labeled as European hake could not be identified.
Additional studies that have established genetic methods to identify Merluccius species were focused on the ability to differentiate species from multiple Gadiformes families. Therefore, this study will be discussed in the mixed gadoids section.
Pollock and codfish Pollock and codfish are members of the Gadidae family, which has been reported to contain 22 to 24 species and is a significant contributor to the worldwide food supply (Froese and Pauly 2006; Rodger 2006). Some prominent members include Alaska or walleye pollock (Theragra chalcogramma), blue whiting (Micromesistius poutassou), Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), pollack (Pollachius pollachius), haddock (Melanogrammus aeglefinus), and whiting (Merlangius merlangus). For 2005, the Alaska pollock and blue whiting capture fisheries ranked as the 2nd and 5th largest in the world, respectively, with reported harvests of 2.8 and 2.1 million metric tons (Johnson 2007). The cod fisheries are also important food sources: U.S. landings of Pacific cod in 2005 were valued at U.S. $151 million and the Atlantic cod fishery ranked 12th worldwide, with a total harvest of 843000 tons (Johnson 2007). Food products from Gadidae fish species are sold commercially in many forms, including fresh/frozen fillets, frozen fillet blocks, surimi blocks, salt-cured or smoked, fish sticks, canned fish, and roe. Increases in the international trade of these processed seafood products have also increased the feasibility of fish species substitution, especially due to the similar appearance of many gadoids.
Alaska pollock are widespread throughout the Bering Sea in the Northern Pacific Ocean and are an important commercial fishery in countries such as the United States, Russia, and Japan. Surimi, which is often made of Alaska pollock, was the leading U.S. seafood export for 2006, worth U.S. $366 million (Johnson 2007). Pollock roe and fillets have also been among the top U.S. seafood exports, with values of U.S. $314 million and U.S. $234 million, respectively, for 2006. Spicy roe products from Alaska pollock are a highly popular seafood item in Asia. However, due to the similar appearance and increased availability of the cheaper codfish roe, these products are sometimes substituted for illegal economic gains. The mt cyt b gene fragment was found to be useful in PCR-RFLP-based differentiation of Alaska pollock from gray cod in spicy pollock/codfish roe products (Aranishi and others 2005b). Furthermore, an improvement to the extraction of DNA from cod and pollock caviar was reported, thereby reducing the time required for species identification (Aranishi and others 2006).
Codfish are often sold commercially for use in generic fish products, such as “fish ‘n’ chips” and frozen fish sticks. Although Atlantic cod is of greater value than Pacific cod, the Atlantic cod fishery has recently experienced a large collapse and is not as widely available as it has been previously (Rodger 2006). A recent paper focused on the ability to identify Alaska pollock, Atlantic cod, and Pacific cod based on polymorphisms in a 558 bp segment of the mt cyt b gene (Aranishi and others 2005a). The authors used universal gadoid primers for PCR amplification of mt cyt b and then carried out RFLP analysis to reveal species-specific restriction patterns. The method was reported to be rapid, simple, and reliable for the differentiation of these 3 species and to be applicable for the routine detection of fraudulent substitution in commercial products. Nevertheless, in a comparison of the ability of PCR-RFLP, PCR-SSCP, and denaturing gradient gel electrophoresis (DGGE) to identify codfish species based on mt cyt b, Comi and others (2005) reported that DGGE was the only method able to differentiate all 8 species being considered.
An alternative method using species-specific TaqMan probes has been reported to allow for the simultaneous detection of Atlantic cod, haddock, and whiting in raw and ethanol-preserved eggs (Taylor and others 2002). This multiplex, real-time PCR TaqMan assay utilized fluorescent probes that were designed based on polymorphisms in the genes coding for adenosine triphosphate (ATP) synthase subunit 6 (ATPase 6) and subunit 8 (ATPase 8) in the 3 species. The analysis could be carried out in a single tube due to the different fluorescent dyes associated with each probe. More than 98% accuracy of sample identification was reported and it was suggested that this method could prove to be a powerful tool for applications such as stock assessment and the detection of commercial fraud. A real-time PCR assay for the quantification of haddock in commercial products has also been developed (Hird and others 2005). This method, which is based on the transferrin gene, can quantify the amount of haddock to within 7% of gravimetric values in raw fish or lightly processed products. The method could be used to identify haddock species in heavily processed products; however, reliable quantification was not possible.
Although the above techniques may prove useful in fish fraud detection, they are limited in the number of species that can be differentiated at one time. For example, if a sample labeled as Atlantic cod is substituted with something other than Pacific cod or Alaska pollock, the analysis may not be adequate for fraud detection. For this reason, Calo-Mata and others (2003) attempted to characterize genetic differences between 16 different gadoid species, which included Atlantic cod, Pacific cod, Greenland cod (Gadus ogac), Alaska pollock, whiting, haddock, and ling (Molva molva). Using FINS analysis on a 464 bp fragment of the mt cyt b gene, the researchers were able to differentiate all species except Greenland cod and Pacific cod. Sequence information from these 16 species allowed for the development of a PCR-RFLP protocol to differentiate 15 of the gadoid species in a faster and less costly fashion than FINS. The PCR-RFLP method was also reported to be reliable for the correct identification of commercial samples of salted cod.
Fragments of the mitochondrial genes 12S rRNA (430 bp) and 16S rRNA (630 bp) were recently investigated as an alternative to using mt cyt b in PCR-RFLP Gadidae species identification (Di Finizio and others 2007). Following digestion with 2 restriction enzymes on the amplified 16S rRNA fragment, the authors were able to differentiate 7 gadoids, including Atlantic cod, Alaska pollock, blue whiting, Spanish ling (Molva macrophthalma), greater forkbeard (Phycis blennoides), and 2 subspecies of poor cod (Trisopterus minutus minutus and Trisopterus minutus capelanus). Overall, this method was found to be a powerful technique as compared to sequencing, and the 16S rRNA fragment was recommended for use in routine gadoid species identification.
Mixed gadoids In addition to developing techniques to distinguish species within a particular gadoid family, current research is also focused on differentiating gadoid species from multiple families. One study investigated the ability to differentiate species in 18 processed products (frozen, smoked, cooked, salted, or dried) containing fish from the families Gadidae and Merlucciidae (Pepe and others 2005). The authors obtained sequence information from GenBank on a 359 bp region of the mt cyt b gene in 7 different gadoid species, including European hake, Atlantic cod, Alaska pollock, Pacific cod, and Greenland cod. The same region was also sequenced from whole fish samples of Argentine hake, European hake, and shallow-water Cape hake. This sequence information was then used to identify species in the processed fish products. The authors found some intraspecies variance when comparing sequences of the products to the sequences listed in GenBank and suggested further genetic studies with the mt cyt b fragment in Merluccius family. All products were determined to contain fish from one of the 2 families, except in the case of 1 sample of smoked salt cod, which was simply reported to be outside of the Gadidae sequence cluster.
In a later study, the same group investigated genetic identification of species in surimi-based products (Pepe and others 2007). Similar to the above-mentioned study, whole fish samples of Argentine hake, European hake, shallow-water Cape hake, and Alaska pollock were obtained and a 359 bp region of the mt cyt b gene was sequenced. Sequence information on this DNA region was also obtained from GenBank for 18 other fish species belonging primarily to the families Merlucciidae, Gadidae, and Lotidae. Nineteen surimi-based products labeled to be Alaska pollock were then purchased and the mt cyt b gene fragment from each product was sequenced. Subsequent phylogenetic analysis showed that mislabeling of surimi products is a common occurrence, with only three of the samples actually containing Alaska pollock. Eleven samples were found to belong to the Merlucciidae family, 2 were from the Gadidae family, 2 were from the Pomacentridae family, and 1 was from the Sparidae family.
A variation of PCR-RFLP using lab-on-a-chip technology recently allowed for the differentiation of 10 white fish species (Dooley and others 2005b). The majority of the fish under study were of the families Gadidae (Atlantic and Pacific cod, haddock, whiting, Alaska pollock, and coley or saithe [Pollachius virens]) and Merluccius (European hake, blue grenadier [Macruronus novaezelandiae], and deep-water Cape hake [Merluccius paradoxus]), with 1 species from the order Pleuronectiformes (European plaice). By exploiting polymorphisms in a fragment of the mt cyt b gene, Dooley and colleagues were able to identify fish species at a level as low as 5% (w/w) in a fish admixture. Species identification was based on PCR-RFLP profiles stored in a database, thereby eliminating the need for reference materials to accompany each analysis. Freeze-dried samples of fish species were also distributed to 5 food control laboratories to assess the feasibility of this method for fraud detection. Species were correctly identified in all samples containing a single species, but only in 6 out of 9 admixture samples. The authors recommended this method for use in verifying the presence of a whitefish species given on a label and also for determining some whitefish species in mixed samples. However, species determination will be limited to fish for which authenticated profiles have already been established.
A genetic method was recently developed to allow for the differentiation of 9 gadoid species, including South Pacific hake, Arctic cod (Arctogadus glacialis), saffron cod (Eleginus gracilis), southern blue whiting (Micromesistius australis), longfin codling (Laemonema longipes), and blue grenadier, in imported dried cod fish products (Akasaki and others 2006b). In this study, a region of mtDNA containing fragments of the genes for tRNAGlu and cyt b was amplified by PCR and then analyzed with RFLP. Phylogenetic analysis on the mt cyt b gene sequence was carried out to give conclusive evidence to support the results of PCR-RFLP. Despite these promising advances in fish fraud research, effective identification of gadoid species in food products will require the need for examination of many individuals and species to reveal any intra- and/or interspecies variances that could potentially complicate results (Akasaki and others 2006b).
An alternative method for gadoid species identification was recently developed involving monoplex and quadruplex PCR systems (Hubalkova and others 2008). In this study, species-specific primers were developed to target partial sequences of the nuclear gene pantophysin I in 6 gadoids: Alaska pollock, blue whiting, whiting, saithe, Atlantic cod, and hake species. All 6 species could be differentiated using monoplex PCR systems that amplify species-specific fragments (201 to 737 bp); however, the system was not able to differentiate individual Merluccius species. A quadruplex PCR system was also developed, which allowed for the simultaneous identification of Alaska pollock, blue whiting, and hake species. When the monoplex and quadruplex methods were applied to species identification in commercial fillets and compressed fish meat blocks, numerous cases of mislabeling were found. For example, 46 products were declared to contain Alaska pollock, but 22 of these products were found to instead contain blue whiting or hake species, and 18 of the products contained a mixture of one of these 2 species with Alaska pollock. Overall, this method was reported to be more convenient, less costly, and less time-consuming than previous PCR-RFLP methods. Furthermore, the use of species-specific primers allowed for straightforward detection of multiple gadoid species in 1 product.
Salmon, trout, and char all belong to the Salmonidae family, under the order Salmoniformes, and are commonly referred to as salmonids. These fish are important worldwide in both wild-capture fisheries and the aquaculture industry and are sold in a variety of forms, including fresh fillets, cold- and hot-smoked salmon, and canned salmon. Wild-caught salmon has limited availability, but species such as Chinook (Oncorhynchus tshawytscha) and sockeye (Oncorhynchus nerka) are highly valued products in the global marketplace. The prices of the different salmon species within the wild-caught fisheries vary widely, from as high as U.S. $5.71/kg (ex-vessel, 2006) for Chinook salmon down to U.S. $0.29/kg (ex-vessel, 2006) for pink salmon (Oncorhynchus gorbuscha) (Johnson 2007). On the other hand, aquaculture provides a large supply of farmed salmonids, such as Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) that are available year-round at competitive prices. For example, in 2006 farmed Atlantic salmon was sold at an average ex-farm price of $3.92/kg (Johnson 2007). Given the wide variation in prices between salmon species, intentional species substitution may be tempting due to the potential for economic gains. The USFDA gives several examples of salmon product mislabeling on the seafood substitution website, such as the replacement of wild salmon with farmed salmon; the substitution of chum salmon (Oncorhynchus keta) with pink salmon; and the substitution of salmon with steelhead (rainbow) trout (USFDA 2006). Based on the potential for salmon substitution cases, DNA-based studies have investigated the differentiation of species in many fresh, frozen, and smoked salmon products (Table 3). However, to differentiate farm-raised and wild-caught salmon within a given species, alternative methods have been developed based on isotopic and fatty acid analyses (Thomas and others 2008).
Table 3—. Genetic targets and methods used in authentication research involving salmonids.
|Atlantic salmon, brown trout, and Atlantic salmon × brown trout hybrids||PCR||Nuclear 5S rRNA||Species-specific||Fresh, frozen, alcohol-preserved, and smoked; from scales, fins, bones, and tissue||Pendas and others 1995|
|Atlantic salmon and trout (brown, brook, and rainbow)||PCR-SSCP||mt cytochrome b||123 bp||Fillet and caviar||Rehbein and others 1997|
| || ||148 bp|| || |
| || ||358 bp|| || |
|8 salmonid species (Atlantic, sockeye, chum, pink, chinook, coho, rainbow trout, and coastal cutthroat trout)||PCR-RFLP (3 enzymes)||GH-2||1007 to 1273 bp||Fresh/frozen, ethanol-preserved, and smoked||McKay and others 1997|
|8 salmonid species (Atlantic, sockeye, chum, pink, chinook, coho, masu, and brown trout)||PCR-RFLP (3 enzymes)||MHC class II (coding + noncoding regions)||809 to 3000 bp, depending on species||Blood, liver, scales, and fins||Withler and others 1997|
|Rainbow trout and Atlantic salmon||PCR-RFLP (2 enzymes)||mt COSII||464 bp||Raw and smoked||Carrera and others 1999a|
|Atlantic salmon and rainbow trout||PCR-RFLP (2 enzymes)||mt 16S rRNA||950 bp||Raw and cold-smoked||Carrera and others 1999b|
|Atlantic salmon, rainbow trout, and Atlantic pomfret||PCR||Nuclear 5S rRNA||Species-specific||Smoked||Carrera and others 2000a|
|Atlantic salmon and rainbow trout||PCR-RFLP (3 enzymes)||Nuclear p53 gene||532 bp (salmon)|
518 bp (trout)
|Raw and smoked||Carrera and others 2000b|
|10 salmonids (Atlantic, chum, coho, pink, sockeye, chinook, rainbow trout, Arctic char, brook trout, and brown trout)||PCR-RFLP (6 enzymes)||mt cytochrome b||464 bp||Raw and boiled for 15 min; commercial samples (smoked, pickled, heat-treated)||Russell and others 2000Hold and others 2001a|
|Chinook, coho, steelhead, and coho/chinook hybrid salmon||Multiplex PCR and GH-2 sequences||Microsatellite isolocus (I-Ots-2) and GH-2||100 to 250 bp||Not specified||Greig and others 2002|
|Atlantic salmon, brown trout, keta salmon, and coho salmon||PCR-RFLP + HPLC (2 enzymes)||mt cytochrome b||463 to 464 bp||Reference samples and cold-smoked salmon||Horstkotte and Rehbein 2003|
|Atlantic salmon and rainbow trout||PCR-RFLP and lab-on-a-chip CE (4 enzymes)||mt cytochrome b||464 bp||Fresh/frozen||Dooley and others 2005a|
|10 salmonids (Atlantic, chum, coho, pink, sockeye, chinook, rainbow trout, Arctic char, brook trout, and brown trout)||PCR-SSCP||mt cytochrome b nuclear parvalbumin and nuclear growth hormone||300 to 460 bp||Raw, cold-smoked, and roe||Rehbein 2005|
|Rainbow trout and Atlantic salmon||PCR-AFLP (2 enzymes)||Species-specific SCAR marker||349 bp||Fresh, pickled, and smoked||Zhang and Cai 2006|
Atlantic salmon and rainbow trout A major focus of genetic species identification research among the salmonids has been differentiating Atlantic salmon and rainbow trout. These 2 species are both very important in aquaculture, ranking among the top 20 world aquaculture species (by weight) in 2005 (Johnson 2007). Atlantic salmon and rainbow trout are often used in smoked products, where the morphological identifiers are removed and the species is very difficult to distinguish (Carrera and others 2000b). Although similar in appearance, rainbow trout is less expensive and has been reported to be illegally substituted for Atlantic salmon in smoked products, particularly in Europe and China (Carrera and others 2000b; Zhang and Cai 2006).
PCR-RFLP has been reported to be an effective method for differentiation of Atlantic salmon and rainbow trout in both raw and smoked products. Successful analyses have taken place using fragments of the following genes: mt cytochrome c oxidase subunit II (COII) (464 bp) (Carrera and others 1999a); mt 16S rRNA (950 bp) (Carrera and others 1999b); and nuclear p53 (518-532 bp) (Carrera and others 2000b). However, Carrera and others (1999b) reported using cold-smoked samples, in which the maximum preparation temperature is 30 °C, and the other 2 studies did not specify whether samples were cold- or hot-smoked. The larger fragments analyzed might be degraded by further processing, such as hot-smoking or canning. A more recent study into the differentiation of these 2 species also utilized the PCR-RFLP methodology, but with the addition of lab-on-a-chip technology (Dooley and others 2005a). In this case, a 464 bp fragment of the mt cyt b gene was isolated from fresh/frozen samples of Atlantic salmon and rainbow trout. The analysis was reported to be more rapid and sensitive than with traditional gel electrophoresis, allowing for the detection of fragments as small as 25 bp. When DNA admixtures of the 2 species were analyzed, Atlantic salmon could be detected with as little as 5% salmon DNA; however, rainbow trout could not be detected when the admixture contained 25% or less of trout DNA. To verify the suitability of this method in detecting fraud, processed samples should also be tested.
Other methods used to differentiate Atlantic salmon from trout include PCR-SSCP and PCR amplification of species-specific DNA fragments. PCR-SSCP on small fragments (123 to 358 bp) of the mt cyt b gene resulted in the production of species-specific patterns of single-stranded DNA that could be utilized to identify samples of Atlantic salmon, rainbow trout, brook trout (Salvelinus fontinalis), and brown trout (Salmo trutta fario) (Rehbein and others 1997). On the other hand, PCR amplification of the nuclear 5S ribosomal ribonucleic acid (rRNA) coding region combined with the nontranscribed spacer region (NTS) is an effective method because the length of the noncoding region of this fragment varies with species (Rasmussen and Morrissey 2008). This method was used in one study to differentiate Atlantic salmon, brown trout, and hybrids of the 2 species (Pendas and others 1995). The study used fresh, frozen, and smoked samples, and was able to isolate DNA from a variety of tissue types, including fins, scales, bones, and tissue. This same method was also used to differentiate cold-smoked samples of Atlantic salmon, rainbow trout, and Atlantic pomfret (Brama brama) (Carrera and others 2000a). Species-specific PCR amplification was reported to be simpler and quicker than PCR-RFLP because it eliminates the need for a restriction digest.
An innovative approach for differentiating Atlantic salmon and rainbow trout is the use of PCR-AFLP to develop a species-specific sequence characterized amplified region (SCAR) marker (Zhang and Cai 2006). The method was carried out on a 349 bp DNA fragment and allowed for the identification of fresh, pickled, and smoked samples of Atlantic salmon and rainbow trout. Additionally, the method was reported to be more sensitive than that used by Dooley and others (2005a) for the detection of rainbow trout in DNA admixtures of the 2 species: rainbow trout could be detected when the admixture contained as little as 1% trout DNA for fresh samples and 10% trout DNA for pickled and smoked samples. However, it would not be possible to detect salmon DNA using this method because the SCAR marker only appears in the presence of rainbow trout. The authors concluded that the method was a rapid and reliable procedure for identifying fraud in salmon products. Nevertheless, to identify fish species besides rainbow trout, additional species-specific SCAR markers would need to be developed.
Multiple salmonids While the above-mentioned studies were focused on differentiating Atlantic salmon and trout, other genetic identification research has been focused on developing methods to differentiate a wider range of salmonid species. In addition to preventing economic fraud, genetic diagnosis of salmon species could help with conservation management efforts. For example, some salmon stocks have been listed as endangered, requiring separately regulated fisheries for different species in countries such as Canada and the United States (Withler and others 2004).
In one study, 8 species of salmonids were identified using PCR-RFLP analysis on a 1007 to 1273 bp portion of the growth hormone type 2 (GH-2) gene (McKay and others 1997). In addition to fresh samples, frozen and smoked samples were also analyzed. The samples included both salmon (Atlantic, sockeye, chum, pink, chinook, coho [Oncorhynchus kisutch]) and trout (rainbow and coastal cutthroat [Oncorhynchus clarkii clarkii]), and were all collected in British Columbia, Canada. Another study investigated the differentiation of 10 salmonid species with PCR-RFLP on an 809 to 3000 bp fragment of the major histocompatibility complex (MHC) class II gene (coding + noncoding regions) (Withler and others 1997). Samples were collected from Canada, the United States, and Japan, and DNA was collected from the blood, liver, scales, or fins. Unique restriction profiles were generated for eight of the species (listed in Table 3), but not for rainbow and coastal cutthroat trout.
In a later study, PCR-RFLP on a 464 bp segment of the mt cyt b gene allowed for the differentiation of 10 salmonid species (Russell and others 2000). The samples were analyzed raw and after being cooked in boiling water for 15 min. A follow-up study was carried out to test the practicality and reproducibility of this method with 5 different European laboratories (Hold and others 2001a). The labs were provided references to authentic species along with 2 unknown samples containing either a single species or a mixture of 2 species. The restriction profiles generated for the authentic species were consistent among all laboratories and all unknowns were correctly identified. Hold and others (2001a) also assessed the practical use of this method by analyzing 70 commercial salmon samples, including smoked, pickled, and heat-treated products. All samples were purchased in the United Kingdom. The analysis confirmed the product label species claims in all but one case, in which chum salmon was substituted for pink salmon. In the products that listed unspecified “salmon” on the label, the analysis was able to report the actual salmon species in all but one case, in which species was not determined. According to Rehbein (2005), this method is now part of the official German method for fish species identification by PCR.
PCR-SSCP has also been investigated as a potential method for species differentiation among the 10 salmonids analyzed by Russell and others (2000). A combination of 3 gene targets (mt cyt b, parvalbumin, and growth hormone) ranging in size from 300 to 460 bp was used to acquire species-specific patterns of single-stranded DNA in native polyacrylamide gels (Rehbein 2005). While parvalbumin and growth hormone were recommended for use with salmon muscle tissue, mt cyt b was more suitable for analysis of salmon roe due to the relatively high levels of mtDNA in fish eggs. Although species identification was possible in raw and lightly processed salmon products, this method was not successful with canned salmon.
To reduce the time and labor involved in species identification of salmonids, Greig and others (2002) developed 2 diagnostic tests for the differentiation of chinook, coho and steelhead that do not require restriction enzyme digests. The 1st test is based on species-specific differences in the allele profiles of a microsatellite isolocus (I-Ots-2), while the 2nd test capitalizes on species-specific differences in sequence length of a portion of the GH-2 gene. Multiplex PCR was used to amplify the DNA fragments, followed by amplicon detection with a laser scanner. The results of the study showed successful diagnosis of the 3 species of salmonids, along with the detection of coho/chinook hybrids. The authors reported that these 2 tests could be used in combination to confirm the identification of fish in special cases, such as with suspected hybrids or fish species thought to be absent from watersheds. However, they may also prove to be advantageous in species diagnosis of commercial salmon products.
Horstkotte and Rehbein (2003) reported the use of RFLP and high-performance liquid chromatography (HPLC) to differentiate Atlantic salmon, brown trout, chum salmon, and coho salmon. PCR amplification was carried out on a 462 to 463 bp fragment of the mt cyt b gene in each species, and the amplicons were digested with 2 restriction enzymes. The resulting digestion fragments were then separated by HPLC instead of the traditional gel electrophoresis method. Commercial products containing cold-smoked salmon were successfully identified using this method. Five of the samples were found to be Atlantic salmon, while the other 2 were identified as coho salmon. These findings were confirmed by the method described in Russell and others (2000). Compared to gel electrophoresis, HPLC was reported to have a shorter analysis time (with a small number of samples), lower detection limits, and an extended linear working range. However, the resolution levels obtained with HPLC were inferior and this method may also require additional time and costs for some aspects of the analysis.
According to Withler and others (2004), DNA-based analysis of salmonid species in British Columbia, Canada, has resulted in several legal convictions for fish fraud, specifically with regard to illegal harvests of endangered species. Use of DNA analysis has been considered to be a cost saving measure, due to the number of guilty pleas made by defendants after becoming aware of the results of genetic testing. It was also helpful in exonerating 2 individuals suspected of illegal sales of endangered salmon. Most species were identified with the method described in Withler and others (1997); however, rainbow and cutthroat trout were differentiated using the method of McKay and others (1997) and Atlantic salmon and brown trout were identified using the method described in Pendas and others (1995). To determine the source populations of salmonids under investigation, genetic stock identification (GSI) methods were carried out based on allele frequencies of microsatellite loci and the MHC gene. Withler and others (2004) reported that, although these methods have generally been in agreement with fishery officers in terms of species and stock identification, there have been some erroneous results in samples from other sources, such as restaurants and fish plants.
Scombroids belong to the suborder Scombroidei (order Perciformes), including the families Scombridae (mackerels, tunas, and bonitos), Trichiuridae (cutlassfish, hairtails, and ribbonfish), Istiophoridae (billfish), and Xiphiidae (swordfish). The majority of genetic identification research with scombroids has been carried out with those in the family Scombridae; however, several studies have also been carried out with fish in the Trichiuridae, Istiophoridae, and Xiphiidae families (Table 4).
Table 4—. Genetic targets and methods used in authentication research involving scombroids.
|11 scombroid species (2 were indistinguishable)||PCR and sequence analysis||mt cytochrome b||123 bp (canned)|
464 bp (frozen)
|Frozen and canned||Unseld and others 1995|
|4 scombroid species (yellowfin, albacore, skipjack, and bonito)||PCR-RFLP (4 enzymes)||mt cytochrome b||<123 bp||Canned and fresh||Ram and others 1996|
|6 scombroid species (albacore, yellowfin, Atlantic bonito, skipjack, bluefin, and bigeye)||PCR-RFLP (3 enzymes)||mt cytochrome b||126 bp||Canned and frozen||Quinteiro and others 1998|
|8 scombroid species (albacore, yellowfin, Atlantic bonito, skipjack, bluefin, bigeye, A. thazard, and little tunny)||PCR-SSCP||mt cytochrome b||123 bp||Canned||Rehbein and others 1999b|
|Altantic bonito and bluefin tuna||Multiplex PCR||mt cytochrome b||207 bp (bluefin)|
225 bp (bonito)
|Fresh/frozen muscle tissue||Lockley and Bardsley 2000|
|Atlantic bluefin and Pacific bluefin||Magnetic Capture Hybridization||mt ATCO||150 bp|
|Frozen and ethanol-preserved muscle tissue||Takeyama and others 2000|
|11 scombroid species||PCR-RFLP (4 enzymes)||mt ATCO||927 bp||Frozen and ethanol-preserved||Takeyama and others 2001|
|Pacific bluefin tuna and southern bluefin tuna||PCR-RFLP (2 enzymes)||mt ATCO||927 bp||Frozen muscle tissue||Smith and others 2001|
|3 scombroid species (albacore, yellowfin, and Atlantic bluefin tuna) and 2 Morone species (striped bass and white bass)||PCR-AFLP||Numerous DNA fragments (160 to 208 bands)||200 to 1000 bp||Blood, fin, clips, and tissue||Han and Ely 2002|
|13 scombroid species||PCR-RFLP (4 to 5 enzymes)||mt cytochrome b||680 bp||Not specified (implies the use of fresh/frozen tissue)||Chow and others 2003|
| ||mt ATCO||927 bp|| || |
|Yellowfin, bigeye, and albacore tuna||PCR-RFLP + HPLC (2 enzymes)||mt cytochrome b||179 bp||Reference samples and canned tuna chunks||Horstkotte and Rehbein 2003|
|Swordfish||Species-specific PCR||mt control region||87 to 357 bp||Fresh, frozen, cooked, sterilized, and dried dressed fried meat||Hsieh and others 2004|
|Albacore, yellowfin, bluefin, bigeye, and skipjack||Nested primer PCR-RFLP (5 enzymes)||mt cytochrome b||276 bp||Frozen and canned reference samples, commercial tuna cans||Pardo and Perez-Villareal 2004|
|Bluefin, albacore, yellowfin, and bigeye||PCR-RFLP (3 enzymes)||mt cytochrome b||376 bp||Uncooked reference samples and commercial tuna fillets (sashimi)||Lin and others 2005|
|Mackerel (chub and Atlantic)||PCR-RFLP (2 enzymes)||Nuclear 5S rRNA||Species-specific||Frozen||Aranishi 2005|
|Albacore tuna and yellowfin tuna||Real-time PCR||mt 16S rRNA||≤100 bp||Fresh, ethanol-preserved, and canned||Lopez and Pardo 2005|
|Trichiurus sp. 2, T. lepturus, and T. japonicus||PCR-RFLP (2 enzymes)||mt 16S rRNA||600 bp||Not specified (implies the use of fresh/frozen fillets)||Chakraborty and others 2005|
|T. lepturus and T. japonicus||PCR-RFLP (1 enzyme)||mt 16S rRNA||600 bp||Fresh, frozen, and ethanol-preserved||Chakraborty and others 2006|
|Trichiurus sp. 2 and T. japonicus||PCR-RFLP (1 enzyme)||mt 16S rRNA||600 bp||64 fillets purchased from supermarkets in Japan||Chakraborty and others 2007|
|Billfish species (swordfish, blue marlin, black marlin, Indo-Pacific sailfish, and striped marlin)||PCR-RFLP (2 enzymes)||mt cytochrome b||348 bp||Fresh/frozen||Hsieh and others 2005|
| || || ||Raw, cooked, canned, and commercial products (raw fillets, frozen meat, and fried meat)||Hsieh and others 2007|
|S. colias||Multiplex PCR||NTS (species-specific)|
5S rRNA (genus-specific)
|159 bp (S. colias)|
196 to 201 (Scomber genus)
|Specimen samples and canned products||Infante and Manchado 2006|
|Skipjack tuna (unsuccessful with albacore, bluefin, and yellowfin)||Species-specific PCR||NADH2||230 bp||Fresh/frozen reference samples||Dalmasso and others 2006|
|6 scombroids (Atlantic bluefin, Pacific bluefin, southern bluefin, yellowfin, bigeye, and albacore tuna)||Direct Force Measurements||mt ATCO||150 bp|
|Frozen muscle tissue||Tanaka and others 2006|
|8 scombroids (bluefin, albacore, bigeye, yellowfin, skipjack, A. thazard, E. affinis, and striped bonito)||PCR-RFLP (5 enzymes)||mt cytochrome b||126 bp|
|Cooked reference samples and commercially canned products||Lin and Hwang 2007|
|4 scombroids (albacore, yellowfin, bluefin, and bigeye)||Real-time PCR||ATPase 6||208 bp||Fresh and canned||Dalmasso and others 2007|
|3 scombroids (yellowfin, bigeye, and skipjack tuna)||Multiplex PCR||mt cyt b||113 to 262 bp||Fresh, cooked, and canned||Michelini and others 2007|
|5 scombroids (albacore, yellowfin, bluefin, bigeye, and skipjack tuna)||Multiplex primer-extension assay||mt cyt b||132 bp||Fresh and canned||Bottero and others 2007|
|5 scombroids (skipjack, A. thazard, A. rochei, E. affinis, and striped bonito)||Species-specific monoplex PCR and multiplex PCR||mt cytochrome b||110 to 156 bp (monoplex)|
143 to 506 bp (multiplex; fragments >320 bp could not be amplified in cooked products)
|Raw and as commercial dried bonito product (monoplex)|
Raw and cooked fillets (multiplex)
|Lin and Hwang 2008a, 2008b|
Mackerels, tunas, and bonitos The Scombridae family consists of 51 species of mackerels, tunas, and bonitos (Nelson 1994). These fish are distributed worldwide in tropical and subtropical seas and vary greatly in size and price. For example, bluefin tuna (genus Thunnus) is highly regarded in Japan and is consumed raw in sushi and sashimi products(Mackie and others 1999; Rodger 2006). In contrast, skipjack tuna (Katsuwonus pelamis, formerly known as Euthynnus pelamis) is much smaller and cheaper and is often sold as a canned product. Although there are regulations on labeling canned tuna and bonito in many countries, enforcement has traditionally been complicated because there was no reliable means of species recognition (Mackie and others 1999). Genetic identification methods are currently being extensively investigated among scombroids for the purpose of both preventing economic fraud and detecting illegal fishing and trading of protected stocks (Takeyama and others 2001). Extensive studies have been carried out to identify species in canned and other heavily processed samples, in which the DNA is severely degraded. Since these studies require the use of smaller gene fragments, the Scombridae section has been divided into 2 subsections: (1) heavily processed products and (2) lightly processed products.
Heavily processed products The severe thermal treatment that occurs during canning degrades tuna DNA to fragments ranging in size from <100 to 360 bp (compared with ≤20000 bp in frozen samples) (Quinteiro and others 1998; Pardo and Perez-Villareal 2004; Chapela and others 2007; Hsieh and others 2007). Taking degradation factors into account, Unseld and others (1995) investigated species identification based on fragments of the mt cyt b gene with lengths of 123 bp (59 bp without primers) and a 464 bp (402 bp without primers). After PCR amplification of DNA from canned tuna samples, the authors were able to detect the 123 bp fragments, but not the 464 bp fragments. Sequencing of the PCR amplicon allowed for the differentiation of 10 out of 11 scombroid species analyzed, including bigeye (Thunnus obesus), southern bluefin tuna (Thunnus maccoyii), and skipjack. Yellowfin tuna (Thunnus albacares) and northern bluefin (Thunnus thynnus) were indistinguishable, and intraspecies polymorphism was detected among samples of Auxis thazard.
Because sequencing is relatively time-consuming and expensive, subsequent studies into scombroid species differentiation focused on the use of PCR-RFLP and PCR-SSCP methods. For example, Ram and others (1996) investigated the use of PCR-RFLP analysis on a <123 bp fragment of the mt cyt b gene to identify raw and canned samples of 5 scombroid species: albacore (Thunnus alalunga), yellowfin, skipjack, Euthynnus affinis, and A. thazard. All scombroids could be differentiated except A. thazard. The small size of the fragment analyzed allowed for successful analysis among most of the heat-sterilized samples. One exception was a sample of canned tuna in water, in which no visible PCR product could be obtained. The authors suggested that more research into intraspecies polymorphism should be carried out before this method is used to authenticate species in commercial products. In a similar study, PCR-RFLP on a 126 bp fragment of the mt cyt b gene allowed for successful differentiation of both frozen and canned samples of albacore, yellowfin, Atlantic bonito (Sarda sarda), skipjack, northern bluefin, and bigeye (Quinteiro and others 1998). This research group later published a study reporting successful differentiation of 8 scombroid species (listed in Table 4), using PCR-SSCP on a 123 bp fragment of the mt cyt b gene (Rehbein and others 1999b). The repeatability of the PCR-SSCP method was tested among 8 European laboratories and in 65 out of 72 cases (90%), the species was correctly identified. However, intraspecies polymorphism was reported for skipjack and Atlantic bonito.
PCR-RFLP combined with HPLC was applied to a 179 bp fragment of the mt cyt b gene in scombroids (Horstkotte and Rehbein 2003). Following a restriction digest with 2 enzymes and HPLC analysis, the reference samples of bigeye tuna and yellowfin tuna were successfully differentiated. Four commercially canned tuna samples were then analyzed with this method. Based on the reference samples and previous PCR-RFLP studies, the canned samples were determined to contain albacore (n = 1), yellowfin (n = 1), and an unknown species (n = 2). The results were confirmed by RFLP and SSCP procedures previously published by the same group (Rehbein and others 1998, 1999b). The use of additional reference samples may allow for identification of the 2 unknown canned tuna samples.
Another study utilized a slightly larger fragment (276 bp) of the mt cyt b gene fragment to differentiate albacore, yellowfin, bluefin, bigeye, and skipjack (Pardo and Perez-Villareal 2004). This study made use of nested primer PCR-RFLP analysis, which requires 2 consecutive PCR assays prior to the restriction digest. The authors tested the method with canned tuna samples that they had prepared and commercially canned samples. After the 1st PCR, DNA from the laboratory-canned tuna could be visualized on an agarose gel; however, DNA from the commercially canned samples, containing tuna that had been smoked or soaked in sauces and spices, was not visible. After a 2nd PCR, the 276 bp fragment was successfully amplified in all samples and a restriction digest with 5 enzymes allowed for the differentiation of the 5 Thunnus species. The authors reported this to be the longest gene fragment obtained from commercially canned tuna. The use of longer gene fragments is advantageous due to the increased likelihood of finding diagnostic sites for identification of other species.
Lin and Hwang (2007) also reported the ability to differentiate Thunnus species in both laboratory-cooked (simulated canning) and commercially canned tuna. PCR-RFLP analysis on 2 fragments of the mt cyt b gene, along with 5 restriction enzymes allowed for the identification of samples of bluefin, albacore, yellowfin, skipjack, bigeye, E. affinis, A. thazard, and striped bonito (Sarda orientalis) in laboratory samples. A 376 bp fragment could not be amplified in the canned tuna, so 2 smaller fragments were used. A 126 bp fragment was sufficient to differentiate all species except bigeye and yellowfin, in which case a slightly longer, 146 bp fragment, had to be utilized. To enhance DNA extraction from canned tuna, the authors employed a novel technique that involves DNA-binding magnetic beads. With this technique, species detection was possible in 18 commercially canned samples that had been processed in a variety of sauces. However, the 146 bp fragment could not be amplified in four of the samples (skipjack and E. affinis) and a 2nd PCR had to be carried out on the 126 bp fragment.
Recently, there has been a trend toward development of real-time and multiplex PCR assays for species identification in canned scombroid products. Both of these techniques are generally more rapid than PCR-RFLP, they are more useful in identifying multiple species in a mixture, and they are less vulnerable to contamination errors (Rasmussen and Morrissey 2008). Due to the potential for Atlantic bonito to be substituted for the more expensive bluefin tuna in processed products, Lockley and Bardsley (2000) developed a species-specific multiplex PCR assay based on mt cyt b. The assay was not tested with canned samples, but the species-specific cyt b fragments were within the appropriate size range for use with heavily processed fish products (<360 bp). A later study investigated the use of multiplex PCR to differentiate bluefin, albacore, yellowfin, and skipjack tuna based on species-specific fragments of the genes coding for reduced nicotinamide adenine dinucleotide (NADH2) and 5S rRNA (Dalmasso and others 2006). However, the method was only able to identify skipjack tuna, based on a 230 bp fragment of the NADH2 gene, and the 5S rRNA primers designed to differentiate the other 3 tuna species proved to be unsuccessful.
Two subsequent publications reported the ability to differentiate several tuna species at once using multiplex PCR assays. In one study, a triplex PCR assay allowed for the differentiation of yellowfin, bigeye, and skipjack tuna based on 246, 262, and 113 bp mt cyt b fragments, respectively (Michelini and others 2007). Numerous specimens were examined for each species to prevent errors arising from intraspecies polymorphism. Furthermore, the method was reported to be successful with a variety of fresh, frozen, and canned fish products. In another study published that same year, a multiplex PCR assay was designed to distinguish 5 tuna species in raw and canned products: albacore, yellowfin, bigeye, bluefin, and skipjack (Bottero and others 2007). This assay was also based on species-specific differences in mt cyt b; however, in this case the DNA fragment was 132 bp for all samples and species were identified using a dideoxynucleotide primer extension reaction, which allows for the detection of single base mutations in a given PCR product. This method allowed for successful species identification in all 30 canned samples that were tested.
A species-specific PCR method was developed for differentiation of 5 species commonly found in a commercial dried bonito product (katsuobushi) traditionally consumed in Taiwan and Japan (Lin and Hwang 2008a). These species were: skipjack tuna, E. affinis, A. thazard, A. rochei, and striped bonito. Due to the extensive processing that the dried bonito product undergoes, the resulting DNA fragments are significantly degraded and it is difficult to find a short consensus sequence that contains sufficient restriction cutting sites. Therefore, species-specific primers were developed that would result in the amplification of mt cyt b fragments with diagnostic lengths, ranging from 110 to 156 bp. With this method, the authors were able to identify the species in 11 out of 14 commercially purchased dried bonito products. To further improve identification of these 5 species, this research group also developed a multiplex PCR assay that combines 5 species-specific primer sets in 1 PCR tube (Lin and Hwang 2008b). The primers used in multiplex PCR were designed to amplify fragments of mt cyt b ranging in size from 143 to 506 bp. Using this method, species could be detected in all 12 raw commercial bonito fillets tested and in 5 out of 8 cooked commercial bonito fillets. The results suggested that fragments larger than about 320 bp could not be amplified from cooked fillets, indicating that this multiplex assay may be limited to analysis of uncooked samples.
Other studies have focused on the differentiation of mackerel species (genus Scomber) in commercial food products. For example, Aranishi (2005) investigated the differentiation of chub mackerel (S. japonicus) and Atlantic mackerel (S. scombrus) using the nuclear 5S rRNA and NTS region. The Atlantic mackerel commands a lower market price in Japan and is often mislabeled as chub mackerel. The 2 species only exhibited a small difference (40 bp) in the lengths of their respective NTS regions, so a restriction digest had to be carried out to differentiate the 2 species. Another mackerel species, Scomber colias, is highly valued in southern Spain and labeling regulations have been developed for its use in canned regional products; however, it is very similar in both appearance and texture to other species in the Scomber genus. Therefore, a multiplex PCR assay was developed for detection of S. colias in canned products (Infante and Manchado 2006). This assay was based on amplification of a species-specific 159 bp fragment in the NTS region, along with a 196 to 201 bp fragment of the 5S rRNA gene as a positive Scomber control. Out of a total of 18 canned products tested with this method, 17 were reported to be positive for S. colias.
In 2005, the 1st real-time PCR method for the detection and relative quantification of tuna species was published (Lopez and Pardo 2005). In this study, 3 TaqMan systems were developed: one system was able to identify the presence of a Scombroidei species, while the other 2 were able to specifically detect albacore tuna and yellowfin tuna. The TaqMan systems were designed to target fragments of the 16S rRNA gene that were 100 bp or shorter to ensure successful species identification with canned tuna products. This method was reported to be suitable for use in the detection of illegal tuna species substitution in canned white and light tuna products. Since then, another real-time PCR assay has been developed for the differentiation of 4 tuna species: bluefin tuna, yellowfin tuna, albacore tuna, and bigeye tuna (Dalmasso and others 2007). This assay utilized melting curve analysis with a fluorescence resonance energy transfer (FRET) probe, which is highly sensitive to differences in probe and template interactions and facilitates interpretation of the results. Based on a 208 bp fragment of the ATPase 6 gene, this assay was able to identify tuna species in both fresh and canned products.
Lightly processed products An early study into genetic identification of scombroids reported the ability to differentiate 4 Thunnus species by PCR sequencing of a 350 bp fragment of the mt cyt b gene (Bartlett and Davidson 1991). However, as mentioned previously, the sequencing method is a relatively time-consuming and costly technique for genetic differentiation. To investigate the feasibility of a PCR-RFLP method developed in a phylogenetic study carried out by Chow and Inoue (1993), a comprehensive survey was conducted involving 11 scombroid species from a variety of regions worldwide (Takeyama and others 2001). PCR-RFLP analysis was carried out on a 927 bp fragment of the flanking region between the COIII and ATPase genes (termed ATCO). Large sequence differences were reported among northern bluefin subspecies from the Atlantic (Thunnus thynnus thynnus) and Pacific (Thunnus thynnus orientalis) oceans (note: these subspecies were later recognized as 2 separate species: Atlantic or northern bluefin [T. thynnus] and Pacific bluefin [T. orientalis][Collete 1999]). Intraspecies differences were also detected among bigeye tuna from the Atlantic and Pacific oceans, demonstrating the importance of comprehensive sampling for establishing genetic identification methods. Species that were successfully differentiated using this method include albacore, blackfin (Thunnus atlanticus), longtail (Thunnus tonggol), southern bluefin, and yellowfin tuna. This method was later used to assess the accuracy of traditional, morphology-based techniques for identifying Pacific bluefin tuna harvested within the New Zealand bluefin tuna fishery between 1990 and 2000 (Smith and others 2001). Out of 69 samples that were previously identified to be Pacific bluefin tuna, 59 were confirmed by the PCR-RFLP method and 10 were found to be southern bluefin.
In a later study, Chow and others (2003) employed PCR-RFLP with both the 927 bp fragment of the mt ATCO flanking region (used in the above-mentioned studies) and a 680 bp fragment of the mt cyt b gene. The use of the mt cyt b gene fragment was an attempt to improve upon previous studies, which had found that a shorter fragment of the mt cyt b gene could not be used to differentiate all Thunnus species (Chow and Inoue 1993; Chow and Kishino 1995). Chow and others (2003) developed standard restriction profiles for 13 different scombroid species, including A. rochei, A. thazard, skipjack, albacore, and striped bonito. Striped bonito and all Thunnus species could be differentiated using PCR-RFLP on mt ATCO with 2 restriction enzymes, but there was intraspecies polymorphism between α and β types of bigeye tuna. On the other hand, PCR-RFLP on mt cyt b required 5 restriction enzymes to differentiate all Thunnus species, and there was no intraspecies polymorphism between α and β types of bigeye tuna. These methods were then used in combination to identify over 900 samples of scombroid larvae and juveniles captured in the Pacific Ocean between 1992 and 1998. The authors found that the reliability of the PCR-RFLP methods used in their study depends solely on the magnitude of intraspecies polymorphism, and determining this requires analysis of a large number of wild samples.
One way to overcome difficulties with intraspecies polymorphism could be to employ PCR-AFLP methods that allow for sampling of the entire genome rather than an isolated fragment. AFLP analysis was carried out with 3 Thunnus species (albacore, yellowfin, and Atlantic bluefin tuna) and 2 Morone species (striped bass [M. saxatilis] and white bass [M. chrysops]) (Han and Ely 2002). The use of 23 primers resulted in isolated genetic fragments ranging in size from 200 to 1000 bp. Unique AFLP profiles were observed for all 5 species under study. The scombroids were found to have relatively high intraspecies polymorphism, but relatively low interspecies polymorphism, suggesting that albacore, yellowfin, and Atlantic bluefin tuna are closely related. Atlantic bluefin was found to have AFLP patterns more similar to yellowfin than albacore, in agreement with previous studies. The authors recommended AFLP analysis as a suitable technique for genetic identification of closely related species.
To enable differentiation of commercial tuna fillets and sashimi, a recent study performed PCR-RFLP analysis on a 376 bp fragment of the mt cyt b gene (Lin and others 2005). Reference samples of bluefin, albacore, yellowfin, and bigeye were obtained from a variety of regions in the Pacific, Atlantic, and Indian oceans. This allowed the authors to determine intraspecies divergences within the gene fragment and avoid these regions when choosing appropriate restriction enzymes for RFLP analysis. After the PCR-RFLP method was established, it was successfully employed to identify Thunnus species in 12 commercially purchased tuna fillets (sashimi). The technique was reported to be quick, cheap, and convenient for the recognition of tuna species.
An alternative approach to tuna species identification has been the detection of mismatched DNA using techniques such as magnetic capture hybridization (Takeyama and others 2000) or direct force measurements (Tanaka and others 2006). Takeyama and others (2000) reported the ability to discriminate between Atlantic and Pacific bluefin tuna through the use of bacterial magnetic particles. These particles were able to detect single- nucleotide and 3-nucleotide differences between DNA probes bound to PCR products (150 and 406 bp) of ATCO originating from the 2 different species of bluefin tuna. Based on these same PCR-amplified DNA fragments, Tanaka and others (2006) utilized atomic force microscopy to obtain direct force measurements revealing DNA mismatches. With this method, the authors were able to differentiate 6 tuna species: Atlantic bluefin, Pacific bluefin, southern bluefin, yellowfin, bigeye, and albacore tuna. Although potentially useful in species identification, this approach requires several laboratory steps, including amplification of 2 PCR products per species, immobilization of probes and PCR products on gold-coated glass slides, and measurement of disruption forces using an atomic force microscope.
Cutlassfish, hairtails, and ribbonfish The family Trichiuridae comprises 9 genera and 32 species of cutlassfish, hairtails, and ribbonfish (Chakraborty and others 2005). Recent studies have investigated the genetic differentiation of 3 closely related species in the genus Trichiurus: T. lepturus, T. japonicus, and Trichiurus sp. 2 (Chakraborty and others 2005, 2006). In Japan, these 3 species are commonly consumed fresh as sashimi products or cooked as boiled or grilled products (Chakraborty and others 2005). Trichiurus sp. 2 is the preferred type, commanding a higher market price due to its high meat quality. T. lepturus and T. japonicus are very similar to each other and have just recently been recognized as separate species. In one study, PCR-RFLP on a 600 bp fragment of the mt 16S rRNA gene resulted in species-specific profiles for T. lepturus and T. japonicus (Chakraborty and others 2006). When 2 restriction enzymes were employed, this same method could be used to differentiate T. lepturus, T. japonicus, and Trichiurus sp 2 (Chakraborty and others 2005). This protocol was recommended as a rapid, cost-effective, and reliable method for identification of these 3 closely related Trichiurus species. To verify the reliability of PCR-RFLP in the differentiation of commercial samples, 64 hairtail fillets were purchased in supermarkets throughout Japan (Chakraborty and others 2007). Commercial hairtail fillets are commonly labeled and marketed as “Tachiuo” in Japan, which generally refers to T. japonicus. However, Trichiurus sp. 2 was found to account for 53% of the fillet samples, while the remaining 47% contained T. japonicus.
Billfish and swordfish Billfish, marlins, sailfish, and spearfish are all members of the Istiophoridae family, while swordfish are found in the Xiphiidae family. Meat from several of these species is highly regarded in Taiwan, where it is consumed as sashimi, dried fish floss, or minced fish products (Hsieh and others 2005). However, due to increases in demand and high prices, these products are sometimes substituted by fish of lesser value. To allow for the detection of fraudulence, recent studies have investigated the ability to differentiate 5 species belonging to the families Istiophoridae and Xiphiidae: swordfish (Xiphias gladius), Atlantic blue marlin (Makaira nigricans), black marlin (Makaira indica), Indo-Pacific sailfish (Istiophorus platypterus), and striped marlin (Tetrapturus audax) (Hsieh and others 2004, 2005, 2007). Several sets of species-specific primers were developed to amplify DNA fragments (87 to 357 bp) present in the control region of swordfish (Hsieh and others 2004). These fragments allowed for the detection of swordfish in a variety of products, including fresh, frozen, cooked, and sterilized samples. Later, PCR-RFLP on a 348 bp fragment of mt cyt b allowed for successful identification of all 5 species listed previously (Hsieh and others 2005). This method was later used to differentiate commercial billfish products consisting of raw fish fillets, frozen fish meats and fried fish meats (Hsieh and others 2007). Two out of 10 commercial samples could not be identified, and the authors suggested that this implied substitution with less valuable fish. Overall, the protocol was reported to be a sensitive, rapid, and reliable method for detecting fraudulent substitutes. Interestingly, this method was also recently employed to detect species in 6 billfish meat samples suspected in 2 incidents of foodborne histamine poisonings (Tsai and others 2007). The poisonings were found to be due to consumption of Atlantic blue marlin and swordfish fillets that had been served in restaurants in southern and central Taiwan.
Rockfish are members of the family Sebastidae, found within the suborder Scorpaenoidei (order Scorpaeniformes). There are approximately 110 species of rockfish in the Sebastes genus, and about 30 of these are commercially harvested along the U.S. West Coast (Rodger 2006). U.S. commercial landings of rockfish totaled over 43000 metric tons in 2007 and were valued at over U.S. $30 million (Voorhees 2008).
One of the most popular and valuable of the rockfish species has historically been the Pacific Ocean perch (Sebastes alutus), however, as demand increased over the years, other rockfish species have also experienced increased popularity (Rodger 2006). As a result, many of these species have been heavily exploited in commercial and recreational fisheries on both the Atlantic and Pacific coasts. However, fisheries management and conservation approaches have been complicated by the strong similarities among many rockfish species, especially during early life stages (Gharrett and others 2001). Studies that have applied DNA-based methods to the identification of rockfish species will be discussed in this section and are summarized in Table 5. Studies that have focused on the differentiation of rockfish species from Northern red snapper (Lutjanus campechanus) will be discussed in the Northern red snapper and substitute species section.
Several studies have focused on the use of mtDNA fragments to identify rockfish species. Rocha-Olivares (1998) developed a multiplex PCR assay to identify starry rockfish (Sebastes constellatus), rosethorn rockfish (Sebastes helvomaculatus), pinkrose rockfish (Sebastes simulator), and swordspine rockfish (Sebastes ensifer). A 750 bp fragment of mt cyt b was screened in more than 60 rockfish species to design the appropriate species-specific primers, along with a primer set specific for the Sebastomus subgenus. Following its development, the multiplex assay was tested with samples of unidentified field-caught larvae and juveniles. The results showed that 3 of the 5 samples tested belonged to the subgenus Sebastomus and none of them was among the 4 species targeted by the multiplex assay. The same 750 bp fragment of mt cyt b was later evaluated in an evolutionary study for 54 Sebastes species (Rocha-Olivares and others 1999). Using the methods developed in this study, rockfish species identification was successfully applied in subsequent studies (Rocha-Olivares and others 2000; Taylor and others 2004). In one study, mt cyt b sequencing was used to identify species of 5 juvenile rockfish (Rocha-Olivares and others 2000). The juveniles were determined to be starry rockfish and swordspine rockfish, and the results were combined with morphological observations to provide descriptions of new developmental stages of both species. In another study, mt cyt b sequencing was applied to characterize the species composition of rockfish larvae collected off the coast of Southern California (Taylor and others 2004). Unambiguous identification was possible for 20 out of 23 rockfish species, and the most abundant species was found to be Sebastes hopkinsi, followed by swordspine rockfish, Sebastes rufus, and Sebastes jordani.
Restriction site analysis of several mtDNA regions has also been investigated as a means of rockfish species identification (Gharrett and others 2001; Li and others 2006). Gharrett and others (2001) carried out restriction digests with 10 enzymes on the mtDNA regions coding for ND-3/ND-4 (2385 bp) and 12S/16S rRNA (2430 bp). Based on the results, they were able to design a simpler scheme that utilizes digestion of 1 to 4 restriction enzymes with the ND-3/ND-4 region to allow for differentiation of 15 rockfish species that are common to Alaskan waters. A later study investigated the use of these gene targets in PCR-RFLP to identify an additional 56 rockfish species (Li and others 2006). As with Gharrett and others (2001), the ND-3/ND-4 region provided better species resolution than the 12S/16S rRNA region. Based on their findings, Li and others (2006) developed a key for species identification based on digestion of the ND-3/ND-4 region with up to 4 restriction enzymes. Using this key, 58 out of the total 71 target species can be identified.
Besides mtDNA, microsatellite markers have also been examined for their use in rockfish species identification. In their study, Pearse and others (2007) utilized microsatellite markers previously characterized in rockfish to develop a multilocus species assignment method. Through the use of a panel of 6 microsatellite markers, most of the 33 Eastern Pacific rockfish species examined could be identified. However, identification of fish in the closely related subgenus Pteropodus required the use of an additional 5 microsatellite markers. When combined, these microsatellite markers allowed for correct species assignment in 97.4% of individuals tested. Furthermore, the analysis is based on smaller DNA fragments (<430 bp) than previous rockfish studies, which increases the potential for applications in evaluating poorly preserved specimens or processed food products.
Fish found in the suborder Percoidei (order Perciformes) are commonly referred to as percoids, a group that includes perches, sunfish, groupers, snappers, and jacks. Genetic identification research carried out with percoids has included fish from many of the families within this suborder, including Apogonidae, Carangidae, Centropomidae, Lutjanidae, Moronidae, Percidae, Polyprionidae, Sciaenidae, Serranidae, and Sparidae (Table 6). This section also includes a discussion of the differentiation of Nothern red snapper (Lutjanus campechanus) from commonly substituted species, such as other snappers, tilapia (order Perciformes, suborder Labroidei), and rockfish.
Table 6—. Genetic targets and methods used in authentication research involving percoids.
|Seabass, Gilthead seabream, shi drum, and common dentex||PCR-RFLP (2 enzymes)||mt cytochrome b||359 bp||Frozen||Cocolin and others 2000|
|Nile perch, E. guaza, and wreckfish||PCR-RFLP (2 enzymes)||mt 12S rRNA||436 bp||Muscle portion of fish (implies the use of uncooked samples)||Asensio and others 2000|
|Nile perch, E. marginatus or E. guaza, and wreckfish||Multiplex PCR||Nuclear 5S rRNA||Species-specific||Not specified (implies the use of fresh/frozen fillets)||Asensio and others 2001|
| || || ||Commercial fish fillets and restaurant grouper meals||Asensio 2008 and Asensio and others 2008|
|Nile perch, E. guaza, and wreckfish||PCR-RAPD||Random amplification of genome to produce “DNA fingerprint”||Various sizes||Not specified (implies the use of fresh/frozen fillets)||Asensio and others 2002|
|Trachurus species (Atlantic horse mackerel, Mediterranean horse mackerel, and blue jack mackerel)||PCR and sequencing||mt 16S rRNA|
mt cytochrome b
|Frozen and ethanol-preserved||Karaiskou and others 2003|
|Snappers and rockfish||PCR sequencing and phylogenetic analysis||mt cytochrome b||953 bp||Retail “red snapper”||Marko and others 2004|
|Nile perch, wreckfish, and 6 species of grouper||Multiplex conventional PCR and real-time PCR||mt 16S rRNA||140 bp (wreckfish)|
230 bp (Nile perch)
300 bp (grouper)
|Frozen muscle tissue||Trotta and others 2005|
|Wild and hatchery-raised red drum||Microsatellite analysis||25 microsatellite markers||Various sizes||Ethanol-preserved fin clips and frozen heart tissues||Renshaw and others 2006|
|Yellow perch and European perch||PCR sequencing||mt cytochrome b||Approximately 400 bp||Breaded and deep-fried fish fillets||Strange and Stepien 2007|
|6 snapper species (scarlet, L. argentimaculatus, L. erythropterus, L. malabaricus, L. leutjanus, and P. pinjalo)||Semi-nested PCR-RFLP (3 to 4 enzymes)||mt 12S rRNA||459 bp||Fresh and salted samples||Zhang and others 2007|
|Snapper species, rockfish species, and tilapia||PCR sequencing and phylogenetic analysis||mt cytochrome b|
mt control region
|Whole fish and fillets||Logan and others 2008|
|Pompano dolphinfish and common dolphinfish||Multiplex PCR||mt cytochrome b||352 bp (pompano)|
397 bp (common)
554 bp (universal)
|Preserved tissue samples||Rocha-Olivares and Chávez-González 2008|
Porgies, temperate basses, and drums The porgies, such as Gilthead seabream (Sparus aurata) and common dentex (Dentex dentex) are part of the family Sparidae; temperate basses, such as European seabass (Dicentrarchus labrax), white bass, and striped bass, are all part of the family Moronidae; and the family Sciaenidae comprises warm-water marine fish, including shi drum (Umbrina cirrosa) and red drum (Sciaenops ocellatus). The Mediterranean fisheries have experienced increased production, economic importance, and trade of a number of marine fish, including seabream and seabass (Cocolin and others 2000). Many of these fish are sold as frozen fillets, making it difficult to identify fraudulent species substitution. To help in the genetic identification of frozen fish fillets, Cocolin and others (2000) developed a PCR-RFLP method for distinguishing seabass, Gilthead seabream, shi drum, and common dentex. A 359 bp fragment of the mt cyt b gene was utilized along with 2 restriction enzymes on 5 to 10 frozen samples of each species. Although no intraspecies polymorphism was reported, a more comprehensive sampling effort may be required to verify the reliability of this method.
In a study described under the Scombroids section, the closely related striped bass and white bass were able to be differentiated with an AFLP method (Han and Ely 2002). Although these 2 species exhibit 97% to 99% nucleotide sequence identity, this study reported the ability to generate distinct banding patterns that allow for reliable species identification. Another study investigated the forensic differentiation of “wild” and hatchery-raised forms of red drum, an economically important fish species in the southern United States (Renshaw and others 2006). The authors were able to optimize a method based on hypervariable microsatellite markers that allowed for satisfactory species differentiation within the mandated forensic confidence level of ≥99%.
Perches, groupers, and wreckfish Several researchers have investigated differentiation of grouper species (Epinephelus guaza and Epinephelus marginatus) from Nile perch (Lates niloticus) and wreckfish (Polyprion americanus) (Asensio and others 2000, 2001, 2002, 2008; Asensio 2008). Due to its lower value, Nile perch is often labeled as grouper or wreckfish and sold for a higher price. Asensio and others (2000) reported the ability to differentiate E. guaza, Nile perch, and wreckfish using PCR-RFLP on a 436 bp fragment of the mt 12S rRNA gene. The same research group also published a study reporting the development of a PCR-random amplified polymorphic DNA (RAPD) method with 2 different primers that allowed for differentiation of these 3 species (Asensio and others 2002). In another study, species-specific primers were created to amplify the nuclear 5S rRNA and NTS region in these 3 species (Asensio and others 2001). PCR amplification followed by gel electrophoresis allowed for visualization of genetic fragments with species-specific sizes. This method was later applied to the identification of grouper meals in the restaurant industry (Asensio 2008), and, together with an enzyme-linked immunosorbent assay (ELISA), to the detection of grouper mislabeling in commercial fish fillets (Asensio and others 2008). Out of 37 purported grouper meals served at cafeterias (school and university) and restaurants, only 9 were determined to contain authentic grouper (E. marginatus). In the case of commercial fish fillets, only 12 out of 70 samples were determined to be grouper (E. marginatus). Of the remaining fillets, 13 were determined to be wreckfish, 34 were determined to be Nile perch, and 11 were undetermined. The multiplex PCR assay proved to be advantageous over ELISA in that it was able to detect the presence of Nile perch; however, both methods were recommended for use in grouper authentication.
A more extensive multiplex PCR assay has also been developed that allows for differentiation of 6 grouper species (Epinephelus aeneus, E. caninus, E. costae, E. marginatus, Mycteroperca fusca, and Mycteroperca rubra) from Nile perch and wreckfish (Trotta and others 2005). In addition to conventional analysis of the fragments with gel electrophoresis, the authors also presented a 2nd approach in which a real-time PCR assay allowed for simultaneous amplification and analysis of the PCR fragments. The suitability and reliability of this method was then tested with 41 additional species belonging to 28 different fish families, with no cross-reactivity observed.
In an investigation into the mislabeling of yellow perch (Perca flavescens) “fish fries,”Strange and Stepien (2007) utilized PCR sequencing of mt cyt b to identify species in breaded and deep-fried fish fillets. Yellow perch, which is harvested in the Great Lakes and served in local restaurants, has experienced declining stocks and increasing prices in recent years. On the other hand, European perch (Perca fluviatilis) is readily available and is sold at lower prices. In this study, 2 out of 5 samples examined were found to be substituted with European perch, implicating the need for increased DNA testing in this area to prevent economic fraud.
Saurels Fish in the genus Trachurus (Carangidae family) are commonly referred to as saurels. There are 15 species in this genus, including three of particular economic importance in Europe: the Atlantic horse mackerel (T. trachurus), Mediterranean horse mackerel (T. mediterraneus), and blue jack mackerel (T. picturatus) (Karaiskou and others 2003). These fish are very similar in appearance and species detection can be difficult when the morphological features cannot be distinguished. Therefore, Karaiskou and others (2003) investigated a method to differentiate these 3 species based on interspecies polymorphisms in a 340 bp segment of the mt cyt b gene and a 570 bp segment of the mt 16S rRNA gene. PCR amplification followed by sequence analysis revealed species-specific differences at several positions within each fragment that could allow for genetic identification. The authors reported this to be a potential method for the detection of commercial fraud. However, alternative methods may be preferred for regular application, as sequence analysis is relatively costly and time-consuming.
Northern red snapper and substitute species There are over 100 species of snapper worldwide, and at least 29 of these inhabit North American waters (Rodger 2006). Most snappers are part of the genus Lutjanus, including the humphead snapper (L. sanguineus), the Southern red snapper (L. purpureus), the Pacific red snapper (L. peru), and the Northern red snapper (L. campechanus). The Northern red snapper is found in the Middle Atlantic and Gulf coasts of the United States and is one of the most economically important fisheries in the Gulf of Mexico, commanding higher prices than any other snapper species (Marko and others 2004). Although L. campechanus is the only fish with the legal common name “red snapper” in the United States, it is often substituted with other snappers, groupers, and rockfish that are similar in appearance. In the USFDA Seafood List, the term red snapper is found within the vernacular names for 9 other snapper species, 13 species of rockfish (vernacular name “Pacific red snapper”), and 1 species in the Centroberyx (formerly known as Trachichthodes) genus (USFDA 2002). The USFDA strongly discourages the use of the vernacular name in commerce, as it may lead to cases of illegal seafood misbranding. However, based on the results of several studies, red snapper mislabeling appears to be a widespread occurrence. For example, in an examination of 81 retail fillets labeled as “red snapper,” 70% of the fillets were found to be mislabeled, with the most commonly substituted species being the humphead snapper (Hsieh and others 1995). In another retail market study, 17 out of 22 (77%) fish labeled as “red snapper” were found to be species other than L. campechanus (Marko and others 2004). The fish were purchased from 9 vendors in 8 states and were identified based on sequencing and phylogenetic analysis. Based on these results, it was estimated that 60% to 94% of fish sold as red snapper in the United States are mislabeled. These estimates were supported by another study using DNA barcoding, in which 7 out of 9 (78%) retail “red snapper” fillets purchased in New York City did not contain L. campechanus (Wong and Hanner 2008). Interestingly, substitution was not limited to a particular species or genus: the 7 mislabeled fillets were substituted with 5 different fish species from different genera. Due to the wide range of species that may be substituted for Northern red snapper, the development of rapid species identification methods may be challenging, especially when samples with degraded DNA are considered.
Another market study utilized sequencing data from mt cyt b and the mt control region to identify species sold as Pacific red snapper at various grocery chains, fish markets, and sushi restaurants throughout the states of California and Washington (Logan and others 2008). A total of 77 whole fish or fillets sold as “Pacific red snapper” were collected from 27 different establishments, and species were identified based on phylogenetic analysis. The majority of these products (90%) contained fish within the Sebastes (rockfish) genus, 7 were found to contain tilapia (genus Oreochromis), and 1 contained Northern red snapper L. campechanus. Of the samples identified as rockfish, 56% contained species that are considered to be overfished. As demonstrated in this study, using the same retail label for such a large number of fish species can lead to consumer confusion, along with complications in terms of conservation efforts and economic deception.
In China, the term “red snapper” applies to multiple fish species in the genus Lutjanus, and the main species that contribute to red snapper products are the humphead snapper, L. argentimaculatus, L. erythropterus, and L. malabaricus (Zhang and others 2007). These species are commonly used in fresh and salted red snapper products; however, they are occasionally substituted with species of lesser value, such as Lethrinus lentjan and Pinjalo pinjalo. Therefore, a seminested PCR-RFLP assay was designed based on a fragment of the 12S rRNA gene to differentiate the major species of red snapper and their substitutes (Zhang and others 2007). The authors were able to discriminate all species using this method, even in the case of salted fish products.
Dolphinfish The dolphinfish family (Coryphaenidae) consists of only 2 members: the common dolphinfish (Coryphaena hippurus) and the pompano dolphinfish (Coryphaena equiselis). These fish are found in tropical and subtropical coastal waters worldwide and are popular in sport and commercial fisheries. The common dolphinfish is often marketed as “mahi mahi” and sold as fresh/frozen fillets or steaks. In Mexico, dolphinfish are reserved for recreational fishing and commercial harvesting is illegal. To facilitate detection of illegal commercial harvests of dolphinfish, Rocha-Olivares and Chávez-González (2008) developed a multiplex PCR assay for dolphinfish species identification. The assay is a tetraplex PCR based on the mt cyt b gene, and its products range in size from 352 bp to 554 bp. The method was developed and optimized using 15 reference samples, and it was then tested with a set of 82 samples from Chiapas, Mexico, that had been morphologically identified as the common dolphinfish. The multiplex PCR assay revealed that all samples were indeed common dolphinfish, except for 2 that were suspected to have been misidentified in the field. While this method shows promise for the detection of dolphinfish species in Mexico, there may be problems due to intraspecies variability. Pompano dolphinfish sequences from the North Atlantic Ocean were found to share a base with the common dolphinfish at a site that is targeted by a species-specific primer. To resolve intraspecies variability at this site, a more extensive reference sample collection from various geographic locations will need to be obtained and analyzed.
Small pelagic fish
Small pelagic fish belong to the order Clupeiformes, which includes the families Engraulidae (anchovies) and Clupeidae (herrings, shads, sardines, menhadens, sprats). These fish represent a major portion of the global seafood supply and many are continuously listed in the top 20 wild-caught species worldwide, including anchoveta (Engraulis ringens), Atlantic herring (Clupea harengus harengus), Japanese anchovy (Engraulis japonicus), European pilchard (Sardina pilchardus), European sprat (Sprattus sprattus sprattus), and South American pilchard (also known as Pacific sardine, Sardinops sagax) (Johnson 2007). Therefore, a number of genetic identification research studies have been carried out with these small pelagic fish (Table 7). Although most small pelagics in North America are used as bait in other fisheries, the European markets often utilize these fish as specialty products, which are hand-filleted, salted, cured, canned, and sold for a high price as semipreserves (Rodger 2006). For example, anchovy semipreserves are highly regarded in Italy for their characteristic taste and muscular texture (Sebastio and others 2001). By Italian law, anchovy semipreserves should contain exclusively European anchovy (Engraulis encrasicolus L.); however, the European anchovy fishery has experienced relatively low harvests. Due to the high demand for anchovy semipreserves, this product may be susceptible to illegal substitution with other species, such as the gilt sardine (Sardinella aurita).
Table 7—. Genetic targets and methods used in authentication research involving small pelagic fish.
|European anchovy and gilt sardine||PCR-RFLP (4 enzymes)||mt cytochrome b||376 bp||Fresh and semipreserved (salt-cured and fillets in oil)||Sebastio and others 2001|
|9 small pelagics (European pilchard, gilt sardine, Atlantic herring, European sprat, European anchovy, Japanese anchovy, S. melanostictus, S. sagax caeruleus, and S. maderensis)||PCR-RFLP (2 enzymes)||mt cytochrome b||142 bp|
|Raw and canned||Jerome and others 2003a|
|14 small pelagics||FINS||mt cytochrome b||103 bp||Canned sardines and sardine-type products||Jerome and others 2003b|
|Anchovy (E. encrasicolus, E. anchoita, E. ringens, and E. japonicus)||PCR-RFLP (2 enzymes) and FINS||mt cytochrome b||284 bp for RFLP|
540 bp for sequencing
|Fresh, frozen, salted, semipreserved, and in vinaigrette sauce||Santaclara and others 2006a|
|16 small pelagics from the orders Clupeiformes and Salmoniformes||Sequencing and phylogenetic analysis||mt 16S rDNA||606 bp||Ethanol-preserved reference samples and imported chirimen samples||Akasaki and others 2006a|
|15 small pelagics (anchovy and sardine-type species)||PCR sequencing||mt cyt b, 16S rRNA, and COI||212 to 274 bp||Frozen, ethanol-preserved, canned, and semi-preserved (salted, marinated, and ripened) products||Jerome and others 2008|
Several studies have focused on genetic species identification to prevent fraudulent production of anchovy specialty products and a database has been created for this purpose (http://anchovyid.jrc.ec.europa.eu). For example, Sebastio and others (2001) investigated a method to distinguish between the European anchovy and the gilt sardine. Successful species diagnosis was obtained in both fresh and semipreserved samples of European anchovy and gilt sardine using PCR-RFLP on a 376 bp fragment of the mt cyt b gene (Sebastio and others 2001). Another study focused on differentiating European anchovy, anchoveta, Japanese anchovy, and Argentine anchovy (Engraulis anchoita), all of which are common anchovy species important in the European marketplace (Santaclara and others 2006a). This study considered the use of both PCR-RFLP and FINS to analyze a fragment of the mt cyt b gene in anchovy samples in a variety of commercial forms. PCR-RFLP was not always successful at differentiating Japanese anchovy and European anchovy; however, FINS was reported to be successful at differentiation of all 4 species and was suggested to be a suitable technique for species identification. A later study described the use of direct sequencing of species-specific mtDNA sequences to identify 15 different anchovy and sardine-type fish species in anchovy-labeled products (Jerome and others 2008). Three sets of primers were developed to amplify DNA fragments from mt cyt b, 16S rRNA, and COI, ranging in size from 212 to 274 bp. Although species diagnosis was possible with just one of the 3 markers, the authors recommended combining all 3 to increase the specificity and resolution power of the diagnosis.
Sardines are consumed throughout Europe, especially in countries such as Spain, France, and Italy (Welch and others 2002). According to Codex Alimentarius, any of 21 listed species of small pelagic fish (including sardines, anchovies, herrings, sprats, and menhaden) may be used in the production of canned sardines or sardine-type products (CODEX STAN 94). When European pilchard is used exclusively in the product, it may be labeled as containing simply “sardines.” However, when the product contains any of the other small pelagics approved by Codex, the label must list a distinctive designation for that fish, such as geographic area, country of origin, or the species or common name. Since the morphological features of these fish are removed during processing, methods for species identification are currently being researched to help with enforcement of this regulation. Phylogenetic analysis on 9 of the 21 listed small pelagic species allowed for the discovery of a species-specific short fragment (<150 bp) of the mt cyt b gene (Jerome and others 2003a). A PCR-RFLP method was then developed that allowed for European pilchard to be differentiated from the other species in both raw and canned products, including gilt sardine, Japanese anchovy, Sardinops melanostictus, Sardinops sagax caeruleus, and Sardinella maderensis. Although European pilchard showed a unique restriction profile, many of the other species tested could not be differentiated from each other. Therefore, direct sequence analysis could prove to be a more reliable method for distinguishing various small pelagics in sardine products. To this effect, FINS was carried out to authenticate species in 26 commercial canned sardine products and 21 sardine-type canned products originating in 13 different countries (Jerome and others 2003b). The analysis was carried out on a 103 bp fragment of the mt cyt b gene using 14 reference samples of Clupeomorpha species, including all 9 species used previously. European pilchard was unequivocally identified in all 26 canned sardine products; however, the authors were not able to identify species in some of the sardine-type canned products. It was noted that the use of more reference samples in a future study would likely allow for these unknown samples to be assigned species names. Also, further research was suggested to investigate the intraspecies polymorphisms among species other than European pilchard on the Codex list.
Another challenge to regulatory authorities is the Japanese specialty product chirimen, whose main ingredient is Clupeiform larvae (Akasaki and others 2006a). According to Japanese regulations, imports of sardines and sardine products belonging to the genera Etrumeus, Sardinops, or Engraulis must be approved in advance. However, chirimen is produced through boiling and drying of the Clupeiform larvae, making species detection difficult. A recent study focused on the identification of boiled and dried larvae and juvenile sardines at the genus level to enhance import screening by Japanese officials (Akasaki and others 2006a). A 606 bp portion of the mt 16S rDNA gene was sequenced for 16 standard samples of fish from the orders Clupeiformes and Salmoniformes. Subsequent phylogenetic analysis on specimens of imported chirimen from 4 different countries revealed the presence of Engraulis spp. in some samples. This method may therefore prove to be useful for customs officials attempting to screen imported chirimen products.
Sturgeons are prehistoric fish belonging to the family Acipenseridae (Rodger 2006). There are 25 different sturgeon species within this family and they are part of one of the most threatened groups of vertebrates (Ludwig 2006). These fish are highly regarded for their eggs, with the best sources of sturgeon caviar considered to be from the Caspian and Black seas. Sturgeon eggs have been deemed as “real” caviar and, according to U.S. law, products containing Acipenseridae eggs can be labeled as caviar, while products containing eggs from other types of fish must include the name of the fish (Rodger 2006). The 4 most important caviar producers/products are beluga sturgeon (Huso huso) which produces beluga caviar; starry sturgeon (Acipenser stellatus) which produces sevruga caviar; Russian sturgeon (Acipenser gueldenstaedtii) producing osietra or ossetra caviar; and Persian sturgeon (Acipenser persicus), which produces asetra caviar (Ludwig 2008). However, all four of these sturgeon species have also been categorized as endangered species under the Intl. Union for the Conservation of Nature and Natural Resources (IUCN) Red List. Due to its limited availability, sturgeon caviar has come to be a highly exclusive and expensive fishery product, with mean import prices reaching as high as U.S. $4290 per kg for beluga caviar in 2005 (Ludwig 2008). The high price and limited supply of sturgeon caviar has resulted in increases in the vulnerability of threatened sturgeon populations and the occurrence of fraudulent substitution and mislabeling. For a summary of the application of genetic methods to sturgeon species identification, see Table 8. Additional information on this topic is available in 2 excellent review articles published by Ludwig (2006, 2008).
Table 8—. Genetic targets and methods used in authentication research involving sturgeon.
|Sturgeon caviar (beluga, Russian, and sevruga)||PCR with species-specific primers||mt cytochrome b, 16S rDNA, and 18S rDNA||Not given||Caviar||DeSalle and Birstein 1996|
|Sturgeon caviar (beluga, Russian, and sevruga)||PCR-SSCP||mt cytochrome b||123 bp|
|Frozen caviar||Rehbein and others 1997|
|Adriatic sturgeon and A. sturio||PCR-RFLP (1 enzyme)||mt 12S rDNA||389 bp (Adriatic)|
400 bp (A. sturio)
|Blood and tissue samples||Ludwig and Kirschbaum 1998|
|Sturgeon caviar (beluga, sevruga, Russian, fringebarbel, and sterlet sturgeon)||PCR-SSCP||mt cytochrome b||116 bp|
|Fresh, frozen and ethanol-preserved blood, caviar, and muscle tissue||Rehbein and others 1999a|
|Sturgeon caviar (beluga, sevruga, Russian, Persian, fringebarbel, sterlet, Siberian, A. naccarii, A. sturio, and amur)||PCR-RFLP (4 enzymes)||mt tRNAGlu- cytochrome b||462 bp||Caviar, blood, and tissue||Wolf and others 1999|
|Starry sturgeon compared with 9 other Acipenseridae spp.||PCR amplification||Microsatellite locus LS-39||108 to 156 bp||Blood, fin clips, and roe samples||Jenneckens and others 2001|
|22 Acipenseriformes spp.||PCR-RFLP (7 enzymes)||mt tRNAGlu- cytochrome b-tRNAThr||34 bp-1221 bp-46 bp||Fresh caviar and alcohol-fixed blood, fin clips, liver, caviar, and sperim||Ludwig and others 2002|
|Beluga, sevruga, and Russian sturgeon||PCR-RFLP + HPLC (2 enzymes)||mt cytochrome b||462 bp||Reference caviar samples and frozen sturgeon fillets||Horstkotte and Rehbein 2003|
|Adriatic sturgeon and A. sturio||PCR amplification and sequence analysis||DNA satellite markers (HindIII and PstI) and mt DNA markers (5S rDNA spacers, 12S rDNA and cyt b)||139 bp (12S rDNA)|
212 bp (cyt b)
|Tissue samples||de la Herran and others 2004|
|8 sturgeon species (beluga, starry, Russian, sterlet, siberian, fringebarbel, kaluga, and amur sturgeon)||Species-specific and multiplex PCR||mtDNA control region (D-loop)||182 to 439 bp||Caviar and tissue samples||Mugue and others 2008|
One of the earlier studies into caviar fraud sequenced fragments of 3 genes (mt cyt b, 16S rDNA and 12S rDNA) in beluga, starry, and Russian sturgeon (DeSalle and Birstein 1996). The authors were able to identify interspecies polymorphism in these sequences and developed species-specific primers to allow for differentiation. This method was then employed to identify species in 23 commercial samples of caviar purchased from gourmet food shops in Manhattan, New York, and 2 samples brought from Russia. Five of the New York samples were found to be fraudulent, and three of these were reported to be particularly alarming due to the fragile status of the substituted fish.
Beluga, starry, and Russian sturgeon caviars have been successfully differentiated using PCR-SSCP analysis on fragments of the mt cyt b gene (Rehbein and others 1997). Since only a few samples of caviar were analyzed and the conditions had not been optimized, a follow-up study was published reporting improvements in the method (Rehbein and others 1999a). In addition to the species already analyzed, the improved PCR-SSCP method also allowed for the successful differentiation of fringebarbel sturgeon (Acipenser nudiventris) and sterlet (Acipenser ruthenus) using a genetic fragment under 150 bp. However, the 2 species considered in the study to be osietra caviar (A. gueldenstaedtii and A. persicus) were found to have identical PCR-SSCP patterns and could not be differentiated from each other.
Wolf and others (1999) developed a PCR-RFLP method for sturgeon differentiation based on a 462 bp portion of the mt tRNAGlu-cyt b region (Wolf and others 1999). The authors reported this method to be suitable for the differentiation of caviar from 10 sturgeon species, including the species listed in the studies above along with Adriatic sturgeon (Acipenser naccarii), Acipenser sturio, amur sturgeon (Acipenser schrenckii), and Siberian sturgeon (Acipenser baerii baerii). However, within the paper, no discernable polymorphisms between Persian sturgeon and Russian sturgeon were reported. Fifteen commercial lots of caviar purchased in Russia and Germany were analyzed, and 1 case of mislabeling was detected. Although this study allowed for more species to be differentiated than in previous research, there was some ambiguity regarding genetic polymorphisms between and within Persian sturgeon, Russian sturgeon, and Siberian sturgeon.
Ludwig and Kirschbaum (1998) reported the ability to differentiate the Adriatic sturgeon and A. sturio using PCR-RFLP on a portion of the mt 12S rDNA gene. Ludwig and others (2002) later investigated genetic identification of 22 different sturgeon species based on polymorphisms in the entire sequence of the mt cyt b gene, flanked by a 34 bp section of mt tRNAGlu and a 46 bp segment of tRNAThr. PCR-RFLP with 7 restriction enzymes allowed for the successful differentiation of 17 species of Acipenseriformes. Species that could not be differentiated included Russian sturgeon, Persian sturgeon, and 3 species from the genus Scaphirhynchus (S. suttkusi, S. platorynchus, and S. albus). Beluga, starry, and Russian sturgeon have been successfully differentiated by an RFLP-HPLC technique described by Horstkotte and Rehbein (2003). Reference samples of caviar from these 3 species were clearly distinguished after PCR-RFLP on a 462 bp fragment of the mt cyt b gene (Horstkotte and Rehbein 2003). Using this method, 3 frozen fillet samples from Romania were then identified to be: beluga (n = 1), starry (n = 1), and Russian sturgeon (n = 1). These results were then confirmed by previously established RFLP and SSCP techniques (Rehbein and others 1999a) and HPLC was deemed to be a useful tool in genetic species identification research. However, molecular techniques such as these may be vulnerable to intentional caviar manipulation. In their study, Wuertz and others (2007) revealed the possibility of contaminating Siberian sturgeon caviar products with artificially amplified DNA (mt cyt b) from A. sturio. To prevent this from occurring in the global caviar trade, the authors recommended incorporation of a DNase treatment into regulatory testing protocols.
Genetic satellite DNA research has also revealed methods for differentiation of sturgeon species (Jenneckens and others 2001; de la Herran and others 2004; Robles and others 2004). For instance, the microsatellite locus LS-39 was studied in 10 Acipenseridae species and was reported to exhibit species-specificity for starry sturgeon (Jenneckens and others 2001). The authors reported this study to be the first to describe a nuclear marker for differentiation of black caviar. To examine the sturgeon species present in the waters of Western Europe, de la Herran and others (2004) utilized a combination of DNA satellite markers (HindIII and PstI) and mt DNA markers (5S rDNA spacers, 12S rDNA, and cyt b). The results of the study showed that the Adriatic sturgeon coexists with A. sturio from the Adriatic Sea to the Iberian Peninsula.
A study explaining some of the complications to date with identification of Russian sturgeon reported that this species consists of 3 genetic forms—the pure A. gueldenstaedtii, an A. baerii-like form, and a rare form whose mtDNA is similar to that of A. naccarii (Birstein and others 2005). The A. baerii-like and the A. naccarii-like forms could not thus far be differentiated from the pure forms of A. baerii and A. naccarii, and species authentication of samples labeled as Russian sturgeon was predicted to be problematic. A subsequent study reported progress in this area with the ability to differentiate the pure A. gueldenstaedtii from its A. baerii-like form (Mugue and others 2008). This study developed species-specific primers to identify these 2 A. gueldenstaedtii forms, along with 7 other sturgeon species, including beluga, Siberian, starry, amur, and kaluga sturgeon (Huso dauricus). The primers were designed to amplify species-specific fragments of the mtDNA control region (D-loop) whose size differences could be visualized on an agarose gel. To increase the robustness and reliability of the test, more than 1400 sturgeon specimens were examined for intraspecies and interspecies polymorphisms. Successful results were obtained for all but one of the species examined, the Persian sturgeon, which could not be differentiated from the Russian sturgeon based on mtDNA analysis. In this case, the authors recommended examination of nuclear markers that could be used to differentiate these 2 species.
Research into genetic identification of eels has been focused on those in the genus Anguilla (family Anguillidae, order Anguilliformes), which comprises 18 species and 3 subspecies (Rehbein and others 2002). This genus includes several eels sold worldwide for human consumption, with some of the most common being European eel (Anguilla anguilla), American eel (Anguilla rostrata), Japanese eel (Anguilla japonica), and shortfin eel (Anguilla australis australis). Eel meat from different species varies in quality and price. For example, on the Japanese market, A. japonica products command a higher market value than A. anguilla products (Itoi and others 2005). Although Japanese law demands that eel species and place of origin be listed on the label, these products become difficult to differentiate when morphological characteristics are removed during processing. Further, some species are illegally harvested and sold commercially, thereby interfering with conservation and management efforts (Lin and others 2002). To prevent the fraudulent sale of eel, several studies have investigated potential genetic methods that could be used to authenticate eel species (Table 9).
Table 9—. Genetic targets and methods used in authentication research involving eels.
|4 eel species (American, Japanese, shortfin, and European)||PCR-SSCP||mt cytochrome b||123 bp|
|Frozen||Rehbein and others 1997|
|18 eel species (genus Anguilla)||PCR-RFLP (6 enzymes)||mt 16S rRNA gene||1300 bp||Raw preserved in ethanol, buffer, and formalin||Aoyama and others 2000|
|4 eel species (American, Japanese, shortfin, and European)||PCR-RFLP (3 enzymes) and SSCP||mt cytochrome b||464 bp (RFLP) 123 bp (SSCP)||Raw and hot-smoked (canned for SSCP only)||Rehbein and others 2002|
|4 eel species (American, Japanese, European, and giant mottled) and predicted results for 14 other species (genus Anguilla)||PCR-RFLP (2 enzymes, 4 species) and PCR with species-specific primers (2 species)||mt cytochrome b and predicted results for 12 S rRNA||1230 bp (cyt b)|
Species-specific fragment sizes not given
|Not specified||Lin and others 2002|
|Japanese eel||Real-time PCR||mt 16S rRNA||153 bp||Fresh/ethanol-preserved liver and muscle tissue||Watanabe and others 2004|
|European eel and Japanese eel||Species-specific primers||mt 16S rRNA||560 bp|
|Frozen and broiled products||Sezaki and others 2005|
|European eel and Japanese eel||SNP-based PCR with TaqMan fluorescent probes and PCR-RFLP (1 enzymes)||mt 16S rRNA|| ||Frozen and broiled||Itoi and others 2005|
|American eel and European eel||PCR with species-specific primers||mt cytochrome b||589 bp|
|Ethanol-preserved gill tissue||Trautner 2006|
|4 eel species (American, Japanese, giant mottled, and European)||PCR amplification and genetic data analysis||4 micro-satellite loci||Various sizes||Ethanol-preserved fin and liver tissue||Maes and others 2006|
|4 eel species (giant mottled eel, A. bicolor bicolor, A. mossambica, and A. bengalensis labiata)||PCR-RFLP (8 enzymes) and Semi-multiplex PCR||mt 16S rRNA||1350 to 1354 bp (PCR-RFLP)|
253 to 537 bp (multiplex)
|Ethanol-preserved tissue||Gagnaire and others 2007|
In one study, PCR-SSCP was employed on short segments of the mt cyt b gene to successfully differentiate uncooked samples of European, American, Japanese, and shortfin eels (Rehbein and others 1997). PCR-RFLP has also been shown to be a successful method for the differentiation of raw eel, with one study using a 398 bp region of the mt cyt b gene (Wakao and others 1999) and another study using a 1300 bp section of the mt 16S rRNA domain (Aoyama and others 2000). Wakao and others (1999) reported the ability to distinguish 6 different eel species, while Aoyama and others (2000) were able to differentiate 18 Anguilla eel species harvested throughout the world with the use of 6 restriction enzymes. To test genetic identification methods with cooked eel products, Rehbein and others (2002) carried out PCR-SSCP and PCR-RFLP on raw, hot-smoked, and canned (PCR-SSCP only) eel samples. A 123 bp fragment of the mt cyt b gene was utilized for PCR-SSCP, while PCR-RFLP was carried out with a 464 bp fragment of the same gene. European, American, Japanese, and shortfin eels were successfully differentiated with both methods. Samples were then sent out to 5 other laboratories to test the reliability of these methods. With samples containing only one species, the laboratories showed 100% identification accuracy; however, mixtures containing European eel combined with either American or shortfin eel did not allow for recognition of the secondary species in 60% of cases. These results demonstrated the need for improved methods to identify eel species in mixed samples.
To this regard, assays utilizing species-specific primers and multiplex PCR, which are useful for species identification in mixed samples, have been developed for the rapid differentiation of eel species (Lin and others 2002; Sezaki and others 2005; Trautner 2006; Gagnaire and others 2007). For example, Lin and others (2002) considered the use of species-specific primers and PCR-RFLP on a portion of either the mt cyt b gene or the mt 12S rRNA gene to differentiate all 18 Anguilla species. Four eel species were tested using these methods: the giant mottled eel (Anguilla marmorata; a protected species in Taiwan), European, American, and Japanese eels. Of 58 samples analyzed, >95% were successfully identified using restriction enzymes and >99% were successfully identified with the species-specific primers. Sezaki and others (2005) designed primers to amplify fragments of the mt 16S rRNA gene, resulting in a PCR product (560 bp) common to both the European and Japanese eels and a PCR product (approximately 390 bp) that was specific for the Japanese eel. In a similar study, Trautner (2006) designed primers to amplify species-specific fragments of the mt cyt b gene in the American eel (589 bp) and the European eel (789 bp). To differentiate 4 eel species common to the southwestern Indian Ocean region, Gagnaire and others (2007) developed both a PCR-RFLP and a semimultiplex PCR assay based on the mt 16S rRNA gene. Each method was found to be useful in eel species identification; however, the PCR-RFLP method would not be practical for use with processed eel products, as it required a relatively large PCR product (>1000 bp) in comparison with the semimultiplex assay. Furthermore, the authors reported that the semimultiplex method has already been adapted to a real-time PCR assay utilizing SYBR® Green probes.
Other real-time PCR assays have also been applied to the detection of eel species. For example, Watanabe and others (2004) developed a rapid method for the detection of Japanese eel using real-time PCR on a 153 bp fragment of mt 16S rRNA. This method was reported to be a convenient and advantageous alternative to previous techniques, especially in regard to the fact that species identification could be achieved while onboard a research vessel. Another real-time PCR approach was developed to distinguish Japanese and European eels based on single nucleotide polymorphisms (SNPs) in the mt 16S rRNA gene (Itoi and others 2005). Species-specific TaqMan Minor Groove Binder probes allowed for the successful identification of both fresh and broiled samples of the 2 species, and the authors reported this method to be a valuable tool for species diagnosis in processed foods.
An alternative method used in eel species discrimination involves the use of nuclear DNA in the form of microsatellite markers. One study reported successful differentiation of 4 species of eel based on genetic analysis of 4 co-dominant microsatellite loci (Maes and others 2006). The method enabled rapid screening of eel samples and gave results with confidence levels of >90% for detection and classification and >95% for species assignment of random samples.
Sharks in the orders Carcharhiniformes (ground sharks) and Lamniformes (mackerel sharks) have been important in genetic species identification (Table 10). These include the silky (Carcharhinus falciformis), blue (Prionace glauca), dusky (Carcharhinus obscurus), great white (Carcharodon carcharias), shortfin mako (Isurus oxyrinchus), porbeagle (Lamna nasus), and longfin mako (Isurus paucus) sharks. Pelagic sharks, such as these, are harvested in direct fisheries or as bycatch in the tuna and billfish fisheries (Shivji and others 2002). The demand for shark fins has been escalating, and has resulted in the exploitation of sharks worldwide. Species identification becomes complicated in the marketplace, where products are sold as detached shark fins and headless, finless shark carcasses (Pank and others 2001). To help promote conservation and management efforts through monitoring of the shark fin trade, methods to detect shark species are being investigated.
Table 10—. Genetic targets and methods used in authentication research involving sharks from the orders Carcharhiniformes and Lamniformes.
|11 shark species (9 carcharhinids and 2 sphyrnids)||PCR-RFLP (7 enzymes)||mt cytochrome b-tRNATHR||394 to 396 bp||Heart, white muscle, and fin tissue either frozen or stored in lysis buffer||Heist and Gold 1999|
|Dusky shark and sandbar shark||4-primer multiplex PCR||Nuclear 5S rRNA-ITS2-28S rRNA||Varies with species||Muscle, fin, heart, and liver tissue||Pank and others 2001|
|6 shark species (silky, blue, shortfin mako, porbeagle, dusky, and longfin mako)||8-primer multiplex PCR||Nuclear 5S rRNA-ITS2-28S rRNA||Varies with species||Fresh muscle tissue preserved in SED or ethanol, and dried fin and muscle tissues||Shivji and others 2002|
|Great white shark and 68 nontarget shark species||Bi-locus 5-primer multiplex PCR||Nuclear 5S rRNA-ITS2-28S rRNA and mt cytochrome b||1340 bp (universal)|
580 bp (ITS2)
511 bp (cyt b)
|Dried fin and ethanol-preserved fins, muscle, liver, heart, and vertebrate with some tissue attached||Chapman and others 2003|
|Great white shark|| || || ||Dried shark fins intended for export to Asian markets||Shivji and others 2005|
|3 hammerhead shark species (scalloped, great, and smooth)||5-primer multiplex PCR||Nuclear 5S rRNA-ITS2-28S rRNA||Varies with species||Dried fins and ethanol-preserved fins, muscle, liver, heart, and vertebrate with some tissue attached||Abercrombie and others 2005|
|35 shark species||PCR sequencing||mt 12S rRNA-tRNAVAL-16S rRNA||1400 bp||Blood, fin, and muscle tissue frozen, ethanol-preserved, or SDS-urea-preserved||Greig and others 2005|
|14 to 15 target shark species + 72 nontarget species||Multiplex PCR||Nuclear 5S rRNA-ITS2-28S rRNA||Varies with species||Reference samples and fin clips obtained in the Hong Kong market||Clarke and others 2006|
|Basking shark and 80 nontarget species||4-primer multiplex PCR||Nuclear 5S rRNA-ITS2-28S rRNA||860 to 1500 bp (universal)|
1100 bp (basking shark)
|Ethanol-preserved fins, muscle, and liver tissue and dried shark fins from the Japanese and Hong Kong markets||Magnussen and others 2007|
|8 shark species (shortfin mako, bull, scalloped hammerhead, dusky, blacktip shark, bigeye thresher, tiger shark, and blacknose shark)||PCR||Nuclear 5S rRNA||Species-specific||Fresh, frozen, and ethanol-preserved fin clips, gills, and muscles||Pinhal and others 2008|
A PCR-RFLP assay was developed to discriminate the 11 species of sharks most frequently landed in the U.S. Atlantic large coastal shark fishery, including the silky shark, dusky shark, lemon shark (Negaprion brevirostris), bignose shark (Carcharhinus altimus), and bull shark (Carcharhinus leucas) (Heist and Gold 1999). Using a 7-enzyme restriction digest with a fragment of mt cyt b, all species could be differentiated, despite some instances of polymorphism among the shark species. A later study reported the investigation of a 1400 bp region of mitochondrial DNA for its use in differentiation of shark species common to the western North Atlantic Ocean, including 20 commercially exploited species and 12 prohibited species (Greig and others 2005). The authors found this segment of DNA, which includes portions of the genes coding for 12S rRNA and 16S rRNA along with the entire sequence for tRNAVAL, to be suitable for species differentiation, exhibiting low intraspecies polymorphism and high interspecies polymorphism in most cases. Furthermore, preliminary work showed that a 400 bp fragment of this sequence would enable species identification in the case of degraded DNA.
As an alternative to PCR-RFLP and sequencing techniques, several studies have been focused on the development of multiplex PCR assays to differentiate shark species. For example, Pank and others (2001) developed a multiplex PCR approach to improve differentiation of the dusky shark and the sandbar shark (Carcharhinus plumbeus). The study employed species-specific primers in a 4-primer multiplex format, which involves the simultaneous digestion of shark DNA with 2 universal shark primers and 2 species-specific primers. The specific DNA fragment analyzed was the nuclear ribosomal internal transcribed spacer-2 locus (ITS-2), located between the 5.8S rDNA and 28S rDNA coding regions. This method was also used in an 8-primer multiplex format to identify 6 shark species common to the pelagic fisheries and the global fin market: silky, blue, shortfin mako, porbeagle, dusky, and longfin mako (Shivji and others 2002). The method was reported to be successful for distinguishing body parts of the 6 species from all but one other species of shark in the North Atlantic fishery. This method was also found to be useful in preliminary species identification tests involving dried fins sold in the commercial markets of Asia and the Mediterranean region.
The great white is the most widely protected member of the subclass Elasmobranchii (skates, rays, and sharks) and is listed in Appendix III of the Convention on International Trade in Endangered Species (CITES); however, monitoring efforts have been complicated by difficulties in identifying shark parts on the market (Chapman and others 2003; Shivji and others 2005). Diagnostic markers for the great white shark were recently developed using a 5-primer multiplex PCR assay with simultaneous analysis of the nuclear ITS-2 and mt cyt b loci (Chapman and others 2003). This method allowed for genetic identification of the great white shark, even when it was present in a mixed sample with DNA from 9 other commercially fished shark species. These diagnostic markers were then used to investigate the species of shark fins that had been confiscated from a seafood warehouse along the U.S. East Coast and were destined for export to the Asian market (Shivji and others 2005). All 21 pectoral fins suspected to be great white shark were confirmed by genetic analysis to indeed belong to this species, illustrating the occurrence of illegal trading of a highly protected shark species.
Another study expanded upon the above methods to investigate the molecular detection of large-bodied hammerhead sharks (order Carcharhiniformes), which have experienced substantial declines despite conservation management efforts (Abercrombie and others 2005). A 5-primer multiplex PCR assay on ITS-2 allowed for identification of the scalloped hammerhead (Sphyrna lewini), the great hammerhead (Sphyrna mokarran), and the smooth hammerhead (Sphyrna zygaena). Subsequent analysis of commercial samples revealed the widespread presence of these species in international trade and in the largest global fin market, located in Hong Kong. Species recognition of fins sold at this market is difficult because the fins are grouped into Chinese-name categories on the basis of their market value (Clarke and others 2006). To elucidate the relationship between market category and species, Clarke and others (2006) drew upon previous work with the ITS-2 locus and determined concordance with hypothesized category/species matches. Around 40% of the auctioned fin weight was found to consist of 14 species, including the shortfin mako, silky, sandbar, and hammerhead. The blue shark was reported to be the dominant species, with an auctioned fin weight amounting to 17% of the total market. The authors mentioned the need to develop diagnostic primers for the basking (Cetorhinus maximus) and whale (Rhincodon typus) sharks, which are listed in Appendix II of CITES and therefore require trade monitoring. To this effect, a 4-primer multiplex PCR assay for the ITS-2 locus was recently established for genetic identification of the basking shark (Magnussen and others 2007). After the method was developed and tested against 80 nontarget species, it was used to analyze shark fins obtained from a major U.S. seafood dealer and markets in Japan and Hong Kong. Basking shark was identified in the 2 fins in possession of the U.S. dealer; 3 out of 5 of the Japanese samples; and 13 out of 14 Hong Kong samples. Since the harvest and trade of this species is prohibited in the United States, this diagnostic test may become a valuable part of future law enforcement investigations.
Another potential gene target for shark species identification is the 5S rRNA gene alongside the variable NTS region. Pinhal and others (2008) exploited species-specific differences in this PCR product to differentiate 8 shark species, including the blacktip shark (Carcharhinus limbatus), the bigeye thresher (Alopias superciliosus), the tiger shark (Galeocerdo cuvier), and the blacknose shark (Carcharhinus acronotus). All 8 species gave distinct band patterns (130 to 1000 bp) in an agarose gel following PCR amplification with 1 primer set. The authors pointed out that this method may be simpler and less costly as compared to previous shark species identification studies that utilized multiplex PCR to simultaneously amplify 2 gene targets.
Fish in the order Lophiiformes are commonly referred to as anglerfish because they contain movable, rod-like structures above their mouths that are used to attract prey. Genetic identification research within this order has been carried out with the Lophiidae family (goosefish) (Table 11).
Table 11—. Genetic targets and methods used in authentication research involving anglerfish.
|Blackfin goosefish||PCR-RAPD||Random amplification of genome to produce “DNA fingerprint”||Fragment patterns vary with primer||Fresh||Ramella and others 2005|
|Angler and black-bellied angler||PCR-RFLP (6 enzymes)||mt tRNAGlu-cytochrome b||486 bp||Muscle tissue (implies the use of uncooked sample)||Sanjuan and others 2002|
Out of the 4 genera in the Lophiidae family, the Lophius genus has been reported to be the most important in terms of economic potential (Ramella and others 2005). For example, the angler (Lophius piscatorius) and black-bellied angler (Lophius budegassa) are important fishing resources in the northeastern Atlantic Ocean (Sanjuan and others 2002). Due to the greater consumer demand for the black-bellied angler, it is sometimes illegally substituted with the angler. Therefore, Sanjuan and others (2002) studied the use of PCR-RFLP on a 486 bp portion of the region coding for mt tRNAGlu-cyt b. Successful detection of the 2 species was reported, with no intraspecies polymorphism found in 15 individuals of each species.
Another anglerfish species of interest is the blackfin goosefish (Lophius gastrophysus), which has enjoyed high acceptance in the international market (Ramella and others 2005). This fish is widely available in the coastal waters of Brazil and has become one of that country's most valuable fisheries. Although the blackfin goosefish is readily identifiable with morphological features, processed products are susceptible to species substitution. To help prevent fraud, Ramella and others (2005) investigated the use of an RAPD protocol for identification of this fish. The optimal conditions for this procedure were developed, and the method was said to be a rapid and accurate tool for exposing commercial fraud.
Mixed fish groups
Several studies have reported genetic methods to differentiate fish from multiple taxonomic orders (Table 12). In the original paper outlining the development of the FINS procedure, Bartlett and Davidson (1992) reported the ability to successfully identify samples of salted cod, partially cooked battered cod, pickled herring, smoked mackerel, canned salmon, and smoked salmon. Identification was based on phylogenetic analysis of a 307 bp fragment of the mt cyt b gene sequence. The actual species of the samples under evaluation were not given in the published paper. However, the authors reported that the method was successful in determining species origin of the samples.
Table 12—. Genetic targets and methods used in authentication research involving mixed fish groups.
|Sardine, herring, cod liver, tuna (6 species), and Atlantic bonito||PCR-SSCP||mt cytochrome b||123 bp|
|Canned and frozen||Rehbein and others 1997|
|23 fish species (including eel, salmonids, gadoids, scombroids, goosefish, flatfish, and percoids)||PCR-RFLP (4 enzymes)||mt tRNAGlu-cytochrome b||464 bp||Frozen||Wolf and others 2000|
|36 fish species (including gadoids, eels, sardines, salmonids, and flatfish)||PCR-RFLP (7 enzymes)||mt cytochrome b||464 bp||Raw and ethanol-preserved or heat-treated, some mixed species samples||Hold and others 2001b|
|10 fish species (tuna sp., blue ling, carp, haddock, mackerel, mackerel shark, saithe, catfish, skipjack tuna, and Alaska pollock)||PCR-SSCP||mt cytochrome b||148 bp||Raw muscle tissue||Weder and others 2001|
|11 fish species (including gadoids, small pelagics, and scombroids)||PCR-RFLP (3 enzymes) and PCR-SSCP||mt cytochrome b||464 bp||Reference specimens and fish meal||Rehbein 2002|
|9 fish species (common dolphinfish, pompano dolphinfish, wahoo, Indo-Pacific sailfish, black marlin, blue marlin, shortbill spearfish, striped marlin, and swordfish)||Multiplex PCR||mt cytochrome b and 16S rRNA (positive control)||90 to 632 bp||White muscle tissue samples, preserved eggs and larvae||Hyde and others 2005|
|5 fish species (whiting, rainbow trout, flathead mullet, Pomatomus saltatrix, and Belone belone)||PCR-RFLP (5 enzymes)||mt cytochrome b||Not given||Frozen muscle tissue||Hisar and others 2006|
|Numerous fish species from multiple families and orders||DNA barcoding||COI||Approximately|
|Fresh, cooked, smoked, and roe||(Smith and others 2008; Wong and Hanner 2008; Yancy and others 2008)|
To improve recognition of canned fish species, Rehbein and others (1997) developed a protocol based on PCR-SSCP. The study investigated commercially canned samples of sardine (S. pilchardus), herring (C. harengus), and cod liver (G. morhua). Additional reference samples included raw cod muscle and canned muscle of bigeye tuna, albacore tuna, yellowfin tuna, Atlantic bonito, skipjack, little tunny (Euthynnus alleteratus), and A. thazard. The authors reported that different SSCP bands were acquired for each of these species; however, some of the bands were quite similar. Sprat (Sprattus sprattus) and Atlantic mackerel were also mentioned in the analysis, but the ability to differentiate between these 2 species was not reported. This protocol was recommended for fish species identification in the case of processed products with heavily degraded DNA, closely related species, and products containing fish tissue other than muscle. This method was later studied with many other fish and animal species, and species identification was possible with blue ling (Molva dypterygia), carp, haddock, mackerel, mackerel shark, saithe, catfish, skipjack tuna, and Alaska pollock (Weder and others 2001). On the other hand, in the case of several other fish species, analytical bands were either weak (for example, with Atlantic cod) or could not be obtained (for example, with Atlantic salmon, herring, European sprat, and European plaice) with PCR-SSCP.
Wolf and others (2000) investigated the ability to use PCR-RFLP to differentiate 23 species of fish, including eel, salmonids, gadoids, scombroids, goosefish, flatfish, and percoids. PCR was carried out with a primer adapted for amplification of a 464 bp fragment of mtDNA coding for tRNAGlu and cyt b in all species. Restriction digests with 4 different enzymes revealed species-specific profiles that allowed all 23 species to be differentiated. The authors described this method as simple and promising for the genetic detection of falsely labeled fish or fish products made of a single species. However, just 1 specimen was analyzed for each species of fish and the method was only tested with frozen samples. Future studies are necessary to investigate intraspecies polymorphism and analysis of cooked samples with degraded DNA.
Another study employed PCR-RFLP on a 464 bp portion of the mt cyt b gene to investigate the differentiation of 36 fish species from 7 families: Merlucciidae, Anguillidae, Clupeidae, Salmonidae, Soleidae, Pleuronectidae, and Scophthalmidae (Hold and others 2001b). One specimen from each fish was used as a reference sample, and the use of 7 restriction enzymes allowed for differentiation of 34 out of the 36 species. The only species that could not be differentiated were the European hake and the Senegalese hake (Merluccius senegalensis). Ten different mixed samples containing up to 3 fish species were also analyzed with this method. The restriction profiles were compared with those of the reference samples and all species were correctly identified. Mixed and single species samples were also analyzed following a heat treatment at 100 °C for 15 min. Compared to the reference samples, these processed samples showed no differences in the expected restriction banding patterns. To test the reproducibility of the method, 10 of the 36 species were then analyzed in a collaborative study with 12 European laboratories. The laboratories were provided with 10 unknown samples and had to identify them by comparison with the restriction profiles of reference samples. Out of 120 unknown samples analyzed 113 (94%) were correctly identified. Of the 7 samples that were not correctly identified 2 failed to undergo PCR amplification, 4 were assigned to the wrong species within the correct family, and 1 was not assigned to the right species or family. The authors recommended this protocol as an effective method for the detection of fraudulent species substitution in commercially sold fish, including processed products. PCR-RFLP analysis on a portion of the mt cyt b gene has also been reported to allow for differentiation of 5 fish species, including rainbow trout, flathead mullet (Mugil cephalus), and whiting (Hisar and others 2006). However, digestion with 5 different restriction enzymes was necessary to verify species.
Rehbein (2002) reported the application of DNA-based species identification methods to detect a wide range of fish commonly used in fish meal, including herring, capelin, anchovy, Atlantic horse mackerel, saithe, blue whiting, lesser sand eel (Ammodytes marinus), and great sand eel (Hyperoplus lanceolatus). Identification was based on amplification of the 464 bp region of mt cyt b, followed by RFLP and SSCP analysis. In 19 out of 22 purported “single-species” fish meal products, identification was possible utilizing a combination of RFLP and SSCP techniques. However, two of the sand eel fish meal products showed additional band patterns besides those expected for the 2 sand eel species, indicating the presence of multiple species.
Hyde and others (2005) developed a multiplex PCR assay to allow for the identification of 6 Indo-Pacific billfish species, 2 dolphinfish species, and wahoo (Acanthocybium solandri). The authors were focused on improving sampling efforts for billfish larvae and therefore a species identification method was developed that could be carried out onboard a ship. To allow for rapid species identification (within 3 h of sample acquisition), DNA was extracted from eggs and larvae through a rapid boiling method and multiplex PCR products were visualized using pre-cast agarose gels. Close to 300 samples of istiophorid larvae and over 200 eggs collected over the course of 2 cruises were analyzed using this method. The overall success rate for larval species identification was 95.2%, and the larval samples were identified as blue marlin and shortbill spearfish (Tetrapturus angustirostris). The success rate was lower for the egg samples (46%), and the samples were identified as swordfish, shortbill spearfish, common dolphinfish, wahoo, blue marlin, and pompano dolphinfish. In addition to showing high potential for use in determination of the spatial and temporal distribution of spawning habitats for these species, the multiplex assay developed here may also be applied to identification of billfish, dolphinfish, and wahoo species in fresh/frozen or lightly processed food products.
The Barcode of Life Campaign (BOL) is focused on sequencing an approximately 650 bp portion of the COI gene in all organisms and then using this sequence information to identify species (Hebert and others 2003a, 2003b). Several studies have been published investigating the potential for COI to be used in fish species identification, including a paper analyzing the use of DNA barcodes within the USFDA Regulatory Fish Encyclopedia (Yancy and others 2008). The authors obtained DNA barcodes for 72 fish species from 27 different families, including Gadidae, Lutjanidae, Salmonidae, and Scombridae, and reported that each species had a unique, diagnostic COI sequence that would allow for species identification. Furthermore, in a blind study of 60 unknown fish muscle samples, DNA barcoding allowed for correct species identification in all cases, indicating the usefulness of this technique in uncooked fish products. In a study examining the usefulness of DNA barcoding for species identification of commercial fish products, the authors tested a variety of samples, including sushi, fresh fillets, cooked fish, and roe (Wong and Hanner 2008). Although the reference library is still being constructed, the BOL database (BOLD) was still able to provide sequence matches of >97% similarity for 90 out of the 91 fish products tested. Over 25% of the products were found to be mislabeled, and the most commonly substituted fish was red snapper. Some other examples of mislabeling include white tuna sushi substituted with the less expensive Mozambique tilapia (Oreochromis mossambicus) and “eco-friendly” Alaskan halibut (that is, Pacific halibut, Hippoglossus stenolepis) substituted with the endangered Atlantic halibut (Hippoglossus hippoglossus).
The use of DNA barcoding to identify species in smoked samples has also been investigated (Smith and others 2008). Smoked products from fish in 10 families and 4 orders (Salmoniformes, Anguilliformes, Gadiformes, and Perciformes) were tested with this method. Barcode sequences (520 to 621 bp) were obtained for all smoked products except 1 hoki (blue grenadier) sample, and species were identified with 99% to 100% sequence similarities to reference specimens in BOLD and GenBank. While DNA barcoding has been shown to be useful for species identification of fresh and lightly processed products, the ability to identify species in heavily processed fish products has not been thoroughly examined. In this regard, a promising study focused on museum specimens reported that shorter DNA sequences (approximately 100 bp) within the barcoding region can be effective for species identification (Hajibabaei and others 2006). These results imply that DNA barcoding may prove to be suitable for species identification of commercially canned fish products, as DNA fragments isolated from these products are generally shorter than 360 bp.
Shellfish Species Substitution
While genetic methods for species identification have been extensively developed for use with a variety of fish species, research with shellfish (that is, mollusks and crustaceans) has shown delayed progress in some areas. One reason for this has been that amplification of DNA from mollusks, which includes gastropods, bivalves, and cephalopods, is complicated by compounds secreted by these organisms that act as PCR inhibitors (Fernandez and others 2000). These compounds, mucopolysaccharides and polyphenolic proteins, have been reported to copurify with DNA and interfere with the enzymatic processing of nucleic acids. Therefore, some of the universal primers that have been successful with fish species have shown limited success with mollusks (Fernandez and others 2000). Despite this setback, numerous studies have reported the successful development of methods for the differentiation of a variety of shellfish species (Table 13 to 16).
Table 13—. Genetic targets and methods used in authentication research involving cephalopods.
|5 cephalopod species (3 Ommastrephidae and 2 Loliginidae)||PCR-RFLP (1 enzyme)||mt 16S rRNA||600 to 700 bp||Frozen||Colombo and others 2002|
|10 cephalopods (1 octopus and 9 squids from families Ommastrephidae and Loliginidae)||FINS||mt 16S rRNA||Universal: 500 bp (for sequencing)|
Designed: 200 bp
|Fresh, frozen, processed, or formalin-fixed and ethanol-preserved||Chapela and others 2002|
|8 cephalopods (families Loliginidae and Ommastrephidae)||FINS and PCR-RFLP (2 enzymes)||mt cytochrome b||297 bp||Fresh/frozen, canned, and “squid rings”||Chapela and others 2003|
|23 cephalopods (families Loliginidae, Ommastrephidae, Sepiidae, and Octopodidae)||FINS and PCR-RFLP (3 enzymes)||mt cytochrome b||208 bp|
|Fresh, frozen, precooked, canned, and ethanol-preserved||Santaclara and others 2007|
Table 14—. Genetic targets and methods used in authentication research involving bivalves.
|3 oyster species (American, Pacific, and C. ariakensis)||PCR-RFLP (2 enzymes)||mt 16S rRNA||Approximately|
|Uncooked muscle tissue||Foighil and others 1995|
|2 oyster species (C. angulata and Pacific)||PCR-RFLP (4 enzymes)||mt COI and mt 16S rRNA||660 bp (COI)||Wild and hatchery-raised oysters||Boudry and others 1998|
|8 oyster species (C. angulata, Pacific, C. ariakensis, American, flat, dwarf, C. sikamea, and Crassostrea gasar)||PCR amplification and sequence analysis||HindIII satellite DNA||166 ± 2 bp||Uncooked muscle tissue||Lopez-Flores and others 2004|
|4 oyster species (C. angulata, Pacific, flat, and dwarf)||6-primer multiplex PCR||Nuclear 5S rRNA||Species-specific||Uncooked mantle tissue||Cross and others 2006|
|5 oyster species (Pacific, C. angulata, C. ariakensis, C. sikamea, and C. hongkongensis) and 4 oyster genera (Crassostrea, Saccostrea, Ostrea, and Hyotissa)||Multiplex PCR||mt COI Nuclear 28S rRNA||166 to 1200 bp||Fresh, ethanol-preserved tissue||Wang and Guo 2008a|
|8 oyster species or species pairs (C. ariakensis, Pacific/C. angulata, flat/O. angasi, C. hongkongensis, C. sikamea, O. conchaphila, American/C. rhizophorae, and S. echinata/S. glomerata)||PCR||ITS-1||500 to 600 bp||Uncooked adductor muscle||Wang and Guo 2008b|
| ||ITS-2|| || || |
|3 clam species (grooved carpet shell, pullet carpet shell, and Japanese carpet shell)||PCR-RFLP (2 enzymes)||Nuclear α-actin||520 bp||Fresh/frozen||Fernandez and others 2000|
| ||ITS-1 and ITS-2||1074 bp|
|Fresh/frozen||Fernandez and others 2001|
|4 clam species (grooved carpet shell, pullet carpet shell, Japanese carpet shell, and banded carpet shell)||PCR-RFLP (2 enzymes)||mt 16S rRNA||502 to 545 bp||Fresh/frozen||Fernandez and others 2002b|
|2 clam species (grooved carpet shell and pullet carpet shell)||PCR-SSCP||Nuclear α-actin||150 bp||Fresh/frozen||Fernandez and others 2002a|
|5 razor clam species (Ensis arcuatus, E. siliqua, E. directus, E. macha, and S. marginatus)||PCR-RFLP (2 enzymes)||Nuclear 5S rRNA||Approximately|
|Fresh, ethanol-preserved tissue, and canned samples||Fernandez-Tajes and Mendez 2007|
|2 razor clam species (E. arcuatus and E. siliqua)||PCR-RFLP (1 enzyme)||Nuclear ITS-1||Approximately|
|Fresh and ethanol-preserved tissue||Freire and others 2008|
|3 mussel species (blue, Mediterranean, and foolish)||PCR||Adhesive protein gene||126 bp|
|Not given (implies the use of fresh/frozen specimens)||Inoue and others 1995|
|5 bivalve species (Atlantic bay scallop, Mercenaria mercenaria, Mulinia lateralis, Spisula solidissima, and Mya arenaria)||Multiplex PCR||COI 18S rRNA||<400 bp (COI)|
430 bp (18S)
|Fresh gill tissue and ethanol-preserved larvae||Hare and others 2000|
|4 mussel species (blue, Chilean blue, Mediterranean, and green-lipped)||PCR-RAPD||Random amplification||Species-specific||Fresh/frozen||Rego and others 2002|
|8 mussel genera (Mytilus, Perna, Aulacomya, Semimytilus, Brachidontes, Choromytilus, and Perumytilus) and species within the genera Perna, Choromytilus, and Mytilus||PCR-RFLP FINS||Nuclear 18S rRNA|
Adhesive protein gene
|167 to 924 bp|
100 to 394 bp
126 to 180 bp
|Fresh/frozen, ethanol-preserved, and canned||Santaclara and others 2006b|
|M. varia, great Atlantic scallop, queen scallop, and C. distorta||PCR-RFLP (1 enzyme)||nuclear 5.8S rRNA, ITS-1, and ITS-2||366 to 767 bp||Fresh, frozen, canned, and ethanol-preserved||Lopez-Pinon and others 2002|
|Scallops (P. jacobaeus and P. maximus) compared with vertebrate fish||PCR with scallop-specific and vertebrate-specific primers||mt 16S rRNA||Not given||Commercial pectinid scallop product (frozen, cut, and seasoned)||Colombo and others 2004|
|M. varia, great Atlantic scallop, and Mytilus spp.||Multiplex PCR||mt 16S rRNA nuclear 18S rRNA||190 to 398 bp||Larvae, fresh/frozen, and ethanol-preserved||Bendezu and others 2005|
|Iceland scallop and American sea scallop||Multiplex PCR||mt COI||459 bp|
|Fresh/frozen||Marshall and others 2007|
|C. farreri, noble scallop, yesso scallop, and Atlantic bay scallop||Species-specific PCR||Microsatellites||94 to 284 bp||Fresh/frozen, canned, and dried||Zhan and others 2008|
Table 15—. Genetic targets and methods used in authentication research involving gastropods.
|Abalone (H. midae, H. spadicea, and H. rubra)||PCR with generic and species-specific primers and PCR-RFLP (1 enzyme)||lysin gene||700 to 1300 bp (fresh, dried, and cooked)|
146 bp (canned)
|Fresh/frozen, dried, cooked, and canned||Sweijd and others 1998|
|Abalone (H. asinina, H. ovina, and H. varia)||PCR with species-specific primers, PCR-RFLP (3 to 4 enzymes) and RAPD||mt 16S rRNA, nuclear 18S rRNA, and RAPD||580 bp (16S)|
900 bp (18S)
|Fresh/frozen||Klinbunga and others 2003|
Table 16—. Genetic targets and methods used in authentication research involving crustaceans.
|Mud crab (S. serrata, S. olivacea, S. paramamosain, and S. tranquebarica)||PCR and PCR-RFLP (1 to 2 enzymes)||mt 16S rRNA nuclear ITS1||Various lengths (ITS1)|
562 bp (16S)
|Not specified (implies the use of fresh/frozen tissue)||Imai and others 2004|
|4 penaeid shrimp species (green tiger prawn, caramote prawn, deep-water rose shrimp, and M. monoceros)||PCR-RFLP (2 enzymes)||mt cytochrome b||356 bp||Fresh/frozen||Hisar and others 2008|
|Tanner crab, queen scallop, and hybrids||SNP genotyping assay||mt 16S rRNA nuclear ITS1||225 bp (ITS1)|
464 bp (16S)
|Fresh muscle tissue||Smith and others 2005|
Cephalopods are members of the mollusk class Cephalopoda, which includes octopus, squid, cuttlefish, and nautilus. Most genetic identification research on cephalopods has been carried out with squids belonging to the families Ommastrephidae (order Decapoda, suborder Oegopsina) and Loliginidae (order Decapoda, suborder Myopsina) (Table 13). Squids are a popular seafood product in Mediterranean countries and are generally eviscerated, skinned, and sold as squid rings (Chapela and others 2003). Loliginidae species, such as the swordtip squid (Loligo edulis) and Patagonian squid (Loligo gahi), are commonly preferred over Ommastrephidae species, which include the Wellington flying squid (Nototodarus sloani), the Argentine shortfin squid (Illex argentinus), and the southern shortfin squid (Illex coindetii). Due to the difference in price of the 2 types of squid, Ommastrephidae species are sometimes illegally substituted for Loliginidae species. To help ensure that species are labeled correctly, several studies have investigated the use of either FINS or PCR-RFLP for cephalopod species identification (Chapela and others 2002, 2003; Colombo and others 2002; Santaclara and others 2007).
In one study, PCR-RFLP on a 600 to 700 bp portion of the mt 16S rRNA gene was carried out with 3 Ommastrephidae species and 2 Loliginidae species (Colombo and others 2002). Digestion with 1 restriction enzyme showed characteristic bands for the 2 families and allowed for the differentiation of the 2 Loligo species (Patagonian squid and swordtip squid). However, species differentiation was not possible for the 3 squids analyzed within the Ommastrephidae family: southern shortfin squid, Todarodes sagittatus, and Todaropsis eblanae. Another study published in the same year employed FINS on a portion of the mt 16S rRNA gene to differentiate 7 species of squids (families Ommastrephidae and Loliginidae) and 1 octopus (Bathypolypus arcticus) (Chapela and others 2002). Two species could not be differentiated: the southern shortfin squid and the northern shortfin squid (Illex illecebrosus). Using this procedure, the cephalopod species in 8 commercial seafood products (7 lots of squid rings and 1 canned “octopus squid”) were then identified. The majority of the squid rings were found to contain Wellington flying squid and the “octopus squid” product contained Argentine shortfin squid. However, use of this region of the 16S rRNA gene in species identification may prove to be problematic because most of the fragment has a well-conserved sequence and it does not allow for the unequivocal detection of all commercially important species of cephalopods (Chapela and others 2003).
To investigate the suitability of the mt cyt b gene over the mt 16S rRNA gene for cephalopod species identification, Chapela and others (2003) carried out both FINS and PCR-RFLP on a 297 bp fragment of the mt cyt b gene for 8 of the 9 squid species analyzed in the 2002 study. Although most of the species could be identified with both FINS and PCR-RFLP, neither method was able to differentiate southern shortfin squid and northern shortfin squid. FINS with the mt cyt b fragment was then used to successfully identify cephalopod species in 17 commercial seafood products: 16 lots of squid rings and 1 canned sample labeled “octopus-squid.” Argentine shortfin squid was identified in the “octopus squid” product and in the majority of squid ring products. As in the previous study by Chapela and others (2002), none of the commercial products analyzed contained species from the more expensive Loliginidae family. A subsequent study reported the differentiation of more than 20 cephalopod species, including squids, octopi, and sepias, using FINS and PCR-RFLP on fragments of mt cyt b (Santaclara and others 2007). Both PCR-RFLP and FINS could be used with a 651 bp fragment to differentiate 21 out of 23 species, whereas identification of these species with a 208 bp fragment was only possible with FINS. None of the techniques was able to differentiate southern shortfin squid and northern shortfin squid, as the mt cyt b sequences were identical for the fragments analyzed. Once the techniques were validated with reference samples, a market study was carried out using a combination of PCR-RFLP and FINS to diagnose species in 15 commercial cephalopod samples, including frozen squid rings and canned octopus. Overall, these research efforts have provided a reliable means for identification of cephalopod species in a variety of processed forms; however, use of an alternative DNA fragment will be necessary for differentiation of the southern shortfin squid and northern shortfin squid.
The mollusk class Bivalvia comprises bivalve organisms such as clams, oysters, mussels, and scallops. Genetic identification research has been carried out with a number of members of this class, including those under the orders Veneroida (Venus clams), Mytiloida (mussels), and Ostreoida (scallops and oysters) (Table 14).
Clams The grooved carpet shell (Ruditapes decussatus), pullet carpet shell (Venerupis pullastra), Japanese carpet shell (Ruditapes philippinarum), and banded carpet shell (Tapes rhomboides) are the 4 most popular clams in the Spanish market (Fernandez and others 2002b). The grooved carpet shell is the most preferred species due to its delicate taste and meat texture; however, it is also the most expensive and is sold at prices up to 4-fold higher than the other 3 species (Fernandez and others 2000). Although whole clams can be identified by morphological properties, species identification is complicated when the shells are removed and the clam meat is mixed with other ingredients. Two studies reported the ability to differentiate the grooved carpet shell, pullet carpet shell, and Japanese carpet shell using PCR-RFLP analysis with 2 restriction enzymes. In one study, the analysis was based on a 520 bp fragment of the nuclear α-actin gene (Fernandez and others 2000) and in the other study, analysis was based on a larger fragment (1074 to 1195 bp) of ITS-1 and ITS-2 (Fernandez and others 2001). Fernandez and others (2000) reported that 4 different DNA extraction methods were attempted before the clam DNA could be successfully amplified by PCR. To provide a simple technique for the differentiation of grooved carpet shell and pullet carpet shell, a PCR-SSCP assay was also developed based on a short fragment (150 bp) of the α-actin gene (Fernandez and others 2002a). This research group also employed PCR-RFLP on a 502 to 545 bp fragment of the mt 16S rRNA gene to distinguish grooved carpet shell, pullet carpet shell, Japanese carpet shell, and banded carpet shell (Fernandez and others 2002b). To overcome complications with PCR inhibitors, the clam DNA was purified with a commercial kit prior to PCR amplification. The method was recommended as a powerful technique for genetic differentiation of these clam species and a less costly alternative to sequencing of PCR products.
The European market for razor clams (genera Ensis and Solen) has shown increased economic importance over the past few years (Fernandez-Tajes and Mendez 2007). Several razor clams are native to Europe, including Solen marginatus, Ensis siliqua, and E. arcuatus. The latter two are the favored razor clam species for use in the canned and fresh markets, respectively (Freire and others 2008). However, these species are very similar to the less valuable, nonnative razor clam species E. directus and E. macha. Therefore, a PCR-RFLP method to differentiate these 5 razor clam species was developed based on the 5S rRNA region of nuclear DNA. Amplification of this fragment with universal primers allowed for the identification of S. marginatus, and digestion with a combination of 2 restriction enzymes then allowed for differentiation of the remaining 4 species. Although the fragments used exceeded previous limits for PCR amplification of DNA from canned fish and seafood, the authors reported the ability to use the method with canned clam products. Based on the agarose gel figures shown in the article, several of the diagnostic bands from canned products were concentrated enough to be readily verified; however, others appeared too faint for reliable species identification. In another study, PCR-RFLP on the ITS-1 region was used to differentiate E. siliqua from E. arcuatus (Freire and others 2008). Despite the importance of E. siliqua in the canning industry, the DNA fragment used in this study was relatively large (560 bp) and the method was not tested with canned clam products.
Mussels The order Mytiloida encompasses the common mussels and includes the families Mytilidae (sea mussels) and Pinnidae (pen shells). Three species within the genus Mytilus account for the majority of commercial activity: the blue mussel (M. edulis), the foolish mussel (M. trossulus), and the Mediterranean mussel (M. galloprovincialis) (Rodger 2006). Most mussels are sold fresh in the shell; however, additional food uses include steamed, canned, baked, deep-fried, or smoked. Although some genetic research studies with mussels have been carried out, most have not focused specifically on preventing fraudulent species substitution. For example, one research group investigated the identification of blue mussel genotypes along the west coast of Canada to study their geographic distribution (Heath and others 1995), while another characterized satellite DNAs found in 4 species of Mytilus mussels (blue mussel, Mediterranean mussel, foolish mussel, and M. californianus) for evolutionary biology purposes (Martinez-Lage and others 2002), and 2 others focused on the population genetics of Mytilus chilensis through the use of RAPD analysis (Toro and others 2004) and allozymic variation (Toro and others 2006). One drawback of early studies on genetic identification of mussels was the occurrence of intrapopulation polymorphisms in both mtDNA and nuclear DNA, which complicates species diagnosis. However, Inoue and others (1995) were able to differentiate the blue mussel, the Mediterranean mussel, and the foolish mussel using small fragments of the gene coding for the nonrepetitive region of an adhesive foot protein. Later, Rego and others (2002) investigated the use of RAPD as an alternative technique to identify 4 different mussel species: the blue mussel, M. chilensis, the Mediterranean mussel, and the New Zealand or green-lipped mussel (Perna canaliculus). This technique did not allow for the differentiation of all species; however, the New Zealand mussel could be distinguished from Mytilus mussels and the Mediterranean mussel could be differentiated from the other 3 mussels.
Santaclara and others (2006b) incorporated previous findings by Inoue and others (1995) into the development of a comprehensive DNA-based protocol for the identification of 8 genera of mussel species, and numerous individual species within three of these genera. This study called for the use of a combination of multiplex PCR, PCR-RFLP, and FINS on 3 different genetic targets: the nuclear 18S rRNA, ITS-1, and the polyphenolic adhesive protein gene. The method was reported to be successful in the determination of mussel genus or species in food products, included canned mussels.
Oysters Oysters that are found on the commercial food market belong to the family Ostreidae and include the Pacific cupped oyster (Crassostrea gigas), American cupped oyster (Crassostrea virginica), and the European flat oyster (Ostrea edulis). Oysters are generally served fresh on the half shell or they are shipped in the form of frozen meats to be used primarily by the restaurant and hotel industry (Rodger 2006). Several studies have been carried out to establish genetic methods for oyster identification; however, most have been focused on monitoring and management efforts. One study reported the characterization of DNA sequence polymorphism for the mt 16S rRNA gene in Pacific cupped oyster, Crassostrea sikamea, and the Olympia flat oyster (Ostrea lurida) (Banks and others 1993). This same gene was later used with PCR-RFLP to discriminate among the American cupped oyster and 2 Asian oyster species: the Pacific cupped oyster and Crassostrea ariakensis (Foighil and others 1995). PCR-RFLP was also reported to enable successful identification of Crassostrea angulata and the Pacific cupped oyster (Boudry and others 1998). Although fragments of both the mt 16S rRNA and mt COI genes were analyzed, only the COI fragment showed sufficient polymorphism for use in a restriction digest. A later study focused on use of the COI gene, along with the nuclear 28S ribosomal RNA gene, to develop multiplex PCR assays for identification of oyster genus and species (Wang and Guo 2008a). Multiplex PCR using fragments of COI allowed for the differentiation of all 5 Crassostrea oyster species found in China, whereas multiplex PCR with fragments of 28S rRNA allowed for identification of 2 Crassostrea species and differentiation of 4 oyster genera (Crassostrea, Saccostrea, Ostrea, and Hyotissa).
Another genetic marker reported to be useful in the genetic study of oysters is HindIII satellite DNA. This satellite DNA was analyzed in 8 different species of Asian, European, American, and African oysters and was reported to allow for differentiation of oyster species, especially in the case of the European flat oyster and Ostreola stentina (Lopez-Flores and others 2004). An 8-primer multiplex PCR assay allowed for the differentiation of 4 species of oyster: flat, dwarf, Pacific, and C. angulata (Cross and others 2006). The analysis was carried out on repetition units of 5S rDNA and resulted in the production of PCR amplicons with species-specific lengths. This method was reported to be quick and efficient, with potential applications in traceability and management efforts.
To increase the number of oyster species that can be analyzed simultaneously, Wang and Guo (2008b) developed a PCR assay based on ITS-1 and ITS-2. Out of 12 oyster species investigated with these genetic markers, the results showed diagnostic bands for 8 different oyster species or species pairs. This assay proved to be advantageous in that only 2 primer sets were required, simultaneous amplification was possible, and a wide range of species could be differentiated. On the other hand, the ITS assay was limited in its ability to differentiate several species pairs, such as the Pacific cupped oyster and C. angulata, which have been successfully identified with other PCR-based assays (Boudry and others 1998; Wang and Guo 2008a).
Scallops There are around 300 species of scallops worldwide, but no more than 20 of these are available in sufficient quantities to be consumed commercially (Zhan and others 2008). Some common scallop species found on the commercial market include the American sea scallop (Placopecten magellanicus), Iceland scallop (Chlamys islandica), and the Atlantic bay scallop (Argopecten irradians). Scallops are widely consumed in Europe, and some of the popular products include scallop adductor muscles and scallop roe. On the other hand, in North American markets, shucked scallop muscles are commonly served breaded and deep-fried (Rodger 2006).
A PCR-RFLP method was developed to identify 4 scallops species that are distributed throughout European coastlines, the first three of which are commercially harvested: Mimachlamys varia, queen scallop (Aequipecten opercularis), great Atlantic scallop (Pecten maximus), and Chlamys distorta (Lopez-Pinon and others 2002). Using a region of ribosomal DNA spanning the 5.8S rRNA gene along with the flanking ITS-1 and IT-2, the authors were able to identify species in both fresh and canned products.
In a study into genetic identification of scallops, the possibility of commercial fraud in frozen mollusk products was investigated (Colombo and others 2004). In this case, a commercial product labeled as pectinid scallop (mainly Pecten jacobaeus and Pecten maximus) was suspected to contain vertebrate fish. A set of primers was designed for the specific amplification of a region of the mt 16S rRNA gene in scallops. The product was tested with these primers along with a set of vertebrate fish primers designed to amplify a portion of the mt cyt b gene. The test results revealed that the product did indeed contain scallop meat, although the exact species was not determined. A subsequent study utilized the mt 16S rRNA gene in a multiplex PCR assay to differentiate Mytilus spp. (M. edulis, M. galloprovincialis, and hybrids of the two) from the great Atlantic scallop and M. varia (Bendezu and others 2005). The queen scallop could not be differentiated based on this gene fragment, and all species gave similar-sized fragments in an additional assay based on the nuclear 18S rRNA gene. Multiplex PCR has also been used with the COI gene to differentiate the American sea scallop and the Iceland scallop (Marshall and others 2007). Both species are commercially harvested in the North Atlantic Ocean; however, there were concerns regarding the possession and retention of sea scallops from a closed fishery as bycatch in the Iceland scallop fishery. The authors utilized the newly developed multiplex PCR assay to analyze over 900 scallops seized from 2 fishing vessels. The results showed excessive amounts of sea scallops from a closed fishery in both cases, indicative of scallop poaching.
Species-specific microsatellite markers (94 to 284 bp) have also been utilized in scallop species identification (Zhan and others 2008). Four species were differentiated with this method: Chlamys farreri, noble scallop (Mimachlamys nobilis), yesso scallop (Patinopecten yessoensis), and the Atlantic bay scallop. These scallops are of economic importance in both aquaculture and fisheries throughout Asia and North America. Utilizing this method, the authors were able to detect scallop species in a variety of processed products, including frozen, dried, and canned scallops. Although species were detected in all dried (n = 80) and canned (n = 30) samples tested, species could not be detected in 20 out of the 30 frozen samples analyzed, possibly due to the presence of additional scallop species not accounted for in this assay. Therefore, future work in this area may call for the development of diagnostic tests that account for a greater number of commercially important scallop species.
Mixed bivalves One study reported the ability to differentiate 5 commercially important bivalve species (Atlantic bay scallop and 4 clam species) through a multiplex PCR assay based on COI (Hare and others 2000). The purpose of the study was to allow for species identification of marine bivalve larvae following collection and sampling. The assay could be carried out successfully in a single tube containing 10 species-specific primers targeting regions of COI (<400 bp) and 2 universal primers targeting a 430 bp fragment of the positive control (18S rRNA). After the assay was optimized with fresh bivalve tissue, it was tested with numerous samples of cultured larvae and larve collected in the field with plankton tows. Overall, the reaction was found to be capable for the differentiation of all 5 bivalve species with 92% accuracy and 1.4% false positives. Although this assay was developed for analysis of larval samples, it may readily be applied for the detection of species substitution in foods, as the target species are commercially important and the genetic markers were small enough to be detected in fresh or lightly processed foods.
Marine organisms in the mollusk class Gastropoda include abalone, limpets, cowries, and conch. Genetic identification research for the prevention of economic fraud with gastropods has primarily been carried out on abalone species (order Archaeogastropoda; Table 15).
Abalone There are at least 20 commercially important species of abalone worldwide, many of which are harvested from natural stocks (Klinbunga and others 2003). Due to increased poaching and exploitation of a local abalone species (Haliotis midae) in South Africa, one study investigated the ability to differentiate this species from 2 other abalone species: Haliotis spadicea and Haliotis rubra (Sweijd and others 1998). PCR with generic and species-specific primers resulted in the amplification of regions of the lysin gene from fresh, dried, cooked, and canned samples of all 3 species. Although the species could be differentiated, this approach relies on a negative result (nonamplification) to show that a species is not present. To promote legal confidence in the test results, a PCR-RFLP procedure was also considered as a potential complement to the PCR approach. Four different restriction enzymes were found to have the ability to differentiate H. midae and H. spadicea (no results reported for H. rubra). Forty individuals were analyzed, with no intraspecies polymorphism observed.
In a phylogenetic study, tandemly repeated satellite DNA was analyzed for 5 species of Eastern Pacific abalone (genus Haliotis): red abalone (H. rufescens), pinto abalone (H. kamtschatkana), pink abalone (H. corrugata), white abalone (H. sorenseni), and flat abalone (H. walallensis) (Muchmore and others 1998). Based on the study results, species-specific consensus sequences were established that allowed for differentiation of the 5 species in the case of population identification, taxonomy, hybrid parentage identification, and forensic studies. A later study involved the use of species-specific PCR, PCR-RFLP, and PCR-RAPD to differentiate 3 species of abalone in Thailand (genus Haliotis): H. asinina, H. ovina, and H. varia (Klinbunga and others 2003). PCR-RFLP was carried out with fragments of both the mt 16S rRNA gene (580 bp) and the nuclear 18S rRNA gene (900 bp). Differentiation of all 3 species could be achieved either by digestion of the 16S rRNA gene fragment with 1 restriction enzyme or by RAPD analysis. Although species-specific PCR on the 16S rRNA gene fragment was reported to be more accurate and convenient than RAPD, it was only successfully developed in H. asinina and H. varia.
Crabs, crayfish, lobster, and shrimp are all members of the order Decapoda, within the subphylum Crustacea. Species identification research in this area has been focused on crabs and shrimp (Table 16).
Crabs Mud crabs (genus Scylla) are an important component of commercial fisheries and aquaculture in the Indo-West Pacific countries. The differentiation of 4 species of mud crab was recently investigated using both PCR-RFLP and species-specific PCR amplification (Imai and others 2004). PCR-RFLP analysis on a 562 bp fragment of the mt 16S rRNA gene allowed for identification of all 4 species. These species could also be identified based on polymorphisms in the ITS-1 region, in which 2 species were differentiated by differences in fragment length and the other 2 were identified following digestion with a restriction enzyme. Although this study was primarily focused on developing genetic markers for hybridization breeding studies, these procedures could readily be applied to species identification for the prevention of mislabeled products.
A method for high-throughput detection of Tanner crabs (Chionoecetes bairdi) illegally harvested in the Alaskan snow crab (queen crab, Chionoecetes opilio) fishery was recently reported (Smith and others 2005). This technique was based on SNPs in the ITS-1 region and the mt 16S rRNA gene and allowed for rapid and successful differentiation of the 2 species of crabs along with their hybrids.
Shrimps and prawns Shrimps and prawns in the families Penaeidae and Pandalidae are commonly found on the international food market. These species are harvested and farmed throughout the world and are consumed in a variety of ways, including peeled and precooked, deep-fried, steamed, and as a flavoring or soup base. Due to their body size, the green tiger prawn (Penaeus semisulcatus) and caramote prawn (Penaeus kerathurus) are more valuable in Turkish markets than the deep-water rose shrimp (Parapenaeus longirostris) and Metapenaeus monoceros. To facilitate species identification in processed shrimp products, these 4 species of penaeid shrimps were recently differentiated using PCR-RFLP on a 356 bp fragment of mt cyt b (Hisar and others 2008). Species were readily identified in uncooked specimens; however, the applicability of this method to heavily processed shrimp products remains to be investigated.
DNA-Based Monitoring of Commercial Whalemeat Products
Over the past 200 y, a number of whale species have been overexploited through unregulated commercial whaling practices (Braham 1984; Clapham and others 2002). These include several closely related species in the Balaenopteridae family, such as the blue whale (Balaenoptera musculus), fin whale (Balaenoptera physalus), humpback whale (Megaptera novaeangliae), and sei whale (Balaenoptera borealis). In 1982, the Intl. Whaling Commission (IWC) voted to impose an international moratorium on commercial whaling, which went into effect in 1986 (Morell 2007). Despite this moratorium, commercial whalemeat can still be found in countries such as Norway, Iceland, Japan, and the Republic of (South) Korea. In Japan, commercial whalemeat products are commonly obtained through a controversial scientific whaling program, which allows for annual harvests of hundreds of minke whales (Balaenoptera acutorostrata) along with smaller numbers of Bryde's (Balaenoptera edeni), sei, and sperm whales (Physeter catodon) (Morell 2007). This program is permitted under a clause in the 1946 Intl. Whaling Convention that allows whales to be taken for scientific research (Normile 2000). An additional route by which whales may enter the commercial market in both Japan and South Korea is through fisheries bycatch. Furthermore, illegal hunting of whales for distribution in the commercial market is believed to occur under the pretext of fisheries bycatch or scientific whaling (Baker and Palumbi 1994; Baker 2008). This type of market fraud carries with it considerable economic incentives, as the wholesale value of a minke whale is reportedly between US$30000 and US$100000 (Palumbi 2007; Baker 2008).
One way to monitor illegal whaling operations is to track individual, legally obtained whales through the marketplace and detect any unregistered individuals through market surveys. However, morphological identification of whale meat, skin, and blubber from closely related species is difficult in itself, and further complications arise with identification of processed commercial whalemeat products. In Japan, these products are generally sold either fresh as “sashimi” or in processed forms that may be smoked, dried, marinated, or canned, and inaccurate or incomplete species labeling is common (Baker and others 1996; Baker 2008). In South Korea, most whalemeat is sold “freshly parboiled” and species names are almost never reported. To help monitor whaling of protected species, a DNA-based approach was developed based on sequencing and phylogenetic analysis of a segment of the mtDNA control region (155 to 378 bp) (Baker and Palumbi 1994). Using this method, Baker and Palumbi (1994) analyzed 16 commercial products purchased in Japan, including dried and salted meat strips, unfrozen sliced meat (sashimi), and meat marinated in sesame oil and soy sauce. The products were all labeled “kujira,” meaning whale, and were found to contain a surprising variety of cetacean species, such as minke whale, fin whale, humpback whale, and 1 or 2 dolphin species. When these results were compared to current whaling activity, the authors found the regulatory system to be inadequate for verifying harvest and trade operations in commercial and scientific whaling. Inconsistencies between catch records and commercial products were also reported in a subsequent study, which took into account commercial whalemeat purchased in South Korea (Baker and others 1996). Using the molecular method previously established (Baker and Palumbi 1994), commercial products in South Korea were found to contain numerous species, including dolphin (Genus Lagenorhynchus), a beaked whale, Northern Hemisphere minke whales, and 2 unrecognized species or subspecies of baleen whales. Out of 13 minke whale samples, at least seven of them originated from different individual whales.
The case of the North Pacific minke whale
In both South Korea and Japan, the North Pacific minke whale (B. acutorostrata scammoni) is the most common whale species that is found on the market as a result of illegal, unreported, or unregulated (IUU) exploitation (Baker 2008). There are 2 sources for North Pacific minke whale products in Japan and Korea: the offshore waters of the western North Pacific (“O” stock) and the Sea of Japan (“J” stock). The “O” stock is known to be a relatively abundant stock and its whales are commonly captured under special permit in Japan's scientific whaling program. On the other hand, the “J” stock is a depleted stock that is subject to fisheries bycatch and some degree of illegal whaling by both Japan and Korea (Baker 2008). In a 2-tiered genetic analysis, phylogenetics were combined with intraspecific haplotype frequency to identify species origin and, in some cases, stock origin of 655 whalemeat products purchased in Japan (Baker and others 2000). The products were found to contain a variety of species, including 8 species or subspecies of baleen whales (six of which are protected), sperm whales, beaked whales, killer whales, dolphins, and even domestic sheep and horses. A total of 81 products were identified as North Pacific minke whale and up to 43% of these samples were estimated to originate from the depleted “J” stock. Another study utilized phylogentic analysis of the mtDNA control region, along with haplotype frequencies and DNA profiling for up to 6 microsatellite loci to identify species, stock origin, and individuals in commercial products purchased on Japanese and Korean markets (Dalebout and others 2002). Out of 99 North Pacific minke whale products purchased in Japan, 86 represented unique individual whales and 33.7% of these were estimated to originate from the “J” stock. In Korea, 34 unique individuals were found in 42 North Pacific minke whale products, and only three of these had haplotypes expected for the “O” stock.
DNA-based identification tools
DNA-based tools that have been developed to assist with molecular genetic identification of whalemeat products include a DNA register for minke whales and the web-based program DNA Surveillance (http://www.dna-surveillance.auckland.ac.nz). The DNA register was created to help control whalemeat trade in Norway, and its effectiveness was tested using 20 whalemeat products purchased on the Norwegian market, along with 2 samples of minke whale that had been beached in Denmark (Palsbøll and others 2006). After genotyping samples at 12 loci, the products could be successfully matched to profiles in the DNA register. The ability to detect illegitimate (unreported) samples was verified with the beached whale samples from Denmark. Despite the promising results of the DNA register, the study did report a number of methodological problems that would need to be addressed before the register could be used to reliably monitor trade.
DNA Surveillance carries out phylogenetic analysis on user-submitted sequences to allow for the DNA-based identification of about 90 species of whales, dolphins, and porpoises (Ross and others 2003). This program is based on species-specific differences in the mtDNA control region and cyt b. DNA Surveillance has been utilized in market surveys to identify species in whalemeat products sold in South Korea during 1999–2005 (Baker and others 2006, 2007). In the 2007 study, Baker and others utilized a combination of microsatellite genotyping, mtDNA haplotype frequencies, and genetic sex identification to determine individual identification of North Pacific minke whale products assumed to originate from the “J” stock. Out of a total of 289 products purchased during 1999–2003, 205 were determined to represent unique individuals. The results of this study were used in a market analysis that estimated the true number of “J” stock minke whales on the South Korean market to be 827, almost twice the number officially reported as bycatch during this time period. Taking into account “J” stock whales that were reportedly killed as bycatch in the Japanese fishery from 1999 to 2003 gives a total of over 1200 individual North Pacific minke whales taken from a depleted stock over a 5-y period (Palumbi 2007). Unfortunately, this exceeds the estimated rate of loss that can be sustained by the “J” stock: a previous population dynamics model determined that a loss of 50 to 150 minke whales per year would result in extinction of the “J” stock by mid-century (Baker and others 2000). These predictions illustrate the importance of increasing monitoring and regulation efforts in order for the North Pacific minke whale to continue to exist in the Sea of Japan.