In the last decade, global seafood supply and trade have increased substantially. This has simultaneously increased the need for seafood safety and authenticity assessment. DNA barcodes or genetic barcodes have been designed for accurate seafood species authentication, which decreases mislabeling and fraud in the seafood industry.
An Introduction to Genetic Barcoding
DNA or genetic barcoding is a method used to detect species based on the identification of a short section of DNA from specific genes, which is referred to as the DNA barcode.1 The working principle of DNA barcoding is based on the comparison of genomic sections of unknown species with a reference library. Therefore, an organism or species can be identified in a manner similar to the method by which a supermarket scanner scans black stripes of the UPC barcode to identify an item against a reference database.2 The key advantage of DNA barcoding is its capacity to identify plants, even in the absence of flowers or fruits or insect larvae that have fewer diagnostic features.
Different gene regions are used as barcodes to identify specific organisms. A section of the cytochrome c oxidase I (COI or COX1) gene, which is located in the mitochondrial DNA, is the most commonly used genetic barcode for animal identification. Besides COX1, the internal transcribed spacer (ITS) rRNA and ribulose bisphosphate carboxylase oxygenase (RuBisCO) are used as a genetic barcode for fungal and plant species identification, respectively. The 16S rRNA gene and 18S rRNA gene are mostly used for the identification of prokaryotes and microbial eukaryotes, respectively.3
There are three main criteria that must be fulfilled to consider a gene region as a DNA barcode. Firstly, the genetic sequence must retain considerable species-level variability. Secondly, the genome must contain conserved flaking sites to develop universal PCR primers for a wider taxonomic application. Finally, the length of the sequence for the DNA barcode must be appropriate for DNA extraction and amplification.4
In 2003, a standardized DNA barcoding technique and terminology were proposed to identify species. Paul D.N. Hebert and his colleagues from the University of Guelph, Canada, were the first to demonstrate the use of the COI gene as a barcode for species identification.5
Why is Seafood Authentication Necessary?
Seafood is a popular and significantly demanded product of the global fish and aquaculture industries. In many developing countries, seafood is the most valued traded commodity, whose net export value could be more than that of tobacco, sugar, and rice combined.
According to the Food and Agriculture Organization of the United Nations, global fish demand has increased on average by 3.1% per year between 1961 and 2017. It has doubled between 1960 and 2018. To meet the global demand for seafood, there is a need to implement novel technologies to enhance production, sustainably.6
A rapid increase in international fish trade invoked the need for better traceability of fishery products.7 In some cases, fish species substitutions have been recorded, which has increased concerns linked to food quality and safety. Fish substitutions are purposely done to increase profits because higher-value fish are replaced with other less well-known, illegal, and often cheaper fish. However, fish substitution may also occur accidentally when fish species share remarkable phenotypic similarities.
Many studies have shown that substituted fish may carry hidden risks to consumers. For instance, if a fish is substituted with cryptic species of contaminated areas without sanitary checks, it may induce many allergic reactions. The consumption of poisonous fish could lead to diarrhea, oxidative stress, and weight loss. Furthermore, it can also lead to a decrease in alkaline phosphatase and alanine aminotransferase concentrations in blood plasma.8 Fish traceability measures play a vital role in avoiding issues linked to fish substitution.
The European Union (EU) associated Common Fisheries Policy and the Common Market Organization have recommended the labelling of fisheries and aquaculture products.9 Many innovative methods and technologies have been implemented to identify taxa accurately. DNA Barcoding is one of the most effective strategies linked with fish traceability. At present, this strategy has been regarded as a golden tool in protecting economies and consumer's health.
DNA Barcode for Seafood
The COI mitochondrial gene has been used as a popular barcode marker in fish. A recently improved COI barcoding protocol has enhanced the species prediction accuracy. In this context, the COI-2 and COI-3 primer cocktails were found to be more effective as mammalian and fish barcodes. Besides the COI mitochondrial gene, rhodopsin and 16S rDNA are also used as DNA barcodes for seafood.
In Italy, a genetic barcode using 650 bp of COI gene was developed to identify seafood fraud. The application of this strategy helped identify several incorrect labels on seafood samples.10 Furthermore, this strategy enabled the identification of mislabelled Lagocephalus spp, a poisonous fish species that has been banned from the EU market, as squid. Similarly, the DNA barcoding technique was implemented in the Bulgarian wholesale fish market, which revealed the prevalence of a high level of species substitution.
Some merchants replace “white tuna” with Lepidocybum flavorunneum (Escolar), a banned fish species in Italian and Japanese markets. Commonly fish species with higher value, such as Solea solea, Merluccius merluccius, and Pleuronectes platessa are mislabelled with less valuable fish, such as Pangasius hypophthalmus.8
DNA barcodes based on cytb sequences revealed the substitution of Lutjanus campechanus (red snapper) with less valuable species of Lutjanidae in the US.11 This technique has effectively determined seafood mislabelling in many other countries, such as the United Kingdom, Spain, Germany, Italy, Greece, France, and Portugal.
Evolution of DNA Barcoding Technologies
DNA barcoding technologies for species identification and authentication have advanced significantly over the years due to the availability and advancements in comprehensive barcode databases, such as GenBank. This database is a part of the International Nucleotide Sequence Database Collaboration (NCBI) that comprises the European Nucleotide Archive (ENA) and DNA DataBank of Japan (DDBJ). In addition, the Barcode of Life Database (BOLD) is a cloud-based data storage that supports the development and applications of genetic barcodes.
The Fish Barcode of Life Initiative (FISH-BOL) was developed in 2005 and contains DNA barcode records of around 8000 fish species.12 Fish-Trace is another European database that contains information on 220 marine species from 75 different families. Aquagene is a free database that offers genetic information on more than 603 marine species.
Evolutionary models, such as the Kimura2-parameter distance method, are used to compute nucleotide variations that offer a correct match between the target and reference sequences.13 Advancements in genomic technologies, such as the development of Restriction Site Associated DNA sequencing, have enabled the differentiation of two closely related species. The rapid progress in Next Generation Sequencing techniques has significantly contributed to the evolution of the genetic barcoding technology.
- Amaral J, Meira L, Oliveira M, Mafra I. Advances in Authenticity Testing for Meat Speciation. Advances in Food Authenticity Testing. 2016; 369-414. https://doi.org/10.1016/B978-0-08-100220-9.00014-X
- Yang F, Ding F, Chen H, et al. DNA Barcoding for the Identification and Authentication of Animal Species in Traditional Medicine. Evid Based Complement Alternat Med. 2018; doi:10.1155/2018/5160254
- Guo M, Yuan C, Tao L. et al. Life barcoded by DNA barcodes. Conservation Genet Resour. 2022; 14, 351–365. https://doi.org/10.1007/s12686-022-01291-2
- Kress WJ, Erickson DL. DNA barcodes: genes, genomics, and bioinformatics. Proc Natl Acad Sci USA. 2008; 105(8):2761-2762. doi:10.1073/pnas.0800476105
- Hebert PD, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Biol Sci. 2003; 270(1512):313-321. doi:10.1098/rspb.2002.2218
- Naylor RL, Kishore A, Sumaila UR, et al. Blue food demand across geographic and temporal scales. Nat Commun. 2021;12(1):5413. doi:10.1038/s41467-021-25516-4
- Vindigni G, Pulvirenti A, Alaimo S, Monaco C, Spina D, Peri I. Bioinformatics Approach to Mitigate Mislabeling in EU Seafood Market and Protect Consumer Health. Int J Environ Res Public Health. 2021;18(14):7497. doi:10.3390/ijerph18147497
- Filonzi L, Ardenghi A, Rontani PM, Voccia A, Ferrari C, Papa R, Bellin N, Nonnis Marzano F. Molecular Barcoding: A Tool to Guarantee Correct Seafood Labelling and Quality and Preserve the Conservation of Endangered Species. Foods. 2023; 12(12):2420. https://doi.org/10.3390/foods12122420
- Armani A. et al. Is raw better? A multiple DNA barcoding approach (full and mini) based on mitochondrial and nuclear markers reveals low rates of misdescription in sushi products sold on the Italian market. Food Control. 2017; 79, 126-133. https://doi.org/10.1016/j.foodcont.2017.03.030
- Dawan J, Ahn J. Application of DNA barcoding for ensuring food safety and quality. Food Sci Biotechnol. 2022;31(11):1355-1364. doi:10.1007/s10068-022-01143-7
- Marval-Rodríguez A, Renán X, Galindo-Cortes G, Acuña-Ramírez S, Jiménez-Badillo MdL, Rodulfo H, Montero-Muñoz JL, Brulé T, De Donato M. Assessing the Speciation of Lutjanus campechanus and Lutjanus purpureus through Otolith Shape and Genetic Analyses. Fishes. 2022; 7(2):85. https://doi.org/10.3390/fishes7020085
- Ward RD. FISH-BOL, a case study for DNA barcodes. Methods Mol Biol. 2012; 858:423-439. doi:10.1007/978-1-61779-591-6_21
- Nishimaki T, Sato K. An Extension of the Kimura Two-Parameter Model to the Natural Evolutionary Process. J Mol Evol. 2019; 87(1):60-67. doi:10.1007/s00239-018-9885-1