DNA‐Based Methods for the Identification of Commercial Fish and Seafood Species

ABSTRACT: The detection of species substitution has become an important topic within the food industry and there is a growing need for rapid, reliable, and reproducible tests to verify species in commercial fish and seafood products. Increases in international trade and global seafood consumption, along with fluctuations in the supply and demand of different fish and seafood species, have resulted in intentional product mislabeling. The effects of species substitution are far‐reaching and include economic fraud, health hazards, and illegal trade of protected species. To improve detection of commercial seafood fraud, a variety of DNA‐based techniques have been developed, including Multiplex PCR, FINS, PCR‐RFLP, PCR‐RAPD, PCR‐AFLP, and PCR‐SSCP, which are all based on polymorphisms in the genetic codes of different species. These techniques have been applied in the differentiation of many types of fish and seafood species, such as gadoids, salmonids, scombroids, and bivalves. Some emerging technologies in this field include the use of real‐time PCR, lab‐on‐a‐chip, and DNA microarray chips. In this review article, the major DNA‐based methods currently employed in the authentication of commercial fish and seafood species are discussed and future trends are highlighted. Examples of commercial applications and the use of online database resources are also considered.     

Implementation of quality control methods (physico-chemical, microbiological and sensory) in conjunction with multivariate analysis towards fish authenticity

Nowadays authenticity of foods and fish in particular has become of crucial importance because of high number of adulteration cases. Authenticity control has gained ground thanks to the development of several rapid physico‐chemical and microbiological methods aiming at distinguishing one species from another based on solid scientific evidence. It has been proven that despite the precision and accuracy of robust analytical and protein and DNA‐based techniques, detection of authenticity could not be claimed without resorting to multivariate analysis. This review summarizes both the most advanced and state of the art used techniques for detecting fish and seafood authenticity (both in terms of species and geographical origin). Another issue reported in this review is the preservation of fish and seafood through the implementation of old and novel techniques (ice, modified atmosphere packaging). Several informative tables were included in this paper referring to the employed quality control and sensory analysis methods and multivariate analysis for fish and seafood.     

Microplastics in coastal areas and seafood: implications for food safety

Microplastics have become ubiquitous in the marine environment. Microplastics have been detected in many coastal environments and species, including commercial seafood. This triggers concern about potential economic impacts and the risks of dietary exposure, especially for coastal communities. However, data regarding the levels of microplastics in coastal seafood and their toxicological effects are still limited. Accordingly, the dietary risk is still poorly explored. This review summarizes and discusses recent scientific findings on (i) the presence of microplastics in coastal waters, (ii) the occurrence of microplastics in coastal seafood and the likelihood of trophic transfer, and (iii) the effects of microplastics on coastal fish and shellfish species. Human toxicity data are also reviewed, but the risks for human health are difficult to determine due to limited data. Based on available worldwide data, the estimation of microplastics intake through seafood consumption shows a huge variation. Additionally, a lack of standardized analytical methods complicates the comparison of results between studies and therefore seriously affects the reliability of risk assessments. It is concluded that more exposure and toxicity data are needed properly to assess human health risks of microplastics in coastal seafood, and the lack of data currently impede the derivation of a risk-based food safety standard. The pros and cons of an interim solution, i.e. setting a provisional action level, are being discussed.     

Marketplace substitution of Atlantic salmon for Pacific salmon in Washington State detected by DNA barcoding

Accurate identification of seafood in the marketplace is an issue of international concern, due to high rates of market substitution of cheaper or more widely available species for expensive or high-demand species. Salmon samples from stores and restaurants throughout western Washington, USA were tested using DNA sequencing of a short section of the mitochondrial cytochrome c oxidase I (COI) gene (DNA barcoding) to identify Atlantic salmon substituted for Pacific salmon. Of 99 salmon samples, 11 (11%) were Atlantic salmon sold as Pacific salmon. More than 38% of restaurant samples were mislabeled to species, while only 7% of store samples were mislabeled. Market substitution rates were significantly greater in restaurants compared to stores, and consistently greater in winter compared to spring, although not significantly. The high market substitution rate in restaurants documents a pressing need for more monitoring and enforcement specifically in restaurants. DNA barcoding is a valuable tool for rapid and definitive authentication of salmon in the marketplace, and should be more widely adopted to discourage market substitution.     

The use of molecular markers in the verification of fish and seafood authenticity and the detection of adulteration

The verification of authenticity and detection of food mislabeling are elements that have been of high importance for centuries. During the last few decades there has been an increasing consumer demand for the verification of food identity and the implementation of stricter controls around these matters. Fish and seafood are among the most easily adulterated foodstuffs mainly due to the significant alterations of the species' morphological characteristics that occur during the different types of processing, which render the visual identification of the animals impossible. Even simple processes, such as filleting remove very important morphological elements and suffice to prevent the visual identification of species in marketed products. Novel techniques have therefore been developed that allow identification of species, the differentiation between species and also the differentiation of individuals that belong to the same species but grow in different populations and regions. Molecular markers have been used during the last few decades to fulfill this purpose and several improvements have been implemented rendering their use applicable to a commercial scale. The reliability, accuracy, reproducibility, and time-and cost-effectiveness of these techniques allowed them to be established as routine methods in the industry and research institutes. This review article aims at presenting the most important molecular markers used for the authentication of fish and seafood. The most important techniques are described, and the results of numerous studies are outlined and discussed, allowing interested parties to easily access and compare information about several techniques and fish/seafood species.     

海产品品种识别

随着捕捞工业发展,捕捞量增加,消费市场中鱼类品种增加。出现低质量海产品冒充高品质产品现象,所以需要找出有效的品种鉴定方法。本文介绍了目前应用于品种鉴定的几种方法,包括蛋白质分子标记技术及DNA分子标记技术。     

How to Determine the Geographical Origin of Seafood?

Traceability of seafood is a much needed service for the seafood industry. Current ways of tracing seafood are minimal while tracing of shellfish is nearly nonexistent. Tracing fish and shellfish are necessary for indicating where the fish and shellfish were fished from, farmed and packed from. This study reviews history of traceability of aquaculture and analytical approaches to verify the origin of seafood. It then describes the new molecular technique of the traceability by using PCR-DGGE to discriminate the geographical origin of fish (cases studies of Pangasius fish from Viet Nam and Sea bass fish from France) by analysis the DNA fragments of microorganisms (bacteria) on fish. This method is based on the assumption that the microbial communities of food are specific to a geographic area.     

Application of barcoding to Amazonian commercial fish labelling

Methods for seafood authentication are increasingly demanded in food sciences. In this study we have detected mislabelling for eight out of 22 Amazonian fish species in two Brazilian markets, employing species-specific DNA markers. Mislabelling was most likely due to inadvertent errors in fish classification. These results emphasize the convenience of applying DNA methodology for seafood authentication.     

DNA barcoding reveals high substitution rate and mislabeling in croaker fillets (Sciaenidae) marketed in Brazil: The case of “pescada branca” (Cynoscion leiarchus and Plagioscion squamosissimus)

The removal of morphological features during fish processing hinders identification to the species level, increasing the chances of species substitution and the mislabeling of marketed products. We used DNA barcoding to assess whether species substitutions occur in croaker (Sciaenidae) fillets labeled as “pescada branca” sold in the Brazilian Amazon, where two species are known under this vernacular name (Cynoscion leiarchus and Plagioscion squamosissimus). A 577-bp cytochrome C oxidase subunit I (COI) sequence was obtained from 137 fillets and compared with the sequences of whole Sciaenidae fish that were identified based on their morphology and the reference sequences of the BOLD and GenBank public databases. DNA barcoding was able to identify 90% of the samples analyzed to the species level, and the results showed a high rate of species substitution in the fillets labeled as “pescada branca”. The substitution rate was 100% if using the criterion that the fillets should be C. leiarchus and 76.6% if using the criterion that they should be P. squamosissimus. Additionally, the results show that “pescada branca” was replaced in most cases by species of lower commercial value, which clearly demonstrates economic fraud aimed at increased profits. Our data confirm that DNA barcoding is a sensitive and reliable tool that can be applied to authenticate processed fish.     

Authentication of raw and processed tuna from Indonesian markets using DNA barcoding,nuclear gene and character-based approach

Establishing seafood authentication methods is an important task for fisheries research laboratories and food control authorities. Nowadays, the extent of fish species substitution is suspected being greater than ever before in commercial markets. In order to provide reliable polymerase chain reaction (PCR)-based authentication systems for tunas, we collected and analyzed authentic tuna reference samples and tuna-food products from Indonesian markets. Our analytical methods mainly relied on identification using the mitochondrial cytochrome c oxidase subunit I (COI) gene, as a genetic marker for “DNA barcoding,” as well as the rhodopsin (RH1) gene as a nuclear marker. Additionally, we identified species-specific nucleotide diagnostic positions (characters) to complete the results obtained basic local alignment search (BLAST) and phylogenetic analysis. Authentication results of tuna-food products showed relatively successful amplification for the COI gene; RH1 acted as an alternative solution for some of the samples, which had failed to react in COI-PCR. Species of the genus Thunnus could not be unambiguously differentiated by BLAST and phylogenetic analysis (neighbor-joining tree) in all cases due to the high similarity of the COI sequences. However, the character-based identification method was found to be helpful for species assignment in case of tuna-food products. Therefore, our findings demonstrated that the COI gene could be more reliable used as a tool for Indonesian commercial tuna products authentication, if the sequencing results were combined with the character-based identification using differences at certain nucleotide positions.     

The use of stable isotopes for authentication of gadoid fish species

The rise of processed seafood in international trade has increased the feasibility of fish species substitution. Gadidae fish species are sold commercially as salted fish, and differences in price between fish of different species may lead to falsification. The present study addresses this falsification issue by attempting to discriminate among salted Atlantic cod and salted saithe using isotope ratio mass spectrometry (IRMS) as well as the stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N). δ¹⁵N in tissues with lower turnover rates (bone and skin) and in tissues with greater turnover rates (muscle) can be used to authenticate the species of salted fish samples when distinguishing between Atlantic cod and saithe.     

Risk‐Benefit Analysis of Seafood Consumption: A Review

Abstract: Seafood, defined here as marine and freshwater fish and shellfish, is recognized as a healthy food choice because it is a low‐fat protein source that provides long‐chain omega‐3 fatty acids important for early development along with eye and heart health. However, seafood is also known to contain certain contaminants, such as methylmercury and persistent organic pollutants, which can have harmful effects on human health and development. In order to limit exposure to contaminants while maximizing the benefits of seafood consumption, a number of quantitative and qualitative risk‐benefit analyses have been conducted for seafood consumption. This review paper provides a brief background on risk‐benefit analysis of foods, followed by a discussion of the risks and benefits associated with fish consumption. Next, risk‐benefit analyses are reviewed in an historical context. While risk‐benefit analysis consists of three main elements (that is, assessment, management, and communication), this review will primarily focus on risk‐benefit assessments. Overall, most studies have found that the benefits far outweigh the risks among the general population, especially when a variety of fish is consumed at least twice per week. However, for certain populations (for example, pregnant women and young children) a more targeted approach is warranted in order to ensure that these groups consume fish that are low in contaminants but high in omega‐3 fatty acids. The potentially harmful unintended consequences of risk‐benefit communication on the general population and certain groups are also discussed.     

Development of a genetic method for the identification of salmon,trout, and bream in seafood products by means of PCR–RFLP and FINS methodologies

In the present study, two alternative methods for identifying 13 salmon, trout and bream species were developed. Both of them are based on polymerase chain reaction (PCR) amplification of a cytochrome b gene fragment. Subsequently, different techniques were assayed to assign the PCR amplicons previously obtained to particular species. The first one is based on the restriction fragment length polymorphism (RFLP) and includes three endonucleases for generating species-specific restriction profiles, while the second one is based on the phylogenetic analysis of DNA sequences. The main novelty of this work lies in the applicability of the developed methods to all kinds of processed products, including those undergoing intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 25 commercial samples including some that had been subjected to intensive thermal treatment, allowing the detection of those incorrectly labeled (16%). Therefore, these methods are useful to check the fulfillment of labeling regulations for seafood products, verify the correct traceability in commercial trade, and for fisheries control.     

Universal mitochondrial 16s rRNA biomarker for mini-barcode to identify fish species in Malaysian fish products

Mislabelling in fish products is a highly significant emerging issue in world fish trade in terms of health and economic concerns. DNA barcoding is an efficient sequencing-based tool for detecting fish species substitution but due to DNA degradation, it is in many cases difficult to amplify PCR products of the full-length barcode marker (~650 bp), especially in severely processed products. In the present study, a pair of universal primers targeting a 198 bp sequence of the mitochondrial 16s rRNA gene was designed for identification of fish species in the processed fish products commonly consumed in Malaysia. The specificity of the universal primers was tested by both in-silico studies using bioinformatics software and through cross-reaction assessment by practical PCR experiments against the DNA from 38 fish species and 22 other non-target species (animals and plants) and found to be specific for all the tested fish species. To eliminate the possibility of any false-negative detection, eukaryotic endogenous control was used during specificity evaluation. The developed primer set was validated with various heat-treated (boiled, autoclaved and microwaved) fish samples and was found to show high stability under all processing conditions. The newly developed marker successfully identified 92% of the tested commercial fish products with 96–100% sequence similarities. This study reveals a considerable degree of species mislabelling (20.8%); 5 out of 24 fish products were found to be mislabelled. The new marker developed in this work is a reliable tool to identify fish species even in highly processed products and might be useful in detecting fish species substitution thus protecting consumers’ health and economic interests.     

A method to detect the parasitic nematodes from the family Anisakidae, in Sardina pilchardus, using specific primers of 18 S DNA gene

Anisakiasis is the infestation of man by the third larval stage of the Anisakidae family parasites, caused by the consumption of raw or undercooked seafood. The hosts of the third larvae parasite are marine fishes, among which Sardina pilchardus is of high commercial interest in many countries. So, the detection of anisakid larvae in fisheries products (fresh and processed) is of great interest to a rapid and feasible diagnosis of parasitism. Currently, there are different types of parasite detection methods but they are all difficult and tedious. The aim of this work was to detect, using DNA-based techniques, the presence of anisakid larvae in the entire fish host. Three pairs of primers were designed in the 18 S DNA gene taking into account the maximum matching between the anisakid species sequences and the minimum matching concerning for both the fish species and some other groups of organisms that parasitize fish (non-anisakid nematodes, trematodes, and cestodes). Experimental mixtures of parasite and fish host were made and the sensitivity of anisakid DNA detection tested by PCR. All the above primers and two others combinations of them were specific for the nematode group within the studied species, and the sensitivity of detection was on the order of 1 part of nematode to 100 000 parts of fish (anisakid weight:fish sample fresh weight = 1:≈100 000).     

Detection of Different DNA Animal Species in Commercial Candy Products

Candy products are consumed all across the world, but there is not much information about their composition. In this study we have used a DNA‐based approach for determining the animal species occurring in 40 commercial candies of different types. We extracted DNA and performed PCR amplification, cloning and sequencing for obtaining species‐informative DNA sequences. Eight species were identified including fish (hake and anchovy) in 22% of the products analyzed. Bovine and porcine were the most abundant appearing in 27 samples each one. Most products contained a mixture of species. Marshmallows (7), jelly‐types, and gummies (20) contained a significantly higher number of species than hard candies (9). We demonstrated the presence of DNA animal species in candy product which allow consumers to make choices and prevent allergic reaction.     

Validation of a method for the detection of five species,serogroups, biotypes and virulence factors of Vibrio by multiplex PCR in fish and seafood

In this work a sequential multiplex PCR system was designed and validated for the detection of most frequent foodborne pathogen Vibrio species in fish and seafood (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginoliticus and Vibrio mimicus). The method proposed functions in a hierarchical way, being composed of an end-point multiplex PCR to detect the presence of DNA belonging to the studied species, followed by multiplex PCR and fragment analysis allowing the viability assessment of the detected strains. The final multiplex PCR step of the method may be applied if identification of the serogroup, biotype and/or virulence factor level is necessary. Forty samples of commercial fish and seafood products were used at the method validation stage. Sixty three marine organism samples obtained from various estuarine areas of Spain including shrimps, crabs, bivalve mollusks and fishes were screened for presence of Vibrio species and 2 mussel samples were found positive for V. parahaemolyticus. On the whole, the proposed method is robust and readily adaptable in routine molecular diagnostic laboratories, allowing monitoring and simultaneous detection of all these bacterial pathogens in seafood samples, reducing the expenses and time consumed by other analytical methods.     

Authentication of the most important species of rockfish by means of fins

In the present work, a method for the authentication of more of 100 scorpaenids species has been developed by means of forensically informative nucleotide sequencing technique, which is based on a PCR followed by a phylogenetic analysis. Due to the different commercial value of the species belonging to this taxonomic group, substitutions between species in seafood products can take place. Two different methodological strategies are proposed, allowing the analysis of fresh, frozen, or highly processed products with total reliability. These analytical systems have been validated and subsequently applied to 25 commercial samples. Therefore, this technique can be used as a routine method to avoid the mislabeling in the marketing of scorpaenid species and it is also suitable to assess the correct seafood traceability of these products.     

POTENTIAL FUTURE CHALLENGES TO THE APPLICATION OF GAMMA IRRADIATION TO SEAFOOD IN TUNISIA

There is an increasing consumer demand for high quality, minimally processed, additive-free and microbiologically safe foods. The future application of irradiation to the seafood industry is developing. In order to investigate the feasibility of using gamma irradiation in the fish and seafood industry, questionnaires were distributed to the managers of 25 fish exporting companies. The cost of irradiation and the additional transport services were discussed and mostly accepted. This survey showed that essential conditions are to be found in Tunisia to introduce irradiation technology into the exporting seafood industry. However, the legal status of seafood irradiation varies in some importing countries (mainly European Union [EU]). Regulators within the EU have taken different approaches to allowing processors to use this technology. Such inconsistency is related to the objection of misinformed consumers. Consequently, commercial trade of irradiated seafood could be impeded.

PRACTICAL APPLICATIONS

This article provides evidence of the importance of industry managers' role in the food chain when testing the feasibility of a new process. Fish shelf-life extension using gamma irradiation is an emerging technology that presents many advantages to all food chain components and allows international trade requirements to be met. The seafood export industry in Tunisia shows the technological need for such a process. Furthermore, this study demonstrated that the economic cost of irradiation in Tunisia is acceptable and that it could enhance fish export capacity. However, the legal status of fish irradiation in the import countries, mainly the European Union, should be modernized in order to assure the feasibility of irradiated fish export from Tunisia.     

Norwegian monitoring programme on the inorganic and organic contaminants in fish caught in the Barents Sea, Norwegian Sea and North Sea, 1994-2001

The results from part of a monitoring programme of contaminant levels in fish and other seafood products initiated by the Directorate of Fisheries in Norway in 1994 are presented. Concentrations of 22 elements (four are presented here: As, Cd, Hg and Pb) and HCB, HCH, PCB 28, 52, 101, 105, 118, 138, 153, 156, 180, pp-DDD, p-DDE, p-DDT, sum DDT and ¹³⁷Cs were determined in 17 species of fish caught in three sampling locations: the Barents Sea, the Norwegian Sea and the North Sea. The fish species analysed in the survey were limited to species of commercial importance for Norway with catching volumes of at least 10 000 metric tons year^-1. The survey started in 1994 and is expected to continue beyond 2010. The analyses were carried out on 25 individual fish from each species and each sampling location, and the locations were representative of commercial fishing grounds for the species in question. The concentrations of contaminants found were considerably lower than the maximum levels permissible in fish set by CODEX and the European Union for contaminants in seafood products.